Oral Gavage Delivery of Stable Isotope Tracer for in Vivo Metabolomics

Stable isotope-resolved metabolomics (SIRM) is a powerful tool for understanding disease. Advances in SIRM techniques have improved isotopic delivery and expanded the workflow from exclusively in vitro applications to in vivo methodologies to study systemic metabolism. Here, we report a simple, minimally-invasive and cost-effective method of tracer delivery to study SIRM in vivo in laboratory mice. Following a brief fasting period, we orally administered a solution of [U-13C] glucose through a blunt gavage needle without anesthesia, at a physiological dose commonly used for glucose tolerance tests (2 g/kg bodyweight). We defined isotopic enrichment in plasma and tissue at 15, 30, 120, and 240 min post-gavage. 13C-labeled glucose peaked in plasma around 15 min post-gavage, followed by period of metabolic decay and clearance until 4 h. We demonstrate robust enrichment of a variety of central carbon metabolites in the plasma, brain and liver of C57/BL6 mice, including amino acids, neurotransmitters, and glycolytic and tricarboxylic acid (TCA) cycle intermediates. We then applied this method to study in vivo metabolism in two distinct mouse models of diseases known to involve dysregulation of glucose metabolism: Alzheimer’s disease and type II diabetes. By delivering [U-13C] glucose via oral gavage to the 5XFAD Alzheimer’s disease model and the Lepob/ob type II diabetes model, we were able to resolve significant differences in multiple central carbon pathways in both model systems, thus providing evidence of the utility of this method to study diseases with metabolic components. Together, these data clearly demonstrate the efficacy and efficiency of an oral gavage delivery method, and present a clear time course for 13C enrichment in plasma, liver and brain of mice following oral gavage of [U-13C] glucose—data we hope will aid other researchers in their own 13C-glucose metabolomics study design

Homozygous loss-of-function mutations in SLC26A7 cause goitrous congenital hypothyroidism.

Defects in genes mediating thyroid hormone biosynthesis result in dyshormonogenic congenital hypothyroidism (CH). Here, we report homozygous truncating mutations in SLC26A7 in 6 unrelated families with goitrous CH and show that goitrous hypothyroidism also occurs in Slc26a7-null mice. In both species, the gene is expressed predominantly in the thyroid gland, and loss of function is associated with impaired availability of iodine for thyroid hormone synthesis, partially corrected in mice by iodine supplementation. SLC26A7 is a member of the same transporter family as SLC26A4 (pendrin), an anion exchanger with affinity for iodide and chloride (among others), whose gene mutations cause congenital deafness and dyshormonogenic goiter. However, in contrast to pendrin, SLC26A7 does not mediate cellular iodide efflux and hearing in affected individuals is normal. We delineate a hitherto unrecognized role for SLC26A7 in thyroid hormone biosynthesis, for which the mechanism remains unclear

APOΕ4 Lowers Energy Expenditure in Females and Impairs Glucose Oxidation by Increasing Flux through Aerobic Glycolysis

BACKGROUND: Cerebral glucose hypometabolism is consistently observed in individuals with Alzheimer\u27s disease (AD), as well as in young cognitively normal carriers of the Ε4 allele of Apolipoprotein E (APOE), the strongest genetic predictor of late-onset AD. While this clinical feature has been described for over two decades, the mechanism underlying these changes in cerebral glucose metabolism remains a critical knowledge gap in the field. METHODS: Here, we undertook a multi-omic approach by combining single-cell RNA sequencing (scRNAseq) and stable isotope resolved metabolomics (SIRM) to define a metabolic rewiring across astrocytes, brain tissue, mice, and human subjects expressing APOE4. RESULTS: Single-cell analysis of brain tissue from mice expressing human APOE revealed E4-associated decreases in genes related to oxidative phosphorylation, particularly in astrocytes. This shift was confirmed on a metabolic level with isotopic tracing of 13C-glucose in E4 mice and astrocytes, which showed decreased pyruvate entry into the TCA cycle and increased lactate synthesis. Metabolic phenotyping of E4 astrocytes showed elevated glycolytic activity, decreased oxygen consumption, blunted oxidative flexibility, and a lower rate of glucose oxidation in the presence of lactate. Together, these cellular findings suggest an E4-associated increase in aerobic glycolysis (i.e. the Warburg effect). To test whether this phenomenon translated to APOE4 humans, we analyzed the plasma metabolome of young and middle-aged human participants with and without the Ε4 allele, and used indirect calorimetry to measure whole body oxygen consumption and energy expenditure. In line with data from E4-expressing female mice, a subgroup analysis revealed that young female E4 carriers showed a striking decrease in energy expenditure compared to non-carriers. This decrease in energy expenditure was primarily driven by a lower rate of oxygen consumption, and was exaggerated following a dietary glucose challenge. Further, the stunted oxygen consumption was accompanied by markedly increased lactate in the plasma of E4 carriers, and a pathway analysis of the plasma metabolome suggested an increase in aerobic glycolysis. CONCLUSIONS: Together, these results suggest astrocyte, brain and system-level metabolic reprogramming in the presence of APOE4, a \u27Warburg like\u27 endophenotype that is observable in young females decades prior to clinically manifest AD

It Costs to Be Clean and Fit: Energetics of Comfort Behavior in Breeding-Fasting Penguins

), seabirds known to fast for up to one month during incubation shifts ashore.A time budget was estimated from focal and scan sampling field observations and the energy cost of comfort activities was calculated from the associated increase in heart rate (HR) during comfort episodes, using previously determined equations relating HR to energy expenditure. We show that incubating birds spent 22% of their daily time budget in comfort behavior (with no differences between day and night) mainly devoted to preening (73%) and head/body shaking (16%). During comfort behavior, energy expenditure averaged 1.24 times resting metabolic rate (RMR) and the corresponding energy cost (i.e., energy expended in excess to RMR) was 58 kJ/hr. Energy expenditure varied greatly among various types of comfort behavior, ranging from 1.03 (yawning) to 1.78 (stretching) times RMR. Comfort behavior contributed 8.8–9.3% to total daily energy expenditure and 69.4–73.5% to energy expended daily for activity. About half of this energy was expended caring for plumage.This study is the first to estimate the contribution of comfort behavior to overall energy budget in a free-living animal. It shows that although breeding on a tight energy budget, king penguins devote a substantial amount of time and energy to comfort behavior. Such findings underline the importance of comfort behavior for the fitness of colonial seabirds

Homozygous loss-of-function mutations in SLC26A7 cause goitrous congenital hypothyroidism

Defects in genes mediating thyroid hormone biosynthesis result in dyshormonogenic congenital hypothyroidism (CH). Here, we report homozygous truncating mutations in SLC26A7 in 6 unrelated families with goitrous CH and show that goitrous hypothyroidism also occurs in Slc26a7-null mice. In both species, the gene is expressed predominantly in the thyroid gland, and loss of function is associated with impaired availability of iodine for thyroid hormone synthesis, partially corrected in mice by iodine supplementation. SLC26A7 is a member of the same transporter family as SLC26A4 (pendrin), an anion exchanger with affinity for iodide and chloride (among others), whose gene mutations cause congenital deafness and dyshormonogenic goiter. However, in contrast to pendrin, SLC26A7 does not mediate cellular iodide efflux and hearing in affected individuals is normal. We delineate a hitherto unrecognized role for SLC26A7 in thyroid hormone biosynthesis, for which the mechanism remains unclear

Phagocytosis of Staphylococcus aureus by Macrophages Exerts Cytoprotective Effects Manifested by the Upregulation of Antiapoptotic Factors

It is becoming increasingly apparent that Staphylococcus aureus are able to survive engulfment by macrophages, and that the intracellular environment of these host cells, which is essential to innate host defenses against invading microorganisms, may in fact provide a refuge for staphylococcal survival and dissemination. Based on this, we postulated that S. aureus might induce cytoprotective mechanisms by changing gene expression profiles inside macrophages similar to obligate intracellular pathogens, such as Mycobacterium tuberculosis. To validate our hypothesis we first ascertained whether S. aureus infection could affect programmed cell death in human (hMDMs) and mouse (RAW 264.7) macrophages and, specifically, protect these cells against apoptosis. Our findings indicate that S. aureus-infected macrophages are more resistant to staurosporine-induced cell death than control cells, an effect partly mediated via the inhibition of cytochrome c release from mitochondria. Furthermore, transcriptome analysis of human monocyte-derived macrophages during S. aureus infection revealed a significant increase in the expression of antiapoptotic genes. This was confirmed by quantitative RT-PCR analysis of selected genes involved in mitochondria-dependent cell death, clearly showing overexpression of BCL2 and MCL1. Cumulatively, the results of our experiments argue that S. aureus is able to induce a cytoprotective effect in macrophages derived from different mammal species, which can prevent host cell elimination, and thus allow intracellular bacterial survival. Ultimately, it is our contention that this process may contribute to the systemic dissemination of S. aureus infection

Pathological chemotherapy response score is prognostic in tubo-ovarian high-grade serous carcinoma: A systematic review and meta-analysis of individual patient data

There is a need to develop and validate biomarkers for treatment response and survival in tubo-ovarian high-grade serous carcinoma (HGSC). The chemotherapy response score (CRS) stratifies patients into complete/near-complete (CRS3), partial (CRS2), and no/minimal (CRS1) response after neoadjuvant chemotherapy (NACT). Our aim was to review current evidence to determine whether the CRS is prognostic in women with tubo-ovarian HGSC treated with NACT.This article is freely available via Open Access. Click on the Publisher URL to access the full-text via the publisher's site

Small RNAs in Aedes aegypti: one giant step for virus control in mosquitoes

2022 Spring.Includes bibliographical references.Aedes aegypti mosquitoes are key vectors of medically relevant arthropod-borne (arbo) viruses such as Zika (ZIKV), dengue (DENV1-4), and yellow fever (YFV). When Ae. aegypti become infected with arboviruses, RNA interference (RNAi) is a critical antiviral immune mechanism that is a key determinant for successful virus transmission. The major antiviral pathway is the RNAi small-interfering RNA (siRNA) pathway, although evidence shows that the Piwi-interacting RNA (piRNA) pathway also acts as an important RNAi mechanism for controlling persistently infective viruses. The overarching goals of this work were twofold: (1) to determine the potency of the Ae. aegypti siRNA pathway against Zika virus and (2) to understand molecular mechanisms underlying piRNA-mediated antiviral immunity and its implications on mosquito vector competence. To achieve these goals, we (1) engineered transgenic Ae. aegypti mosquitoes that synthetically triggered the endogenous siRNA pathway against ZIKV and then quantified virus resistance in these mosquitoes, (2) sequenced small RNAs (sRNAs) of the mosquito virome that may impact vector competence and virus persistence, and (3) characterized structural features of Piwi4, an antiviral protein, involved in sRNA binding and subcellular localization to gain insights on its role in the piRNA and siRNA pathways. A major challenge in the fight against arboviruses is the lack of effective vaccines and limited therapeutic options. Vector control remains the primary method of preventing disease, and integrated vector management (IVM), including the genetic control of mosquitoes, is imperative to prevent emerging arboviral diseases. To this end, we designed an antiviral effector gene – a ZIKV-specific double stranded (ds) RNA –that synthetically triggered the mosquito's siRNA pathway after a bloodmeal in transgenic Ae. aegypti. Small RNA analyses in transgenic midguts revealed ZIKV-specific 21 nucleotide (nt) siRNAs 24 hours after a non-infectious bloodmeal. Nearly complete (90%) inhibition of ZIKV replication was found 7-to-14 days post-infection (dpi); furthermore, significantly fewer transgenic mosquitoes contained ZIKV in their salivary glands (p = 0.001), which led to a reduction in the number of ZIKV-containing saliva samples as measured by transmission assay. Our work shows that the siRNA pathway can be synthetically exploited to generate ZIKV-resistant Ae. aegypti mosquitoes. In the context of gene drive, antiviral effectors expressed in transgenic Ae. aegypti will be an invaluable tool for a population replacement vector control approach. piRNA-mediated antiviral immunity involves an endogenous viral element (EVE) – viral derived cDNA (vDNA) integrated into host genomes – as well as infection with a cognate virus, which together trigger piRNA amplification and lead to virus silencing. EVEs are from viruses that infected a population in previous generations, and most are derived from insect-specific viruses (ISVs) that persistently infect Ae. aegypti. We hypothesized that ISVs and ISV-derived piRNA populations, like EVEs, have geographic structure and impact vector competence to arboviruses. To test this hypothesis, we sequenced sRNAs from geographically distinct Ae. aegypti and characterized virus-derived sRNAs (vsRNAs). Overall, the distribution of total sRNAs was highly variable. Small RNAs derived from ISVs were diverse and dependent on geographic origin. We next infected Ae. aegypti from Poza Rica, Mexico with DENV2 and analyzed changes in the sRNA virome. DENV2 intrathoracic inoculation resulted in DENV2-specific siRNAs and piRNAs. We also found increased loads of sRNAs against the ISVs verdadero (Partitiviridae: unclassified), Aedes anphevirus (Xinmoviridae: Anphevirus), and chaq-like virus (Partitiviridae: unclassified) after DENV2 infection compared to ISV-derived sRNAs in controls. Overall, our study highlights the diversity of infective ISVs and the complexity of the sRNA virome across Ae. aegypti populations, which likely has consequences on sRNA crosstalk, virus replication, and vector competence. To gain insights on how Piwis, piRNA-binding proteins, are involved in virus control, we characterized structural features of an antiviral Piwi, Piwi4, involved in RNA binding and subcellular localization. We found that Piwi4 PAZ (Piwi/Argonaute/Zwille), the domain that binds the 3'-terminal ends of piRNAs, bound to mature (3'-terminal 2'-O-methylated) and 3'-terminal unmethylated RNAs with similar micromolar affinities (KD = 1.7 ± 0.8 μM and KD of 5.0 ± 2.2 μM, respectively) in a sequence independent manner. Through site-directed mutagenesis studies, we identified highly conserved residues involved in RNA binding and found that subtle changes in the amino acids flanking the binding pocket across PAZ proteins had significant impacts on binding behaviors, likely by impacting protein secondary structure. We also found that Piwi4 was both cytoplasmic and nuclear in mosquito tissues, and we identified a Piwi4 nuclear localization signal in the N-terminal region of the protein. These studies provide insights on the dynamic role of Piwi4 in RNAi and pave the way for future studies aimed at understanding Piwi4 interactions with diverse RNA populations

The Antiviral Small-Interfering RNA Pathway Induces Zika Virus Resistance in Transgenic Aedes aegypti

The resurgence of arbovirus outbreaks across the globe, including the recent Zika virus (ZIKV) epidemic in 2015–2016, emphasizes the need for innovative vector control methods. In this study, we investigated ZIKV susceptibility to transgenic Aedes aegypti engineered to target the virus by means of the antiviral small-interfering RNA (siRNA) pathway. The robustness of antiviral effector expression in transgenic mosquitoes is strongly influenced by the genomic insertion locus and transgene copy number; we therefore used CRISPR/Cas9 to re-target a previously characterized locus (Chr2:321382225) and engineered mosquitoes expressing an inverted repeat (IR) dsRNA against the NS3/4A region of the ZIKV genome. Small RNA analysis revealed that the IR effector triggered the mosquito’s siRNA antiviral pathway in bloodfed females. Nearly complete (90%) inhibition of ZIKV replication was found in vivo in both midguts and carcasses at 7 or 14 days post-infection (dpi). Furthermore, significantly fewer transgenic mosquitoes contained ZIKV in their salivary glands (p = 0.001), which led to a reduction in the number of ZIKV-containing saliva samples as measured by transmission assay. Our work shows that Ae. aegypti innate immunity can be co-opted to engineer mosquitoes resistant to ZIKV