Multiomics Characterization of a Less Invasive Microfluidic-Based Cell Sorting Technique.

Fluorescence-activated cell sorting (FACS) is a specialized technique to isolate specific cell subpopulations with a high level of recovery and accuracy. However, the cell sorting procedure can impact the viability and metabolic state of cells. Here, we performed a comparative study and evaluated the impact of traditional high-pressure charged droplet-based and microfluidic chip-based sorting on the metabolic and phosphoproteomic profile of different cell types. While microfluidic chip-based sorted cells more closely resembled the unsorted control group for most cell types tested, the droplet-based sorted cells showed significant metabolic and phosphoproteomic alterations. In particular, greater changes in redox and energy status were present in cells sorted with the droplet-based cell sorter along with larger shifts in proteostasis. 13C-isotope tracing analysis on cells recovering postsorting revealed that the sorter-induced suppression of mitochondrial TCA cycle activity recovered faster in the microfluidic chip-based sorted group. Apart from this, amino acid and lipid biosynthesis pathways were suppressed in sorted cells, with minimum impact and faster recovery in the microfluidic chip-based sorted group. These results indicate microfluidic chip-based sorting has a minimum impact on metabolism and is less disruptive compared to droplet-based sorting.

Notch signaling in thyrocytes is essential for adult thyroid function and mammalian homeostasis.

The thyroid functions as an apex endocrine organ that controls growth, differentiation and metabolism1, and thyroid diseases comprise the most common endocrine disorders2. Nevertheless, high-resolution views of the cellular composition and signals that govern the thyroid have been lacking3,4. Here, we show that Notch signalling controls homeostasis and thermoregulation in adult mammals through a mitochondria-based mechanism in a subset of thyrocytes. We discover two thyrocyte subtypes in mouse and human thyroids, identified in single-cell analyses by different levels of metabolic activity and Notch signalling. Therapeutic antibody blockade of Notch in adult mice inhibits a thyrocyte-specific transcriptional program and induces thyrocyte defects due to decreased mitochondrial activity and ROS production. Thus, disrupting Notch signalling in adult mice causes hypothyroidism, characterized by reduced levels of circulating thyroid hormone and dysregulation of whole-body thermoregulation. Inducible genetic deletion of Notch1 and 2 in thyrocytes phenocopies this antibody-induced hypothyroidism, establishing a direct role for Notch in adult murine thyrocytes. We confirm that hypothyroidism is enriched in children with Alagille syndrome, a genetic disorder marked by Notch mutations, suggesting that these findings translate to humans.

LC-HRMS-based targeted metabolomics for high-throughput and quantitative analysis of 21 growth inhibition-related metabolites in Chinese hamster ovary cell fed-batch cultures.

Chinese hamster ovary (CHO) cells have been widely used in the biopharmaceutical industry for production of therapeutic proteins. CHO cells in fed-batch cultures produce various amino acid-derived intermediate metabolites. These small molecule metabolic byproducts have proven to be critical to cell growth, culture performance, and, more interestingly, antibody drug productivity. Herein, we developed an LC-HRMS-based targeted metabolomics approach for comprehensive quantification of total 21 growth inhibition-related metabolites generated from 14 different amino acids in CHO cell fed-batch cultures. High throughput derivatization procedures, matrix-matched calibration curves, stable isotope-labeled internal standards, and accurate mass full MS scan were utilized to achieve our goal for a wide range of metabolite screening as well as validity and reliability of metabolite quantification. We further present a novel analytical strategy for extending the assay's dynamic range by utilizing naturally occurring isotope M + 1 ion as a quantification analog in the circumstances where the principal M ion is beyond its calibration range. The integrated method was qualified for selectivity, sensitivity, linearity, accuracy, precision, isotope analysis, and other analytical aspects to demonstrate assay robustness. We then applied this metabolomics approach to characterize metabolites of interest in a CHO cell-based monoclonal antibody (mAb) production process with fed-batch bioreactor culture mode. Absolute quantification combined with multivariate statistical analysis illustrated that our target analytes derived from amino acids, especially from branched-chain amino acids, closely correlated with cell viability and significantly differentiated cellular stages in production process.

In vivo partial reprogramming alters age-associated molecular changes during physiological aging in mice.

Partial reprogramming by expression of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) for short periods of time restores a youthful epigenetic signature to aging cells and extends the life span of a premature aging mouse model. However, the effects of longer-term partial reprogramming in physiologically aging wild-type mice are unknown. Here, we performed various long-term partial reprogramming regimens, including different onset timings, during physiological aging. Long-term partial reprogramming lead to rejuvenating effects in different tissues, such as the kidney and skin, and at the organismal level; duration of the treatment determined the extent of the beneficial effects. The rejuvenating effects were associated with a reversion of the epigenetic clock and metabolic and transcriptomic changes, including reduced expression of genes involved in the inflammation, senescence and stress response pathways. Overall, our observations indicate that partial reprogramming protocols can be designed to be safe and effective in preventing age-related physiological changes. We further conclude that longer-term partial reprogramming regimens are more effective in delaying aging phenotypes than short-term reprogramming.

Mitochondrial Redox Metabolism: The Epicenter of Metabolism during Cancer Progression.

Mitochondrial redox metabolism is the central component in the cellular metabolic landscape, where anabolic and catabolic pathways are reprogrammed to maintain optimum redox homeostasis. During different stages of cancer, the mitochondrial redox status plays an active role in navigating cancer cells' progression and regulating metabolic adaptation according to the constraints of each stage. Mitochondrial reactive oxygen species (ROS) accumulation induces malignant transformation. Once vigorous cell proliferation renders the core of the solid tumor hypoxic, the mitochondrial electron transport chain mediates ROS signaling for bringing about cellular adaptation to hypoxia. Highly aggressive cells are selected in this process, which are capable of progressing through the enhanced oxidative stress encountered during different stages of metastasis for distant colonization. Mitochondrial oxidative metabolism is suppressed to lower ROS generation, and the overall cellular metabolism is reprogrammed to maintain the optimum NADPH level in the mitochondria required for redox homeostasis. After reaching the distant organ, the intrinsic metabolic limitations of that organ dictate the success of colonization and flexibility of the mitochondrial metabolism of cancer cells plays a pivotal role in their adaptation to the new environment.

Stable Isotope Tracing Metabolomics to Investigate the Metabolic Activity of Bioactive Compounds for Cancer Prevention and Treatment.

A major hallmark of cancer is the metabolic reprogramming of cancer cells to fuel tumor growth and proliferation. Various plant-derived bioactive compounds efficiently target the metabolic vulnerabilities of cancer cells and exhibit potential as emerging therapeutic agents. Due to their safety and common use as dietary components, they are also ideal for cancer prevention. However, to render their use as efficient as possible, the mechanism of action of these phytochemicals needs to be well characterized. Stable isotope tracing is an essential technology to study the molecular mechanisms by which nutraceuticals modulate and target cancer metabolism. The use of positionally labeled tracers as exogenous nutrients and the monitoring of their downstream metabolites labeling patterns enable the analysis of the specific metabolic pathway activity, via the relative production and consumption of the labeled metabolites. Although stable isotope tracing metabolomics is a powerful tool to investigate the molecular activity of bioactive compounds as well as to design synergistic nutraceutical combinations, this methodology is still underutilized. This review aims to investigate the research efforts and potentials surrounding the use of stable isotope tracing metabolomics to examine the metabolic alterations mediated by bioactive compounds in cancer.

4-Coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase.

Lignin biosynthesis is evolutionarily conserved among higher plants and features a critical 3-hydroxylation reaction involving phenolic esters. However, increasing evidence questions the involvement of a single pathway to lignin formation in vascular plants. Here we describe an enzyme catalyzing the direct 3-hydroxylation of 4-coumarate to caffeate in lignin biosynthesis as a bifunctional peroxidase that oxidizes both ascorbate and 4-coumarate at comparable rates. A combination of biochemical and genetic evidence in the model plants Brachypodium distachyon and Arabidopsis thaliana supports a role for this coumarate 3-hydroxylase (C3H) in the early steps of lignin biosynthesis. The subsequent efficient O-methylation of caffeate to ferulate in grasses is substantiated by in vivo biochemical assays. Our results identify C3H as the only non-membrane bound hydroxylase in the lignin pathway and revise the currently accepted models of lignin biosynthesis, suggesting new gene targets to improve forage and bioenergy crops.

Local and Systemic Metabolic Responses during Light-Induced Rapid Systemic Signaling.

Plants evolved multiple signaling pathways that transduce light-related signals between leaves. These are thought to improve light stress acclimation in a process termed systemic acquired acclimation. Although responses to light stress have been studied extensively in local leaves, and to a lesser degree in systemic leaves, little is known about the responses that occur in the different tissues that connect the local to the systemic leaves. These could be important in defining the specificity of the systemic response as well as in supporting the generation of different systemic signals. Here, we report that local application of light stress to one rosette leaf of bolting Arabidopsis (Arabidopsis thaliana) plants resulted in a metabolic response that encompassed local, systemic and transport tissues (stem tissues that connect the local to the systemic tissues), demonstrating a high degree of physical and metabolic continuity between different tissues throughout the plant. Our results further indicate that the response of many of the systemically altered metabolites is associated with the function of the reactive oxygen species wave and that the levels of eight different metabolites are altered in a similar manner in all tissues tested (local, systemic, and transport). These compounds could define a core metabolic signature for light stress that propagates from the local to the systemic leaves. Our findings suggest that metabolic changes occurring in cells that connect the local and systemic tissues play an important role in systemic acquired acclimation and could convey specificity to the rapid systemic response of plants to light stress.

Rapid Accumulation of Glutathione During Light Stress in Arabidopsis.

Environmental stress conditions can drastically affect plant growth and productivity. In contrast to soil moisture or salinity that can gradually change over a period of days or weeks, changes in light intensity or temperature can occur very rapidly, sometimes over the course of minutes or seconds. We previously reported that in response to rapid changes in light intensity (0-60 s), Arabidopsis thaliana plants mount a large-scale transcriptomic response that includes several different transcripts essential for light stress acclimation. Here, we expand our analysis of the rapid response of Arabidopsis to light stress using a metabolomics approach and identify 111 metabolites that show a significant alteration in their level during the first 90 s of light stress exposure. We further show that the levels of free and total glutathione accumulate rapidly during light stress in Arabidopsis and that the accumulation of total glutathione during light stress is associated with an increase in nitric oxide (NO) levels. We further suggest that the increase in precursors for glutathione biosynthesis could be linked to alterations in photorespiration, and that phosphoenolpyruvate could represent a major energy and carbon source for rapid metabolic responses. Taken together, our analysis could be used as an initial road map for the identification of different pathways that could augment the rapid response of plants to abiotic stress. In addition, it highlights the important role of glutathione in these responses.

Reactive oxygen species, abiotic stress and stress combination.

Reactive oxygen species (ROS) play a key role in the acclimation process of plants to abiotic stress. They primarily function as signal transduction molecules that regulate different pathways during plant acclimation to stress, but are also toxic byproducts of stress metabolism. Because each subcellular compartment in plants contains its own set of ROS-producing and ROS-scavenging pathways, the steady-state level of ROS, as well as the redox state of each compartment, is different at any given time giving rise to a distinct signature of ROS levels at the different compartments of the cell. Here we review recent studies on the role of ROS in abiotic stress in plants, and propose that different abiotic stresses, such as drought, heat, salinity and high light, result in different ROS signatures that determine the specificity of the acclimation response and help tailor it to the exact stress the plant encounters. We further address the role of ROS in the acclimation of plants to stress combination as well as the role of ROS in mediating rapid systemic signaling during abiotic stress. We conclude that as long as cells maintain high enough energy reserves to detoxify ROS, ROS is beneficial to plants during abiotic stress enabling them to adjust their metabolism and mount a proper acclimation response.

Evaluation of immune responses to an oral typhoid vaccine, Ty21a, in children from 2 to 5 years of age in Bangladesh.

Young children are very susceptible to typhoid fever, emphasizing the need for vaccination in under five age groups. The parenteral Vi polysaccharide vaccine is not immunogenic in children under 2 years and the oral Ty21a vaccine (Vivotif) available in capsular formulation is only recommended for those over 5 years. We studied immune responses to a liquid formulation of Ty21a in children 2-5 years of age. Since children in developing countries are in general hypo responsive to oral vaccines, the study was designed to determine if anti-helminthic treatment prior to vaccination, improves responses. In a pilot study in 20 children aged 4-5 years, the immune responses in plasma and in antibody in lymphocyte secretions (ALS) to the enteric coated capsule formulation of Ty21a was found to be comparable to a liquid formulation (P>0.05). Based on this, children (n=252) aged ≥ 2-<3 years and ≥3-<5 years were randomized to receive a liquid formulation of Ty21a with and without previous anti-helminthic treatment. The vaccine was well tolerated with only a few mild adverse events recorded in <1% of the children. De-worming did not improve immune responses and both age groups developed 32-71% IgA, IgG, and IgM responses in plasma and 63-86% IgA responses in ALS and stool specimens to a membrane preparation (MP) of Ty21a. An early MP specific proliferative T cell response was also seen. We recommend that safety and efficacy studies with a liquid formulation of the vaccine are carried out in children under five, including those less than two years of age to determine if Ty21a is protective in these age groups and applicable as a public health tool for controlling typhoid fever in high prevalence areas of typhoid fever including Bangladesh.