Caenorhabditis elegans as a Convenient Animal Model for Microbiome Studies.

Microbes constitute the most prevalent life form on Earth, yet their remarkable diversity remains mostly unrecognized. Microbial diversity in vertebrate models presents a significant challenge for investigating host-microbiome interactions. The model organism Caenorhabditis elegans has many advantages for delineating the effects of host genetics on microbial composition. In the wild, the C. elegans gut contains various microbial species, while in the laboratory it is usually a host for a single bacterial species. There is a potential host-microbe interaction between microbial metabolites, drugs, and C. elegans phenotypes. This mini-review aims to summarize the current understanding regarding the microbiome in C. elegans. Examples using C. elegans to study host-microbe-metabolite interactions are discussed.

Bradykinin promotes murine melanoma cell migration and invasion through endogenous production of superoxide and nitric oxide.

Spatial confinement and temporal regulation of signaling by nitric oxide (NO) and reactive oxygen species (ROS) occurs in cancer cells. Signaling mediated by NO and ROS was investigated in two sub clones of the murine melanoma B16F10-Nex2 cell line, Nex10C and Nex8H treated or not with bradykinin (BK). The sub clone Nex10C, similar to primary site cells, has a low capacity for colonizing the lungs, whereas the sub clone Nex8H, similar to metastatic cells, corresponds to a highly invasive melanoma. BK-treated Nex10C cells exhibited a transient increase in NO and an inhibition in basal O2- levels. Inhibition of endogenous NO production by l-NAME resulted in detectable levels of O2-. l-NAME promoted Rac1 activation and enhanced Rac1-PI3K association. l-NAME in the absence of BK resulted in Nex10C cell migration and invasion, suggesting that NO is a negative regulator of O2- mediated cell migration and cell invasion. BK-treated Nex8H cells sustained endogenous NO production through the activation of NOS3. NO activated Rac1 and promoted Rac1-PI3K association. NO stimulated cell migration and cell invasion through a signaling axis involving Ras, Rac1 and PI3K. In conclusion, a role for O2- and NO as positive regulators of Rac1-PI3K signaling associated with cell migration and cell invasion is proposed respectively for Nex10C and Nex8H murine melanoma cells.

Knockdown of the inducible nitric oxide synthase (NOS2) splicing variant S3 promotes autophagic cell death from nitrosative stress in SW480 human colon cancer cells.

Low levels of nitric oxide (NO) produced by constitutively expressed inducible NO synthase (NOS2) in tumor cells may be an important factor in their development. NOS2 expression is associated with high mortality rates for various cancers. Alternative splicing of NOS2 down-regulates its enzymatic activity, resulting in decreased intracellular NO concentrations. Specific probes to detect alternative splicing of NOS2 were used in two isogenic human colon cancer cell lines derived either from the primary tumor (SW480) or from a lymph node metastasis (SW620). Splicing variant of NOS2 S3, lacking exons 9, 10, and 11, was overexpressed in SW480 cells. NOS2 S3 was silenced in SW480 cells. Flow-cytometry analysis was used to estimate the intracellular NO levels and to analyze the cell cycle of the studied cell lines. Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine apoptosis and autophagy markers. SW480 and SW620 cells expressed NOS2 S3. Overexpression of the NOS2 S3 in SW480 cells downregulated intracellular NO levels. SW480 cells with knocked down NOS2 S3 (referred to as S3C9 cells) had higher intracellular levels of NO compared to the wild-type SW480 cells under serum restriction. Higher NO levels resulted in the loss of viability of S3C9 cells, which was associated with autophagy. Induction of autophagy by elevated intracellular NO levels in S3C9 cells under serum restriction, suggests that autophagy operates as a cytotoxic response to nitrosative stress. The expression of NOS2 S3 plays an important role in regulating intracellular NO production and maintaining viability in SW480 cells under serum restriction. These findings may prove significant in the design of NOS2/NO-based therapies for colon cancer.

Redox and Metabolic Regulation of Intestinal Barrier Function and Associated Disorders.

The intestinal epithelium forms a physical barrier assembled by intercellular junctions, preventing luminal pathogens and toxins from crossing it. The integrity of tight junctions is critical for maintaining intestinal health as the breakdown of tight junction proteins leads to various disorders. Redox reactions are closely associated with energy metabolism. Understanding the regulation of tight junctions by cellular metabolism and redox status in cells may lead to the identification of potential targets for therapeutic interventions. In vitro and in vivo models have been utilized in investigating intestinal barrier dysfunction and in particular the free-living soil nematode, Caenorhabditis elegans, may be an important alternative to mammalian models because of its convenience of culture, transparent body for microscopy, short generation time, invariant cell lineage and tractable genetics.

Glucose-6-Phosphate Dehydrogenase, Redox Homeostasis and Embryogenesis.

Normal embryogenesis requires complex regulation and precision, which depends on multiple mechanistic details. Defective embryogenesis can occur by various mechanisms. Maintaining redox homeostasis is of importance during embryogenesis. NADPH, as produced from the action of glucose-6-phosphate dehydrogenase (G6PD), has an important role in redox homeostasis, serving as a cofactor for glutathione reductase in the recycling of glutathione from oxidized glutathione and for NADPH oxidases and nitric oxide synthases in the generation of reactive oxygen (ROS) and nitrogen species (RNS). Oxidative stress differentially influences cell fate and embryogenesis. While low levels of stress (eustress) by ROS and RNS promote cell growth and differentiation, supra-physiological concentrations of ROS and RNS can lead to cell demise and embryonic lethality. G6PD-deficient cells and organisms have been used as models in embryogenesis for determining the role of redox signaling in regulating cell proliferation, differentiation and migration. Embryogenesis is also modulated by anti-oxidant enzymes, transcription factors, microRNAs, growth factors and signaling pathways, which are dependent on redox regulation. Crosstalk among transcription factors, microRNAs and redox signaling is essential for embryogenesis.

tert-Butyl Hydroperoxide (tBHP)-Induced Lipid Peroxidation and Embryonic Defects Resemble Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in C. elegans.

G6PD is required for embryonic development in animals, as severe G6PD deficiency is lethal to mice, zebrafish and nematode. Lipid peroxidation is linked to membrane-associated embryonic defects in Caenorhabditis elegans (C. elegans). However, the direct link between lipid peroxidation and embryonic lethality has not been established. The aim of this study was to delineate the role of lipid peroxidation in gspd-1-knockdown (ortholog of g6pd) C. elegans during reproduction. tert-butyl hydroperoxide (tBHP) was used as an exogenous inducer. Short-term tBHP administration reduced brood size and enhanced germ cell death in C. elegans. The altered phenotypes caused by tBHP resembled GSPD-1 deficiency in C. elegans. Mechanistically, tBHP-induced malondialdehyde (MDA) production and stimulated calcium-independent phospholipase A2 (iPLA) activity, leading to disturbed oogenesis and embryogenesis. The current study provides strong evidence to support the notion that enhanced lipid peroxidation in G6PD deficiency promotes death of germ cells and impairs embryogenesis in C. elegans.

Nitric oxide and interactions with reactive oxygen species in the development of melanoma, breast, and colon cancer: A redox signaling perspective.

Cancer development is closely related to chronic inflammation, which is associated with identifiable markers of tumor progression, such as uncontrolled cell proliferation, angiogenesis, genomic instability, chemotherapeutic resistance, and metastases. Redox processes mediated by reactive oxygen species (ROS) and nitric oxide (NO) within the inflammatory tumor microenvironment play an essential role in directly influencing intercellular and intracellular signaling. These reactive species originating in the cancer cell or its microenvironment, mediate the epithelial-mesenchymal transition (EMT) and the mesenchymal-epithelial transition (MET). However, intracellular interactions between NO and ROS must be controlled to prevent cell death. Melanoma, breast, and colon cancer cells have developed a mechanism to survive and adapt to oxidative and nitrosative stress. The mechanism involves a spatial-temporal fine adjustment of the intracellular concentrations of NO and ROS, thereby guaranteeing the successful development of cancer cells. Physiological concentrations of NO and supra physiological concentrations of ROS are prevalent in cancer cells at the primary site. The situation reverses in cancer cells undergoing the EMT prior to being released into the blood stream. Intracellular supra physiological concentrations of NO found in circulating cancer cells endow them with anoikis resistance. When the anoikis-resistant cancer cells arrive at a metastatic site they undergo the MET. Endogenous supra physiological concentrations of ROS and physiological NO concentrations are prevalent in these cells. Understanding tumor progression from the perspective of redox signaling permits the characterization of new markers and approaches to therapy. The synthesis and use of compounds with the capacity of modifying intracellular concentrations of NO and ROS may prove effective in disrupting a redox homeostasis operative in cancer cells.

Ras, Rac1, and phosphatidylinositol-3-kinase (PI3K) signaling in nitric oxide induced endothelial cell migration.

The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras - ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (Ras(N17)) or with the non-nitrosatable mutant of Ras (Ras(C118S)); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK.

Endothelium-derived nitric oxide (NO) activates the NO-epidermal growth factor receptor-mediated signaling pathway in bradykinin-stimulated angiogenesis.

Nitric oxide (NO) is involved in angiogenesis and stimulates the EGF-R signaling pathway. Stimulation of different endothelial cell lines with bradykinin (BK) activates the endothelial NO synthase (eNOS) and promotes EGF-R tyrosine phosphorylation. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues and S-nitrosylation of the EGF-R. NO-mediated stimulatory effects on tyrosine phosphorylation of the EGF-R, where cGMP independent. Inhibition of soluble guanylyl cyclase followed by BK stimulation of human umbilical vein endothelial cells (HUVECs) did not change tyrosine phosphorylation levels of EGF-R. BK-stimulation of HUVEC promoted S-nitrosylation of the phosphatase SHP-1 and of p21Ras. Phosphorylation and activation of the ERK1/2 MAP kinases mediated by BK was dependent on the activation of the B2 receptor, of the EGF-R, and of p21 Ras. Inhibition of BK-stimulated S-nitrosylation prevented the activation of the ERK1/2 MAP kinases. Furthermore, activated ERK1/2 MAP kinases inhibited internalization of EGF-R by phosphorylating specific Thr residues of its cytoplasmic domain. BK-induced proliferation of endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). BK stimulated the expression of vascular endothelial growth factor (VEGF). VEGF expression was dependent on the activation of the EGF-R, the B2 receptor, p21Ras, and on NO generation. A Matrigel®-based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Additionally, pre-treatment of BK-stimulated HUVEC with L-NAME, PD98059, and with SU5416, a specific inhibitor of VEGFR resulted in inhibition of in vitro angiogenesis. Our findings indicate that BK-mediated angiogenesis in endothelial cells involves the induction of the expression of VEGF associated with the activation of the NO/EGF-R/p21Ras/ERK1/2 MAP kinases signaling pathway.

Epidemiological profiles and pathogenicity of Vancomycin-resistant Enterococcus faecium clinical isolates in Taiwan.

The emerging Vancomycin-resistant Enterococcus faecium (VRE-fm) is an opportunistic pathogen causing nosocomial infections. The identification of VRE-fm is important for successful prevention and control in healthcare settings. VRE-fm clinical isolates obtained from regional hospitals in northern Taiwan were characterized for antimicrobial susceptibility, virulence genes and biofilm production. Most isolates exhibited multi-drug resistance and carried the virulence genes, esp and hyl. While all isolates produce biofilms, those isolates that carried esp exhibited greater biofilm production. Isolates with different virulence gene carriages were examined for pathogenicity by using a nematode model, Caenorhabditis elegans, for determining microbial-host interactions. The survival assay showed that C. elegans was susceptible to Linezolid-resistant VRE-fm isolates with hyl. Combining the molecular epidemiological profiles regarding pathogenesis in C. elegans can serve as a guide for physicians in limiting opportunistic infections caused by VRE-fm.

Impaired immune response and barrier function in GSPD-1-deficient C. elegans infected with Klebsiella pneumoniae.

gspd-1-RNAi knockdown Caenorhabditis elegans was used as an immune-compromised model to investigate the role of G6PD in host-pathogen interactions. A shorted lifespan, increased bacterial burden and bacterial translocation were observed in gspd-1-knockdown C. elegans infected with Klebsiella pneumoniae (KP). RNAseq revealed that the innate immune pathway, including clc-1 and tsp-1, was affected by gspd-1 knockdown. qPCR confirmed that tight junction (zoo-1, clc-1) and immune-associated genes (tsp-1) were down-regulated in gspd-1-knockdown C. elegans and following infection with KP. The down-regulation of antimicrobial effector lysozymes, including lys-1, lys-2, lys-7, lys-8, ilys-2 and ilys-3, was found in gspd-1-knockdown C. elegans infected with KP. Deletion of clc-1, tsp-1, lys-7, and daf-2 in gspd-1-knockdown C. elegans infected with KP abolished the shorten lifespan seen in the Mock control. GSPD-1 deficiency in C. elegans resulted in bacterial accumulation and lethality, possibly due to a defective immune response. These findings indicate that GSPD-1 has a protective role in microbial defense in C. elegans by preventing bacterial colonization through bacterial clearance.

Thiol-Based Antioxidants and the Epithelial/Mesenchymal Transition in Cancer.

Significance: The epithelial/mesenchymal transition (EMT) is commonly associated with tumor metastasis. Oxidative and nitrosative stress is maintained in cancer cells and is involved in the EMT. Cancer cells are endowed with high levels of enzymatic and nonenzymatic antioxidants, which counteract the effects of oxidative and nitrosative stress. Thiol-based antioxidant systems such as the thioredoxin/thioredoxin reductase (Trx/TrxR) and glutathione/glutaredoxin (GSH/Grx) are continually active in cancer cells, while the thioredoxin-interacting protein (Txnip), the negative regulator of the Trx/TrxR system, is downregulated. Recent Advances: Trx/TrxR and GSH/Grx systems play a major role in maintaining EMT signaling and cancer cell progression. Critical Issues: Enhanced stress conditions stimulated in cancer cells inhibit EMT signaling. The elevated expression levels of the Trx/TrxR and GSH/Grx systems in these cells provide the antioxidant protection necessary to guarantee the occurrence of the EMT. Future Directions: Elevation of the intracellular reactive oxygen species and nitric oxide concentrations in cancer cells has been viewed as a promising strategy for elimination of these cells. The development of inhibitors of GSH synthesis and of the Trx/TrxR system together with genetic-based strategies to enhance Txnip levels may provide the necessary means to achieve this goal. Antioxid. Redox Signal. 36, 1037-1050.

Reelin is a platelet protein and functions as a positive regulator of platelet spreading on fibrinogen.

Abnormalities of platelet functions have been linked to reelin-impaired neuronal disorders. However, little attention has been given to understanding the interplay between reelin and platelet. In this study, reelin was found to present in the human platelets and megakaryocyte-like leukemic cells. Reelin-binding assays revealed that extracellular reelin can interact with platelets through the receptor belonging to the low density lipoprotein receptor gene family. The reelin-to-platelet interactions enhance platelet spreading on fibrinogen concomitant with the augmentation of lamellipodia formation and F-actin bundling. In contrast, reelin has no effect on integrin alphaIIbbeta3 activation and agonist-induced platelet aggregation. Molecular analysis revealed that the up-regulation of Rac1 activity and the inhibition of protein kinase C delta-Thr505 phosphorylation are important for reelin-mediated enhancement of platelet spreading on fibrinogen. These findings demonstrate for the first time that reelin is present in platelets and the reelin-to-platelet interactions play a novel role in platelet signaling and functions.

G6PD: A hub for metabolic reprogramming and redox signaling in cancer.

Metabolic hubs play a major role in the initiation and development of cancer. Oncogenic signaling pathways drive metabolic reprogramming and alter redox homeostasis. G6PD has potential oncogenic activity and it plays a pivotal role in cell proliferation, survival and stress responses. Aberrant activation of G6PD via metabolic reprogramming alters NADPH levels, leading to an antioxidant or a pro-oxidant environment which can either enhance DNA oxidative damage and genomic instability or initiate oncogenic signaling. Nutrient deprivation can rewire metabolism, which leads to mutations that determine a cancer cell's fate. Deregulated G6PD status and oxidative stress form a vicious cycle, which paves the way for cancer progression. This review aims to update and focus the potential role of G6PD in metabolic reprogramming and redox signaling in cancer.

G6PD deficiency, redox homeostasis, and viral infections: implications for SARS-CoV-2 (COVID-19).

The COVID-19 pandemic has so far affected more than 45 million people and has caused over 1 million deaths worldwide. Infection with SARS-CoV-2, the pathogenic agent, which is associated with an imbalanced redox status, causes hyperinflammation and a cytokine storm, leading to cell death. Glucose-6-phosphate dehydrogenase (G6PD) deficient individuals may experience a hemolytic crisis after being exposed to oxidants or infection. Individuals with G6PD deficiency are more susceptible to coronavirus infection than individuals with normally functioning G6PD. An altered immune response to viral infections is found in individuals with G6PD deficiency. Evidence indicates that G6PD deficiency is a predisposing factor of COVID-19.

Impaired inflammasome activation and bacterial clearance in G6PD deficiency due to defective NOX/p38 MAPK/AP-1 redox signaling.

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway that modulates cellular redox homeostasis via the regeneration of NADPH. G6PD-deficient cells have a reduced ability to induce the innate immune response, thus increasing host susceptibility to pathogen infections. An important part of the immune response is the activation of the inflammasome. G6PD-deficient peripheral blood mononuclear cells (PBMCs) from patients and human monocytic (THP-1) cells were used as models to investigate whether G6PD modulates inflammasome activation. A decreased expression of IL-1ß was observed in both G6PD-deficient PBMCs and PMA-primed G6PD-knockdown (G6PD-kd) THP-1 cells upon lipopolysaccharide (LPS)/adenosine triphosphate (ATP) or LPS/nigericin stimulation. The pro-IL-1ß expression of THP-1 cells was decreased by G6PD knockdown at the transcriptional and translational levels in an investigation of the expression of the inflammasome subunits. The phosphorylation of p38 MAPK and downstream c-Fos expression were decreased upon G6PD knockdown, accompanied by decreased AP-1 translocation into the nucleus. Impaired inflammasome activation in G6PD-kd THP-1 cells was mediated by a decrease in the production of reactive oxygen species (ROS) by NOX signaling, while treatment with hydrogen peroxide (H2O2) enhanced inflammasome activation in G6PD-kd THP-1 cells. G6PD knockdown decreased Staphylococcus aureus and Escherichia coli clearance in G6PD-kd THP-1 cells and G6PD-deficient PBMCs following inflammasome activation. These findings support the notion that enhanced pathogen susceptibility in G6PD deficiency is, in part, due to an altered redox signaling, which adversely affects inflammasome activation and the bactericidal response.

SOMOSAT: Utility of a web-based self-assessment tool in undergraduate medical education.

BACKGROUND: Relatively few studies have rigorously assessed the effectiveness of computer-based self-assessment in medical education. AIM: To assess whether an online self-assessment tool can be an effective adjunct to a traditional curriculum for second-year medical students. METHODS: The NYU School of Medicine Online Self-Assessment Tool (SOMOSAT) consists of >450 multiple-choice questions spanning disciplines of internal medicine, administered as separate modules focused on individual organ systems. Questions are coded on multiple dimensions, permitting second-year medical students to receive low-stakes, highly specific feedback regarding their knowledge and performance. Students can also review their answers to guide future study. We employed data collected during SOMOSAT operation to assess its utility and effectiveness. RESULTS: Overall, SOMOSAT accurately predicted student performance on future exams. SOMOSAT participants generally performed better than non-participants on subsequent graded course examinations (p < 0.05). Students using SOMOSAT subsequently experienced greater improvement in areas in which they initially performed poorly, compared with those in which they initially performed well. Students reported that SOMOSAT was most helpful in filling knowledge gaps, and providing opportunities to practice exam-style questions. CONCLUSION: The ability of SOMOSAT to enhance learning and exam performance suggests that web-based self-assessment tools can be effective adjuncts to traditional educational methods.

S-nitrosothiols and H2S donors: Potential chemo-therapeutic agents in cancer.

Nitric Oxide (NO) and Hydrogen Sulfide (H2S) are components of an "interactome", which is defined as a redox system involving the interactions of RSS, RNS and ROS. Chemical interaction by these species is common and is characterized by one and two electron oxidation, nitrosylation, nitration and sulfuration/polysulfidation reactions. NO and H2S are gases that penetrate cell membranes, are synthesized by specific enzymes, are ubiquitous, regulate protein activities through post-translational modifications and participate in cell signaling. The two molecules at high concentrations compared to physiological concentrations may result in cellular damage particularly through their interaction with other reactive species. NO and H2S can interact with each other and form a variety of molecular species which may have constructive or destructive behavior depending on the cell type, the cellular environment (ex. oxygen tension, pH, redox state), where the products are produced and in what concentrations. Cross talk exists between NO and H2S, whereby they can influence the generation and signaling behavior of each other. Given the above mentioned properties of NO and H2S and studies in cancer cells and animal models employing NO and H2S donors that generate higher than physiological concentrations of NO and H2S and are effective in killing cancer cells but not normal cells, lend credence to the possibility of the utility of these donors in an approach to the treatment of cancer.

Reflex and habituation behavior of Caenorhabditis elegans assessed by a mechanical vibration system and image analysis.

BACKGROUND: The nematode Caenorhabditis elegans is an emerging invertebrate animal model for investigating neuronal functions in behavioral assays. C. elegans mechanosensation was characterized by the use of a constant mechanical stimulation transmitter followed by quantitative imaging. NEW METHOD: C. elegans reflex and habituation behaviors were characterized by mechanical vibration followed by image analysis. A custom-designed system consists of an aluminum alloy Petri dish holder frame coupled with a mechanical vibration buzzer delivering adjustable pulsed vibration to an agar plate. The basal and evoked movements of C. elegans were recorded by a microscopic digital camera followed by quantitative analysis using microscopic imaging software. RESULTS: Application of the platform in C. elegans was demonstrated with three proof-of-concept experiments: (1) Evaluation of the reflex response stimulated by tapping and mechanical vibration with a mechano-sensation defective mutant. (2) Comparison of the reflex response stimulated by mechanical vibration between wild type and aging mutants. (3) Assessment of the efficacy of the mechanical vibration system on long-term memory for habituation. COMPARISON WITH EXISTING METHODS: Conventional C. elegans mechanosensation techniques depend on stimulation either by manually touching a single animal or tapping the Petri dish followed by scoring via visual observation from the examiner. The mechanical vibration method has greater capacity compared to conventional methods which are labor-intensive, have low throughput and lack quantifiable parameters. CONCLUSIONS: The mechanical vibration system followed by image analysis is a convenient and integrated platform for investigatingC. elegans reflex and habituation in aging and neural behavioral assays.

IDH-1 deficiency induces growth defects and metabolic alterations in GSPD-1-deficient Caenorhabditis elegans.

NADPH is a reducing equivalent that maintains redox homeostasis and supports reductive biosynthesis. Lack of major NADPH-producing enzymes predisposes cells to growth retardation and demise. It was hypothesized that double deficiency of the NADPH-generating enzymes, GSPD-1 (Glucose-6-phosphate 1-dehydrogenase), a functional homolog of human glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, and IDH-1 (isocitrate dehydrogenase-1) affect growth and development in the nematode, Caenorhabditis elegans (C. elegans). The idh-1;gspd-1(RNAi) double-deficient C. elegans model displayed shrinkage of body size, growth retardation, slowed locomotion, and impaired molting. Global metabolomic analysis was employed to address whether or not metabolic pathways were altered by severe NADPH insufficiency by the idh-1;gspd-1(RNAi) double-deficiency. The principal component analysis (PCA) points to a distinct metabolomic profile of idh-1;gspd-1(RNAi) double-deficiency. Further metabolomic analysis revealed that NADPH-dependent and glutamate-dependent amino acid biosynthesis were significantly affected. The reduced pool of amino acids may affect protein synthesis, as indicated by the absence of NAS-37 expression during the molting process. In short, double deficiency of GSPD-1 and IDH-1 causes growth retardation and molting defects, which are, in part, attributed to defective protein synthesis, possibly mediated by altered amino acid biosynthesis and metabolism in C. elegans.