RNAi-mediated Gene Knockdown Research Articles

Cysteine-rich 61 inhibition attenuates hepatic insulin resistance and improves lipid metabolism in high-fat diet fed mice and HepG2 cells.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is strongly associated with insulin resistance development. Hepatic lipid accumulation and inflammation are considered the main drivers of hepatic insulin resistance in MASLD. Cysteine-rich 61 (Cyr61 also called CCN1), a novel secretory matricellular protein, is implicated in liver inflammation, and its role in MASLD is not clearly understood. Therefore, we investigated the role of Cyr61 in hepatic insulin resistance and lipid metabolism as major factors in MASLD pathogenesis. In high-fat diet (HFD)-fed C57BL/6J mice, Cyr61 was downregulated or upregulated via viral transduction. Measurements of glucose homeostasis, histological assessment of liver tissues, and gene expression and signaling pathways of lipogenesis, fatty acid oxidation, and inflammation were performed using liver samples from these mice. Cyr61 levels in HepG2 cells were reduced using RNAi-mediated gene knockdown. Inflammation and insulin resistance were evaluated using real-time polymerase chain reaction and western blotting. HFD/AAV-shCyr61 mice exhibited enhanced glucose tolerance via the protein kinase B pathway, reduced hepatic inflammation, decreased lipogenesis, and increased fatty acid oxidation. Notably, HFD/AAV-shCyr61 mice showed elevated protein expression of sirtuin 6 and phosphorylated-AMP-activated protein kinase. Invitro experiments demonstrated that inhibition of Cyr61 downregulated pro-inflammatory cytokines such as interleukin-1 beta, IL-6, and tumor necrosis factor-alpha via the nuclear factor kappa B/c-Jun N-terminal kinase pathway, and alleviated insulin resistance. Cyr61 affected hepatic inflammation, lipid metabolism, and insulin resistance. Inhibition of Cyr61 reduced inflammation, recovered insulin resistance, and altered lipid metabolism invivo and invitro. Therefore, Cyr61 is a potential therapeutic target in MASLD.

Mitoferrin2 is a synthetic lethal target for chromosome 8p deleted cancers

BackgroundSomatic copy number alterations are a hallmark of cancer that offer unique opportunities for therapeutic exploitation. Here, we focused on the identification of specific vulnerabilities for tumors harboring chromosome 8p deletions.MethodsWe developed and applied an integrative analysis of The Cancer Genome Atlas (TCGA), the Cancer Dependency Map (DepMap), and the Cancer Cell Line Encyclopedia to identify chromosome 8p-specific vulnerabilities. We employ orthogonal gene targeting strategies, both in vitro and in vivo, including short hairpin RNA-mediated gene knockdown and CRISPR/Cas9-mediated gene knockout to validate vulnerabilities.ResultsWe identified SLC25A28 (also known as MFRN2), as a specific vulnerability for tumors harboring chromosome 8p deletions. We demonstrate that vulnerability towards MFRN2 loss is dictated by the expression of its paralog, SLC25A37 (also known as MFRN1), which resides on chromosome 8p. In line with their function as mitochondrial iron transporters, MFRN1/2 paralog protein deficiency profoundly impaired mitochondrial respiration, induced global depletion of iron-sulfur cluster proteins, and resulted in DNA-damage and cell death. MFRN2 depletion in MFRN1-deficient tumors led to impaired growth and even tumor eradication in preclinical mouse xenograft experiments, highlighting its therapeutic potential.ConclusionsOur data reveal MFRN2 as a therapeutic target of chromosome 8p deleted cancers and nominate MFNR1 as the complimentary biomarker for MFRN2-directed therapies.

Exploring the efficacy of RNAi-mediated gene knock-down via oral delivery of dsRNA in the Colorado potato beetle (Leptinotarsa decemlineata Say)

Exploring the efficacy of RNAi-mediated gene knock-down via oral delivery of dsRNA in the Colorado potato beetle (Leptinotarsa decemlineata Say)

Juvenile hormone regulates the photoperiodic plasticity of elytra coloration in the ladybird Harmonia axyridis.

Many animals, including insects, exhibit plasticity of body colour in response to environmental changes. Varied expression of carotenoids, major cuticle pigments, significantly contributes to body colour flexibility. However, the molecular mechanisms by which environmental cues regulate carotenoid expression remain largely unknown. In this study, we used the ladybird Harmonia axyridis as a model to investigate the photoperiodic-responsive plasticity of elytra coloration and its endocrine regulation. It was found that H.axyridis females under long-day conditions develop elytra that are much redder than those under short-day conditions, resulting from the differential accumulation of carotenoids. Exogenous hormone application and RNAi-mediated gene knockdown indicate that carotenoid deposition was directed through the juvenile hormone (JH) receptor-mediated canonical pathway. Moreover, we characterized an SR-BI/CD36 (SCRB) gene SCRB10 as the carotenoid transporter responding to JH signalling and regulating the elytra coloration plasticity. Taken together, we propose that JH signalling transcriptionally regulates the carotenoid transporter gene for the photoperiodic coloration plasticity of elytra in the beetles, which reveals a novel role of the endocrine system in the regulation of carotenoid-associated animal body coloration under environmental stimuli.

Haem transporter HRG-1 is essential in the barber's pole worm and an intervention target candidate.

Parasitic roundworms (nematodes) have lost genes involved in the de novo biosynthesis of haem, but have evolved the capacity to acquire and utilise exogenous haem from host animals. However, very little is known about the processes or mechanisms underlying haem acquisition and utilisation in parasites. Here, we reveal that HRG-1 is a conserved and unique haem transporter in a broad range of parasitic nematodes of socioeconomic importance, which enables haem uptake via intestinal cells, facilitates cellular haem utilisation through the endo-lysosomal system, and exhibits a conspicuous distribution at the basal laminae covering the alimentary tract, muscles and gonads. The broader tissue expression pattern of HRG-1 in Haemonchus contortus (barber's pole worm) compared with its orthologues in the free-living nematode Caenorhabditis elegans indicates critical involvement of this unique haem transporter in haem homeostasis in tissues and organs of the parasitic nematode. RNAi-mediated gene knockdown of hrg-1 resulted in sick and lethal phenotypes of infective larvae of H. contortus, which could only be rescued by supplementation of exogenous haem in the early developmental stage. Notably, the RNAi-treated infective larvae could not establish infection or survive in the mammalian host, suggesting an indispensable role of this haem transporter in the survival of this parasite. This study provides new insights into the haem biology of a parasitic nematode, demonstrates that haem acquisition by HRG-1 is essential for H. contortus survival and infection, and suggests that HRG-1 could be an intervention target candidate in a range of parasitic nematodes.

The molecular basis of socially induced egg-size plasticity in honey bees.

Reproduction involves the investment of resources into offspring. Although variation in reproductive effort often affects the number of offspring, adjustments of propagule size are also found in numerous species, including the Western honey bee, Apis mellifera. However, the proximate causes of these adjustments are insufficiently understood, especially in oviparous species with complex social organization in which adaptive evolution is shaped by kin selection. Here, we show in a series of experiments that queens predictably and reversibly increase egg size in small colonies and decrease egg size in large colonies, while their ovary size changes in the opposite direction. Additional results suggest that these effects cannot be solely explained by egg-laying rate and are due to the queens' perception of colony size. Egg-size plasticity is associated with quantitative changes of 290 ovarian proteins, most of which relate to energy metabolism, protein transport, and cytoskeleton. Based on functional and network analyses, we further study the small GTPase Rho1 as a candidate regulator of egg size. Spatio-temporal expression analysis via RNAscope and qPCR supports an important role of Rho1 in egg-size determination, and subsequent RNAi-mediated gene knockdown confirmed that Rho1 has a major effect on egg size in honey bees. These results elucidate how the social environment of the honey bee colony may be translated into a specific cellular process to adjust maternal investment into eggs. It remains to be studied how widespread this mechanism is and whether it has consequences for population dynamics and epigenetic influences on offspring phenotype in honey bees and other species.

Mitochondrial protease ClpP supplementation ameliorates diet-induced NASH in mice.

Mitochondrial dysfunction is considered a pathogenic linker in the development of non-alcoholic steatohepatitis (NASH). Inappropriate mitochondrial protein-quality control, possibly induced by insufficiency of the mitochondrial matrix caseinolytic protease P (ClpP), can potentially cause mitochondrial dysfunction. Herein, we aimed to investigate hepatic ClpP levels in a diet-induced model of NASH and determine whether supplementation of ClpP can ameliorate diet-induced NASH. NASH was induced by a high-fat/high-fructose (HF/HFr) diet in C57BL/6J mice. Stress/inflammatory signals were induced in mouse primary hepatocytes (MPHs) by treatment with palmitate/oleate (PA/OA). ClpP levels in hepatocytes were reduced using the RNAi-mediated gene knockdown technique but increased through the viral transduction of ClpP. ClpP activation was induced by administering a chemical activator of ClpP. Hepatic ClpP protein levels in C57BL/6J mice fed a HF/HFr diet were lower than the levels in those fed a normal chow diet. PA/OA treatment also decreased the ClpP protein levels in MPHs. Overexpression or activation of ClpP reversed PA/OA-induced mitochondrial dysfunction and stress/inflammatory signal activation in MPHs, whereas ClpP knockdown induced mitochondrial dysfunction and stress/inflammatory signals in these cells. On the other hand, ClpP overexpression or activation improved HF/HFr-induced NASH characteristics such as hepatic steatosis, inflammation, fibrosis, and injury in the C57BL/6J mice, whereas ClpP knockdown further augmented steatohepatitis in mice fed a HF/HFr diet. Reduced ClpP expression and subsequent mitochondrial dysfunction are key to the development of diet-induced NASH. ClpP supplementation through viral transduction or chemical activation represents a potential therapeutic strategy to prevent diet-induced NASH. Western diets, containing high fat and high fructose, often induce non-alcoholic steatohepatitis (NASH). Mitochondrial dysfunction is considered pathogenically linked to diet-induced NASH. We observed that the mitochondrial protease ClpP decreased in the livers of mice fed a western diet and supplementation of ClpP ameliorated western diet-induced NASH.

Full Functional Sex Reversal Achieved Through Silencing of MroDmrt11E Gene in Macrobrachium rosenbergii: Production of All-Male Monosex Freshwater Prawn.

The freshwater prawn Macrobrachium rosenbergii is one kind of important economic aquaculture species and displays remarkable sexual dimorphism. The molecular mechanism of sexual differentiation in M. rosenbergii has been primarily unraveled through the research efforts of the androgenic gland and its related genes. However, the understanding of conserved genes involved in the molecular mechanism underpinning sex determination and sexual differentiation of M. rosenbergii is still fragmentary. MroDmrt11E is a member of the doublesex and mab-3-related transcription factor (Dmrt) gene family and is prominently expressed in the testis. In the present study, in vivo knockdown of MroDmrt11E at the postlarva stage in male prawn induced a complete and functional sex reversal and achieved the production of an all-male monosex population. Furthermore, a great deal of new information of upregulated and downregulated transcriptions involved in sexual differentiation of MroDmrt11E knockdown was enriched by comparative transcriptomic analysis. The effects of RNAi-mediated gene knockdown of MroDmrt11E on the differentially expressed and sex-related candidate genes, such as transformer, fruitless, feminization, insulin-like androgenic gland gene, Dmrt gene family, were primarily focused on, and their possible molecular regulatory relationships in sexual differentiation were analyzed. Meanwhile, the response of primary Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways was investigated to expound the potential roles of MroDmrt11E in male sexual differentiation, which provided a deeper understanding of the molecular regulatory network underlying sexual differentiation of M. rosenbergii. The finding provided a novel sexual manipulation technique through silencing of Dmrt gene family for achieving a complete and functional sex reversal and offered a new insight regarding the mechanism of the Dmrt gene family in the sexual differentiation of crustaceans.

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro.

Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided into conventional DCs (cDCs) and plasmacytoid DCs (pDCs). cDCs specializes in presenting antigens to and activate naïve T cells. On the other hand, pDCs can produce large quantities of type I interferons (IFN-I) during viral infection. The specification of DCs occurs at an early stage of DC progenitors in the bone marrow (BM) and is defined by a network of transcription factors (TFs). For example, cDCs highly express ID2, while pDCs highly express E2-2. Since more and more subsets of DCs are being identified, there is a growing interest in understanding specific TFs controlling DC development. Here, we establish a method to screen TFs critical for DCs differentiation in vitro by delivering lentivirus carrying short hairpin RNA (shRNA) into an immortalized hematopoietic stem and progenitor cell (iHSPCs) line. After the selection and in vitro differentiation, cDC and pDC potential of the stable knockdown cell lines are analyzed by flow cytometry. This approach provides a platform to identify genes potentially governing DC fates from progenitors in vitro.

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line <em>In vitro</em>

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line <em>In vitro</em>

Inhibiting USP14 ameliorates inflammatory responses in trophoblast cells by suppressing MAPK/NF-κB signaling.

BackgroundPreeclampsia can cause severe consequences for pregnant women and infants, and developing effective medicine or methods to prevent or treat patients with preeclampsia is urgently needed. Ubiquitin‐specific protease 14 (USP14) has emerged as a critical regulator in the development of human cancers and neurodegenerative diseases. However, its role in preeclampsia remains elusive.MethodsThe expression of USP14 in placental tissues from healthy donors and preeclampsia patients were determined by quantitative reverse transcription PCR assay. The protein levels of targeted genes were evaluated by Western blotting assay. Small interfering RNA‐mediated gene knockdown was used to reduce USP14 expression in trophoblast cell lines.ResultsThe expression levels of USP14 and proinflammatory cytokine were substantially upregulated in placental tissues from preeclampsia patients. Knockdown or inhibition of USP14 significantly abrogated hypoxia/reoxygenation‐induced upregulation of nuclear factor kappa B (NF‐κB) activation and proinflammatory cytokine production.ConclusionOur results suggested that USP14 promotes proinflammatory cytokine production through activation of NF‐κB. Developing drugs targeting USP14 may be beneficial for the prevention or treatment of patients with preeclampsia.

Genome-wide assessment and development of molecular diagnostic methods for imidacloprid-resistance in the brown planthopper, Nilaparvata lugens (Hemiptera; Delphacidae).

The brown planthopper, Nilaparvata lugens (Stål), is one of the most notorious pests of rice throughout Asia. The brown planthopper has developed high resistance to imidacloprid, a member of neonicotinoid insecticides. Several genes and mutations conferring imidacloprid resistance in N. lugens, especially in eastern and southeastern Asia populations, have been reported. Thus, the key mechanisms of imidacloprid resistance need to be examined. RNA-seq analyses revealed that only one cytochrome P450 monooxygenase gene, CYP6ER1, was commonly upregulated in the five resistant strains tested. Sequences of CYP6ER1, which were highly expressed in the imidacloprid-resistant strains, contained a three-nucleotide deletion in the coding region, and amino acid substitutions and deletion, compared to that in an imidacloprid-susceptible strain. RNAi-mediated gene knockdown of CYP6ER1 increased imidacloprid susceptibility in a resistant strain. Further, we established two simple and convenient PCR-based molecular diagnostic methods to detect the CYP6ER1 locus with the three-nucleotide deletion. Using these methods, the resistance of F2 progenies derived from the crosses of F1 siblings from susceptible and resistant parents was analyzed, showing that the imidacloprid resistance had a relationship to the CYP6ER1 locus with the three-nucleotide deletion. The overexpression of a variant CYP6ER1 with amino acid substitutions and deletion was involved in imidacloprid resistance in N. lugens. Based on these findings, molecular diagnostic methods have been developed and are promising tools for monitoring imidacloprid resistance in paddy fields. © 2020 Society of Chemical Industry.

Recent developments in targeting genes and pathways by RNAi-based approaches in colorectal cancer.

A wide spectrum of genetic and epigenetic variations together with environmental factors has made colorectal cancer (CRC), which involves the colon and rectum, a challenging and heterogeneous cancer. CRC cannot be effectively overcomed by common conventional therapies including surgery, chemotherapy, targeted therapy, and hormone replacement which highlights the need for a rational design of novel anticancer therapy. Accumulating evidence indicates that RNA interference (RNAi) could be an important avenue to generate great therapeutic efficacy for CRC by targeting genes that are responsible for the viability, cell cycle, proliferation, apoptosis, differentiation, metastasis, and invasion of CRC cells. In this review, we underline the documented benefits of small interfering RNAs and short hairpin RNAs to target genes and signaling pathways related to CRC tumorigenesis. We address the synergistic effects of RNAi-mediated gene knockdown and inhibitors/chemotherapy agents to increase the sensitivity of CRC cells to common therapies. Finally, this review points new delivery systems/materials for improving the cellular uptake efficiency and reducing off-target effects of RNAi.

A role for ceramide glycosylation in resistance to oxaliplatin in colorectal cancer

There is growing evidence to support a role for the ceramide-metabolizing enzyme, glucosylceramide synthase (GCS), in resistance to a variety of chemotherapeutic agents. Whether GCS contributes to oxaliplatin resistance in colorectal cancer (CRC) has not yet been determined. We have addressed this potentially important clinical issue by examining GCS function in two panels of oxaliplatin-resistant, isogenic CRC cell lines. Compared to parental cell lines, oxaliplatin-resistant cells have increased expression of GCS protein associated with increased levels of the pro-survival ceramide metabolite, glucosylceramide (GlcCer). Inhibition of GCS expression by RNAi-mediated gene knockdown resulted in a reduction in cellular GlcCer levels, with restored sensitivity to oxaliplatin. Furthermore, oxaliplatin-resistant CRC cells displayed lower ceramide levels both basally and after treatment with oxaliplatin, compared to parental cells. GlcCer, formed by GCS-mediated ceramide glycosylation, is the precursor to a complex array of glycosphingolipids. Differences in cellular levels and species of gangliosides, a family of glycosphingolipids, were also seen between parental and oxaliplatin-resistant CRC cells. Increased Akt activation was also observed in oxaliplatin-resistant CRC cell lines, together with increased expression of the anti-apoptotic protein survivin. Finally, this study shows that GCS protein levels are greatly increased in human CRC specimens, compared to matched, normal colonic mucosa, and that high levels of UGCG gene expression are significantly associated with decreased disease-free survival in colorectal cancer patients. These findings uncover an important cellular role for GCS in oxaliplatin chemosensitivity and may provide a novel cellular target for augmenting chemotherapeutic drug effectiveness in CRC.

Ectonucleotide pyrophosphatase 2 (ENPP2) plays a crucial role in myogenic differentiation through the regulation by WNT/β-Catenin signaling.

Ectonucleotide pyrophosphate phosphodiesterase type II (ENPP2), also known as Autotaxin (ATX), is an enzyme present in blood circulation that converts lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA). While LPA has been demonstrated to play diverse roles in skeletal myogenesis, mainly through in vitro studies, the role of ENPP2 in skeletal myogenesis has not been determined. We previously found that Enpp2 is induced by a positive WNT/β-Catenin signaling regulator, R-spondin2 (RSPO2), in C2C12 myoblast cells. As RSPO2 promotes myogenic differentiation via the WNT/β-Catenin signaling pathway, we hypothesized that ENPP2 may act as a key mediator for the crosstalk between WNT and LPA signaling during myogenic differentiation. Herein, we found that ENPP2 function is essential for myogenic differentiation in C2C12 cells. Pharmacological ENPP2 inhibitors or RNAi-mediated Enpp2 gene knockdown severely impaired the myogenic differentiation, including the cell fusion process, whereas administration of the recombinant ENPP2 protein enhanced myogenic differentiation. Consistent with the in vitro results, mice lacking the Enpp2 gene showed a disrupted muscle regeneration after acute muscle injury. The size of newly regenerated myofibers in Enpp2 mutant muscle was significantly reduced compared with wild-type regenerated muscle. Modified expression patterns of myogenic markers in Enpp2 mutant muscle further emphasized the impaired muscle regeneration process. Finally, we convincingly demonstrate that the Enpp2 gene is a direct transcriptional target for WNT/β-Catenin signaling. Functional TCF/LEF1 binding sites within the upstream region of Enpp2 gene were identified by chromatin immunoprecipitation using anti-β-Catenin antibodies and reporter assay. Our study reveals that ENPP2 is regulated by WNT/β-Catenin signaling and plays a key positive role in myogenic differentiation.

Targeted genomic profiling identifies frequent deleterious mutations in FAT4 and TP53 genes in HBV-associated hepatocellular carcinoma

BackgroundHepatitis B virus (HBV) is the major risk factor for hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV-associated HCC pathogenesis is still unclear. Genetic alterations in cancer-related genes have been linked to many human cancers. Here, we aimed to explore genetic alterations in selected cancer-related genes in patients with HBV-associated HCC.MethodsTargeted sequencing was used to analyze six cancer-related genes (PIK3CA, TP53, FAT4, IRF2, HNF4α and ARID1A) in eight pairs of HBV-associated HCC tumors and their adjacent non-tumor tissues. Sanger sequencing, quantitative PCR, Western-blotting and RNAi-mediated gene knockdown were used to further validate findings.ResultsTargeted sequencing revealed thirteen non-synonymous mutations, of which 9 (69%) were found in FAT4 and 4 (31%) were found in TP53 genes. Non-synonymous mutations were not found in PIK3CA, IRF2, HNF4α and ARID1A. Among these 13 non-synonymous mutations, 12 (8 in FAT4 and 4 in TP53) were predicted to have deleterious effect on protein function by in silico analysis. For TP53, Y220S, R249S and P250R non-synonymous mutations were solely identified in tumor tissues. Further expression profiling of FAT4 and TP53 on twenty-eight pairs of HCC tumor and non-tumor tissues confirmed significant downregulation of both genes in HCC tumors compared with their non-tumor counterparts (P < 0.001 and P < 0.01, respectively). Functional analysis using RNAi-mediated knockdown of FAT4 revealed an increased cancer cell growth and proliferation, suggesting the putative tumor suppressor role of FAT4 in HCC.ConclusionsThis study highlights the importance of FAT4 and TP53 in HCC pathogenesis and identifies new genetic variants that may have potentials for development of precise therapy for HCC.

Down-regulation of Survivin by BIX-01294 Pretreatment Overcomes Resistance of Hepatocellular Carcinoma Cells to TRAIL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cancer-selective, cell-death-inducing agent with little toxicity to normal cells. However, various human cancers and cancer cell lines have been reported to be resistant to TRAIL. Molecular clarification of resistance mechanism is needed. Compound screening, proliferation assays, western blotting, and flow cytometry were used to examine the sensitizer activity of methyl transferase inhibitor BIX-01294 in combination with TRAIL, in hepatocellular carcinoma (HCC) cells. RNA sequencing analysis and single guide (sg)RNA-mediated gene deletion were used to investigate the role of survivin in sensitization. In HCC cells, BIX-01294 enhanced TRAIL sensitivity by reducing survivin expression at the RNA level. Small interference RNA-mediated gene knockdown demonstrated the mechanism of sensitization to be via the reduction of survivin. Euchromatin histone methyltransferase 2 (EHMT2) inhibition by BIX-01294 may be a potent anti-tumor therapeutic strategy for human HCC.

Loss-of-function approaches in comparative physiology: is there a future for knockdown experiments in the era of genome editing?

Loss-of-function technologies, such as morpholino- and RNAi-mediated gene knockdown, and TALEN- and CRISPR/Cas9-mediated gene knockout, are widely used to investigate gene function and its physiological significance. Here, we provide a general overview of the various knockdown and knockout technologies commonly used in comparative physiology and discuss the merits and drawbacks of these technologies with a particular focus on research conducted in zebrafish. Despite their widespread use, there is an ongoing debate surrounding the use of knockdown versus knockout approaches and their potential off-target effects. This debate is primarily fueled by the observations that, in some studies, knockout mutants exhibit phenotypes different from those observed in response to knockdown using morpholinos or RNAi. We discuss the current debate and focus on the discrepancies between knockdown and knockout phenotypes, providing literature and primary data to show that the different phenotypes are not necessarily a direct result of the off-target effects of the knockdown agents used. Nevertheless, given the recent evidence of some knockdown phenotypes being recapitulated in knockout mutants lacking the morpholino or RNAi target, we stress that results of knockdown experiments need to be interpreted with caution. We ultimately argue that knockdown experiments should not be discontinued if proper control experiments are performed, and that with careful interpretation, knockdown approaches remain useful to complement the limitations of knockout studies (e.g. lethality of knockout and compensatory responses).

Non‐Polarized mpkCCD Cell Model for Aquaporin‐2 Phosphorylation and Membrane Trafficking Study

Aquaporin‐2 (AQP2) is a water channel protein expressed in the kidney collecting duct principal cells responsible for water reabsorption. AQP2's ability to regulate water reabsorption depends on its phosphorylation states and trafficking to and from the apical plasma membrane regulated by the peptide hormone vasopressin. The mpkCCD cells have been an instrumental collecting duct cell model for studying AQP2 phosphorylation and trafficking. However, the cells require a time‐consuming polarization process before they can respond to vasopressin. We sought to circumvent this issue by examining AQP2 phosphorylation and trafficking in non‐polarized AQP2‐overexpressing mpkCCD cells treated with forskolin, which elevates intracellular cyclic AMP (cAMP), a second messenger of vasopressin. We found that forskolin induced AQP2 phosphorylation at serine 269 (S269) by 1.6 folds as observed in polarized mpkCCD and isolated rat inner medullary collecting duct cells treated with vasopressin. Before forskolin stimulation, most AQP2 was intracellular and was not phosphorylated at S269 by confocal immunofluorescence microscopy. In response to forskolin, AQP2 was found in the plasma membrane and was phosphorylated at S269, similar to those observed in the polarized mpkCCD cells and native collecting duct cells stimulated with vasopressin. As a proof of principle, we found that small hairpin RNA‐mediated protein kinase A knockdown reduced AQP2 phosphorylation at S269 and membrane localization in the non‐polarized mpkCCD cells treated with forskolin. Thus, non‐polarized AQP2‐overexpressing mpkCCD cells can be used in lieu of polarized mpkCCD cells when time is a limiting factor including transient small hairpin RNA‐mediated gene knockdown experiments.Support or Funding InformationThis work was supported by the Ministry of Science and Technology, TAIWAN (MOST 107‐2320‐B‐002‐057‐MY3).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interplay between mitochondria and diet mediates pathogen and stress resistance in Caenorhabditis elegans.

Diet is a crucial determinant of organismal biology; interactions between the host, its diet, and its microbiota are critical to determining the health of an organism. A variety of genetic and biochemical means were used to assay stress sensitivity in C. elegans reared on two standard laboratory diets: E. coli OP50, the most commonly used food for C. elegans, or E. coli HT115, which is typically used for RNAi-mediated gene knockdown. We demonstrated that the relatively subtle shift to a diet of E. coli HT115 had a dramatic impact on C. elegans’s survival after exposure to pathogenic or abiotic stresses. Interestingly, this was independent of canonical host defense pathways. Instead the change arises from improvements in mitochondrial health, likely due to alleviation of a vitamin B12 deficiency exhibited by worms reared on an E. coli OP50 diet. Increasing B12 availability, by feeding on E. coli HT115, supplementing E. coli OP50 with exogenous vitamin B12, or overexpression of the B12 transporter, improved mitochondrial homeostasis and increased resistance. Loss of the methylmalonyl-CoA mutase gene mmcm-1/MUT, which requires vitamin B12 as a cofactor, abolished these improvements, establishing a genetic basis for the E. coli OP50-incurred sensitivity. Our study forges a mechanistic link between a dietary deficiency (nutrition/microbiota) and a physiological consequence (host sensitivity), using the host-microbiota-diet framework.