Pharmacological and functional similarities of the human neuropeptide Y system in C. elegans challenges phylogenetic views on the FLP/NPR system

Abstract Background The neuropeptide Y system affects various processes, among others food intake, and is frequently discussed in the context of targeting obesity. Studies in model organisms are indispensable to enable molecular studies in a physiological context. Although the NPY system is evolutionarily conserved in all bilaterians, in the widely used model Caenorhabditis elegans there is controversy on the existence of NPY orthologous molecules. While the FMRFamide-like peptide (FLP)/Neuropeptide receptor-Resemblance (NPR) system in the nematode was initially suggested to be orthologous to the mammalian NPY system, later global phylogenetic studies indicate that FLP/NPR is protostome-specific. Methods We performed a comprehensive pharmacological study of the FLP/NPR system in transfected cells in vitro, and tested for functional substitution in C. elegans knockout strains. Further, we phenotypically compared different flp loss-of-function strains. Differences between groups were compared by ANOVA and post-hoc testing (Dunnett, Bonferroni). Results Our pharmacological analysis of the FLP/NPR system including formerly functionally uncharacterized NPY-like peptides from C. elegans demonstrates that G protein-coupling and ligand requirements for receptor activation are similar to the human NPY system. In vitro and in vivo analyses show cross-reactivity of NPY with the FLP/NPR system manifesting in the ability of the human GPCRs to functionally substitute FLP/NPR signaling in vivo. The high pharmacological/functional similarities enabled us to identify C. elegans FLP-14 as a key molecule in avoidance behavior. Conclusions Our data demonstrate the pharmacological and functional similarities of human NPY and C. elegans NPR systems. This adds a novel perspective to current phylogenetic reconstructions of the neuropeptide Y system. NPY and NPR receptors are pharmacologically so similar that the human receptors can functionally compensate for the C. elegans ones, suggesting orthologous relationships. This is also underlined by the presence of NPY-like peptides and parallels in peptide requirements for receptor activation. Further, the results presented here highlight the potential of this knowledge for physiological as well as molecular studies on neuropeptide GPCRs such as the NPY system in the future.

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A novel treatment strategy for lapatinib resistance in a subset of HER2-amplified gastric cancer

BMC Cancer ◽

10.1186/s12885-021-08283-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Gang Ning ◽

Qihui Zhu ◽

Wonyoung Kang ◽

Hamin Lee ◽

Leigh Maher ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Lines ◽

Target Genes ◽

Kinase Inhibitor ◽

Pharmacological Study ◽

Mapk Signaling ◽

Her2 Overexpression ◽

Loss Of Function

Abstract Background Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Human epidermal growth factor receptor 2 (HER2) amplification occurs in approximately 13–23% of all GC cases and patients with HER2 overexpression exhibit a poor prognosis. Lapatinib, a dual EGFR/HER2 tyrosine kinase inhibitor, is an effective agent to treat HER2-amplified breast cancer but it failed in gastric cancer (GC) clinical trials. However, the molecular mechanism of lapatinib resistance in HER2-amplified GC is not well studied. Methods We employed an unbiased, genome-scale screening with pooled CRISPR library on HER2-amplified GC cell lines to identify genes that are associated with resistance to lapatinib. To validate the candidate genes, we applied in vitro and in vivo pharmacological tests to confirm the function of the target genes. Results We found that loss of function of CSK or PTEN conferred lapatinib resistance in HER2-amplified GC cell lines NCI-N87 and OE19, respectively. Moreover, PI3K and MAPK signaling was significantly increased in CSK or PTEN null cells. Furthermore, in vitro and in vivo pharmacological study has shown that lapatinib resistance by the loss of function of CSK or PTEN, could be overcome by lapatinib combined with the PI3K inhibitor copanlisib and MEK inhibitor trametinib. Conclusions Our study suggests that loss-of-function mutations of CSK and PTEN cause lapatinib resistance by re-activating MAPK and PI3K pathways, and further proved these two pathways are druggable targets. Inhibiting the two pathways synergistically are effective to overcome lapatinib resistance in HER2-amplified GC. This study provides insights for understanding the resistant mechanism of HER2 targeted therapy and novel strategies that may ultimately overcome resistance or limited efficacy of lapatinib treatment for subset of HER2 amplified GC.

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Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology

Non-Coding RNA ◽

10.3390/ncrna5010012 ◽

2019 ◽

Vol 5 (1) ◽

pp. 12 ◽

Cited By ~ 7

Author(s):

Nils R. Hansmeier ◽

Pia J. M. Widdershooven ◽

Sajjad Khani ◽

Jan-Wilhelm Kornfeld

Keyword(s):

Gene Targeting ◽

Mouse Models ◽

Noncoding Rna ◽

Genome Engineering ◽

Model Organisms ◽

Specific Gene ◽

Loss Of Function ◽

Mouse Lines

In recent years, long noncoding RNAs (lncRNAs) have emerged as multifaceted regulators of gene expression, controlling key developmental and disease pathogenesis processes. However, due to the paucity of lncRNA loss-of-function mouse models, key questions regarding the involvement of lncRNAs in organism homeostasis and (patho)-physiology remain difficult to address experimentally in vivo. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 platform provides a powerful genome-editing tool and has been successfully applied across model organisms to facilitate targeted genetic mutations, including Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus. However, just a few lncRNA-deficient mouse lines have been created using CRISPR/Cas9-mediated genome engineering, presumably due to the need for lncRNA-specific gene targeting strategies considering the absence of open-reading frames in these loci. Here, we describe a step-wise procedure for the generation and validation of lncRNA loss-of-function mouse models using CRISPR/Cas9-mediated genome engineering. In a proof-of-principle approach, we generated mice deficient for the liver-enriched lncRNA Gm15441, which we found downregulated during development of metabolic disease and induced during the feeding/fasting transition. Further, we discuss guidelines for the selection of lncRNA targets and provide protocols for in vitro single guide RNA (sgRNA) validation, assessment of in vivo gene-targeting efficiency and knockout confirmation. The procedure from target selection to validation of lncRNA knockout mouse lines can be completed in 18–20 weeks, of which <10 days hands-on working time is required.

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567 MDK1319/MDK-701: A potent fully efficacious peptidyl agonist of IL-7Rαγc, designed with no reference to cytokine or receptor structure and unrelated to IL-7, fused to an Fc-domain for PK enhancement

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0567 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A601-A601

Author(s):

William Dower ◽

Alice Bakker ◽

Steven Cwirla ◽

Prarthana Joshi ◽

Praechompoo Pongtornpipat ◽

...

Keyword(s):

T Cell ◽

Neutralizing Antibodies ◽

Human Lymphocytes ◽

Receptor Activation ◽

Biological Properties ◽

Cross Reactivity ◽

Lymphocyte Subpopulation ◽

Minimal Risk

BackgroundIL-7 receptor activation is essential for the proper development and homeostasis of T-cell subpopulations, and maintenance of the TCR clonal repertoire. Emerging evidence indicates potential clinical utility of IL-7 for immunotherapy of lymphopenia, oncology, and other indications. Here we report the discovery of MDK1319, a small novel peptidyl agonist of IL-7R. This peptide is structurally unrelated to IL-7, with a MW less than 5000D. To improve in vivo properties, we fused MDK1319 to an IgG-Fc-domain to construct MDK-701, which exhibits biological properties similar to those of IL-7 in vitro, and when administered to non-human primates.MethodsPeptides were selected from peptide libraries by screens designed to identify molecules binding simultaneously to the Rα and γc subunits of the human IL-7 receptor. Synthetic peptides, and peptides fused to IgG Fc-domains were evaluated for efficacy, potency, and quality of signaling in IL-7-responsive cell lines and human lymphocytes. PK/PD properties in non-human primates were also determined.ResultsMDK1319 and MDK-701 activate the major IL-7R signaling pathways, JAK-STAT (pSTAT5), and PI3K (pAKT), and induce proliferation in human PBMCs, exhibiting lymphocyte subpopulation selectivity, kinetics, efficacy, and potency similar to those of IL-7. Agonism is attributable to direct activation of IL-7R, as shown by dependence on the presence of the IL-7Rα subunit for response in test cells, and insensitivity to IL-7 neutralizing antibodies. MDK1319 and MDK-701 do not activate nor inhibit any other (off target) Rγc family receptors at concentrations 100-fold greater than required for maximal IL-7R activation. MDK-701 administered to cynomologous macaques (single dose, IV at 1 mg/kg) exhibits a circulating terminal half life of ~32 hr; and induces peripheral lymphocyte profiles similar to IL-7 treatment, including initial reduction (tissue migration), followed by sustained elevation of peripheral lymphocytes remaining above baseline for 29 days, with no observed adverse effects.ConclusionsIn addition to the utility of Fc-fusion MDK-701 for monotherapy, the small peptidyl nature of the active peptide MDK1319, fusable to recombinant protein partners, offers opportunities for incorporation into bispecific molecules, linking IL-7 activity to a variety of useful functions. These include synergistic cytokine activities, checkpoint blockade, and tissue targeting. Cells engineered to secrete MDK1319 display autocrine stimulation potentially useful in T-cell therapeutics. The structural novelty of MDK1319 substantially decreases risk of cross reactivity of any anti-drug immune response with endogenous IL-7, and may provide a safer alternative to modified forms of IL-7 reported to produce significant anti-IL-7 immunogenicity.Ethics ApprovalAnimal studies were performed by Envol Biomedical or Charles Rivers Laboratories, as approved by the Institution Ethics Boards with the following study and approval numbers:Envol Biomedical 7037-20 MDK2002; 7037-20: PK/PD Cynomolgus monkeysCharles Rivers Laboratories 20200121001K; US19001: PK Mouse The use of human PBMC in this study was authorized under Minimal Risk Research Related Activities at Stanford Blood Center (SQL 79075)

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A homozygous loss-of-function CAMK2A mutation causes growth delay, frequent seizures and severe intellectual disability

eLife ◽

10.7554/elife.32451 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 13

Author(s):

Poh Hui Chia ◽

Franklin Lei Zhong ◽

Shinsuke Niwa ◽

Carine Bonnard ◽

Kagistia Hana Utami ◽

...

Keyword(s):

Intellectual Disability ◽

Germline Mutation ◽

Growth Delay ◽

Global Developmental Delay ◽

Loss Of Function ◽

C Elegans ◽

Calcium Calmodulin Dependent ◽

Severe Intellectual Disability

Calcium/calmodulin-dependent protein kinase II (CAMK2) plays fundamental roles in synaptic plasticity that underlies learning and memory. Here, we describe a new recessive neurodevelopmental syndrome with global developmental delay, seizures and intellectual disability. Using linkage analysis and exome sequencing, we found that this disease maps to chromosome 5q31.1-q34 and is caused by a biallelic germline mutation in CAMK2A. The missense mutation, p.His477Tyr is located in the CAMK2A association domain that is critical for its function and localization. Biochemically, the p.His477Tyr mutant is defective in self-oligomerization and unable to assemble into the multimeric holoenzyme.In vivo, CAMK2AH477Y failed to rescue neuronal defects in C. elegans lacking unc-43, the ortholog of human CAMK2A. In vitro, neurons derived from patient iPSCs displayed profound synaptic defects. Together, our data demonstrate that a recessive germline mutation in CAMK2A leads to neurodevelopmental defects in humans and suggest that dysfunctional CAMK2 paralogs may contribute to other neurological disorders.

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Promoters of the dhs-21 gene encoding dicarbonyl/l-xylulose reductase in Caenorhabditis elegans

Vietnam Journal of Biotechnology ◽

10.15625/1811-4989/15/2/12353 ◽

2018 ◽

Vol 15 (2) ◽

pp. 359-365

Author(s):

Lê Thọ Sơn ◽

Joohong Ahnn ◽

Jeong Hoon Cho ◽

Nguyễn Huy Hoàng

Keyword(s):

Caenorhabditis Elegans ◽

Model Organism ◽

Biological Research ◽

Loss Of Function ◽

Wild Type ◽

C Elegans ◽

Larval Stages ◽

Vital Functions

Dicarbonyl/L-xylulose (DCXR) was identified as a dehydrogenase. This type of enzyme was presented in various forms of lives including bacteria, fungi, plants and animals. Generally, it converts L-xylulose to xylitol in the presence of either cofactor NADH or NADPH in vitro. Previous studies reported the biochemistry properties and crystal structure but largely uncovered biological roles of DCXRs. It was impossible to dissect the functions in mice or human cells that had many DCXR homologs in their genomes. Interestingly, the wild-type Caenorhabditis elegans, a well-known model organism in biological research, has only nuclear genomic dhs-21 that encodes a unique homologous DCXR. Thus Ce.dhs-21 and the host C. elegans were relevant for investigation of the physiologically-vital functions of the DCXR. This research aimed to the expression of dhs-21 in vivo. We defined three promoters , manipulated three relative reporter-constructs that conjugated the dhs-21 gene and Green Flouresent Protein (known as GFP) one. The construct vectors were transferred into wild-type C. elegans N2 and as well as the hermaphroditic loss of function dhs-21(jh129) by microinjection. In the results, we found that the expression pattern of dhs-21 under the only p2-promoter construct was stable and similar to immunogold Electric Microscopy (EM) images. The dhs-21 gene was expressed in both sexes of at all larval stages till the deaths of worms. DHS-21 was expressed in the cytosol of the intestinal, gonad sheath and uterous seam cell (utse).

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Sigma-1 Receptor Activation Induces Autophagy and Increases Proteostasis Capacity In Vitro and In Vivo

Cells ◽

10.3390/cells8030211 ◽

2019 ◽

Vol 8 (3) ◽

pp. 211 ◽

Cited By ~ 15

Author(s):

Maximilian Christ ◽

Heike Huesmann ◽

Heike Nagel ◽

Andreas Kern ◽

Christian Behl

Keyword(s):

Clinical Investigation ◽

Receptor Activation ◽

Autophagic Flux ◽

Neuroprotective Effects ◽

Sigma 1 Receptor ◽

C Elegans ◽

Protein Clearance ◽

Sigma 1

Dysfunction of autophagy and disturbed protein homeostasis are linked to the pathogenesis of human neurodegenerative diseases and the modulation of autophagy as the protein clearance process has become one key pharmacological target. Due to the role of sigma-1 receptors (Sig-1R) in learning and memory, and the described pleiotropic neuroprotective effects in various experimental paradigms, Sig-1R activation is recognized as one potential approach for prevention and therapy of neurodegeneration and, interestingly, in amyotrophic lateral sclerosis associated with mutated Sig-1R, autophagy is disturbed. Here we analyzed the effects of tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine hydrochloride (ANAVEX2-73), a muscarinic receptor ligand and Sig-1R agonist, on autophagy and proteostasis. We describe, at the molecular level, for the first time, that pharmacological Sig-1R activation a) enhances the autophagic flux in human cells and in Caenorhabditis elegans and b) increases proteostasis capacity, ultimately ameliorating paralysis caused by protein aggregation in C. elegans. ANAVEX2-73 is already in clinical investigation for the treatment of Alzheimer’s disease, and the novel activities of this compound on autophagy and proteostasis described here may have consequences for the use and further development of the Sig-1R as a drug target in the future. Moreover, our study defines the Sig-1R as an upstream modulator of canonical autophagy, which may have further implications for various conditions with dysfunctional autophagy, besides neurodegeneration.

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A Long Lost Key Opens an Ancient Lock: Drosophila Myb Causes a Synthetic Multivulval Phenotype in Nematodes

10.1101/788265 ◽

2019 ◽

Author(s):

Paul J. Vorster ◽

Paul Goetsch ◽

Tilini U. Wijeratne ◽

Keelan Z. Guiley ◽

Laura Andrejka ◽

...

Keyword(s):

Cell Fate ◽

Cell Cycle Progression ◽

Target Genes ◽

Suppressor Gene ◽

Last Common Ancestor ◽

Loss Of Function ◽

Core Complex ◽

C Elegans

ABSTRACTThe five-protein MuvB core complex (LIN9/Mip130, LIN37/Mip40, LIN52, LIN54/Mip120, and LIN53/p55CAF1/RBBP4) has been highly conserved during the evolution of animals. This nuclear complex interacts with proteins encoded by the RB tumor suppressor gene family and its associated E2F-DP transcription factors to form DREAM complexes that repress the expression of genes that regulate cell cycle progression and cell fate. The MuvB core complex also interacts with proteins encoded by the Myb oncogene family to form the Myb-MuvB complexes that activate many of the same target genes. We show that animal-type Myb genes and proteins are present in Bilateria, Cnidaria, and Placozoa, the latter including some of the simplest known animal species. However, bilaterian nematode worms appear to have lost their animal-type Myb genes hundreds of millions of years ago. Nevertheless, the amino acids in the LIN9 and LIN52 proteins that directly interact with the MuvB-binding domains of human B-Myb and Drosophila Myb are conserved in C. elegans. Here we show that, despite greater than 500 million years since their last common ancestor, the Drosophila melanogaster Myb protein can bind to the nematode LIN9 and LIN52 family proteins in vitro and can cause a synthetic multivulval (synMuv) phenotype in vivo. This phenotype is similar to that caused by loss-of-function mutations in C. elegans synMuvB class genes including those that encode homologs of the MuvB core, RB, E2F, and DP. Furthermore, amino acid substitutions in the MuvB-binding domain of Drosophila Myb that disrupt its functions in vitro and in vivo also disrupt its activity in C. elegans. We speculate that nematodes and other animals may contain another protein that can bind to LIN9 and LIN52 in order to activate transcription of genes repressed by DREAM complexes.

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TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽

10.1186/s12885-021-08562-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Chengwu Xiao ◽

Wei Zhang ◽

Meimian Hua ◽

Huan Chen ◽

Bin Yang ◽

...

Keyword(s):

Cell Lines ◽

Prognostic Indicator ◽

The Cancer Genome Atlas ◽

Mrna Levels ◽

Loss Of Function ◽

Protein Levels ◽

Kaplan Meier ◽

Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

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β-elemene alleviates airway stenosis via the ILK/Akt pathway modulated by MIR143HG sponging miR-1275

Cellular & Molecular Biology Letters ◽

10.1186/s11658-021-00261-0 ◽

2021 ◽

Vol 26 (1) ◽

Author(s):

Guoying Zhang ◽

Cheng Xue ◽

Yiming Zeng

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Cell Model ◽

Protective Efficacy ◽

Rabbit Model ◽

Akt Pathway ◽

Loss Of Function ◽

Airway Stenosis

Abstract Background We have previously found that β-elemene could inhibit the viability of airway granulation fibroblasts and prevent airway hyperplastic stenosis. This study aimed to elucidate the underlying mechanism and protective efficacy of β-elemene in vitro and in vivo. Methods Microarray and bioinformatic analysis were used to identify altered pathways related to cell viability in a β-elemene-treated primary cell model and to construct a β-elemene-altered ceRNA network modulating the target pathway. Loss of function and gain of function approaches were performed to examine the role of the ceRNA axis in β-elemene's regulation of the target pathway and cell viability. Additionally, in a β-elemene-treated rabbit model of airway stenosis, endoscopic and histological examinations were used to evaluate its therapeutic efficacy and further verify its mechanism of action. Results The hyperactive ILK/Akt pathway and dysregulated LncRNA-MIR143HG, which acted as a miR-1275 ceRNA to modulate ILK expression, were suppressed in β-elemene-treated airway granulation fibroblasts; β-elemene suppressed the ILK/Akt pathway via the MIR143HG/miR-1275/ILK axis. Additionally, the cell cycle and apoptotic phenotypes of granulation fibroblasts were altered, consistent with ILK/Akt pathway activity. In vivo application of β-elemene attenuated airway granulation hyperplasia and alleviated scar stricture, and histological detections suggested that β-elemene's effects on the MIR143HG/miR-1275/ILK axis and ILK/Akt pathway were in line with in vitro findings. Conclusions MIR143HG and ILK may act as ceRNA to sponge miR-1275. The MIR143HG/miR-1275/ILK axis mediates β-elemene-induced cell cycle arrest and apoptosis of airway granulation fibroblasts by modulating the ILK/Akt pathway, thereby inhibiting airway granulation proliferation and ultimately alleviating airway stenosis.

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Previous Humoral Immunity to the Endemic Seasonal Alphacoronaviruses NL63 and 229E Is Associated with Worse Clinical Outcome in COVID-19 and Suggests Original Antigenic Sin

Life ◽

10.3390/life11040298 ◽

2021 ◽

Vol 11 (4) ◽

pp. 298

Author(s):

Daniele Focosi ◽

Angelo Genoni ◽

Ersilia Lucenteforte ◽

Silvia Tillati ◽

Antonio Tamborini ◽

...

Keyword(s):

Humoral Immunity ◽

Cross Reactivity ◽

Clinical Severity ◽

World Health ◽

Laboratory Findings ◽

Cellular And Humoral Immunity ◽

Rate Ratios ◽

Original Antigenic Sin

Antibody-dependent enhancement (ADE) of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) infection has been hypothesized. However, to date, there has been no in vitro or in vivo evidence supporting this. Cross-reactivity exists between SARS CoV-2 and other Coronaviridae for both cellular and humoral immunity. We show here that IgG against nucleocapsid protein of alphacoronavirus NL63 and 229E correlate with the World Health Organization’s (WHO) clinical severity score ≥ 5 (incidence rate ratios was 1.87 and 1.80, respectively, and 1.94 for the combination). These laboratory findings suggest possible ADE of SARS CoV-2 infection by previous alphacoronavirus immunity.

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