Knockout Lines Research Articles

β-1,3-GLUCANASE10 regulates tomato development and disease resistance by modulating callose deposition.

β-1,3-Glucanases are considered key regulators responsible for the degradation of callose in plants, yet little is known about the role and mode of action of their encoding genes in tomato (Solanum lycopersicum). In the present study, we identified the β-1,3-glucanase encoding gene β-1,3-GLUCANASE10 (SlBG10) and revealed its regulation in tomato pollen and fruit development, seed production, and disease resistance by modulating callose deposition. Compared to wild-type or SlBG10 overexpressing (SlBG10-OE) lines, knockout of SlBG10 caused pollen arrest and failure to set fruit with reduced male rather than female fecundity. Further analyses showed that SlBG10-knockout promoted callose deposition in anther at the tetrad-to-microspore stages, resulting in pollen abortion and male sterility. Moreover, loss-of-function SlBG10 delayed degradation of endosperm cell wall calloses during cellularization and impeded early seed development. We also uncovered that Botrytis cinerea infection induces SlBG10 expression in wild-type tomato, and the knockout lines showed increased callose accumulation in fruit pericarps, reduced susceptibility to B. cinerea and enhanced antioxidant capacity to maintain tomato fruit quality. However, expression of genes encoding cell wall hydrolases decreased in SlBG10-knockout tomatoes and thus led to an increase in pericarp epidermal thickness, enhancement in fruit firmness, reduction of fruit water loss and extension of tomato shelf life. These findings not only expand our understanding of the involvement of β-1,3-glucanases as callose regulators in multiple developmental processes and pathogen resistance, but also provide additional insight into the manipulation of multi-agronomic traits for targeted tomato breeding.

Disruption of the amino acid transporter CsAAP2 inhibits auxin-mediated root development in cucumber.

Amino acid transporters are the principal mediators of organic nitrogen distribution within plants and are essential for plant growth and development. Despite this importance, relatively few amino acid transporter genes have been explored and elucidated in cucumber (Cucumis sativus). Here, a total of 86 amino acid transporter genes were identified in the cucumber genome. We further identified Amino Acid Permease (AAP) subfamily members that exhibited distinct expression patterns in different tissues. We found that the CsAAP2 as a candidate gene encoding a functional amino acid transporter is highly expressed in cucumber root vascular cells. CsAAP2 knockout lines exhibited arrested development of root meristem, which then caused the delayed initiation of lateral root and the inhibition of root elongation. What is more, the shoot growth of aap2 mutants was strongly retarded due to defects in cucumber root development. Moreover, aap2 mutants exhibited higher concentrations of amino acids and lignin in roots. We found that the mutant roots had a stronger ability to acidize medium. Furthermore, in the aap2 mutants, polar auxin transport was disrupted in the root tip, leading to high auxin levels in roots. Interestingly, slightly alkaline media rescued their severely reduced root growth by stimulating auxin pathway.

Sugar transporter ZmSWEET1b is responsible for assimilate allocation and salt stress response in maize

Sugar efflux transporter SWEET family is involved in multiple biological processes, from nectar secretion, pollen fertility to seed filling. Although roles of SWEETs in abiotic stress adaption have been revealed mainly in reference organism Arabidopsis, cereal crops SWEETs responses to abiotic stimulation remain largely elusive. Here, we report the characterization of maize SWEET family member ZmSWEET1b, with emphasis on its response to salinity stress. ZmSWEET1b is a canonical sugar transporter, characteristic of seven transmembrane helices and plasma membrane localization. ZmSWEET1b and its rice ortholog OsSWEET1b in phylogenetic clade I underwent convergent selection during evolution. Two independent knockout lines were created by the CRISPR/Cas9 method to functionally characterized ZmSWEET1b. Sucrose and fructose contents are significantly decreased in ZmSWEET1b knockout lines. Mature leaves of ZmSWEET1b-edited lines exhibit chlorosis, reminiscent of senescence-like phenotype. Ears and seeds of ZmSWEET1b knockout lines are small. Upon salinity treatment, ZmSWEET1b-edited lines become more wilted. Transcriptional abundance of genes for Na+ efflux from roots to the rhizosphere, including ZmSOS1, ZmH+-ATPASE 2, and ZmH+-ATPASE 8, is decreased in salt-treated ZmSWEET1b knockout lines. These findings indicate that convergently selected sugar transporter ZmSWEET1b is important for maize plant development and responses to salt stress. The manipulation of ZmSWEET1b may represent a feasible way forward in the breeding of salinity tolerant ideotypes through the optimization of assimilate allocation.

Auxin signaling module OsSK41-OsIAA10-OsARF regulates grain yield traits in rice.

Auxin is an important phytohormone in plants, and auxin signaling pathways in rice play key roles in regulating its growth, development, and productivity. To investigate how rice grain yield traits are regulated by auxin signaling pathways and to facilitate their application in rice improvement, we validated the functional relationships among regulatory genes such as OsIAA10, OsSK41, and OsARF21 that are involved in one of the auxin (OsIAA10) signaling pathways. We assessed the phenotypic effects of these genes on several grain yield traits across two environments using knockout and/or overexpression transgenic lines. Based on the results, we constructed a model that showed how grain yield traits were regulated by OsIAA10 and OsTIR1, OsAFB2, and OsSK41 and OsmiR393 in the OsSK41-OsIAA10-OsARF module and by OsARF21 in the transcriptional regulation of downstream auxin response genes in the OsSK41-OsIAA10-OsARF module. The population genomic analyses revealed rich genetic diversity and the presence of major functional alleles at most of these loci in rice populations. The strong differentiation of many major alleles between Xian/indica and Geng/japonica subspecies and/or among modern varieties and landraces suggested that they contributed to improved productivity during evolution and breeding. We identified several important aspects associated with the genetic and molecular bases of rice grain and yield traits that were regulated by auxin signaling pathways. We also suggested rice auxin response factor (OsARF) activators as candidate target genes for improving specific target traits by overexpression and/or editing subspecies-specific alleles and by searching and pyramiding the 'best' gene allelic combinations at multiple regulatory genes in auxin signaling pathways in rice breeding programs.

Biomechanical and biochemical assessment of YB-1 expression in A375 melanoma cell line: Exploratory study.

Malignant melanoma is the most lethal form of skin cancer. Y-box binding protein 1 (YB-1) plays a prominent role in mediating metastatic behavior by promoting epithelial-to-mesenchymal transition (EMT). Migratory melanoma cells exhibit two major migration modes: elongated mesenchymal or rounded amoeboid. Using A375 melanoma cell line and the YB-1 knock-out model, we aimed to elucidate biochemical and biomechanical changes in migration signaling pathways in the context of melanoma metastases. We subjected A375 YB-1 knock-out and parental cells to atomic force microscopy (stiffness determination), immunolabelling, and proteome analysis. We found that YB-1 expressing cells were significantly stiffer compared to the corresponding YB-1 knock-out cell line. Our study demonstrated that the constitutive expression of YB-1 in A375 melanoma cell line appears to be closely related to known biomarkers of epithelial-to-mesenchymal transition, nestin, and vimentin, resulting in a stiffer phenotype, as well as a wide array of proteins involved in RNA, ribosomes, and spliceosomes. YB-1 knock-out resulted in nestin depletion and significantly lower vimentin expression, as well as global upregulation of proteins related to the cytoskeleton and migration. YB-1 knock-out cells demonstrated both morphological features and biochemical drivers of mesenchymal/ameboid migration. Melanoma is a highly plastic, adaptable, and aggressive tumor entity, capable of exhibiting characteristics of different migratory modes.

Claudin-4-adhesion signaling drives breast cancer metabolism and progression via liver X receptor β

BackgroundCell adhesion is indispensable for appropriate tissue architecture and function in multicellular organisms. Besides maintaining tissue integrity, cell adhesion molecules, including tight-junction proteins claudins (CLDNs), exhibit the signaling abilities to control a variety of physiological and pathological processes. However, it is still fragmentary how cell adhesion signaling accesses the nucleus and regulates gene expression.MethodsBy generating a number of knockout and rescued human breast cell lines and comparing their phenotypes, we determined whether and how CLDN4 affected breast cancer progression in vitro and in vivo. We also identified by RNA sequencing downstream genes whose expression was altered by CLDN4-adhesion signaling. Additionally, we analyzed by RT-qPCR the CLDN4-regulating genes by using a series of knockout and add-back cell lines. Moreover, by immunohistochemistry and semi-quantification, we verified the clinicopathological significance of CLDN4 and the nuclear receptor LXRβ (liver X receptor β) expression in breast cancer tissues from 187 patients.ResultsWe uncovered that the CLDN4-adhesion signaling accelerated breast cancer metabolism and progression via LXRβ. The second extracellular domain and the carboxy-terminal Y197 of CLDN4 were required to activate Src-family kinases (SFKs) and the downstream AKT in breast cancer cells to promote their proliferation. Knockout and rescue experiments revealed that the CLDN4 signaling targets the AKT phosphorylation site S432 in LXRβ, leading to enhanced cell proliferation, migration, and tumor growth, as well as cholesterol homeostasis and fatty acid metabolism, in breast cancer cells. In addition, RT-qPCR analysis showed the CLDN4-regulated genes are classified into at least six groups according to distinct LXRβ- and LXRβS432-dependence. Furthermore, among triple-negative breast cancer subjects, the "CLDN4-high/LXRβ-high" and "CLDN4-low and/or LXRβ-low" groups appeared to exhibit poor outcomes and relatively favorable prognoses, respectively.ConclusionsThe identification of this machinery highlights a link between cell adhesion and transcription factor signalings to promote metabolic and progressive processes of malignant tumors and possibly to coordinate diverse physiological and pathological events.

The cytokine receptor CRLF3 is a human neuroprotective EV-3 (Epo) receptor.

The evolutionary conserved orphan cytokine receptor-like factor 3 (CRLF3) has been implicated in human disease, vertebrate hematopoiesis and insect neuroprotection. While its specific functions are elusive, experimental evidence points toward a general role in cell homeostasis. Erythropoietin (Epo) is a major regulator of vertebrate hematopoiesis and a general cytoprotective cytokine. Erythropoietic functions mediated by classical Epo receptor are understood in great detail whereas Epo-mediated cytoprotective mechanisms are more complex due to involvement of additional Epo receptors and a non-erythropoietic splice variant with selectivity for certain receptors. In the present study, we show that the human CRLF3 mediates neuroprotection upon activation with the natural Epo splice variant EV-3. We generated CRLF3 knock-out iPSC lines and differentiated them toward the neuronal lineage. While apoptotic death of rotenone-challenged wild type iPSC-derived neurons was prevented by EV-3, EV-3-mediated neuroprotection was absent in CRLF3 knock-out neurons. Rotenone-induced apoptosis and EV-3-mediated neuroprotection were associated with differential expression of pro-and anti-apoptotic genes. Our data characterize human CRLF3 as a receptor involved in Epo-mediated neuroprotection and identify CRLF3 as the first known receptor for EV-3.

Abstract 4824: Intracellular ferritin expression levels regulate growth and resistance to ferroptosis in pancreatic cancer cell lines

Abstract Background: Pancreatic cancer is one of the cancers with the highest mortality rate, and it is important to develop effective treatments and elucidate prognostic factors. It has been reported that elevated serum ferritin is associated with the prognosis of pancreatic cancer. However, there are few reports on how ferritin in pancreatic cancer tissue acts in pancreatic cancer. Ferritin heavy chain (FTH) is a major component of ferritin, which stores iron and plays a role in suppressing iron-induced generation of reactive oxygen species. In this study, we aimed to examine the expression level of FTH in pancreatic cancer cells and its prognostic and cell biological roles. Methods: We performed immunostaining of FTH in pancreatic cancer tissue specimens operated on at our hospital and classified the expression level of FTH by scoring the immunostaining intensity and staining rate. The expression levels and prognosis were then evaluated. The overexpression and knockout strains of FTH were generated using a human pancreatic cancer cell line (MIAPaCa-2), and their proliferative and stress tolerance abilities were compared with those of the control group. Results: Immunostaining of 149 pancreatic cancer surgical specimens showed that the group with high FTH had a significantly shorter overall survival rate than the group with low FTH (5-year survival rate; 16.8% vs. 36.9%, p = 0.018). In multivariate analysis, high FTH expression was also a prognostic factor: in vitro, cell lines overexpressing FTH had enhanced proliferative potential compared to controls, while knockout lines were suppressed. In addition, the knockout lines showed decreased resistance to RSL-3, an inducer of ferroptosis. Additional studies are currently underway to examine resistance to other drugs and the production of reactive oxygen species in the cells. Conclusion: In pancreatic cancer, high ferritin expression is associated with worse prognosis and may enhance cell proliferation and stress tolerance, and might be a new treatment or biomarker. Citation Format: Masataka Maruno, Yo-hei Kanamori, Takashi Matsumoto, Yuta Shiraishi, Toru Takematsu, Tatsunori Miyata, Kosuke Mima, Shigeki Nakagawa, Hidetoshi Nitta, Hiromitsu Hayashi, Toshiro Moroishi, Hideo Baba. Intracellular ferritin expression levels regulate growth and resistance to ferroptosis in pancreatic cancer cell lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4824.

Abstract 6030: Asparagine metabolism is linked to sex differences in colorectal cancer growth

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in men and women in the United States. We have shown that tumors from female CRC patients have increased asparagine (Asn) which is positively associated with poorer overall survival for female patients only (n=196, stage I-III). Furthermore, analysis of asparagine synthetase (ASNS) expression shows that ASNS is similarly associated with poorer survival for females with CRC only. Currently it is not understood why there are sex-specific differences in tumor metabolism of Asn, or the roles of ASNS in CRC progression. Therefore, we hypothesize that limiting ASNS-driven Asn production is a viable target to suppress CRC development in a sex-specific manner. Therefore, we used a combination of in vitro and in vivo models with human CRC ASNS knock out (KO) cells to generate additional hypotheses that may reveal mechanisms that underlie this finding. Using a 3D spheroid model, we cultured HCT116 ASNS KO and isogenic HCT116 ASNS WT cell lines and modulated Asn supply. During 7 days of growth, depletion of Asn slowed the growth of ASNS KO cells compared to WT, whereas Asn supplementation (400 μM) recovered growth in both cell lines; however, spheroid growth was less in the KO cells. Subcutaneous xenograft of these cell lines in male and female mice (n=10/genotype/sex) showed that loss of ASNS reduced tumor burden in both male and female mice, and improved survival in female mice alone. Pathological assessment of tumors indicated poorly differentiated carcinoma and presence of viable tumor mass in both groups. However, the mitotic activity index, and cellular proliferation (measured by Ki67 staining) was significantly reduced in female ASNS KO mice compared to ASNS WT, showing that the tumor cells with ASNS KO are growing slower than those of ASNS WT counterparts. A multi-omics approach was then used to investigate metabolic pathways and signaling networks altered between the tumor genotypes. Tumor tissue metabolomics revealed significant decreases in nucleotides, amino acids, fatty acids within the ASNS KO compared to WT tumors. Using RNA-Seq, gene set enrichment and Ingenuity Pathway Analysis (IPA), data were integrated revealing disruption to Wnt/B-catenin signaling and immune responses associated with ASNS KO in female mice. Serum metabolomics also revealed a higher ratio of circulating glutamine to glutamate in the female mice with ASNS KO xenografts compared to WT, and a decreased expression of the tumor aspartate-glutamate transporter SLC1A3, therefore tumors with ASNS KO could have decreased ability to transport metabolites needed for Asn generation. In summary we identified that lack of ASNS suppresses tumor progression in a pre-clinical CRC model, and significantly improves survival in female mice only. Validation and further mechanistic experiments will reveal the robustness of this finding. Citation Format: Caroline Johnson, Oladimeji Aladelokun, Sajid Khan. Asparagine metabolism is linked to sex differences in colorectal cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6030.

Abstract 2966: Exploring the therapeutic potential of a novel Siglec-6 targeted bispecific antibody to selectively kill leukemia cells

Abstract Objectives of the Study: Bispecific antibodies (BsAbs) are a class of immunotherapy drugs that facilitate targeted killing of cancer cells. Here, we report therapeutic potential of a novel Siglec-6/CD3 targeted bsAb against chronic lymphocytic leukemia (CLL) cells. We show here for the first time the functional and therapeutic relevance of Siglec-6 in CLL cell adhesion and migration. Mechanistically, we have shown Siglec-6 mediated cell adhesion and migration through Siglec-6 ligand (sialyl Tn) mediated DOCK8 dependent activation of Cdc42 associated with actin polymerization. To evaluate the therapeutic potential of Siglec-6, we generated a human (hu) Siglec-6 transgenic mouse model and crossed it with the TCL1 CLL mouse model to obtain Siglec-6 x TCL1 mice which develop Siglec-6+ leukemia. A pilot in-vivo study with anti-Siglec-6/CD3 targeted bsAb showed a survival benefit in humanized CD3 (huCD3) mice engrafted with Siglec-6+ leukemic cells. Methods used: Flow cytometry was used to analyze cell surface expression of Siglec-6 and sialyl Tn (sTn). Mass spectrometry analysis was performed to identify Siglec-6 interacting proteins. CRISPR/Cas9 technique was used to generate knock-out (KO) cell lines for mechanistic studies. Phalloidin staining followed by confocal imaging was used to examine actin polymerization. For in-vivo BsAb treatment experiments, huCD3 mice were engrafted with 5 million Siglec-6+ leukemic cells isolated from Siglec-6 x TCL1 mouse splenocytes. Weekly treatment (i.v) was started after mice display 5% circulating leukemia. Results and Conclusion: Compared to Siglec-6+ CLL cells, Siglec-6- CLL cells exhibited significant reduction in adhesion to (~50%) and migration towards (~50%) CLL-BMSCs. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells and primary CLL cells to the spleen and bone marrow in NSG mice (~35%). Mass spectrometry and co-immunoprecipitation analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation and WASP protein recruitment, followed by increased actin polymerization, that were compromised in Siglec-6 or DOCK8 KO MEC1 cells. Siglec-6+ leukemia engrafted huCD3 mice treated with anti-Siglec-6/CD3 BsAb has so far demonstrated a survival benefit with median survival of 18 weeks versus a median survival of 8 weeks in the control group that received a non-targeting BsAb (n=4/group). Significance: We have for the first time shown Siglec-6 dependent recruitment of DOCK8 leading to migration and adhesion of B-CLL cells. An anti-Siglec-6/CD3 BsAb provides a survival benefit against Siglec-6+ leukemia, paving the way for development of Siglec-6 targeted therapeutics to treat CLL. Citation Format: Jessica Nunes, Charlene Mao, Matthew Purcell, Elizabeth Perry, Liwen Zhang, Xiaokui Mo, Matthew Cyr, Meixiao Long, John Byrd, Christoph Rader, Natarajan Muthusamy. Exploring the therapeutic potential of a novel Siglec-6 targeted bispecific antibody to selectively kill leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2966.

Abstract 6143: Novel multiplexed assays of ferroptosis, pyroptosis and necroptosis biomarkers for translational studies

Abstract Background: The recent discovery that multiple chemo- and immuno-therapies activate non-apoptotic cell death pathways such as necroptosis, pyroptosis, and ferroptosis in cancer cells has necessitated development of pharmacodynamic biomarker assays to study their novel mechanisms of action. We describe the development, analytical validation, and proof-of-concept demonstration of assays for potential biomarkers of ferroptosis, pyroptosis and necroptosis pathways to support anti-cancer drug development. Methods: We developed multiplex sandwich immunoassays on the Luminex platform to quantify transferrin receptor (TfR), glutathione peroxidase 4 (GPx4), active caspase-3, cleaved and full-length gasdermins D and E (GSDMD, GSDME), MLKL oligomers, and MLKL heterodimers with RIPK1 and RIPK3. Novel antibodies to gasdermin cleavage neoepitopes were generated and their specificity validated with knockout (GSDMD) or non-expressing (GSDME) cell lines. We utilized well-established in vitro cancer cell line models (NCI-H522, THP-1, MV411, MB231, HT29) and inducers/inhibitors of pyroptosis, necroptosis and ferroptosis to test for expected directional changes of biomarker modulation. Results: Analytical validity of the measurements is demonstrated with acceptable reproducibility, precision, dilutional linearity, dilutional recovery, spike recovery, sample freeze-thaw and temperature stability. The fitness-for-purpose of the assays is demonstrated by increased cleavage of GSDME, loss of full-length GSDME, and activation of caspase-3 in NCI-H522 cells treated with doxorubicin; similarly, cleavage of GSDMD and loss of full-length GSDMD is shown in THP-1 cells primed with LPS and treated with nigericin, and in MV-4-11 cells treated with val-boroPro. HT29 cells treated with ZVAD + birinapant + TNFα were used to demonstrate increased MLKL oligomerization or heterodimer formation with RIPK1/RIPK3. Modulation of ferroptosis biomarkers TfR and GPx4 (SU-DHL-5, MDA-MB-231) in response to imidazole ketone erastin (IKE) or RSL3 was cell line-dependent. Studies are underway to validate modulation of select ferroptosis biomarkers using orthogonal LC-MS methods. Conclusions: We describe development and translational readiness of novel multiplex assays to interrogate drugs inducing ferroptosis, pyroptosis and necroptosis cell death pathways. Our data demonstrate utility of biomarkers of pyroptosis and necroptosis, whereas biomarkers of ferroptosis require further appraisal. In vivo pharmacodynamic studies of pyroptosis, ferroptosis and necroptosis inducers are ongoing to verify assay fitness-for-purpose with tumor biopsy specimens. This project was funded with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: William G. Herrick, Jeevan Govindharajulu, Ralph E. Parchment, James H. Doroshow, Apurva K. Srivastava. Novel multiplexed assays of ferroptosis, pyroptosis and necroptosis biomarkers for translational studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6143.

Exploring the metabolic and physiological roles of HQT in S. lycopersicum by gene editing.

The most abundant phenolic compound in Solanaceous plants is chlorogenic acid (CGA), which possesses protective properties such as antimicrobial and antioxidant activities. These properties are particularly relevant when plants are under adverse conditions, such as pathogen attack, excess light, or extreme temperatures that cause oxidative stress. Additionally, CGA has been shown to absorb UV-B light. In tomato and potato, CGA is mainly produced through the HQT pathway mediated by the enzyme hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase. However, the absence of natural or induced mutants of this gene has made it unclear whether other pathways contribute to CGA production and accumulation. To address this question, we used CRISPR technology to generate multiple knock-out mutant lines in the tomato HQT gene. The resulting slhqt plants did not accumulate CGA or other caffeoylquinic acids (CQAs) in various parts of the plant, indicating that CQA biosynthesis depends almost entirely on the HQT pathway in tomato and, likely, other Solanaceous crops. We also found that the lack of CGA in slhqt plants led to higher levels of hydroxycinnamoyl-glucose and flavonoids compared to wild-type plants. Gene expression analysis revealed that this metabolic reorganization was partly due to flux redirection, but also involved modulation of important transcription factor genes that regulate secondary metabolism and sense environmental conditions. Finally, we investigated the physiological role of CGA in tomato and found that it accumulates in the upper epidermis where it acts as a protector against UV-B irradiation.

Generation of two homozygous SHOX2 knock-out human induced pluripotent stem cell lines using CRISPR/Cas9

SHOX2 is a homeobox transcription factor associated with atrial fibrillation (AF) and sinus node dysfunction. Here, we generated two homozygous SHOX2 knock-out hiPSC lines from a healthy control line and a corrected AF patient line (disease-specific SHOX2 mutation corrected to WT) using CRISPR/Cas9. These cell lines maintained pluripotency, an ability to differentiate into all three germlayers and a normal karyotype, presenting a valuable tool to investigate the impact of a full SHOX2 knock-out with respect to arrhythmogenic diseases on a cellular level.

Indian Himalayan natural Arabidopsis thaliana accessions with abolished miR158 levels exhibit robust miR173-initiated trans-acting cascade silencing.

Small RNAs (sRNAs) such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) are short 20-24-nucleotide non-coding RNAs. They are key regulators of gene expression in plants and other organisms. Several 22-nucleotide miRNAs trigger biogenesis cascades of trans-acting secondary siRNAs, which are involved in various developmental and stress responses. Here we show that Himalayan Arabidopsis thaliana accessions having natural mutations in the miR158 locus exhibit robust cascade silencing of the pentatricopeptide repeat (PPR)-like locus. Furthermore, we show that these cascade sRNAs trigger tertiary silencing of a gene involved in transpiration and stomatal opening. The natural deletions or insertions in MIR158 led to improper processing of miR158 precursors, thereby blocking synthesis of mature miR158. Reduced miR158 levels led to increased levels of its target, a pseudo-PPR gene that is targeted by tasiRNAs generated by the miR173 cascade in other accessions. Using sRNA datasets derived from Indian Himalayan accessions, as well as overexpression and knockout lines of miR158, we show that absence of miR158 led to buildup of pseudo-PPR-derived tertiary sRNAs. These tertiary sRNAs mediated robust silencing of a gene involved in stomatal closure in Himalayan accessions lacking miR158 expression. We functionally validated the tertiary phasiRNA that targets NHX2, which encodes a Na+ -K+ /H+ antiporter protein, thereby regulating transpiration and stomatal conductance. Overall, we report the role of the miRNA-TAS-siRNA-pseudogene-tertiary phasiRNA-NHX2 pathway in plant adaptation.

Additional polgD257A mutation (mutator) does not influence dopaminergic neurodegeneration in aged parkin-deficient mice

Background: Parkinson’s disease is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra pars compacta. Among the first identified causes of autosomal recessive Parkinson’s disease were mutations in the parkin gene. Independently, we and other groups have developed various parkin knockout mice, and none displayed dopaminergic degeneration in the substantia nigra. Interestingly, dopaminergic degeneration in the substantia nigra has been reported in a parkin knockout line (exon 3 deletion) carrying an additional mutation (D257A) in the mitochondrial DNA polymerase (polg) gene (mutator). The mutator mice show accelerated mutation rates in mitochondrial DNA resulting in a premature-aging phenotype. Methods: To verify this finding, we crossed our parkin-deficient mice with the mutator mice, and characterized phenotypic changes of the parkin/mutator double mutant mice up to one year of age. We examined their locomotion and motor coordination behaviors by using the open field, the rotarod, and the pole test, subsequently investigating their nigrostriatal axis by counting TH-positive cells in every tenth section throughout the entire substantia nigra pas compacta and their termini in the striatum. Results: The double mutants did not display additional deficiencies in locomotion in our behavior tests. We could also not detect dopaminergic neurodegeneration in the substantia nigra pars compacta of aged double mutants measured by levels of tyrosine hydroxylase positive neurons in the substantia nigra pars compacta as well as in striatal terminals. Conclusion: Our results do not support the hypothesis that the polgD257A mutation contributes to the age-related vulnerability of dopaminergic neurons in parkin-deficient mice. Keywords: Parkin, neurodegeneration, polgD257A, mutator, substantia nigra

The role of the mitochondrial outer membrane protein SLC25A46 in mitochondrial fission and fusion.

Mutations in SLC25A46 underlie a wide spectrum of neurodegenerative diseases associated with alterations in mitochondrial morphology. We established an SLC25A46 knock-out cell line in human fibroblasts and studied the pathogenicity of three variants (p.T142I, p.R257Q, and p.E335D). Mitochondria were fragmented in the knock-out cell line and hyperfused in all pathogenic variants. The loss of SLC25A46 led to abnormalities in the mitochondrial cristae ultrastructure that were not rescued by the expression of the variants. SLC25A46 was present in discrete puncta at mitochondrial branch points and tips of mitochondrial tubules, co-localizing with DRP1 and OPA1. Virtually, all fission/fusion events were demarcated by a SLC25A46 focus. SLC25A46 co-immunoprecipitated with the fusion machinery, and loss of function altered the oligomerization state of OPA1 and MFN2. Proximity interaction mapping identified components of the ER membrane, lipid transfer proteins, and mitochondrial outer membrane proteins, indicating that it is present at interorganellar contact sites. SLC25A46 loss of function led to altered mitochondrial lipid composition, suggesting that it may facilitate interorganellar lipid flux or play a role in membrane remodeling associated with mitochondrial fusion and fission.

Comparative analysis of two new zebrafish models: The cyp1a low-expression line and cyp1a knockout line under PAHs exposure

Cytochrome P450 1 (CYP1) is an important enzyme family involved in the metabolism of pollutants, and used as a biomarker to monitor environmental pollution. In this study, a fluorescence-labeled cyp1a zebrafish line, named as KI (cyp1a+/+-T2A-mCherry) (KICM), was originally constructed to monitor dioxin-like compounds in the environment. However, the cyp1a gene expression in the KICM line was inhibited by the fluorescence labeling, thus leading to a significantly increased sensitivity of KICM zebrafish line to PAHs. Then, a cyp1a knockout zebrafish line, named KOC, were constructed for comparative analysis with the cyp1a low-expression line. Interestingly, knockout of the cyp1a gene did not increase the sensitivity of zebrafish to PAHs as significantly as the cyp1a low-expression line. So, the expression levels of related genes in the aryl hydrocarbon receptor pathway were analyzed and the results showed that the expression level of cyp1b in KOC group was significantly higher than that of wild type and KICM under the same PAH exposure. This indicated that the effect of losing cyp1a was compensated by inducing expression of cyp1b. In conclusion, two new zebrafish models including cyp1a low-expression line and cyp1a knockout line were constructed in this study, which may provide a convenient model for subsequent studies on the toxicity mechanism of PAHs and the role of cyp1a in detoxification.

Overexpression of a GIPC glycosyltransferase gene, OsGMT1, suppresses plant immunity and delays heading time in rice

Glycosylinositol phosphorylceramides (GIPCs) are the major sphingolipids in the plant plasma membrane. In Arabidopsis, mutations of genes involved in the synthesis of GIPCs affect many physiological aspects of plants, including growth, pollen fertility, defense, and stress signaling. Loss of function of the GIPC MANNOSYL-TRANSFERASE1 (AtGMT1) results in GIPC misglycosylation and induces plant immune responses accompanied by a severely dwarfed phenotype, thus indicating that GIPCs play important roles in plant immunity. Here, we investigated the enzymatic activity and phenotypes of transgenic lines of OsGMT1, the ortholog of AtGMT1. Sphingolipidomic analysis indicated that OsGMT1 retained the enzymatic activity of GIPC hexose (Hex) glycosylation, but the knockout lines did not accumulate H2O2. In contrast, the OsGMT1 overexpression lines showed significant down-regulation of several defense-associated or cell wall synthesis-associated genes, and enhanced sensitivity to rice blast. Furthermore, we first demonstrated the sensitivity of rice cells to MoNLP1 protein through calcein AM release assays using rice protoplasts, thus legitimizing the presence of MoNLPs in rice blast fungus. In addition, yeast two-hybrid screens using OsGMT1 as bait revealed that OsGMT1 may regulate heading time through the OsHAP5C signaling pathway. Together, our findings suggested clear physiological functional differentiation of GMT1 orthologs between rice and Arabidopsis.

Ablation of pigment epithelium-derived factor receptor (PEDF-R/Pnpla2) causes photoreceptor degeneration

Photoreceptor cells express the patatin-like phospholipase domain-containing 2 (PNPLA2) gene that codes for pigment epithelium-derived factor receptor (PEDF-R) (also known as ATGL). PEDF-R exhibits phospholipase activity that mediates the neurotrophic action of its ligand PEDF. Because phospholipids are the most abundant lipid class in the retina, we investigated the role of PEDF-R in photoreceptors by generating CRISPR Pnpla2 knock-out mouse lines in a retinal degeneration-free background. Pnpla2−/− mice had undetectable retinal Pnpla2 gene expression and PEDF-R protein levels as assayed by RT-PCR and immunofluorescence, respectively. The photoreceptors of mice deficient in PEDF-R had deformities as examined by histology and transmission electron microscopy. Pnpla2 knockdown diminished the PLA2 enzymatic activity of PEDF-R in the retina. Lipidomic analyses revealed the accumulation of lysophosphatidyl choline-DHA and lysophosphatidyl ethanolamine-DHA in PEDF-R-deficient retinas, suggesting a possible causal link to photoreceptor dysfunction. Loss of PEDF-R decreased levels of rhodopsin, opsin, PKCα, and synaptophysin relative to controls. Pnpla2−/− photoreceptors had surface-exposed phosphatidylserine, and their nuclei were TUNEL positive and condensed, revealing an apoptotic onset. Paralleling its structural defects, PEDF-R deficiency compromised photoreceptor function in vivo as indicated by the attenuation of photoreceptor a- and b-waves in Pnpla2−/− and Pnpla2+/− mice relative to controls as determined by electroretinography. In conclusion, ablation of PEDF-R in mice caused alteration in phospholipid composition associated with malformation and malperformance of photoreceptors. These findings identify PEDF-R as an important component for photoreceptor structure and function, highlighting its role in phospholipid metabolism for retinal survival and its consequences.

Roles of miR-196a and miR-196b in Zebrafish Motor Function.

The exertion of motor function depends on various tissues, such as bones and muscles. miR-196 has been widely studied in cancer and other fields, but its effect on bone and skeletal muscle is rarely reported. In order to explore the role of miR-196 family in bone and skeletal muscle, we used the previously successfully constructed miR-196a-1 and miR-196b gene knockout zebrafish animal models for research. The behavioral trajectories of zebrafish from 4 days post-fertilization (dpf) to 7 dpf were detected to analyze the effect of miR-196a-1 and miR-196b on motor ability. Hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM) were used to detect the dorsal muscle tissue of zebrafish. The bone tissue of zebrafish was detected by microcomputed tomography (micro-CT). Real-time PCR was used to detect the expression levels of related genes, including vcp, dpm1, acta1b, mylpfb, col1a1a, bmp8a, gdf6a, and fgfr3. The behavioral test showed that the total behavioral trajectory, movement time, and movement speed of zebrafish larvae were decreased in the miR-196a-1 and miR-196b gene knockout lines. Muscle tissue analysis showed that the structure of muscle fibers in the zebrafish lacking miR-196a-1 and miR-196b was abnormal and was characterized by vacuolar degeneration of muscle fibers, intranuclear migration, melanin deposition, and inflammatory cell infiltration. Bone CT examination revealed decreased bone mineral density and trabecular bone number. The real-time PCR results showed that the expression levels of vcp, dpm1, gdf6a, fgfr3, and col1a1a were decreased in the miR-196b gene knockout group. The expression levels of dpm1, acta1b, mylpfb, gdf6a, and col1a1a were decreased, and the expression level of fgfr3 was increased in the miR-196b gene knockout group compared with the wild-type group. miR-196a-1 and miR-196b play an important role in muscle fiber structure, bone mineral density, and bone trabecular quantity by affecting the expression of vcp, dpm1, acta1b, mylpfb, gdf6a, fgfr3, and col1a1a and then affect the function of the motor system.