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The genome of Hepatitis B virus (HBV) persists in infected hepatocytes as a nuclear episome (cccDNA) that is responsible for the transcription of viral genes and viral rebound, following antiviral treatment arrest in chronically infected patients. There is currently no clinically approved therapeutic strategy able to efficiently target cccDNA (Lucifora J 2016). The development of alternative strategies aiming at permanently abrogating HBV RNA production requires a thorough understanding of cccDNA transcriptional and post-transcriptional regulation. In a previous study, we discovered that 1C8, a compound that inhibits the phosphorylation of some cellular RNA-binding proteins, could decrease the level of HBV RNAs. Here, we aimed at identifying kinases responsible for this effect. Among the kinases targeted by 1C8, we focused on DYRK1A, a dual-specificity kinase that controls the transcription of cellular genes by phosphorylating transcription factors, histones, chromatin regulators as well as RNA polymerase II. The results of a combination of genetic and chemical approaches using HBV-infected hepatocytes, indicated that DYRK1A positively regulates the production of HBV RNAs. In addition, we found that DYRK1A associates with cccDNA, and stimulates the production of HBV nascent RNAs. Finally, reporter gene assays showed that DYRK1A up-regulates the activity of the HBV enhancer 1/X promoter in a sequence-dependent manner. Altogether, these results indicate that DYRK1A is a proviral factor that may participate in the HBV life cycle by stimulating the production of HBx, a viral factor absolutely required to trigger the complete cccDNA transcriptional program., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pastor et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: This study aimed to determine the roles of interleukin (IL)-17, TAO kinase 1 (TAOK1), and NOD-like receptor protein 3 (NLRP3) in cardiomyocyte pyroptosis and proliferation., Methods: The IL-17-treated H9C2 cells were used as in vitro heart failure (HF) models. These cells were subjected to TAOK1 overexpression or knockdown and treated with BMS-986299 (NLRP3 inflammasome agonist), MCC950 (NLRP3 inflammasome inhibitor), or verteporfin (Yes-associated protein [YAP] inhibitor). Thereafter, their pyroptosis, proliferative capacity, and gene and protein expression levels were detected. Doxorubicin-induced HF rats were used as in vivo models and subjected to TAOK1 overexpression. Thereafter, their myocardial pathology, NLRP3 inflammasome-mediated pyroptosis, and YAP/TEAD pathway function were evaluated., Results: IL-17 treatment increased the pyroptosis and decreased the proliferative capacity of H9C2 cells. Additionally, IL-17 treatment inducedto the activation of the NLRP3 inflammasomes and inhibition of the YAP/TEAD pathway in the H9C2 cells. Moreover, the IL-17-mediated effects on the H9C2 cells were alleviated by TAOK1 overexpression and augmented by TAOK1 knockdown. Furthermore, treatment with BMS-986299 or verteporfin affected the pyroptosis, proliferative capacity, and NLRP3 inflammasome activation of the H9C2 cells independently of TAOK1 expression. In the doxorubicin-induced HF rat model, TAOK1 overexpression mitigated myocardial injury, suppressed NLRP3 inflammasome pathway activation, and restored the YAP/TEAD pathway activity., Conclusion: TAOK1 played a crucial role in regulating IL-17-mediated increase in the pyroptosis and decrease in the proliferation of cardiomyocytes by regulating the activities of the NLRP3 inflammasomes and the YAP/TEAD pathway., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Neurofibromatosis type 2 is a genetic disorder that results in the formation and progressive growth of schwannomas, ependymomas, and/or meningiomas. The NF2 gene encodes the Merlin protein, which links cell cortical elements to the actin cytoskeleton and regulates a number of key enzymes including Group I p21-activated kinases (PAKs), the Hippo-pathway kinase LATS, and mTORC. While PAK1 and PAK2 directly bind Merlin and transmit proliferation and survival signals when Merlin is mutated or absent, inhibition of Group 1 PAKs alone has not proven sufficient to completely stop the growth of NF2-deficient meningiomas or schwannomas in vivo, suggesting the need for a second pathway inhibitor. As the Hippo pathway is also activated in NF2-deficient cells, several inhibitors of the Hippo pathway have recently been developed in the form of YAP-TEAD binding inhibitors. These inhibitors prevent activation of pro-proliferation and anti-apoptotic Hippo pathway effectors. In this study, we show that PAK inhibition slows cell proliferation while TEAD inhibition promotes apoptotic cell death. Finally, we demonstrate the efficacy of PAK and TEAD inhibitor combinations in several NF2-deficient Schwannoma cell lines., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Benton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: This study aimed to identify the associations between individual KRAS, STK11, KEAP1, or TP53 mutations, as well as the comutation status of these genes, and the tumor mutation burden (TMB) with clinical outcomes of lung adenocarcinoma patients treated with immune checkpoint inhibitors (ICIs)., Methods: We collected data from patients with lung adenocarcinoma treated with ICIs from the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) database between June 2019 and August 2023. The main endpoints were the treatment response and overall survival (OS)., Results: Among 343 patients with lung adenocarcinoma, 61 (18%), 69 (20%), 41 (12%), and 222 (65%) patients had KRAS, STK11, KEAP1, and TP53 mutations, respectively. An overall objective response was observed in 94 of 338 patients (28%), including 2 (1%) who achieved a complete response and 92 (27%) who achieved a partial response. Patients with STK11, KEAP1, or TP53 mutations had a significantly greater TMB (P<0.001). According to the univariate analysis, the treatment response was significantly correlated with TP53 mutation in both the general (P = 0.041) and KRAS wild-type (P = 0.009) populations. KEAP1 and TP53 mutations were associated with worse OS among assessable patients (hazard ratio (HR) = 2.027, P = 0.002; HR = 1.673, P = 0.007, respectively) and among patients without KRAS mutations (HR = 1.897, P = 0.012; HR = 1.908, P = 0.004, respectively). According to the multivariate analysis, KEAP1 (HR = 1.890, P = 0.008) and TP53 (HR = 1.735, P = 0.011) mutations were found to be independent factors for OS., Conclusions: STK11, KEAP1, and TP53 mutations are significantly associated with a high TMB. TP53 mutation could affect the treatment response to some degree, and both KEAP1 and TP53 mutations resulted in inferior OS in the general patient population and in those with KRAS-wild-type lung adenocarcinoma, indicating that KEAP1 and TP53 mutations might act as prognostic factors for ICI treatment in lung adenocarcinoma patients., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Yuichi Ando reports research funds and personal fees from Chugai Pharmaceutical Co., Ltd., research funds from BeiGene. Ltd and Geo Holdings Corporation, and personal fees from Bayer Holding Ltd. The other authors have no conflicts of interest to declare., (Copyright: © 2024 Liang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mesenchymal stem cells (MSC) can differentiate into chondrocytes. Epstein-Barr virus-induced gene 3 (EBI3) is differentially expressed during chondrogenic differentiation and can be produced by MSC. EBI3 is also a subunit of interleukin (IL)-27 and IL-35, and it accumulates in the endoplasmic reticulum (ER) when its partners, such as IL-27 p28 and IL-35 p35, are insufficient. ER stress induced by protein accumulation is responsible for chondrogenic differentiation. However, the role of EBI3 and its relevance to the ER stress in chondrogenic differentiation of MSC have never been addressed. Here, we demonstrate that EBI3 protein is expressed in the early stage of chondrogenic differentiation of MSC. Additionally, knockdown, overexpression, or induction of EBI3 through IL-1β inhibits chondrogenesis. We show that EBI3 localizes and accumulates in the ER of MSC after overexpression or induction by IL-1β and TNF-α, whereas ER stress inhibitor 4-phenylbutyric acid decreases its accumulation in MSC. Moreover, EBI3 modulates ER stress sensor inositol-requiring enzyme 1 α (IRE1α) after induced by IL-1β, and MSC-like cells coexpress EBI3 and IRE1α in rheumatoid arthritis (RA) synovial tissue. Altogether, these data demonstrate that intracellular EBI3 commits to chondrogenic differentiation by regulating ER stress sensor IRE1α., Competing Interests: Y.T. has received speaking fees and/or honoraria from Daiichi-Sankyo, Astellas, Chugai, Eli Lilly, Pfizer, Abbvie, YL Biologics, Bristol-Myers, Takeda, Mitsubishi-Tanabe, Novartis, Eisai, Janssen, Teijin and has received research grants from Asahi-kasei, Mitsubishi-Tanabe, Chugai, Takeda, Sanofi, Bristol-Myers, UCB, Daiichi-Sankyo, Eisai, Ono. S.N. has received consulting fees, speaking fees, and/or honoraria from Bristol-Myers, Pfizer, GlaxoSmithKline, Sanofi, Chugai, Astellas, Asahi-kasei, Boehringer Ingelheim and has received research grants from Mitsubishi-Tanabe, Novartis and MSD. All other authors declare no conflicts of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2022 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Increased intrahepatic bile acids cause endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) is activated to maintain homeostasis. UPR dysregulation, including the inositol-requiring enzyme 1α/X-box protein 1 (IRE1α/XBP1) pathway, is associated with adult liver diseases but has not been characterized in pediatric liver diseases. We evaluated hepatic UPR expression in pediatric cholestatic liver disease (CLD) explants and hypothesize that an inability to appropriately activate the hepatic IRE1α/XBP1 pathway is associated with the pathogenesis of CLD., Methods: We evaluated 34 human liver explants, including: pediatric CLD (Alagille, ALGS, and progressive familial intrahepatic cholestasis, PFIC), pediatric non-cholestatic liver disease controls (autoimmune hepatitis, AIH), adult CLD, and normal controls. We performed RNA-seq, quantitative PCR, and western blotting to measure expression differences of the hepatic UPR and other signaling pathways., Results: Pathway analysis demonstrated that the KEGG 'protein processing in ER' pathway was downregulated in pediatric CLD compared to normal controls. Pediatric CLD had decreased hepatic IRE1α/XBP1 pathway gene expression and decreased protein expression of phosphorylated IRE1α compared to normal controls. IRE1α/XBP1 pathway gene expression was also decreased in pediatric CLD compared to AIH disease controls., Conclusions: Pediatric CLD explants have decreased expression of the protective IRE1α/XBP1 pathway and down-regulated KEGG protein processing in the ER pathways. IRE1α/XBP1 pathway expression differences occur when compared to both normal and non-cholestatic disease controls. Attenuated expression of the IRE1α/XBP1 pathway is associated with cholestatic diseases and may be a target for future therapeutics., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Kriegermeier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The protein kinase Gcn2 is present in virtually all eukaryotic cells. It is best known for its role in helping cells cope with amino acid starvation. Under starvation, Gcn2 phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF2α), to stimulate a signal transduction pathway that allows cells to cope and overcome starvation. Gcn2 has been implicated in many additional biological functions. It appears that for all functions, Gcn2 must directly bind to its effector protein Gcn1, mediated via a region in Gcn1 called the RWD binding domain (RWDBD). Arg-2259 in this region is important for Gcn2 binding. Overexpression of a Gcn1 fragment only encompassing the RWDBD binds Gcn2, thereby disrupting endogenous Gcn1-Gcn2 interaction which dampens Gcn2 activation. Consequently, cells are unable to increase eIF2α phosphorylation under starvation conditions, visible by impaired growth. This dominant negative phenotype is reverted by the R2259A substitution, again allowing Gcn1-Gcn2 interaction and enhanced eIF2α phosphorylation. We have found that the amino acid substitutions, R2289A, R2297A, and K2301A, also reverted the dominant negative phenotype as well as allowed enhanced eIF2α phosphorylation, as found previously for the R2259A substitution. This suggests that the respective amino acids are relevant for the overexpressed RWDBD to disrupt Gcn1-Gcn2 interaction and impair Gcn2 activation, supporting the idea that in Gcn1 these amino acids mediate Gcn2-binding. Our findings suggest that two helices in Gcn1 constitute a Gcn2 binding site. We serendipitously found amino acid substitutions that enhanced the dominant negative phenotype that correlated with a further reduction in eIF2α-P levels, suggesting that the respective RWDBD variants are more potent in disrupting Gcn1-Gcn2 interaction., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Gottfried et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The propensity of viruses to co-opt host cellular machinery by reprogramming the host's RNA-interference machinery has been a major focus of research, however, regulation of host defense mechanisms by virus-encoded miRNA, is an additional regulatory realm gaining momentum in the arena of host-viral interactions. The Human Cytomegalovirus (HCMV) miRNAs, regulate many cellular pathways alone or in concordance with HCMV proteins, thereby paving a conducive environment for successful infection in the human host. We show that HCMV miRNA, hcmv-miR-UL148D inhibits staurosporine-induced apoptosis in HEK293T cells. We establish that ERN1 mRNA is a bonafide target of hcmv-miR-UL148D and its encoded protein IRE1α is translationally repressed by the overexpression of hcmv-miR-UL148D resulting in the attenuation of apoptosis. Unlike the host microRNA seed sequence (6-8 nucleotides), hcmv-miR-UL148D has long complementarity to 3' UTR of ERN1 mRNA resulting in mRNA degradation. The repression of IRE1α by the hcmv-miR-UL148D further downregulates Xbp1 splicing and c-Jun N-terminal kinase phosphorylation thus regulating ER-stress and ER-stress induced apoptotic pathways. Strikingly, depletion of ERN1 attenuates staurosporine-induced apoptosis which further suggests that hcmv-miR-UL148D functions through regulation of its target ERN1. These results uncover a role for hcmv-miR-UL148D and its target ERN1 in regulating ER stress-induced apoptosis., Competing Interests: The authors have declared that no competing interests exists.

Colorectal cancer (CRC) ranks the third in cancers and the second in the reasons of cancer-related death. More evidence indicates that long non-coding RNA participates in tumor initiation and progression. It's known that cancer susceptibility candidate 9 is an oncogenic long non-coding RNA in CRC. miR-542-3p is a negative regulator of CRC, while integrin-linked kinase could contribute to tumor progression and chemoresistance. However, the correlation among long non-coding RNA cancer susceptibility candidate 9, miR-542-3p and integrin-linked kinase in CRC is still unclear. We demonstrated long non-coding RNA cancer susceptibility candidate 9 in CRC specimens and cell lines overexpressed via real-time quantitative polymerase chain reaction. Once long non-coding RNA cancer susceptibility candidate 9 was knocked down, it significantly inhibited proliferation, invasion, and migration of CRC cells in real-time quantitative polymerase chain reaction, cell counting kit-8, 5-ethynyl-2'-deoxyuridine, and transwell assays, which also was validated in vivo. Long non-coding RNA cancer susceptibility candidate 9 negatively regulates miR-542-3p in a targeted manner, and the function of up-regulated miR-542-3p was confirmed similarly. While miR-542-3p negatively regulates integrin-linked kinase. Thus, we further verified that overexpression of integrin-linked kinase on down-regulated long non-coding RNA cancer susceptibility candidate 9 or up-regulated miR-542-3p significantly restored CRC cell proliferation via bioinformatic analysis, dual-luciferase report assay, real-time quantitative polymerase chain reaction, RNA immunoprecipitation, and western blot. This study testified that silencing long non-coding RNA cancer susceptibility candidate 9 could inhibit proliferation and invasion of CRC cells by miR-542-3p/integrin-linked kinase., Competing Interests: The authors have declared that no competing interests exist.

Objective: Stem cell therapy is a promising approach for diabetes via promoting the differentiation of insulin-producing cells (IPCs). This study aimed to screen the differentially expressed miRNAs (DEmiRNAs) during the differentiation of muscle-derived stem cells (MDSCs) into IPCs, and uncover the underlying function and mechanism of a specific DEmiRNA, miR-708-5p., Methods: MDSCs were successfully isolated from the leg muscle of rats, and were induced for IPCs differentiation through a five-stage protocol. miRNA microarray assay was performed for screening DEmiRNAs during differentiation. The features of MDSCs-derived IPCs were identified by qRT-PCR, flow cytometry, and immunofluorescence staining. The targeting of STK4 by miR-708-5p was examined by luciferase assay. The protein expression of STK4, YAP1, and p-YAP1 was determined by Western blot and immunofluorescence staining., Results: MDSCs were successfully isolated and differentiated into IPCs. A total of 12 common DEmiRNAs were obtained during five-stage differentiation. Among them, miR-708-5p that highly expressed in MDSCs-derived IPCs was selected. Overexpression of miR-708-5p upregulated some key transcription factors (Pdx1, Ngn3, Nkx2.2, Nkx6.1, Gata4, Gata6, Pax4, and Pax6) involving in IPCs differentiation, and increased insulin positive cells. In addition, STK4 was identified as the target gene of miR-708-5p. miR-708-5p overexpression downregulated the expression of STK4 and the downstream phosphorylated YAP1., Conclusions: There were 12 DEmiRNAs involved in the differentiation of MDSCs into IPCs. miR-708-5p promoted MDSCs differentiation into IPCs probably by targeting STK4-mediated Hippo-YAP1 signaling pathway., Competing Interests: The authors have declared that no competing interests exist.

Bone abnormalities affect all individuals with Down syndrome (DS) and are linked to abnormal expression of DYRK1A, a gene found in three copies in people with DS and Ts65Dn DS model mice. Previous work in Ts65Dn male mice demonstrated that both genetic normalization of Dyrk1a and treatment with ~9 mg/kg/day Epigallocatechin-3-gallate (EGCG), the main polyphenol found in green tea and putative DYRK1A inhibitor, improved some skeletal deficits. Because EGCG treatment improved mostly trabecular skeletal deficits, we hypothesized that increasing EGCG treatment dosage and length of administration would positively affect both trabecular and cortical bone in Ts65Dn mice. Treatment of individuals with DS with green tea extract (GTE) containing EGCG also showed some weight loss in individuals with DS, and we hypothesized that weights would be affected in Ts65Dn mice after EGCG treatment. Treatment with ~20 mg/kg/day EGCG for seven weeks showed no improvements in male Ts65Dn trabecular bone and only limited improvements in cortical measures. Comparing skeletal analyses after ~20mg/kg/day EGCG treatment with previously published treatments with ~9, 50, and 200 mg/kg/day EGCG showed that increased dosage and treatment time increased cortical structural deficits leading to weaker appendicular bones in male mice. Weight was not affected by treatment in mice, except for those given a high dose of EGCG by oral gavage. These data indicate that high doses of EGCG, similar to those reported in some treatment studies of DS and other disorders, may impair long bone structure and strength. Skeletal phenotypes should be monitored when high doses of EGCG are administered therapeutically., Competing Interests: The authors have declared that no competing interests exist.

Skin cutaneous melanoma (SKCM) is a common cancer of which mortality is increasing continuously. Our study conducted a series of analyses on the clinical significance of Serine/threonine kinase 17B (STK17B) in SKCM to provide a new biomarker for diagnosis and treatment. The RNA-sequence data were obtained from The Cancer Genome Atlas and Genotype-Tissue Expression databases. The data of 468 SKCM patients were divided into STK17B high- and low-expression groups and analyzed by Bioconductor package to identify the differential expressed genes. The R package of "clusterProfiler" was used for Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene-Set Enrichment Analysis analyses. A protein-protein interaction network and immune infiltration landscape were respectively constructed via STRING database and ssGSEA. STK17B had lower expression in SKCM than normal tissues. Besides, STK17B expression was significantly related to some clinicopathological characteristics in SKCM patients including T stage, Breslow depth, radiation therapy, melanoma Clark level, and pathologic stage. The Kaplan-Meier curve analyses revealed that the low expression of STK17B was correlated with poor overall survival and disease-specific survival. We constructed nomograms to predict the 1-, 3-, and 5-year survival of SKCM patients. The function enrichment analyses showed STK17B-related differential expressed genes were enriched in cellular differentiation and immune-related progress. STK17B expression level were positively correlated with infiltrating level of immune cells. In this study, we found that STK17B, which played an important role in immune infiltration, could be a new biomarker for diagnosis and prognosis in SKCM patients., Competing Interests: We declare that they have no conflicts of Interest.

Rice production is affected by many biotic and abiotic stresses; among them, bacterial blight (BB) and blast diseases and low soil phosphorous stress cause significant yield losses. The present study was carried out with the objective of combining the BB resistance gene, Xa21, the blast resistance gene, Pi54, and the low soil phosphorous tolerance QTL/gene, Pup1, into the genetic background of the Indian mega-rice variety, MTU1010 (Cottondora Sannalu), through marker-assisted pedigree breeding. RP5973-20-9-8-24-12-7 [a near isogenic line (NIL) of MTU1010 possessing Pup1] and RP6132 [a NIL of Akshayadhan possessing Xa21 + Pi54] were crossed and 'true' F1s were identified, using the target gene-specific markers and selfed. F2 plants, which are homozygous for all the three target genes/QTLs, were identified using PCR based markers and were advanced further through the pedigree method of breeding, with selection based on phenotypic traits specific for MTU1010. At the F5 generation, a set of 15 promising triple positive homozygous lines were identified and screened for their resistance against BB and blast diseases and tolerance to low soil P. Among them, two lines (LPK 30-18-16 and LPK 49-15-22) showed higher yields as compared to MTU1010, along with the desirable long slender grain type in both low soil P and normal soil P plots, and also exhibited high levels of resistance against BB and blast diseases, with lesser grain shattering as compared to MTU1010. These lines are being advanced for multi-location trials for validating their performance., Competing Interests: The authors have declared that no competing interests exist.

Phosphorylation by serine-threonine and tyrosine kinases is critical for determining protein function. Array-based platforms for measuring reporter peptide signal levels allow for differential phosphorylation analysis between conditions for distinct active kinases. Peptide array technologies like the PamStation12 from PamGene allow for generating high-throughput, multi-dimensional, and complex functional proteomics data. As the adoption rate of such technologies increases, there is an imperative need for software tools that streamline the process of analyzing such data. We present Kinome Random Sampling Analyzer (KRSA), an R package and R Shiny web-application for analyzing kinome array data to help users better understand the patterns of functional proteomics in complex biological systems. KRSA is an All-In-One tool that reads, formats, fits models, analyzes, and visualizes PamStation12 kinome data. While the underlying algorithm has been experimentally validated in previous publications, we demonstrate KRSA workflow on dorsolateral prefrontal cortex (DLPFC) in male (n = 3) and female (n = 3) subjects to identify differential phosphorylation signatures and upstream kinase activity. Kinase activity differences between males and females were compared to a previously published kinome dataset (11 female and 7 male subjects) which showed similar global phosphorylation signals patterns., Competing Interests: R.H. and T.T. are employed by PamGene International B.V., and F.N. was employed by PamGene International B.V. during the manuscript development. The remaining authors have declared that no conflicts of interests exist. We do not receive any direct funding from PamGene International - they are collaborators. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The development of B and T lymphocytes critically depends on RAG1/2 endonuclease activity to mediate antigen receptor gene assembly by V(D)J recombination. Although control of RAG1/2 activity through cell cycle- and ubiquitin-dependent degradation of RAG2 has been studied in detail, relatively little is known about mechanisms regulating RAG1 stability. We recently demonstrated that VprBP/DCAF1, a substrate adaptor for the CRL4 E3 ubiquitin ligase complex, is required to maintain physiological levels of RAG1 protein in murine B cells by facilitating RAG1 turnover. Loss of VprBP/DCAF1 in vivo results in elevated RAG1 expression, excessive V(D)J recombination, and immunoglobulin light chain repertoire skewing. Here we show that RAG1 is constitutively degraded when ectopically expressed in a human fibroblast cell line. Consistent with our findings in murine B cells, RAG1 turnover under these conditions is sensitive to loss of VprBP, as well as CRL4 or proteasome inhibition. Further evidence indicates that RAG1 degradation is ubiquitin-dependent and that RAG1 association with the CRL4VPRBP/DCAF1 complex is independent of CUL4 activation status. Taken together, these findings suggest V(D)J recombination co-opts an evolutionarily conserved and constitutively active mechanism to ensure rapid RAG1 turnover to restrain excessive RAG activity., Competing Interests: The authors have declared that no competing interests exist.

CCRK/CDK20 was reported to interact with BROMI/TBC1D32 and regulate ciliary Hedgehog signaling. In various organisms, mutations in the orthologs of CCRK and those of the kinase ICK/CILK1, which is phosphorylated by CCRK, are known to result in cilia elongation. Furthermore, we recently showed that ICK regulates retrograde ciliary protein trafficking and/or the turnaround event at the ciliary tips, and that its mutations result in the elimination of intraflagellar transport (IFT) proteins that have overaccumulated at the bulged ciliary tips as extracellular vesicles, in addition to cilia elongation. However, how these proteins cooperate to regulate ciliary protein trafficking has remained unclear. We here show that the phenotypes of CCRK-knockout (KO) cells closely resemble those of ICK-KO cells; namely, the overaccumulation of IFT proteins at the bulged ciliary tips, which appear to be eliminated as extracellular vesicles, and the enrichment of GPR161 and Smoothened on the ciliary membrane. The abnormal phenotypes of CCRK-KO cells were rescued by the exogenous expression of wild-type CCRK but not its kinase-dead mutant or a mutant defective in BROMI binding. These results together indicate that CCRK regulates the turnaround process at the ciliary tips in concert with BROMI and probably via activating ICK., Competing Interests: No authors have competing interests.

Aberrant NF-κB signaling fuels tumor growth in multiple human cancer types including both hematologic and solid malignancies. Chronic elevated alternative NF-κB signaling can be modeled in transgenic mice upon activation of a conditional NF-κB-inducing kinase (NIK) allele lacking the regulatory TRAF3 binding domain (NT3). Here, we report that expression of NT3 in the mesenchymal lineage with Osterix (Osx/Sp7)-Cre or Fibroblast-Specific Protein 1 (FSP1)-Cre caused subcutaneous, soft tissue tumors. These tumors displayed significantly shorter latency and a greater multiple incidence rate in Fsp1-Cre;NT3 compared to Osx-Cre;NT3 mice, regardless of sex. Histological assessment revealed poorly differentiated solid tumors with some spindled patterns, as well as robust RelB immunostaining, confirming activation of alternative NF-κB. Even though NT3 expression also occurs in the osteolineage in Osx-Cre;NT3 mice, we observed no bony lesions. The staining profiles and pattern of Cre expression in the two lines pointed to a mesenchymal tumor origin. Immunohistochemistry revealed that these tumors stain strongly for alpha-smooth muscle actin (αSMA), although vimentin staining was uniform only in Osx-Cre;NT3 tumors. Negative CD45 and S100 immunostains precluded hematopoietic and melanocytic origins, respectively, while positive staining for cytokeratin 19 (CK19), typically associated with epithelia, was found in subpopulations of both tumors. Principal component, differential expression, and gene ontology analyses revealed that NT3 tumors are distinct from normal mesenchymal tissues and are enriched for NF-κB related biological processes. We conclude that constitutive activation of the alternative NF-κB pathway in the mesenchymal lineage drives spontaneous sarcoma and provides a novel mouse model for NF-κB related sarcomas., Competing Interests: The authors have declared that no competing interests exist.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms., Competing Interests: The authors have declared that no competing interests exist.

Unlike the majority of cancers, survival for lung cancer has not shown much improvement since the early 1970s and survival rates remain low. Genetically engineered mice tumor models are of high translational relevance as we can generate tissue specific mutations which are observed in lung cancer patients. Since these tumors cannot be detected and quantified by traditional methods, we use micro-computed tomography imaging for longitudinal evaluation and to measure response to therapy. Conventionally, we analyze microCT images of lung cancer via a manual segmentation. Manual segmentation is time-consuming and sensitive to intra- and inter-analyst variation. To overcome the limitations of manual segmentation, we set out to develop a fully-automated alternative, the Mouse Lung Automated Segmentation Tool (MLAST). MLAST locates the thoracic region of interest, thresholds and categorizes the lung field into three tissue categories: soft tissue, intermediate, and lung. An increase in the tumor burden was measured by a decrease in lung volume with a simultaneous increase in soft and intermediate tissue quantities. MLAST segmentation was validated against three methods: manual scoring, manual segmentation, and histology. MLAST was applied in an efficacy trial using a Kras/Lkb1 non-small cell lung cancer model and demonstrated adequate precision and sensitivity in quantifying tumor growth inhibition after drug treatment. Implementation of MLAST has considerably accelerated the microCT data analysis, allowing for larger study sizes and mid-study readouts. This study illustrates how automated image analysis tools for large datasets can be used in preclinical imaging to deliver high throughput and quantitative results., Competing Interests: All authors are or were full- or part-time employees of Pfizer, Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Angiosperm cell adhesion is dependent on interactions between pectin polysaccharides which make up a significant portion of the plant cell wall. Cell adhesion in Arabidopsis may also be regulated through a pectin-related signaling cascade mediated by a putative O-fucosyltransferase ESMERALDA1 (ESMD1), and the Epidermal Growth Factor (EGF) domains of the pectin binding Wall associated Kinases (WAKs) are a primary candidate substrate for ESMD1 activity. Genetic interactions between WAKs and ESMD1 were examined using a dominant hyperactive allele of WAK2, WAK2cTAP, and a mutant of the putative O-fucosyltransferase ESMD1. WAK2cTAP expression results in a dwarf phenotype and activation of the stress response and reactive oxygen species (ROS) production, while esmd1 is a suppressor of a pectin deficiency induced loss of adhesion. Here we find that esmd1 suppresses the WAK2cTAP dwarf and stress response phenotype, including ROS accumulation and gene expression. Additional analysis suggests that mutations of the potential WAK EGF O-fucosylation site also abate the WAK2cTAP phenotype, yet only evidence for an N-linked but not O-linked sugar addition can be found. Moreover, a WAK locus deletion allele has no effect on the ability of esmd1 to suppress an adhesion deficiency, indicating WAKs and their modification are not a required component of the potential ESMD1 signaling mechanism involved in the control of cell adhesion. The WAK locus deletion does however affect the induction of ROS but not the transcriptional response induced by the elicitors Flagellin, Chitin and oligogalacturonides (OGs)., Competing Interests: The authors have declared that no competing interests exist.

HASPIN is a serine/threonine kinase that regulates mitosis by phosphorylating histone H3 at threonine 3. The expression levels of HASPIN in various cancers are associated with tumor malignancy and poor survival, suggesting that HASPIN inhibition may suppress cancer growth. As HASPIN mRNA levels are elevated in human breast cancer tissues compared with adjacent normal tissues, we examined the growth-suppressive effects of CHR-6494, a potent HASPIN inhibitor, in breast cancer cell lines in vitro and in vivo. We found that HASPIN was expressed in breast cancer cells of all molecular subtypes, as well as in immortalized mammary epithelial cells. HASPIN expression levels appeared to be correlated with the cell growth rate but not the molecular subtype of breast cancer. CHR-6494 exhibited potent antiproliferative effects against breast cancer cell lines and immortalized mammary epithelial cells in vitro, but failed to inhibit the growth of MDA-MB-231 xenografted tumors under conditions that have significant effects in a colorectal cancer model. These results imply that CHR-6494 does have antiproliferative effects in some situations, and further drug screening efforts are anticipated to identify more potent and selective HASPIN inhibition for use as an anticancer agent in breast cancer patients., Competing Interests: The authors have declared that no competing interests exist.

Nonsense-mediated mRNA decay (NMD) controls eukaryotic mRNA quality, inducing the degradation of faulty transcripts. Key players in the NMD pathway were originally identified, through genetics, in Caenorhabditis elegans as smg (suppressor with morphological effect on genitalia) genes. Using forward genetics and fluorescence-based NMD reporters, we reexamined the genetic landscape underlying NMD. Employing a novel strategy for mapping sterile mutations, Het-Map, we identified clk-2, a conserved gene previously implicated in DNA damage signaling, as a player in the nematode NMD. We find that CLK-2 is expressed predominantly in the germline, highlighting the importance of auxiliary factors in tissue-specific mRNA decay. Importantly, the human counterpart of CLK-2/TEL2, TELO2, has been also implicated in the NMD, suggesting a conserved role of CLK-2/TEL2 proteins in mRNA surveillance. Recently, variants of TELO2 have been linked to an intellectual disability disorder, the You-Hoover-Fong syndrome, which could be related to its function in the NMD., Competing Interests: The authors have declared that no competing interests exist.

Intellectual disability (ID) is a developmental disorder that includes both intellectual and adaptive functioning deficits in conceptual, social, and practical domains. Although evidence-based interventions for patients have long been desired, their progress has been hindered due to various determinants. One of these determinants is the complexity of the origins of ID. The ceramide transport protein (CERT) encoded by CERT1 mediates inter-organelle trafficking of ceramide for the synthesis of intracellular sphingomyelin. Utilizing whole exome sequencing analysis, we identified a novel CERT variant, which substitutes a serine at position 135 (S135) for a proline in a patient with severe ID. Biochemical analysis showed that S135 is essential for hyperphosphorylation of a serine-repeat motif of CERT, which is required for down-regulation of CERT activity. Amino acid replacements of S135 abnormally activated CERT and induced an intracellular punctate distribution pattern of this protein. These results identified specific ID-associated CERT1 mutations that induced gain-of-function effects on CERT activity. These findings provide a possible molecular basis for not only new diagnostics but also a conceivable pharmaceutical intervention for ID disorders caused by gain-of-function mutations in CERT1., Competing Interests: The authors have declared that no competing interests exist.

Paclitaxel is a representative anticancer drug that induces chemotherapy-induced peripheral neuropathy (CIPN), a common side effect that limits many anticancer chemotherapies. Although PINK1, a key mediator of mitochondrial quality control, has been shown to protect neuronal cells from various toxic treatments, the role of PINK1 in CIPN has not been investigated. Here, we examined the effect of PINK1 expression on CIPN using a recently established paclitaxel-induced peripheral neuropathy model in Drosophila larvae. We found that the class IV dendritic arborization (C4da) sensory neuron-specific expression of PINK1 significantly ameliorated the paclitaxel-induced thermal hyperalgesia phenotype. In contrast, knockdown of PINK1 resulted in an increase in thermal hypersensitivity, suggesting a critical role for PINK1 in sensory neuron-mediated thermal nociceptive sensitivity. Interestingly, analysis of the C4da neuron morphology suggests that PINK1 expression alleviates paclitaxel-induced thermal hypersensitivity by means other than preventing alterations in sensory dendrites in C4da neurons. We found that paclitaxel induces mitochondrial dysfunction in C4da neurons and that PINK1 expression suppressed the paclitaxel-induced increase in mitophagy in C4da neurons. These results suggest that PINK1 mitigates paclitaxel-induced sensory dendrite alterations and restores mitochondrial homeostasis in C4da neurons and that improvement in mitochondrial quality control could be a promising strategy for the treatment of CIPN., Competing Interests: The authors declare that there is no conflict of interest.

Background: Mutations in STK11 (STK11m) and frequently co-occurring KRAS mutations (KRASm/STK11m) are associated with poor survival in metastatic NSCLC (mNSCLC) immuno-oncology trials. There are limited data regarding the prognostic significance of these mutations in a real-world setting., Methods: This retrospective cohort study analyzed de-identified electronic medical records from the Flatiron Clinico-Genomic database to identify patients with mNSCLC who had initiated first-line immunotherapy (IO; alone or in combination) or chemotherapy under routine care between January 1, 2013 and June 30, 2017. The primary objectives were to assess the prevalence of STK11m and KRASm/STK11m and to determine associations of these mutations with overall and progression-free survival (OS, PFS)., Results: Of 2407 patients with mNSCLC, STK11m and KRASm/STK11m were present in 13.6% and 6.5% of patients, respectively. Worse OS outcomes were observed in patients with STK11m versus STK11wt mNSCLC receiving IO (first-line, HR [95% CI], 1.4 [0.9-2.3; p = 0.1]; second-line [subset of first-line cohort], HR, 1.6 [1.3-2.0; p = 0.0002]) or chemotherapy (first-line, HR, 1.4 [1.2-1.6; p < 0.0001]); PFS outcomes showed similar trends. KRASm/STK11m double mutations were associated with worse OS and PFS outcomes versus KRASwt/STK11wt with IO and chemotherapy, similar to the single mutation (STK11m vs STK11wt) findings., Conclusions: This large observational genomic study among patients receiving routine care highlights the negative prognostic impact of STK11m in patients with mNSCLC treated with IO or chemotherapy. These results complement previous clinical trial data and provide further evidence in the real world of a patient population that would benefit from new treatment options., Competing Interests: Norah J. Shire and Robert McEwen are employees and stockholders of AstraZeneca. Asieh Golozar, Alyssa B. Klein, and Todd Hembrough were employees of AstraZeneca at the time of the study. Jenna M. Collins, Kathy H. Fraeman, and Beth L. Nordstrom are employees of Evidera, and Evidera received funding from AstraZeneca for this study. Naiyer A. Rizvi has received personal fees from AbbVie, Apricity, AstraZeneca, Boehringer Ingelheim, Calithera, Dracen, Editas, EMD Serono, G1 Therapeutics, Genentech, Gilead, GlaxoSmithKline, Illumina, Lilly, Merck, Neogenomics, Novartis, and Takeda; personal fees and stock options from Brooklyn ImmunoTherapeutics, Bellicum, Arcus, and Gritstone; stock from Gritstone; and royalties related to a patent filed by Memorial Sloan Kettering Cancer Center for identifying determinants of cancer response to immunotherapy (PCT/US2015/062208) licensed to Personal Genome Diagnostics. The commercial affiliation, consultancy, employment, patents noted for the authors do not alter adherence to PLOS ONE policies on sharing data and materials relevant to the current manuscript.

Cattle temperament is a complex and economically relevant trait. The objective of this study was to identify genomic regions and genes associated with cattle temperament. From a Brahman cattle population of 1,370 animals evaluated for temperament traits (Exit velocity-EV, Pen Score-PS, Temperament Score-TS), two groups of temperament-contrasting animals were identified based on their EV-average values ±1/2 standard deviation (SD). To be considered in the calm group, the EV of females ranged between 0.16-1.82 m/s (n = 50) and the EV of males ranged between 0.4-1.56 m/s (n = 48). Females were classified as temperamental if their EV ranged between 3.13-7.66 m/s (n = 46) and males were classified as temperamental if their EV ranged between 3.05-10.83 m/s (n = 45). Selected animals were genotyped using a total of 139,376 SNPs (GGP-HD-150K), evaluated for their association with EV. The Genome-Wide Association analysis (GWAS) identified fourteen SNPs: rs135340276, rs134895560, rs110190635, rs42949831, rs135982573, rs109393235, rs109531929, rs135087545, rs41839733, rs42486577, rs136661522, rs110882543, rs110864071, rs109722627, (P<8.1E-05), nine of them were located on intergenic regions, harboring seventeen genes, of which only ACER3, VRK2, FANCL and SLCO3A1 were considered candidate associated with bovine temperament due to their reported biological functions. Five SNPs were located at introns of the NRXN3, EXOC4, CACNG4 and SLC9A4 genes. The indicated candidate genes are implicated in a wide range of behavioural phenotypes and complex cognitive functions. The association of the fourteen SNPs on bovine temperament traits (EV, PS and TS) was evaluated; all these SNPs were significant for EV; only some were associated with PS and TS. Fourteen SNPs were associated with EV which allowed the identification of twenty-one candidate genes for Brahman temperament. From a functional point of view, the five intronic SNPs identified in this study, are candidates to address control of bovine temperament, further investigation will probe their role in expression of this trait., Competing Interests: The authors have declared that no competing interests exist.

ERECTA gene family encodes leucine-rich repeat receptor-like kinases that control major aspects of plant development such as elongation of aboveground organs, leaf initiation, development of flowers, and epidermis differentiation. To clarify the importance of ERECTA signaling for the development of soybean (Glycine max), we expressed the dominant-negative ERECTA gene from Arabidopsis thaliana that is truncated in the kinase domain (AtΔKinase). Expression of AtΔKinase in soybean resulted in the short stature, reduced number of leaves, reduced leaf surface area and enhanced branching in the transgenic plants. The transgenic AtΔKinase soybean plants exhibited increased tolerance to water deficit stress due to the reduction of total leaf area and reduced transpiration compared to the wild-type plants. Production of seeds in AtΔKinase lines was higher compared to wild type at regular conditions of cultivation and after exposure to drought stress. Transgenic seedlings expressing AtΔKinase were also able to withstand salt stress better than the wild-type. Established results demonstrated the significance of native soybean genes (GmER and GmERL) in development and stress response of soybean, and suggested that the truncated ERECTA gene of Arabidopsis thaliana can be used to manipulate the growth and stress response of different crop species., Competing Interests: The authors have declared that no competing interests exist.

Background: Evidence from multiple studies suggests metabolic abnormalities play an important role in lung cancer. Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer. The present study aimed to explore differences in the global metabolic response between male and female patients in LUAD and to identify the metabolic genes associated with lung cancer susceptibility., Methods: Transcriptome and clinical LUAD data were acquired from The Cancer Genome Atlas (TCGA) database. Information on metabolic genes and metabolic subsystems were collected from the Recon3D human metabolic model. Two validation datasets (GSE68465 and GSE72094) were downloaded from the Gene Expression Omnibus (GEO) database. Differential expression analysis, gene set enrichment analysis and protein-protein interaction networks were used to identified key metabolic pathways and genes. Functional experiments were used to verify the effects of genes on proliferation, migration, and invasion in lung cancer cells in vitro., Results: Samples of tumors and adjacent non-tumor tissue from both male and female patients exhibited distinct global patterns of gene expression. In addition, we found large differences in methionine and cysteine metabolism, pyruvate metabolism, cholesterol metabolism, nicotinamide adenine dinucleotide (NAD) metabolism, and nuclear transport between male and female LUAD patients. We identified 34 metabolic genes associated with lung cancer susceptibility in males and 15 in females. Most of the metabolic cancer-susceptibility genes had high prediction accuracy for lung cancer (AUC > 0.9). Furthermore, both bioinformatics analysis and experimental results showed that TAOK2 was down-regulated and ASAH1 was up-regulated in male tumor tissue and female tumor tissue in LUAD. Functional experiments showed that inhibiting ASAH1 suppressed the proliferation, migration, and invasion of lung cancer cells., Conclusions: Metabolic cancer-susceptibility genes may be used alone or in combination as diagnostic markers for LUAD. Further studies are required to elucidate the functions of these genes in LUAD., Competing Interests: The authors have declared that no competing interests exist.

The integrated stress response (ISR) is one of the most important cytoprotective mechanisms and is integrated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Four eIF2α kinases, heme-regulated inhibitor (HRI), double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and general control nonderepressible 2 (GCN2), are activated in response to several stress conditions. We previously reported that nanosecond pulsed electric fields (nsPEFs) are a potential therapeutic tool for ISR activation. In this study, we examined which eIF2α kinase is activated by nsPEF treatment. To assess the responsible eIF2α kinase, we used previously established eIF2α kinase quadruple knockout (4KO) and single eIF2α kinase-rescued 4KO mouse embryonic fibroblast (MEF) cells. nsPEFs 70 ns in duration with 30 kV/cm electric fields caused eIF2α phosphorylation in wild-type (WT) MEF cells. On the other hand, nsPEF-induced eIF2α phosphorylation was completely abolished in 4KO MEF cells and was recovered by HRI overexpression. CM-H2DCFDA staining showed that nsPEFs generated reactive oxygen species (ROS), which activated HRI. nsPEF-induced eIF2α phosphorylation was blocked by treatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Our results indicate that the eIF2α kinase HRI is responsible for nsPEF-induced ISR activation and is activated by nsPEF-generated ROS., Competing Interests: The authors have declared that no competing interests exist.

Testis-specific serine/threonine kinase 1-like (TSSK1-like), which plays important roles in late-phase spermatogenesis and male fertility, was characterized in Pacific abalone Haliotis discus hannai, an important commercial marine gastropod. Further, its expression patterns were assessed in diploid and induced triploid males showing differential degrees of testis maturation. Abalone TSSK1-like shared conserved structural features with mammalian TSSK1s and other potential metazoan orthologs, especially regarding the catalytic STKc domain. Phylogenetically, abalone TSSK1-like displayed a genetic affiliation with its molluscan TSSK1-like orthologs and human TSSK1. Additionally, abalone TSSK1-like gene showed a tetrapartite exon-intron organization, unlike the intronless structure of most amniotic tetrapodian TSSK1s. Molecular phylogenetic analysis in the metazoan lineage suggested a possible revision in the origin of the earliest ancestral TSSK1. Further, abalone TSSK1-like showed testis-predominant expression, which was significantly influenced by both age and seasonal reproductive cycles. Comparative expression analyses between diploid and triploid abalone males suggested that robust TSSK1-like expression occurred primarily at the post-meiotic stage. Additionally, RT-PCR assay indicates that mature abalone sperms retain TSSK1-like transcripts after release. Taken together, this study provides useful insights for further studies to assess male reproduction and sterility and/or partial fertility of induced male triploidy in abalone species., Competing Interests: The authors have declared that no competing interests exist.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by pathological hallmarks of neurofibrillary tangles and amyloid plaques. The plaques are formed by aggregation and accumulation of amyloid β (Aβ), a cleavage fragment of amyloid precursor protein (APP). Enhanced neuronal activity and seizure events are frequently observed in AD, and elevated synaptic activity promotes Aβ production. However, the mechanisms that link synaptic hyperactivity to APP processing and AD pathogenesis are not well understood. We previously found that Polo-like kinase 2 (Plk2), a homeostatic repressor of neuronal overexcitation, promotes APP β-processing in vitro. Here, we report that Plk2 stimulates Aβ production in vivo, and that Plk2 levels are elevated in a spatiotemporally regulated manner in brains of AD mouse models and human AD patients. Genetic disruption of Plk2 kinase function reduces plaque deposits and activity-dependent Aβ production. Furthermore, pharmacological Plk2 inhibition hinders Aβ formation, synapse loss, and memory decline in an AD mouse model. Thus, Plk2 links synaptic overactivity to APP β-processing, Aβ production, and disease-relevant phenotypes in vivo, suggesting that Plk2 may be a potential target for AD therapeutics., Competing Interests: The authors have declared that no competing interests exist.

Aim: In this study, we investigated the role and mechanism of Salt-induced kinase 1 (SIK1) in regulation of hepatic glucose and lipid metabolism in a high-fat food (HFD) and streptozocin (STZ)-induced type 2 diabetes mellitus (T2DM) rat model., Methods: A diabetic rat model treated with HFD plus low-dose STZ was developed and was transduced to induce a high expression of SIK1 in vivo via a tail-vein injection of a recombinant adenoviral vector. The effects on hepatic glucogenetic and lipogenic gene expression, systemic metabolism and pathological changes were then determined., Results: In T2DM rats, SIK1 expression was reduced in the liver. Overexpression of SIK1 improved hyperglycaemia, hyperlipidaemia and fatty liver, reduced the expression of cAMP-response element binding protein (CREB)-regulated transcription co-activator 2 (CRTC2), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), pS577 SIK1, sterol regulatory element binding-protein-1c (SREBP-1c) and its target genes, including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), and increased the expression of SIK1, pT182 SIK1 and pS171 CRTC2 in diabetic rat livers with the suppression of gluconeogenesis and lipid deposition., Conclusion: SIK1 plays a crucial role in the regulation of glucose and lipid metabolism in the livers of HFD/STZ-induced T2DM rats, where it suppresses hepatic gluconeogenesis and lipogenesis by regulating the SIK1/CRTC2 and SIK1/SREBP-1c signalling pathways. Strategies to activate SIK1 kinase in liver would likely have beneficial effects in patients with T2DM and nonalcoholic fatty liver disease (NAFLD)., Competing Interests: The authors have declared that no competing interests exist.

IMPACT, a highly conserved protein, is an inhibitor of the eIF2α kinase GCN2. In mammals, it is preferentially expressed in neurons. Knock-down of IMPACT expression in neuronal cells increases basal GCN2 activation and eIF2α phosphorylation and decreases translation initiation. In the mouse brain, IMPACT is particularly abundant in the hypothalamus. Here we describe that the lack of IMPACT in mice affects hypothalamic functions. Impact-/- mice (Imp-KO) are viable and have no apparent major phenotypic defect. The hypothalamus in these animals shows increased levels of eIF2α phosphorylation, as expected from the described role of IMPACT in inhibiting GCN2 and from its abundance in this brain region. When fed a normal chow, animals lacking IMPACT weight slightly less than wild-type mice. When fed a high-fat diet, Imp-KO animals gain substantially less weight due to lower food intake when compared to wild-type mice. STAT3 signaling was depressed in Imp-KO animals even though leptin levels were identical to the wild-type mice. This finding supports the observation that Imp-KO mice have defective thermoregulation upon fasting. This phenotype was partially dependent on GCN2, whereas the lean phenotype was independent of GCN2. Taken together, our results indicate that IMPACT contributes to GCN2-dependent and -independent mechanisms involved in the regulation of autonomic functions in response to energy availability., Competing Interests: The authors have declared that no competing interests exist.

Immunotherapy has fundamentally changed the landscape of cancer treatment. Despite the encouraging results with the checkpoint modulators, response rates vary widely across tumor types, with a majority of patients exhibiting either primary resistance without a significant initial response to treatment or acquired resistance with subsequent disease progression. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator in T cells and dendritic cells (DC). While HPK1 gene knockout (KO) studies suggest its role in anti-tumor immune responses, the involvement of kinase activity and thereof its therapeutic potential remain unknown. To investigate the potential of pharmacological intervention using inhibitors of HPK1, we generated HPK1 kinase dead (KD) mice which carry a single loss-of-function point mutation in the kinase domain and interrogated the role of kinase activity in immune cells in the context of suppressive factors or the tumor microenvironment (TME). Our data provide novel findings that HKP1 kinase activity is critical in conferring suppressive functions of HPK1 in a wide range of immune cells including CD4+, CD8+, DC, NK to Tregs, and inactivation of kinase domain was sufficient to elicit robust anti-tumor immune responses. These data support the concept that an HPK1 small molecule kinase inhibitor could serve as a novel agent to provide additional benefit in combination with existing immunotherapies, particularly to overcome resistance to current treatment regimens., Competing Interests: The commercial affiliation with BMS does not alter our adherence to all PLOS ONE policies on sharing data and materials.

We and others have demonstrated that various abnormalities of the bone marrow (BM) mesenchymal stromal cells (MSCs) such as aberrant cytokine expression, abnormal hedgehog signaling, and impaired miRNA biogenesis are observed in patients with acute myeloid leukemia (AML). However, underlying mechanisms to induce the dysfunction of BM MSCs have not yet been clarified. We previously showed that AML cells release abundant exosomal miR-7977, which, in turn, enters BM mesenchymal stromal cells (MSCs). However, the precise function of miR-7977 is not known. In this study, we performed transduction of a miR-7977 mimic into MSCs, compared transcriptomes between control-transduced (n = 3) and miR-7977-transduced MSCs (n = 3), and conducted pathway analysis. The array data revealed that the expression of 0.05% of genes was reduced 2-fold and the expression of 0.01% of genes was increased 2-fold. Interestingly, approximately half of these genes possessed a miR-7977 target site, while the other genes did not, suggesting that miR-7977 regulates the gene expression level directly and indirectly. Gene set enrichment analysis showed that the gene sets of Yes-associated protein 1 (YAP1) &#95;up were significantly enriched (p<0.001, q<0.25), suggesting that miR-7977 modulates the Hippo-YAP signaling pathway. Visualization of pathway and network showed that miR-7977 significantly reduced the expression of Hippo core kinase, STK4. miR-7977 inactivated the Hippo-YAP signaling pathway as proven by GFP-tagged YAP nuclear trans localization and TEAD reporter assay. The miR-7977-transduced MSC cell line, HTS-5, showed elevated saturation density and enhanced entry into the cell cycle. These results suggest that miR-7977 is a critical factor that regulates the Hippo-YAP signaling pathway in BM-MSCs and may be involved in the upregulation of leukemia-supporting stroma growth., Competing Interests: The authors have declared that no competing interests exist.

Many of the small ribosomal subunit proteins are required for the stabilisation of late small ribosomal subunit (SSU) precursors and for final SSU rRNA processing in S. cerevisiae. Among them are ribosomal proteins (r-proteins) which form a protein cluster around rpS0 (uS2) at the "neck" of the SSU (S0-cluster) and others forming a nearby protein cluster around rpS3 (uS3) at the SSU "beak". Here we applied semi-quantitative proteomics together with complementary biochemical approaches to study how incomplete assembly of these two r-protein clusters affects binding and release of SSU maturation factors and assembly of other r-proteins in late SSU precursors in S. cerevisiae. For each of the two clusters specific impairment of the local r-protein assembly state was observed in Rio2 associated SSU precursors. Besides, cluster-specific effects on the association of biogenesis factors were detected. These suggested a role of S0-cluster formation for the efficient release of the two nuclear export factors Rrp12 and Slx9 from SSU precursors and for the correct incorporation of the late acting biogenesis factor Rio2. Based on our and on previous results we propose the existence of at least two different r-protein assembly checkpoints during late SSU maturation in S. cerevisiae. We discuss in the light of recent SSU precursor structure models how r-protein assembly states might be sensed by biogenesis factors at the S0-cluster checkpoint., Competing Interests: The authors have declared that no competing interests exist.

Overexpression of the Dual-specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A) gene contributes to the retardation, craniofacial anomalies, cognitive impairment, and learning and memory deficits associated with Down Syndrome (DS). DCAF7/HAN11/WDR68 (hereafter WDR68) binds DYRK1A and is required for craniofacial development. Accumulating evidence suggests DYRK1A-WDR68 complexes enable proper growth and patterning of multiple organ systems and suppress inappropriate cell growth/transformation by regulating the balance between proliferation and differentiation in multiple cellular contexts. Here we report, using engineered mouse C2C12 and human HeLa cell lines, that WDR68 is required for normal levels of DYRK1A. However, Wdr68 does not significantly regulate Dyrk1a mRNA expression levels and proteasome inhibition did not restore DYRK1A in cells lacking Wdr68 (Δwdr68 cells). Overexpression of WDR68 increased DYRK1A levels while overexpression of DYRK1A had no effect on WDR68 levels. We further report that WDR68 is similarly required for normal levels of the closely related DYRK1B kinase and that both DYRK1A and DYRK1B are essential for the transition from proliferation to differentiation in C2C12 cells. These findings reveal an additional role of WDR68 in DYRK1A-WDR68 and DYRK1B-WDR68 complexes., Competing Interests: The authors have declared that no competing interests exist.

Vaccinia-related kinase 1 (VRK1) is a pro-proliferative nuclear kinase. Mice engrafted with VRK1-depleted MDA-MB-231 breast cancer cells have been shown to develop fewer distal metastases than controls, suggesting VRK1 might play a role in cell migration, invasion, and/or colonization. In work described herein, we investigated the impact of VRK1 overexpression on human mammary epithelial cells. In 2D culture, VRK1 overexpression diminishes cell migration and invasion and impairs the migration-associated processes of cell spreading and cytoskeletal rearrangement. VRK1-overexpressing cells show reduced accumulation of the mesenchymal marker vimentin and increased accumulation of the epithelial markers E-cadherin and claudin-1. VRK1 overexpression also leads to reduced levels of the transcriptional repressors snail, slug, and twist1. Cumulatively, these data indicate that VRK1 overexpression augments the epithelial properties of both MCF10a and MDA-MB-231 cells. We further studied the impact of VRK1 on the epithelial properties of MCF10a cells in 3D matrigel culture, in which cells proliferate and form epithelial sheets that mature into hollow spherical acini. VRK1 overexpression significantly accelerates the initial stages of cell proliferation, leading to larger acini that nevertheless differentiate and mature. Our analysis of human tumor tissue microarrays (TMAs) revealed that VRK1 protein levels are higher in lymph node metastases than in patient-matched mammary tumors. Using public databases, we determined that VRK1 is among the top 10% of overexpressed transcripts in multiple subtypes of invasive breast cancer, and that high levels of VRK1 expression are correlated with decreased relapse-free survival. In sum, overexpression of VRK1, by regulating the transcription repressors snail, slug, and twist1, can promote a mesenchymal-to-epithelial transition (MET) in cell culture. VRK1-mediated MET might facilitate the colonization of distal sites by metastatic breast cancer cells, providing some insight into the frequent association of VRK1 overexpression with breast malignancies and the correlation between VRK1 overexpression and poor clinical outcome., Competing Interests: The authors have declared that no competing interests exist.

Maintaining goal representations is a critical component of cognitive control and is required for successful performance in many daily activities. This is particularly important when goal-relevant information needs to be maintained in working memory (WM), updated in response to changing task demands or internal goal states, and protected from interference by inhibiting counter-goal behaviors. Modulation of fronto-striatal dopamine is critical for updating and maintaining goals and representations. Here we test the hypothesis that a genetic predisposition (C957T T+ and DRD2/ANKK1-TaqIA A+) for reduced striatal D2 receptor availability would facilitate goal maintenance using the AX-continuous performance task (AX-CPT), on a sample of 196 adults (25-67 y). We demonstrate that carriers of two polymorphisms that have been linked to reduced striatal D2 receptor density show increased performance on context-dependent (BX) trials, and that the effect of these polymorphisms was only significant for long ISI trials where the demand for goal maintenance is high. The current results add further knowledge to the role of D2 receptor functioning in cognitive stability and flexibility, and could have implications for understanding cognitive deficits in patients characterized by altered dopamine functioning., Competing Interests: The authors have declared that no competing interests exist.

The mitis group, a member of the genetically diverse viridans group streptococci, predominately colonizes the human oropharynx. This group has been shown to cause a wide range of infectious complications in humans, including bacteremia in patients with neutropenia, orbital cellulitis and infective endocarditis. Hydrogen peroxide (H2O2) has been identified as a virulence factor produced by this group of streptococci. More importantly, it has been shown that Streptococcus oralis and S. mitis induce epithelial cell and macrophage death via the production of H2O2. Previously, H2O2 mediated killing was observed in the nematode Caenorhabditis elegans in response to S. oralis and S. mitis. The genetically tractable model organism C. elegans is an excellent system to study mechanisms of pathogenicity and stress responses. Using this model, we observed rapid H2O2 mediated killing of the worms by S. gordonii in addition to S. mitis and S. oralis. Furthermore, we observed colonization of the intestine of the worms when exposed to S. gordonii suggesting the involvement of an infection-like process. In response to the H2O2 produced by the mitis group, we demonstrate the oxidative stress response is activated in the worms. The oxidative stress response transcription factor SKN-1 is required for the survival of the worms and provides protection against H2O2 produced by S. gordonii. We show during infection, H2O2 is required for the activation of SKN-1 and is mediated via the p38-MAPK pathway. The activation of the p38 signaling pathway in the presence of S. gordonii is not mediated by the endoplasmic reticulum (ER) transmembrane protein kinase IRE-1. However, IRE-1 is required for the survival of worms in response to S. gordonii. These finding suggests a parallel pathway senses H2O2 produced by the mitis group and activates the phosphorylation of p38. Additionally, the unfolded protein response plays an important role during infection., Competing Interests: The authors have declared that no competing interests exist.

The innate immune system plays a critical role in the initial antiviral response. However, the timing and duration of these responses must be tightly regulated during infection to ensure appropriate immune cell activation and anti-viral defenses. Here we demonstrate that during antiviral response, a negative regulator miR-221 was also induced in an ELF4-dependent manner. We further show that ELF4 promotes miR-221 expression through direct binding to its promoter. Overexpression and knockdown assay show that miR-221 can negatively regulate IFNβ production in time of virus infection. RNA-seq analysis of miR-221 overexpressed cells revealed multiple candidate targets. Taken together, our study identified a novel negative microRNA regulator of innate antiviral response, which is dependent on ELF4., Competing Interests: The authors have declared that no competing interests exist.

Chemogenomic approaches involving highly annotated compound sets and cell based high throughput screening are emerging as a means to identify novel drug targets. We have previously screened a collection of highly characterized kinase inhibitors (Khan et al., Journal of General Virology, 2016) to identify compounds that increase or decrease expression of a human cytomegalovirus (HCMV) protein in infected cells. To identify potential novel anti-HCMV drug targets we used a machine learning approach to relate our phenotypic data from the aforementioned screen to kinase inhibition profiling of compounds used in this screen. Several of the potential targets had no previously reported role in HCMV replication. We focused on one potential anti-HCMV target, MAPK4K, and identified lead compounds inhibiting MAP4K4 that have anti-HCMV activity with little cellular cytotoxicity. We found that treatment of HCMV infected cells with inhibitors of MAP4K4, or an siRNA that inhibited MAP4K4 production, reduced HCMV replication and impaired detection of IE2-60, a viral protein necessary for efficient HCMV replication. Our findings demonstrate the potential of this machine learning approach to identify novel anti-viral drug targets, which can inform the discovery of novel anti-viral lead compounds., Competing Interests: The authors have declared that no competing interests exist.

Activation of the β2 integrin lymphocyte function-associated antigen-1 (LFA-1) in T cells induces stabilization of proinflammatory AU-rich element (ARE)-bearing mRNAs, by triggering the nuclear-to-cytoplasmic translocation of the mRNA-binding and -stabilizing protein HuR. However, the mechanism by which LFA-1 engagement controls HuR localization is not known. Here, we identify and characterize four key regulators of LFA-1-induced changes in HuR activity: the p38 pathway kinase MK2 and the constitutive nuclear proteins hnRNPs C, H1 and K. LFA-1 engagement results in rapid, sequential activation of p38 and MK2. Post-LFA-1 activation, MK2 inducibly associates with both hnRNPC and HuR, resulting in the dissociation of HuR from hnRNPs C, H1 and K. Freed from the three hnRNPs, HuR translocates from the nucleus to the cytoplasm, and mediates the stabilization of labile cytokine transcripts. Our results suggest that the modulation of T cell cytokine mRNA half-life is an intricate process that is negatively regulated by hnRNPs C, H1 and K and requires MK2 as a critical activator., Competing Interests: The authors have declared that no competing interests exist.

The human genetic diversity around the world was studied through several high variable genetic markers. In South America the demic consequences of admixture events between Native people, European colonists and African slaves have been displayed by uniparental markers variability. The mitochondrial DNA (mtDNA) has been the most widely used genetic marker for studying American mixed populations, although nuclear markers, such as microsatellite loci (STRs) commonly used in forensic science, showed to be genetically and geographically structured. In this work, we analyzed DNA from buccal swab samples of 296 individuals across Peru: 156 Native Amazons (Ashaninka, Cashibo and Shipibo from Ucayali, Huambiza from Loreto and Moche from Lambayeque) and 140 urban Peruvians from Lima and other 33 urban areas. The aim was to evaluate, through STRs and mtDNA variability, recent migrations in urban Peruvian populations and to gain more information about their continental ancestry. STR data highlighted that most individuals (67%) of the urban Peruvian sample have a strong similarity to the Amazon Native population, whereas 22% have similarity to African populations and only ~1% to European populations. Also the maternally-transmitted mtDNA confirmed the strong Native contribution (~90% of Native American haplogroups) and the lower frequencies of African (~6%) and European (~3%) haplogroups. This study provides a detailed description of the urban Peruvian genetic structure and proposes forensic STRs as a useful tool for studying recent migrations, especially when coupled with mtDNA., Competing Interests: The authors have declared that no competing interests exist.

In a variety of species, reduced food intake, and in particular protein or amino acid (AA) restriction, extends lifespan and healthspan. However, the underlying epigenetic and/or transcriptional mechanisms are largely unknown, and dissection of specific pathways in cultured cells may contribute to filling this gap. We have previously shown that, in mammalian cells, deprivation of essential AAs (methionine/cysteine or tyrosine) leads to the transcriptional reactivation of integrated silenced transgenes, including plasmid and retroviral vectors and latent HIV-1 provirus, by a process involving epigenetic chromatic remodeling and histone acetylation. Here we show that the deprivation of methionine/cysteine also leads to the transcriptional upregulation of endogenous retroviruses, suggesting that essential AA starvation affects the expression not only of exogenous non-native DNA sequences, but also of endogenous anciently-integrated and silenced parasitic elements of the genome. Moreover, we show that the transgene reactivation response is highly conserved in different mammalian cell types, and it is reproducible with deprivation of most essential AAs. The General Control Non-derepressible 2 (GCN2) kinase and the downstream integrated stress response represent the best candidates mediating this process; however, by pharmacological approaches, RNA interference and genomic editing, we demonstrate that they are not implicated. Instead, the response requires MEK/ERK and/or JNK activity and is reproduced by ribosomal inhibitors, suggesting that it is triggered by a novel nutrient-sensing and signaling pathway, initiated by translational block at the ribosome, and independent of mTOR and GCN2. Overall, these findings point to a general transcriptional response to essential AA deprivation, which affects the expression of non-native genomic sequences, with relevant implications for the epigenetic/transcriptional effects of AA restriction in health and disease., Competing Interests: This study was funded by Ajinomoto Co., Inc., Japan and one of the authors [M.B.] is an employee of this commercial funder. No other employment or consultancy relationships exist with the commercial funder, and no patents, products in development, or marketed products result from this study. The authors declare that no competing interests exist and that the commercial affiliation of one of the authors does not alter the adherence of authors to all PLOS ONE policies on sharing data and materials.

Background: Many studies have proven the importance of SnRK1 in the regulation of carbohydrate metabolism and plant development. Compared to Arabidopsis, much less is known about SnRK1 complexes in crop plants, and therefore, more work needs to be done to identify SnRK1 genes and to investigate their function in crop plants., Methods: In this study we identified five SnRK1-related genes in potato by analyzing the potato genome through BLAST, which encode one α-subunit isoform (stKIN), two β-subunit isoforms (stKINβ1 and stKINβ2) and two γ-subunit isoforms (stKINγ and stKINβγ). To investigate the functions of SnRK1 in the tuber development of potato, we further made overexpression and RNAi transgenic plants of these five genes. Based on these overexpression transgenic plants, the Fast protein liquid chromatography (FPLC) were employed to purify SnRK1 complexes, which were tracked by western-blot., Results: Experiments in vivo and in vitro showed that these five proteins in potato are functional SNF1/AMPK/SnRK1-related proteins. The SnRK1 activity decreased by 60% in the RNAi transgenic lines of stKIN; the starch content increased by 25% in the overexpression transgenic lines of stKIN, compared to that in the wild-type lines, whereas there is no significant difference in SnRK1 activity and starch content in the RNAi transgenic or overexpression lines of stKINβ1, stKINβ2, stKINγ and stKINβγ. In addition, we found that a few different SnRK1 complexes are present in potato by partially purifying SnRK1 complexes from these overexpression transgenic plants., Conclusions: Five functional SnRK1-related genes were identified in potato, including three novel genes, which encode one α-subunit isoform (stKIN), two β-subunit isoforms (stKINβ1 and stKINβ2) and two γ-subunit isoforms (stKINγ and stKINβγ). We found that a few SnRK1 related genes are present in potato tuber, which form several different SnRK1 isoenzymes. We found that stKIN is the primary α subunit of SnRK1 in potato tuber and plays important roles in the development of potato tubers., Competing Interests: The authors have declared that no competing interests exist.

Two-pore domain K+ channels (K2P) are responsible for background K+ currents and regulate the resting membrane potential and cellular excitability. Their activity is controlled by a large variety of physicochemical factors and intracellular signaling pathways. The majority of these effects converge on the intracellular C-terminus of the channels, resulting in the modification of the gating at the selectivity filter. Another gating mechanism, the activation gate at the helix bundle crossing is also well documented in other K+ channel families, however, it remains uncertain whether this type of gating is functional in K2P channels. The regulation of TWIK-related spinal cord K+ channel (TRESK) is different from the other K2P channels. Regulatory factors acting via the C-terminus are not known, instead channel activity is modified by the phosphorylation/dephosphorylation of the unusually long intracellular loop between the 2nd and 3rd transmembrane segments. These unique structural elements of the regulation lead us to examine channel gating at the bundle crossing region. Ba2+ was applied to the intracellular side of excised membrane patches and the characteristics of the channel block were determined. We compared the kinetics of the development of Ba2+ block when the channels were phosphorylated (inhibited) or dephosphorylated (activated) and also in different mutants mimicking the two functional states. Neither the phosphorylation/dephosphorylation nor the point mutations influenced the development of Ba2+ block, suggesting that the conformational changes of the bundle crossing region do not contribute to the phosphorylation-dependent gating of TRESK.

Mutations in the Cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders. Recently we have generated Cdkl5 KO mice by targeting exon 2 on the C57BL/6N background, and demonstrated postsynaptic overaccumulation of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors in the hippocampus. In the current study, we subjected the Cdkl5 KO mice to a battery of comprehensive behavioral tests, aiming to reveal the effects of loss of CDKL5 in a whole perspective of motor, emotional, social, and cognition/memory functions, and to identify its undetermined roles. The neurological screen, rotarod, hot plate, prepulse inhibition, light/dark transition, open field, elevated plus maze, Porsolt forced swim, tail suspension, one-chamber and three-chamber social interaction, 24-h home cage monitoring, contextual and cued fear conditioning, Barnes maze, and T-maze tests were applied on adult Cdkl5 -/Y and +/Y mice. Cdkl5 -/Y mice showed a mild alteration in the gait. Analyses of emotional behaviors revealed significantly enhanced anxiety-like behaviors of Cdkl5 -/Y mice. Depressive-like behaviors and social interaction of Cdkl5 -/Y mice were uniquely altered. The contextual and cued fear conditioning of Cdkl5 -/Y mice were comparable to control mice; however, Cdkl5 -/Y mice showed a significantly increased freezing time and a significantly decreased distance traveled during the pretone period in the altered context. Both acquisition and long-term retention of spatial reference memory were significantly impaired. The morphometric analysis of hippocampal CA1 pyramidal neurons revealed impaired dendritic arborization and immature spine development in Cdkl5 -/Y mice. These results indicate that CDKL5 plays significant roles in regulating emotional behaviors especially on anxiety- and fear-related responses, and in both acquisition and long-term retention of spatial reference memory, which suggests that focus and special attention should be paid to the specific mechanisms of these deficits in the CDKL5 deficiency disorder.

The ability to express a gene of interest in a spatio-temporal manner using Gal4-UAS system has allowed the use of Drosophila model to study various biological phenomenon. During Drosophila eye development, a synchronous wave of differentiation called Morphogenetic furrow (MF) initiates at the posterior margin resulting in differentiation of retinal neurons. This synchronous differentiation is also observed in the differentiating retina of vertebrates. Since MF is highly dynamic, it can serve as an excellent model to study patterning and differentiation. However, there are not any Gal4 drivers available to observe the gain- of- function or loss- of- function of a gene specifically along the dynamic MF. The decapentaplegic (dpp) gene encodes a secreted protein of the transforming growth factor-beta (TGF-beta) superfamily that expresses at the posterior margin and then moves with the MF. However, unlike the MF associated pattern of dpp gene expression, the targeted dpp-Gal4 driver expression is restricted to the posterior margin of the developing eye disc. We screened GMR lines harboring regulatory regions of dpp fused with Gal4 coding region to identify MF specific enhancer of dpp using a GFP reporter gene. We employed immuno-histochemical approaches to detect gene expression. The rationale was that GFP reporter expression will correspond to the dpp expression domain in the developing eye. We identified two new dpp-Gal4 lines, viz., GMR17E04-Gal4 and GMR18D08-Gal4 that carry sequences from first intron region of dpp gene. GMR17E04-Gal4 drives expression along the MF during development and later in the entire pupal retina whereas GMR18D08-Gal4 drives expression of GFP transgene in the entire developing eye disc, which later drives expression only in the ventral half of the pupal retina. Thus, GMR18D08-Gal4 will serve as a new reagent for targeting gene expression in the ventral half of the pupal retina. We compared misexpression phenotypes of Wg, a negative regulator of eye development, using GMR17E04-Gal4, GMR18D08-Gal4 with existing dpp-Gal4 driver. The eye phenotypes generated by using our newly identified MF specific driver are not similar to the ones generated by existing dpp-Gal4 driver. It suggests that misexpression studies along MF needs revisiting using the new Gal4 drivers generated in our studies.