Id Genes Research Articles

Id2 and Id4 are not the major negative regulators of oligodendrocyte differentiation during early central nervous system development.

Myelin sheathes ensure the rapid conduction of neural impulse and provide nutritional support for neurons. Myelin sheathes are formed by differentiated oligodendrocytes (OLs) in the central nervous system. During OL development, the differentiation of oligodendrocyte progenitor cells (OPCs) into mature OLs is controlled by both positive differentiation factors (drivers) and negative regulatory factors (brakes). Previous studies have suggested Id2 and Id4 as the key negative factors for OL differentiation. However, these conclusions were mainly based on in vitro studies and the reported OL phenotype in Id4 mutants appear to be mild. In this study, we systematically investigated the in vivo function of Id2 and Id4 genes in OL differentiation in their genetic mutants and in embryonic chicken spinal cord. Our results showed that disruption of Id4 has no effect on OL differentiation and maturation, whereas Id2 mutants and Id2/Id4 compound mutants display a mild and transient precocity of OL differentiation. In agreement with these loss-of-function studies, Id2, but not Id4, is weakly expressed in OPCs. Despite their minor roles in OL differentiation, forced expression of Id2 and Id4 in embryonic chicken spinal cords strongly inhibit the differentiation of OPCs. Taken together, our detailed functional and expressional studies strongly suggest that Id2 and Id4 are not the major in vivo repressors of OPC differentiation during animal development, shedding new light on the molecular regulation of early OL development.

Identification of liver-derived bone morphogenetic protein (BMP)-9 as a potential new candidate for treatment of colorectal cancer.

Colorectal cancer (CRC) is a high‐incidence malignancy worldwide which still needs better therapy options. Therefore, the aim of the present study was to investigate the responses of normal or malignant human intestinal epithelium to bone morphogenetic protein (BMP)‐9 and to find out whether the application of BMP‐9 to patients with CRC or the enhancement of its synthesis in the liver could be useful strategies for new therapy approaches. In silico analyses of CRC patient cohorts (TCGA database) revealed that high expression of the BMP‐target gene ID1, especially in combination with low expression of the BMP‐inhibitor noggin, is significantly associated with better patient survival. Organoid lines were generated from human biopsies of colon cancer (T‐Orgs) and corresponding non‐malignant areas (N‐Orgs) of three patients. The N‐Orgs represented tumours belonging to three different consensus molecular subtypes (CMS) of CRC. Overall, BMP‐9 stimulation of organoids promoted an enrichment of tumour‐suppressive gene expression signatures, whereas the stimulation with noggin had the opposite effects. Furthermore, treatment of organoids with BMP‐9 induced ID1 expression (independently of high noggin levels), while treatment with noggin reduced ID1.In summary, our data identify the ratio between ID1 and noggin as a new prognostic value for CRC patient outcome. We further show that by inducing ID1, BMP‐9 enhances this ratio, even in the presence of noggin. Thus, BMP‐9 is identified as a novel target for the development of improved anti‐cancer therapies of patients with CRC.

TAMI-79. THERAPEUTIC REVERSAL OF PRENATAL PONTINE ID1 SIGNALING IN DIPG

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor with rare survival beyond two years. This poor prognosis is largely due to the tumor's highly infiltrative and invasive nature. Nearly 80% of DMGs harbor K27M mutation in the genes encoding histone H3.1 (H3F3A) or H3.3 (HISTIH3B), often with concurrent ACVR1 mutation. Inhibitor of DNA-binding (ID) proteins are key transcriptional regulators of genes involved in lineage commitment and are associated with invasiveness and poor clinical outcomes in multiple human cancers. Introduction of H3K27M and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been confirmed in human tumors or targeted therapeutically. We developed an in-utero electroporation (IUE) murine H3K27M-driven tumor model, which demonstrates increased ID1 expression in H3K27M- and ACVR1-mutated tumor cells. Exome and transcriptome sequencing analysis of multi-focal DMG tumors (n=52) and normal brain tissue revealed that increased ID1 expression is associated with H3K27M/ACVR1-mutation and brainstem location, and correlates with poor survival in patients. ChIP-sequencing for H3K27ac and H3K27me3 in multiple DMG tumors (n=5) revealed that the ID1 gene is epigenetically active, which matches the epigenetic state of murine prenatal hindbrain cells. Higher ID1-expressing astrocyte-like DIPG cells share a similar transcriptional program with ID1+/SPARCL1+ positive oligo/astrocyte-precursor (OAPC) cells from the developing human brain and demonstrate upregulation of gene sets involved in regulation of cell migration. Both genetic and pharmacologic [cannabidiol (CBD)] suppression of ID1 result in decreased DIPG cell invasion/migration in vitro and invasion/tumor growth in multiple in vivo models. Mechanistically, CBD reduces proliferation through production of reactive oxygen species. Further, DIPG patients treated off-trial with CBD (n=15) displayed reduced ID1 tumor expression and improved overall survival. In summary, ID1 is upregulated in DIPG through K27M-mediated epigenetic reactivation of a developmental OAPC-like transcriptional state, and ID1-driven invasiveness of DIPG is therapeutically targetable with CBD.

DDRE-30. CELL-PENETRATING PEPTIDE ANTAGONIST OF CCAAT/ENHANCER BINDING PROTEIN ß DISPLAYS POTENT ANTI-GLIOBLASTOMA ACTIVITY

Abstract CCAAT/Enhancer Binding Protein Beta (C/EBPß) is a transcription factor overexpressed in glioblastoma (GBM). Mechanistically, C/EBPß is a master regulator of mesenchymal transition in GBM, and its increased expression correlates with mesenchymal differentiation and predicts poor clinical outcome. C/EBPß activity requires dimerization with co-factors such as CREB/ATF family members via leucine zipper interactions. ST101 is a novel peptide antagonist of C/EBPß currently being evaluated in a Phase 1/2 clinical study in patients with advanced unresectable and metastatic solid tumors. ST101 binds to the C/EBPß leucine zipper, thereby preventing dimer formation and inhibiting its transcriptional activity, resulting in selective tumor cell cytotoxicity. Here, we describe ST101 non-clinical anti-tumor activity against GBM. In vitro studies in T98G and U251 cells demonstrate ST101 dose-dependent impact of cell viability (EC50 of 2.2 and 1.2 μM, respectively), accompanied by significant impact on C/EBPß-mediated gene expression as determined by qPCR analysis. In contrast, normal human mononuclear and epithelial cells were not sensitive to ST101 (EC50 > 80 μM). In vivo, ST101 displayed significant anti-tumor activity in a U251 GBM subcutaneous xenograft model, resulting in 81.4% tumor growth inhibition (TGI) vs. control and undetectable tumors in 50% of animals. Following ST101 exposure tumors displayed reduced BIRC3 and ID2 gene expression, and significantly increased cleaved caspase 3 immunostaining indicative of cell death induction. In U251 tumors, subtherapeutic ST101 (< 5% TGI) in combination with temozolomide (< 5% TGI) resulted in 52.8% TGI, significantly greater than either single-agent alone. Similarly, in a temozolomide-refractory T98G GBM subcutaneous xenograft model, ST101 (41.6% TGI) in combination with TMZ (< 5% TGI) resulted in significant anti-GBM response (72.4% TGI). These data emphasize the potential of ST101 as a potent peptide therapeutic for GBM.

Inhibitor of Differentiation-2 Protein Ameliorates DSS-Induced Ulcerative Colitis by Inhibiting NF-κB Activation in Neutrophils.

The loss of inhibitor of differentiation-2 (ID2) could lead to the development of colitis in mice, supplementation with exogenous ID2 protein might be a potential strategy to ameliorate colitis. In this study, the effects of ID2 protein supplementation on Dextran sodium sulfate (DSS)-induced colitis were investigated. Firstly, we confirmed that the expression of ID2 was reduced in the colon tissues of DSS-induced colitis mice and patients with ulcerative colitis (UC). Then, we constructed a recombinant plasmid containing the human Id2 gene and expressed it in Escherichia coli (E. coli) successfully. After purification and identification, purified hID2 could ameliorate DSS-induced colitis efficiently in mice by improving disease symptoms, decreasing the levels of proinflammatory cytokines in colon tissues, maintaining the integrity of intestinal barrier and reducing the infiltration of neutrophils and macrophages in the colon. Further study showed that hID2 could be endocytosed efficiently by neutrophils and macrophages, and hID2 lost its protection function against colitis when neutrophils were depleted with an anti-Gr-1 antibody. hID2 decreased the mRNA levels of IL-6, IL-1β and TNF-α in lipopolysaccharides (LPS)-stimulated neutrophils and efficiently inhibited the activation of NF-κB signalling pathway in neutrophils. Interestingly, hID2 showed a synergistic role in inhibition of NF-κB activation with pyrrolidine dithiocarbamic acid (PDTC), an inhibitor of NF-κB activation. Therefore, this study demonstrated the potential use of hID2 to treat UC, and hID2 protein might be a promising anti-inflammatory agent that targets the NF-κB signalling pathway in neutrophils.

Screening of host genes regulated by ID1 and ID3 proteins during foot-and-mouth disease virus infection

Foot-and-mouth disease virus (FMDV) is an important pathogen that harms cloven-hoofed animals and has caused serious losses to livestock production since its discovery. Furthermore, inhibitor of DNA binding (ID) proteins have been thoroughly studied in tumorigenesis, differentiation and metastasis, but its role in viral infection is rarely known. In this study, three gene knockout cell lines ID1 KO, ID3 KO, ID1/3 KO were obtained based on BHK-21 cells. We found that ID1 and ID3 genes single or double knockout promote the replication of FMDV. Moreover, compared with negative control cells during virus infection, there were 551 up-regulated genes and 1222 down-regulated genes in the ID1 KO cell line; 916 up-regulated genes and 1845 down-regulated genes in the ID3 KO cell line; 810 up-regulated genes and 1566 down-regulated genes in ID1/3 KO cell line. Further genes expression patterns verification results also showed a good correlation between the data of RT-qRCR and RNA-seq. These findings provide a basis for studying the relevant mechanisms between host genes and ID genes during FMDV infection.

Integration and gene co-expression network analysis of scRNA-seq transcriptomes reveal heterogeneity and key functional genes in human spermatogenesis

Spermatogenesis is a complex process of cellular division and differentiation that begins with spermatogonia stem cells and leads to functional spermatozoa production. However, many of the molecular mechanisms underlying this process remain unclear. Single-cell RNA sequencing (scRNA-seq) is used to sequence the entire transcriptome at the single-cell level to assess cell-to-cell variability. In this study, more than 33,000 testicular cells from different scRNA-seq datasets with normal spermatogenesis were integrated to identify single-cell heterogeneity on a more comprehensive scale. Clustering, cell type assignments, differential expressed genes and pseudotime analysis characterized 5 spermatogonia, 4 spermatocyte, and 4 spermatid cell types during the spermatogenesis process. The UTF1 and ID4 genes were introduced as the most specific markers that can differentiate two undifferentiated spermatogonia stem cell sub-cellules. The C7orf61 and TNP can differentiate two round spermatid sub-cellules. The topological analysis of the weighted gene co-expression network along with the integrated scRNA-seq data revealed some bridge genes between spermatogenesis’s main stages such as DNAJC5B, C1orf194, HSP90AB1, BST2, EEF1A1, CRISP2, PTMS, NFKBIA, CDKN3, and HLA-DRA. The importance of these key genes is confirmed by their role in male infertility in previous studies. It can be stated that, this integrated scRNA-seq of spermatogenic cells offers novel insights into cell-to-cell heterogeneity and suggests a list of key players with a pivotal role in male infertility from the fertile spermatogenesis datasets. These key functional genes can be introduced as candidates for filtering and prioritizing genotype-to-phenotype association in male infertility.

Targeted Disruption of the Inhibitor of DNA Binding 4 (Id4) Gene Alters Photic Entrainment of the Circadian Clock.

Inhibitor of DNA binding (Id) genes comprise a family of four helix–loop–helix (HLH) transcriptional inhibitors. Our earlier studies revealed a role for ID2 within the circadian system, contributing to input, output, and core clock function through its interaction with CLOCK and BMAL1. Here, we explore the contribution of ID4 to the circadian system using a targeted disruption of the Id4 gene. Attributes of the circadian clock were assessed by monitoring the locomotor activity of Id4−/− mice, and they revealed disturbances in its operation. Id4-mutant mice expressed a shorter circadian period length, attenuated phase shifts in responses to continuous and discrete photic cues, and an advanced phase angle of entrainment under a 12:12 light:dark cycle and under short and long photoperiods. To understand the basis for these properties, suprachiasmatic nucleus (SCN) and retinal structures were examined. Anatomical analysis reveals a smaller Id4−/− SCN in the width dimension, which is a finding consistent with its smaller brain. As a result of this feature, anterograde tracing in Id4−/− mice revealed retinal afferents innovate a disproportionally larger SCN area. The Id4−/− photic entrainment responses are unlikely to be due to an impaired function of the retinal pathways since Id4−/− retinal anatomy and function tested by pupillometry were similar to wild-type mice. Furthermore, these circadian characteristics are opposite to those exhibited by the Id2−/− mouse, suggesting an opposing influence of the ID4 protein within the circadian system; or, the absence of ID4 results in changes in the expression or activity of other members of the Id gene family. Expression analysis of the Id genes within the Id4−/− SCN revealed a time-of-day specific elevated Id1. It is plausible that the increased Id1 and/or absence of ID4 result in changes in interactions with bHLH canonical clock components or with targets upstream and/or downstream of the clock, thereby resulting in abnormal properties of the circadian clock and its entrainment.

Combining Gene Mutation with Gene Expression Data Improves Outcomes Prediction in Acute Promyelocytic Leukemia

INTRODUCTIONIn the context of all-trans retinoic acid (ATRA) and anthracycline-based chemotherapy, heterogeneity of clinical outcomes of patients with acute promyelocytic leukemia (APL) may be higher than expected, mainly outside well-controlled clinical trials. Although recent clinical studies suggest that such heterogeneity could be significantly reduced or even abolished in light of the current arsenic trioxide (ATO)-based therapies, this compound is still not available for most reference centers in low- and middle-income countries (LMIC), and, at least for a near future, alternative strategies for predicting outcomes and improve risk stratification in APL should be tested. Here, we combined recurrent mutations with aberrant expression of genes previously associated with poor prognosis in both APL and acute myeloid leukemia, and proposed an integrative score in APL (ISAPL) for outcomes prediction. METHODS: As a learning set, diagnostic bone marrow samples from 150 adult patients with newly diagnosed APL who were enrolled in the IC-APL study were analyzed. The treatment protocol was identical to that of the LPA2005 trial reported by the Programa Español de Tratamiento en Hematologia/Dutch-Belgian Hemato-Oncology Cooperative Group (PETHEMA/HOVON), except for the replacement of idarubicin by daunorubicin due to its better availability and lower cost in the participating centers. Integer weights for the risk score were derived from Cox proportional hazard model, identical to that reported by Damm et al. (Blood. 2011 Apr 28;117(17):4561-8), using OS as primary endpoint. Variables with P -values lower than 0.05 were included in the model. Variables considered for the model inclusion were the following: FLT3 -ITD status, gene expression profile of ∆Np73/TAp73 ratio and transcript levels of KMT2E, BAALC, ID1, and WT1 genes. Other candidates, such as PIM2, PRAME, ERG, and IDH1 rs11554137 and WT1 rs16754 polymorphisms, were not associated with lower OS and not included in the score. Hazard ratios (HR) for OS were calculated for each variable separately. The HR was converted to integer weights according to the following: variables with HR < 1 were excluded from analyses; variables with HR > 1 and < 1.5 were assigned a weight of 1; variables with HR > 1.5 and < 2.5 were assigned a weight of 2; variables with HR > 2.5 were assigned a weight of 3. The final score was the sum of these integer weights. RESULTS: Complete data for ISAPL modeling were available for 99 of 150 patients (median score: 5, range: 0-16). The median age of the learning set was 35 years (range: 18-65 years) with 44 males (44%). According to PETHEMA/GIMEMA criteria for predicting relapse, 14%, 41%, 27% and 60% of patients assigned to the first, second, third and fourth ISAPL quartiles were deemed high-risk patients (P=0.036). Except for a higher leukocyte count at diagnosis in patients assigned to the fourth-ISAPL quartile (P=0.033), no significant differences in baseline features were found among groups. Early mortality (P=0.005), complete remission (P=0.002), overall survival (P <0.001; Figure 1A), disease-free survival (P=0.017; Figure 1B), event-free survival (P <0.001; Figure 1C), and cumulative incidence of relapse (P=0.03; Figure 1D) rates were significantly different among patients assigned to the first-, second-, third- and fourth-ISAPL quartiles. CONCLUSIONS: In summary, ISAPL resulted in the separation of four distinct groups of patients, with significant differences for hematologic recovery, relapse, and survival. We will next validate these findings in internal and external validation cohorts. [Display omitted] DisclosuresBittencourt:Janssen, Takeda: Honoraria. Pagnano:Roche: Speakers Bureau; Amgen: Consultancy; Bristol-Meirs Squibb: Consultancy, Speakers Bureau; Novartis: Consultancy. Lo Coco:Novartis: Honoraria, Speakers Bureau; Lundbeck: Honoraria, Speakers Bureau; TEVA: Honoraria, Speakers Bureau.

Transient Reduction of Transferrin Saturation Contributes to Hepcidin Inhibition Independently of Erfe in Mice Treated with Erythropoietin

IntroductionErythropoiesis is the major consumer of iron for hemoglobin synthesis. Erythroid cells release erythroferrone (ERFE), an erythropoietin (EPO) target gene, to signal their iron needs to the liver through hepcidin inhibition (Kautz et al., 2014). Since hepcidin degrades the iron exporter ferroportin, thus reducing body iron concentration, its inhibition by ERFE increases iron availability for erythroid cells. Hepcidin is mainly controlled by the BMP-SMAD-pathway. We demonstrated that ERFE inhibits hepcidin only when the BMP-SMAD signaling is attenuated (Nai et al., 2016). However, how the BMP-SMAD pathway is reduced in a physiologic context of increased erythropoiesis remains unknown. The major source of iron for erythroid cells is the circulating diferric-transferrin (holo-TF) that is uptaken by TFR1, highly expressed by erythroid cells. Since holo-TF, measured as transferrin saturation, is involved in hepcidin activation by the BMP-SMAD pathway, we hypothesized that increase iron uptake by EPO-stimulated erythropoiesis reduces transferrin saturation, thus inhibiting hepcidin earlier than and independently of ERFE.MethodsC57BL/6 wild type and Erfe KO male mice (8-9 week-old) were challenged with a single EPO injection (200 U/mouse) and sacrificed at early time points (3-15 hrs) post-treatment. To analyze erythroid differentiation and Tfr1 levels at short time points after EPO injection (3 and 6 hrs post-treatment), Ter119+ cells were separated from bone marrow (BM) and spleen according to Forward Scatter (FSC) value and CD44 expression, as described(Liu et al., 2013). Tfr1 was measured by flow cytometry in isolated population and expressed as MFI. Expression of BM and spleen Erfe, GypA and Tfr1 was evaluated by qRT-PCR. Hamp, Id1 and Atoh8 were measured from total liver of EPO- or saline-treated mice. Serum iron, transferrin saturation (TS) and liver iron content were evaluated by standard methods (Pagani et al., 2011).ResultsWild type mice were analyzed at different time point (3, 6, 9 and 15 hrs) after EPO injection. EPO-treated mice upregulate Erfe mRNA both in BM and spleen, starting from 3 hrs post-injection. Hepcidin and the BMP-SMAD target genes Id1 and Atoh8 are significantly decreased at 9 hrs post-EPO. Although Id1 and Atoh8 start to increase 15 hrs post-EPO, hepcidin remains low due to Erfe inhibition. EPO treatment transiently (at 6 and 9 hrs) reduces both serum iron and TS that are increased thereafter (15 hrs) due to hepcidin inhibition. Since EPO stimulates Tfr1 expression in erythroid cells, we measured Tfr1mRNA and protein levels in BM and spleen. Tfr1 mRNA is slightly increased in the spleen and shows a trend towards increase in the BM at 6 hrs post-EPO, whereas it is comparable to untreated mice at 9 and 15 hrs post-EPO. However, flow cytometry analysis shows that, despite the number of erythroid precursors expressing Tfr1 is comparable in untreated and EPO treated mice, membrane Tfr1 is strongly increased at 3 and 6 hrs post-EPO, indicating that increased erythroid uptake accounts for serum iron/TS reduction shortly after EPO treatment. To demonstrate that increased Tfr1 expression by EPO in erythroid cells downregulates the liver BMP-SMAD pathway and hepcidin through a transient reduction in circulating iron/TS and independently of Erfe, we analyzed EPO-injected Erfe KO mice. Both in wild type and Erfe KO animals Id1 is strongly inhibited at 6-9 hrs post EPO. Interestingly, Erfe KO animals show a trend towards a reduction in hepcidin at 9 hrs post-EPO, and a significant decrease at 15 hrs, with low serum iron and TS.The mild but significant effect observed in Erfe KO mice suggests that downregulation of the BMP-SMAD pathway due to transient reduction of TS contributes to hepcidin inhibition in an erythroid-dependent, but Erfe-independent, manner.ConclusionsEPO stimulates erythroid expression of membrane Tfr1 at short time point after treatment, causing a transient decreased in transferrin saturation and a downregulation of the hepatic BMP-SMAD pathway that contributes to hepcidin inhibition independently of Erfe. DisclosuresFerrari:GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Camaschella:Vifor Pharma: Membership on an entity's Board of Directors or advisory committees.

EPCT-07. ID1 IS A KEY TRANSCRIPTIONAL REGULATOR OF DIPG INVASION AND IS TARGETABLE WITH CANNABIDIOL

Diffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors with no effective therapies beyond radiation. The highly invasive nature of DIPG is key to its aggressive phenotype, but the factors and mechanisms contributing to this aggressive invasion are unknown. Inhibitor of DNA binding (ID) proteins, key regulators of lineage commitment during embryogenesis, are implicated in tumorigenesis in multiple human solid tumors. Prior work showed that recurrent H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes. However, the impact and targetability of ID1 have not been explored in human DIPG. Exome and transcriptome sequencing analyses of multi-focal DIPG tumors and normal brain tissue from autopsy (n=52) revealed that ID1 expression is significantly elevated in DIPG samples. Higher ID1 expression correlates with reduced survival in DIPG patients and increased regional invasion in multi-focal autopsy samples. Analyses of developing mouse brain RNA/ChIP-Seq data revealed high ID1 expression and H3K27ac promoter binding in prenatal hindbrain compared to all other prenatal and postnatal brain regions. ChIP-qPCR for H3K27ac and H3K27me3 revealed that ID1 gene regulatory regions are epigenetically poised for upregulation in DIPG tissues compared to normal brain, regardless of H3/ACVR1 mutational status. These data support that the developing pons is regionally poised for ID1 activation. Genetic (shRNA) ID1 knockdown of primary human H3.3K27M-DIPG cells (DIPG007) resulted in significantly reduced invasion/migration and significantly improved survival of K27M-DIPG mice. Knockdown of ID1 in DIPG cells also resulted in down-regulation of the WNK1-NKCC1 pathway, which regulates tumor cell electrolyte homeostasis and migration. Finally, treatment of DIPG007 cells with cannabidiol (CBD) reduced ID1 levels, viability of DIPG cells and significantly improved survival of K27M-DIPG mice. In summary, our findings indicate that multifactorial (genetic and regional) epigenetic upregulation of ID1 drives DIPG invasiveness; and that targeting ID1 with CBD could potentially be an effective therapy for DIPG.

WGS-Based Analysis of Carbapenem-Resistant Acinetobacter baumannii in Vietnam and Molecular Characterization of Antimicrobial Determinants and MLST in Southeast Asia.

Carbapenem-resistant Acinetobacter baumannii (A. baumannii, CRAb) is an emerging global threat for healthcare systems, particularly in Southeast Asia. Next-generation sequencing (NGS) technology was employed to map genes associated with antimicrobial resistance (AMR) and to identify multilocus sequence types (MLST). Eleven strains isolated from humans in Vietnam were sequenced, and their AMR genes and MLST were compared to published genomes of strains originating from Southeast Asia, i.e., Thailand (n = 49), Myanmar (n = 38), Malaysia (n = 11), Singapore (n = 4) and Taiwan (n = 1). Ten out of eleven Vietnamese strains were CRAb and were susceptible only to colistin. All strains harbored ant(3”)-IIa, armA, aph(6)-Id and aph(3”) genes conferring resistance to aminoglycosides, and blaOXA-51 variants and blaADC-25 conferring resistance to ß-lactams. More than half of the strains harbored genes that confer resistance to tetracyclines, sulfonamides and macrolides. The strains showed high diversity, where six were assigned to sequence type (ST)/2, and two were allocated to two new STs (ST/1411-1412). MLST analyses of 108 strains from Southeast Asia identified 19 sequence types (ST), and ST/2 was the most prevalent found in 62 strains. A broad range of AMR genes was identified mediating resistance to ß-lactams, including cephalosporins and carbapenems (e.g., blaOXA-51-like, blaOXA-23, blaADC-25, blaADC-73, blaTEM-1, blaNDM-1), aminoglycosides (e.g., ant(3”)-IIa, aph(3”)-Ib, aph(6)-Id, armA and aph(3’)-Ia), phenicoles (e.g., catB8), tetracyclines (e.g., tet.B and tet.39), sulfonamides (e.g., sul.1 and sul.2), macrolides and lincosamide (e.g., mph.E, msr.E and abaF). MLST and core genome MLST (cgMLST) showed an extreme diversity among the strains. Several strains isolated from different countries clustered together by cgMLST; however, different clusters shared the same ST. Developing an action plan on AMR, increasing awareness and prohibiting the selling of antibiotics without prescription must be mandatory for this region. Such efforts are critical for enforcing targeted policies on the rational use of carbapenem compounds and controlling AMR dissemination and emergence in general.

ID1 and ID3 are Negative Regulators of TGFβ2-Induced Ocular Hypertension and Compromised Aqueous Humor Outflow Facility in Mice.

PurposeIn POAG, elevated IOP remains the major risk factor in irreversible vision loss. Increased TGFβ2 expression in POAG aqueous humor and in the trabecular meshwork (TM) amplifies extracellular matrix (ECM) deposition and reduces ECM turnover in the TM, leading to a decreased aqueous humor (AH) outflow facility and increased IOP. Inhibitor of DNA binding proteins (ID1 and ID3) inhibit TGFβ2-induced fibronectin and PAI-1 production in TM cells. We examined the effects of ID1 and ID3 gene expression on TGFβ2-induced ocular hypertension and decreased AH outflow facility in living mouse eyes.MethodsIOP and AH outflow facility changes were determined using a mouse model of Ad5-hTGFβ2C226S/C288S-induced ocular hypertension. The physiological function of ID1 and ID3 genes were evaluated using Ad5 viral vectors to enhance or knockdown ID1/ID3 gene expression in the TM of BALB/cJ mice. IOP was measured in conscious mice using a Tonolab impact tonometer. AH outflow facilities were determined by constant flow infusion in live mice.ResultsOver-expressing ID1 and ID3 significantly blocked TGFβ2-induced ocular hypertension (P < 0.0001). Although AH outflow facility was significantly decreased in TGFβ2-transduced eyes (P < 0.04), normal outflow facility was preserved in eyes injected concurrently with ID1 or ID3 along with TGFβ2. Knockdown of ID1 or ID3 expression exacerbated TGFβ2-induced ocular hypertension.ConclusionsIncreased expression of ID1 and ID3 suppressed both TGFβ2-elevated IOP and decreased AH outflow facility. ID1 and/or ID3 proteins thus may show promise as future candidates as IOP-lowering targets in POAG.

Involvement of NR5A1 and NR5A2 in the Regulation of Steroidogenesis by Clock Gene and BMPs by Human Granulosa Cells

We previously reported that the expression levels of Clock gene are linked to the expression levels of steroidogenetic enzymes in human granulosa cells (EJ 2019). However, the downstream molecules of the Clock gene actions in the regulation of ovarian steroidogenesis have yet to be elucidated. In the present study, we investigated the roles of the transcription factors, NR5A1 (also known as SF-1) and NR5A2 (LRH-1), which play key roles in the reproductive function as well as steroidogenesis by focusing on the functional link between Clock gene and bone morphogenetic protein (BMP) signaling using human granulosa KGN cells. First of all, we examined the effects of BMPs/growth differentiation factor (GDF) on forskolin (FSK)-induced steroidogenesis. As a result, FSK-induced mRNA levels of StAR and P450scc, but not P450arom, were potently suppressed by treatments with BMP-6, -9, -15 and GDF-9. The expression levels of NR5A1 and NR5A2 mRNA were also upregulated by FSK treatment, while the BMP-target gene Id-1 mRNA levels were stimulated by the treatment with BMPs. Of interest, treatments with BMPs/GDF increased FSK-induced NR5A1 mRNA levels but suppressed FSK-induced NR5A2 mRNA levels by granulosa cells. The expression levels of NR5A1 mRNA were positively correlated with the changes of P450arom and 3βHSD mRNA, whereas the expression levels of NR5A2 mRNA were correlated with that of StAR and P450scc mRNA. In addition, the expression levels of NR5A1 and NR5A2 mRNAs were positively correlated with the levels of Clock mRNA. In particular, Clock mRNA levels showed highly positive correlation with the levels of NR5A2 mRNA compared with NR5A1 mRNA. Of note, Id-1 mRNA levels were positively correlated with the levels of NR5A1 mRNA, but negatively correlated with that of NR5A2 mRNA. Furthermore, the inhibition of Clock gene expression by siRNA attenuated the expression levels of NR5A1 and NR5A2 mRNA, resulting in decreased mRNA levels of StAR and P450arom in the presence of FSK. Thus, the present results suggested a novel mechanism by which Clock expression is functionally linked to the expression of NR5A1 and NR5A2, the latter of which is further regulated by BMP signaling by granulosa cells. The interaction among Clock, NR5A1/NR5A2 and BMPs may be involved in the fine tuning of steroidogenesis by ovarian follicles.

Involvement of BMP-15 in Glucocorticoid Actions on Ovarian Steroidogenesis by Rat Granulosa Cells

Glucocorticoid receptor (GR) are known to be expressed in the ovary and glucocorticoids are shown to exert direct effects on granulosa cell functions. In the clinical setting, menstrual abnormality, amenorrhea and hypermenorrhea can be shown in patients with glucocorticoid excess. On the other hand, glucocorticoids can also be used for the treatment of PCOS with hyperandrogenism. However, the effects of glucocorticoids on the reproductive system have not been fully elucidated. In the present study, we investigated the influence of glucocorticoids on follicular steroidogenesis using primary culture of rat granulosa cells, by focusing on the ovarian bone morphogenetic proteins (BMPs) acting as a luteinizing inhibitor. Granulosa cells isolated from female immature rats were treated with follicle-stimulating hormone (FSH) in the presence of dexamethasone (Dex) in serum-free conditions. After treatment with Dex for 48 h, the changes of estradiol (E2) and progesterone (P4) production and cAMP synthesis induced by FSH treatments were measured by ELISA. Total RNAs of granulosa cells treated with FSH, Dex and BMPs were extracted and mRNA levels of steroidogenetic factors and enzymes, BMP receptors and Id-1 were quantified by real-time RT-PCR. Phosphorylation of Smad1/5/9 induced by BMPs was evaluated by Western blotting using cell lysates in the presence or absence of Dex. As a result, it was revealed that Dex treatment decreased FSH-induced E2 production by granulosa cells. In accordance with the steroid results, Dex suppressed FSH-induced P450arom mRNA expression as well as FSH-induced cAMP synthesis by granulosa cells. By contrast, Dex treatment augmented FSH-induced P4 production by granulosa cells in a concentration-dependent manner. Dex treatment was found to enhance basal and FSH-induced mRNA levels of P4-synthetic enzymes including P450scc and 3βHSD. Of note, Dex treatment activated the BMP target gene Id-1 transcription and Smad1/5/9 phosphorylation, in particular, induced by BMP-15 among various BMP ligands including BMP-2, -4, -6, -7, -9 and -15. It was also revealed that Dex treatment increased mRNA levels of ALK-6, a type-I receptor for BMP-15, and that BMP-15 treatment in turn upregulated GR mRNA levels expressed by granulosa cells. Given that BMP-15 acts as an inhibitor for P4 production by suppressing FSH-receptor actions, it was suggested that glucocorticoid is functionally linked to the enhancement of endogenous BMP-15, leading to the negative feedback toward the P4 overproduction induced by FSH and Dex in granulosa cells. Collectively, it was revealed that glucocorticoids elicit differential effects on the ovarian steroidogenesis of E2 and P4, in which GR and BMP-15 actions are mutually enhanced in granulosa cells.

Involvement of BMP-15 in glucocorticoid actions on ovarian steroidogenesis by rat granulosa cells

To elucidate the impact of glucocorticoids on ovarian steroidogenesis and its molecular mechanism by focusing on bone morphogenetic proteins (BMPs), we examined the effect of dexamethasone (Dex) on estradiol and progesterone synthesis by using primary culture of rat granulosa cells. It was revealed that Dex treatment dose-dependently decreased estradiol production but increased progesterone production induced by follicle-stimulating hormone (FSH) by granulosa cells. In accordance with the effects of Dex on estradiol synthesis, Dex suppressed P450arom mRNA expression and cAMP synthesis induced by FSH. Dex treatment in turn enhanced basal as well as FSH-induced levels of mRNAs encoding the enzymes for progesterone synthesis including P450scc and 3βHSD but not StAR and 20αHSD. Of note, Dex treatment significantly upregulated transcription of the BMP target gene Id-1 and Smad1/5/9 phosphorylation in the presence of BMP-15 among the key ovarian BMP ligands. It was also found that Dex treatment increased the expression level of BMP type-I receptor ALK-6 among the type-I and –II receptors for BMP-15. Inhibitory Smad6/7 expression was not affected by Dex treatment. On the other hand, BMP-15 treatment upregulated glucocorticoid receptor (GR) expression in granulosa cells. Collectively, it was revealed that glucocorticoids elicit differential effects on ovarian steroidogenesis, in which GR and BMP-15 actions are mutually enhanced in granulosa cells.

Inhibitor of DNA Binding 2 (ID2): A Novel Marker for Lymph Node Metastasis in Head and Neck Squamous Cell Carcinoma.

Although aggressive invasion and sequential lymph node metastasis (LNM) significantly affect the prognosis of patients with head and neck squamous cell carcinoma (HNSCC), studies on identifying the factors that regulate this process remain scarce. This study found an inhibitor of DNA binding 2 (ID2) as a novel molecule involved in the regulation of invasion and LNM of HNSCC and further verified its functional role. The study examined the translational significance between ID2 expression levels and the presence of LNM as well as the prognosis for 119 patients with HNSCC after treatment. In addition, in vitro and in vivo experiments were performed using ID2 gene-modulated HNSCC cell lines to determine the functional role of ID2 in the invasion and LNM of HNSCC. Elevated levels of ID2 expression were closely associated with the presence of LNM in 119 patients with HNSCC, resulting in a poor prognosis. Overexpression of ID2-induced invasion and LNM of HNSCC cells was observed in vitro and in vivo. By contrast, knockdown of the ID2 gene diminished invasion and LNM of HNSCC cells. In addition, the ID2 expression level increased the expression level of matrix metalloproteinase 1 (MMP1), a molecule downstream to ID2. Furthermore, silencing of MMP1 in ID2-overexpressed HNSCC cells rescued the elevated invasion and LNM capabilities of these cells, suggesting that ID2 enhances invasion and LNM partly via MMP1 activation. In the invasion and LNM of HNSCC, ID2 plays an important role by modulating MMP1 expression, suggesting ID2-MMP1 axis to be a novel alternative therapeutic target for invasion and LNM of HNSCC.

Dissecting the impact of regional identity and the oncogenic role of human-specific NOTCH2NL in an hESC model of H3.3G34R-mutant glioma

H3.3G34R-mutant gliomas are lethal tumors of the cerebral hemispheres with unknown mechanisms of regional specificity and tumorigenicity. We developed a human embryonic stem cell (hESC)-based model of H3.3G34R-mutant glioma that recapitulates the key features of the tumors with cell-type specificity to forebrain interneuronal progenitors but not hindbrain precursors. We show that H3.3G34R, ATRX, and TP53 mutations cooperatively impact alternative RNA splicing events, particularly suppression of intron retention. This leads to increased expression of components of the Notch pathway, notably NOTCH2NL, a human-specific gene family. We also uncover a parallel mechanism of enhanced NOTCH2NL expression via genomic amplification of its locus in some H3.3G34R-mutant tumors. These findings demonstrate a novel mechanism whereby evolutionary pathways that lead to larger brain size in humans are co-opted to drive tumor growth.

Regulation of ID4 In Vivo for Efficient Magnetothermal Therapy of Breast Cancer

AbstractMagnetic nanoparticles (MNPs) mediated magnetothermal therapy (MTT) has been proven as a promising modality for tumor treatment. However, the low therapeutic efficacy presents a major obstacle to its mass adoption. The Authors proposed to increase the therapeutic efficacy of MTT through the manipulation of magnetothermal‐related gene expression. ID4, a member of inhibitors of differentiation (ID) protein family, is identified to be downregulated in intracellular MTT (in‐MTT)‐treated MCF‐7 breast cancer cells according to the whole transcriptome sequencing. Interestingly, this occurrence is not observed in the exogenous MTT‐treated cells. More importantly, ID4‐silenced tumor cells show significantly decreased cell viability after in‐MTT, whereas ID4‐overexpressed cells exhibit more resistance to the MTT. This effect is also observed in other malignant breast cells derived from the human mammary gland. Furthermore, ID4 siRNA‐loaded superparamagnetic iron oxide nanoparticles are developed and delivered them to breast cancer xenografted nude mice. It is found that the synergistic combination of ID4 gene silencing and in‐MTT greatly inhibited the tumor growth and the efficacy of tumor volume inhibition is &gt; 98%. This study, for the first time, provides the possibility of the sensitization of breast cancer cells for efficient MTT via molecular manipulation of cell signaling.

Study on the Mechanism of Targeted Poly(lactic-coglycolic acid) Nano-Delivery Carriers in the Treatment of Hemangiomas.

Hemangiomas, also called infantile hemangiomas (IH), are the most common congenital benign vascular tumors in infants and young children. At present, there are many treatment methods for proliferative hemangiomas, which have different effects and lack predictability. Propranolol has gradually replaced glucocorticoids as the first-line treatment for infants and young children with hemangiomas. However, premature discontinuation is prone to relapse, and the efficacy and safety of medication need to be further studied and determined. The exact pathogenesis of hemangiomas is still unclear. Therefore, in this study, poly(lactic-co-glycolic acid) (PLGA) nanoparticles were used as drug delivery carriers, propranolol was encapsulated, and PLGA-propranolol (PLGA-PP) nanodelivery preparations were prepared and targeted. Anisotropy and pharmacokinetics were preliminary studied. At the same time, after the treatment of HemECs cells with PLGA-PP in gradient concentration in vitro, CCK-8 method was used to detect the cell proliferation, and Anyixin-V/PI double staining method was used to detect the apoptosis rate of cells. The effect of PLGA-PP nano-delivery vector on hemangioma was studied by western blot method to detect the expression level of Id-1 protein in HemECs. The results showed that after PLGA-PP treated HemECs for 24 h, PLGA-PP significantly inhibited HeECs proliferation and promoted their apoptosis, and the intracellular Id-1 protein expression was also reduced. Therefore, this study believes that the mechanism of PLGA-PP nano-targeted delivery preparations in the treatment of hemangiomas is achieved by down-regulating the Id-1 gene, thereby inhibiting the colonization of HemECs and promoting its apoptosis effect.