Id Genes Research Articles

LncRNA MYOSLID contributes to PH via targeting BMPR2 signaling in pulmonary artery smooth muscle cell.

The pathogenesis and vascular remodeling during pulmonary hypertension (pH) have been associated with dysregulation of bone morphogenetic protein receptor type 2 (BMPR2) and transforming growth factor-β (TGF-β) signaling in pulmonary artery smooth muscle cells (PASMCs). Evidence suggests that the human-specific lncRNA MYOSLID is a transcriptional target of the TGF-β/SMAD pathway. In this study, we investigated the involvement of MYOSLID in the pathogenesis of PH. Lung tissues from PH patients and rat PH models were analyzed to assess clinical relevance. RNA-Seq was performed to identify target genes. Pulmonary artery smooth muscle cells (PASMCs) were used to evaluate function and underlying mechanisms. RNA-Seq analysis of PASMCs stimulated by TGF-β1 revealed significantly dysregulated lncRNAs. MYOSLID expression was markedly elevated in lung tissues from PH patients and in PASMCs stimulated with TGF-β1. Mechanistically, loss of MYOSLID inhibited the TGF-β pathway by reducing SMAD2/3 pHosphorylation and activated the BMPR2 pathway by enhancing SMAD1/5/9 phosphorylation and increasing ID genes expression in PASMCs. DAZAP2, a target gene of MYOSLID, functions as an inhibitor of BMPR2 signaling. Moreover, DAZAP2 expression was significantly elevated in lung tissues from PH patients and rat PH models. Functionally, knockdown of MYOSLID and DAZAP2 reduced proliferation, migration, and apoptosis resistance in PASMCs. The activation of the MYOSLID-DAZAP2-BMPR2 axis contributes to pulmonary vascular remodeling, and targeting MYOSLID and DAZAP2 may represent novel therapeutic strategies for PH treatment.

Gene regulatory mechanisms of T cell exhaustion in diffuse large B cell lymphoma based on single-cell transcriptome data

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous and aggressive B cell malignancy that accounts for about 30 % of non-Hodgkin lymphomas. The current standard treatment for DLBCL is rituximab plus chemotherapy, but many patients are refractory or relapse, indicating the need for improved understanding of its molecular pathology. T cell exhaustion is a state of dysfunction or impairment that occurs in chronic infections or cancers, and is associated with poor prognosis in DLBCL. However, the molecular mechanisms of T cell exhaustion in DLBCL are poorly understood. In this study, we performed a comprehensive analysis of T cell exhaustion in DLBCL using public single-cell transcriptome data. We identified different subtypes of T cells and characterized their gene expression features. We found that DLBCL had a significantly higher proportion of exhausted T cells than normal tonsil, and that exhausted T cells had distinct gene expression signatures from non-exhausted T cells. These signatures included genes related to inhibitory receptors, cytokines, transcription factors and metabolic enzymes. We also found that ID3 gene was significantly upregulated in exhausted T cells in DLBCL, which may play a key role in T cell exhaustion. We constructed a protein-protein interaction network, identifying major hub proteins involved in T cell exhaustion or migration. We also performed KEGG and GO enrichment analysis for the differentially expressed genes between exhausted and non-exhausted T cells, and found important signaling pathways related to T cell exhaustion in DLBCL. Our results provide new insights into the molecular mechanisms underlying T cell exhaustion and offer novel therapeutic targets for this complex disease.

Culture and amplification-free nanopore sequencing for rapid detection of pathogens and antimicrobial resistance genes from urine.

Urinary Tract Infections (UTIs) are among the most prevalent infections globally. Every year, approximately 150million people are diagnosed with UTIs worldwide. The current state-of-the-art diagnostic methods are culture-based and have a turnaround time of 2-4 days for pathogen identification and susceptibility testing. This study first establishes an optical density culture-based method for spiking healthy urine samples with the six most prevalent uropathogens. Urine samples were spiked at clinically significant concentrations of 103-105 CFU/ml. Three DNA extraction kits (BioStic, PowerFood, and Blood and Tissue) were investigated based on the DNA yield, average processing time, elution volume, and the average cost incurred per extraction. After DNA extraction, the samples were sequenced using MinION and Flongle flow cells. The Blood and Tissue kit outperformed the other kits based on the investigated parameters. Using nanopore sequencing, all the pathogens and corresponding genes were only identified at a spike concentration of 105 CFU/ml, achieved after 10min and 3 hours of sequencing, respectively. However, some pathogens and antibiotic-resistance genes (ARG) could be identified from spikes at 103 colony formation units (CFU/mL). The overall turnaround time was five hours, from sample preparation to sequencing-based identification of pathogen ID and antimicrobial resistance genes. This study demonstrates excellent promise in reducing the time required for informed antibiotic administration from 48 to 72h to five hours, thereby reducing the number of empirical doses and increasing the chance of saving lives.

Strong Activation of ID1, ID2, and ID3 Genes Is Coupled with the Formation of Vasculogenic Mimicry Phenotype in Melanoma Cells.

Gene expression patterns are very sensitive to external influences and are reflected in phenotypic changes. It was previously described that transferring melanoma cells from a plastic surface to Matrigel led to formation of de novo vascular networks-vasculogenic mimicry-that are characteristic to a stemness phenotype in aggressive tumors. Up to now there was no detailed data about the gene signature accompanying this process. Here, we show that this transfer shortly led to extremely strong epigenetic changes in gene expression in the melanoma cells. We observed that on Matrigel numerous genes controlling ribosome biogenesis were upregulated. However, most of the activated genes were inhibitors of the differentiation genes (ID1, ID2, and ID3). At the same time, the genes that control differentiation were downregulated. Both the upregulated and the downregulated genes are simultaneously targeted by different transcription factors shaping sets of co-expressed genes. The specific group of downregulated genes shaping contacts with rDNA genes are also associated with the H3K27me3 mark and with numerous lincRNAs and miRNAs. We conclude that the stemness phenotype of melanoma cells is due to the downregulation of developmental genes and formation of dedifferentiated cells.

ID2-ETS2 axis regulates the transcriptional acquisition of pro-tumoral microglia phenotype in glioma

Glioblastoma is a highly aggressive brain tumour that creates an immunosuppressive microenvironment. Microglia, the brain’s resident immune cells, play a crucial role in this environment. Glioblastoma cells can reprogramme microglia to create a supportive niche that promotes tumour growth. However, the mechanisms controlling the acquisition of a transcriptome associated with a tumour-supportive microglial reactive state are not fully understood. In this study, we investigated changes in the transcriptional profile of BV2 microglia exposed to C6 glioma cells. RNA-sequencing analysis revealed a significant upregulation of microglial inhibitor of DNA binding 1 (Id1) and Id2, helix-loop-helix negative transcription regulatory factors. The concomitant regulation of microglial ETS proto-oncogene 2, transcription factor (ETS2)-target genes, i.e., Dusp6, Fli1, Jun, Hmox1, and Stab1, led us to hypothesize that ETS2 could be regulated by ID proteins. In fact, ID2-ETS2 protein interactions increased in microglia exposed to glioma cells. In addition, perturbation of the ID2-ETS2 transcriptional axis influenced the acquisition of a microglial tumour-supportive phenotype. ID2 and ETS2 genes were found to be expressed by the tumour-associated microglia isolated from human glioblastoma tumour biopsies. Furthermore, ID2 and ETS2 gene expressions exhibited inverse prognostic values in patients with glioma in cohorts from The Cancer Genome Atlas. Collectively, our findings indicate that the regulation of ETS2 by ID2 plays a role in the transcriptional regulation of microglia in response to stimuli originating from glioblastoma cells, information that could lead to developing therapeutic strategies to manipulate microglial tumour-trophic functions.

Assessment of ID family proteins expression in colorectal cancer of Iraqi patients.

Colorectal cancer (CRC) is the second most deathly worldwide and third most common cancer, CRC is a very heterogeneous disease where tumors can form by both environmental and genetic risk factors and includes epigenetic and genetic alternations. Inhibitors of DNA binding proteins (ID) are a class of helix-loop-helix transcription regulatory factors; these proteins are considered a family of four highly preserved transcriptional regulators (ID1-4), shown to play significant roles in many processes that are associated with tumor development. ID family plays as negatively dominant antagonists of other essential HLH proteins, concluding the creation of non-functional heterodimers and regulation of the transcription process. 120 Fresh tissue and blood samples Forty (40) samples of fresh tissue and blood were collected from patients diagnosed with CRC, twenty (20) samples were collected from a patient diagnosed as healthy. The (qRT-PCR) method is a sensitive technique for the quantifying of steady-state mRNA levels that used to evaluation the expression levels of ID (1-4) gene. The findings indicate downregulation in ID1 in tissue with a highly significant change between patients and control groups, where upregulation in the ID1 gene is shown in blood samples.ID2 gene also demonstrated high significant change where show upregulation in tissue and downregulation in blood sample. ID3 and ID4 genes show downregulation in tissue and blood samples with a significant change in ID3 blood samples between patient and blood groups. Because of the regulation function of the ID family in many processes, the up or down regulation of IDs genes in tumors Proves how important its tumor development, and therefore those proteins can be used as an indicator for CRC.

The prognostic impact of pathogenic stromal cell-associated genes in lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) stands as the most prevalent subtype among lung cancers. Interactions between stromal and cancer cells influence tumor growth, invasion, and metastasis. However, the regulatory mechanisms of stromal cells in the lung adenocarcinoma tumor microenvironment remain unclear. This study seeks to elucidate the regulatory connections among critical pathogenic genes and their associated expression variations within distinct stromal cell subtypes. MethodAnalysis and investigation were conducted on a total of 114,019 single-cell RNA data and 346 The Cancer Genome Atlas (TCGA) LUAD-related samples using bioinformatics and statistical algorithms. Differential gene expression analysis was performed for tumor samples and controls, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Differential genes between stromal cells and other cell clusters were identified and intersected with the differential genes from TCGA. We employed a combination of LASSO regression and multivariable Cox regression to identify the ultimate set of pathogenic gene. Survival models were trained to predict the relationship between patient survival and these pathogenic genes. Analysis of transcription factor (TF) cell specificity and pseudotime trajectories within stromal cell subpopulations revealed that vascular endothelial cells (ECs) and matrix cancer-associated fibroblasts (CAFs) are key in regulation of the prognosis-associated genes CAV2, COL1A1, TIMP1, ETS2, AKAP12, ID1 and COL1A2. ResultsSeven pathogenic genes associated with LUAD in stromal cells were identified and used to develop a survival model. High expression of these genes is linked to a greater risk of poor survival. Stromal cells were categorized into eight subtypes and one unannotated cluster. Mesothelial cells, vascular endothelial cells (ECs), and matrix cancer-associated fibroblasts (CAFs) showed cell-specific regulation of the pathogenic genes. ConclusionsThe seven disease-causing genes in vascular ECs and matrix CAFs can be used to detect the survival status of LUAD patients, providing new directions for future targeted drug design.

E3 ubiquitin ligase RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα

IL-3/STAT5 signaling pathway is crucial for the development and activation of immune cells, contributing to the cellular response to infections and inflammatory stimuli. Dysregulation of the IL-3/STAT5 signaling have been associated with inflammatory and autoimmune diseases characterized by inflammatory cell infiltration and organ damage. IL-3 receptor α (IL-3Rα) specifically binds to IL-3 and initiates intracellular signaling, resulting in the phosphorylation of STAT5. However, the regulatory mechanisms of IL-3Rα remain unclear. Here, we identified the E3 ubiquitin ligase RNF128 as a negative regulator of IL-3/STAT5 signaling by targeting IL-3Rα for lysosomal degradation. RNF128 was shown to selectively bind to IL-3Rα, without interacting with the common beta chain IL-3Rβ, which shares the subunit with GM-CSF. The deficiency of Rnf128 had no effect on GM-CSF-induced phosphorylation of Stat5, but it resulted in heightened Il-3-triggered activation of Stat5 and increased transcription of the Id1, Pim1, and Cd69 genes. Furthermore, we found that RNF128 promoted the K27-linked polyubiquitination of IL-3Rα in a ligase activity-dependent manner, ultimately facilitating its degradation through the lysosomal pathway. RNF128 inhibited the activation and chemotaxis of macrophages in response to LPS stimulation, thereby attenuating excessive inflammatory responses. Collectively, these results reveal that RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα. This study uncovers E3 ubiquitin ligase RNF128 as a novel regulator of the IL-3/STAT5 signaling pathway, providing potential molecular targets for the treatment of inflammatory diseases.

The H3K4 demethylase JMJ1 is required for proper timing of flowering in Brachypodium distachyon.

Flowering is a key developmental transition in the plant life cycle. In temperate climates, flowering often occurs in response to the perception of seasonal cues such as changes in day-length and temperature. However, the mechanisms that have evolved to control the timing of flowering in temperate grasses are not fully understood. We identified a Brachypodium distachyon mutant whose flowering is delayed under inductive long-day conditions due to a mutation in the JMJ1 gene, which encodes a Jumonji domain-containing protein. JMJ1 is a histone demethylase that mainly demethylates H3K4me2 and H3K4me3 in vitro and in vivo. Analysis of the genome-wide distribution of H3K4me1, H3K4me2, and H3K4me3 in wild-type plants by chromatin immunoprecipitation and sequencing combined with RNA sequencing revealed that H3K4m1 and H3K4me3 are positively associated with gene transcript levels, whereas H3K4me2 is negatively correlated with transcript levels. Furthermore, JMJ1 directly binds to the chromatin of the flowering regulator genes VRN1 and ID1 and affects their transcription by modifying their H3K4me2 and H3K4me3 levels. Genetic analyses indicated that JMJ1 promotes flowering by activating VRN1 expression. Our study reveals a role for JMJ1-mediated chromatin modification in the proper timing of flowering in B. distachyon.

Prognostic impact of inhibitors of DNA binding proteins1 and inhibitors of DNA binding proteins4 genes expression on adult Egyptian patients with acute myeloid leukemia

Background Acute myeloid leukemia (AML) pathogenesis and treatment are currently being better understood at an accelerated rate. Determining genetic and epigenetic changes that can identify patients who are at risk of poor outcomes is therefore desired to optimize treatment options. Many solid tumors have been reported to overexpress Inhibitors of DNA binding proteins (ID1), but few research has looked at the clinical significance of ID1 expression in AML. Additionally, little research has been focused on the direct role of ID4 in myeloid malignancies, as well as its expression and methylation patterns. The aim of the current study was to assess ID1 and ID4 gene expression in bone marrow (BM) aspiration specimens of 91 AML patients, compared with 14 control donors of bone marrow transplantation (BMT), using real-time polymerase chain reaction (RT-PCR). Data were correlated with patients’ clinicopathological features, response to treatment, disease-free survival (DFS), and overall survival (OS) rates. Results ID1 transcript level was significantly increased in AML bone marrow samples compared with normal controls (P = 0.002), while ID4 gene expression showed a nonsignificant difference (P = 0.717). In addition, there was a significant increase in ID1 gene expression in fms-like tyrosine kinase 3 (FLT3) mutant group than fms-like tyrosine kinase 3 wild group (P = 0.010). The total leukocytic count (TLC) was significantly higher in patients with high ID1 expression (P = 0.038) and patients with undetected ID4 expression (P = 0.025). No significant associations were detected between ID1 and ID4 expression levels and patients’ clinicopathological characteristics and OS rates. Conclusion In contrast to ID4, overexpressed ID1 can be adopted as a genetic biomarker for diagnosing AML. ID1 and ID4 expressions did not affect the patients’ OS or DFS.

Silibinin is a suppressor of the metastasis-promoting transcription factor ID3

BackgroundID3 (inhibitor of DNA binding/differentiation-3) is a transcription factor that enables metastasis by promoting stem cell-like properties in endothelial and tumor cells. The milk thistle flavonolignan silibinin is a phytochemical with anti-metastatic potential through largely unknown mechanisms. Hypothesis/purposeWe have mechanistically investigated the ability of silibinin to inhibit the aberrant activation of ID3 in brain endothelium and non-small cell lung cancer (NSCLC) models. MethodsBioinformatic analyses were performed to investigate the co-expression correlation between ID3 and bone morphogenic protein (BMP) ligands/BMP receptors (BMPRs) genes in NSCLC patient datasets. ID3 expression was assessed by immunoblotting and qRT-PCR. Luciferase reporter assays were used to evaluate the gene sequences targeted by silibinin to regulate ID3 transcription. In silico computational modeling and LanthaScreen TR-FRET kinase assays were used to characterize and validate the BMPR inhibitory activity of silibinin. Tumor tissues from NSCLC xenograft models treated with oral silibinin were used to evaluate the in vivo anti-ID3 effects of silibinin. ResultsAnalysis of lung cancer patient datasets revealed a top-ranked positive association of ID3 with the BMP9 endothelial receptor ACVRL1/ALK1 and the BMP ligand BMP6. Silibinin treatment blocked the BMP9-induced activation of the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Constitutive, acquired, and adaptive expression of ID3 in NSCLC cells were all significantly downregulated in response to silibinin. Silibinin blocked ID3 transcription via BMP-responsive elements in ID3 gene enhancers. Silibinin inhibited the kinase activities of BMPRs in the micromolar range, with the lower IC50 values occurring against ACVRL1/ALK1 and BMPR2. In an in vivo NSCLC xenograft model, tumoral overexpression of ID3 was completely suppressed by systematically achievable oral doses of silibinin. ConclusionsID3 is a largely undruggable metastasis-promoting transcription factor. Silibinin is a novel suppressor of ID3 that may be explored as a novel therapeutic approach to interfere with the metastatic dissemination capacity of NSCLC.

The RNA-binding protein Cpeb4 regulates splicing of the Id2 gene in osteoclast differentiation.

Cytoplasmic polyadenylation element-binding protein 4 (Cpeb4) is an RNA-binding protein that regulates posttranscriptional regulation, such as regulation of messengerRNA stability and translation. In the previous study, we reported that Cpeb4 localizes to nuclear bodies upon induction of osteoclast differentiation by RANKL. However, the mechanisms of the localization of Cpeb4 and osteoclastogenesis by Cpeb4 remain unknown. Here, we show that Cpeb4 localizes to the nuclear bodies by its RNA-binding ability and partially regulates normal splicing during osteoclast differentiation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisanalysis with Phos-tag® revealed that the phosphorylation levels of Cpeb4 were already high in the RAW264.7 cells and were not altered by RANKL treatment. Immunofluorescence showed that exogenous Cpeb4 in HEK293T cells without RANKL stimulation localized to the same foci as shown in RANKL-stimulated RAW264.7 cells. Furthermore, when nuclear export was inhibited by leptomycin B treatment, Cpeb4 accumulated throughout the nucleus. Importantly, RNA recognition motif (RRM) 7 of Cpeb4 was essential for the localization. In contrast, the intrinsically disordered region, RRM1, and zinc finger domain CEBP_ZZ were not necessary for the localization. The mechanistic study showed that Cpeb4 co-localized and interacted with the splicing factors serine/arginine-rich splicing factor5 (SRSF5) and SRSF6, suggesting that Cpeb4 may be involved in the splicing reaction. RNA-sequencing analysis revealed that the expression of genes related to cell proliferation processes, such as mitotic cell cycle and regulation of cell cycle processes, was elevated in osteoclasts depleted of Cpeb4. Interestingly, the splicing pattern of the inhibitor of DNA binding 2 (Id2) gene, which suppresses osteoclast differentiation, was altered by the depletion of Cpeb4. These results provide new insight into the role of Cpeb4 as a player of normal splicing of Id2 in osteoclast differentiation.

Functional Enrichment Analysis of Tumor Microenvironment-Driven Molecular Alterations That Facilitate Epithelial-to-Mesenchymal Transition and Distant Metastasis.

Nowadays, hepatocellular carcinoma (HCC) is the second leading cause of cancer deaths, and identifying the effective factors in causing this disease can play an important role in its prevention and treatment. Tumors provide effective agents for invasion and metastasis to other organs by establishing appropriate communication between cancer cells and the microenvironment. Epithelial-to-mesenchymal transition (EMT) can be mentioned as one of the effective phenomena in tumor invasion and metastasis. Several factors are involved in inducing this phenomenon in the tumor microenvironment, which helps the tumor survive and migrate to other places. It can be effective to identify these factors in the use of appropriate treatment strategies and greater patient survival. This study investigated the molecular differences between tumor border cells and tumor core cells or internal tumor cells in HCC for specific EMT genes. Expression of NOTCH1, ID1, and LST1 genes showed a significant increase at the HCC tumor border. Targeting these genes can be considered as a useful therapeutic strategy to prevent distant metastasis in HCC patients.

The NRF2/ID2 Axis in Vascular Smooth Muscle Cells: Novel Insights into the Interplay between Vascular Calcification and Aging.

Vascular calcification (VC) increases with age and markedly exacerbates the risk of cardiovascular morbidity and mortality. However, effective pharmaceutical interventions are lacking and the molecular mechanisms linking aging to VC remain elusive. This study explored the role of nuclear factor erythroid 2-related factor 2 (NRF2) in age-associated VC, specifically focusing on vascular smooth muscle cell (VSMC) senescence. Using a chronologically aging mouse model, we noted a significant decline in the expression of NRF2 in the aged mice aortas, coinciding with increased VC. Administering NRF2 activators effectively reduced calcification. By establishing adenine-and vitamin D-induced VC models in VSMC-specific Nrf2 knockout (Nrf2SMCKO) mice, there was an increase in VC with increased VSMC senescence. Aortic rings and primary VSMCs from Nrf2SMCKO mice also showed increased VC under high-phosphate conditions. Furthermore, Nrf2 overexpression inhibited VSMC calcification with decreased VSMC senescence and an osteogenic phenotype, whereas Nrf2 silencing aggravated calcification. Transcriptome RNA-seq analysis of the aortas from Nrf2SMCKO and control mice revealed that inhibitor of DNA binding 2 (Id2) is a core downstream gene of NRF2. Id2 overexpression alleviated NRF2 knockdown-induced VC and VSMC senescence, while silencing Id2 negated the protective effects of NRF2. Moreover, results of a dual luciferase reporter assay indicated that NRF2 promotes the transcriptional activity of the Id2 gene promoter region. This study emphasizes the critical role of age-related NRF2 dysfunction in the nexus between VSMC senescence and VC. The NRF2-ID2 axis in VSMCs has been proposed as a promising therapeutic target for reducing VC and mitigating age-related cardiovascular diseases.

Single-Cell Transcriptional Analysis of Lamina Propria Lymphocytes in the Jejunum Reveals Innate Lymphoid Cell-like Cells in Pigs.

Pigs are the most suitable model to study various therapeutic strategies and drugs for human beings, although knowledge about cell type-specific transcriptomes and heterogeneity is poorly available. Through single-cell RNA sequencing and flow cytometry analysis of the types in the jejunum of pigs, we found that innate lymphoid cells (ILCs) existed in the lamina propria lymphocytes (LPLs) of the jejunum. Then, through flow sorting of live/dead-lineage (Lin)-CD45+ cells and single-cell RNA sequencing, we found that ILCs in the porcine jejunum were mainly ILC3s, with a small number of NK cells, ILC1s, and ILC2s. ILCs coexpressed IL-7Rα, ID2, and other genes and differentially expressed RORC, GATA3, and other genes but did not express the CD3 gene. ILC3s can be divided into four subgroups, and genes such as CXCL8, CXCL2, IL-22, IL-17, and NCR2 are differentially expressed. To further detect and identify ILC3s, we verified the classification of ILCs in the porcine jejunum subgroup and the expression of related hallmark genes at the protein level by flow cytometry. For systematically characterizing ILCs in the porcine intestines, we combined our pig ILC dataset with publicly available human and mice ILC data and identified that the human and pig ILCs shared more common features than did those mouse ILCs in gene signatures and cell states. Our results showed in detail for the first time (to our knowledge) the gene expression of porcine jejunal ILCs, the subtype classification of ILCs, and the markers of various ILCs, which provide a basis for an in-depth exploration of porcine intestinal mucosal immunity.

Doxorubicin-induced modulation of TGF-β signaling cascade in mouse fibroblasts: insights into cardiotoxicity mechanisms

Doxorubicin (DOX)-induced cardiotoxicity has been widely observed, yet the specific impact on cardiac fibroblasts is not fully understood. Additionally, the modulation of the transforming growth factor beta (TGF-β) signaling pathway by DOX remains to be fully elucidated. This study investigated DOX’s ability to modulate the expression of genes and proteins involved in the TGF-β signaling cascade in mouse fibroblasts from two sources by assessing the impact of DOX treatment on TGF-β inducible expression of pivotal genes and proteins within fibroblasts. Mouse embryonic fibroblasts (NIH3T3) and mouse primary cardiac fibroblasts (CFs) were treated with DOX in the presence of TGF-β1 to assess changes in protein levels by western blot and changes in mRNA levels by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Our results revealed a dose-dependent reduction in cellular communication network factor 2 (CCN2) protein levels upon DOX treatment in both NIH3T3 and CFs, suggesting an antifibrotic activity by DOX in these fibroblasts. However, DOX only inhibited the TGF-β1 induced expression of COL1 in NIH3T3 cells but not in CFs. In addition, we observed that DOX treatment reduced the expression of BMP1 in NIH3T3 but not primary cardiac fibroblasts. No significant changes in SMAD2 protein expression and phosphorylation in either cells were observed after DOX treatment. Finally, DOX inhibited the expression of Atf4 gene and increased the expression of Cdkn1a, Id1, Id2, Runx1, Tgfb1, Inhba, Thbs1, Bmp1, and Stat1 genes in NIH3T3 cells but not CFs, indicating the potential for cell-specific responses to DOX and its modulation of the TGF-β signaling pathway.

Changes in Expression in BMP2 and Two Closely Related Genes in Guinea Pig Retinal Pigment Epithelium during Induction and Recovery from Myopia.

We previously reported differential gene expression of the bone morphogenetic protein 2 (Bmp2) in guinea pig retinal pigment epithelium (RPE) after 1 day of hyperopic defocus, imposed with a negative contact lens (CLs). The study reported here sought to obtain insights into the temporal profiles of gene expression changes in Bmp2, as well as those of two closely related genes, the inhibitor of DNA binding 3 (Id3) and Noggin (Nog), both during myopia induction and when the CL treatment was terminated to allow recovery from induced myopia. To induce myopia, 2-week-old pigmented guinea pigs (New Zealand strain, n = 8) wore monocular -10 diopter (D) rigid gas-permeable (RGP) CLs for one week, while the other eye served as a control. Ocular measurements were made at baseline, 3 days, and 7 days after the initiation of CL wear, with treatment then being terminated and additional measurements being made after a further 3 days, 1 week, and 2 weeks. Spherical equivalent refractive errors (SERs), axial length (AL), choroidal thickness (ChT), and scleral thickness (ScT) data were collected using retinoscopy, optical biometry (Lenstar), and spectral domain optical coherence tomography (SD-OCT), respectively. RPE samples were collected from both eyes of the guinea pigs after either 1 day or 1 week of CL wear or 1 day or 2 weeks after its termination, and RNA was subsequently isolated and subjected to quantitative real-time PCR (qRT-PCR) analyses, targeting the Bmp2, Id3, and Nog genes. Mean interocular differences (treated-control) in AL and SER were significantly different from baseline after 3 and 7 days of CL wear, consistent with induced myopia (p < 0.001 for all cases). Termination of CL wear resulted in the normalization (i.e., recovery) of the ALs and SERs of the treated eyes within 7 days, and the earlier significant ChT thinning with CL wear (p = 0004, day 7) was replaced by rapid thickening, which remained significant on day 7 (p = 0.009) but had normalized by day 14. The ChT changes were much smaller in magnitude than the AL changes in both phases. Interocular differences in the ScT showed no significant changes. The Bmp2 and Id3 genes were both significantly downregulated with CL wear, after 1 day (p = 0.012 and 0.016) and 7 days (p = 0.002 and 0.005), while Bmp2 gene expression increased and Nog gene expression decreased after the termination of CL wear, albeit transiently, which was significant on 1 day (p = 0.004 and 0.04) but not 2 weeks later. No change in Id3 gene expression was observed over the latter period. Conclusions: The above patterns of myopia induction and recovery validate this negative RGP-CL model as an alternative to traditional spectacle lens models for guinea pigs. The defocus-driven, sign-dependent changes in the expression of the Bmp2 gene in guinea pig RPE are consistent with observations in chicks and demonstrate the important role of BMP2 in eye growth regulation.

Inhibitor of DNA Binding Protein 3 (ID3) and Nuclear Respiratory Factor 1 (NRF1) Mediated Transcriptional Gene Signatures are Associated with the Severity of Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is a degenerative vasculopathy. We have previously shown that transcription regulating proteins- inhibitor of DNA binding protein 3 (ID3) and the nuclear respiratory factor 1 (NRF1) contribute to vascular dysregulation. In this study, we have identified sex specific ID3 and NRF1-mediated gene networks in CAA patients diagnosed with Alzheimer's Disease (AD). High expression of ID3 mRNA coupled with low NRF1 mRNA levels was observed in the temporal cortex of men and women CAA patients. Low NRF1 mRNA expression in the temporal cortex was found in men with severe CAA. High ID3 expression was found in women with the genetic risk factor APOE4. Low NRF1 expression was also associated with APOE4 in women with CAA. Genome wide transcriptional activity of both ID3 and NRF1 paralleled their mRNA expression levels. Sex specific differences in transcriptional gene signatures of both ID3 and NRF1 were observed. These findings were further corroborated by Bayesian machine learning and the GeNIe simulation models. Dynamic machine learning using a Monte Carlo Markov Chain (MCMC) gene ordering approach revealed that ID3 was associated with disease severity in women. NRF1 was associated with CAA and severity of this disease in men. These findings suggest that aberrant ID3 and NRF1 activity presumably plays a major role in the pathogenesis and severity of CAA. Further analyses of ID3- and NRF1-regulated molecular drivers of CAA may provide new targets for personalized medicine and/or prevention strategies against CAA.

Id2 GABAergic interneurons comprise a neglected fourth major group of cortical inhibitory cells.

Cortical GABAergic interneurons (INs) represent a diverse population of mainly locally projecting cells that provide specialized forms of inhibition to pyramidal neurons and other INs. Most recent work on INs has focused on subtypes distinguished by expression of Parvalbumin (PV), Somatostatin (SST), or Vasoactive Intestinal Peptide (VIP). However, a fourth group that includes neurogliaform cells (NGFCs) has been less well characterized due to a lack of genetic tools. Here, we show that these INs can be accessed experimentally using intersectional genetics with the gene Id2. We find that outside of layer 1 (L1), the majority of Id2 INs are NGFCs that express high levels of neuropeptide Y (NPY) and exhibit a late-spiking firing pattern, with extensive local connectivity. While much sparser, non-NGFC Id2 INs had more variable properties, with most cells corresponding to a diverse group of INs that strongly expresses the neuropeptide CCK. In vivo, using silicon probe recordings, we observed several distinguishing aspects of NGFC activity, including a strong rebound in activity immediately following the cortical down state during NREM sleep. Our study provides insights into IN diversity and NGFC distribution and properties, and outlines an intersectional genetics approach for further study of this underappreciated group of INs.

Highly drug resistant clone of Salmonella Kentucky ST198 in clinical infections and poultry in Zimbabwe

A highly multidrug-resistant strain of Salmonella enterica serotype Kentucky (S. Kentucky) of sequence type (ST)198 emerged in North Africa and has since spread widely. To investigate the genetic diversity and phylogenetic relationship of S. Kentucky in Zimbabwe and identify potential sources of infection, the whole-genome sequence of 37 S. Kentucky strains isolated from human clinical infections and from poultry farms between 2017 and 2020 was determined. Of 37 S. Kentucky isolates, 36 were ST198 and one was ST152. All ST198 isolates had between six and fifteen antimicrobial resistance (AMR) genes, and 92% carried at least ten AMRs. All ST198 isolates harbored the Salmonella genomic island K-Israel variant (SGI1-KIV) integrated into the chromosome with aac(3)-ld, aac(6)-laa, aadA7, blaTEM-1, sul1, and tetA genes, with occasional sporadic loss of one or more genes noted from five isolates. All ST198 isolates also had mutations in the quinolone resistance-determining region of the gyrA and parC genes. The blaCTX-M-14.1 and fosA3 genes were present in 92% of the ST198 isolates, conferring resistance to extended-spectrum cephalosporins and fosfomycin, respectively, were present on an IncHI2 plasmid with the aadA2b, aadA1, aph(3’)-Ib, aph(6’)-Id, cmlA1 and sul3 AMR genes. S. Kentucky ST198 isolates from Zimbabwe formed a closely related phylogenetic clade that emerged from a previously reported global epidemic population. The close genetic relationship and population structure of the human clinical and poultry isolates of ST198 in Zimbabwe are consistent with poultry being an important source of highly resistant strains of S. Kentucky in Zimbabwe.