Effect of hyperglycemia on tbx5a and nppa gene expression and its correlation to structural and functional changes in developing zebrafish heart.

The objective of the current study is to analyze the effects of gestational diabetes on structural and functional changes in correlation with these two essential regulators of developing hearts in vivo using zebrafish embryos. We employed fertilized zebrafish embryos exposed to a hyperglycemic condition of 25 mM glucose for 96 h postfertilization. The embryos were subjected to various structural and functional analyses in a time-course manner. The data showed that exposure to high glucose significantly affected the embryo's size, heart length, heart rate, and looping of the heart compared to the control. Further, we observed an increased incidence of ventricular standstill and valvular regurgitation with a marked reduction of peripheral blood flow in the high glucose-exposed group compared to the control. In addition, the histological data showed that the high-glucose exposure markedly reduced the thickness of the wall and the number of cardiomyocytes in both atrium and ventricles. We also observed striking alterations in the pericardium like edema, increase in diameter with thinning of the wall compared to the control group. Interestingly, the expression of tbx5a and nppa was increased in the early development and found to be repressed in the later stage of development in the hyperglycemic group compared to the control. In conclusion, the developing heart is more susceptible to hyperglycemia in the womb, thereby showing various developmental defects possibly by altering the expression of crucial gene regulators such as tbx5a and nppa.

Dietary zinc supplementation rescues fear-based learning and synaptic function in the Tbr1+/- mouse model of autism spectrum disorders.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by a dyad of behavioural symptoms-social and communication deficits and repetitive behaviours. Multiple aetiological genetic and environmental factors have been identified as causing or increasing the likelihood of ASD, including serum zinc deficiency. Our previous studies revealed that dietary zinc supplementation can normalise impaired social behaviours, excessive grooming, and heightened anxiety in a Shank3 mouse model of ASD, as well as the amelioration of synapse dysfunction. Here, we have examined the efficacy and breadth of dietary zinc supplementation as an effective therapeutic strategy utilising a non-Shank-related mouse model of ASD-mice with Tbr1 haploinsufficiency. METHODS: We performed behavioural assays, amygdalar slice whole-cell patch-clamp electrophysiology, and immunohistochemistry to characterise the synaptic mechanisms underlying the ASD-associated behavioural deficits observed in Tbr1+/- mice and the therapeutic potential of dietary zinc supplementation. Two-way analysis of variance (ANOVA) with Sídák's post hoc test and one-way ANOVA with Tukey's post hoc multiple comparisons were performed for statistical analysis. RESULTS: Our data show that dietary zinc supplementation prevents impairments in auditory fear memory and social interaction, but not social novelty, in the Tbr1+/- mice. Tbr1 haploinsufficiency did not induce excessive grooming nor elevate anxiety in mice. At the synaptic level, dietary zinc supplementation reversed α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) hypofunction and normalised presynaptic function at thalamic-lateral amygdala (LA) synapses that are crucial for auditory fear memory. In addition, the zinc supplemented diet significantly restored the synaptic puncta density of the GluN1 subunit essential for functional NMDARs as well as SHANK3 expression in both the basal and lateral amygdala (BLA) of Tbr1+/- mice. LIMITATIONS: The therapeutic effect of dietary zinc supplementation observed in rodent models may not reproduce the same effects in human patients. The effect of dietary zinc supplementation on synaptic function in other brain structures affected by Tbr1 haploinsufficiency including olfactory bulb and anterior commissure will also need to be examined. CONCLUSIONS: Our data further the understanding of the molecular mechanisms underlying the effect of dietary zinc supplementation and verify the efficacy and breadth of its application as a potential treatment strategy for ASD.

Phase 1 open-label trial of intravenous administration of MVA-BN-brachyury-TRICOM vaccine in patients with advanced cancer.

BACKGROUND: MVA-BN-brachyury-TRICOM is a recombinant vector-based therapeutic cancer vaccine designed to induce an immune response against brachyury. Brachyury, a transcription factor overexpressed in advanced cancers, has been associated with treatment resistance, epithelial-to-mesenchymal transition, and metastatic potential. MVA-BN-brachyury-TRICOM has demonstrated immunogenicity and safety in previous clinical trials of subcutaneously administered vaccine. Preclinical studies have suggested that intravenous administration of therapeutic vaccines can induce superior CD8+ T cell responses, higher levels of systemic cytokine release, and stronger natural killer cell activation and proliferation. This is the first-in-human study of the intravenous administration of MVA-BN-brachyury-TRICOM. METHODS: Between January 2020 and March 2021, 13 patients were treated on a phase 1, open-label, 3+3 design, dose-escalation study at the National Institutes of Health Clinical Center. The study population was adults with advanced solid tumors and was enriched for chordoma, a rare sarcoma of the notochord that overexpresses brachyury. Vaccine was administered intravenously at three DLs on days 1, 22, and 43. Blood samples were taken to assess drug pharmacokinetics and immune activation. Imaging was conducted at baseline, 1 month, and 3 months post-treatment. The primary endpoint was safety and tolerability as determined by the frequency of dose-limiting toxicities; a secondary endpoint was determination of the recommended phase 2 dose. RESULTS: No dose-limiting toxicities were observed and no serious adverse events were attributed to the vaccine. Vaccine-related toxicities were consistent with class profile (ie, influenza-like symptoms). Cytokine release syndrome up to grade 2 was observed with no adverse outcomes. Dose-effect trend was observed for fever, chills/rigor, and hypotension. Efficacy analysis of objective response rate per RECIST 1.1 at the end of study showed one patient with a partial response, four with stable disease, and eight with progressive disease. Three patients with stable disease experienced clinical benefit in the form of improvement in pain. Immune correlatives showed T cell activation against brachyury and other tumor-associated cascade antigens. CONCLUSIONS: Intravenous administration of MVA-BN-brachyury-TRICOM vaccine was safe and tolerable. Maximum tolerated dose was not reached. The maximum administered dose was 109 infectious units every 3 weeks for three doses. This dose was selected as the recommended phase 2 dose. TRIAL REGISTRATION NUMBER: NCT04134312.

T-box transcription factor TBX1, targeted by microRNA-6727-5p, inhibits cell growth and enhances cisplatin chemosensitivity of cervical cancer cells through AKT and MAPK pathways.

Cervical cancer (CC) is the fourth most common cancers among women worldwide. T-box transcription factor 1 (TBX1), a member of the T-box family, has anti-tumor effects in some types of cancer, but its role in CC is yet unknown. The aim of this study is to investigate the functions and underlying mechanisms of TBX1 in CC. Online database UALCAN showed that TBX1 was down-regulated in CC tissues compared with normal tissues and patients with lower TBX1 expression level had a poor prognosis. TBX1 overexpression significantly decreased the proliferation, migration, and invasion of Hela and SiHa cells. Conversely, cell apoptosis and chemosensitivity to cisplatin were promoted in TBX1-overexpressing CC cells. Moreover, up-regulation of TBX1 inhibited both AKT and MAPK signaling pathways. Furthermore, dual luciferase report assay indicated that TBX1 could directly bind to miR-6727-5p. In addition, TBX1 expression was inhibited by miR-6727-5p mimic and up-regulated by miR-6727-5p inhibitor. Knockdown of TBX1 reversed the inhibitory effect of the miR-6727-5p inhibitor on CC cells. This study demonstrates that TBX1, a target gene of miR-6727-5p, acts as a tumor suppressor in CC, indicating that TBX1 may be a new target for CC therapy.

Tbx6 induces cardiomyocyte proliferation in postnatal and adult mouse hearts.

Cardiovascular disease is a leading cause of death worldwide. Mammalian cardiomyocytes (CMs) proliferate during embryonic development, whereas they largely lose their regenerative capacity after birth. Defined factors expressed in cardiac progenitors or embryonic CMs may activate the cell cycle and induce CM proliferation in postnatal and adult hearts. Here, we report that the overexpression of Tbx6, enriched in the cardiac mesoderm (progenitor cells), induces CM proliferation in postnatal and adult mouse hearts. By screening 24 factors enriched in cardiac progenitors or embryonic CMs, we found that only Tbx6 could induce CM proliferation in primary cultured postnatal rat CMs. Intriguingly, it did not induce the proliferation of cardiac fibroblasts. We next generated a recombinant adeno-associated virus serotype 9 vector encoding Tbx6 (AAV9-Tbx6) for transduction into mouse CMs in vivo. The subcutaneous injection of AAV9-Tbx6 into neonatal mice induced CM proliferation in postnatal and adult mouse hearts. Mechanistically, Tbx6 overexpression upregulated multiple cell cycle activators including Aurkb, Mki67, Ccna1, and Ccnb2 and suppressed the tumor suppressor Rb1. Thus, Tbx6 promotes CM proliferation in postnatal and adult mouse hearts by modifying the expression of cell cycle regulators.

Intranuclear delivery of the transcription modulation domain of Tbet-improved lupus nephritis in (NZB/NZW) F1 lupus-prone mice.

Excessive expression of Tbet and IFNγ is evidence of systemic lupus erythematosus (SLE) in lupus patients. In this study, the nucleus-transducible form of Transcription Modulation Domain (TMD) of Tbet (ntTbet-TMD), which is a fusion protein between Protein Transduction Domain Hph-1 (Hph-1-PTD) and the TMD of Tbet comprising DNA binding domain and isotype-specific domain, was generated to inhibit Tbet-mediated transcription in the interactomic manner. ntTbet-TMD was effectively delivered into the nucleus of the cells and specifically inhibited Tbet-mediated transcription without influencing the differentiation of other T cell subsets and signaling events for T cell activation. The severity of nephritis was significantly reduced by ntTbet-TMD as effectively as methylprednisolone in lupus-prone mice. The number of Th1, Th2 or Th17 cells and the secretion of their cytokines substantially decreased in the spleen and kidney of lupus-prone mice by ntTbet-TMD treatment. In contrast to methylprednisolone, the marked increase of Treg cells and the secretion of their immunosuppressive cytokine were detected in the spleen of (NZB/NZW) F1 mice treated with ntTbet-TMD. Thus, ntTbet-TMD can improve nephritis in lupus-prone mice by modulating the overall proinflammatory microenvironment and rebalancing T cell subsets, leading to new immune therapeutics for Th1-mediated autoimmune diseases.

A cis-regulatory module upstream of deltaC regulated by Ntla and Tbx16 drives expression in the tailbud, presomitic mesoderm and somites.

Somites form by an iterative process from unsegmented, presomitic mesoderm (PSM). Notch pathway components, such as deltaC (dlc) have been shown to play a role in this process, while the T-box transcription factors Ntla and Tbx16 regulate somite formation upstream of this by controlling supply and movement of cells into the PSM during gastrulation and tailbud outgrowth. In this work, we report that Ntla and Tbx16 play a more explicit role in segmentation by directly regulating dlc expression. In addition we describe a cis-regulatory module (CRM) upstream of dlc that drives expression of a reporter in the tailbud, PSM and somites during somitogenesis. This CRM is bound by both Ntla and Tbx16 at a cluster of T-box binding sites, which are required in combination for activation of the CRM.

Transcriptional suppression of connexin43 by TBX18 undermines cell-cell electrical coupling in postnatal cardiomyocytes.

T-box transcription factors figure prominently in embryonic cardiac cell lineage specifications. Mesenchymal precursor cells expressing Tbx18 give rise to the heart's pacemaker, the sinoatrial node (SAN). We sought to identify targets of TBX18 transcriptional regulation in the heart by forced adenoviral overexpression in postnatal cardiomyocytes. Neonatal rat cardiomyocytes (NRCMs) transduced with GFP showed sarcolemmal, punctate Cx43 expression. In contrast, TBX18-transduced NRCMs exhibited sparse Cx43 expression. Both the transcript and protein levels of Cx43 were greatly down-regulated within 2 days of TBX18 transduction. Direct injection of TBX18 in the guinea pig heart in vivo inhibited Cx43 expression. The repressor activity of TBX18 on Cx43 was highly specific; protein levels of Cx45 and Cx40, which comprise the main gap junctions in the SAN and conduction system, were unchanged by TBX18. A reporter-based promoter assay demonstrated that TBX18 directly represses the Cx43 promoter. Phenotypically, TBX18-NRCMs exhibited slowed intercellular calcein dye transfer kinetics (421 ± 54 versus control 127 ± 43 ms). Intracellular Ca(2+) oscillations in control NRCM monolayers were highly synchronized. In contrast, TBX18 overexpression led to asynchronous Ca(2+) oscillations, demonstrating reduced cell-cell coupling. Decreased coupling led to slow electrical propagation; conduction velocity in TBX18 NRCMs slowed by more than 50% relative to control (2.9 ± 0.5 versus 14.3 ± 0.9 cm/s). Taken together, TBX18 specifically and directly represses Cx43 transcript and protein levels. Cx43 suppression leads to significant electrical uncoupling, but the preservation of other gap junction proteins supports slow action potential propagation, recapitulating a key phenotypic hallmark of the SAN.

Tbx12 regulates eye development in Xenopus embryos.

The regulation of vertebrate eye development requires the activity of many transcription factors. In this report, we demonstrate that the T-box factor Tbx12 is necessary for normal development of the retina. Tbx12 is expressed during early stages of retinal development in multiple species of vertebrate embryos. We injected mRNAs encoding wild type and mutant forms of Tbx12 into Xenopus embryos. The Tbx12 injected embryos exhibit multiple defects in eye development including reduced eye size and disruption of normal retinal laminar organization. Tbx12 appears to function as a repressor of transcription during eye development. Our results indicate that Tbx12 activity is required for the proper generation and organization of retinal cells in the vertebrate eye.

Microarray analysis of Tbx2-directed gene expression: a possible role in osteogenesis.

Tbx2 is a member of the developmentally important transcriptional regulatory T-box gene family, whose target genes have not been well characterized. In an attempt to identify genes that may be regulated by Tbx2, mouse cDNA microarrays were used to analyze differential gene expression profiles, comparing stably transfected NIH3T3 cells overexpressing Tbx2 and vector-transfected controls. Among 8734 genes, 107 genes were up-regulated by 2-fold or greater, and 66 genes were down-regulated by 2-fold or greater. Caveolin, pleiotrophin (osf-1), osteoblast-specific factor-2 (osf-2) and collagen type I alpha were among the genes upregulated in the Tbx2-overexpressing cells, whereas cadherin 3, tenascin C, and insulin-like growth factor binding protein 10/CYR61 (IBP10) were among the genes downregulated. Northern blot analysis confirmed the correlation of expression of several genes, including IBP10 and osf-2, in fibroblast NIH3T3 and rat osteosarcoma ROS17/2.8 cells differentially expressing Tbx2. In ROS17/2.8 cells transfected with antisense Tbx2, osf-2 was downregulated, whereas transfection of sense Tbx2 upregulated this gene. Interestingly, the expression of pleiotrophin (osf-1) and collagen I alpha with Tbx2 transfection showed an inverse regulatory correlation between NIH3T3 and ROS17/2.8 cells. Thus, Tbx2 can act as both a repressor and activator, and the cellular context can influence the effect on gene expression. Although the data do not address whether Tbx2 directly mediates the transcriptional effect, a number of candidate genes possess putative T-box gene regulatory elements. The results support the hypothesis that Tbx2 may be an important modulator of bone development. Further functional cluster analysis indicates that Tbx2 might also be involved in the regulation of cell cycle and cell adhesion.