Baf-mediated transcriptional regulation of teashirt is essential for the development of neural progenitor cell lineages.

Accumulating evidence hints heterochromatin anchoring to the inner nuclear membrane as an upstream regulatory process of gene expression. Given that the formation of neural progenitor cell lineages and the subsequent maintenance of postmitotic neuronal cell identity critically rely on transcriptional regulation, it seems possible that the development of neuronal cells is influenced by cell type-specific and/or context-dependent programmed regulation of heterochromatin anchoring. Here, we explored this possibility by genetically disrupting the evolutionarily conserved barrier-to-autointegration factor (Baf) in the Drosophila nervous system. Through single-cell RNA sequencing, we demonstrated that Baf knockdown induces prominent transcriptomic changes, particularly in type I neuroblasts. Among the differentially expressed genes, our genetic analyses identified teashirt (tsh), a transcription factor that interacts with beta-catenin, to be closely associated with Baf knockdown-induced phenotypes that were suppressed by the overexpression of tsh or beta-catenin. We also found that Baf and tsh colocalized in a region adjacent to heterochromatin in type I NBs. Notably, the subnuclear localization pattern remained unchanged when one of these two proteins was knocked down, indicating that both proteins contribute to the anchoring of heterochromatin to the inner nuclear membrane. Overall, this study reveals that the Baf-mediated transcriptional regulation of teashirt is a novel molecular mechanism that regulates the development of neural progenitor cell lineages.

Discovery of novel amidobenzimidazole derivatives as orally available small molecule modulators of stimulator of interferon genes for cancer immunotherapy.

Stimulator of interferon genes (STING) agonists show promise as immunomodulatory agents for cancer therapy. In this study, we report the discovery of a novel orally available STING agonist, SAP-04, that exhibits potent immunomodulatory effects for cancer therapy. By optimizing the amidobenzimidazole core with various pyridine-based heterocyclic substituents, we identified a monomeric variant that displayed more efficient STING agonistic activity than the corresponding dimer. SAP-04 efficiently induced cytokine secretion related to innate immunity by directly binding of the compound to the STING protein, followed by sequential signal transduction for the STING signaling pathway and type I interferon (IFN) responses. Further pharmacological validation in vitro and in vivo demonstrated the potential utility of SAP-04 as an immunomodulatory agent for cancer therapy in vivo. The in vivo anticancer effect was observed in a 4T1 breast tumor syngeneic mouse model through oral administration of the compound. Our findings suggest a possible strategy for developing synthetically accessible monomeric variants as orally available STING agonists.

Effect of speech-stimulus degradation on phoneme-related potential.

Auditory evoked potential (AEP) has been used to evaluate the degree of hearing and speech cognition. Because AEP generates a very small voltage relative to ambient noise, a repetitive presentation of a stimulus, such as a tone, word, or short sentence, should be employed to generate ensemble averages over trials. However, the stimulation of repetitive short words and sentences may present an unnatural situation to a subject. Phoneme-related potentials (PRPs), which are evoked-responses to typical phonemic stimuli, can be extracted from electroencephalography (EEG) data in response to a continuous storybook. In this study, we investigated the effects of spectrally degraded speech stimuli on PRPs. The EEG data in response to the spectrally degraded and natural storybooks were recorded from normal listeners, and the PRP components for 10 vowels and 12 consonants were extracted. The PRP responses to a vocoded (spectrally-degraded) storybook showed a statistically significant lower peak amplitude and were prolonged compared with those of a natural storybook. The findings in this study suggest that PRPs can be considered a potential tool to evaluate hearing and speech cognition as other AEPs. Moreover, PRPs can provide the details of phonological processing and phonemic awareness to understand poor speech intelligibility. Further investigation with the hearing impaired is required prior to clinical application.

Development of Potent Immune Modulators Targeting Stimulator of Interferon Genes Receptor.

Stimulator of interferon genes (STING) is an endoplasmic reticulum-membrane protein that plays important roles in cancer immunotherapy by activating innate immune responses. We designed and synthesized STING modulators and characterized compounds 4a and 4c that share a crucial amidobenzimidazole moiety. In vitro STING binding and cell-based activity assays demonstrated the potency and efficacy of the compounds that function as direct STING agonists by stimulating STING downstream signaling and promoting type I interferon immune responses. In vitro metabolic studies and the pharmacokinetic properties of the compounds led us to investigate their anticancer activity in an in vivo syngeneic model. Intravenous injection of compounds 4a and 4c significantly decreased tumor volume in a CT26 murine colorectal carcinoma model, and the immunological memory-derived cancer inhibition was observed in 4c-treated mouse models. The present results suggest the therapeutic potential of the compounds for cancer immunotherapy via STING-mediated immune activation.

Development of Small-Molecule STING Activators for Cancer Immunotherapy.

In cancer immunotherapy, the cyclic GMP-AMP synthase-stimulator of interferon genes (STING) pathway is an attractive target for switching the tumor immunophenotype from 'cold' to 'hot' through the activation of the type I interferon response. To develop a new chemical entity for STING activator to improve cyclic GMP-AMP (cGAMP)-induced innate immune response, we identified KAS-08 via the structural modification of DW2282, which was previously reported as an anti-cancer agent with an unknown mechanism. Further investigation revealed that direct STING binding or the enhanced phosphorylation of STING and downstream effectors were responsible for DW2282-or KAS-08-mediated STING activity. Furthermore, KAS-08 was validated as an effective STING pathway activator in vitro and in vivo. The synergistic effect of cGAMP-mediated immunity and efficient anti-cancer effects successfully demonstrated the therapeutic potential of KAS-08 for combination therapy in cancer treatment.

Block and Random Practice: A Wii Fit Dynamic Balance Training in Older Adults.

Purpose: To compare the effectiveness of blocked and random practice schedules of balance training in dynamic balance abilities of older adults using Wii Fit balance game tasks. Method: Forty-one participants who were not receiving hospice care or living in a nursing home participated. Three Wii Fit balance tasks (tasks A, B, and C) were selected for training, and one task (task D) was selected as the transfer test among the nine tasks in the Wii Fit balance game software. Scores for tasks A and D were evaluated. Completion times for tasks B and C were evaluated. Moved distance for the functional reach test (FRT), completion time for the timed up and go test (TUG), and performance score for the Tinetti performance-oriented mobility assessment (POMA) were also tested as clinical balance assessment outcomes. Results: The training significantly improved the performance outcomes of clinical balance assessments and task D. There were no significant group × time interaction effects and no significant main effects by group during the acquisition and retention periods of tasks A, B, and C. However, significant main effects by time were observed for tasks A, B, and C. Conclusions: When dynamic balance training such as the Wii Fit balance system is administered to older adults in a clinical setting, either a block or a random practice schedule can be effectively used to improve the dynamic balance skills. Wii Fit-based balance training is clinically effective for improving the dynamic balance ability.

Mitochondrial dysfunction and activation of iNOS are responsible for the palmitate-induced decrease in adiponectin synthesis in 3T3L1 adipocytes.

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress are considered the key determinants of insulin resistance. Impaired mitochondrial function in obese animals was shown to induce the ER stress response, resulting in reduced adiponectin synthesis in adipocytes. The expression of inducible nitric oxide synthase (iNOS) is increased in adipose tissues in genetic and dietary models of obesity. In this study, we examined whether activation of iNOS is responsible for palmitate-induced mitochondrial dysfunction, ER stress, and decreased adiponectin synthesis in 3T3L1 adipocytes. As expected, palmitate increased the expression levels of iNOS and ER stress response markers, and decreased mitochondrial contents. Treatment with iNOS inhibitor increased adiponectin synthesis and reversed the palmitate-induced ER stress response. However, the iNOS inhibitor did not affect the palmitate-induced decrease in mitochondrial contents. Chemicals that inhibit mitochondrial function increased iNOS expression and the ER stress response, whereas measures that increase mitochondrial biogenesis (rosiglitazone and adenoviral overexpression of nuclear respiratory factor-1) reversed them. Inhibition of mitochondrial biogenesis prevented the rosiglitazone-induced decrease in iNOS expression and increase in adiponectin synthesis. These results suggest that palmitate-induced mitochondrial dysfunction is the primary event that leads to iNOS induction, ER stress, and decreased adiponectin synthesis in cultured adipocytes.

NF-kappaB activation in hypothalamic pro-opiomelanocortin neurons is essential in illness- and leptin-induced anorexia.

Anorexia and weight loss are prevalent in infectious diseases. To investigate the molecular mechanisms underlying these phenomena, we established animal models of infection-associated anorexia by administrating bacterial and viral products, lipopolysaccharide (LPS) and human immunodeficiency virus-1 transactivator protein (Tat). In these models, we found that the nuclear factor-kappaB (NF-kappaB), a pivotal transcription factor for inflammation-related proteins, was activated in the hypothalamus. In parallel, administration of LPS and Tat increased hypothalamic pro-inflammatory cytokine production, which was abrogated by inhibition of hypothalamic NF-kappaB. In vitro, NF-kappaB activation directly stimulated the transcriptional activity of pro-opiomelanocortin (POMC), a precursor of anorexigenic melanocortin, and mediated the stimulatory effects of LPS, Tat, and pro-inflammatory cytokines on POMC transcription, implying the involvement of NF-kappaB in controlling feeding behavior. Consistently, hypothalamic injection of LPS and Tat caused a significant reduction in food intake and body weight, which was prevented by blockade of NF-kappaB and melanocortin. Furthermore, disruption of I kappaB kinase-beta, an upstream kinase of NF-kappaB, in POMC neurons attenuated LPS- and Tat-induced anorexia. These findings suggest that infection-associated anorexia and weight loss are mediated via NF-kappaB activation in hypothalamic POMC neurons. In addition, hypothalamic NF-kappaB was activated by leptin, an important anorexigenic hormone, and mediates leptin-stimulated POMC transcription, indicating that hypothalamic NF-kappaB also serves as a downstream signaling pathway of leptin.

Does the mitral annulus shrink or enlarge during systole? A real-time 3D echocardiography study.

This study was conducted to explore the geometrical changes of the mitral annulus during systole. The 3D shape of the mitral annulus was reconstructed in 13 normal subjects who had normal structure of the mitral apparatus using real-time 3D echocardiography (RT3DE) and 3D computer software. The two orthogonal (antero-posterior and commissure-commissure) dimensions, the areas (2D projected and 3D surface) and the non-planarity of the mitral annulus were estimated during early, mid and late systole. We demonstrated that the MA had a "saddle shape" appearance and it consistently enlarged mainly in the antero-posterior direction from early to late systole with lessening of its non-planarity, as was determined by 3D reconstruction using RT3DE and 3D computer software.

Essential role of mitochondrial function in adiponectin synthesis in adipocytes.

OBJECTIVE: Adiponectin is an important adipocytokine that improves insulin action and reduces atherosclerotic processes. The plasma adiponectin level is paradoxically reduced in obese individuals, but the underlying mechanism is unknown. This study was undertaken to test the hypothesis that mitochondrial function is linked to adiponectin synthesis in adipocytes. RESEARCH DESIGN AND METHODS: We examined the effects of rosiglitazone and the measures that increase or decrease mitochondrial function on adiponectin synthesis. We also examined the molecular mechanism by which changes in mitochondrial function affect adiponectin synthesis. RESULTS: Adiponectin expression and mitochondrial content in adipose tissue were reduced in obese db/db mice, and these changes were reversed by the administration of rosiglitazone. In cultured adipocytes, induction of increased mitochondrial biogenesis (via adenoviral overexpression of nuclear respiratory factor-1) increased adiponectin synthesis, whereas impairment in mitochondrial function decreased it. Impaired mitochondrial function increased endoplasmic reticulum (ER) stress, and agents causing mitochondrial or ER stress reduced adiponectin transcription via activation of c-Jun NH(2)-terminal kinase (JNK) and consequent induction of activating transcription factor (ATF)3. Increased mitochondrial biogenesis reversed all of these changes. CONCLUSIONS: Mitochondrial function is linked to adiponectin synthesis in adipocytes, and mitochondrial dysfunction in adipose tissue may explain decreased plasma adiponectin levels in obesity. Impaired mitochondrial function activates a series of mechanisms involving ER stress, JNK, and ATF3 to decrease adiponectin synthesis.

Assessment of left ventricular asynchrony using volume-time curves of 16 segments by real-time 3 dimensional echocardiography: comparison with tissue Doppler imaging.

BACKGROUND: Recent technical developments with high-resolution real-time 3 dimensional echocardiography (RT3DE) facilitate the acquisition of high quality images and the analysis of segmental volume-time curves (VTCs). AIMS: To assess left ventricular (LV) asynchrony using the VTCs of 16 segments by RT3DE, and to evaluate accuracy compared to tissue Doppler imaging (TDI). METHODS: Twenty-three heart failure (HF) patients (LVEF: 25+/-6%, age: 60+/-13 years) and 16 normal controls underwent TDI and RT3DE. The standard deviation (SD3) of the end systolic time reaching minimal systolic volume for the 16 segments on VTCs was obtained by RT3DE. The standard deviation (SD2) of the electromechanical coupling time for the 8 segments was measured using TDI. RESULTS: SD3 was markedly higher in HF patients than in controls (7.7+/-2.5 vs 1.5+/-1.0%, P<0.01) and increased as LVEF decreased (r=-0.85, P<0.01). SD2 was also significantly higher in HF patients (27.0+/-8.6 vs 12.6+/-5.0 ms, P<0.01) and had a good negative correlation with LVEF (r=-0.72, P<0.01). SD3 was well correlated to SD2 (r=0.66, P<0.01). CONCLUSIONS: We suggest that analysis of VTCs in 16 segments using RT3DE may be a useful alternative to TDI for the evaluation of LV asynchrony.

3D geometry of a normal tricuspid annulus during systole: a comparison study with the mitral annulus using real-time 3D echocardiography.

AIM: This study was done to explore the 3D geometry of the normal tricuspid annulus and compare it with the mitral annulus (MA), using real-time 3D echocardiography (RT3DE) and newly developed 3D computer software. METHODS: Thirteen left ventricular (LV) and 13 right ventricular (RV) volumetric images were obtained using RT3DE from normal subjects. LV and RV volumetric data were segmented into 16 rotational apical planes (angle increment=11.25 degrees ) around the rotational axis from the apex through the center of two annuli, using newly developed 3D software (TomTec, Co., Munich, Germany). Two hinge points of the MA and the TA were traced in each plane during early and late systole. The MA and the TA were then automatically reconstructed with those 32 traced points. 3D surface (3DMAA, 3DTAA) and 2D projected areas (2DMAA, 2DTAA) of the annuli were calculated automatically as well, from those fitted data. For a comparison between the two annuli, the 3D and 2D area measurements of both annuli were corrected (c) according to the height of each patient (c2DMAA, c2DTAA, c3DMAA, and c3DTAA). Non-planarity of the annuli was estimated by their non-planar angles (MA: the angle between the anterior and posterior parts of the annulus; TA: the angle between the septal and free wall parts of the annulus). RESULTS: The TA revealed a less non-planar saddle shape than did the MA, which had a significantly wider non-planar angle (167.7+/-3.2 vs 145.5+/-6.1 degrees , p<0.01) and a round or oval appearance on the projected view. The c2DTAA was significantly larger than the c2DMAA during early systole (p<0.01). However, there was no significant difference between the c3DTAA and c3DMAA during early and late systole (p>0.01). CONCLUSION: We assessed the 3D geometry of the MA and the TA by 3D reconstruction using RT3DE and newly developed computer software. The TA had a less non-planar shape compared with the MA, with either a round or oval appearance on the projected view.

CCAAT/enhancer-binding protein delta activates the Runx2-mediated transcription of mouse osteocalcin II promoter.

CCAAT/enhancer-binding proteins (C/EBPs) are involved in the regulation of cell proliferation, differentiation, and control of metabolic function. Although the roles of C/EBPs in osteoblasts are largely unknown, both C/EBPbeta and -delta have been shown to enhance rat osteocalcin promoter activity through the synergistic activation of Runx2 at the C/EBP element. Here we show that in the mouse, C/EBPdelta increases the expression of osteocalcin whereas C/EBPbeta does not. This increased expression was found to occur at the transcriptional level, as demonstrated by the increased transcriptional activity from mouse osteocalcin II (OG2) promoter by C/EBPdelta. Although we found three putative C/EBP sites in the -637/+/-34 region of the OG2 promoter, none of these sites showed binding activity with in vitro translated C/EBP proteins. Notably, we show that C/EBPdelta physically interacts with Runx2 and that C/EBPdelta overexpression increases binding between the Runx2-C/EBPdelta complex and the OSE2 element, a critical osteoblast-specific cis-acting element in the OG2 promoter. Consistent with these DNA binding data, a mutation in OSE2 abrogated the stimulatory effect of C/EBPdelta on this promoter activity. Finally, chromatin immunoprecipitation analysis in MC3T3-E1 cells showed in vivo occupancy of the OG2 promoter by Runx2 and C/EBPdelta. In conclusion, C/EBPdelta was found to regulate mouse osteocalcin OG2 promoter activity indirectly by interacting with Runx2 in the context of the OSE2 element and this subsequently resulted in the cooperative activation of the OG2 promoter.

AMPK activation increases fatty acid oxidation in skeletal muscle by activating PPARalpha and PGC-1.

AMP-activated protein kinase (AMPK) activation increases fatty acid oxidation in skeletal muscle by decreasing malonyl CoA concentrations. However, this may not explain the long-term effects of AMPK activation. Here we show that AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) increases mRNA expression of PPARalpha target genes and PGC-1 in cultured muscle cells and mouse skeletal muscle, and that inhibition of PPARalpha and PGC-1 by siRNAs prevents AICAR-stimulated increase in fatty acid oxidation. These data suggest that a novel transcriptional regulatory mechanism involving PPARalpha and PGC-1 exists that is responsible for long-term stimulation of fatty acid oxidation in skeletal muscle by AICAR.

Activation of peroxisome proliferator-activated receptor-gamma inhibits the Runx2-mediated transcription of osteocalcin in osteoblasts.

Mesenchymal cells are able to differentiate into several distinct cell types, including osteoblasts and adipocytes. The commitment to a particular lineage may be regulated by specific transcription factors. Peroxisome proliferator-activated receptor-gamma (PPARgamma), acting in conjunction with CCAAT/enhancer-binding protein-alpha, has been suggested as a key regulator of adipogenic differentiation. Previous studies have shown that the activation of PPARgamma in osteoblasts suppresses osteoblast differentiation and the expression of osteocalcin, an osteoblast-specific protein. However, the mechanism of this inhibition remains unclear. We investigated the effect of PPARgamma activation on the expression of osteocalcin and analyzed the molecular mechanism. Mouse osteoblastic MC3T3-E1 cells expressed PPARgamma, which was transcriptionally active, whereas rat osteosarcoma ROS 17/2.8 cells did not. Treatment of MC3T3-E1 osteoblasts and ROS 17/2.8 cells stably transfected with PPARgamma2 with the PPARgamma activator 15-deoxy-Delta12,14-prostaglandin J2 inhibited the mRNA expression of osteocalcin and Runx2, the latter of which is a key transcription factor in osteoblast differentiation. This decreased expression of osteocalcin and Runx2 was partly explained by the decreased level of Runx2 resulting from the suppressed transcription from the Runx2 promoter. However, in addition to this indirect effect, the activation of PPARgamma by 15-deoxy-Delta12,14-prostaglandin J2 directly suppressed the Runx2-mediated induction of the activities of the osteocalcin promoter and the artificial promoter p6OSE2, which contains six tandem copies of osteoblast-specific element-2, the Runx2-binding promoter sequence. This inhibition was mediated by a physical interaction between PPARgamma and Runx2 and the subsequent repression of the transcriptional activity at the osteoblast-specific element-2 sequence. Thus, this study demonstrates that the activation of PPARgamma inhibits osteocalcin expression both by suppressing the expression of Runx2 and by interfering with the transactivation ability of Runx2.