Relationship of Metabolic Dysfunction-Associated Steatohepatitis-Related Hepatocellular Carcinoma with Oral and Intestinal Microbiota: A Cross-Sectional Pilot Study.

Background and Objectives: The incidence of metabolic dysfunction-associated steatohepatitis (MASH)-related hepatocellular carcinoma (HCC) is increasing worldwide, alongside the epidemic of obesity and metabolic syndrome. Based on preliminary reports regarding the potential association of HCC and periodontitis, this study aimed to analyze the involvement of periodontal bacteria as well as the oral and intestinal bacterial flora in MASH-related HCC (MASH-HCC). Materials and Methods: Forty-one patients with MASH and nineteen with MASH-HCC participated in the study, completing survey questionnaires, undergoing periodontal examinations, and providing samples of saliva, mouth-rinsed water, feces, and peripheral blood. The oral and fecal microbiome profiles were analyzed by 16S ribosomal RNA sequencing. Bayesian network analysis was used to analyze the causation between various factors, including MASH-HCC, examinations, and bacteria. Results: The genus Fusobacterium had a significantly higher occupancy rate (p = 0.002) in the intestinal microflora of the MASH-HCC group compared to the MASH group. However, Butyricicoccus (p = 0.022) and Roseburia (p < 0.05) had significantly lower occupancy rates. The Bayesian network analysis revealed the absence of periodontal pathogenic bacteria and enteric bacteria affecting HCC. However, HCC directly affected the periodontal bacterial species Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, and Prevotella intermedia in the saliva, as well as the genera Lactobacillus, Roseburia, Fusobacterium, Prevotella, Clostridium, Ruminococcus, Trabulsiella, and SMB53 in the intestine. Furthermore, P. gingivalis in the oral cavity directly affected the genera Lactobacillus and Streptococcus in the intestine. Conclusions: MASH-HCC directly affects periodontal pathogenic and intestinal bacteria, and P. gingivalis may affect the intestinal bacteria associated with gastrointestinal cancer.

Size-reduced embryos reveal a gradient scaling-based mechanism for zebrafish somite formation.

Little is known about how the sizes of animal tissues are controlled. A prominent example is somite size, which varies widely both within an individual and across species. Despite intense study of the segmentation clock governing the timing of somite generation, how it relates to somite size is poorly understood. Here, we examine somite scaling and find that somite size at specification scales with the length of the presomitic mesoderm (PSM) despite considerable variation in PSM length across developmental stages and in surgically size-reduced embryos. Measurement of clock period, axis elongation speed and clock gene expression patterns demonstrate that existing models fail to explain scaling. We posit a 'clock and scaled gradient' model, in which somite boundaries are set by a dynamically scaling signaling gradient across the PSM. Our model not only explains existing data, but also makes a unique prediction that we confirm experimentally - the formation of periodic 'echoes' in somite size following perturbation of the size of one somite. Our findings demonstrate that gradient scaling plays a central role in both progression and size control of somitogenesis.

Over-expression of Nicotinamide phosphoribosyltransferase in mouse cells confers protective effect against oxidative and ER stress-induced premature senescence.

A main feature of aged organisms is the accumulation of senescent cells. Accumulated senescent cells, especially stress-induced premature senescent cells, in aged organisms lead to the decline of the regenerative potential and function of tissues. We recently reported that the over-expression of NAMPT, which is the rate-limiting enzyme in mammalian NAD+ salvage pathway, delays replicative senescence in vitro. However, whether Nampt-overexpressing cells are tolerant of stress-induced premature senescence remains unknown. Here, we show that primary mouse embryonic fibroblasts derived from Nampt-overexpressing transgenic mice (Nampt Tg-MEF cells) possess resistance against stress-induced premature senescence in vitro. We found that higher oxidative or endoplasmic reticulum (ER) stress is required to induce premature senescence in Nampt Tg-MEF cells compared to wild-type cells. Moreover, we found that Nampt Tg-MEF cells show acute expression of unfolded protein response (UPR)-related genes, which in turn would have helped to restore proteostasis and avoid cellular senescence. Our results demonstrate that NAMPT/NAD+ axis functions to protect cells not only from replicative senescence, but also from stress-induced premature senescence in vitro. We anticipate that in vivo activation of NAMPT activity or increment of NAD+ would protect tissues from the accumulation of premature senescent cells, thereby maintaining healthy aging.

Relationship between surrounding tissue morphology and directional collective migration of the posterior lateral line primordium in zebrafish.

Collective cell migration, in which cells assemble and move together, is an essential process in embryonic development, wound healing and cancer metastasis. Chemokine signaling guides cell assemblies to their destinations. In zebrafish posterior lateral line primordium (PLLP), a model system for collective cell migration, it has been proposed that the chemokine ligand Cxcl12a secreted from muscle pioneer cells (MPs) and muscle fast fibers (MFFs), which are distributed along with the horizontal midline, binds to the receptor Cxcr4b in PLLP and that Cxcl12a-Cxcr4b signaling guides the anterior-to-posterior migration of PLLP along the horizontal midline. However, how the surrounding tissues affect PLLP migration remains to be elucidated. Here, we investigated the relationship between the PLLP and the surrounding tissues and found that a furrow between the dorsal and ventral myotomes is generated by Sonic hedgehog (Shh) signaling-dependent MP and MFF differentiation and that the PLLP migrates in this furrow. When transient inhibition of Shh signaling impaired both the furrow formation and differentiation of cxcl12a-expressing MPs/MFFs, directional PLLP migration was severely perturbed. Furthermore, when differentiated MPs and MFFs were ablated by femtosecond laser irradiations, the furrow remained and PLLP migration was relatively unaffected. These results suggest that the furrow formation between the dorsal and ventral myotomes is associated with the migratory behavior of PLLP.

Somite boundary determination in normal and clock-less vertebrate embryos.

Vertebrate segments called somites are generated by periodic segmentation of the presomitic mesoderm (PSM). In the most accepted theoretical model for somite segmentation, the clock and wavefront (CW) model, a clock that ticks to determine particular timings and a wavefront that moves posteriorly are presented in the PSM, and somite positions are determined when the clock meets the posteriorly moving wavefront somewhere in the PSM. Over the last two decades, it has been revealed that the molecular mechanism of the clock and wavefront in vertebrates is based on clock genes including Hes family transcription factors and Notch effectors that oscillate within the PSM to determine particular timings and fibroblast growth factor (FGF) gradients, acting as the posteriorly moving wavefront to determine the position of somite segmentation. A clock-less condition in the CW model was predicted to form no somites; however, irregularly sized somites were still formed in mice and zebrafish, suggesting that this was one of the limitations of the CW model. Recently, we performed interdisciplinary research of experimental and theoretical biological studies and revealed the mechanisms of somite boundary determination in normal and clock-less conditions by characterization of the FGF/extracellular signal-regulated kinase (ERK) activity dynamics. Since features of the molecular clock have already been described in-depth in several reviews, we summarized recent findings regarding the role of FGF/ERK signaling in somite boundary formation and described our current understanding of how FGF/ERK signaling contributes to somitogenesis in normal and clock-less conditions in this review.

Noise-resistant developmental reproducibility in vertebrate somite formation.

The reproducibility of embryonic development is remarkable, although molecular processes are intrinsically stochastic at the single-cell level. How the multicellular system resists the inevitable noise to acquire developmental reproducibility constitutes a fundamental question in developmental biology. Toward this end, we focused on vertebrate somitogenesis as a representative system, because somites are repeatedly reproduced within a single embryo whereas such reproducibility is lost in segmentation clock gene-deficient embryos. However, the effect of noise on developmental reproducibility has not been fully investigated, because of the technical difficulty in manipulating the noise intensity in experiments. In this study, we developed a computational model of ERK-mediated somitogenesis, in which bistable ERK activity is regulated by an FGF gradient, cell-cell communication, and the segmentation clock, subject to the intrinsic noise. The model simulation generated our previous in vivo observation that the ERK activity was distributed in a step-like gradient in the presomitic mesoderm, and its boundary was posteriorly shifted by the clock in a stepwise manner, leading to regular somite formation. Here, we showed that this somite regularity was robustly maintained against the noise. Removing the clock from the model predicted that the stepwise shift of the ERK activity occurs at irregular timing with irregular distance owing to the noise, resulting in somite size variation. This model prediction was recently confirmed by live imaging of ERK activity in zebrafish embryos. Through theoretical analysis, we presented a mechanism by which the clock reduces the inherent somite irregularity observed in clock-deficient embryos. Therefore, this study indicates a novel role of the segmentation clock in noise-resistant developmental reproducibility.

Targeting E3 Ubiquitin Ligases and Deubiquitinases in Ciliopathy and Cancer.

Cilia are antenna-like structures present in many vertebrate cells. These organelles detect extracellular cues, transduce signals into the cell, and play an essential role in ensuring correct cell proliferation, migration, and differentiation in a spatiotemporal manner. Not surprisingly, dysregulation of cilia can cause various diseases, including cancer and ciliopathies, which are complex disorders caused by mutations in genes regulating ciliary function. The structure and function of cilia are dynamically regulated through various mechanisms, among which E3 ubiquitin ligases and deubiquitinases play crucial roles. These enzymes regulate the degradation and stabilization of ciliary proteins through the ubiquitin-proteasome system. In this review, we briefly highlight the role of cilia in ciliopathy and cancer; describe the roles of E3 ubiquitin ligases and deubiquitinases in ciliogenesis, ciliopathy, and cancer; and highlight some of the E3 ubiquitin ligases and deubiquitinases that are potential therapeutic targets for these disorders.

Presomitic mesoderm-specific expression of the transcriptional repressor Hes7 is controlled by E-box, T-box, and Notch signaling pathways.

Somites are a pair of epithelial spheres beside a neural tube and are formed with an accurate periodicity during embryogenesis in vertebrates. It has been known that Hes7 is one of the core clock genes for somitogenesis, and its expression domain is restricted in the presomitic mesoderm (PSM). However, the molecular mechanism of how Hes7 transcription is regulated is not clear. Here, using transgenic mice and luciferase-based reporter assays and in vitro binding assays, we unravel the mechanism by which Hes7 is expressed exclusively in the PSM. We identified a Hes7 essential region residing -1.5 to -1.1 kb from the transcription start site of mouse Hes7, and this region was indispensable for PSM-specific Hes7 expression. We also present detailed analyses of cis-regulatory elements within the Hes7 essential region that directs Hes7 expression in the PSM. Hes7 expression in the PSM was up-regulated through the E-box, T-box, and RBPj-binding element in the Hes7 essential region, presumably through synergistic signaling involving mesogenin1, T-box6 (Tbx6), and Notch. Furthermore, we demonstrate that Tbx18, Ripply2, and Hes7 repress the activation of the Hes7 essential region by the aforementioned transcription factors. Our findings reveal that a unified transcriptional regulatory network involving a Hes7 essential region confers robust PSM-specific Hes7 gene expression.

Nicotinamide phosphoribosyltransferase delays cellular senescence by upregulating SIRT1 activity and antioxidant gene expression in mouse cells.

Senescent cells accumulate in tissues of aged animals and deteriorate tissue functions. The elimination of senescent cells from aged mice not only attenuates progression of already established age-related disorders, but also extends median lifespan. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD+ salvage pathway, has shown a protective effect on cellular senescence of human primary cells. However, it still remains unclear how NAMPT has a protective impact on aging in vitro and in vivo. In this study, we found that primary mouse embryonic fibroblast (MEF) cells undergo progressive decline of NAMPT and NAD+ contents during serial passaging before becoming senescent. Furthermore, we showed that constitutive Nampt over-expression increases cellular NAD+ content and delays cellular senescence of MEF cells in vitro. We further found that constitutive Nampt over-expression increases SIRT1 activity, increases the expression of antioxidant genes, superoxide dismutase 2 and catalase and promotes resistance against oxidative stress. These findings suggest that Nampt over-expression in MEF cells delays cellular senescence by the mitigation of oxidative stress via the upregulation of superoxide dismutase 2 and catalase gene expressions by SIRT1 activation.

ERK Activity Dynamics during Zebrafish Embryonic Development.

During vertebrate development, extracellular signal-regulated kinase (ERK) is activated by growth factors such as fibroblast growth factor (FGF), and it regulates the formation of tissues/organs including eyes, brains, somites, limbs, and inner ears. However, an experimental system to monitor ERK activity dynamics in the entire body of the vertebrate embryo is lacking. We recently studied ERK activity dynamics in the pre-somitic mesoderm of living zebrafish embryos injected with mRNAs encoding a Förster resonance energy transfer (FRET)-based ERK biosensor. In this study, transgenic zebrafish stably and ubiquitously expressing the ERK biosensor were generated to monitor ERK activity dynamics throughout embryonic development. The system allowed the identification of ERK activation domains in embryos from the late blastula to the late segmentation stage, consistent with immunostaining patterns obtained using anti-phosphorylated ERK antibody. A spatiotemporal map of ERK activity in the entire body during zebrafish embryogenesis was generated, and previously unidentified activation dynamics and ERK domains were identified. The proposed system is the first reported method to monitor ERK activity dynamics during vertebrate embryogenesis, providing insight into the role of ERK activity in normal and abnormal development in living vertebrate embryos.

Retinoic acid controls proper head-to-trunk linkage in zebrafish by regulating an anteroposterior somitogenetic rate difference.

During vertebrate development, the primary body axis elongates towards the posterior and is periodically divided into somites, which give rise to the vertebrae, skeletal muscles and dermis. Somites form periodically from anterior to posterior, and the anterior somites form in a more rapid cycle than the posterior somites. However, how this anteroposterior (AP) difference in somitogenesis is generated and how it contributes to the vertebrate body plan remain unclear. Here, we show that the AP difference in zebrafish somitogenesis originates from a variable overlapping segmentation period between one somite and the next. The AP difference is attributable to spatiotemporal inhibition of the clock gene her1 via retinoic acid (RA) regulation of the transcriptional repressor ripply1. RA depletion thus disrupts timely somite formation at the transition, eventually leading to the loss of one somite and the resultant cervical vertebra. Overall, our results indicate that RA regulation of the AP difference is crucial for proper linkage between the head and trunk in the vertebrate body plan.

An anterior limit of FGF/Erk signal activity marks the earliest future somite boundary in zebrafish.

Vertebrate segments called somites are generated by periodic segmentation of the anterior extremity of the presomitic mesoderm (PSM). During somite segmentation in zebrafish, mesp-b determines a future somite boundary at position B-2 within the PSM. Heat-shock experiments, however, suggest that an earlier future somite boundary exists at B-5, but the molecular signature of this boundary remains unidentified. Here, we characterized fibroblast growth factor (FGF) signal activity within the PSM, and demonstrated that an anterior limit of downstream Erk activity corresponds to the future B-5 somite boundary. Moreover, the segmentation clock is required for a stepwise posterior shift of the Erk activity boundary during each segmentation. Our results provide the first molecular evidence of the future somite boundary at B-5, and we propose that clock-dependent cyclic inhibition of the FGF/Erk signal is a key mechanism in the generation of perfect repetitive structures in zebrafish development.

Celf1 regulation of dmrt2a is required for somite symmetry and left-right patterning during zebrafish development.

RNA-binding proteins (RBPs) bind to numerous and diverse mRNAs to control gene expression post-transcriptionally, although the in vivo functions of specific RBP-mRNA interactions remain largely unknown. Here, we show that an RBP named Cugbp, Elav-like family member 1 (Celf1) controls expression of a gene named doublesex and mab-3 related transcription factor 2a (dmrt2a), which is essential for somite symmetry and left-right patterning during zebrafish development. Celf1 promotes dmrt2a mRNA decay by binding to UGU repeats in the 3'UTR of dmrt2a mRNA such that celf1 overexpression reduces the amount of dmrt2a mRNA, leading to asymmetric somitogenesis and laterality defects. Furthermore, blocking the Celf1-dmrt2a mRNA interaction by a target protector morpholino alleviates failures in somite symmetry and left-right patterning that are caused by celf1 overexpression. Our results therefore demonstrate that Celf1-dependent fine-tuning of dmrt2a expression is essential for generating bilateral symmetry of somites and left-right asymmetric patterning during zebrafish development.

Canopy1, a positive feedback regulator of FGF signaling, controls progenitor cell clustering during Kupffer's vesicle organogenesis.

The assembly of progenitor cells is a crucial step for organ formation during vertebrate development. Kupffer's vesicle (KV), a key organ required for the left-right asymmetric body plan in zebrafish, is generated from a cluster of ~20 dorsal forerunner cells (DFCs). Although several genes are known to be involved in KV formation, how DFC clustering is regulated and how cluster formation then contributes to KV formation remain unclear. Here we show that positive feedback regulation of FGF signaling by Canopy1 (Cnpy1) controls DFC clustering. Cnpy1 positively regulates FGF signals within DFCs, which in turn promote Cadherin1-mediated cell adhesion between adjacent DFCs to sustain cell cluster formation. When this FGF positive feedback loop is disrupted, the DFC cluster fails to form, eventually leading to KV malformation and defects in the establishment of laterality. Our results therefore uncover both a previously unidentified role of FGF signaling during vertebrate organogenesis and a regulatory mechanism underlying cell cluster formation, which is an indispensable step for formation of a functional KV and establishment of the left-right asymmetric body plan.

Association of Elevated Serum Branched-chain Amino Acid Levels With Longitudinal Skeletal Muscle Loss.

Context: Branched-chain amino acids (BCAA) are substrates for protein synthesis. Although their intake may contribute to an increase in skeletal muscle mass, elevated serum BCAA levels have been reported to be associated with insulin resistance, potentially resulting in decreased skeletal muscle mass. Objective: This study aimed to explore the association between elevated serum BCAA levels and longitudinal skeletal muscle loss. Design and Setting: A cohort analysis was conducted, in which serum amino acids were analyzed in healthy individuals who underwent a medical health checkup at Kameoka Municipal Hospital (HOZUGAWA study), Japan. Patients: Seventy-one participants (37 men and 34 women) underwent follow-up checkups after the baseline visit. The follow-up duration was 1.2 ± .4 years. Main Outcome Measures: The relationship between fasting baseline serum BCAA levels and lifestyle factors, body composition, blood test results, dietary history, and changes in skeletal muscle mass was evaluated. Results: In both men and women, serum BCAA levels were positively correlated with body weight, body mass index, skeletal muscle mass index (SMI), and serum triglycerides but inversely correlated with serum high-density lipoprotein cholesterol. In men, fasting serum BCAA levels were inversely associated with the rate of change in SMI (adjusted ß = -.529, P = .006), and elevated BCAA levels were independently associated with a longitudinal decrease in skeletal muscle mass (odds ratio: 1.740; 95% confidence interval: 1.023-2.960 per 50 nmol/mL serum BCAAs increase). Conclusion: Increased circulating BCAAs could be an indicator of skeletal muscle loss in men.

Left-right asymmetry in zebrafish.

In vertebrates, internal organs are positioned asymmetrically across the left-right (LR) axis, placing them in a defined area within the body. This LR asymmetric placement is a conserved feature of the vertebrate body plan. Events determining LR asymmetry occur during embryonic development, and are regulated by the coordinated action of genetic mechanisms that are evolutionarily conserved among vertebrates. Recent studies using zebrafish have provided new insights into how the Kupffer's vesicle organizer region is generated, and how it relays LR asymmetry information to the lateral plate mesoderm. In this review, we summarize recent advances in zebrafish and describe our current understanding of the mechanisms underlying these processes.

Quantitative analysis of vitreous and plasma concentrations of brilliant blue g after use as a surgical adjuvant in chromovitrectomy.

PURPOSE: To measure the concentration of brilliant blue G (BBG) in vitreous and plasma after use as a surgical adjuvant for staining and peeling of the internal limiting membrane to determine potential systemic adverse effects. METHODS: This study was designed as a prospective, interventional, clinical, case series. Five eyes from five patients with macular hole or epiretinal membrane underwent BBG-assisted internal limiting membrane and epiretinal membrane removal. The vitreous samples were obtained and stored at the end of surgery in all five cases. The plasma specimens were extracted and stored at the end of the operation, after 4 hours, and after 7 days post operation. For BBG analysis of plasma and vitreous, high-performance liquid chromatography coupled with tandem mass spectrometric detection was used. RESULTS: Brilliant blue G was not detected in plasma from all five cases at the three points of measurement. The mean vitreous BBG concentration was 34.5 ± 23.7 ng/mL (range, 11.3-70.9 ng/mL). Postoperative progress was good, and adverse effects were not observed in any of the five cases. CONCLUSION: Brilliant blue G, which remained at low levels in the vitreous cavity, was not found in the systemic blood flow after the operation. Thus, any adverse effects of systemic BBG would be avoided.

Clinical evaluation and feasibility of changing intraoperative visibility with a novel viewing filter system for human eye.

PURPOSE: The study was conducted to develop a new viewing system as a clinical prototype that enables visibility during surgery. METHODS: The system was composed of several filters attached to the microscope. This nonrandomized, retrospective, observational case series study involved 33 eyes from 32 patients who presented with various diseases and underwent surgery. The authors evaluated the changes in visualization focusing on controlling intraoperative visibility under air infusion and enhancing Brilliant Blue G staining focusing a sharp-cut filter Y (SCY). Visibility was compared under various surgical conditions, including cataract surgery, both with and without this system. Quantitative analysis of changes in intraoperative reflection including halation under air infusion and Brilliant Blue G intensity was carried out using the International Commission on Illumination 1976 (L*, a*, b*) color space method. RESULTS: A SCY reduced the reflection and halation by a maximum of 69.6%, when compared with use of no filter under air infusion (P < 0.01). The color difference between Brilliant Blue G-stained and nonstained areas was improved by 127.8% relative to values with no filter and using SCY (P < 0.01) in macular hole cases. Furthermore, in cataract surgery with corneal opacity, improvement of visibility was observed by SCY insertion. CONCLUSION: The system improved intraoperative visibility under air infusion and the Brilliant Blue G staining intensity by use of SCY during vitrectomy.

Symptomatic and morphological differences between choroidal excavations.

PURPOSE: The purpose of this report is to describe the morphological and clinical features of two patients with focal choroidal excavation in an attempt to understand more about this rare condition. CASE REPORT: Spectral-domain optical coherence tomography (SD-OCT), fluorescein angiography, and indocyanine green angiography were used to assess the morphological characteristics of the patients' choroidal excavations. Both patients showed the following features on SD-OCT: (1) the retinal pigment epithelium band and inner/outer segment junction followed the contour of the choroidal excavation, which involved the outer nuclear layers up to the outer limiting membrane; (2) the sclerochoroidal junction was smooth and undisturbed, but large choroidal vessels were present beneath each excavation. The patient with metamorphopsia showed separation between the photoreceptor outer segment and the retinal pigment epithelium as well as disturbance of the inner/outer segment junction on SD-OCT volume scans and hyperfluorescence and hypofluorescence in the foveal region on indocyanine green angiography. CONCLUSIONS: Symptomatic and morphological differences between focal choroidal excavations suggested anatomical alterations between the photoreceptor tips and the retinal pigment epithelium or location of choroidal excavation as the cause of metamorphopsia. We speculate that the pathogenesis of focal choroidal excavation involves outward traction on the macula caused by choroidal vascular abnormalities because of embryonic developmental failure of the choroid.

Celf1 is required for formation of endoderm-derived organs in zebrafish.

We recently reported that an RNA binding protein called Cugbp Elav-like family member 1 (Celf1) regulates somite symmetry and left-right patterning in zebrafish. In this report, we show additional roles of Celf1 in zebrafish organogenesis. When celf1 is knocked down by using an antisense morpholino oligonucleotides (MO), liver buds fail to form, and pancreas buds do not form a cluster, suggesting earlier defects in endoderm organogenesis. As expected, we found failures in endoderm cell growth and migration during gastrulation in embryos injected with celf1-MOs. RNA immunoprecipitation revealed that Celf1 binds to gata5 and cdc42 mRNAs which are known to be involved in cell growth and migration, respectively. Our results therefore suggest that Celf1 regulates proper organogenesis of endoderm-derived tissues by regulating the expression of such targets.