Macrophages enhance regeneration of lateral line neuromast derived from interneuromast cells through TGF-ß in zebrafish.

Macrophages play a pivotal role in the response to injury, contributing significantly to the repair and regrowth of damaged tissues. The external lateral line system in aquatic organisms offers a practical model for studying regeneration, featuring interneuromast cells connecting sensory neuromasts. Under normal conditions, these cells remain dormant, but their transformation into neuromasts occurs when overcoming inhibitory signals from Schwann cells and posterior lateral line nerves. The mechanism enabling interneuromast cells to evade inhibition by Schwann cells remains unclear. Previous observations suggest that macrophages physically interact with neuromasts, nerves, and Schwann cells during regeneration. This interaction leads to the regeneration of neuromasts in a subset of zebrafish with ablated neuromasts. To explore whether macrophages achieve this effect through secreted cytokines, we conducted experiments involving tail amputation in zebrafish larvae and tested the impact of cytokine inhibitors on neuromast regeneration. Most injured larvae remarkably regenerated a neuromast within 4 days post-amputation. Intriguingly, removal of macrophages and inhibition of the anti-inflammatory cytokine transforming growth factor-beta (TGF-ß) significantly delayed neuromast regeneration. Conversely, inhibition of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) had minor effects on the regeneration process. This study provides insights into how macrophages activate interneuromast cells, elucidating the pathways underlying neuromast regeneration.

Diversity and composition of gut microbiota in healthy individuals and patients at different stages of hepatitis B virus-related liver disease.

BACKGROUND: Hepatitis B virus (HBV) causes chronic hepatitis B (CHB), liver cirrhosis, and hepatocellular carcinoma. The evolution of human gut microbiota during the progression of HBV-related liver diseases remains unclear. Therefore, we prospectively enrolled patients with HBV-related liver diseases and healthy individuals. Through 16S ribosomal RNA amplicon sequencing, we characterized the gut microbiota of the participants and predicted the functions of microbial communities. RESULTS: We analyzed the gut microbiota of 56 healthy controls and 106 patients with HBV-related liver disease [14 with resolved HBV infection, 58 with CHB, and 34 with advanced liver disease (15 with liver cirrhosis and 19 with hepatocellular carcinoma)]. Patients with HBV-related liver disease exhibited a higher degree of bacterial richness (all P < 0.05) than did healthy controls. Beta diversity analyses revealed a distinct clustering pattern between healthy controls and patients with HBV-related liver disease (all P < 0.05). The composition of bacteria (from the phylum level to the genus level) varied across the stages of liver disease. Linear discriminant analysis effect size revealed multiple taxa that differ significantly in abundance between healthy controls and patients with HBV-related liver disease; however, fewer differences were observed among patients with resolved HBV infection, those with CHB, and those with advanced liver disease. The ratio of Firmicutes to Bacteroidetes was increased in all three patient groups compared with the ratio in healthy controls (all P < 0.001). The analysis of the sequencing data by using PICRUSt2 revealed the changes in microbial functions with disease progression. CONCLUSIONS: The diversity and composition of gut microbiota appear to vary significantly between healthy controls and patients at different stages of HBV-related liver disease. The understanding of gut microbiota may provide novel therapeutic options in these patients.

Serum cytokine profiles predict outcomes of chronic hepatitis B patients discontinuing entecavir or tenofovir therapy.

BACKGROUND/PURPOSE: Distinct hepatitis relapse has been observed after discontinuing entecavir (ETV) or tenofovir disoproxil fumarate (TDF) therapy in chronic hepatitis B (CHB) patients. End-of-therapy (EOT) serum cytokines were compared and used for outcome prediction. METHODS: A total of 80 non-cirrhotic CHB patients in a tertiary medical center in Taiwan who discontinued ETV (n = 51) or TDF (n = 29) therapy after fulfilling the APASL guidelines were prospectively enrolled. Serum cytokines were measured at EOT and 3rd month afterwards. Multivariable analysis was performed to predict virological relapse (VR, HBV DNA >2000 IU/mL), clinical relapse (CR, VR and alanine aminotransferase > 2-fold upper limit of normal) and hepatitis B surface antigen (HBsAg) seroclearance. RESULTS: Compared with TDF group, ETV stoppers had greater interleukin 5 (IL-5), IL-12 p70, IL-13, IL-17 A and tumor necrosis factor alpha (TNF-alpha) (all P < 0.05) at EOT. Older age, TDF use, higher EOT HBsAg and IL-18 (Hazard ratio [HR], 1.01; 95% CI, 1.00-1.02) levels at EOT predicted VR, while older age, higher EOT HBsAg and IL-7 (HR, 1.25; 95% CI, 1.00-1.56) levels predicted CR. In TDF stoppers, higher IL-7 (HR, 1.29; 95% CI, 1.05-1.60) and IL-18 (HR, 1.02; 95% CI, 1.00-1.04) levels predicted VR, while IL-7 (HR, 1.34; 95% CI, 1.08-1.65) and interferon-gamma (IFN-gamma) (HR, 1.08; 95% CI, 1.02-1.14) levels predicted CR. A lower EOT HBsAg level was associated with HBsAg seroclearance. CONCLUSION: Distinct cytokine profiles were observed after stopping ETV or TDF. Higher EOT IL-7, IL-18, and IFN-gamma could be probable predictors for VR and CR in patients discontinuing NA therapies.

Macrophages Break Interneuromast Cell Quiescence by Intervening in the Inhibition of Schwann Cells in the Zebrafish Lateral Line.

In the zebrafish lateral line system, interneuromast cells (INCs) between neuromasts are kept quiescent by underlying Schwann cells (SWCs). Upon severe injuries that cause the complete loss of an entire neuromast, INCs can occasionally differentiate into neuromasts but how they escape from the inhibition by SWCs is still unclear. Using a genetic/chemical method to ablate a neuromast precisely, we found that a small portion of larvae can regenerate a new neuromast. However, the residual regeneration capacity was hindered by inhibiting macrophages. Using in toto imaging, we further discovered heterogeneities in macrophage behavior and distribution along the lateral line. We witnessed the crawling of macrophages between the injured lateral line and SWCs during regeneration and between the second primordium and the first mature lateral line during development. It implies that macrophages may physically alleviate the nerve inhibition to break the dormancy of INCs during regeneration and development in the zebrafish lateral line.

Stathmin-like 4 is critical for the maintenance of neural progenitor cells in dorsal midbrain of zebrafish larvae.

A delicate balance between proliferating and differentiating signals is necessary to ensure proper growth and neuronal specification. By studying the developing zebrafish brain, we observed a specific and dynamic expression of a microtubule destabilizer gene, stathmin-like 4 (stmn4), in the dorsal midbrain region. The expression of stmn4 was mutually exclusive to a pan-neuronal marker, elavl3 that indicates its role in regulating neurogenesis. We showed the knockdown or overexpression of stmn4 resulted in premature neuronal differentiation in dorsal midbrain. We also generated stmn4 maternal-zygotic knockout zebrafish by the CRISPR/Cas9 system. Unexpectedly, only less than 10% of stmn4 mutants showed similar phenotypes observed in that of stmn4 morphants. It might be due to the complementation of the increased stmn1b expression observed in stmn4 mutants. In addition, time-lapse recordings revealed the changes in cellular proliferation and differentiation in stmn4 morphants. Stmn4 morphants displayed a longer G2 phase that could be rescued by Cdc25a. Furthermore, the inhibition of Wnt could reduce stmn4 transcripts. These results suggest that the Wnt-mediated Stmn4 homeostasis is crucial for preventing dorsal midbrain from premature differentiation via the G2 phase control during the neural keel stage.

D-501036, a novel selenophene-based triheterocycle derivative, exhibits potent in vitro and in vivo antitumoral activity which involves DNA damage and ataxia telangiectasia-mutated nuclear protein kinase activation.

D-501036 [2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl-N-methylpyrrole] is herein identified as a novel antineoplastic agent with a broad spectrum of antitumoral activity against several human cancer cells and an IC(50) value in the nanomolar range. The IC(50) values for D-501036 in the renal proximal tubule, normal bronchial epithelial, and fibroblast cells were >10 mumol/L. D-501036 exhibited no cross-resistance with vincristine- and paclitaxel-resistant cell lines, whereas a low level of resistance toward the etoposide-resistant KB variant was observed. Cell cycle analysis established that D-501036 treatment resulted in a dose-dependent accumulation in S phase with concomitant loss of both the G(0)-G(1) and G(2)-M phase in both Hep 3B and A-498 cells. Pulsed-field gel electrophoresis showed D-501036-induced, concentration-dependent DNA breaks in both Hep 3B and A-498 cells. These breaks did not involve interference with either topoisomerase-I and topoisomerase-II function or DNA binding. Rapid reactive oxygen species production and formation of Se-DNA adducts were evident following exposure of cells to D-501036, indicating that D-501036-mediated DNA breaks were attributable to the induction of reactive oxygen species and DNA adduct formation. Moreover, D-501036-induced DNA damage activated ataxia telangiectasia-mutated nuclear protein kinase, leading to hyperphosphorylation of Chk1, Chk2, and p53, decreased expression of CDC25A, and up-regulation of p21(WAF1) in both p53-proficient and p53-deficient cells. Collectively, the results indicate that D-501036-induced cell death was associated with DNA damage-mediated induction of ataxia telangiectasia-mutated activation, and p53-dependent and -independent apoptosis pathways. Notably, D-501036 shows potent activity against the growth of xenograft tumors of human renal carcinoma A-498 cells. Thus, D-501036 is a promising anticancer compound that has strong potential for the management of human cancers.