Cell and Tissue Research最新文献

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer, with existing therapies largely ineffective for advanced stages. Ankyrin 3 (ANK3), a protein involved in various cellular processes, has been associated with poor prognosis and immune infiltration in ccRCC. However, its role in ccRCC development and progression remains poorly understood. In the present study, ANK3-knockdown 786-O cells were generated using a shRNA approach. Cellular assays revealed that ANK3 knockdown significantly enhanced cell proliferation, migration, and invasion compared to shControl cells. ANK3-knockdown cells also displayed increased nuclear size and accumulation of G2/M phase of the cell cycle. Proteomic analysis identified 59 significantly altered proteins in ANK3-knockdown cells, primarily involved in cell adhesion and structural integrity. Among these, 10 proteins were significantly associated with overall survival in ccRCC patients. Morphological analysis showed that ANK3-knockdown cells exhibited a more spindle-like shape and increased cellular protrusions (filopodia). Western blot analysis further demonstrated altered expression levels of junctional and cytoskeleton proteins, including decreased E-cadherin and increased ZO-1, ICAM-1, vimentin, and β-actin. Immunofluorescence analysis revealed the accumulation of F-actin in the cytoplasm and filopodia in ANK3-knockdown cells. Additionally, ANK3 knockdown enhanced cell adhesion and aggregation capabilities. Finally, a decrease in ANK3 expression was confirmed in ccRCC tissues (grades I and II) compared to adjacent normal tissues. These findings suggest that ANK3 acts as a potential modulator of cell adhesion and cytoskeleton remodeling in ccRCC and may represent a promising therapeutic target for this malignancy.

The brown planthopper (Nilaparvata lugens) has a high reproductive rate, posing a significant challenge to biological control of rice pests. This is largely due to the physiological and functional traits of its telotrophic ovarioles, which feature a centralized nutrient supply to ensure each oocyte obtains essential basic nutrients. Current studies on insect ovarioles primarily focus on hormonal regulation and gene expression; the cellular composition and spatial relationships within N. lugens ovariole tissues remain poorly understood. To illustrate the internal architecture of ovarioles and the intricate oogenesis process at nanoscale resolution, this study employed focused ion beam-scanning electron microscopy for 3D volume reconstruction, overcoming limitations of traditional 2D electron microscopy. Using this advanced imaging technique, we systematically characterized key ovariole components including terminal filaments, trophocytes, germ cell clusters, three types of follicular cells, nutritive cords, and oocytes. Our findings reveal that rather than maintaining a continuous linkage, the nutritive cord transiently connects to the oocytes during specific developmental stages to facilitate nutrient transfer. An assembly of unique, cake-like phospholipoglycoproteins was identified within the oocytes, suggesting specialized energy storage mechanisms. The detailed 3D model elucidates the spatial relationship and relative position between various parts of the ovariole, offering novel insights into the mechanisms of oocyte growth and nutrient acquisition. These findings advance foundational knowledge of insect reproductive biology and provide a valuable framework for future research on pest control strategies targeting N. lugens reproduction.

Since human inner ear hair cells do not regenerate, the current treatments of hereditary deafness depend on hearing aids or cochlear implant. However, uncovering the functions of genes responsible for hereditary hearing loss is not only useful for their diagnosis but also for developing therapies. The pathogenetic mechanism of human non-syndromic deafness DFNB77 without morphological defects in the inner year caused by LOXHD1 mutations is not fully understood. We introduced zebrafish because the lateral line hair cells are structurally and physiologically similar to the human inner ear hair cells and mutations involved in non-symptomatic hearing loss can be assessed by their swimming behavior. The knock-out (KO) of LOXHD1b gene which is expressed in the lateral line hair cells was generated using the CRISPR-Cas9 system in zebrafish, and its morphological and functional changes were evaluated. As with human patients the LOXHD1b KO zebrafish larvae did not exhibit detectable morphological defects, but showed prolonged water flow sensing time. These results suggest that LOXHD1b plays pivotal roles for the hair cell neural activity and its KO zebrafish mutant serves as a useful model for revealing the molecular mechanisms linking LOXHD with hair cell function and for a drug screening to rescue the swimming phenotype.

There is intense public interest in the potential of antioxidant supplements as a preventative treatment for conditions associated with increased oxidative stress, such as type 2 diabetes mellitus. However, there is limited evidence regarding the effects of antioxidants on beta cells during physiological and pathological conditions. We examined the direct effects of N-acetyl-L-cysteine (NAC) supplementation on the MIN6 beta cell line under physiological and fatty acid stress conditions. MIN6 cells were cultured in growth medium with or without NAC (physiological conditions) or growth medium plus palmitate with or without NAC (fatty acid stress conditions). We observed that MIN6 cells receiving NAC, under physiological or fatty acid stress conditions, displayed significantly reduced cellular ATP content and oxidative stress without changes to markers of cell proliferation or apoptosis. Regardless of the treatment conditions, NAC lowered basal insulin release with no change to cellular insulin content, yet improved high glucose-stimulated insulin secretion (GSIS) was only observed under physiological conditions. Importantly, phosphorylated AKT was significantly reduced with NAC supplementation under physiological and fatty acid stress conditions, which was inversely proportional to ERK1/2^{Thr202/Tyr204} phosphorylation during fatty acid stress. Our findings provided evidence that NAC directly impacts ATP production and insulin signaling pathways in MIN6 cells. This study highlights the importance of investigating ROS balance in pancreatic beta cells under physiological and pathological conditions to determine if antioxidant therapies can improve functionality without interfering with essential signaling processes.

In the gastrointestinal tract, several modulators are involved in generating complex motility patterns, including smooth muscle cells (SMCs), interstitial cells of Cajal (ICCs), and PDGFRα+ cells. Notably, ICCs are excitable cells that generate characteristic oscillations in pacemaker activity mediated by the cytoplasmic Ca²⁺ concentration. Since the primary source of cytoplasmic Ca²⁺ is the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR), its distribution and localisation in the cytoplasm may indicate characteristic aspects that help in understanding its physiological functions. Furthermore, caveolae, which are invaginations of the plasma membrane (PM) observed in both ICCs and SMCs, are key sites for Ca²⁺ sparks, and their functions have been well studied in muscle cells. However, the mechanism by which caveolae and ER interact to regulate Ca²⁺ in ICCs remains unclear compared with that in SMCs. In this study, microdomains comprising the PM, caveolae, and ER/SR were analysed using focused ion beam/scanning electron microscopy (FIB/SEM) to provide new insights into ICC functional analysis. Additionally, as mitochondria could regulate local Ca²⁺ concentration, mitochondria-associated membrane (MAM) contacts with the ER were also analysed in ICCs. Novel ER ultrastructures with distinct characteristics and distributions were identified in each cell type by FIB/SEM. Furthermore, reconstructed three-dimensional (3D) images enabled measurement of the distance between the PM and ER, as well as the MAM areas, thus contributing to a better understanding of ICC physiological features. These new morphological insights may help resolve controversial interpretations in physiological and pharmacological studies of ICCs.

To limit the damage caused by pathogenic bacteria, host organisms possess different defense systems and mechanisms for preventing infection, combating the pathogens, and creating a memory that will avert recurrent infections. Pathogens, on the other hand, have developed countermeasures to enable their replication and spreading. For intracellular pathogenic bacteria, the battleground is localized at the cellular level. Different cell types, including phagocytic, epithelial, and endothelial cells, fibroblasts, and trophoblasts, not only are equipped with diverse defense tools, but also provide different microenvironments, such as varying oxygen tension, pH levels, tonicity, and nutrient supply. The outcome of the infection depends on these conditions in conjunction with microbe-derived virulence factors and bacterial microenvironment needs. Here, we will review the current knowledge on how eukaryotic cells fight obligate intracellular bacteria and how these pathogens counteract the host cell defenses, focusing on cell death pathways. Whereas common cellular strategies for dealing with intracellular bacteria exist, there are also unique approaches adjusted to the individual properties of the pathogen.

Infection induces the aggregation of hemocytes on the dorsal vessel of mosquitoes. These hemocytes, called periostial hemocytes, phagocytose pathogens and produce immune factors on the abdominal portion of the dorsal vessel, called the heart. One of these immune factors, nitric oxide, is a pleiotropic free radical that is an antimicrobial and a heartbeat reducer. But nitric oxide is not just produced by hemocytes. It is also synthesized by pericardial cells that flank the heart, and other tissues. To determine whether it is the periostial hemocytes or the pericardial cells that modulate the heart following infection, we chemically ablated the hemocytes using clodronate liposomes and measured immune responses and heart physiology. We demonstrate that clodronate liposomes ablate the sessile hemocytes, including the periostial hemocytes, while leaving the pericardial cells and heart integrity unaffected. Moreover, ablating hemocytes abolishes the phagocytosis of bacteria, alters the deposition of melanized bacteria, and decreases nitric oxide synthase activity on the heart. Importantly, hemocyte ablation eliminates the infection induced reduction of the heart rate, mainly by modifying the anterograde heart rate. Therefore, periostial hemocytes drive immune responses on the heart and infection-induced changes to circulatory physiology.

Glaucoma remains the leading cause of irreversible blindness worldwide, with elevated intraocular pressure (IOP) being the only modifiable risk factor in primary open-angle glaucoma (POAG). Despite adequate IOP control, many patients continue to progress to irreversible optic neuropathy, emphasising the need for alternate treatments. Transforming growth factor-beta (TGF-β) promotes extracellular matrix (ECM) production and fibrosis at the optic nerve head (ONH) in glaucoma. A disintegrin and metalloprotease-12 and metalloprotease-19 (ADAM12 and ADAM19) are implicated in fibrosis. Recent studies have explored miRNA-based manipulation of the TGF-β signalling pathway as a potential therapeutic strategy in fibrosis. This study investigates whether miR-29b modulation affects ADAM12, ADAM19, and ECM gene expression in human lamina cribrosa (LC) cells. Primary human normal lamina cribrosa (NLC) and glaucoma LC (GLC) cells were treated with TGF-β1 and transfected with either a miR-29b mimic or control. Gene expression levels of ADAM12, ADAM19, miR-29b, and several ECM genes were quantified using real-time RT-qPCR, and protein expression levels by Western blotting. ADAM12 and ADAM19 expression was elevated in untreated GLC cells, and treatment with TGF-β1 in both NLC and GLC cells increased ADAM12 and ADAM19 expression. The expression of miR-29b was significantly reduced in both GLC- and TGF-β1-treated NLC and GLC cells. Transfection with miR-29b resulted in a marked reduction in ADAM12 and ADAM19 mRNA expression in TGF-β1-treated NLC and GLC cells. Additionally, miR-29b transfection reduced ECM gene expression in both NLC and GLC under TGF-β1 stimulation. Our results demonstrate that miR-29b plays a crucial role in fibrotic remodelling at the LC by antagonising the effects of TGF-β1 on ADAM and ECM gene expression, representing a novel therapeutic target in glaucoma.

Familial neurohypophysial diabetes insipidus (FNDI) is an autosomal dominant disorder caused by mutations in the arginine vasopressin (AVP) gene. In AVP neurons in a mouse model of FNDI, aggregates of mutant AVP precursors accumulate within a specific compartment of the endoplasmic reticulum (ER). However, as FNDI mice aged, or were exposed to repeated water deprivation, the ER lumen dilated and mutant aggregates dispersed throughout the ER. Meanwhile, autophagic isolation membranes, known as phagophores, emerged to envelop ER containing these aggregates, indicating induction of ER-phagy. Previous in vitro studies showed that phagophores originate from ER membranes, but the structural relationship between phagophores and the ER membrane in vivo remains unknown. In this study, we used serial block-face scanning electron microscopy to investigate the structural relationship between phagophores, ER membranes, and protein aggregates within dilated ER of AVP neurons from FNDI mice subjected to intermittent water deprivation for 4 weeks. Three-dimensional analysis revealed that phagophores enveloped aggregates located within the dilated ER. Serial imaging further demonstrated a physical connection between these phagophores and intact ER membranes. This study provides the first in vivo evidence of the structural continuity between phagophores and the ER membrane in AVP neurons in a mouse model of FNDI.