FEBS Open Bio最新文献

In this study, we aimed to investigate the effect of salubrinal (SAL) on endoplasmic reticulum stress via an experimental in vitro heat stress model (HSM) of spermatogenic cells. In order to achieve this, mouse spermatogonium (GC1) and spermatocyte (GC2) cell lines were used. The IC50 dose of SAL was calculated using an MTT assay. Each cell line was separated into four different groups: control (GC1C, GC2C), SAL-treated (GC1SAL, GC2SAL), experimental HSM (GC1HSM, GC2HSM), and SAL-treated HSM (GC1HSMSAL, GC2HSMSAL). Control cells were incubated under standard culture conditions. HSM group cells were incubated at 43 °C for 60 min. In the SAL group, cells were incubated with 20 μm SAL-containing culture medium for 24 h. Following treatment, all groups were stained with immunofluorescence probes for p-PERK, ATF6, GRP78, p-IRE1α, p-eIF2α, and HSP70 antibodies. Moreover, the mRNA levels of GRP78, PERK, and eIF2α were evaluated via qRT-PCR. We observed that HSM cells showed cytotoxic effects as all markers showed elevated immunoreactivity levels, which were attributed to ER stress. SAL treatment decreased levels of ER stress. Furthermore, GRP78, PERK, and eIF2α mRNA levels were upregulated in the HSM group and although there was a downregulation following SAL treatment, the difference was not statistically significant. In light of these findings, we concluded that heat stress triggers ER stress in spermatogenic cells, and SAL might affect ER stress markers. Further studies on ER-related stress mechanisms in spermatogenic cells will be critical in developing therapeutic strategies with advanced molecular analyses in the future.

Advanced microscopy techniques, combined with a diverse set of fluorescent probes, provide valuable tools for uncovering insights into biological systems and addressing fundamental research questions. However, the need to develop and use genetic tags and probe markers presents notable challenges. Coherent Raman scattering microscopy offers a label-free alternative, enabling live-cell imaging of cellular structures without the need for labeling. Leveraging the benefits of Raman microscopy, we aim to analyze the pancreas in living zebrafish larvae and to evaluate chemical changes in pancreatic exocrine and endocrine compartments following exocrine damage. Here, we present a protocol for Raman-based label-free microscopic analysis of the pancreas in living zebrafish larvae. Using forward stimulated Raman scattering (F-SRS) and epi coherent anti-Stokes Raman scattering (E-CARS), zebrafish pancreatic structures were analyzed and validated. Vibrational Raman spectra between 450 and 3100 cm^-1 were acquired to identify chemical structural features within pancreatic regions. Raman imaging allows discrimination of distinct structures at 2850 and 2934 cm^-1 in pancreatic exocrine and endocrine regions, which could mainly correspond to lipids and proteins, respectively. Exocrine damage causes a significant reduction in both the number and size of exocrine granules. Moreover, changes at 2934 cm^-1 suggested chemical alterations in both exocrine and beta-cell regions. In conclusion, SRS and CARS provide a powerful, label-free approach for live-cell imaging and chemical analysis in islet biology. Given the relative straightforward applicability in the pancreas, we anticipate broad implementation of Raman microscopy in other organs and across various biomedical research fields.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder caused by mitochondrial DNA mutations in complex I of the respiratory chain, leading to impaired ATP production, mitochondrial fragmentation, and oxidative stress that contribute to vision loss. This study investigated the potential repurposing of metformin, a widely used antidiabetic drug, in fibroblasts from LHON patients carrying the m.11778G>A mutation. Fibroblasts from LHON patients and healthy individuals were treated with metformin, and mitochondrial function was assessed using high-content imaging, biochemical assays, immunoblotting, immunofluorescence, and Seahorse analysis. Metformin reduced mitochondrial fragmentation, increased network length, stabilized mitochondrial membrane potential, enhanced ATP production, and lowered ROS accumulation under oxidative stress. Metformin significantly increased mitophagy and autophagic flux, as shown by LC3B puncta quantification with and without chloroquine, and activated AMPK signaling through increased AMPKα1/2 phosphorylation and AMPKβ1 Ser182 phosphorylation. In addition, metformin promoted PGC-1α nuclear translocation, indicating stimulation of mitochondrial biogenesis, while maintaining mtDNA copy number and supporting oxidative phosphorylation. These findings suggest that metformin, at clinically relevant concentrations, enhances mitochondrial health and function in LHON fibroblasts, supporting its potential as an affordable and safe therapeutic option to mitigate vision loss in LHON.

The sirtuin SIRT4 has been implicated in the control of autophagy and mitochondrial quality control via mitophagy. However, the role of SIRT4 in regulating autophagy/mitophagy induced by different stressors is unclear. Here, we show that cells expressing SIRT4(H161Y), a catalytically inactive, dominant-negative mutant of SIRT4, fail to upregulate LC3B-II. These cells also exhibit a reduced autophagic flux upon treatment with different inducers of mitophagy/autophagy, that is, CoCl₂-triggered pseudohypoxia, CCCP (carbonyl cyanide 3-chlorophenylhydrazone)/oligomycin-mediated respiratory chain inhibition, or rapamycin treatment. Interestingly, SIRT4(H161Y) expression upregulated protein levels of HDAC6, which is involved in mitochondrial trafficking and autophagosome-lysosome fusion, and inhibited the conversion of OPA1-L to OPA1-S, which is associated with increased mitochondrial fusion and decreased mitophagy. Both HDAC6 and OPA1 are SIRT4 interactors. However, the pharmacological inhibition of HDAC6 using Tubacin or of OPA1 using MYLS22 did not restore the stress-induced upregulation of LC3B-II levels upon autophagy/mitophagy treatment in SIRT4(H161Y)-expressing cells. Remarkably, inhibition of autophagosome-lysosome fusion and thus disruption of late autophagic flux by BafA1 treatment also failed to restore LC3B-II levels upon autophagy/mitophagy treatment, suggesting an inhibitory effect of SIRT4(H161Y) on the initiation/early phase of autophagy. Consistent with this, we demonstrate that SIRT4(H161Y) promotes the phosphorylation of ULK1 at S638 and S758 (mTORC1 targets), both of which mediate an important inhibitory regulation of autophagy initiation. Thus, our data suggest a positive regulatory function of SIRT4 in the ULK1-dependent early regulation/initiation of stress-induced autophagic flux, presumably via modulation of AMPK/mTORC1 signaling.

Chemotherapy-induced alopecia (CIA) is a major unresolved adverse effect in clinical oncology. We have previously shown that the Sonic hedgehog (Shh) signaling pathway is targeted by cyclophosphamide (CYP) treatment, but the detailed mechanism by which this chemotherapy drug induces alopecia still remains largely unknown. To answer this question, in the present study, we used Shh-GFP^+/- mice and analyzed Shh-expressing cells (Shh⁺ cells) in hair follicles at different times post-CYP treatment. Through flow cytometry assays, we showed that Shh⁺ cells decreased significantly after CYP treatment. To investigate the molecular events involved in this decrease, we carried out gene set enrichment analysis of RNA sequencing data of Shh⁺ cells, which revealed that the expression levels of most Janus-activated kinase/signal transducer and activator of transcription 1 (JAK/STAT1) signaling pathway-related genes were upregulated compared to the controls. Furthermore, through a chromatin immunoprecipitation assay, we showed that STAT1 could bind to the promoter of the Shh gene during CIA in hair follicles and the binding strength increased upon CYP treatment. Treatment with JAK inhibitors also rescued hair loss and upregulated Shh expression in mice with CIA, further supporting the role of the JAK/STAT1 signaling pathway in the regulation of Shh during CIA. Taken together, our results provide new insights into the molecular mechanisms of CIA.

Engineering antibodies to elicit antibody-dependent cell-mediated cytotoxicity (ADCC) can be used to eliminate target cells. Here, we described how single domain VHH arms on a bispecific antibody (BsAb) format can be engineered to modulate cell binding and ADCC activity. The BsAbs, comprising two anti-epidermal growth factor receptor (EGFR) nanobodies, 7D12 and EGA1, were engineered onto a human IgG1 Fc domain in monovalent, bivalent, and tandem formats. While 7D12 had a stronger ADCC activity than EGA1, the tandem 7D12-EGA1 mediated a significantly stronger ADCC activity without significant changes in cell binding in numerous cancer cell lines. In addition, we present how the molecular design of the tandem 7D12 and EGA1 nanobodies could cross-link two different EGFR molecules to obtain stronger ADCC activity.

Anthracycline-induced cardiotoxicity remains a major limitation in cancer therapy, affecting long-term cardiovascular health in survivors. Dietary nitrate supplementation has shown cardioprotective effects in preclinical models of doxorubicin (Dox)-induced and ischemia-reperfusion injury, but it is unclear whether nitrate and/or nitrite (NOx) would have adverse effects on the anticancer efficacy of the drug. To evaluate Dox efficacy against triple-negative breast cancer (TNBC) in the presence of dietary nitrate and nitrite, tumor-bearing BALB/c mice (N = 5 mice per group, 10 mice total) were treated with four weekly intravenous doses of Dox with or without NOx supplementation of their drinking water. Cardiac tissue from the NOx-treated mice exhibited less fibrosis and lower levels of 4-hydroxynonenal-modified proteins, a marker of lipid oxidation and oxidative stress. Tumor sizes varied, but most regressed by the final Dox dose. Importantly, NOx supplementation did not compromise the antitumor efficacy of Dox nor did it promote pulmonary metastasis; instead, a trend toward fewer metastatic lesions was observed. These findings support the potential clinical use of dietary nitrate and nitrite as adjuncts to Dox treatment to mitigate cardiotoxicity without impairing anticancer outcomes.

RETRACTION: F. Chen, K. Zhang, Y. Huang, F. Luo, K. Hu, and Q. Cai, “SPC25 May Promote Proliferation and Metastasis of Hepatocellular Carcinoma via p53,” FEBS Open Bio 10, no. 7 (2020): 1261-1275. https://doi.org/10.1002/2211-5463.12872.

The above article, published online on 29 April 2020 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, Miguel De la Rosa; the Federation of European Biochemical Societies; and John Wiley & Sons Ltd., UK. A third party reported that images in Figures 11A, 11B, and 11C had been published in other articles both prior to and after the publication of this article and are also listed as representing different experiments [Ling et al. 2019 (https://doi.org/10.3892/ijo.2019.4926)]; [Ke et al. 2020 (https://doi.org/10.1186/s12935-020-01474-7)]; and [Ling et al. 2021 (https://doi.org/10.18632/aging.203318)]. Additionally, the Migration and Invasion images for si-SPC25 contain an overlapping section. An investigation by the journal confirmed these instances of image duplication and also discovered that the HepG2 and SMMC-7721 cell lines discussed in this article are known to be misclassified and contaminated, respectively. The authors did not respond to an inquiry and request for original data by the publisher.

The retraction has been agreed to because the evidence of image duplication across multiple publications, in which the images are reported as different samples by different author groups, fundamentally compromises the editors’ confidence in the results presented. As a result, the Editor-in-Chief, FEBS Press, and John Wiley and Sons Ltd. have determined that a retraction is necessary. The authors did not respond to our notice regarding the retraction.

Polyamines are present in all living cells and are implicated in various crucial cellular processes such as proliferation, apoptosis and autophagy. In contrast, excess amounts of polyamines can be toxic to cells. ATP13A2 was recently identified as a mammalian polyamine transporter in neuronal cells. In this study, we attempted to characterize the function of ATP13A2 in cultured human lung epithelial cells. The data demonstrated that ATP13A2 is endogenously expressed in mouse lungs, and in cultured lung epithelial cells, the expression level of ATP13A2 drastically changes over culture time and peaks in the logarithmic phase during the proliferation curve. ATPase activity in ATP13A2-enriched microsomes from lung cells showed polyamine dependence, as previously reported for other cell types and species. Overexpression of ATP13A2 caused a moderate increase in total cellular polyamine content, whereas ATP13A2 knockdown resulted in a decrease in cellular polyamine levels. These findings provide novel information regarding the cellular function of ATP13A2 in lungs and contribute to our understanding of cellular polyamine transport systems.

Salty taste is mediated through two distinct transduction pathways: the amiloride-sensitive and amiloride-insensitive (AI). Transmembrane channel-like 4 (TMC4), a voltage-dependent chloride channel, has recently been identified as playing a critical role in AI-mediated salt taste responses. Although its functional properties have been demonstrated, the specific taste cell types that express TMC4 have not been fully characterized. To investigate the cellular localization of TMC4, we generated Tmc4-EGFP knock-in mice and performed immunohistochemical analyses of the taste papillae. By performing multiple fluorescent immunostaining with cell-type-specific markers, such as KCNQ1 (type I-III), Gustducin and PLCβ2 (type II), and AADC (type III), together with EGFP, we found that over 98% of EGFP-positive cells overlapped with the signal of all three taste cell type markers in the circumvallate papillae of Tmc4-EGFP mice. In the fungiform papillae, 95.9% of EGFP-positive cells colocalized with KCNQ1. These results demonstrate that TMC4 is broadly expressed across all three taste cell types, and suggest the possible involvement of multiple, functionally distinct taste cell populations in AI-mediated salty taste transduction.