FEBS Open Bio最新文献

Herbal and dietary supplements (HDS) are popular among consumers seeking a 'natural' approach for improving their health; however, at present, there is a lack of evidence to support the claims of efficacy and safety for most of these products. Herbal weight loss supplements (WLS) are a group of HDS that are frequently implicated in cases of toxicity; however, the causative substances often remain unknown due to the complex chemical nature of such supplements. This study aimed to analyse the in vitro safety (in human liver carcinoma (HepG2) cells and colon carcinoma (Caco-2) cells) of 12 active compounds commonly found in WLS, first with safety screening using the MTT cytotoxicity assay, followed by metabolic profiling with ¹H NMR spectroscopy. Of the phytochemicals evaluated, epigallocatechin-3,0-gallate (EGCG) was the only compound that caused a significant reduction in the viability of both cell lines (25.3% in HepG2 cells and 18.5% in Caco-2 cells), and this decrease was potentiated by CYP450 induction with rifampicin. Subsequent ¹H NMR analysis showed changes in key metabolites such as amines, amino acids, carboxylic acids, and glucose that were indicative of protein degradation and disrupted energy and lipid metabolism. While the remaining 11 active compounds analysed did not demonstrate significant toxicity in isolation, these require further assessment to determine their safety when used in combination with other phytochemicals. Given that the majority of WLS contain multiple herbal ingredients, each with a complex chemical composition, it is important to understand the role of interactions in adverse events.

Fluorescence recovery after photobleaching (FRAP) is a quantitative technique to study the dynamics of fluorescently tagged proteins in living cells. Current FRAP workflows are limited in throughput because of the requirement for human interaction. Here, we present RoboMic, a fully automated confocal microscopy platform for high-throughput imaging assays such as FRAP. We demonstrate its capabilities using two complementary approaches: sequential FRAP (sFRAP) and a novel parallel FRAP (pFRAP). The latter enables simultaneous photobleaching and monitoring of multiple cells within one imaging cycle, increasing throughput by approximately five- to 10-fold while maintaining spatiotemporal resolution. The protocol consists of microscope control software for automated, AI-based selection and segmentation of cell nuclei, sub-nuclear ROI definition, photobleaching, and time-lapse imaging. As proof of concept, we examined the nuclear dynamics of the androgen receptor and the cohesin complex under diverse conditions, demonstrating that RoboMic generates robust and reproducible data. In a single session, the platform yields hundreds of FRAP measurements, thereby increasing statistical power and scalability for large-scale studies of protein mobility. While we focus here on FRAP, RoboMic can be readily applied to a wide range of quantitative functional imaging assays.

N⁶-methyladenosine (m⁶A) modifications accelerate microRNA (miRNA) biogenesis by promoting the processing of m⁶A-modified primary miRNAs (pri-miRNAs). However, the regulatory mechanism of m⁶A modification of pri-miRNA remains unclear. Here, we found that NF90-NF45 acts as a negative regulator of the m⁶A modification of pri-miRNA by methyltransferase-like 3/14 (METTL3/14). Using overexpression constructs, METTL3/14 promoted the biogenesis of miR-7, whereas NF90-NF45 suppressed miR-7 biogenesis. METTL3/14 overexpression relieved the inhibition of miR-7 biogenesis by NF90-NF45. NF90-NF45 attenuated m⁶A modification of pri-mir-7-1 in vitro; however, it had no effect on the m⁶A modification of pri-mir-200a because of the lower binding affinity of pri-mir-200a to NF90. Furthermore, NF90-NF45 did not interact with METTL3/14, according to immunoprecipitation analysis. These findings suggest that the m⁶A modification of pri-miRNAs by METTL3/14 is regulated by NF90-NF45 competing for pri-miRNA binding.

Poly(ADP-ribose) glycohydrolase (PARG) is a key enzyme involved in poly(ADP-ribose) (PAR) degradation and is considered a potential anticancer target. We previously investigated resistance mechanisms to the PARG inhibitor PDD00017273 in human colorectal cancer HCT116 cells and established an acquired PDD00017273-resistant HCT116R^PDD cell line. In this study, we analyzed the protein levels of enzymes associated with PAR metabolism in both parental HCT116 cells and resistant HCT116R^PDD cells using western blotting. PARG expression levels were similar between HCT116R^PDD and HCT116 cells. However, the levels of PARP1 and ARH3 were reduced in HCT116R^PDD cells compared to HCT116 cells. Nevertheless, intracellular PAR levels were elevated in HCT116R^PDD cells. Interestingly, HCT116R^PDD cells exhibited greater sensitivity to γ-ray irradiation and the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866 than the parental HCT116 cells, yet showed comparable sensitivity to 5-FU, cisplatin, and PARP inhibitors olaparib, talazoparib, and veliparib. Furthermore, we observed that HCT116R^PDD cells tended to maintain slightly higher levels of intracellular NAD⁺/NADH and ATP compared to parental HCT116 cells. These findings suggest that cancer cells employ a mechanism to regulate NAD⁺ and ATP levels, thereby avoiding cell death from intracellular PAR accumulation through coordinated PARP-PARG regulation.

Previously, we reported that netoglitazone, a thiadolidinedione, enhanced both adipogenesis and osteoblastogenesis, and that fatty acid synthase knockdown could selectively repress the adipogenic effect. Here, molecular evidence further demonstrated that pioglitazone enhanced osteoblastic differentiation at least through the protein kinase A/glycogen synthase kinase 3β signaling. (-)-Epigallocatechin gallate (EGCG), a fatty acid synthase inhibitor, selectively retained pioglitazone's pro-osteoblastic effect. Cultures of aged human bone marrow mesenchymal stem cells (bmMSCs) in alginate scaffolds revealed that pioglitazone and EGCG cooperatively enhanced osteoblastic differentiation, biological apatite production, and bone-like tissue maturation. These findings demonstrate that the combination of pioglitazone and EGCG is a promising strategy to enhance osteogenic performance in aged bmMSCs for the development of advanced bone graft materials.

Colorectal cancer (CRC) incidence and mortality continue to rise globally and new prognostic biomarkers are required for the development of targeted therapies. Several studies have suggested that tissue kallikrein-related peptidases (KLKs), including KLK7, contribute to tumorigenesis. We previously demonstrated KLK7's tumor-promoting role both in vitro and in vivo, but its role in CRC metastasis remains unclear. Here, using the Cancer Genome Atlas (TCGA), we confirmed that KLK7 expression is upregulated in advanced stages of CRC and its association with shorter progression-free survival (PFS) of patients. To further understand the role of KLK7 in CRC metastasis, we assessed its expression in ascites from CRC patients with peritoneal metastasis (PM), investigated cell behavior following KLK7 overexpression, and examined its role in metastasis using a mouse model. High KLK7 levels were found in malignant ascites, but not in benign ascites. In xenograft models, KLK7-overexpressing cells increased PM and exhibited higher Peritoneal Cancer Index (PCI) scores compared to controls. In vitro, KLK7 overexpression in HT29-D4 human colon cancer cells significantly enhanced cell proliferation, colony formation, migration, spheroid formation, and adhesion to extracellular matrix proteins. Additionally, KLK7 overexpression altered cell morphology, upregulated moesin (MSN) and integrin subunits, suggesting cytoskeletal remodeling and matrix interactions. Taken together, these findings suggest that KLK7 is a driver of CRC progression and could serve as a potential prognostic marker for aggressive forms of CRC.

The genetic basis underlying nontuberculous mycobacteria (NTM) pathogenesis remains poorly understood. This gap in knowledge has been partially filled over the years through the generation of novel and efficient genetic tools, including the recently developed CRISPR interference (CRISPRi) technology. Our group recently capitalized on the well-established mycobacteria-optimized dCas9_Sth1-mediated gene knockdown system to develop a new subset of fluorescence-based CRISPRi vectors that enable simultaneous controlled genetic repression and fluorescence imaging. In this Research Protocol, we use Mycobacterium smegmatis (M. smeg) and Mycobacterium abscessus (M. abs) as NTM model species and provide simple procedures to assess CRISPRi effectiveness. We describe how to evaluate the efficacy of gene silencing when targeting essential genes but also genes involved in smooth-to-rough envelope transition, a critical feature in NTM pathogenesis. This protocol will have a broad utility for mycobacterial functional genomics and phenotypic assays in NTM species.

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.