Optical microscopes are essential equipment for observing and analysing cultured cells. In observations using a normal optical microscope, as the contrast of cells is rather low, phase contrast and differential interference microscopes are commonly used. In some cases, specific proteins in cells are fluorescently labelled and observed using a fluorescence microscope. In such observations, cells are generally cultured on a glass surface and observed through the glass. Therefore, spherical aberration occurs due to the refraction of light on the glass surface. This spherical aberration becomes larger when using an objective lens with higher magnification and higher numerical aperture, resulting in a decrease of spatial resolution and contrast. Here, we found that by observing cells cultured on an extremely thin silicon nitride (SiN) film (50 nm thick) under a conventional optical microscope, we could directly observe the cells at high resolution without spherical aberration. This improvement of spatial resolution was confirmed with both inverted and upright optical microscopes. Using a normal 100 × objective lens without oil immersion, we were able to directly observe melanosomes in melanoma cells and analyse their dynamic movement. We believe that using our original sample holder with thin SiN film will make it easy to observe live cells at high resolution and that this method will contribute to a wide range of biological research.

Quercetin alleviates imatinib-induced premature ovarian insufficiency by regulating mitophagy via the ROS/JNK/c-JUN pathway.

Mechanistic exploration of methylglyoxal-induced hepatotoxicity involving oxidative stress, apoptosis, and gluconeogenic modulation.

The cinnamaldehyde-thiosemicarbazone-zinc (II) complex induces apoptosis in CAL-27 cells.

The mechanism study of isoorientin regulating neuroinflammation after subarachnoid hemorrhage through AKT/GSK3β.

Therapeutic targeting of oxidative-inflammatory crosstalk by Apelin-12 in neonatal hyperoxia-induced lung injury.

The in vitro effect of omentin-1 on the global proteome of granulosa cells from normal weight Large White and fat Meishan pigs.

Lawsonia Intracellularis (L. intracellularis) is an obligate intracellular bacterium that causes porcine proliferative enteropathy. In this study, a pan-RV (reverse vaccinology based on pangenome analysis) approach was applied to analyze the whole-genome sequences of 9 L. intracellularis strains downloaded from the National Center of Biotechnology Information (NCBI) server. Pangenome analysis revealed a closed pangenome and a core genome consisting of 1372 genes, while reverse vaccinology further revealed 16 candidate proteins with higher in silico immunogenicity parameters, including predicted antigenicity, "outer membrane protein" or "extracellular protein" by subcellular localization analysis and no more than 2 transmembrane regions by transmembrane helices prediction. Three of the 16 proteins that showed no homology with proteins of other species according to BLAST (Basic Local Alignment Search Tool) were selected for serological validation. The Western blotting results revealed that the 3 proteins did not cross-react with anti-Shigella and anti-Salmonella sera, with 1 protein (LAW_RS03650) showed antigenicity when it reacted with positive wild-type anti-L. intracellularis serum. The immunofluorescence of infected cells employing anti-LAW_RS03650 serum indicated a wide distribution of LAW_RS03650 protein in the host cells and L. intracellularis itself. This study identified a novel L. intracellularis antigen, LAW_RS03650, as a candidate for future recombinant vaccine development or species-specific serodiagnostic reagents.

This study investigated the molecular mechanisms by which ginsenoside Rg3 combined with ranibizumab alleviates diabetic macular edema (DME), focusing on antagonizing ANGPTL4/VEGF and regulating the NRP/RhoA pathway to reduce vascular permeability. Transcriptomic sequencing compared blood samples from DME patients and healthy controls, followed by GO/KEGG enrichment analysis. In vitro, human retinal microvascular endothelial cells (HRMECs) were treated with ginsenoside Rg3 (5, 10, 20 μM) alone or combined with ranibizumab (1 mg/mL); cell viability, permeability, and protein expression were assessed. In vivo, diabetic rats received intraperitoneal ginsenoside Rg3 and ranibizumab; ocular pathology, angiogenesis, inflammation, and key protein expression/activity were evaluated. DME patients exhibited significant upregulation of VEGF, ANGPTL4, NRP1 (logFC = 1.9, P < 0.01), and RhoA, associated with angiogenesis/migration/inflammation pathways. In vitro, 10 μM ginsenoside Rg3 optimally reduced HRMEC permeability and suppressed ANGPTL4. Combination therapy further decreased VEGF and ANGPTL4 expression. In vivo, combined treatment significantly reduced retinal edema, angiogenesis, and vascular permeability. It markedly inhibited NRP1 expression and reduced RhoA/ROCK activity. The combination of ginsenoside Rg3 and ranibizumab effectively antagonizes ANGPTL4 and VEGF and regulates the NRP/RhoA pathway, significantly reducing vascular permeability in DME through synergistic action. This provides crucial theoretical support for novel DME combination therapy.

Black rhinoceros are critically endangered due to poaching in the wild (in situ). Globally, fewer than 200 animals are maintained as an ex situ insurance population. Unfortunately, the ex situ population faces major sustainability challenges from disease syndromes characterized by high inflammatory burdens and diverse manifestations of immunometabolic dysfunction, not known to be present among their wild counterparts. Overlapping ex situ disease phenotypes limit diagnostic specificity and highlight the need to define underlying disease mechanisms. In the present study, using a cohort of presumed clinically healthy and inflammatory black rhinoceros, we generated the first immunoproteomic profile of any endangered mammal species and identified 1,311 immune cell proteins. However, no significant differences were detected among clinical phenotypes. Therefore, we applied unsupervised machine learning approaches to detect molecular features suggestive of healthy versus inflammatory phenotypes. Forty-three proteins associated with inflammatory pathways were differentially expressed in a cohort of samples derived from both presumed healthy and inflammatory phenotypes. Results suggest subclinical disease may be relatively widespread ex situ, and that animals experience temporal fluctuations in inflammatory state over time. Findings implicate neutrophil degranulation and dysregulation of the oral-gut-liver axis as drivers of disease syndromes of ex situ black rhinoceros. The forty-three proteins associated with inflammatory pathways represent candidate inflammatory biomarkers to be assessed for clinical applications in future validation studies. Upon validation, these candidate biomarkers may guide management practices to strengthen long-term population sustainability.

CFIm25, a key component of the cleavage factor Im (CFIm) complex needed for mRNA 3' end processing, shows increased protein expression during monocyte-to-macrophage differentiation despite stable mRNA levels. We demonstrate that poly(C)-binding protein 1 (PCBP1) suppresses CFIm25 translation in monocytes by binding to its long 3' untranslated region (UTR). During differentiation, alternative polyadenylation generates a shorter CFIm25 3'UTR lacking PCBP1 binding sites. RNA immunoprecipitation confirms PCBP1 binding to the long 3'UTR, while ribosome association analysis shows enhanced ribosome recruitment upon PCBP1 depletion. PCBP1 knockdown increases CFIm25 protein in undifferentiated cells and induces macrophage differentiation markers without stimulation. These findings reveal how alternative polyadenylation controls CFIm25 expression during immune cell differentiation by modulating RNA-binding protein interactions and provide insight into post-transcriptional regulation of RNA processing factors. Impact statement This work reveals how a key regulator of mRNA processing is itself controlled through a previously uncharacterized mechanism during immune cell differentiation. Our findings provide insights into the molecular circuits governing macrophage development and identify potential therapeutic targets for inflammatory disorders where myeloid cell differentiation is dysregulated.

We propose an innovative technology based on the combination of Raman microspectroscopy and deep learning to classify the Mechanism of Action (MoA) of antimicrobials and predict their novelty. Raman microspectroscopy provides chemical and physical signatures of molecular structures in bacteria to reveal phenotypic responses to antimicrobials. Deep learning techniques are powerful tools to extract discriminative features from complex Raman spectra and classify them. We developed the RaMoA technology and assessed its performance exclusively on the wild-type Escherichia coli (E. coli) ATCC 25922, after 1 h of treatment with 27 antibiotics representing 5 conventional functional classes (i.e., 12 cell wall synthesis inhibitors, 9 protein synthesis inhibitors, 3 DNA replication inhibitors, 2 RNA synthesis inhibitors, and 1 cell membrane function inhibitor). First, using preprocessed Raman spectra as input to a 1D Convolutional Neural Network, we classified Raman spectra of the treated bacteria into the correct MoA class with 96% accuracy. Aggregation of spectra predictions led to the correct MoA assignment for 100% of the antibiotics. Second, we showed how such a reference spectral dataset and autoencoder architecture could address a more difficult task: assessing the novelty of the MoA of a candidate antibiotic. After moving the single cell membrane inhibitor (i.e., colistin), one of the cell wall (i.e., cefazolin), and one of the protein (i.e., chloramphenicol) synthesis inhibitors to the test set, our tool successfully assigned colistin as a novel MoA, while cefazolin and chloramphenicol were rightly identified as cell wall and protein synthesis inhibitors, respectively.

Single cell protein profiling of focal cortical dysplasia in a patient requiring multiple resections.

Amino acid metabolism, demand and supply in Chinese Hamster ovary cell culture - A comprehensive literature review.

The in-situ upcycling of decentralized methane and nitrogen gas (N2)-derived ammonia via methanotrophic bacteria is highly attractive. However, the toxic intermediate generated from ammonia oxidation significantly inhibits cell growth, thereby hindering efficient bioproduction. Herein, by integrating transcriptomic analysis, we develop rational metabolic engineering strategies and an optimized fed-batch fermentation to enhance ammonia utilization in a methanotrophic bacterium of Methylotuvimicrobium sanxanigenens. The modified M. sanxanigenens overexpressing hydroxylamine reductase efficiently co-assimilates methane and ammonia for cell protein synthesis, with an 18-fold increase in productivity. The resulting methanotrophic cell protein (MCP) not only exhibits an ideal essential amino acid profile but also contains bioactive nutrients, including polysaccharides and peptides. Oral administration of this nutritional MCP significantly ameliorates colitis symptoms in male mice by attenuating inflammatory progression and restoring the intestinal barrier. Moreover, MCP treatment maintains gut microbiota homeostasis and promotes the excretion of beneficial metabolites, thereby protecting the intestinal microenvironment. Hence, this study provides a promising biological approach for the local bio-valorization of decentralized CH4 and air into functional feed additives. This biotechnology not only facilitates advancements in developing carbon-negative gas-to-value pathways but also drives green transformations in animal husbandry by reducing the use of antibiotics and vaccines.

The development of rCHO cell lines that stably express therapeutic proteins is crucial for pharmaceutical protein industrial production. In this study, a systematic method was established to identify genomic hotspots for exogenous protein expression in CHO cells and construct stable recombinant CHO cell strains. Four stable monoclonal cell lines (1b7, 1d2, 2d9, and 2f7) were obtained by using the lentiviral random integration reporter gene. Chromosome mapping analysis found four stable integration sites: chr1_0 (7,30,83,299-7,32,45,508 bp) in 1b7, chr1_0 (17,69,68,187-17,69,68,191 bp) in 1d2, chr3 (4,08,81,262-4,08,99,858 bp) in 2d9, and chr5 (1,69,77,575-1,70,61,744 bp) in 2f7. Based on these sites, we developed recombinant CHO cells capable of long-term stable expression of foreign proteins through the combined application of CRISPR/Cas9 technology and Bxb1 recombinase-mediated cassette exchange. Utilizing "promoter capture technology", all screened LP cell monoclonal lines can express exogenous proteins, with the entire construction process completed in just 2∼3 weeks.

Cinnamaldehyde (CA), a bioactive compound from cinnamon, exhibits diverse pharmacological activities including anti-inflammatory and anticancer effects. However, comprehensive exploration of the target landscape of CA at site-specific resolution remains challenging. Herein, we developed MOSCAT (MethOxyamine-enabled Site-specific Cinnamaldehyde Tagging), a probe-free chemical proteomic strategy for mapping cinnamaldehyde-targeted proteins in living cells. Unlike existing probe-based approaches requiring synthetic CA derivatives, MOSCAT directly captures native CA-protein adducts by exploiting the intrinsic aldehyde functionality of CA, providing unbiased target profiling with residue-level resolution. Using MOSCAT, we identified 632 CA-modification sites across 480 proteins in human cells. Remarkably, over 70% of these sites overlap with other post-translational modifications, particularly S-nitrosylation and S-sulfenylation, revealing mechanistic links to the anti-inflammatory activity of CA. Notably, we discovered that CA covalently modifies Cys93 of GPX4, a conserved residue critical for ferroptosis regulation. This modification triggers proteasome-mediated GPX4 degradation, identifying a specific covalent engagement site associated with CA-induced ferroptosis. Our findings demonstrate MOSCAT as a powerful platform for elucidating molecular mechanisms of electrophilic natural products and highlight GPX4 Cys93 as a promising druggable site for CA-based therapeutic interventions.

Plant derived exosome-like nanoparticles (PELNs) are emerging as a powerful tool for treating cancers. Among them, PELNs derived from traditional Chinese medicines have shown great potential in treating various cancers. However, many Chinese medicines remain to be explored, and currently, there are no reports on the use of PELNs for treating prostate cancer. In this study, we extracted Huangqi derived exosome-like nanoparticles (HELNs) from fresh Huangqi (Astragalus membranaceus) and systematically explored the effects and mechanisms of HELNs in treating prostate cancer through an integrated approach of single-cell sequencing, 16S rDNA sequencing, and bulk-RNA sequencing. We found that HELNs demonstrated robust cytotoxic effects against prostate cancer both in vitro and in vivo. HELNs reverse the polarization of M2 macrophages, promote their M1-like polarization, and increase the abundance of anti-tumor probiotics in the gut to exert anti-tumor effects. In terms of direct effects, HELNs downregulate the expression level of GPX4 protein in prostate cancer cells, thereby inducing ferroptosis. Finally, we loaded siGPX4 into HELNs to construct a new nanoparticle siGPX4@HELNs. siGPX4@HELNs induce more pronounced ferroptosis in prostate cancer cells, achieving better therapeutic outcomes while maintaining safety.

Brazzein is a sweet-tasting protein with high stability across a wide range of pH and temperature conditions. This study aimed to develop a simplified peptone-based medium (PSM) for the recombinant expression of brazzein in Pichia pastoris X-33 and to evaluate the effect of two inoculum concentrations (5%, 10%, and 15%) on cell growth and protein production in flask fermentations. Subsequently, fermentation was scaled up to a 2 L bioreactor using PSM and a 10% inoculum, achieving a yield of 0.196 g·L−1 after 216 h of induction. These results demonstrate that the PSM medium promotes robust biomass growth and efficient brazzein expression, representing a cost-effective alternative to conventional complex media. Additionally, the effect of pH (5.0, 5.5, and 6.0) and temperature (20, 25, and 28 °C) on brazzein production was evaluated, revealing that fermentation at pH 5.0 and 28 °C resulted in the highest protein concentration (0.422 g·L−1, unpurified). Finally, kinetic models based on the Monod and Luedeking–Piret equations were developed to describe the relationship between biomass formation, substrate consumption, and recombinant protein production.

Drosophila melanogaster S2 cells are widely used as an in vitro model system and have undergone extensive adaptation during long-term culture. Understanding how their transcriptional programs differ from in vivo tissues is essential for interpreting their biological characteristics and experimental utility. This study aimed to characterize transcriptomic differences between Drosophila S2 cells and newly hatched larval tissues, with a focus on identifying metabolic, regulatory, and proliferative features associated with the long-term maintenance of S2 cells in vitro. RNA sequencing was performed on S2 cells and newly hatched larvae. Differentially expressed genes (DEGs) were identified using edgeR (FDR < 0.05, |log2FC| ≥ 1). Gene Ontology (GO) and KEGG enrichment analyses were used to investigate functional changes. Protein-protein interaction (PPI) networks were constructed based on STRING data to identify hub genes, and selected genes were validated using quantitative real-time PCR (qRT-PCR). A total of 5,937 DEGs were detected between S2 cells and larval tissues. S2 cells displayed pronounced upregulation of genes linked to amino acid metabolism, lipid biosynthesis, cell cycle progression, protein turnover, and RNA interference pathways, whereas genes associated with development and differentiation were broadly downregulated. PPI analysis highlighted 10 hub genes-including P5CS, GluProRS, ND-ACP, Ubi-p63E, and Dcr-2-that represent central nodes in metabolic regulation, protein homeostasis, transcriptional control, and stress response. These features collectively reflect a transcriptional state shaped by long-term in vitro adaptation. This comparative analysis provides a comprehensive overview of transcriptomic and regulatory differences between S2 cells and in vivo larval tissues. The results clarify key molecular characteristics of S2 cells and offer a useful reference for their application in functional genomics, metabolism research, and cell-based assays.