Cryo electron tomography (cryo-ET) enables three-dimensional visualization of biological macromolecules in their near-native environments, bridging the gap between structural and cellular biology. In this study, we evaluated high-resolution subtomogram averaging (STA) using a cryo-TEM (CRYO ARM^TM 300 II, JEOL) and PACEtomo software system for both single-particle analysis (SPA) type grids and lamellae prepared by cryo-focused ion beam (cryo-FIB) milling (JIB-4700F, JEOL). Using mouse apoferritin and E. coli ribosomes in thin vitreous ice film, resolutions of 2.04 Å and 2.86 Å were achieved, respectively. Using cryo-FIB lamellae of Salmonella cells, in situ ribosome structures were resolved to 8.2 Å resolution.

The determination of protein secondary structure constitutes a fundamental step in understanding protein function, folding mechanisms, and disease pathology. This comprehensive review presents a systematic methodological comparison of the principal biophysical techniques employed for secondary structure elucidation, spanning seven decades of technological development from Pauling and Corey’s foundational discoveries to contemporary high-resolution methods. We examine spectroscopic approaches, including circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy, which provide rapid global quantification of secondary structure content with minimal sample requirements, alongside high-resolution techniques, including nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and cryo-electron microscopy (cryo-EM), which offer atomic-level structural details. For each technique, we analyze theoretical foundations, experimental protocols, deconvolution algorithms, accuracy benchmarks, and optimal application domains. Comparative analysis reveals that no single technique achieves optimal performance across all secondary structure classes: FTIR generally provides improved accuracy for β-sheet-rich systems, particularly when sheet topology discrimination is required. NMR uniquely provides residue-level resolution with dynamics information across multiple timescales, whereas crystallography and cryo- EM define atomic-resolution standards for static structures. Complementary techniques, including small-angle X-ray scattering (SAXS), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and Förster resonance energy transfer (FRET), provide essential orthogonal information on global shape, protection patterns, and distance constraints. We present detailed decision frameworks for technique selection based on sample characteristics and research objectives, propose integrative multi- technique workflows, and identify critical gaps in current methodology, including the need for comprehensive multi-method benchmarking and standardized protocols for challenging protein systems such as membrane proteins and intrinsically disordered proteins. This review provides practical guidance for researchers designing experimental strategies for protein secondary structure characterization in the modern era of integrative structural biology.

The emergence of antibiotic-resistant bacteria highlights the need for novel natural antimicrobial agents. This study aimed to evaluate the chemical composition, antimicrobial activity, and redox-modulatory effects of Tanacetum argyrophyllum essential oil (EO) against Escherichia coli wild-type (E. coli K12) and kanamycin-resistant (E. coli pARG-25) strains. EO extraction yielded 0.1% (v/w), with eucalyptol (35%), camphor (24%), and camphene (17%) as major constituents, thus defining a eucalyptol–camphor chemotype. The minimum inhibitory concentrations were 100 µL/mL for both strains. Biophysical analyses revealed that EO exposure delayed the decline in the extracellular oxidation-reduction potential (ORP) and altered the pH dynamics, thus reflecting interference with bacterial kinetics, metabolic activity, and organic acid production. Biochemical assays indicated strain-specific oxidative stress responses: malondialdehyde formation increased by ~50% in the resistant strain, accompanied by substantial upregulation of superoxide dismutase and catalase activities, whereas the wild-type strain showed moderate changes. These data suggest that T. argyrophyllum EO disrupts bacterial redox homeostasis, likely via the modulation of antioxidant defenses, thus resulting in reduced viability and enhanced oxidative stress in resistant cells. The findings provide some influence modes into EO-mediated bacteriostatic effects and support its potential as a natural agent that targets kanamycin-resistant bacteria through redox-dependent pathways.

Rice bran is rich in bioactive compounds, but its potential for cosmeceutical applications remains insufficiently explored. In this study, we investigated the anti-melanogenic effects of a 70% ethanol extract of rice bran prepared by ultrasonication in distilled water. The extract was evaluated for DPPH radical scavenging activity, cytotoxicity, tyrosinase inhibition, melanin production, and associated signaling pathways in B16F10 murine melanoma cells. The rice bran extract exhibited DPPH radical scavenging activity and showed no cytotoxicity in B16F10 cells. It also inhibited tyrosinase activity in a dose-dependent manner and suppressed melanin synthesis in B16F10 cells. Mechanistically, the extract reduced the TRP1, TRP2, and MITF protein levels, while increasing the phosphorylation of Erk and Akt in B16F10 cells. In addition, treatment with PD98059 or LY294002 attenuated the extract-induced downregulation of MITF and altered MITF phosphorylation. These findings suggest that rice bran extract may serve as a potential cosmeceutical ingredient for controlling melanogenesis, warranting further cosmeceutical investigation.

Approximately 1000 to 4000 genes that encode small proteins (less than 100 amino acids) are still officially uncataloged in the human proteome. These sequences, often called smORFs (small open reading frames), have eluded identification for decades, which has relegated them to the simple category of “noncoding RNA” [1]. These sequences have eluded identification for three main reasons: 1. Arbitrary criteria: Historically, bioinformaticians have set a minimum threshold of 100 amino acids to exclude genetic “noise”. 2. Technical difficulties: Small proteins degrade rapidly and are difficult to detect using standard mass spectrometry. 3. Misclassification: Many are hidden within regions previously classified as “noncoding RNA” (ncRNA) or in segments overlapping with other genes. Today, we know that this “genetic noise” hides precision regulators critical to molecular biology, development, and metabolism [2]. Integrating polymer physics (physical-chemical properties) and deep learning (AlphaFold) is one of the few ways to scale up the study of the 1000−4000 missing microproteins. Without these models, we would have to study them one by one with crystallography, which would take centuries.

Modern nanotechnology focuses on developing environmentally friendly methods for synthesizing nanomaterials. Among these, the biosynthesis of nanoparticles using biological microorganisms has emerged as a promising strategy. In this study, silver nanoparticles (AgNPs) were synthesized using the extracellular secretions of the fungus Aspergillus niger. The fungal strain was successfully isolated from rice wine yeast and identified as Aspergillus niger QNUGT6 based on morphological characterization and sequencing of the internal transcribed spacer (ITS) gene region. The formation of AgNPs was confirmed by a visible color change and the appearance of a characteristic surface plasmon resonance (SPR) band at 410 nm. X-ray diffraction (XRD) analysis revealed diffraction peaks corresponding to crystalline AgNPs. Fourier-transform infrared (FTIR) spectroscopy indicated the involvement of various functional groups in the culture medium responsible for the reduction of Ag⁺ ions to Ag⁰. Scanning electron microscopy (SEM) analysis confirmed that the synthesized AgNPs were spherical with an average size of 26.1 ± 7.8 nm. Moreover, the synthesized AgNPs exhibited inhibitory activity against Bacillus cereus bacterial pathogens. Thus, our study provides further evidence for the potential use of Aspergillus fungi to control the properties of AgNPs.

Epigenetic alterations are increasingly being evaluated as diagnostic biomarkers and risk-stratification tools in surgical pathology and cytopathology. The clinical implementation of epigenetic biomarkers requires more than the demonstration of disease-associated molecular changes. DNA methylation, histone modifications, chromatin remodeling, and non-coding RNAs may provide clinically relevant information, but their clinical value depends on reproducibility, specimen adequacy, analytical robustness, interpretability, and suitability for clinical reporting. These requirements are particularly relevant in cytopathology and molecular testing of body fluid specimens, where specimens may have low cellularity or suboptimal nucleic acid quality, and morphological interpretation may be equivocal. Urothelial carcinoma represents an informative model because urine-based methylation assays illustrate both the potential and the limitations of epigenetic translation. These assays have been explored as adjunctive tools to cytology in selected diagnostic and surveillance settings, without replacing guideline-based cystoscopic follow-up. This editorial introduces the Special Issue “Epigenetics in Diagnostic Pathology: Translational Biomarkers, Technologies, and Clinical Impact” and supports a translational approach grounded in diagnostic pathology, in which epigenetic biomarkers are evaluated according to their capacity to generate reliable diagnostic evidence in routine clinical specimens.