Heterogeneity in gene expression largely stems from the discontinuous nature of transcription, with transcripts being produced in bursts with defined frequencies. This cell-to-cell variability in transcription within isogenic cell populations is a known phenomenon across numerous genes. Multiple gene regulatory and epigenetic factors have been identified as key contributors to this pulsatile gene activity. Understanding the effects of epigenetic modulation on transcriptional cell-to-cell variability and kinetics of transcriptional activity is crucial for interpreting changes in treatment responsiveness. We present a detailed protocol that guides the assessment of fluctuations in gene expression induced by epigenetic modulation using single-molecule RNA in situ hybridization (smRNA FISH) combined with confocal microscopy imaging, data analysis, and quantification in breast cancer cells. Through smRNA FISH labeling, both mature and nascent transcripts are identified. Subsequently, the number of mature transcripts and the intensity and frequency of nascent transcripts are quantified, and these measurements are used to calculate the burst size and frequency for the labeled gene. By following this step-by-step methodology, insights are obtained into the intricate relationship between epigenetic alterations and the dynamic nature of gene expression in breast cancer cells.

Lipid droplets (LDs) are organelles that are central to lipid and energy homeostasis across all eukaryotes. In the malaria-causing parasite Plasmodium falciparum the roles of LDs in lipid acquisition from its host cells and their metabolism are poorly understood, despite the high demand for lipids in parasite membrane synthesis. We systematically characterised LD size, composition and dynamics across the disease-causing blood infection. Applying split fluorescence emission analysis and three-dimensional (3D) focused ion beam-scanning electron microscopy (FIB-SEM), we observed a decrease in LD size in late schizont stages. LD contraction likely signifies a switch from lipid accumulation to lipid utilisation in preparation for parasite egress from host red blood cells. We demonstrate connections between LDs and several parasite organelles, pointing to potential functional interactions. Chemical inhibition of triacylglyerol (TAG) synthesis or breakdown revealed essential LD functions for schizogony and in counteracting lipid toxicity. The dynamics of lipid synthesis, storage and utilisation in P. falciparum LDs might provide a target for new anti-malarial intervention strategies.

Mitochondria are dynamic bioenergetic hubs that become compromised with age. In neurons, declining mitochondrial axonal transport has been associated with reduced cellular health. However, it is still unclear to what extent the decline of mitochondrial transport and function observed during ageing are coupled, and if somal and axonal mitochondria display compartment-specific features that make them more susceptible to the ageing process. It is also not known whether the biophysical state of the cytoplasm, thought to affect many cellular functions, changes with age to impact mitochondrial trafficking and homeostasis. Focusing on the mouse peripheral nervous system, we show that age-dependent decline in mitochondrial trafficking is accompanied by reduction of mitochondrial membrane potential and intramitochondrial viscosity, but not calcium buffering, in both somal and axonal mitochondria. Intriguingly, we observe a specific increase in cytoplasmic viscosity in the neuronal cell body, where mitochondria are most polarised, which correlates with decreased cytoplasmic diffusiveness. Increasing cytoplasmic crowding in the somatic compartment of DRG neurons grown in microfluidic chambers reduces mitochondrial axonal trafficking, suggesting a mechanistic link between the regulation of cytoplasmic viscosity and mitochondrial dynamics. Our work provides a reference for studying the relationship between neuronal mitochondrial homeostasis and the viscoelasticity of the cytoplasm in a compartment-dependent manner during ageing.

The mystery of how human DNA is compactly packaged into a nucleus-a space a hundred thousand times smaller-while still allowing for the regulation of gene function, has long been one of the greatest enigmas in cell biology. This puzzle is gradually being solved, thanks in part to the advent of new technologies. Among these, innovative genome-labeling techniques combined with high-resolution imaging methods have been pivotal. These methods facilitate the visualization of DNA within intact nuclei and have significantly contributed to our current understanding of genome organization. This review will explore various labeling and imaging approaches that are revolutionizing our understanding of the three-dimensional organization of the genome, shedding light on the relationship between its structure and function.

In this study, gold nanoparticles (AuNPs) were bioreduced from Ajuga bracteosa, a medicinal herb known for its therapeutic properties against various diseases. Different fractions of the plant extract were used, including the methanolic fraction (ABMF), the n-hexane fraction (ABHF), the chloroform fraction (ABCF), and the aqueous extract for AuNPs synthesis. The characterization of AuNPs was performed using UV-Vis spectrophotometry, FT-IR, XRD, EDX, and TEM. UV-Vis spectroscopy confirmed the formation of AuNPs, with peaks observed at 555 nm. FT-IR analysis indicated strong capping of phytochemicals on the surface of AuNPs, which was supported by higher total phenolic contents (TPC) and total flavonoid contents (TFC) in AuNPs. XRD results showed high crystallinity and a smaller size distribution of AuNPs. TEM analysis revealed the spherical shape of AuNPs, with an average size of 29 ± 10 nm. The biologically synthesized AuNPs exhibited superior antibacterial, antioxidant, and cytotoxic activities compared to the plant extract fractions. The presence of active biomolecules in A. bracteosa, such as neoclerodan flavonol glycosides, diterpenoids, phytoecdysone, and iridoid glycosides, contributed to the enhanced biological activities of AuNPs. Overall, this research highlights the potential of A. bracteosa-derived AuNPs for various biomedical applications due to their remarkable therapeutic properties and effective capping by phytochemicals. RESEARCH HIGHLIGHTS: This research underscores the growing significance of herbal medicine in contemporary healthcare by exploring the therapeutic potential of Ajuga bracteosa and gold nanoparticles (AuNPs). The study highlights the notable efficacy of A. bracteosa leaf extracts and AuNPs in treating bacterial infections, demonstrating their bactericidal effects on a range of strains. The anti-inflammatory properties of plant extracts and nanoparticles are evidenced through paw edema method suggesting their applicability in managing inflammatory conditions. These findings position A. bracteosa and AuNPs as potential candidates for alternative and effective approaches to modern medication.

WNT/CTNNB1 signaling plays a critical role in the development of all multicellular animals. Here, we include both the embryonic stages, during which tissue morphogenesis takes place, and the postnatal stages of development, during which tissue homeostasis occurs. Thus, embryonic development concerns lineage development and cell fate specification, while postnatal development involves tissue maintenance and regeneration. Multiple tools are available to researchers who want to investigate, and ideally visualize, the dynamic and pleiotropic involvement of WNT/CTNNB1 signaling in these processes. Here, we discuss and evaluate the decisions that researchers need to make in identifying the experimental system and appropriate tools for the specific question they want to address, covering different types of WNT/CTNNB1 reporters in cells and mice. At a molecular level, advanced quantitative imaging techniques can provide spatio-temporal information that cannot be provided by traditional biochemical assays. We therefore also highlight some recent studies to show their potential in deciphering the complex and dynamic mechanisms that drive WNT/CTNNB1 signaling.

Measuring the mechanical properties (i.e., elasticity in terms of Young\'s modulus) of biological samples using Atomic Force Microscopy (AFM) indentation at the nanoscale has opened new horizons in studying and detecting various pathological conditions at early stages, including cancer and osteoarthritis. It is expected that AFM techniques will play a key role in the future in disease diagnosis and modeling using rigorous mathematical criteria (i.e., automated user-independent diagnosis). In this review, AFM techniques and mathematical models for determining the spatial variability of elastic properties of biological materials at the nanoscale are presented and discussed. Significant issues concerning the rationality of the elastic half-space assumption, the possibility of monitoring the depth-dependent mechanical properties, and the construction of 3D Young\'s modulus maps are also presented.

Microscopy has long played a pivotal role in the field of assisted reproductive technology (ART). The advent of artificial intelligence (AI) has opened the door for new approaches to sperm and oocyte assessment and selection, with the potential for improved ART outcomes.

Duodenal gastrinomas (DGASTs) are neuroendocrine tumors that develop in the submucosa of the duodenum and produce the hormone gastrin. Surgical resection of DGASTs is complicated by the small size of these tumors and the tendency for them to develop diffusely in the duodenum. Endoscopic mucosal resection of DGASTs is an increasingly popular method for treating this disease due to its low complication rate but suffers from poor rates of pathologically negative margins. Multiphoton microscopy can capture high-resolution images of biological tissue with contrast generated from endogenous fluorescence (autofluorescence [AF]) through two-photon excited fluorescence (2PEF). Second harmonic generation is another popular method of generating image contrast with multiphoton microscopy (MPM) and is a light-scattering phenomenon that occurs predominantly from structures such as collagen in biological samples. Some molecules that contribute to AF change in abundance from processes related to the cancer disease process (e.g., metabolic changes, oxidative stress, and angiogenesis).
MPM was used to image 12 separate patient samples of formalin-fixed and paraffin-embedded duodenal gastrinoma slides with a second-harmonic generation (SHG) channel and four 2PEF channels. The excitation and emission profiles of each 2PEF channel were tuned to capture signal dominated by distinct fluorophores with well-characterized fluorescent spectra and known connections to the physiologic changes that arise in cancerous tissue.
We found that there was a significant difference in the relative abundance of signal generated in the 2PEF channels for regions of DGASTs in comparison to the neighboring tissues of the duodenum. Data generated from texture feature extraction of the MPM images were used in linear discriminant analysis models to create classifiers for tumor versus all other tissue types before and after principal component analysis (PCA). PCA improved the classifier accuracy and reduced the number of features required to achieve maximum accuracy. The linear discriminant classifier after PCA distinguished between tumor and other tissue types with an accuracy of 90.6%-93.8%.
These results suggest that multiphoton microscopy 2PEF and SHG imaging is a promising label-free method for discriminating between DGASTs and normal duodenal tissue which has implications for future applications of in vivo assessment of resection margins with endoscopic MPM.

The COVID-19 pandemic that began in March 2020 continues in many countries. The ongoing pandemic makes early diagnosis a crucial part of efforts to prevent the spread of SARS-CoV-2 infections. As such, the development of a rapid, reliable, and low-cost technique with increased sensitivity for detection of SARS-CoV-2 is an important priority of the scientific community. At present, nucleic acid-based techniques are primarily used as the reference approach for the detection of SARS-CoV-2 infection. However, in several cases, false positive results have been observed with these techniques. Due to the drawbacks associated with existing techniques, the development of new techniques for the diagnosis of COVID-19 is an important research activity. We provide an overview of novel diagnostic methods for SARS-CoV-2 diagnosis that integrate photonic technology with artificial intelligence. Recent developments in emerging diagnostic techniques based on the principles of advanced molecular spectroscopy and microscopy are considered.