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PubMed2025

Free energy spectroscopy reveals the mechanistic landscape of chromatin compaction.

Eukaryotic genomic DNA is repeatedly wrapped into nucleosome spools: the basic building block of chromatin. This organization regulates the physical accessibility of the genome to gene transcription, replication, and repair regulatory factors. Chromatin compaction is controlled by multivalent weak interactions, resulting in a complicated conformational landscape that remains challenging to characterize. This work reports a method for characterizing chromatin compaction, Free Energy Spectroscopy (FES), which is based on DNA nanotechnology and transmission electron microscopy. This method experimentally determines the chromatin compaction free energy landscape in terms of end-to-end distance and nucleosome stacking interactions. By deconvolving the free energy landscapes of partially and fully compact tetranucleosomes, FES revealed three separate mechanisms by which linker histones reshape the compaction energetics to condense chromatin. This study establishes FES as a method with the potential to help answer a broad range of mechanistic questions about genome and epigenome function.

DNA nanotechnology↗

PubMed2025

Nanoscale Epigenetic Profiling of Colorectal Cancer Cell-Derived Exosomes via Single-Vesicle Nanoscopy.

Exosomes play critical roles in cancer diagnosis and treatment as they carry molecular information that reflects the epigenetic state of their parent cells. For the first time, nanoscale epigenetic profiling of individual exosomes derived from colorectal cancer cell lines is demonstrated via photo-induced force microscopy (PiFM). Exosomes from three cell lines with distinct CpG island methylator phenotype (CIMP) status are analyzed at the single-vesicle level. The nano-IR method provides simultaneous high-resolution topographical and spectroscopic data, revealing detailed vibrational signatures that distinguish CIMP-high (HCT116 and HT29) exosomes from CIMP-negative (SW480) ones. Notably, exosomes from CIMP-high cells exhibit red-shifted amide I and nucleic acid region compared to those from CIMP-negative cells, a shift attributed to increased 5-methylcytosine (5mC) modifications, as verified by quantum chemical calculations. Furthermore, these measurements reveal heterogeneity among individual exosomes, suggesting the presence of distinct subpopulations with unique epigenetic profiles, demonstrating the importance of single-vesicle resolution to detect molecular variations that remain obscured in ensemble studies. These findings present the potential of PiFM-based single-vesicle analysis to identify epigenetic markers in exosomes, laying the groundwork for its application in refined cancer diagnostics and targeted therapeutic strategies.

Humans↗

PubMed2026

High-Purity Monovalent Functionalization of Carbon Nanotubes.

Single-walled carbon nanotubes (SWCNTs) show promise for probing molecular interactions at single-molecule resolution, yet generating SWCNT populations bearing a single defined functional tag remains challenging because surface functionalization is inherently stochastic. Here, we present a batch-scale strategy to produce predominantly singly tagged SWCNTs by leveraging the stochastic adsorption of single-stranded DNA (ssDNA). Specifically, SWCNTs are dispersed using a mixture of unmodified ssDNA (um-ssDNA) and a minor fraction of modified ssDNA (m-ssDNA) carrying an affinity handle. We developed a probabilistic ssDNA-SWCNT binding model that predicts the distribution of m-ssDNA per nanotube as a function of the input minor-strand fraction p = m-ssDNA/total ssDNA, enabling selection of conditions that maximize single-tag purity. Using magnetic-bead capture via a biotin affinity interaction and subsequent release, we isolate SWCNTs with 97.6% predicted single-tag purity at 2% recovery. Single-molecule fluorescence imaging further supports predominantly single-label occupancy under the model-selected conditions. Thus, this approach provides a general route to SWCNTs bearing a single molecular handle for downstream conjugation and assembly, supporting diverse future applications in SWCNT-based nanotechnologies.

Nanotubes, Carbon↗

PubMed2026

Insertion of CG repeats and 3' terminus overhangs drive B-to-Z transition: A case study with NF-κB bearing DNA nanostructures.

Z-DNA, a non-canonical helical structure of DNA plays a vital role in various biological processes, including transcription and genomic stability. Though low concentration of trivalent cations is known to induce B-Z transition, the effect of short CG repeats, overhangs sequences, loop length and order of nucleotides on Z-DNA formation in larger DNA is utterly unknown. Earlier, a series of self-assembled branched DNA (bDNA) nanostructures having 5T in the loop are reported to be resistant to B-to-Z DNA transition irrespective of the overhang sequences. Since the presence of alternative purine/pyrimidine sequences and direction of oligonucleotides play a vital role during replication and transcription, we hypothesize that the insertion of a small number of CG repeats, or a change in direction of overhang sequences may influence the B-to-Z DNA transition. Here, we show that Z-DNA formation was induced by inserting CG repeats into bDNA structures that were previously resistant to B-Z transition. Moreover, B-Z transition was also observed when overhangs were introduced at the 3' terminus. The generality of the approach of B-Z transition was demonstrated in a series of bDNA structures including the bDNA having NF-kβ sequences. Different dye binding experiments suggest the formation of Z-DNA in bDNA having overhangs at the 3' terminus against the control of bDNA with 5' overhangs. Interestingly, the melting temperature (Tm) was substantially reduced to 55 °C in the Z-DNA as compared to the LaCl3-induced condensed DNA having Tm of 77 °C. Fluorescence study also supports the presence of minor groove in Z-DNA which binds Hoechst. ITC indicates an entropy- and enthalpy-driven favorable binding between lanthanide cations and bDNA. Thus, the present study establishes a synthetic bDNA nanotechnology platform for systematically investigating how local sequence architecture, including the insertion of CG repeats, loop length, and overhang orientation influences B-to-Z conformational switching under controlled experimental conditions.

B-Z transition↗

PubMed2025

Advances in diagnosis of diseases causing diarrhea in newborn calves.

Diarrhea in newborn calves is a serious global health problem. It poses challenges for animal industry, veterinarians and researchers due to the rapid onset of dehydration. Mixed infections make treatment complicated, and many young calves suffer high rates of illness and death from this condition. Numerous enteropathogens are associated with diarrhea in newborn calves, encompassing viruses, bacteria, parasites, and protozoa. Their occurrence differs by region, yet the most prevalent infections include E. coli, Salmonella species, Clostridium perfringens, Clostridium difficile, Rotavirus, Coronavirus, Cryptosporidium, Toxocara, Giardia and Eimeria. This review outlines the diagnostic techniques for diseases that lead to diarrhea in newborn calves. Diagnosis is based on clinical manifestations; however, the laboratory identification of etiological items is the only valid way for detecting the illness's aetiology and initiating treatment protocols. Classic methods such as bacterial culturing, fecal flotation, direct microscopy, and virus isolation help us understand pathogens better. Immunological assays like ELISA and immunochromatography are fast, accurate, affordable, and useful for on-farm detection. They help identify specific antigens or antibodies efficiently. Molecular methods including PCR (standard, multiplex, real time and digital), LAMP assays, DNA microarrays and whole-genome sequencing allow highly accurate and sensitive detection. They can identify pathogens effectively, even at very low levels. Nanotechnology-based assays introduce a novel level of sensitivity and specificity, often yielding quick results with minimal sample volumes. In conclusion, accurate and rapid diagnosis using advanced techniques is critical for managing and preventing diseases that lead to diarrhea in newborn calves.

Animals↗

PubMed2026

Novel immunotherapeutic strategies for colorectal cancer treatment: Advances, challenges, and future directions.

Immunotherapy has reshaped the treatment landscape of colorectal cancer (CRC), with the clearest and most durable benefit established in mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) disease. However, framing CRC immunotherapy simply as "MSI-H responsive versus microsatellite stable (MSS) resistant" is no longer sufficient. Recent studies indicate that a subset of proficient mismatch repair (pMMR) colon cancers, particularly in the neoadjuvant setting, can mount clinically meaningful responses to immune checkpoint blockade, suggesting that disease stage, local immune organization, and treatment timing critically influence immunotherapy sensitivity. In parallel, emerging evidence has expanded the relevant immune landscape beyond the tumor bed itself, showing that spatially organized stromal and adipose niches can actively divert tumor-reactive lymphocytes and promote immune escape. These advances shift the central challenge in CRC immunotherapy from simply identifying new agents to defining when and in whom immune resistance is reversible, and which biological bottlenecks-such as vascular dysfunction, myeloid suppression, and spatial immune exclusion-must be overcome. In this context, alternative checkpoint inhibitors, bispecific antibodies, cellular therapies, vaccines, nanotechnology-enabled platforms, and microbiome-targeted approaches remain important, but their translational maturity and evidentiary support differ substantially. Biomarker development is likewise evolving from static genomic classification toward dynamic and mechanism-informed stratification incorporating circulating tumor DNA (ctDNA), chromosomal instability, immune architecture, and treatment-induced response trajectories. This review synthesizes recent advances in CRC immunotherapy while emphasizing evidence hierarchy, biomarker-guided patient selection, and the mechanistic basis of combination strategies. We argue that the next phase of CRC immunotherapy will depend less on the indiscriminate addition of novel agents and more on the rational deployment of immunotherapy across molecularly, spatially, and temporally defined disease states.

Humans↗

PubMed2025

Biogenic Synthesis and Characterization of Hypecoum pendulum Mediated Silver Nanoparticles: Revealed Outstanding Anticancer and Genotoxic Potentials.

Fabrication of silver nanoparticles by green approach is the most effective and eco-friendly technique in recent technologies. The current study aimed to generate a simple, valid, and justifiable method for biogenic synthesis of silver nanoparticles (HP-AgNPs) using aqueous extract of Hypecoum pendulum L.(HP) and to assess their in vitro anticancer and genotoxic potentials on baby hamster kidney cell (BHK-21) and human blood lymphocytes using 3-(4,5-dimethylthiazol-2-yl-)-2,5-di-phenyltetrazolium bromide (MTT) and alkaline comet assay, respectively. HP-AgNP characterization was done using UV-vis spectrometry, EDX, SEM, XRD, and FTIR techniques. The crystalline nature of HP-AgNPs with a particle size of 36.3 nm was assessed using the XRD technique. The surface morphologies with a particle size of 80 nm were verified by SEM analysis. UV spectroscopy verified the existence of HP-AgNPs by yielding a sharp peak at 417 nm with an absorbance intensity of 1.54. FTIR assessment revealed the existence of different functional moieties that contribute to the HP-AgNPs stabilization and reduction. Similarly, EDX analysis revealed Ag as a principal element (49%). MTT assay showed significant cytotoxicity by Doxorubicin and HP-AgNPs with a smaller IC50 value of 104.21 ± 4.33 and 134.91 ± 6.33 μg/mL correlated to HP extract (229.84 ± 4.66 μg/mL). The outcomes of the comet assay revealed potential DNA damage in a positive trend with concentration (25-600 μg/mL). HP-AgNP-treated lymphocytes showed higher DNA damage as compared to HP extract-treated cells, but less damage as compared to a positive control, H2O2. These outcomes showed that HP-AgNPs have demonstrated promising anticancer and genotoxic action than HP extract due to their size and shape.

Silver↗

PubMed2024

Gradient-Elution Nanoflow Liquid Chromatography Without a Binary Pump: Smoothed Step Gradients Enable Reproducible, Sensitive, and Low-Cost Separations for Single-Cell Proteomics.

Mass spectrometry-based proteome profiling of trace analytes including single cells benefits from liquid chromatography separations operated at low flow rates (e.g., <50 nl/min). However, high-pressure binary pumps needed to achieve such flow rates are not commercially available, and instead require splitting of the gradient flow to achieve low-nanoliter-per-minute flow rates. Gradient flow splitting can waste solvent and lead to flow inconsistencies. To address this, we have developed a method for creating gradients by combining segments of mobile phase having increasing solvent strength together in an open capillary, and then relying on Taylor dispersion to form the desired smooth gradient profile. Our method dramatically reduces costs, as only a single isocratic high-pressure pump is required. Following development of gradient profiles for both 10- and 20-min active gradients, we measured 200 pg injections of HeLa digest using a timsTOF mass spectrometer. Finally, we investigated differences in protein expression between single cells originating from two different colonies of ATG-KO HeLa cells. Thousands of proteins were quantified, and a potential mechanism explaining differential immune responses of these two colonies upon exposure to viral DNA treatment was determined.

Humans↗