Search Results (342)

Modified oligonucleotides (oligos) are widely used as convenient tools in many scientific fields, including biomedical applications and therapies. In particular, oligos with lipophilic groups attached to the backbone ensure penetration of the cell membrane without the need for transfection. This study examines the interaction between amphiphilic DNA duplexes, in which one of the chains contains a lipophilic substituent, and several DNA repair proteins, particularly DNA-damage-dependent PARPs, using various biochemical approaches. DNA with a lipophilic substituent (LS-DNA) demonstrates more efficient binding with DNA damage activated poly(AD-ribose) polymerases 1-3 (PARP1, PARP2, PARP3) and DNA polymerase β. Chemically reactive LS-DNA derivatives containing a photoactivatable nucleotide (photo-LS-DNAs) or a 5′ deoxyribose phosphate (dRP) group in the vicinity of double-strand breaks (DSBs) are used for the affinity labelling of PARPs and other proteins in several whole-cell extracts of human cells. In particular, photo-LS-DNAs are used to track the level of Ku antigen in the extracts of neuron-like differentiated SH-SY5Y, undifferentiated SH-SY5Y, and olfactory epithelial cells. In vitro, PARP1–PARP3 are shown to be able to slowly excise the 5′ dRP group at DSBs. LS-DNAs can activate PARP1 and PARP2 for autoPARylation, albeit less effectively than regular DNA duplexes. Full article

Prion diseases such as chronic wasting disease involve the conformational conversion of the cellular prion protein (PrP^C) into its misfolded, β-rich isoform (PrP^Sc). While chemical denaturants such as guanidine hydrochloride (GdnHCl) and urea are commonly used to study this process in vitro, their distinct molecular effects on native and misfolded PrP conformers remain incompletely understood. In this study, we employed 500 ns all-atom molecular dynamics simulations and essential collective dynamics analysis to investigate the differential effects of GdnHCl and urea on a composite PrP^C/PrP^Sc system, where white-tailed deer PrP^C interfaces with a corresponding PrP^Sc conformer. GdnHCl was found to preserve interfacial alignment and enhance β-sheet retention in PrP^Sc, while urea promoted partial β-strand dissolution and interfacial destabilization. Both denaturants formed transient contacts with PrP, but urea displaced water hydrogen bonds more extensively. Remarkably, we also observed long-range dynamical coupling across the PrP^C/PrP^Sc interface and between transiently bound solutes and distal protein regions. These findings highlight distinct, denaturant-specific mechanisms of protein destabilization and suggest that localized interactions may propagate non-locally via mechanical or steric pathways. Our results provide molecular-scale insights relevant to prion conversion mechanisms and inform experimental strategies using GdnHCl and urea to modulate misfolding processes in vitro. Full article

A key feature that differentiates Gram-positive and Gram-negative bacteria is the outer membrane, an asymmetric membrane composed of lipopolysaccharides, phospholipids, lipoproteins and integral proteins, including the outer-membrane proteins (OMPs). By being in direct contact with the extracellular milieu, the outer membrane and OMPs participate in multiple functions in Gram-negative bacteria, including controlling nutrient and molecule access to the cytoplasm, membrane vesicle formation and resistance to environmental stresses. OMPs have a characteristic barrel shape formed by antiparallel β-strands, with or without channels that allow diffusion of substrates through the outer membrane. The marine bacterium Vibrio cholerae is responsible for non-invasive gastroenteritis and cholera disease by consumption of contaminated water or food. Its OMPs, besides having a porin function, contribute to resistance to osmotic pressure and antimicrobial agents, intracellular signaling, adhesion to host cells and biofilm formation, amongst other functions. In this review, in addition to quickly reviewing the general structure of the outer membrane, the OMPs and how they reach the outer membrane, the functions attributed to these proteins are compiled. The mechanisms used by each of the described OMP to accomplish these functions in the marine pathogenic bacterium V. cholerae are discussed. Potential clinical and bioengineering applications of OMPs, such as diagnostic tools, vaccine development, and targeted antimicrobial or anti-virulence strategies are presented. What is known about the OMPs of V. cholerae is presented below. Full article

Objectives: Beta-emitting radionuclide therapy, exemplified by ¹⁷⁷Lu-Oxodotreotide (Lutathera^®), enables targeted treatment of neuroendocrine tumors by delivering β-radiation to tumor cells. However, the dose-dependent molecular mechanisms underlying cellular damage remain insufficiently characterized. This study aimed to investigate the phenotypic changes in IMR-32 human neuroblastoma cells following Lutathera exposure, with a focus on the dose-dependent relationship between radiation and cellular damage. Methods: IMR-32 cells were allocated to control, low- (0.05 MBq/mL), medium- (0.5 MBq/mL), and high-dose (5 MBq/mL) groups and treated with ¹⁷⁷Lu-Oxodotreotide for 24 h. Flow cytometry was employed to assess cell viability, apoptosis, mitochondrial membrane potential, γ-H2AX expression (a marker of DNA damage), and proliferation. Results: Lutathera induced dose-dependent cytotoxic effects. Cell viability declined linearly with increasing dose (control: 100% vs. high-dose: 13.48%; r = −0.955, p < 0.001). Apoptosis was significantly elevated (control: 35.34% vs. high-dose: 88.12%; r = 0.999), accompanied by increased γ-H2AX levels (control: 5.26 × 10⁴ vs. high-dose: 13.13 × 10⁴; r = 0.930), indicating DNA double-strand breaks. Mitochondrial membrane potential decreased (control: 6.06 × 10⁴ vs. high-dose: 46.27 × 10⁴; r = 0.999), and proliferation was suppressed (control: 91.10 × 10⁴ vs. high-dose: 103.84 × 10⁴; r = 0.954), both showing strong dose correlations (p < 0.001). Conclusions: ¹⁷⁷Lu-Oxodotreotide exerts dose-dependent cytotoxicity in IMR-32 cells via DNA damage, mitochondrial dysfunction, and apoptosis induction. These findings underscore the necessity of optimizing dosing regimens to balance therapeutic efficacy and safety in clinical settings, providing a foundation for personalized β-emitter therapies. Full article

Previously, we developed a porcine bronchial epithelial cell line designated as PBE cells and demonstrated that this cell line possesses functional Toll-like receptor 3 (TLR3), triggering the expressions of interferons (IFNs), antiviral factors, and inflammatory cytokines after its stimulation with the synthetic double-stranded ARN poly(I:C). In this work, we aimed to further characterize the PBE cell line as a reliable in vitro model for investigating swine influenza virus (SIV) infection and immunity. We evaluated the capacity of two SIV subtypes, H1N1 and H3N2, to replicate and induce cytopathic effects in PBE cells and to modulate the expressions of IFNs, antiviral factors, inflammatory cytokines, and negative regulators of the TLR signaling. We demonstrated that PBE cells are susceptible to both H1N1 and H3N2. SIV infected PBE cells inducing notable cytopathic effects as shown by the alteration of transepithelial electrical resistance (TEER) and cilia. Both SIV subtypes replicated in PBE cells in similar proportion and altered TEER values in comparable magnitudes. However, SIV H3N2 induced higher alterations of cilia than H1N1. SIV infection induced changes in all the immune factors evaluated in PBE cells. We detected quantitative differences when the subtypes H1N1 and H3N2 were compared. The fold expression changes of IFN-β, Mx1, Mx2, IFITM1, OAS1, OAS2, and OASL were higher in PBE cells infected with H3N2 than in cells challenged with H1N1. In addition, although both subtypes stimulated IL-8 expression, only the H3N2 induced IL-6 in infected PBE cells. SIV H1N1 and H3N2 also upregulated the expressions of the negative regulators A20, BCL-3, and MKP-1, while only H1N1 increased SIGIRR and Tollip. Immortalized respiratory cell lines from pigs can be useful in vitro systems for the study of viral infections and immune responses. These studies are of importance in the context of influenza infections not only for the agricultural field because pigs are natural hosts of these viruses but also because these animals serve as intermediate reservoirs of viruses that can threaten humans’ health. We demonstrated here that the PBE cell line can be a useful in vitro model to study SIV infection and immunity. Full article

We investigated the interaction of HspB7 and its α-crystallin domain with the wild-type (WT) C-terminal fragment of human filamin C (FLNC), containing immunoglobulin-like domains 22–24 and its three mutants associated with cardio- and myopathies. The physicochemical properties of the WT FLNC fragment and its three mutants, p.Glu2472_Asn2473delinsAsp (EN/D) located in the 22nd domain, p.P2643_L2645del (ΔPGL), and p.W2710X (Wmut) both located in the 24th immunoglobulin-like domain were analyzed. Although all FLNC fragments had similar secondary structures, WT FLNC and its EN/D and ΔPGL mutants formed dimers, whereas Wmut formed either monomers or aggregates. The surface hydrophobicity of EN/D, ΔPGL, and especially Wmut mutants was larger than that of the WT fragment. Size exclusion chromatography, native gel electrophoresis, and chemical crosslinking indicated that the efficiency of interaction with HspB7 or its α-crystallin domain decreased in the order WT~EN/D > ΔPGL. Wmut was unable to interact with either HspB7 or its α-crystallin domain. Modeling via Alphafold 3 indicated that EN/D mutation affected the orientation of two loops connecting β-strands in the 22nd domain, while the ΔPGL and Wmut mutations exposed a hydrophobic groove in the 24th domain thereby reducing their interaction with HspB7. These findings reveal the molecular mechanisms underlying filaminopathies associated with three mutations in the C-terminal region of filamin C. Full article

HSP110 is a ubiquitous chaperone contributing to proteostasis. It has a disaggregation activity and can refold denatured proteins. It can regulate fundamental signaling pathways involved in oncogenesis, such as Wnt/β-catenin, NF-κB and STAT3 signaling pathways. In gastric and colorectal cancer, HSP110 has been detected in the nucleus, and nuclear expression has been associated with the resistance of cells to 5-FU chemotherapy. Nuclear translocation of HSP110 is promoted by the exposure of cells to DNA-damaging agents. In a previous work, we demonstrated that nuclear HSP110 participates in the NHEJ DNA repair pathway by facilitating the recruitment of DNA-PKcs to Ku70/80 heterodimers at the site of DNA double-strand breaks. In the present work, analysis of HSP110s’ nuclear interactome revealed an enrichment of components from SWI/SNF chromatin remodeling complexes. We demonstrate that HSP110 is strongly associated with chromatin in temozolomide- and oxaliplatin-treated cells and directly interacts with the core subunit SMARCC2, thereby facilitating the assembly of SWI/SNF complexes. This work expands upon the role of HSP110, which regulates not only proteostasis but also the assembly of critical nuclear macromolecular complexes involved in the adaptive stress response. Full article

Activation of cGAS, a cytosolic receptor recognizing double-stranded DNA, in macrophages is important in sepsis (a life-threatening condition caused by infection). The responses against sepsis induced by subcutaneous implantation of the Pseudomonas-contaminated catheters in cGAS-deficient (cGAS^−/−) mice were lower than in wild-type (WT) mice as indicated by liver enzymes, white blood cell count, cytokines, and M1-polarized macrophages in the spleens. Likewise, a lethal dose of lipopolysaccharide (LPS) induced less severe sepsis severity as determined by mortality, organ injury, cell-free DNA, and serum cytokines. Patterns of the transcriptome of lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages were clearly different between cGAS^−/− and WT cells. Gene set enrichment analysis (GSEA; a computational statistical determination of the gene set) indicated more prominent enrichment of oxidative phosphorylation (OXPHOS; the mitochondrial function) and mTORC1 pathways in LPS-activated cGAS^−/− macrophages compared with WT. Meanwhile, LPS upregulated cGAS and increased cGAMP (a cGAS inducer) only in WT macrophages along with less severe inflammation in cGAS^−/− macrophages, as indicated by supernatant cytokines, pro-inflammatory molecules (nuclear factor kappa B; NF-κB), M1 polarization (IL-1β, CD80, and CD86), and macrophage extracellular traps (METs; web-like structures composed of DNA, histones, and other proteins) through the detection of citrullinated histone 3 (CitH3) in supernatant and immunofluorescent visualization. In conclusion, less prominent pro-inflammatory responses of cGAS^−/− macrophages than WT were demonstrated in mice (catheter-induced sepsis and LPS injection model) and in vitro (transcriptomic analysis, macrophage polarization, and METs). Full article

Introduction: Coronavirus (CoV) is an extremely contagious, enveloped positive-single-stranded RNA virus, which has become a global pandemic that causes several illnesses in humans and animals. Hence, it is necessary to investigate viral-induced reactions across diverse hosts. Herein, we propose utilizing naturally secreted extracellular vesicles (EVs), mainly focusing on exosomes to examine virus–host responses following CoV infection. Exosomes are small membrane-bound vesicles originating from the endosomal pathway, which play a pivotal role in intracellular communication and physiological and pathological processes. We suggested that CoV could impact EV formation, content, and diverse immune responses in vitro. Methods: In this study, we infected A-72, which is a canine fibroblast cell line, with a feline coronavirus (FCoV) and canine coronavirus (CCoV) independently in an exosome-free media at 0.001 multiplicity of infection (MOI), with incubation periods of 48 and 72 h. The cell viability was significantly downregulated with increased incubation time following FCoV and CCoV infection, which was identified by performing the 3-(4,5-dimethylthiazo-1-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After the infection, EVs were isolated through ultracentrifugation, and the subsequent analysis involved quantifying and characterizing the purified EVs using various techniques. Results: NanoSight particle tracking analysis (NTA) verified that EV dimensions fell between 100 and 200 nm at both incubation periods. At both periods, total protein and RNA levels were significantly upregulated in A-72-derived EVs following FCoV and CCoV infections. However, total DNA levels were gradually upregulated with increased incubation time. Dot blot analysis indicated that the expression levels of ACE2, IL-1β, Flotillin-1, CD63, caspase-8, and Hsp90 were modified in A-72-derived EVs following both CoV infections. Conclusions: Our results indicated that FCoV and CCoV infections could modulate the EV production and content, which could play a role in the development of viral diseases. Investigating diverse animal CoV will provide in-depth insight into host exosome biology during CoV infection. Hence, our findings contribute to the comprehension and characterization of EVs in virus–host interactions during CoV infection. Full article

Chapparvoviruses (ChPVs) comprise a divergent lineage of the Parvoviridae ssDNA virus family and evolved to infect vertebrate animals independently from the Parvovirinae subfamily. Despite being pathogenic and widespread in environmental samples and metagenomic assemblies, their structural characterization has proven challenging. Here, we report the first structural analysis of a ChPV, represented by the fish pathogen, Syngnathus scovelli chapparvovirus (SsChPV). We show through the SsChPV structure that the lineage harbors a surface morphology, subunit structure, and multimer interactions that are unique among parvoviruses. The SsChPV capsid evolved a threefold-related depression of α-helices that is analogous to the β-annulus pore of denso- and hamaparvoviruses and may play a role in monomer oligomerization during assembly. As interacting β-strands are absent from the twofold symmetry axis, the viral particle lacks the typical stability and resilience of parvovirus capsids. Although all parvoviruses thus far rely on the threading of large, flexible N-terminal domains to the capsid surface for their intracellular trafficking, our results show that ChPVs completely lack any such N-terminal sequences. This led to the subsequent degradation of their fivefold channel, the site of N-terminus externalization. These findings suggest that ChPVs harbor an infectious pathway that significantly deviates from the rest of the Parvoviridae. Full article

Because of the involvement of π-electron cyclic constituents in their side chains, the so-called aromatic residues give rise to a number of strong, narrow, and well-resolved lines spread over the middle wavenumber (1800–600 cm⁻¹) region of the Raman spectra of peptides and proteins. The number of characteristic aromatic markers increases with the structural complexity (Phe → Tyr → Trp), herein referred to as (F_i = 1, …, 6) in Phe, (Y_i = 1, …, 7) in Tyr, and (W_i = 1, …, 8) in Trp. Herein, we undertake an overview of these markers through the analysis of a representative data base gathered from the most structurally simple tripeptides, Gly-Xxx-Gly (where Xxx = Phe, Tyr, Trp). In this framework, off-resonance Raman spectra obtained from the aqueous samples of these tripeptides were jointly used with the structural and vibrational data collected from the density functional theory (DFT) calculations using the M062X hybrid functional and 6-311++G(d,p) atomic basis set. The conformation dependence of aromatic Raman markers was explored upon a representative set of 75 conformers, having five different backbone secondary structures (i.e., β-strand, polyproline-II, helix, classic, and inverse γ-turn), and plausible side chain rotamers. The hydration effects were considered upon using both implicit (polarizable solvent continuum) and explicit (minimal number of 5–7 water molecules) models. Raman spectra were calculated through a multiconformational approach based on the thermal (Boltzmann) average of the spectra arising from all calculated conformers. A subsequent discussion highlights the conformational landscape of conformers and the wavenumber dispersion of aromatic Raman markers. In particular, a new interpretation was proposed for the characteristic Raman doublets arising from Tyr (~850–830 cm⁻¹) and Trp (~1360–1340 cm⁻¹), definitely excluding the previously suggested Fermi-resonance-based assignment of these markers through the consideration of the interactions between the aromatic side chain and its adjacent peptide bonds. Full article

Auxin response factor (ARF) is a plant-specific transcription factor that responds to changes in auxin levels, regulating various biological processes in plants such as flower development, senescence, lateral root formation, stress response, and secondary metabolite accumulation. In this study, we identified the ARF gene family in Populus euphratica Oliv. using bioinformatics analysis, examining their conserved structural domains, gene structure, expression products, and evolutionary relationships. We found that the 34 PeARF genes were unevenly distributed on 19 chromosomes of P. euphratica. All 56 PeARF proteins were hydrophilic and unstable proteins localized in the nucleus, with secondary structures containing α-helices, extended strands, random coils, and β-turns but lacking transmembrane helices (TM-helices) and signal peptides. Evolutionary analysis divided the PeARF proteins into five subfamilies (A–E), with high conservation observed in the order and number of motifs, domains, gene structure, and other characteristics within each subfamily. Expression pattern analysis revealed that 17 PeARF genes were upregulated during cell growth and heterophylly development. This comprehensive analysis provides insights into the molecular mechanisms of ARF genes in P. euphratica growth, development, and stress response, serving as a basis for further studies on the auxin signaling pathway in P. euphratica. Full article

The aim of this study was to investigate the structural characteristics of four polysaccharides derived from Citri grandis fructus immaturus and their antioxidant and expectorant activities. ECP1 fraction passing through a 500 kDa dialysis bag (ECP1A) and ECP2 fraction retained in a 300 kDa dialysis bag (ECP2B) had molecular weights of 340 and 1217 kDa, respectively. All four polysaccharides were composed of six monosaccharides, including l-rhamnose, d-arabinose, d-xylose, d-mannose, d-glucose, and d-galactose, with molar ratios of 1.99:52.38:6.99:2.64:5.15:31.15 for ECP1A and 1.54:65.13:6.34:2.51:3.58:22.07 for ECP2B. ECP1A had an α/β-glucopyranose ring, and the glycosyl groups were linked mainly by 1→4, 1→2, or 1→6 glycosidic bonds. It likely adopted a single-stranded helical conformation. ECP2B had a β-glucopyranose ring, and the glycosyl groups were linked mainly by 1→4, 1→2, or 1→6 glycosidic bonds. Furthermore, in vitro experiments showed that ECP1A displayed excellent antioxidant activity (IC₅₀ = 0.4614 mg/mL). ECP2B significantly inhibited MUC5AC mucin content expression in the mucus hypersecretion model of BEAS-2B cells, thus exerting an expectorant effect. A significant negative correlation was observed between the molecular weight of Citri grandis fructus immaturus polysaccharides and their antioxidant activity, and the uronic acid and d-arabinose contents of these polysaccharides exhibited strong negative trends with both antioxidant and expectorant activities. This study shows the potential for developing and utilizing polysaccharides from Citri grandis fructus immaturus as an antioxidant and expectorant agent. Full article

α-Synuclein (αS) interacts with lipid membranes in neurons to form amyloid fibrils that contribute to Parkinson’s disease, and its non-amyloid-β component domain is critical in the fibrillation. In this study, the salt (NaCl) effect on the membrane interaction and fibril formation of αS_57–102 peptide (containing the non-amyloid-β component domain) was characterized at the molecular level because the αS_57–102 fibrils exhibited structural polymorphism with two morphologies (thin and thick) in the presence of NaCl but showed one morphology (thin) in the absence of NaCl. The membrane-bound conformation (before fibrillation) of αS_57–102 had two helical regions (first and second) on the membrane regardless of salt, but the length of the first region largely shortened when NaCl was present, exposing its hydrophobic area to the solvent. The exposed region induced two distinct pathways of fibril nucleation, depending on the molar ratios of free and membrane-bound αS_57–102: one from the association of free αS_57–102 with membrane-bound αS_57–102 and the other from the assembly among membrane-bound αS_57–102. The differences mainly affected the β-strand orientation and helical content within the fibril conformations, probably contributing to the thickness degree, leading to structural polymorphism. Full article

B30.2 domains, sometimes referred to as PRY/SPRY domains, were originally identified by sequence profiling methods at the gene level. The B30.2 domain comprises a concanavalin A-like fold consisting of two twisted seven-stranded anti-parallel β-sheets. B30.2 domains are present in about 150 human and 700 eukaryotic proteins, usually fused to other domains. The B30.2 domain represents a scaffold, which, through six variable loops, binds different unrelated peptides or endogenous low-molecular-weight compounds. At the cellular level, B30.2 proteins engage in supramolecular assemblies with important signaling functions. In humans, B30.2 domains are often found in E3-ligases, such as tripartite motif (Trim) proteins, SPRY domain-containing SOCS box proteins, Ran binding protein 9 and −10, Ret-finger protein-like, and Ring-finger proteins. The B30.2 protein recognizes the target and recruits the E2-conjugase by means of the fused domains, often involving specific adaptor proteins. Further well-studied B30.2 proteins are the methyltransferase adaptor protein Ash2L, some butyrophilins, and Ryanodine Receptors. Although the affinity of an isolated B30.2 domain to its ligand might be weak, it can increase strongly due to avidity effects upon recognition of oligomeric targets or in the context of macromolecular machines. Full article