Geroprotection aims at extending healthspan by delaying age-associated pathologies. Polyamines including spermine and spermidine are interconvertible metabolites whose longevity-promoting effects have traditionally been attributed to autophagy induction. In addition, recent evidence identifies spermine as an endogenous Fe2+ chelator that suppresses ferroptosis, thereby complementing the autophagy-inducing activity of spermidine. Indeed, spermidine inhibits EP300 acetyltransferase activity and supports hypusination-dependent activation of TFEB, both leading to autophagy. However, enhanced autophagic flux may increase susceptibility to ferroptosis through ferritinophagy and lipid remodeling. In parallel, polyamine catabolism generates H2O2 and acrolein, both of which facilitate lipid peroxidation and ferroptotic demise. The discovery that spermine directly chelates redox-active Fe2+ closes a conceptual gap by explaining how polyamine supplementation can promote longevity while avoiding excessive ferroptotic cell loss. Multiple lines of evidence including metabolomics, isotope tracing, cell-free lipid peroxidation systems, Fe2+-binding biophysics, mass spectrometry, Raman spectroscopy, nuclear magnetic resonance and disease models demonstrate that spermine limits labile iron and ferroptosis. Together, these findings support a unified model in which spermidine-driven autophagy and spermine-mediated ferroptosis inhibition cooperate to preserve tissue homeostasis and healthspan.
Toxic hepatitis is characterized by enhanced oxidative stress and disruption of antioxidant defense mechanisms in the liver mitochondria. In this study, we investigated the effects of polyphenolic extracts (Helmar-1 and Helmar-2) obtained from Helichrysum maracandicum on mitochondrial antioxidant systems in rats with experimentally induced toxic hepatitis. Experimental toxic hepatitis was established in rats through intraperitoneal administration of carbon tetrachloride (CCl₄) diluted in olive oil (50%, 1 mL/kg), administered twice weekly over a two-week period. Liver injury was verified by elevated plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The animals were subsequently treated with Helmar-1 and Helmar-2 extracts at a dose of 20 mg/kg per day for 10 consecutive days. The activities of key antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase), malondialdehyde (MDA) content, and mitochondrial respiration and oxidative phosphorylation parameters (V₂, V₃, V₄, RCR, and ADP/O ratio) were assessed in liver mitochondria. Treatment with both Helmar-1 and Helmar-2 extracts resulted in a significant enhancement of antioxidant enzyme activities, a marked reduction in MDA levels, and substantial improvement in mitochondrial respiration and oxidative phosphorylation parameters compared to untreated toxic hepatitis groups. Overall, these results confirm that Helichrysum maracandicum polyphenol extracts improve mitochondrial respiration, oxidative phosphorylation efficiency, antioxidant defense, and membrane stability in toxic hepatitis.
Spinal muscular atrophy is a neuromuscular disorder primarily caused by mutations in the SMN1 (Survival of Motor Neuron 1) gene. SMN1 is ubiquitously expressed and encodes a protein essential for the assembly of small nuclear ribonucleoproteins, key components of pre-mRNA splicing. The SMN protein also participates in several other fundamental cellular processes, including RNA transport, regulation of actin dynamics, transcription, and translation. While multiple hypotheses have been put forward to explain the selective motor neurons (MNs) vulnerability to SMN deficiency, the precise mechanisms involved remain incompletely understood. In this study, we used neuron-specific smn-1 RNAi silencing in D-type MNs or in touch receptor neurons in C. elegans In touch receptor neurons, smn-1 silencing caused distinct defects in neuronal process morphology. Our results reveal pronounced neuron-specific differences in sensitivity within the neurons of C. elegans, providing a robust framework to dissect the mechanisms underlying selective neuronal vulnerability of spinal cord MNs in spinal muscular atrophy.
Resistance training in rodent models is widely used to investigate muscular and cardiovascular adaptations. Traditional models often attach loads to the tail. The present study aimed to develop and validate a resistance training model for rats using a pulley system combined with a load-bearing vest, allowing progressive overload without tail attachment. Spontaneously hypertensive rats were assigned to control (CTR, n = 4) or resistance training (RT, n = 6) groups for a 10-week ladder climbing protocol. Functional and morphometric adaptations were evaluated in the soleus and flexor hallucis longus (FHL) muscles. Resistance training resulted in a significant increase in relative maximum load carried (p = 0.0007), corresponding to a 79.5% improvement in strength capacity. The RT group also showed increased relative mass of the FHL muscle (p = 0.002). Gene expression analyses did not show statistically significant differences between groups (p > 0.05). The pulley-vest system proved to be a viable and effective resistance training model for rats, allowing controlled overload while avoiding tail load attachment and potential vascular interference. This model represents a refined experimental approach for resistance training studies in rodent models, particularly in conditions where tail circulation must be preserved.
DNA helicases are ATP-dependent motor proteins that catalyze duplex DNA unwinding and are involved in DNA repair, recombination, and replication restart. Prominent members of the non-hexameric SF1A UvrD-family helicases are Escherichia coli UvrD, Rep, Bacillus stearothermophilus PcrA, and Mycobacterium tuberculosis (Mtb) UvrD1. SF1A monomers are processive 3'-to-5' single-stranded DNA translocases but need to be activated to become DNA helicases. One mechanism of activation is dimerization. Whereas Rep, UvrD, and PcrA form non-covalent dimers, the Mtb UvrD1 helicase forms a redox-dependent covalent dimer. Dimerization of Mtb UvrD1 occurs between the same regulatory domain (2B) within each subunit stabilized by a disulfide bond formed between the same cysteine (Cys451) within each subunit. Dimerization relieves an inhibitory interaction between the 2B domain and duplex DNA within the monomer-DNA complex. We show here that Rep, UvrD, and PcrA dimerize using the same 2B-2B interface. By placing a Cys residue within the 2B domains of Rep, UvrD, and PcrA in the structurally equivalent position occupied by Cys451 of Mtb UvrD1, all three enzymes form redox-dependent covalent dimers that are constitutively active helicases with increased processivity compared to the non-covalent dimers. Hence, the 2B domain is a general dimerization domain for UvrD-family SF1A helicases.
N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (NAGPA), also known as the uncovering enzyme, catalyzes the final step in mannose-6-phosphate (M6P) signal generation for lysosomal enzyme trafficking. Despite its central role, the enzyme's architecture, catalytic mechanism, and the contribution of its C-terminal region remain incompletely defined. Here, we combine solution biophysics, cryo-electron microscopy, structural modeling, and a quantitative cell-based assay to characterize human NAGPA. We show that NAGPA forms a noncovalent dimer in solution, resolving prior uncertainty regarding disulfide-linked higher-order assemblies. The structure reveals an elongated dimer composed of two catalytic cores and two C-terminal EGF-like stalks, semi-rigid in nature, that likely position the catalytic domains ∼5 nm from the membrane. A structure determined in the presence of the substrate analog GlcNAc-1-phosphate captures GlcNAc and phosphate in the active site, identifying an invariant DGGGS motif that is critical for substrate recognition and enzyme catalysis. Based on these observations, we propose a substrate-assisted SNi-like mechanism for cleavage of the glycosidic C-O bond between GlcNAc and M6P. Functional assays show that the membrane-tethered full-length NAGPA is more active than the isolated catalytic core, and that mutations in the hinge linking the catalytic domain to the C-terminal stalk reduce activity. Together, these findings establish a structural and mechanistic framework for understanding NAGPA function in lysosomal enzyme targeting.
Although plants are exposed to many environmental stressors (heat, drought, salinity, and pathogen), they survive by rapidly reprogramming their gene expression. Recent findings show that many regulatory mechanisms are maintained within liquid-liquid phase separation (LLPS) formed biomolecular condensates, instead of maintaining these mechanisms within membrane-bound organelles. LLPS has been shown to exist in plant cells; however, the integration of LLPS with functional stress adaptations and gene regulations is still unclear from the studies that have been published thus far. In this review, we discuss recent research that demonstrates how these biomolecular condensates act as dynamic regulatory centers by linking environmental stress perception to transcriptional and post-transcriptional regulation of genes in plants. We describe the biophysical principles that govern the occurrence of LLPS in a plant cellular environment and provide an overview of how both abiotic (e.g., drought, heat) and biotic (i.e., pathogens) stressors initiate the LLPS and remodel nuclear and cytosolic condensate structures. The focus of this work will be on the effects of stress-induced changes (both qualitatively and quantitatively) in the condensate composition and material properties on transcription factor activity (either as an activator or repressor) and the organization of chromatin, RNA stability, and selective translation. We will also discuss post-translational modifications (PTMs) that modulate condensate dynamics and allow for reversibility. Collectively, these findings suggest that LLPS likely contributes significantly to gene regulation and stress adaptation in plants, although the degree of mechanistic validation varies among different condensate systems.
Regarded as one of the most complex membrane protein folds, CLCs form a large family of membrane proteins that function as anion channels and secondary active anion/proton transporters. Despite low sequence similarity, available structures are remarkably similar, maintaining the same inverted topology and fold, with subunits assembled as dimers in wild-type structures. Because of these strong structural features, CLC-ec1, a prokaryotic homologue from E. coli, has become a highly valuable model system for studying membrane protein folding and oligomerization assembly. Associating via a membrane-embedded dimerization interface, the subunits participate in a dynamic equilibrium between monomers and dimers in lipid bilayers, enabling investigation of the physical driving forces underlying the formation of stable membrane complexes. Like soluble protein assembly, studies indicate that CLC-ec1 dimerization is driven by a solvophobic force arising from the free energy gained by burying lipid bilayer defects. Dimerization stability is influenced by lipid composition and pH, which, in turn, provide a mechanism for functional regulation through oligomerization. In this review, I provide an overview of how the exploration of CLC folding and oligomerization advances our understanding of membrane protein self-assembly in general and the role of oligomerization in regulating function.
Many bacteria, and most archaea, have a paracrystalline protein surface layer (S-layer) encapsulating their outer membrane or cell wall. For pathogenic Rickettsia species, an S-layer has been hypothesized to be comprised of the outer membrane protein OmpB. We used cryo-electron tomography (cryoET) to image the Rickettsia parkeri cell surface and observed a repetitive S-layer structure proximal to the outer membrane of the bacterium that is absent in an ompB STOP ::tn mutant. Our data reveal that OmpB is essential for the Rickettsia S-layer and suggest that we can leverage cryoET to examine rickettsial S-layer structure and function.
NanT and NanX are bacterial transporters that import the sialic acids, N-acetylneuraminate and 2,7-anhydro-n-acetylneuraminate, respectively. Here, we used complementary biophysical and computational approaches to structurally characterise Escherichia coli NanX. Size exclusion chromatography, analytical ultracentrifugation and low-resolution cryo-electron microscopy reveal that NanX exists in both monomeric and dimeric states following purification. Molecular modelling and substrate docking identify key residues likely involved in 2,7-anhydro-n-acetylneuraminate recognition. Using this information, we engineered a mutant NanX transporter that can import the NanT-specific substrate, N-acetylneuraminate, which we verified using a bacterial growth assay. These data identify amino acids involved in major facilitator superfamily mediated sialic acid transport and offer a new research perspective of its metabolism.
The study proposes an experimental approach to determining the rates of individual stages of a reaction catalyzed by bacterial luciferase, based on the tryptophan fluorescence of the protein and the stopped-flow technique. The relationship between the fluorescence intensity of tryptophan residues in luciferase and the presence of substrates and reaction products in the active site of the enzyme is substantiated. The non-steady-state kinetics of bioluminescence in the reaction of two bacterial luciferases with aliphatic aldehydes of different chain lengths, as well as the kinetics of enzyme fluorescence intensity during the reaction, were analyzed. The obtained results confirmed the relationship between the rate of the two kinetic stages of enzyme luminescence and the processes of flavin substrate binding and enzyme activity recovery after the catalytic act.
This research aims to analyze and compare the biomechanical and histological characteristics of temporal fascia (TF) and dura mater grafts (DM); both obtained from adult human cadavers preserved in 10% formalin solution. TF and DM samples (bilateral, n = 3 strips per tissue) were obtained from 12 adult human cadavers (6 males, 6 females, age 46-86, mean 73) preserved in 10% formalin solution. Biomechanical tensile test was used to measure the maximum force, stiffness, energy absorption, and maximum deformation, maximum stress, maximum strain, elastic (Young's) modulus, and toughness. Histological analysis assessed collagen and elastic fiber densities using standard staining methods via Pentachrome, Masson Trichrome and Verhoeff staining. The elastic modulus, ultimate stress, and toughness were higher in formalin-fixed temporalis fascia than in formalin-fixed dura mater, indicating a stiffer material behavior. Histologically, collagen fiber and elastic fiber densities of grafts were similar. Formalin-fixed temporal fascia exhibited higher stiffness and tensile strength than formalin-fixed dura mater, likely due to fixation-induced structural alterations rather than differences in elastic fiber content.
A fundamental understanding of catalyst deactivation and regeneration is essential for developing sustainable catalytic pyrolysis processes for lignocellulosic biomass conversion. Herein, the effect of alumina regeneration on the catalytic pyrolysis of ferulic acid, a lignin-derived model compound, was investigated using kinetic analysis combined with FTIR, TPD-MS, XPS, TEM, XRD, thermogravimetry, and DFT calculations. Comparative kinetic analysis showed that the apparent activation energies of key reactions, including decarboxylation, demethoxylation, and the formation of 4-vinylguaiacol, guaiacol, phenol, and other aromatic products, increase by 4-23 kJ mol- 1 after catalyst regeneration, indicating partial modification of the alumina surface. DFT calculations revealed that 4-vinylguaiacol formation proceeds preferentially via a surface-assisted acidic decarboxylation pathway involving alumina-bound intermediates, whereas alternative surface interactions generate strongly bound species that may contribute to catalyst deactivation. Combined experimental and theoretical evidence indicates that surface-bound phenolate complexes act as precursors to carbon deposition. These species undergo styrene-like polymerization on alumina, forming a polymeric carbonaceous layer that decomposes upon heating, releasing aromatic products and progressively forming extended polyaromatic carbon domains. The proposed mechanism is supported by TEM, FTIR, XPS, and TPD-MS results. These findings establish a unified mechanistic framework linking catalytic decarboxylation, carbon-layer formation, and catalyst deactivation during biomass conversion.
The eukaryotic DNA damage and replication stress checkpoint is initiated by activation of the apical kinase complex ATR-ATRIP on RPA-coated ssDNA. In Saccharomyces cerevisiae, the Mec1-Ddc2 (hATR-ATRIP) activator and checkpoint mediator Dpb11 (hTopBP1) is recruited to the 9-1-1 checkpoint clamp (another Mec1-Ddc2 activator) at 5' ss-dsDNA junctions. It remains unclear how Mec1-Ddc2 encounters its activators on damaged DNA due to their differential DNA binding preferences. Using real-time single-molecule imaging, we show that Dpb11 binds to ssDNA directly and localizes to ss-dsDNA junctions in an RPA-dependent manner. Furthermore, Dpb11 recruits Mec1-Ddc2 to ss-dsDNA junctions. Single-molecule force spectroscopy was used to demonstrate that Dpb11 forms bridges on ssDNA, both alone and in the presence of RPA, reducing the end-to-end distance of gapped DNA. These data support a model in which Dpb11 facilitates Mec1-Ddc2 colocalization with its activators directly by recruiting Mec1-Ddc2 to gap junctions and indirectly by decreasing the effective gap length.
The effect of a pencil scanning proton beam in two regions of the Bragg curve with different linear energy transfer (LET) relative to X-ray radiation on the induction of micronuclei (MN) in cytochalasin-blocked binucleate lymphocytes (CBBLs) during in vitro irradiation of human peripheral blood at doses ranging from 0 to 2.0 Gy was studied. A nonlinear dose-response relationship was observed in the dose range of 0 to 1.0 Gy. The frequency of MN in CBBLs during proton irradiation was 2 times lower than during X-rays and did not depend on the LET value. In the dose range above 1 Gy, the dose dependences were linear, the value of the relative biological effectiveness depended on LET and was equal to 0.76 before the Bragg peak, and 1.16 at the peak.
TATA-box binding protein-associated factor 15 (TAF15) is an RNA-binding protein and the primary fibrillar constituent in a subset of frontotemporal lobar degeneration (FTLD) cases. However, the molecular determinants underlying TAF15 aggregation remain unclear. Here, we show that TAF15 forms amyloid fibrils under physiological conditions and develop a cellular biosensor to monitor its propagation. Both recombinant TAF15 fibrils and pathological aggregates extracted from FTLD patient brains selectively seed TAF15 biosensor cells, demonstrating prion-like properties. The closely related protein FUS does not seed TAF15 aggregation, revealing a cross-seeding barrier, but partially incorporates into inclusions during TAF15-induced seeding, potentially explaining their pathological overlap in FTLD. Computational and peptide-based mapping identifies aggregation-prone motifs within the low-complexity domain that stabilize ex vivo fibril cores and drive TAF15 propagation. These findings establish TAF15 as an amyloid-forming, prion-like protein and define sequence determinants underlying its self-assembly, providing a mechanistic framework for FTLD-TAF15 and potential therapeutic targets.
Mutations in the splice-regulator RBM20 cause heart failure with reduced ejection fraction (HFrEF), typically manifesting as dilated cardiomyopathy (DCM). Mutations at position 634 in the RS-domain cause DCM with (R634L) or without (R634W) left ventricular non-compaction (LVNC). However, the mechanisms underlying phenotype variability and personalized therapy beyond HFrEF remain unclear. We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), 3D-cardiospheres and engineered myocardial tissues from patients with RBM20 mutations R634L (LVNC) or R634W (DCM). Using CRISPR/Cas9, we created isogenic rescue and mutation-insertion lines, identifying RBM20 mis-localization, splicing errors in TTN and RYR2, and sarcomere irregularities in both. DCM-CM showed increased resting Ca2+ leak and reduced Ca2+ transient amplitude, typical of HFrEF, and spatial disorganization of sarcoplasmic reticulum and mitochondria. In contrast, LVNC-CM exhibited elevated Ca2+ transient amplitude with faster kinetics, driven by elevated cAMP and mis-spliced, hyperactive CAMK2D, leading to PLN-hyperphosphorylation and increased metabolic respiration. Further, LVNC showed desmosomal derangement potentially from mis-splicing of Junction plakoglobin and reduced 3D cardiosphere compaction. Despite distinct mechanisms, contractile force was reduced in both. Isogenic controls confirm mutation causality. Drug intervention with verapamil partially improved selected abnormal Ca2+ handling and contractile phenotypes in LVNC- and DCM-CM, whereas the CAMK2D inhibitor AIP improved systolic Ca2+ handling predominantly in LVNC-CM. In conclusion, different amino acid substitutions at the same RBM20-residue induce opposing Ca2+-handling and structural phenotypes. While DCM features impaired Ca2+ handling, LVNC shows defective cell-cell coupling and activated Ca2+ handling and metabolism, yet insufficient to compensate for organ-level dysfunction. This supports personalized pharmacological therapies in early HF, and potential CRISPR/Cas9 repair for RBM20 cardiomyopathy.
Oral squamous cell carcinoma (OSCC) represents 90% of all head and neck cancers. Despite decades of research, the 5-year survival rate is 50%. Strikingly, the overall incidence rate is projected to increase by 30% in the next 10 years, which may result in a sharp increase in mortality. Two fundamental aspects of OSCC are that it progresses via the inactivation and mutation of tumor suppressor genes (TSGs) and has a "cold" tumor microenvironment (TME). A major barrier in the treatment of OSCC is the lack of novel therapies clinicians have at their disposal that are designed to disrupt tumor progression by reshaping the cold tumors into inflammatory "hot" tumors. To overcome these obstacles, we employed a lipid nanoparticle (LNP) that co-encapsulates p53 mRNA and the small molecule ciclopirox (CPX). We demonstrate that both drugs have innate chemotherapeutic properties by facilitating caspase activation. Moreover, these therapies can create a less immunosuppressive TME in part by repolarizing tumor-associated macrophages (TAMs) to M1-like phenotypes. When formulated together, our platform provides an all-in-one approach for OSCC, effective in both p53-therapy-susceptible and p53-therapy-resistant models. Additionally, this work offers a template for a delivery platform capable of tackling multiple mechanisms of OSCC progression and survival.
To evaluate the association between bevacizumab use and fistula occurrence in patients with metastatic or persistent cervical cancer (CC) treated with definitive chemoradiotherapy (CRT). We retrospectively analyzed 101 patients with FIGO stage IIB-IVB CC treated with curative-intent CRT between 2017 and 2023. The primary outcome was the incidence of fistula formation, with a focus on the impact of bevacizumab exposure. Fistulas occurred in 7 of 101 patients (6.9%). Among the 13 patients treated with bevacizumab, 4 (30.8%) developed fistulas compared with 3 (3.4%) of 88 not receiving bevacizumab. Univariable analysis showed significant associations with bevacizumab use (OR = 12.59, 95% CI: 2.43-65.38, p = 0.003), and FIGO stage IVB (OR = 21.90, p = 0.001). In multivariable analysis, bevacizumab (AOR = 13.5, 95% CI: 1.84-99.5, p = 0.011) and FIGO stage IV (AOR = 26.7, 95% CI: 2.59-274.4, p = 0.006) remained independent predictors. No fistulas occurred after 2021, coinciding with institutional discontinuation of bevacizumab in metastatic or persistent disease after CRT (p = 0.043). Bevacizumab use and FIGO stage IV disease were associated with increased risk of fistula formation after definitive CRT. These findings highlight the need for careful treatment planning when considering bevacizumab in previously irradiated patients, particularly in the current era of multimodal therapy.
A new family of genes encoding potential antimicrobial peptides with compact and elegant structure has been found in the genomes of several Fungi and some arthropod species. Their expression products are constituted of about 85 amino acids, including a signal peptide, and are folded into two α-helical segments connected by a short unstructured coil. Three conserved disulphide bridges between cysteines located in symmetrically mirrored positions connect the two helical domains. These peptides, here named as Hairpin Loop Peptides (HLPs), have been found in the genomes of many Fungi species but only in selected clades. Orthologues have also been discovered in the genomes of some insects, notably Hemiptera, a few other arthropods and other organisms. They are not found in plants, that however express smaller peptides of similar topology with HLPs, but different amino acidic composition and physicochemical properties. They appear to have originated in Fungi and then migrated to insects through horizontal gene transfer. The antimicrobial activity of HLPs is predicted by several software programmes, but this aspect needs to be supported by experimental evidence. The occurrence of HLPs in several edible mushrooms may suggest potential uses of these peptides in food preservation and possibly also in medical applications. Their simple and nearly rigid structure can be easily modified to improve specificity, stability and solubility, thus making these peptides suitable for a variety of different applications.