Native Structure Research Articles

Designing proteins: Mimicking natural protein sequence heterogeneity.

This study presents an enhanced protein design algorithm that aims to emulate natural heterogeneity of protein sequences. Initial analysis revealed that natural proteins exhibit a permutation composition lower than the theoretical maximum, suggesting a selective utilization of the 20-letter amino acid alphabet. By not constraining the amino acid composition of the protein sequence but instead allowing random reshuffling of the composition, the resulting design algorithm generates sequences that maintain lower permutation compositions in equilibrium, aligning closely with natural proteins. Folding free energy computations demonstrated that the designed sequences refold to their native structures with high precision, except for proteins with large disordered regions. In addition, direct coupling analysis showed a strong correlation between predicted and actual protein contacts, with accuracy exceeding 82% for a large number of top pairs (>4L). The algorithm also resolved biases in previous designs, ensuring a more accurate representation of protein interactions. Overall, it not only mimics the natural heterogeneity of proteins but also ensures correct folding, marking a significant advancement in protein design and engineering.

Computational engineering of water-soluble human potassium ion channels through QTY transformation.

Transmembrane potassium ion channels are crucial for ion transport, metabolism, and signaling, and serve as promising targets for anti-cancer therapies. However, their hydrophobic transmembrane nature requires detergents, posing a major bottleneck for experimental handling. In this paper, we present a structural bioinformatics study of six experimentally determined and twelve modeled potassium channel structures, in which hydrophobic amino acids (L, I/V, and F) were systematically replaced with neutral hydrophilic ones (Q, T, and Y), making the proteins more water-soluble. QTY (computationally predicted) and native (experimental and repredicted) variants show remarkable structural similarity (RMSD: ~0.50 Å - ~2.14 Å) despite significant sequence differences. QTY variants, both rigid and refined with MD simulations, maintain comparable to native variants stability, solvent-accessible surface area (SASA), and ionic, aromatic, and van der Waals interactions but differ in the grand average of hydropathy (GRAVY), solubility, and hydrophobic contacts. Overall, our study presents a computational approach for designing hydrophilic potassium ion channels while maintaining the native global structure that could potentially simplify their practical use by eliminating the need for detergents.

A Brief Review of Radiolabelling Nucleic Acid-Based Molecules for Tracking and Monitoring.

The rise of nucleic acid-based therapeutics continues apace. At the same time, the need for radiolabelled oligonucleotides for determination of spatial distribution is increasing. Complex molecular structures with mostly multiple charges and low solubility in organic solvents increase the challenge of integrating radionuclides. In preclinical research, it is important to understand the fate of new drug candidates in biodistribution studies, target binding or biotransformation studies. Depending on a specific question, the selection of a respective radiolabelling strategy is crucial. Radiometals for molecular imaging with positron emission tomography or single-photon computed tomography generally require an attached chelating agent for stable complexation of the metal with the oligonucleotide, whereas labelling using carbon-11/-14 or tritium allows incorporation of the radioisotope into the native structure without altering it. Moreover, the suitability of direct radiolabelling of the oligonucleotide of interest or indirect radiolabelling, for example, by a two-step pretargeting approach, for the study design requires consideration. This review focuses on the challenges of radiolabelling nucleic acid-based molecules with beta-plus, gamma and beta-minus emitters and their use for tracking and monitoring.

Influence of Cataract Causing Mutations on αA-Crystallin: A Computational Approach.

The αA-crystallin protein plays a vital role in maintaining the refractive index and transparency of the eye lens. Significant clinical studies have emerged as the αA-crystallin is prone to aggregation, resulting in the formation of cataracts with varied etiologies due to mutations. This work aims to comprehend the structural and functional role of cataract-causing mutations in αA-crystallin, particularly at N-Terminal and α-Crystallin Domains, using in-silico approaches including molecular dynamics simulation. About 19 mutants of αA-crystallin along with native structure were simulated for 100 ns and the post-simulations analyses reveal pronounced dynamics of αA-crystallin due to the enhanced structure flexibility as its native compactness was lost and is witnessed mainly by the mutants R12L, R21L, R21Q, R54L, R65Q, R116C and R116H. It is observed that αA-crystallin discloses the NTD motions as the dominant one and the same was endorsed by the linear variation between Rg and the center-of-mass of αA-crystallin. Interestingly, such enhanced dynamics of αA-crystallin mutants associated with the structure flexibility is internally modulated by the dynamic exchange of secondary structure elements β-sheets and coils (R2 = 0.619) during simulation. Besides, the observed pronounced dynamics of dimer interface region (β3-L6-β4 segment) of ACD along with CTD dynamics also gains importance. Particularly, the highly dynamic mutants are also characterized by enhanced non-covalent and hydrophobic interactions which renders detrimental effects towards its stability, and favours possible protein unfolding mechanisms. Overall, this study highlights the mutation-mediated structural distortions in αA-crystallin and demands the need for further potential development of inhibitors against cataract formation.

Imaging with a twist: Three-dimensional insights of the chiral nematic phase of cellulose nanocrystals via SHG microscopy.

Cellulose nanocrystals (CNCs) are bio-based nanoparticles that, under the right conditions, self-align into chiral nematic liquid crystals with a helical pitch. In this work, we exploit the inherent confocal effect of second-harmonic generation (SHG) microscopy to acquire highly resolved three-dimensional (3D) images of the chiral nematic phase of CNCs in a label-free manner. An in-depth analysis revealed a direct link between the observed variations in SHG intensity and the pitch. The highly contrasted 3D images provided unprecedented detail into liquid crystal's native structure. Local alignment, morphology, as well as the presence of defects are readily revealed, and a provisional framework relating the SHG response to the orientational distribution of CNC nanorods within the liquid crystal structure is presented. This paper illustrates the numerous benefits of using SHG microscopy for visualizing CNC chiral nematic systems directly in the suspension-liquid phase and paves the road for using SHG microscopy to characterize other types of aligned CNC structures, in wet and dry states.

Strategic alteration of arabinoxylan feruloylation enables selective shaping of the human gut microbiota

Arabinoxylans (AXs) are promising prebiotic candidates abundant in cereals. While AX feruloylation impacts properties, its effects on long chains mimicking native structures are unclear. This study revealed a dose-dependent impact of long chain AX feruloylation on gut microbial composition and function. In vitro fecal fermentation with varying AX feruloylation showed increased Bacteroidetes and propionate along with decreased Firmicutes and butyrate. Distinct Bacteroides populations were enriched under different feruloylation levels, suggesting specialized adaptation. Community dynamics and co-occurrence networks highlighted intricate taxon-specific responses, underscoring the unique microbial profiles shaped by individual variations. This study lays the foundation for elucidating the metabolic pathways and enzymatic machineries enabling utilization of feruloylated AXs based on the dose-dependent enrichment of specific taxa. This knowledge can inform rational design of customized prebiotics through precision nutrition. This work provides novel insights into tailoring cereal biomass fermentation and microbiome structure-function relationships. It establishes a platform for developing optimized feruloylated prebiotics to deliberately modulate gut ecology and address intestinal dysbiosis.

A Novel Mutation of FOXC1 (P136L) in an Axenfeld-Rieger Syndrome Patient With a Systematized Delusion of Jealousy: A Case Report and Literature Review.

The main features of Axenfeld-Rieger Syndrome (ARS) are ocular, auditory, neurological, and morphological brain abnormalities. Mutations in forkhead box protein C1 (FOXC1) are among the responsible genes causing ARS, but neuropsychiatric features have rarely been reported. The case of an ARS patient (a 77-year-old man) with delusions of jealousy and impairment of working memory, in addition to the main clinical features, glaucoma and leukoencephalopathy, is presented. The mutation in the patient's genome was found with whole exome sequencing and in silico analysis using PolyPhen-2 and SIFT. Furthermore, AlphaFold2 and PyMOL were used to predict the protein structure based on the mutation. A novel mutation at the forkhead domain of FOXC1 gene (c.408C>A, p.Phe136Leu) was found and confirmed in the patient's family, and it was predicted to cause protein damage; the SIFT score was 0, meaning deleterious, and the PolyPhen2 result also indicated damaging (score: 0.997). The predicted protein structure based on the novel mutation was different from that of the native structure. In the literature review, 6 of 95 (6.3%) cases showed neuropsychiatric features. Of them, 5 of 6 (83.3%) mutations were located in the forkhead domain. A novel mutation was found in the FOXC1 gene (c.408C>A, p.Phe136Leu), which possibly induces delusions of jealousy and impairment of working memory, as well as features of ARS, by changing the protein structure. Mutations in that domain of the FOXC1 gene may be important not only for ocular abnormalities but also for brain function.

Establishing Benchmarks for Airway Replacement: Long-Term Outcomes of Tracheal Autografts in a Large Animal Model.

Airway replacement is a challenging surgical intervention and remains an unmet clinical need. Due to the risk of airway stenosis, anastomotic separation, poor vascularization, and necrosis, it is necessary to establish the gold-standard outcomes of tracheal replacement. In this study, we use a large animal autograft model to assess long-term outcomes following tracheal replacement. Four New Zealand White rabbits underwent tracheal autograft surgery and were observed for 6 months. Clinical and radiographic surveillance were recorded, and grafts were analyzed histologically and radiographically at endpoint. All animals survived to the endpoint with minimal respiratory symptoms and normal growth rates. No complications were observed. Computed tomography scans of the post-surgical airway demonstrated graft patency at all time points. Histological sections showed no sign of stenosis or necrosis with preservation of the native structure of the trachea. We established benchmarks for airway replacement. Our findings suggest that a rabbit model of tracheal autograft with direct reimplantation is feasible and does not result in graft stenosis or airway collapse.

Biologic Augmentation of Rotator Cuff Repair: Current Concepts Review.

Rotator cuff tears are common in an aging population. Thus far, primary repairs have shown high re-tear rates suggesting the need for improved healing modalities. Current augmentations of rotator cuff repairs include synthetic and biological scaffolds, surgical bone marrow venting, and infusing the repair with a variety of stem cells and growth factors aimed at restoring the native cellular structure and function of the repaired tissue. This current concepts review discusses the anatomy, physical presentation, diagnosis, and treatment of rotator cuff tears; biological adjuvants for rotator cuff repairs; and the current literature on outcomes after biologically augmented rotator cuff repairs. [Orthopedics. 202x;4x(x):xx-xx.].

The Inhibition of Serum Amyloid A Protein Aggregation by a Five-Residue Peptidomimetic: Structural and Morphological Insights.

Serum amyloid A (SAA) is a small protein consisting of 104 residues and, under physiological conditions, exists mainly in hexameric form. It belongs to the positive acute-phase proteins, which means that its plasma concentration increases rapidly in response to injury, inflammation, and infection. The accumulation of SAA molecules promotes the formation of amyloid aggregates, which deposit extracellularly in many organs, causing their dysfunction. In our previous work, we successfully designed a peptidomimetic that inhibited the aggregation of amyloidogenic SAA fragments. In the present paper, we show how the same inhibitor, named saa3Dip, affects the oligomerization and aggregation processes of MetSAA1.1 protein. The thioflavin T assay showed that saa3Dip inhibited its fibrillization. The measurement of the internal fluorophore fluorescence (Trp) showed differences that occurred in the tertiary structure of MetSAA1.1 in the presence of the inhibitor, which was also confirmed by CD spectra in the aromatic range. FTIR results suggested that saa3Dip could stabilize some fragments of the native structure of MetSAA1.1, which was confirmed by determining the melting temperature (Tm) of the protein-inhibitor complex. AFM images demonstrated that the presence of saa3Dip prevented the formation of large SAA aggregates. Our results suggest that saa3Dip stabilizes the native conformation of MetSAA1.1.

In-situ Forming Multipolymeric Glucose-Responsive Hydrogels.

Stimuli-responsive hydrogels (HGs) have shown promise for smart drug delivery applications. Specifically, glucose-responsive HGs having phenylboronic acid (PBA) functional groups are extensively pursued for insulin delivery in hyperglycemia. Current polymeric glucose-responsive HGs are cumbersome to fabricate and show a limited insulin release profile. Herein, we develop a straightforward fabrication of glucose-responsive multipolymer HGs (MPHGs) using a three-component in situ mixing. Molecular cargo, such as insulin, was loaded during the gelation. Heterobifunctional formylphenylboronic acid (FPBA) crosslinkers were used to interconnect polyvinyl alcohol (PVA) and branched polyethyleneimine (PEI) via boronate ester and imine bonds, respectively. Three positional isomers of FPBA (2FPBA, 3FPBA, and 4FPBA) resulted in HGs with distinct viscoelastic behaviors under the same conditions. HGs derived from 4FPBA exhibited more solid-like properties compared to 2FPBA and 3FPBA due to a higher crosslinking density. All the HGs exhibited glucose-responsive dissolution and release of embedded insulin cargo without disrupting the native structure. Insulin release profiles show a higher glucose-responsive release from 4FPBA-derived MPHGs. All the HGs were injectable, self-healing, and noncytotoxic below 10 μg/ml concentrations. The MPHGs developed in this study uncover new directions in creating glucose-responsive matrices for self-regulating drug delivery applications. In the future, detailed in vivo studies will be performed for clinical applications.

The RARCrec: A rapid riparian vegetation assessment method for use in river systems undergoing vegetative and geomorphic recovery

SummaryMonitoring the condition of returning vegetation over time is essential for effectively managing riparian vegetation. Rapid riparian assessment methods are standard tools for capturing a suite of variables relevant to river health and are accessible to a range of users with different levels of experience. Monitoring efforts are particularly important for rivers undergoing degrading impacts and subsequent management interventions. In coastal catchments of New South Wales, Australia, vegetation is returning to rivers that were historically cleared for agriculture and are now experiencing geomorphic recovery. We present a rapid riparian assessment method for rivers undergoing vegetative and geomorphic recovery in this region. It addresses a gap in available rapid assessments by providing a methodology that can be used at sites with no historical analogue. Our assessment method adapts and extends a widely used method called the Rapid Assessment of Riparian Condition (RARC) to capture the quality of returning riparian vegetation along recovering rivers (rec), including native species diversity and vegetation structure (hence the name RARCrec). The RARCrec incorporates four sub‐indices – vegetation cover, native species richness, proportion of native to exotic species, and features of vegetation health – to produce an overall vegetation condition score that classifies sites as poor, moderate, or good condition. Scores for each sub‐index are assessed for the vegetation strata (groundcover, graminoids, midstorey, vines, and canopy) on the landform units of river bars, benches, and floodplains. The RARCrec has been designed to be sufficiently general for use in the main Vegetation Formations in coastal New South Wales and has been trialled at 36 sites across this region. Assessments can be conducted within half a day by two technically competent users and can be used to monitor vegetation condition over time to inform where to prioritise implementation of management activities. Field methodology and data sheets, including a scoring system, are provided to support the implementation of the RARCrec.

Interface-packing analysis of F1-ATPase using integral equation theory and manifold learning

It has been shown that the translational entropy of water plays a key role in biological processes such as protein folding and ligand binding. Under the physiological condition, tightly packed protein conformations like native structures are achieved so that the translational entropy of water is maximized. In this study, we investigate the rotation mechanism of a rotary protein motor, F1-ATPase, by analyzing the packing at the interfaces between the subunits. The packing at the interface between a subunit pair is analyzed using the change in the solvent entropy upon forming subunit pair, ∆S. It is found that as the γ subunit rotates, the ∆S value of a α-β subunit pair decrease because the interface packing becomes loose. However, because the interface packing of another α-β subunit pair becomes tighter upon the rotation, ∆S of this α-β subunit pair increases, leading to a compensation of the decrease in ∆S. Such compensation would be necessary to maximize the solvent entropy of F1-ATPase. In this study, packing at the interfaces between the subunits is also analyzed using a manifold-learning technique, and it is suggested a possibility that a qualitative estimation of the ∆S values of some α-β subunit pairs can be predicted using a manifold-learning technique.

Multidimensional study of starch co‐plasticized with glycerol and isosorbide in TPS/PLA blends: Morphological, mechanical, thermal, and structural properties

AbstractBiodegradable blends of polylactic acid (PLA) and thermoplastic starch (TPS) co‐plasticized with glycerol (G) and isosorbide (i) in different ratios were prepared by extrusion in a single screw extruder. The G/isosorbide ratio varied within the range 27.5/2.5 to 15/15. The resulting materials were obtained through blown film extrusion and characterized using a variety of techniques. The plasticizing effect was identified through SEM and FTIR observations. In the SEM images, it was evident that most of the native crystalline structures were destructured. Furthermore, SEM observations showed phase separation of TPS and PLA in all samples, with phase dispersion differences depending on the co‐plasticizer ratio. The FTIR spectra of all TPS/PLA films showed that isosorbide caused changes in the absorption bands that suggested a reduction in the crystallinity of the native starch, agreeing with the XRD results that indicated the formation of different crystalline structures (EH type). The XRD results revealed a change in the crystal structure of the TPS phase influenced by the amount of isosorbide. Isosorbide increased tensile strength and reduced elongation due to strong interactions between the plasticizer and the starch chains through hydrogen bonds, con‐firmed by FTIR analysis. In general, it was established that isosorbide and G co‐plasticization, with small amounts of isosorbide (up to a ratio of 25:5 G/isosorbide), can be employed as an effective plasticizer without significantly affecting the tensile mechanical properties.

Template switching enables chemical probing of native RNA structures.

RNAs are often studied in non-native sequence contexts to facilitate structural studies. However, seemingly innocuous changes to an RNA sequence may perturb the native structure and generate inaccurate or ambiguous structural models. To facilitate the investigation of native RNA secondary structure by selective 2' hydroxyl acylation analyzed by primer extension (SHAPE), we engineered an approach that couples minimal enzymatic steps to RNA chemical probing and mutational profiling (MaP) reverse transcription (RT) methods - a process we call template switching and mutational profiling (Switch-MaP). In Switch-MaP, RT templates and additional library sequences are added post-probing through ligation and template switching, capturing reactivities for every nucleotide. For a candidate SAM-I riboswitch, we compared RNA structure models generated by the Switch-MaP approach to those of traditional primer-based MaP, including RNAs with or without appended structure cassettes. Primer-based MaP masked reactivity data in the 5' and 3' ends of the RNA, producing ambiguous ensembles inconsistent with the conserved SAM-I riboswitch secondary structure. Structure cassettes enabled unambiguous modeling of an aptamer-only construct but introduced non-native interactions in the full length riboswitch. In contrast, Switch-MaP provided reactivity data for all nucleotides in each RNA and enabled unambiguous modeling of secondary structure, consistent with the conserved SAM-I fold. Switch-MaP is a straightforward alternative approach to primer-based and cassette-based chemical probing methods that precludes primer masking and the formation of alternative secondary structures due to non-native sequence elements.

Understanding Polyproline's Unusual Thermoresponsive Properties Using a Polyproline-Based Double Hydrophilic Block Copolymer.

Polyproline is a unique thermoresponsive polymer characterized by large thermal and conformational hysteresis. This article employs polyproline-based double hydrophilic block copolymers (PNIPAMn-b-PLPm) to gain insight into polyproline's thermoresponsive mechanism. The amine-terminated poly(N-isopropylacrylamide) (NH2-PNIPAMm) was used as the macroinitiator for ring-opening polymerization of proline-NCA monomers, resulting in various block copolymers (PNIPAMn-b-PLPm) with varying PLP block lengths. Block copolymers' thermal phase transitions were compared with their homopolymer counterparts using turbidimetry, variable-temperature NMR, dynamic light scattering, and circular dichroism spectroscopy. These experiments revealed that regardless of their compositions, all block copolymers exhibited a two-stage collapse (TCP(PLP) > TCP(PNIPAM)) during the heating cycle. In contrast, only one clearing temperature (TCL) was observed during cooling. The observed clearing temperature is closely correlated to the clearing temperature of PNIPAM blocks, suggesting the role of water-soluble PNIPAM blocks in resolving the PLP blocks. Moreover, thermal and conformational hysteresis related to the polyproline block is significantly suppressed in the presence of a PNIPAM block. Linking PNIPAM blocks has two significant effects on PLP segments' thermoresponsive behavior. For example, during the heating cycle, the precollapsed PNIPAM chains (as TCP(PNIPAM) < TCP(PLP)) prevent orderly aggregation within the PLP block. Meanwhile, during the cooling cycle below the clearing temperature of the PNIPAM block, the PNIPAM chains impart water solubility (as TCL(PNIPAM) > TCL(PLP)) to the collapsed PLP chains. Overall, the PNIPAM block imparts water solubility and perturbs PLP chains to form the native aggregate structure, suppressing the hysteresis effect. Accordingly, the large thermal and conformational hysteresis associated with native PLP chains appears to result from a noninterfering aggregation above the critical temperature.

Exploring the unfolding pathways of protein families using Elastic Network Model

We explore how a protein’s native structure determines its unfolding process. We examine how the local structural features, like shear, and the global structural properties, like the number of soft modes, change during unfolding. Simulations are performed using a Gaussian Network Model (GNM) with bond breaking for both thermal and force-induced unfolding scenarios. We find that unfolding starts in areas of high shear in the native structure and progressively spreads to the low shear regions. Interestingly, analysis of single domain protein families (Chymotrypsin inhibitor and Barnase) reveal that proteins with distinct unfolding pathways exhibit divergent behavior of the number of soft modes during unfolding. This suggests that the number of soft modes might be a valuable tool for understanding thermal unfolding pathways. Additionally, we found a strong link between a protein’s overall structural similarity (TM-score) and its unfolding pathways, highlighting the importance of the native structure in determining how a protein unfolds.

Mechanisms of microbial diversity modulation of mineral black clay to achieve ecological restoration of open-pit mine dump

The harsh climatic conditions and severe scarcity of surface soil present significant challenges to ecological restoration in open-pit mine dumps within China's type II plant cold resistance zone. To address the topsoil shortage, mineral black clay was used to create synthetic soil. This study explored the application of an ecological restoration bacteria (ERB) consortium to accelerate the ecological restoration of synthetic soil-covered areas by enhancing soil ecosystem construction. The results demonstrated that ERB significantly influenced the native bacterial community structure in mixed black clay. Specifically, ERB disrupted the inhibitory effects of the Actinobacterota phylum on the development of native bacterial diversity, leading to an increase in unclassified_o_Solirubrobacterales sp., norank_f_norank_o_norank_c_KD4-96 sp., Sphingomonas sp., Luteitalea sp., norank_f_Vicinamibacteraceae sp., and other aerobic and anaerobic bacteria. These alterations in soil microbial structure directly impacted soil composition and vegetation diversity. The plant diversity survey and metabolomics analysis revealed that the reduction of harmful substances, such as HPED, HODE, and HOME, in black clay soil improved the growth and distribution of Salsola collina Pall. and Medicago sativa L. This increase facilitated the cycling of key nutrients, such as nitrogen (N) and phosphorus (P), and promoted the establishment of symbiotic relationships between plants, microorganisms, and soil. Ultimately, the ecological remediation of the synthetic soil was achieved through the synergistic effects of ERB, which included the degradation of inhibitory soil components, enhanced nutrient consumption by microbiota and plants, and the overall promotion of ecosystem stability in the reclamation area.

Field-scale gene flow of fungicide resistance in Pyrenophora teres f. teres and the effect of selection pressure on the population structure.

The effectiveness of fungicides to control foliar fungal crop diseases is being diminished by the increasing spread of resistances to fungicides. One approach that may help to maintain efficacy is remediation of resistant populations by sensitive ones. However, the success of such approaches can be compromised by re-incursion of resistance through aerial spore dispersal; although, knowledge of localized gene flow is lacking. Here, we report on a replicated mark-release-recapture field experiment with several treatments set up to study spore-dispersal-mediated gene flow of a mutated allele that confers demethylase inhibitor resistance in Pyrenophora teres f. teres (Ptt). Artificial inoculation of the host, barley (Hordeum vulgare), was successful across the 12-ha trial, where the introduced sensitive- and resistant-populations were, respectively, 6- and 13-fold the DNA concentration of the native Ptt population. Subsequent disease pressure remained low which hampered spread of the epidemic to such extent that gene flow was not detected at, or beyond 2.5 m from source points. In the absence of gene flow, plots were assessed for treatment effects; fungicide applied to populations that contained 14.3% of allele mutation increased in frequency to 24.5%, whereas sensitive populations had no change in structure. Untreated controls of native Ptt population remained genetically stable, yet untreated controls that were inoculated with sensitive Ptt had half the resistance frequency of the native population structure. The trial demonstrates the potential for management to remediate fungicide resistant pathogen populations, where localized gene flow is minimal; to safeguard chemical crop protection into the future.

Double-crosslinked dECM bioink to print a self-sustaining 3D multi-layered aortic-like construct

Cardiovascular disease is the leading cause of death worldwide, with related mortality increasing from 12.1 million to 18.6 million in the past 30 years.To address the supply limitation of autologous vascular grafts and overcome the limits of current treatment options, 3D bioprinting techniques have been investigated.This study aimed at introducing a self-supporting and multi-layered 3D bioprinted construct as a promising alternative for large-blood vessel replacement. To this end, we developed an alginate-gelatin bioink enriched with decellularized extracellular matrix (dECM) of porcine aorta combined with a two-step crosslinking process. We investigated the feasibility of achieving structural stability and shape fidelity of the bioprinted construct over time through rheological characterization, printability tests, and degradation tests.According to the results of rheology and printability tests, dECM-enriched bioink combined with the double-crosslinking process (internal and external crosslink) showed good printability and high shape fidelity, withstanding more than 35 layers without the need for support. Moreover, the bioprinted construct preserved its structural stability over time, retaining a wall thickness comparable to that of the native aorta. Finally, immortalized mouse fibroblasts embedded in the bioink were well adhered to the bioink and alive over time. The double-crosslinked bioink represents an impactful strategy to produce an alternative conduit with the native hierarchical structure of the large blood vessels.