Engineering Matrix-Free Drug Protein Nanoparticles with Promising Penetration through Biobarriers for Treating Corneal Neovascularization.

Protein drugs have been widely used in treating various clinical diseases because of their high specificity, fewer side effects, and favorable therapeutic effect, but they greatly suffer from their weak permeability through tissue barriers, high sensitivity to microenvironments, degradation by proteases, and rapid clearance by the immune system. Herein, we disrupted the standard protocol where protein drugs must be delivered as the cargo via a delivery system and innovatively developed a free entrapping matrix strategy by simply mixing bevacizumab (Beva) with zinc ions to generate Beva-NPs (Beva-Zn2+), where Beva is coordinatively cross-linked by zinc ions with a loading efficiency as high as 99.2% ± 0.41%. This strategy was universal to generating various protein NPs, with different metal ions (Cu2+, Fe3+, Mg2+, Sr2+). The synthetic conditions of Beva-NPs were optimized, and the generated mechanism was investigated in detail. The entrapment, releasing profile, and the bioactivities of released Beva were thoroughly studied. By using in situ doping of the fourth-generation polyamindoamine dendrimer (G4), the Beva-G4-NPs exhibited extended ocular retention and penetration through biobarriers in the anterior segment through transcellular and paracellular pathways, effectively inhibiting corneal neovascularization (CNV) from 91.6 ± 2.03% to 13.5 ± 1.87% in a rat model of CNV. This study contributes to engineering of protein NPs by using a facile strategy for overcoming the weaknesses of protein drugs and protein NPs, such as weak tissue barrier permeability, low encapsulation efficiency, poor loading capacity, and susceptibility to inactivation.

Corrigendum to "Emulsifying Lipiodol with pH-Sensitive DOX@HmA nanoparticles for hepatocellular carcinoma TACE treatment eliminate metastasis" Mater. Today Bio, 23, 2023, 100873, ISSN 2590-0064.

[This corrects the article DOI: 10.1016/j.mtbio.2023.100873.].

Mi-BMSCs alleviate inflammation and fibrosis in CCl4-and TAA-induced liver cirrhosis by inhibiting TGF-ß/Smad signaling.

Cirrhosis is an aggressive disease, and over 80 % of liver cancer patients are complicated by cirrhosis, which lacks effective therapies. Transplantation of mesenchymal stem cells (MSCs) is a promising option for treating liver cirrhosis. However, this therapeutic approach is often challenged by the low homing ability and short survival time of transplanted MSCs in vivo. Therefore, a novel and efficient cell delivery system for MSCs is urgently required. This new system can effectively extend the persistence and duration of MSCs in vivo. In this study, we present novel porous microspheres with microfluidic electrospray technology for the encapsulation of bone marrow-derived MSCs (BMSCs) in the treatment of liver cirrhosis. Porous microspheres loaded with BMSCs (Mi-BMSCs) exhibit good biocompatibility and demonstrate better anti-inflammatory properties than BMSCs alone. Mi-BMSCs significantly increase the duration of BMSCs and exert potent anti-inflammatory and anti-fibrosis effects against CCl4 and TAA-induced liver cirrhosis by targeting the TGF-ß/Smad signaling pathway to ameliorate cirrhosis, which highlight the potential of Mi-BMSCs as a promising therapeutic approach for early liver cirrhosis.

Recent Progress in Protein-Polyphenol Assemblies for Biomedical Applications.

Nowadays, natural materials as smart building blocks for assembling functional materials have aroused extensive interest in the scientific community. Proteins and polyphenols are typical natural building blocks that are widely used. On the one hand, proteins are one of the most versatile classes of biomolecules, serving as catalysts, signaling molecules, transporters, receptors, scaffolds that maintain the integrity of cell and tissue, and more. On the other hand, the facile adhesion of naturally abundant polyphenols with other substances and their potential biomedical applications have been highly attractive for functional biomaterials fabrication. Additionally, there are a variety of interactions between the proteins and polyphenols, mainly hydrogen bonding, hydrophobic, and ionic interactions. These reversible dynamic interactions enable proteins and polyphenols to form stable protein-polyphenol assemblies and maintain their inherent structures and biological activities in the assemblies. Therefore, protein-polyphenol assemblies can be applied to design a variety of advanced functional materials for biomedical applications. Herein, recent progress in protein-polyphenol particles, capsules, coatings, and hydrogels is summarized, the preparation and application of these assemblies are introduced in detail, and the future of the field is prospected.

Responsively Degradable Nanoarmor-Assisted Super Resistance and Stable Colonization of Probiotics for Enhanced Inflammation-Targeted Delivery.

Manipulation of the gut microbiota using oral microecological preparations has shown great promise in treating various inflammatory disorders. However, delivering these preparations while maintaining their disease-site specificity, stability, and therapeutic efficacy is highly challenging due to the dynamic changes associated with pathological microenvironments in the gastrointestinal tract. Herein, a superior armored probiotic with an inflammation-targeting capacity is developed to enhance the efficacy and timely action of bacterial therapy against inflammatory bowel disease (IBD). The coating strategy exhibits suitability for diverse probiotic strains and has negligible influence on bacterial viability. This study demonstrates that these armored probiotics have ultraresistance to extreme intraluminal conditions and stable mucoadhesive capacity. Notably, the HA-functionalized nanoarmor equips the probiotics with inflamed-site targetability through multiple interactions, thus enhancing their efficacy in IBD therapy. Moreover, timely "awakening" of ingested probiotics through the responsive transferrin-directed degradation of the nanoarmor at the site of inflammation is highly beneficial for bacterial therapy, which requires the bacterial cells to be fully functional. Given its easy preparation and favorable biocompatibility, the developed single-cell coating approach provides an effective strategy for the advanced delivery of probiotics for biomedical applications at the cellular level.

Gelatin-based dynamic response antioxidant, anti-inflammatory multifunctional hydrogel for enhanced diabetic wound repair.

Diabetic wound therapy presents significant challenges in the clinical environment, where persistent bleeding, disturbed inflammatory regulation, impaired cellular proliferation, and impaired tissue remodeling are major features of diabetic wound healing. However, current treatment strategies need to be considered in the context of the dynamic and complex needs of chronic wound healing. Here, multifunctional dynamic boronic acid cross-linked hydrogels were prepared by the reaction of gelatin (Gel) inoculated with 5-carboxy 3-nitrophenylboronic acid (NPBA) and Epigallocatechin gallate (EGCG) to achieve rapid gelation at pH = 7.4, EGCG could interact electrostatically with cationic antimicrobial peptides (AMP) to achieve the effective loading of AMP in the hydrogels. This hydrogel can be injected and adhered to skin defects in diabetic patients to provide a barrier and rapid hemostasis. In a high glucose microenvironment, the rapid release of AMP effectively kills bacteria, while the responsive release of EGCG eliminates reactive oxygen species (ROS) and promotes macrophage M2 polarization. In addition, the hydrogel had excellent biocompatibility and degradability properties, degraded completely after 3 days of subcutaneous injection, and was non-toxic in H&E staining of major organs and serum liver function indices in mice. This multifunctional injectable hydrogel accelerates diabetic skin wound repair and is a promising dressing for the precise treatment of diabetic wounds.

Local Spinal Cord Injury Treatment Using a Dental Pulp Stem Cell Encapsulated H2S Releasing Multifunctional Injectable Hydrogel.

Spinal cord injury (SCI) commonly induces nerve damage and nerve cell degeneration. In this work, a novel dental pulp stem cells (DPSCs) encapsulated thermoresponsive injectable hydrogel with sustained hydrogen sulfide (H2S) delivery is demonstrated for SCI repair. For controlled and sustained H2S gas therapy, a clinically tested H2S donor (JK) loaded octysilane functionalized mesoporous silica nanoparticles (OMSNs) are incorporated into the thermosensitive hydrogel made from Pluronic F127 (PF-127). The JK-loaded functionalized MSNs (OMSF@JK) promote preferential M2-like polarization of macrophages and neuronal differentiation of DPSCs in vitro. OMSF@JK incorporated PF-127 injectable hydrogel (PF-OMSF@JK) has a soft consistency similar to that of the human spinal cord and thus, shows a high cytocompatibility with DPSCs. The cross-sectional micromorphology of the hydrogel shows a continuous porous structure. Last, the PF-OMSF@JK composite hydrogel considerably improves the in vivo SCI regeneration in Sprague-Dawley rats through a reduction in inflammation and neuronal differentiation of the incorporated stem cells as confirmed using western blotting and immunohistochemistry. The highly encouraging in vivo results prove that this novel design on hydrogel is a promising therapy for SCI regeneration with the potential for clinical translation.

Facile General Injectable Gelatin/Metal/Tea Polyphenol Double Nanonetworks Remodel Wound Microenvironment and Accelerate Healing.

Treating the most widespread complication of diabetes: diabetic wounds poses a significant clinical obstacle due to the intricate nature of wound healing in individuals with diabetes. Here a novel approach is proposed using easily applicable injectable gelatin/metal/tea polyphenol double nanonetworks, which effectively remodel the wound microenvironment and accelerates the healing process. The gelatin(Gel) crosslink with metal ions (Zr4+ ) through the amino acids, imparting advantageous mechanical properties like self-healing, injectability, and adhesion. The nanonetwork's biological functions are further enhanced by incorporating the tea polyphenol metal nanonetwork through in situ doping of the epigallocatechin gallate (EGCG) with great antibacterial, self-healing, antioxidant, and anticancer capabilities. The in vitro and in vivo tests show that this double nanonetworks hydrogel exhibits faster cell migration and favorable anti-inflammatory and antioxidant properties and can greatly reshape the microenvironment of diabetic wounds and accelerate the wound healing rate. In addition, this hydrogel is completely degraded after subcutaneous injection for 7 days, with nondetectable cytotoxicity in H&E staining of major mice organs and the serum level of liver function indicators. Considering the above-mentioned merits of this hydrogel, it is believed that the injectable gelatin/metal/tea polyphenol double nanonetworks have broad biomedical potential, especially in diabetic wound repair and tissue engineering.

Self-Report Amphiphilic Polymer-Based Drug Delivery System with ROS-Triggered Drug Release for Osteoarthritis Therapy.

The development of drug delivery systems with real-time cargo release monitoring capabilities is imperative for optimizing nanomedicine performance. Herein, we report an innovative self-reporting drug delivery platform based on a ROS-responsive random copolymer (P1) capable of visualizing cargo release kinetics via the activation of an integrated fluorophore. P1 was synthesized by copolymerization of pinacol boronate, PEG, and naphthalimide monomers to impart ROS-sensitivity, hydrophilicity, and fluorescence signaling, respectively. Detailed characterization verified that P1 self-assembles into 11 nm micelles with 10 µg mL-1 CMC and can encapsulate hydrophobic curcumin with 79% efficiency. Fluorescence assays demonstrated H2O2-triggered disassembly and curcumin release with concurrent polymer fluorescence turn-on. Both in vitro and in vivo studies validated the real-time visualization of drug release and ROS scavenging, as well as the therapeutic effect on osteoarthritis (OA). Overall, this nanotheranostic polymeric micelle system enables quantitative monitoring of drug release kinetics for enhanced treatment optimization across oxidative stress-related diseases.

Emulsifying Lipiodol with pH-sensitive DOX@HmA nanoparticles for hepatocellular carcinoma TACE treatment eliminate metastasis.

Lipiodol-based transcatheter arterial chemoembolization (TACE) is currently the predominant and first-line treatment option recommended by the global standard for unresectable hepatocellular carcinoma (HCC). However, the unstable emulsion of Lipiodol causes a substantial proportion of chemotherapy drugs to enter the circulation system, leading to poor accumulation in cancer tissues and unexpected side effects of chemotherapy drugs. Herein, we emulsified Lipiodol with a pH-sensitive drug delivery system assembled from hexahistidine and zinc ions (HmA) with a super-high loading capacity of doxorubicin (DOX) and a promising ability to penetrate bio-barriers for the effective treatment of HCC by TACE. In vitro tests showed that DOX@HmA was comparable to free DOX in killing HCC cells. Impressively, during the in vivo TACE treatment, the anti-tumor efficacy of DOX@HmA was significantly greater than that of free DOX, indicating that DOX@HmA increased the accumulation of DOX in tumor. Emulsifying Lipiodol with pH-sensitive DOX@HmA significantly inhibited cell regeneration and tumor angiogenesis and decreased the systemic side effects of chemotherapy, especially by suppressing pulmonary metastasis in liver VX2 tumors in rabbits by inhibiting epithelial-mesenchymal transition (EMT). Emulsifying tumor microenvironment-responsive drug delivery systems (DDSs) with Lipiodol could be a new strategy for clinical TACE chemotherapy with potentially enhanced HCC treatment.

Construction of multifunctional coating with cationic amino acid-coupled peptides for osseointegration of implants.

Osseointegration is an important indicator of implant success. This process can be improved by coating modified bioactive molecules with multiple functions on the surface of implants. Herein, a simple multifunctional coating that could effectively improve osseointegration was prepared through layer-by-layer self-assembly of cationic amino acids and tannic acid (TA), a negatively charged molecule. Osteogenic growth peptide (OGP) and the arginine-glycine-aspartic acid (RGD) functional polypeptides were coupled with Lys6 (K6), the two polypeptides then self-assembled with TA layer by layer to form a composite film, (TA-OGP@RGD)n. The surface morphology and biomechanical properties of the coating were analyzed in gas and liquid phases, and the deposition process and kinetics of the two peptides onto TA were monitored using a quartz crystal microbalance. In addition, the feeding consistency and adsorption ratios of the two peptides were explored by using fluorescence visualization and quantification. The (TA-OGP@RGD)n composite membrane mediated the early migration and adhesion of cells and significantly promoted osteogenic differentiation and mineralization of the extracellular matrix in vitro. Additionally, the bifunctional peptide exhibited excellent osteogenesis and osseointegration owing to the synergistic effect of the OGP and RGD peptides in vivo. Simultaneously, the (TA-OGP@RGD)n membrane regulated the balance of reactive oxygen species in the cell growth environment, thereby influencing the complex biological process of osseointegration. Thus, the results of this study provide a novel perspective for constructing multifunctional coatings for implants and has considerable application potential in orthopedics and dentistry.

Physical Properties of the Second Type of Aciniform Spidroin (AcSp2) from Neoscona theisi Reveal a pH-Dependent Self-Assembly Repetitive Domain.

Orb-weaving spiders can use an array of specialized silks with diverse mechanical properties and functions for daily survival. Of all spider silk types, aciniform silk is the toughest silk fiber that combines high strength and elasticity. Although aciniform spidroins (AcSp) are the main protein in aciniform silks, their complete genes have rarely been characterized until now. Moreover, the structural and physical properties of AcSp variant proteins within the species are also unclear. Here, we present three full-length AcSp genes (named AcSp1A, AcSp1B, and AcSp2) from the orb-weaving spider Neoscona theisi and investigate the structural and mechanical features of these three AcSp repetitive domains. We demonstrate that all three AcSp proteins have mainly α-helical structural features in neutral solution and high thermal stability. Significantly, the AcSp2 repetitive domain shows a pH-dependent structural transition from α to ß conformations and can self-assemble into amyloid fibrils under acidic conditions, which is the first reported AcSp repetitive domain with pH-dependent self-assembly capacity. Compared with the other two AcSp spidroins, AcSp2 demonstrated the lowest expression level in the aciniform gland but had the highest strength for its silk fiber. Collectively, our findings provide new insight into the physical properties of each component of aciniform silk and expand the repertoire of known spidroin sequences for the synthesis of artificial silk materials.

pH-Responsive Hexa-Histidine Metal Assembly (HmA) with Enhanced Biocatalytic Cascades as the Vehicle for Glucose-Mediated Long-Acting Insulin Delivery.

Diabetes has been listed as one of the three major diseases that endanger human health. Accurately injecting insulin (Ins) depending on the level of blood glucose (LBG) is the standard treatment, especially controlling LBG in the long-term by a single injection. Herein, the pH-responsive hexa-histidine metal assembly (HmA) encapsulated with enzymes (GOx and CAT) and Ins (HmA@GCI) is engineered as the vehicle for glucose-mediated insulin delivery. HmA not only shows high proteins loading efficiency, but also well retained proteins activity and protect proteins from protease damage. Within HmA, the biocatalytic activities of enzymes and the efficiency of the cascade reaction between GOx and CAT are enhanced, leading to a super response to the change of LBG with insulin release and efficient clearance of harmful byproducts of GOx (H2 O2 ). In the treatment of diabetic mice, HmA@GCI reduces LBG to normal in half an hour and maintains for more than 5 days by a single subcutaneous injection, and nearly 24 days with four consecutive injections. During the test period, no symptoms of hypoglycemia and toxicity to tissues and organs are observed. These results indicate that HmA@GCI is a safe and long-acting hypoglycemic agent with prospective clinical application.

Hydrazone-Based Amphiphilic Brush Polymer for Fast Endocytosis and ROS-Active Drug Release.

Due to the high reactivity of reactive oxygen species (ROS), it is essential to sweep them away in time. In this study, ClO--responsible amphiphilic brush polymers were prepared by free radical polymerization using two monomers consisting of polyethylene glycol as the hydrophilic part, and an alkyl chain connected by hydrazone as the hydrophobic part. The macromolecules assemble into particles with nanoscaled dimensions in a neutral buffer, which ensures quick cellular internalization. The polymer has a low critical micellization concentration and can encapsulate hydrophobic drug molecules up to 19% wt. The micelles formed by the polymer disassemble in a ClO--rich environment and release 80% of their cargo within 2 h, which possesses a faster release rate compared to the previous systems. The relatively small size and the quick response of hydrazone toward ClO- ensure a quick uptake and elimination of ROS in vitro and in vivo.

Transformation of the shape and shrinking the size of acid-resistant metal-organic frameworks (MOFs) for use as the vehicle of oral proteins.

The oral delivery of protein-based drugs is of great significance, but faces various obstacles, including the deactivation of proteins by the low pH in the stomach and the high concentration of protease, poor transport through intestinal bio-barriers, etc. Herein, we present an acid-resistant metal-organic framework (MOF), NU-1000, in which insulin (Ins, a model protein) was loaded with high capacity (Ins@NU-1000) through the pseudo second-order kinetic model and Langmuir isotherm model. Ins@NU-1000 protects Ins from deactivation in the stomach acid environment and releases it in the intestine through the transformation of the micro-sized rod particles into spherical nanoparticles. Interestingly, the rod particles exhibit long-term retention in the intestine, and Ins is efficiently transported by the shrunk nanoparticles through intestinal bio-barriers and released into the blood, resulting in significant oral hypoglycemic effects (lasting more than 16 h after a single oral administration). Our findings demonstrate that switching the physical properties of the delivery vehicle, such as the shape and size, can contribute to the success of oral protein administration.

Role of Driving Force on Engineering Layer-by-Layer Protein/Polyphenol Coating with Flexible Structures and Properties.

Protein-based coatings are of immense interest due to their rich biological functions. Layer-by-layer (LbL) assembly, as a powerful means of transferring protein functions to the material surface, has received widespread attention. However, the assembly mechanism of protein-based LbL coatings is still far from being explained, not only because of protein structure and function diversity but also characterization limitations. Herein, we monitored in situ the LbL assembly process of tannic acid (TA) and lysozyme (Lyz), a classic pair of polyphenol and protein, by combining quartz crystal microbalance with dissipation monitoring (QCM-D) and spectroscopic ellipsometry (SE). The water content, morphology, mechanical properties, antioxidant activity, and the driving force of TA-Lyz coating engineered under different pH values were analyzed in detail by various techniques. The water content, a key factor in TA-Lyz coatings, increased with increasing assembled pH values, which resulted in a porous morphology, inhomogeneous mechanical distribution, faster assembly growth, and better antioxidant activity in both acellular and cellular levels. In addition, high water content is unfavorable to both entropy and enthalpy changes, and the thermodynamic driving force of TA and Lyz assembly mainly comes from the enthalpy change brought by the noncovalent interaction between TA and Lyz. These results provide new insights into engineering the structure, function, and assembly mechanisms of protein-based coatings.

Complete gene sequence and mechanical property of the fourth type of major ampullate silk protein.

Spiders spin a great diversity of silk types for daily survival and reproduction. Of the six orb-weaver silk types, the dragline silk forming orb web frame attracts the most attention because of its extremely high tensile strength and toughness. So far, four types of major ampullate silk proteins (MaSp1-4) that make up dragline silk have been identified. These MaSp types have diversified amino acid motifs that underlie the impressive mechanical property of dragline silk by forming particular structures. Existing knowledge of MaSp4 proteins is fragmented, making it difficult to illuminate the structure and function of MaSp4. Here, we report the full-length MaSp4 gene with 11,334 bp from the orb-weaving spider Araneus ventricosus. Removing the only intron, the spliced complete transcript of MaSp4 gene is 6897 bp and encodes 2298 amino acids. Analysis of the primary structure of A. ventricosus MaSp4 protein reveals the repetitive region lacks poly-A and GGX motifs but has the unique GPGPQ motifs. Quantitative real-time PCR analyses show high levels of MaSp4 mRNA were detected in major ampullate gland. Structural characterization using CD- and FTIR sepctroscopy reveals a mainly α-helical solution conformation and a very high ß-turn content within fibers. Collectively, our new findings provide complete template for recombinant silk protein with specific properties and support that the GPGPQ motif found in MaSp4 could increase flexibility in dragline silk by packing in more ß-turns, expanding the repertoire of sequences known to form ß-turn that is available for artificial chimeric silk fibers. STATEMENT OF SIGNIFICANCE: Dragline silk forming orb web frame attracts the most attention because of its extremely high tensile strength and toughness. So far, four types of major ampullate silk proteins (MaSp1-4) that make up dragline silk have been identified. Existing knowledge of MaSp4 proteins is fragmented, making it difficult to illuminate the structure and function of MaSp4. Here, we report the full-length MaSp4 gene from the orb-weaving spider Araneus ventricosus. We further identify the sequence, structure, and mechanical property of MaSp4 protein, providing a new insight into the structure-funtion relationships associated with MaSp4. Collectively, our new findings provide complete template for recombinant silk protein with specific properties and support that the GPGPQ motif found in MaSp4 could increase flexibility in dragline silk by packing in more ß-turns, expanding the repertoire of sequences known to form ß-turn that is available for artificial chimeric silk fibers.

Precisely controlling the surface roughness of silica nanoparticles for enhanced functionalities and applications.

HYPOTHESIS: Colloids with rough topography demonstrate more complex interactions and tremendous potential in industrial applications. However, relevant studies suffer from a range of challenges, including cumbersome synthesis, complex characterization, and very limited functionalities. A comprehensive study of rough nanoparticles can not only broaden our understanding of rough colloids, but also help to avoid some of their detrimental impacts in real life (e.g., clogging and pumping failures in slurry processing). EXPERIMENTS: A facile route to precisely control the surface roughness of silica nanoparticles and a highly efficient method to characterize the surface roughness were developed respectively. The fabricated particles can be applied for the immobilization of metal nanostructures; their cytotoxic effects and the capability to be used as a drug-delivery vehicle were also evaluated. FINDINGS: Modifying the addition time of precursors (i.e., TEOS and MPTMS) can precisely control the surface roughness of silica nanoparticles. The developed characterization method based on TEM observations allows statistical analyses on a large number of particles, and therefore features very reasonable accuracy. These rough particles behave like microporous materials, where the loading strategy is closely related to their surface roughness. Medium rough particles are promising carriers of metal nanostructures, while the roughest ones are excellent candidate for doxorubicin delivery to cancer cells.

Injectable, Self-Healing and Multiple Responsive Histamine Modified Hyaluronic Acid Hydrogels with Potentialities in Drug Delivery, Antibacterial and Tissue Engineering.

Hydrogels are 3D network structures composed of physically or chemically crosslinked, hydrophilic molecules. Compared with conventional hydrogels with static and permanent network structures, injectable and responsive hydrogels generated from dynamic networks, have attracted increasing attention from various disciplines due to their wide-ranging applications in tissue engineering, drug delivery, soft robotics, etc. Herein, an injectable self-healing and multiple-responsive hyaluronic acid (HA)- histamine (His)/metal hydrogel is developed by modifying His onto HA and the subsequent, dynamic coordination between imidazole and metal ions. The pH-responsive and mechanical behaviors exhibited by the HA-His/metal hydrogels are tunable with the kinds and the concentrations of metal ions. The HA-His/Zr4+ hydrogels demonstrate a moldable capability at a neutral pH and a multi-stimulus-responsive capability when exposed to a weak alkaline environment and hyaluronidase, which inhibits bacterial growth and biofilm formation. Biocompatibilities and accelerated wound healing are demonstrated in vitro and in vivo and are thoroughly investigated and well characterized. The HA-His/Zr4+ hydrogel has great potential in various biomedical applications, such as pH- and hyaluronidase-responsive sustained release, antibacterial, and implantable materials for tissue engineering.

Characterization of two full-length tubuliform silk gene sequences from Neoscona theisi reveals intragenic concerted evolution and multiple copies in genome.

Orb-web weaving spiders use a variety of silk types for particular tasks, and each silk type is composed of at least two spider silk proteins (spidroins). In the early stage of divergence, however, the molecular evolutionary processes act on spidroin variants are still unclear because of a lack of knowledge for full-length paralogous and orthologous gene sequences among closely related species. Here, we present two complete gene sequences encoding the tubuliform spidroin TuSp1 variants (TuSp1-v2 and TuSp1-v3) from orb-weaving spider Neoscona theisi. Both N. theisi TuSp1-v2 and TuSp1-v3 genes contain a single enormous exon (14,139 bp for TuSp1-v2 and 13,152 bp for TuSp1-v3) and dozens of tandemly arrayed repeats (25 repeats for TuSp1-v2 and 23 repeats for TuSp1-v3) with extreme intragenic homogenization. The pattern of expression for these two spidroins revealed that the level of TuSp1-v3 mRNA is ~3-fold higher than that of TuSp1-v2 in tubuliform gland. Phylogenetic analyses of spidroins not only show the occurrence of a gene duplication event for TuSp1-v2 and TuSp1-v3 in the common ancestor of the Neoscona and Araneus lineage but reinforce the role of concerted evolution for the extreme homogenization of TuSp1 repeats.