High-strength and ultra-tough supramolecular polyamide spider silk fibers assembled via specific covalent and reversible hydrogen bonds.

Achieving ultra-high tensile strength and exceptional toughness is a longstanding goal for structural materials. However, previous attempts using covalent and non-covalent bonds have failed, leading to the belief that these two properties are mutually exclusive. Consequently, commercial fibers have been forced to compromise between tensile strength and toughness, as seen in the differences between nylon and Kevlar. To address this challenge, we drew inspiration from the disparate tensile strength and toughness of nylon and Kevlar, both of which are polyamide fibers, and developed an innovative approach that combines specific intermolecular disulfide bonds and reversible hydrogen bonds to create ultra-strong and ultra-tough polyamide spider silk fibers. Our resulting Supramolecular polyamide spider silk, which has a maximum molecular weight of 1084 kDa, exhibits high tensile strength (1180 MPa) and extraordinary toughness (433 MJ/m3), surpassing Kevlar's toughness 8-fold. This breakthrough presents a new opportunity for the sustainable development of spider silk as an environmentally friendly alternative to synthetic commercial fibers, as spider silk is composed of amino acids. Future research could explore the use of these techniques and fundamental knowledge to develop other super materials in various mechanical fields, with the potential to improve people's lives in many ways. STATEMENT OF SIGNIFICANCE: • By emulating synthetic commercial fibers such as nylon and polyethylene, we have successfully produced supramolecular-weight polyamide spider silk fibers with a molecular weight of 1084 kDa through a unique covalent bond-mediated linear polymerization reaction of spider silk protein molecules. This greatly surpasses the previous record of a maximum molecular weight of 556 kDa. • We obtained supramolecular polyamide spider silk fibers with both high-tensile strength and toughness. The stress at break is 1180 MPa, and the toughness is 8 times that of kevlar, reaching 433 MJ/m3. • Our results challenge the notion that it is impossible to manufacture fibers with both ultra-high tensile strength and ultra-toughness, and provide theoretical guidance for developing environmentally friendly and sustainable structural materials that meet industrial needs.

Disabling spidroin N-terminal homologs' reverse reaction unveils why its intermolecular disulfide bonds have not evolved for 380 million years.

Spiders, ubiquitous predators known for their powerful silks, rely on spidroins that self-assemble from high-concentration solutions stored in silk glands, which are mediated by the NT and CT domains. CT homodimers containing intermolecular disulfide bonds enhance silk performance, promoting spider survival and reproduction. However, no NT capable of forming such disulfide bonds has been identified. Our study reveals that NT homodimers with sulfur substitution can form under alkaline conditions, shedding light on why spiders have not evolved intermolecular disulfide bonds in the NT module during their 380 million years of evolution. This discovery significantly advances our comprehension of spider evolution and silk spinning mechanisms, while also providing novel insights into protein storage, assembly, as well as the mechanisms and therapeutic strategies for neurodegenerative diseases associated with protein aggregation.

Comparative Proteome Analysis of Serum Uncovers Differential Expression of Proteins in Donkeys (Equus Asinus) With Endometritis Caused by Escherichia Coli.

Endometritis is a common disease in donkeys that causes economic losses to donkey farms and the common cause is bacterial infection. Uterine flush fluid proteomics has been used to study protein biomarkers associated with endometritis in mares. As a convenient diagnostic tool, serum proteomics has not been studied yet in equine species with endometritis. This study is aiming to evaluate the serum proteomics in jennies with and without endometritis and identify potential proteins as biomarker for endometritis diagnosis. Nine donkeys recruited into this study were diagnosed of bacterial (Escherichia coli) endometritis and nine healthy jennies were selected as control. Blood samples of each donkey was collected, and serum was separated from each sample. Peptides samples extracted from the serum were analyzed using nano-ultrahigh-performance liquid chromatography-tandem mass spectrometry in data-independent acquisition mode. Protein identification and quantification were performed followed by differential and functional analysis. Of 579 proteins identified in all jennies, 12 proteins were exclusively identified in jennies with endometritis (group E) including myeloperoxidase and Ras-related protein Rab-1B, which might be associated with bacterial infection. There were 11 differentially expressed proteins detected between the two groups of jennies with 4 downregulated proteins and 7 upregulated proteins in jennies with endometritis. Some upregulated proteins along with the GO and KEGG annotation indicated inflammatory response against uterine infection. Characteristic serum proteins identified in jennies with endometritis were associated with inflammation or bacterial infection. These proteins might be potential biomarkers for endometritis diagnosis in jennies.

Endometrial and vaginal microbiome in donkeys with and without clinical endometritis.

Endometrial and vaginal microbiomes are critical in the study of endometritis, which is an important cause of infertility in donkeys. Our objective was to investigate the difference of the endometrial and vaginal microbiomes between healthy donkey jennies (group C) and jennies with endometritis (group E). Endometrial and vaginal swab samples were collected, and the 16 s rRNA gene amplicon high-throughput sequencing technique was applied to identify the microbial composition in the samples. A similar microbial composition pattern was found between endometrial and vaginal samples, which indicated the impact of the vaginal microbiome on the endometrial microbial environment and health. There was a significant difference of endometrial and vaginal swab samples between the two groups. Ruminococcaceae and Lachnospiraceae were significantly more abundant in endometrial and vaginal microbiomes of group E than in group C. Their dominance was consistent with increased anaerobic bacterial taxa in the functional analysis, which might be associated with the pathogenesis of endometritis in donkeys. Sphingomonadaceae, a bacterial family reported in bovine semen, was statistically more abundant in endometrial microbiome of group E than in group C, which might suggest an association between high abundance of Sphingomonadaceae possibly due to uncleared semen and donkey endometritis. Our study revealed the composition of the vaginal and endometrial microbiomes in healthy and endometritis donkeys. These findings will provide more insights into the pathogenesis of donkey endometritis.

Antimicrobial Susceptibility of Bacterial Isolates from Donkey Uterine Infections, 2018-2021.

BACKGROUND: Endometritis is a common reproductive disease in equine animals. No investigation about the bacterial characteristics and antimicrobial susceptibility pattern of donkeys with endometritis has thus far been reported. OBJECTIVES: To determine the common uterine bacterial isolates from donkeys with endometritis and to evaluate their susceptibility to antimicrobials used for the treatment thereof. STUDY DESIGN: Retrospective case-series. METHODS: Medical records at an equine clinical diagnostic center were retrospectively reviewed to identify submissions from donkeys with bacterial endometritis between 2018 and 2021. Data were extracted and analyzed descriptively in terms of the frequency of bacterial species, susceptibility to antimicrobials and multidrug resistance. RESULTS: A total of 73 isolates were identified from 30 donkeys, of which 92% of the isolates were Gram-negative bacteria. Mixed cultures were found in 90% of the donkeys. The most common isolates were Escherichiacoli (31.5%) and Acinetobacter spp. (21.9%). Susceptibility testing revealed that amikacin (98%), cefoxitin (95%), trimethoprim-sulfamethoxazole (78%) and gentamicin (74%) were the most efficient agents for donkeys. Multidrug resistance (MDR) was found in 20% of all bacterial isolates, of which all Pseudomonas aeruginosa isolates showed a multidrug resistance profile. Main limitations: The sample size was relatively small, which means a bias of selection may exist. The antimicrobial resistance and MDR of agents without break points were not calculated, which means the relative results may be underestimated in our study. CONCLUSIONS: Severe infections were detected in donkeys with endometritis. Antimicrobial resistance and MDR bacteria are not rare in our study. This study demonstrated that bacteria identification and antimicrobial susceptibility testing are highly recommended before the treatment of uterine infections in donkeys. Further studies, including the epidemiological investigation of bacterial endometritis of donkeys, should be conducted to provide a better understanding of this critical problem.

Tough synthetic spider-silk fibers obtained by titanium dioxide incorporation and formaldehyde cross-linking in a simple wet-spinning process.

Due to its unique mechanical properties, spider silk shows great promise as a strong super-thin fiber in many fields. Although progress has been made in the field of synthesizing spider-silk fiber from recombinant spidroin (spider silk protein) in the last few decades, methods to obtain synthetic spider-silk fibers as tough as natural silk from small-sized recombinant protein with a simple spinning process have eluded scientists. In this paper, a recombinant spidroin (MW: 93.4 kDa) was used to spin tough synthetic spider-silk fibers with a simple wet-spinning process. Titanium oxide incorporation and formaldehyde cross-linking were used to improve the mechanical properties of synthetic spider-silk fibers. Fibers treated with incorporation or/and cross-linking varied in microstructure, strength and extensibility while all exhibited enhanced strength and toughness. In particular, one fiber possessed a toughness of 249 ± 22 MJ/m3. This paper presents a new method to successfully spin tough spider-silk fibers in a simple way.

Tensile properties of synthetic pyriform spider silk fibers depend on the number of repetitive units as well as the presence of N- and C-terminal domains.

Spiders can spin seven different types of silk, some of which are well characterized, but studies on natural and synthetic pyriform silks are few. In this study, recombinant spidroins composed of one to three pyriform repeat units from Araneus ventricosus, in some cases flanked with non-repetitive N- and C-terminal domains (NT and CT), were produced and spun into continuous silk fibers using a wet-spinning process in organic solvents. All the fibers showed high and similar tensile strain (60-80%), but the Young's modulus, stress and toughness of fibers increased with increasing number of repeat units and in the presence of NT and CT as well. Systematic studies of the secondary structure contents of the different spinning dopes and spun fibers revealed no major differences between the different types of recombinant spidroins. This suggests that optimal tensile properties of artificial spider silks require the presence of several repetitive units as well as terminal domains and that secondary structure content of silk dope and fibers have limited correlation with mechanical behaviors.

Wet-Spinning Synthetic Fibers from Aggregate Glue: Aggregate Spidroin 1 (AgSp1).

Spidroin has the potential of wide applications in the biomedicine field as a natural biomaterial. Various synthetic fibers with outstanding mechanical properties have been produced from different spidroins. However, studies on the structural analysis or biomimetic exploration of aggregate spidroin (AgSp) remain scarce. Here, three recombinant AgSp1 spidroins (1RP, 1RC, 3RP) were constructed and expressed in Escherichia coli, followed by purification via coupling heating and ammonium sulfate precipitation. Circular dichroism (CD) spectrum-based secondary structural analysis shows that 1RP and 3RP have similar structures (mainly random coil) in water and PB buffer, while 1RC is mainly composed of α-helix structure and HFIP can change all of the recombinant AgSp1 into helix structure. Through the wet-spinning method, six types of synthetic fibers were produced from these three recombinant AgSp1 spidroins. Subsequently, the properties and structures of synthetic fibers were characterized by mechanical testing and ATR-FTIR. Synthetic fibers spun from 3RP have considerable tensile strength and extensibility (∼37.56 MPa and ∼4.5%, respectively). To the best of our knowledge, this is the first synthetic fiber obtained from AgSp spidroin. Our results demonstrated that AgSp1 can be regarded as an available source of spidroin for silklike fiber production and may provide valuable perspectives on the AgSp1 biomimetic process for certain applications.

Molecular cloning and analysis of the full-length aciniform spidroin gene from Araneus ventricosus.

Orb-web spiders produce more than seven different protein-based silks/glues by specialized abdominal glands for different uses. Prey-wrapping silk is secreted by aciniform glands for wrapping prey and forming the inner layer of egg case, and is almost twice as tough as other silks because of high strength and extensibility. So far, only two complete gene sequences have been obtained for aciniform spidroins (AcSp1). Here we describe the AcSp1 full-length gene sequence from the spider species Araneus ventricosus, using a long-distance PCR (LD-PCR) approach. The full-length AcSp1 gene is a single enormous exon of 10,338 bp in size, and the predicted protein sequence is 3445 amino acids long and consists of a conserved nonrepetitive N-terminal domain, a central predominantly repetitive region composed of fifteen repeat units, and a conserved nonrepetitive C-terminal domain.