Chromatographic separation by RPLC-ESI-MS of all hydroxyproline isomers for the characterization of collagens from different sources.

During collagen biosynthesis, proline is post-translationally converted to hydroxyproline by specific enzymes. This amino acid, unique to collagen, plays a crucial role in stabilizing the collagen triple helix structure and could serve as an important biomarker for collagen content and quality analysis. Hydroxyproline has four isomers, depending on whether proline is hydroxylated at position 4 or 3 and on whether the cis- or trans- conformation is formed. Moreover, as extensive hydrolysis of collagen is required for its amino acid analysis, epimerization may also occur, although to a lesser extent, giving a total of eight possible isomers. The aim of the present study was to develop a reversed-phase high-performance liquid chromatography-UV-mass spectrometry (RPLC-UV-MS) method for the separation and quantification of all eight hydroxyproline isomers. After the chiral derivatization of the hydroxyproline isomers with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA), to enable their UV detection, the derivatized diastereoisomers were separated by testing different C18 column technologies and morphologies and optimizing operative conditions such as the mobile phase composition (solvent, additives), elution mode, flow rate and temperature. Baseline resolution of all eight isomers was achieved on a HALO® ES-C18 reversed-phase column (150×1.5 mm, 2.7 µm, 160 Å) using isocratic elution and MS-compatible mobile phase. The optimized method was validated for the quantification of hydroxyproline isomers and then applied to different collagen hydrolysates to gain insight and a deeper understanding of hydroxyproline abundances in different species (human, chicken) and sources (native, recombinant).

Coexisting Th1 and Th2 cytokines in patients with collagenous gastritis and implications for its pathogenesis.

OBJECTIVES: Collagenous gastritis (CG) is a rare cause of refractory dyspepsia and anemia that frequently affects children and young adults and whose histological hallmark is chronic mucosal inflammation with a subepithelial collagen band. The etiology remains obscure, and no established treatments exist. We investigated the pathogenesis of CG by determining the expression profiles of genes related to immunity and inflammation in index biopsies. METHODS: Gastric biopsies from 10 newly diagnosed patients with CG were evaluated using the NanoString nCounter assay. Gastric biopsies from 14 normal individuals served as controls. The gene expression ratios for CG versus controls were determined in pooled samples and confirmed in individual samples by quantitative reverse transcription polymerase chain reaction. The results were compared with previously reported expression data from a cohort of patients with collagenous colitis, a colonic disorder with similar morphology, including subepithelial collagen band. RESULTS: CG biopsies featured enhanced expression of key genes encoding both Th1 (IFNγ, TNF-α, IL-2, IL-10, IL-12A, IL-12B, and IL-18) and Th2 cytokines (IL-3, IL-4, IL-5, IL-6, and IL-13). In contrast, biopsies from patients with CC exhibited upregulated Th1 cytokines only. CONCLUSIONS: We show in this first published gene expression profiling study that CG involves simultaneous upregulation of Th1 and Th2 cytokines. This finding is unique, contrasting with other types of chronic gastritis as well as with collagenous colitis, which shares the presence of a collagen band. Involvement of Th2 immunity in CG would support further investigation of potential dietary, environmental, or allergic factors to guide future therapeutic trials.

Detrimental Effects of Chlorhexidine on Articular Cartilage Viability, Matrix, and Mechanics.

BACKGROUND: Chlorhexidine gluconate (CHG) solution is commonly used as an antiseptic irrigation for bacterial decontamination during orthopaedic surgery. Although the chondrotoxicity of CHG on articular cartilage has been reported, the full extent of CHG-related chondrotoxicity and its effects on the extracellular matrix and mechanical properties are unknown. PURPOSE: To investigate the in vitro effects of a single 1-minute CHG exposure on the viability, biochemical content, and mechanics of native articular cartilage explants. STUDY DESIGN: Controlled laboratory study. METHODS: Articular cartilage explants (6 per group) were harvested from femoral condyles of the porcine stifle and sectioned at tidemark. Explants were bathed in CHG solution (0.05% CHG in sterile water) at varying concentrations (0% control, 0.01% CHG, and 0.05% CHG) for 1 minute, followed by complete phosphate-buffered saline wash and culture in chondrogenic medium. At 7 days after CHG exposure, cell viability, matrix content (collagen and glycosaminoglycan [GAG]), and compressive mechanical properties (creep indentation testing) were assessed. RESULTS: One-minute CHG exposure was chondrotoxic to explants, with both 0.05% CHG (2.6% ± 4.1%) and 0.01% CHG (76.3% ± 8.6%) causing a decrease in chondrocyte viability compared with controls (97.5% ± 0.6%; P < .001 for both). CHG exposure at either concentration had no significant effect on collagen content, while 0.05% CHG exposure led to a significant decrease in mean GAG per wet weight compared with the control group (2.6% ± 1.7% vs 5.2% ± 1.9%; P = .029). There was a corresponding weakening of mechanical properties in explants treated with 0.05% CHG compared with controls, with decreases in mean aggregate modulus (177.8 ± 90.1 kPa vs 280.8 ± 19.8 kPa; P < .029) and shear modulus (102.6 ± 56.5 kPa vs 167.9 ± 16.2 kPa; P < .020). CONCLUSION: One-minute exposure to CHG for articular cartilage explants led to dose-dependent decreases in chondrocyte viability, GAG content, and compressive mechanical properties. This raises concern for the risk of mechanical failure of the cartilage tissue after CHG exposure. CLINICAL RELEVANCE: Clinicians should be judicious regarding the use of CHG irrigation at these concentrations in the presence of native articular cartilage.

Distinct spectral signatures unfold ECM stiffness-triggered biochemical changes in breast cancer cells.

Cancer progression often accompanies the stiffening of extracellular matrix (ECM) in and around the tumor, owing to extra deposition and cross-linking of collagen. Stiff ECM has been linked with poor prognosis and is known to fuel invasion and metastasis, notably in breast cancer. However, the underlying biochemical or metabolic changes and the cognate molecular signatures remain elusive. Here, we explored Raman spectroscopy to unveil the spectral fingerprints of breast cancer cells in response to extracellular mechanical cues. Using stiffness-tuneable hydrogels, we showed that cells grown on stiff ECM displayed morphological changes with high proliferation. We further demonstrated that Raman Spectroscopy, a label-free and non-invasive technique, could provide comprehensive information about the biochemical environment of breast cancer cells in response to varying ECM stiffness. Raman spectroscopic analysis classified the cells into distinct clusters based on principal component-based linear discriminant analysis (PC-LDA). Multivariate curve resolution-alternating least squares (MCR-ALS) analysis indicated that cells cultured on stiff ECM exhibited elevated nucleic acid content and lesser lipids. Interestingly, increased intensity of Raman bands corresponding to cytochrome-c was also observed in stiff ECM conditions, suggesting mitochondrial modulation. The key findings harboured by spectral profiles were also corroborated by transmission electron microscopy, confirming altered metabolic status as reflected by increased mitochondria number and decreased lipid droplets in response to ECM stiffening. Collectively, these findings not only give the spectral signatures for mechanoresponse but also provide the landscape of biochemical changes in response to ECM stiffening.

Functional Assessment of Human Articular Cartilage Using Second Harmonic Generation (SHG) Imaging: A Feasibility Study.

Many arthroscopic tools developed for knee joint assessment are contact-based, which is challenging for in vivo application in narrow joint spaces. Second harmonic generation (SHG) laser imaging is a non-invasive and non-contact method, thus presenting an attractive alternative. However, the association between SHG-based measures and cartilage quality has not been established systematically. Here, we investigated the feasibility of using image-based measures derived from SHG microscopy for objective evaluation of cartilage quality as assessed by mechanical testing. Human tibial plateaus harvested from nine patients were used. Cartilage mechanical properties were determined using indentation stiffness (Einst) and streaming potential-based quantitative parameters (QP). The correspondence of the cartilage electromechanical properties (Einst and QP) and the image-based measures derived from SHG imaging, tissue thickness and cell viability were evaluated using correlation and logistic regression analyses. The SHG-related parameters included the newly developed volumetric fraction of organised collagenous network (Φcol) and the coefficient of variation of the SHG intensity (CVSHG). We found that Φcol correlated strongly with Einst and QP (ρ = 0.97 and - 0.89, respectively). CVSHG also correlated, albeit weakly, with QP and Einst, (|ρ| = 0.52-0.58). Einst and Φcol were the most sensitive predictors of cartilage quality whereas CVSHG only showed moderate sensitivity. Cell viability and tissue thickness, often used as measures of cartilage health, predicted the cartilage quality poorly. We present a simple, objective, yet effective image-based approach for assessment of cartilage quality. Φcol correlated strongly with electromechanical properties of cartilage and could fuel the continuous development of SHG-based arthroscopy.

Acellular Extrinsic Fiber Cementum Is Invariably Present in the Superficial Layer of Apical Cementum in Mouse Molar.

The cementum is a highly mineralized tissue that covers the tooth root. The regional differences among the types of cementum, especially in the extrinsic fibers that contribute to tooth support, remain controversial. Therefore, this study used second harmonic generation imaging in conjunction with automated collagen extraction and image analysis algorithms to facilitate the quantitative examination of the fiber characteristics and the changes occurring in these fibers over time. Acellular extrinsic fiber cementum (AEFC) was invariably observed in the superficial layer of the apical cementum in mouse molars, indicating that this region of the cementum plays a crucial role in supporting the tooth. The apical AEFC exhibited continuity and fiber characteristics comparable with the cervical AEFC, suggesting a common cellular origin for their formation. The cellular intrinsic fiber cementum present in the inner layer of the apical cementum showed consistent growth in the apical direction without layering. This study highlights the dynamic nature of the cementum in mouse molars and underscores the requirement for re-examining its structure and roles. The findings of the present study elucidate the morphophysiological features of cementum and have broader implications for the maintenance of periodontal tissue health.

Detailed study of collagen, vasculature, and innervation in the human cardiac conduction system.

BACKGROUND: The cardiac conduction system (CCS) creates and propagates electrical signals generating the heartbeat. This study aimed to assess the collagen content, vasculature, and innervation in the human sinoatrial and atrioventricular CCS, and surrounding tissue. MATERIALS AND METHODS: Ten sinoatrial and 17 atrioventricular CCS samples were collected from 17 adult human autopsied hearts. Masson trichrome stain was used to examine collagen, cardiomyocytes, and fat proportions. Immunohistochemically, vessels and lymphatics were studied by CD31 (pan-endothelial marker) and D2-40 (lymphatic endothelium marker) antibodies. General nerve densities were assessed by S100, while sympathetic nerves were studied using tyrosine hydroxylase, parasympathetic nerves with choline acetyltransferase, and GAP43 (neural growth marker) antibodies looked at these components. All components were quantified with QuPath software (Queens University, Belfast, Northern Ireland). RESULTS: Interstitial collagen was more than two times higher in the sinoatrial vs. atrioventricular CCS (55% vs. 22%). The fat content was 6.3% in the sinoatrial CCS and 6.5% in the atrioventricular CCS. The lymphatic vessel density was increased in the sinoatrial and atrioventricular CCS compared to the surrounding tissue and was lower in the sinoatrial vs. atrioventricular CCS (P=.043). The overall vasculature density did not differ between the SA and AV CCS. The overall innervation and neural growth densities were significantly increased in the CCS compared to the surrounding tissue. The overall innervation was higher in the atrial vs. ventricular CCS (P=.018). The neural growth was higher in the atrial vs. ventricular CCS (P=.018). The sympathetic neural supply was dominant in all the studied regions with the highest density in the sinoatrial CCS. CONCLUSIONS: Our results provide new insights into the unique morphology of the human CCS collagen, fat, vasculature, and innervation. A deeper understanding of the CCS anatomical components and morphologic substrates' role will help in elucidating the causes of cardiac arrhythmias and provide a basis for further therapeutic interventions.

Structure and properties of the acellular porcine cornea irradiated with 60Co-γ and electron beam and its histocompatibility.

Acellular porcine cornea (APC) has been used in corneal transplantation and treatment of the corneal diseases. Sterilization is a key step before the application of graft, and irradiation is one of the most commonly used methods. In this paper, APC was prepared by the physical freeze-thawing combined with biological enzymes, and the effects of the electron beam (E-beam) and cobalt 60 (60Co-γ) at the dose of 15 kGy on the physicochemical properties, structure, immunogenicity, and biocompatibility of the APC were investigated. After decellularization, the residual DNA was 20.86 ± 1.02 ng/mg, and the α-Gal clearance rate was more than 99%. Irradiation, especially the 60Co-γ, reduced the cornea's transmittance, elastic modulus, enzymatic hydrolysis rate, swelling ratio, and cross-linking degree. Meanwhile, the diameter and spacing of the collagen fibers increased. In the rat subcutaneous implantation, many inflammatory cells appeared in the unirradiated APC, while the irradiated had good histocompatibility, but the degradation was faster. The lamellar keratoplasty in rabbits indicated that compared to the E-beam, the 60Co-γ damaged the chemical bond of collagen to a larger extent, reduced the content of GAGs, and prolonged the complete epithelization of the grafts. The corneal edema was more serious within 1 month after the surgery. After 2 months, the thickness of the APC with the two irradiation methods tended to be stable, but that in the 60Co-γ group became thinner. The pathological results showed that the collagen structure was looser and the pores were larger, indicating the 60Co-γ had a more extensive effect on the APC than the E-beam at 15 kGy.

Refractive index of human articular cartilage varies with tissue structure and composition.

Optical properties of biological tissues, such as refractive index, are fundamental properties, intrinsically linked to a tissue's composition and structure. This study aims to investigate the variation of refractive index (RI) of human articular cartilage along the tissue depth (via collagen fibril orientation and optical density) and integrity (based on Mankin and Osteoarthritis Research Society International (OARSI) scores). The results show the relationship between RI and PG content (p=0.042), collagen orientation (p=0.037), and OARSI score (p=0.072). When taken into account, the outcome of this study suggests that the RI of healthy cartilage differs from that of pathological cartilage (p=0.072). This could potentially provide knowledge on how progressive tissue degeneration, such as osteoarthritis, affects changes in cartilage RI, which can, in turn, be used as a potential optical biomarker of tissue pathology.

Assessment of collagen content in fish skin - development of a flow analysis method for hydroxyproline determination.

This work describes the development of a flow injection method to determine hydroxyproline (HYP), one of collagen's most abundant amino acids. Collagen is a protein with several applications and high nutritional value. Evaluating the feasibility of using collagen from fish skin over its mammalian source is essential. The determination of HYP requires the pre-treatment and hydrolysis of the fish skin to break down collagen into its amino acids, and the HYP value quantified relates to the collagen content. The determination was based on the HYP oxidation with permanganate in an alkaline medium and the consequent decrease of colour intensity registered. Under optimal conditions, the developed method enables the determination of the HYP within the dynamic range of 23.8 to 500 mg L-1, with a limit of detection (LOD) of 2.6 mg L-1 and a limit of quantification (LOQ) of 23.8 mg L-1. Different samples were processed, and the digests were analysed by the proposed method and with the conventional procedure with good correlation (relative error < 7%). Moreover, the analyte quantification is performed faster, simpler, and more accurately, with less toxic solutions. The reproducibility of the developed method was also evaluated by calculating the relative standard deviation of the calibration curve slope (RSD < 1%).

The early lives of the islanders: Stable isotope analysis of incremental dentine collagen from the prehispanic period of the Canary Islands.

OBJECTIVES: This study presents isotopic information for incremental dentine collagen and bone bulk collagen from individuals from the Canary Islands (Tenerife and Gran Canaria) to explore dietary differences during childhood life. MATERIALS AND METHODS: Eight individuals have been studied, which comprises 122 δ15 N and δ13 C incremental dentine measurements and eight bulk bone collagen analyses. A baseline of potentially consumed food sources has been developed for comparative purposes. A food reconstruction using isotopic transferred signals (FRUITS) model of probable contributions of each food source towards the diet of each individual has been developed. All samples but one belongs to the later period of indigenous occupation of the archipelago. RESULTS: The dentine collagen data are presented in correlated δ13 C and δ15 N plots per individual, showing the isotopic changes throughout time. δ15 N values for each individual tend to be variable whereas δ13 C data are generally more stable with a range of +9.1 to +14‰ for δ15 N and -17.4 to -20.8‰ for δ13 C. CONCLUSION: The isotopic analysis allows for the reconstruction of eight dietary profiles, which allow us to estimate the different dietary protein sources. The FRUITS model shows different percentages of the primary food sources for each individual. Where both δ13 C and δ15 N are elevated, this could be indicative of a higher marine contribution to the diet. There appear to be two main dietary profiles identifiable in the dataset and these may be related to changes in status or place of residence. Short-term variations in δ13 C and δ15 N and opposing co-variance of isotopic values can be indicative of nutritional stress, although metabolic changes during growth are also considered.

On the identification of the ultra-structural organization of elastic fibers and their effects on the integrity of annulus fibrosus.

Due to the complicated structure of the elastic fiber network in annulus fibrosus, existing in-silico studies either simplified or just overlooked its distribution pattern. Nonetheless, experimental and simulation results have proven that elastic fibers are of great importance to maintaining the structural integrity of annulus fibrosus and therefore to ensuring the load-bearing ability of intervertebral discs. Such needs call for a fine model. This work aims at developing a biphasic annulus fibrosus model by incorporating the accurate distribution pattern of collagen and elastic fibers. Both the structural parameters and intrinsic mechanical parameters were successfully identified using single lamella and inter-lamella microscopy anatomy and micromechanical testing data. The proposed model was then used to implement finite element simulations on various anterior and posterolateral multi-lamellae annulus fibrosus specimens. In general, simulation results agree well with available experimental and simulation data. On this basis, the effects of elastic fibers on the integrity of annulus fibrosus were further investigated. It was found that elastic fibers significantly influence the free swelling, radial stretching and circumferential shear performances of annulus fibrosus. Nonetheless, no significant effects were found for the circumferential stretching capability. The proposed biphasic model considers for the first time the distribution characteristics of elastic fibers at two scales, including both the principal orientations of all fiber families and the detailed distribution pattern within each family. Better understandings on the functions of collagen and elastic fibers can therefore be realized. To further enhance its prediction capability, the current model can be extended in the future by taking the fiber-matrix interaction as well as progressive damages into consideration.

Influence of Staphylococcus epidermidis on Collagen Crimp Patterns of Soft Tissue Allograft.

BACKGROUND: Postoperative infections, commonly from Staphylococcus epidermidis, may result in anterior cruciate ligament graft failure and necessitate revision surgery. In biomechanical studies, S. epidermidis has been shown to establish biofilms on tendons and reduce graft strength. PURPOSE/HYPOTHESIS: The goal of this study was to determine the effect of bacterial bioburden on the collagen structure of tendon. It was hypothesized that an increase in S. epidermidis biofilm would compromise tendon crimp, a pattern necessary for mechanical integrity, of soft tissue allografts. STUDY DESIGN: Controlled laboratory study. METHODS: Cultures of S. epidermidis were used to inoculate tibialis anterior cadaveric tendons. Conditions assessed included 5 × 105 colony-forming units or concentrated spent media from culture (no living bacteria). Incubation times of 30 minutes, 3 hours, 6 hours, and 24 hours were utilized. Second-harmonic generation imaging allowed for visualization of collagen autofluorescence. Crimp lengths were determined using ImageJ and compared based on incubation time. RESULTS: Incubation time positively correlated with increasing S. epidermidis bioburden. Both fine and coarse crimp patterns lengthened with increasing incubation time. Significant coarse crimp changes were observed after only 30-minute incubations (P < .029), whereas significant fine crimp lengthening occurred after 6 hours (P < .0001). No changes in crimp length were identified after incubation in media lacking living bacteria. CONCLUSION: The results of this study demonstrate that exposure to S. epidermidis negatively affects collagen crimp structure. Structural alterations at the collagen fiber level occur within 30 minutes of exposure to media containing S. epidermidis. CLINICAL RELEVANCE: Our study highlights the need for antimicrobial precautions to prevent graft colonization and maximize graft mechanical strength.

Determination of Interstitial Collagen Deposition and Related Topography Using the Second Harmonic Generation-Based HistoIndex Platform.

Interstitial fibrosis is characterized by the increased deposition of extracellular matrix (ECM) components within the interstitial space of various organs, such as the kidneys, heart, lungs, liver, and skin. The primary component of interstitial fibrosis-related scarring is interstitial collagen. Therefore, the therapeutic application of anti-fibrotic medication hinges on the accurate measurement of interstitial collagen levels within tissue samples. Current histological measurement techniques for interstitial collagen are generally semi-quantitative in nature and only provide a ratio of collagen levels within tissues. However, the Genesis™ 200 imaging system and supplemental image analysis software, FibroIndex™, from HistoIndex™, is a novel, automated platform for imaging and characterizing interstitial collagen deposition and related topographical properties of the collagen structures within an organ, in the absence of any staining. This is achieved by using a property of light known as second harmonic generation (SHG). Using a rigorous optimization protocol, collagen structures in tissue sections can be imaged with a high degree of reproducibility and ensures homogeneity across all samples while minimizing the introduction of any imaging artefacts or photobleaching (decreased tissue fluorescence due to prolonged exposure to the laser). This chapter outlines the protocol that should be undertaken to optimize HistoIndex scanning of tissue sections, and the outputs that can be measured and analyzed using the FibroIndex™ software.

Probiotic Fermented Goat's and Sheep's Milk: Effect of Type and Dose of Collagen on Survival of Four Strains of Probiotic Bacteria during Simulated In Vitro Digestion Conditions.

Microbial tolerance of digestive stresses depends not only on the bacterial strain but also on the structure and physicochemical properties of the supply chain and the foods that contain it. In the present study, we aimed to evaluate the effects of the type of milk (ovine, caprine) and the type and dose of collagen on the viability of four probiotic strains, Lacticaseibacillus paracasei L-26, Lacticaseibacillus casei 431, Lactobacillus acidophilus LA-5, and Lacticaseibacillus rhamnosus Lr-32, during in vitro gastrointestinal digestion. The highest survival rate under simulated in vitro digestion conditions compared to the number of cells before digestion was found in two strains, L. casei and L. paracasei, where survival rates were greater than 50% in each batch. The survival rate of the L. rhamnosus strain ranged from 41.05% to 64.23%. In caprine milk fermented by L. acidophilus, a higher survival rate was found in milk with 1.5% hydrolysate than the control, by about 6%. Survival of the L. rhamnosus strain was favorably affected by the 3% addition of bovine collagen in caprine milk, which increased survival by about 14% compared to the control sample. Adding 3% of hydrolysate to sheep's and goat's milk enhanced the survival of the L. rhamnosus strain by 3% and 19%, respectively. This study reports that fermented caprine and ovine milk may be suitable matrices for the probiotic supply of commercial dairy starter cultures and promote gut homeostasis.

Skeletal calcification patterns of batoid, teleost, and mammalian models: Calcified cartilage versus bone matrix.

This study compares the skeletal calcification pattern of batoid Raja asterias with the endochondral ossification model of mammalians Homo sapiens and teleost Xiphias gladius. Skeletal mineralization serves to stiffen the mobile elements for locomotion. Histology, histochemistry, heat deproteination, scanning electron microscopy (SEM)/EDAX analysis, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and Fourier transform infrared spectrometry (FTIR) have been applied in the study. H. sapiens and X. gladius bone specimens showed similar profiles, R. asterias calcified cartilage diverges for higher water release and more amorphous bioapatite. In endochondral ossification, fetal calcified cartilage is progressively replaced by bone matrix, while R. asterias calcified cartilage remains un-remodeled throughout the life span. Ca2+ and PO4 3- concentration in extracellular matrix is suggested to reach the critical salts precipitation point through H2 O recall from extracellular matrix into both chondroblasts or osteoblasts. Cartilage organic phase layout and incomplete mineralization allow interstitial fluids diffusion, chondrocytes survival, and growth in a calcified tissue lacking of a vascular and canalicular system. HIGHLIGHTS: Comparative physico-chemical characterization (TGA, DTG and DSC) testifies the mass loss due to water release, collagen and carbonate decomposition of the three tested matrices. R. asterias calcified cartilage water content is higher than that of H. sapiens and X. gladius, as shown by the respectively highest dehydration enthalpy values. Lower crystallinity degree of R. asterias calcified cartilage can be related to the higher amount of collagen in amorphous form than in bone matrix. These data can be discussed in terms of the mechanostat theory (Frost, 1966) or by organic/inorganic phase transformation in the course evolution from fin to limbs. Mineral analysis documented different charactersof R. asterias vs H. sapiens and X. gladius calcified matrix.

Crystal structure and induced stability of trimeric BxpB: implications for the assembly of BxpB-BclA complexes in the exosporium of Bacillus anthracis.

The outermost exosporium layer of Bacillus anthracis spores, the causative agents of anthrax, is comprised of a basal layer and an external hair-like nap. The nap includes filaments composed of trimers of the collagen-like glycoprotein BclA. Essentially all BclA trimers are attached to the spore in a process in which part of the 38-residue amino-terminal domain (NTD) of BclA forms an extremely stable interaction with the basal layer protein BxpB. Evidence indicates that the BclA-BxpB interaction is direct and requires trimeric BxpB. To further investigate the nature of the BclA-BxpB interaction, we determined the crystal structure of BxpB. The structure was trimeric with each monomer consisting of 11 ß strands with connecting loops. The structure did not include apparently disordered amino acids 1-19, which contain the only two cysteine residues of the 167-residue BxpB. The orientation of the structure reveals regions of BxpB that could be involved in interacting with the BclA NTD and with adjacent cysteine-rich proteins in the basal layer. Furthermore, the BxpB structure closely resembles that of the 134-residue carboxyl-terminal domain of BclA, which forms trimers that are highly resistant to heat and detergent. We demonstrated that BxpB trimers do not share this resistance. However, when BxpB trimers are mixed with a peptide containing residues 20-38 of BclA, they form a complex that is as stable as BclA-BxpB complexes extracted from spores. Together, our results provide new insights into the mechanism of BclA-BxpB attachment and incorporation into the exosporium. IMPORTANCE The B. anthracis exosporium plays major roles in spore survival and infectivity, but the complex mechanism of its assembly is poorly understood. Key steps in this process are the stable attachment of collagen-like BclA filaments to the major basal layer structural protein BxpB and the insertion of BxpB into an underlying basal layer scaffold. The goal of this study is to further elucidate these interactions thereby advancing our understanding of exosporium assembly, a process shared by many spore-forming bacteria including important human pathogens.

Multifilament Collagen Fiber Bundles with Tendon-like Structure and Mechanical Performance.

Collagen multifilament bundles comprised of thousands of monofilaments are prepared by multipin contact drawing of an entangled polymer solution consisting of collagen and poly(ethylene oxide) (PEO). The multifilament bundles are hydrated in graded concentrations of PEO and phosphate buffered saline (PBS) to promote assembly of collagen fibrils within each monofilament while preserving the structure of the multifilament bundle. Multiscale structural characterization reveals that the hydrated multifilament bundle contains properly folded collagen molecules packed in collagen fibrils containing microfibrils, staggered by exactly one-sixth of the microfibril D-band spacing to produce a periodicity of 11 nm. Sequence analysis predicts that in this structure, phenylalanine residues are close enough within and between microfibrils to become ultraviolet C (UVC) crosslinked. In agreement with this analysis, the ultimate tensile strength (UTS) and Young's modulus of the hydrated collagen multifilament bundles crosslinked by UVC radiation increase nonlinearly with total UVC energy to reach values in the range of native tendons without damage to the collagen molecules. This fabrication method recapitulates the structure of a tendon across multiple length scales and offers tunability in tensile properties using only collagen molecules and no other chemical additives in addition to PEO, which is almost entirely removed during the hydration process.

Ultraviolet light induces mechanical and structural changes in full thickness human skin.

While the detrimental health effects of prolonged ultraviolet (UV) irradiation on skin health have been widely accepted, the biomechanical process by which photoaging occurs and the relative effects of irradiation with different UV ranges on skin biomechanics have remained relatively unexplored. In this study, the effects of UV-induced photoageing are explored by quantifying the changes in the mechanical properties of full-thickness human skin irradiated with UVA and UVB light for incident dosages up to 1600 J/cm2. Mechanical testing of skin samples excised parallel and perpendicular to the predominant collagen fiber orientation show a rise in the fractional relative difference of elastic modulus, fracture stress, and toughness with increased UV irradiation. These changes become significant with UVA incident dosages of 1200 J/cm2 for samples excised both parallel and perpendicular to the dominant collagen fiber orientation. However, while mechanical changes occur in samples aligned with the collagen orientation at UVB dosages of 1200 J/cm2, statistical differences in samples perpendicular to the collagen orientation emerge only for UVB dosages of 1600 J/cm2. No notable or consistent trend is observed for the fracture strain. Analyses of toughness changes with maximum absorbed dosage reveals that no one UV range is more impactful in inducing mechanical property changes, but rather these changes scale with maximum absorbed energy. Evaluation of the structural characteristics of collagen further reveals an increase in collagen fiber bundle density with UV irradiation, but not collagen tortuosity, potentially linking mechanical changes to altered microstructure.

Microbial community on industrial salty bovine hides: From the slaughterhouse to the salting.

The leather-making industry is an age-old industry and desiccation with salt has been one of the most used methodologies for obtaining valuable skins. However, halophiles may proliferate and affect the integrity of the hide-collagen structure, as well as leading to undesirable red colorations or less-frequent purple stains. To understand the basis of these industrial hide contaminations, the microbial community from raw hide samples, salt-cured samples and four different industrial salts, was analyzed by 16S rRNA gene metabarcoding together with standard cultivation methods. Comparison of raw hides and correctly cured hides revealed a core microbiome that was absent from contaminated hides. In addition, archaea were missing from well-cured hides, whereas Psychrobacter and Acinetobacter were highly represented (23 % and 17.4 %, respectively). In damaged hides, only a few operational taxonomic units (OTUs), from among the hundreds detected, were able to proliferate and, remarkably, a single Halomonas OTU represented 57.66 % of the reads. Halobacteria, mainly Halovenus, Halorubrum and Halovivax, increased by up to 36.24-39.5 % in the red- and purple-affected hides. The major contaminants were isolated and hide infections, together with collagenase activity, were evaluated. The results showed that hides enriched with the non-pigmented isolate Halomonas utahensis COIN160 damaged the collagen fibers similarly to Halorubrum, and together they were considered to be one of the major causes. Putative degrading inhibitors were also identified from among the Alkalibacillus isolates. It was concluded that hide contaminations were driven by clonal outbreaks of a few specific microbes, which may have been non-pigmented collagen degraders. Acinetobacter and Alkalibacillus, members of the core microbiome of raw and well-cured salted hides, are suggested as hide contaminant inhibitors that need further analysis.