Endogenous Collagen Research Articles

Hydroxyprolyl-Glycine in 24 H Urine Shows Higher Correlation with Meat Consumption than Prolyl-Hydroxyproline, a Major Collagen Peptide in Urine and Blood.

Background. Urinary collagen peptides, the breakdown products of endogenous collagen, have been used as biomarkers for various diseases. These non-invasive biomarkers are easily measured via mass spectrometry, aiding in diagnostics and therapy effectiveness. Objectives. The objective of this study was to investigate the effects of consuming collagen-containing meat on collagen peptide composition in human blood and urine. Methods. Ten collagen peptides in 24 h urine were quantified. Results. Prolyl-hydroxyproline (Pro-Hyp) was the most abundant peptide. Except for hydroxyprolyl-glycine (Hyp-Gly), levels of other minor collagen peptides showed high correlation coefficients with Pro-Hyp (r = 0.42 vs. r > 0.8). Notably, 24 h urinary Hyp-Gly showed a correlation coefficient of r = 0.72 with meat consumption, significantly higher than the coefficient for Pro-Hyp (r = 0.37). Additionally, the levels of Pro-Hyp and Hyp-Gly in the blood of seven young women participants increased similarly after consuming fish meat, while before ingestion, only negligible amounts of Hyp-Gly were present. To examine which peptides are generated by the degradation of endogenous collagen, mouse skin was cultured. The amount of Pro-Hyp released from the skin was approximately 1000-fold higher than that of Hyp-Gly. Following consumption of collagen-containing meat, both Pro-Hyp and Hyp-Gly are released in blood and excreted into urine, although Pro-Hyp is primarily generated from endogenous collagen even under physiological conditions. Conclusions. Therefore, in 24 h urine samples, the non-negligible fraction of Pro-Hyp is contributed by endogenous collagen, making 24 h urine Hyp-Gly level a potential biomarker for evaluating meat consumption on the day.

Lactate triggers KAT8-mediated LTBP1 lactylation at lysine 752 to promote skin rejuvenation by inducing collagen synthesis in fibroblasts

Decreased collagen synthesis by fibroblasts is a key aspect of skin aging. Poly-L-Lactic Acid (PLLA) is a bioabsorbable material that can release lactate continuously, stimulating endogenous collagen synthesis in the skin. Herein, this study aimed to investigate the impact of PLLA-released lactate on collagen production in fibroblasts for skin rejuvenation. Human fibroblasts were exposed to varying concentrations of PLLA in vitro, while PLLA was injected into the back skin of aged mice in vivo. Safety and efficacy of PLLA on collagen synthesis and skin rejuvenation were evaluated through Calcein-AM/PI staining, EdU proliferation assay, and analysis of collagen I and collagen III expression in fibroblasts using western blotting and immunofluorescence. To elucidate the underlying mechanisms, lactate contents in cell-free supernatant and cell lysates from PLLA-treated fibroblasts, as well as total lysine lactylation (Pan Kla) levels were measured. Additionally, we found that fibroblasts can uptake extracellular lactate released from PLLA through monocarboxylate transporter-1 (MCT1) to facilitate latent-transforming growth factor beta-binding protein 1 (LTBP1) lactylation at lysine 752 (K752) via a KAT8-dependent mechanism, then increases the protein levels of collagen I and collagen III in fibroblasts. Overall, this study highlights a valuable insight into lactylation modification of non-histone protein for skin rejuvenation.

In Vivo Photoacoustic Ultrasound (PAUS) Assay for Monitoring Tendon Collagen Compositional Changes during Injury and Healing.

Tendon injury and healing involve significant changes to tissue biology and composition. Current techniques often require animal sacrifice or tissue destruction, limiting assessment of dynamic changes in tendons, including treatment response, disease development, rupture risk, and healing progression. Changes in tendon composition, such as altered collagen content, can significantly impact tendon mechanics and function. Analyses of compositional changes typically require ex vivo techniques with animal sacrifice or destruction of the tissue. In vivo evaluation of tendons is critical for longitudinal assessment. We hypothesize that photoacoustic ultrasound detects differences in collagen concentration throughout healing. We utilized photoacoustic ultrasound, a hybrid imaging modality that combines ultrasound and laser-induced photoacoustic signals to create detailed and high-resolution images of tendons, to identify its endogenous collagen composition. We correlated the photoacoustic signal to picrosirius red staining. The results show that the photoacoustic ultrasound-estimated collagen content in tendons correlates well with picrosirius red staining. This study demonstrates that photoacoustic ultrasound can assess injury-induced compositional changes within tendons and is the first study to image these targets in rat Achilles tendon in vivo.

Endoplasmic reticulum exit sites are segregated for secretion based on cargo size

TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion.

Bioprinting of human dermal microtissues precursors as building blocks for endogenous in vitro connective tissue manufacturing

The advent of 3D bioprinting technologies in tissue engineering has unlocked the potential to fabricate in vitro tissue models, overcoming the constraints associated with the shape limitations of preformed scaffolds. However, achieving an accurate mimicry of complex tissue microenvironments, encompassing cellular and biochemical components, and orchestrating their supramolecular assembly to form hierarchical structures while maintaining control over tissue formation, is crucial for gaining deeper insights into tissue repair and regeneration. Building upon our expertise in developing competent three-dimensional tissue equivalents (e.g. skin, gut, cervix), we established a two-step bottom-up approach involving the dynamic assembly of microtissue precursors (μTPs) to generate macroscopic functional tissue composed of cell-secreted extracellular matrix (ECM). To enhance precision and scalability, we integrated extrusion-based bioprinting technology into our established paradigm to automate, control and guide the coherent assembly of μTPs into predefined shapes. Compared to cell-aggregated bioink, our μTPs represent a functional unit where cells are embedded in their specific ECM. μTPs were derived from human dermal fibroblasts dynamically seeded onto gelatin-based microbeads. After 9 days, μTPs were suspended (50% v/v) in Pluronic-F127 (30% w/v) (µTP:P30), and the obtained formulation was loaded as bioink into the syringe of the Dr.INVIVO-4D6 extrusion based bioprinter. µTP:P30 bioink showed shear-thinning behavior and temperature-dependent viscosity (gel at T > 30 °C), ensuring µTPs homogenous dispersion within the gel and optimal printability. The bioprinting involved extruding several geometries (line, circle, and square) into Pluronic-F127 (40% w/v) (P40) support bath, leveraging its shear-recovery property. P40 effectively held the bioink throughout and after the bioprinting procedure, until µTPs fused into a continuous connective tissue. µTPs fusion dynamics was studied over 8 days of culture, while the resulting endogenous construct underwent 28 days culture. Histological, immunofluorescence analysis, and second harmonic generation reconstruction revealed an increase in endogenous collagen and fibronectin production within the bioprinted construct, closely resembling the composition of the native connective tissues.

The proprotein convertase BLI-4 promotes collagen secretion prior to assembly of the Caenorhabditis elegans cuticle.

Some types of collagens, including transmembrane MACIT collagens and C. elegans cuticle collagens, are N-terminally cleaved at a dibasic site that resembles the consensus for furin or other proprotein convertases of the subtilisin/kexin (PCSK) family. Such cleavage may release transmembrane collagens from the plasma membrane and affect extracellular matrix assembly or structure. However, the functional consequences of such cleavage are unclear and evidence for the role of specific PCSKs is lacking. Here, we used endogenous collagen fusions to fluorescent proteins to visualize the secretion and assembly of the first collagen-based cuticle in C. elegans and then tested the role of the PCSK BLI-4 in these processes. Unexpectedly, we found that cuticle collagens SQT-3 and DPY-17 are secreted into the extraembryonic space several hours before cuticle matrix assembly. Furthermore, this early secretion depends on BLI-4/PCSK; in bli-4 and cleavage-site mutants, SQT-3 and DPY-17 are not efficiently secreted and instead form large intracellular puncta. Their later assembly into cuticle matrix is reduced but not entirely blocked. These data reveal a role for collagen N-terminal processing in intracellular trafficking and the control of matrix assembly in vivo. Our observations also prompt a revision of the classic model for C. elegans cuticle matrix assembly and the pre-cuticle-to-cuticle transition, suggesting that cuticle layer assembly proceeds via a series of regulated steps and not simply by sequential secretion and deposition.

Bioprinting a skin patch with dual-crosslinked gelatin (GelMA) and silk fibroin (SilMA): An approach to accelerating cutaneous wound healing.

Clinical settings often face significant obstacles in treating large acute wounds. The alternative of therapeutic approach is needed urgently. Hydrogels derived from natural or synthetic materials may be designed to perform a variety of functions for promoting wound healing. Herein, a 3D bioprinted hydrogel patch is designed for accelerating acute wound healing, which is fabricated with methacryloyl-substituted gelatin (GelMA) and silk fibroin (SilMA) dual-cross-linked by ultraviolet (UV) light. The GelMA with added silk fibroin (GelSilMA) shows improved biodegradation and mechanical properties. Furthermore, SilMA hydrogel can maintain a moisturized healing environment in wound area persistently with adequate degradation capacity. In vivo, GelSilMA (G-S) hydrogel can help to speed wound closure by the improved microenvironment for epidermal tissue regeneration and endogenous collagen generation accordingly. In summary, the G-S hydrogel patch can accelerate acute wound healing efficiently in a relatively simple and inexpensive manner.

Применение инъекционного коллагена при остеоартрите у мультиморбидных пациентов

It has been established that osteoarthritis (OA) refers to diseases with high comorbidity. According to available data, OA is most often combined with arterial hypertension (AH) and other cardiovascular diseases (CVD). The combination of OA and CVD causes certain difficulties when choosing drug therapy. Traditionally, with pain syndrome, the choice falls on NSAIDs, since this allows in most cases to get a quick response to treatment. However, the use of NSAIDs may be accompanied by an increase in average blood pressure by 5 mm Hg or more in patients with arterial hypertension (AH) and significantly reduces the antihypertensive effectiveness of beta-blockers and ACE inhibitors. Symptomatic effect of the use of symptom-modifying drugs of delayed action (SYSADOA - Symptomatic slow acting drugs for osteoarthritis) develops approximately 8-12 weeks after the start of administration. Limitations in the use of NSAIDs in patients with OA and CVD due to increased cardiovascular risk and a long waiting period for the onset of the effect when using drugs of the SYSADOA group dictate the need to search for new approaches in the treatment of OA, on the one hand, with rapid effectiveness, and on the other - meeting all the requirements of cardiovascular safety. It is known that local injection therapy with the use of different groups of drugs has high efficiency in OA: glucocorticosteroids (GCS), hyaluronic acid, chondroitin, etc. Currently, microinduction collagen preparations with a high safety profile have joined them. Special attention in OA deserves the drug Plexatron, which has in its composition a type 1 tropocollagen with a molecular weight of 300 kD and calcium phosphate 1 mcg, and is intended for intra-articular and periarticular administration. Injectable collagen preparations are capable of inducing the regeneration of damaged collagen fibers by stimulating the migration of fibroblasts to the sites of damage, the release of growth factors and the activation of a key enzyme for the induction of endogenous collagen synthesis - lysine hydroxylase

A Phase 1, Open-Label, Safety, Tolerability, and Efficacy Study of NC525 in Subjects with Advanced Myeloid Neoplasms

Background With the exception of certain select subtypes, Acute Myeloid Leukemia (AML) remains incurable for a majority of patients with current therapies. Despite recent drug approvals targeting actionable molecular mutations and BCL-2, relapse remains inevitable for most patients with AML due to leukemia-initiating cells and progenitors {i.e., leukemic stem cells (LSCs)} which are resistant to conventional cytotoxic drugs. Novel agents that preferentially target LSCs to lessen or eliminate the risk for relapse while avoiding significant myelosuppression by sparing normal hematopoietic stem cells (HSCs), are highly sought-after anti-leukemia therapeutics. Leukocyte Associated Immunoglobulin-like Receptor 1 (LAIR-1) is an immune inhibitory receptor that is present on most immune cell subsets, including T cells, NK cells, monocytes, macrophages, dendritic cells, and granulocytes. LAIR-1, a surface leukocyte receptor containing two immune receptor tyrosine-based inhibitory motifs (ITIM), binds to endogenous collagen ligands and plays an important role in inducing an immunosuppressive microenvironment. In AML, the engagement of LAIR-1 inhibits intracellular downstream survival signals, leading to decreased GM-CSF-mediated survival and proliferation of AML blasts. LAIR-1 is expressed on AML blasts and LSCs regardless of FAB subtypes, cytogenetics or molecular mutations. Recent work demonstrates that myeloid leukemia cell lines, primary AML blasts, monoblasts and monocytes, and MDS-EB-2 blasts all express LAIR-1. Critically, while high expression of LAIR-1 is characteristic of LSCs, this receptor is minimally detectable on normal HSCs, making it an ideal anti-leukemic target. NC525 is a humanized immunoglobulin gamma 1, kappa (IgG1κ) monoclonal antibody that targets LAIR-1-expressing AML blasts and LSCs. Extensive IND-enabling preclinical studies have shown the following features of NC525: (i) Inhibition of colony formation of AML CD34+ bone marrow cells; (ii) sparing of colony formation and differentiation ability of healthy CD34+ HSCs; (iii) direct ex vivo killing of AML blasts without killing healthy leukocytes; (iv) suppression of AML in both MV4-11 and THP-1 CDX models; (v) restriction of MV4-11 in a humanized murine model, without depletion or suppression of human T cells; (vi) restriction of MV4-11 tumor growth in murine blood, spleen, and bone marrow; and (vii) reduction of AML disease burden in primary patient-derived AML xenograft (PDX) models. Finally, NC525 completely inhibits leukemia propagation in secondary transplant models, suggesting that NC525 is capable of eradicating LSCs. Furthermore, NC525 destroys leukemic cells by ligation-mediated apoptosis of leukemic blasts and stem cells without evidence of an immunomodulatory effect on other immune subsets. Methods This is an open-label, non-randomized, Phase 1 dose escalation study to determine the safety and tolerability of NC525 in adult patients with advanced myeloid neoplasms. This study will also assess the clinical benefit of NC525. Eligible patients must have relapsed or refractory AML, myelodysplastic syndrome (MDS), or chronic myelomonocytic leukemia (CMML) that has progressed after standard therapy. Participants will receive NC525 by intravenous infusion on Days 1 and 15 of each 28-day cycle. During Phase 1, the safety and tolerability of NC525 will be evaluated by exploring escalating dose levels of NC525. In addition, the pharmacologically active dose (PAD), defined as the dose that provides a maximal biologic effect, such as achieving a complete remission (CR), CR with partial hematological recovery (CRh) or CR with incomplete hematological recovery (CRi), and the maximum tolerated dose (MTD) of NC525 will be determined. Safety and tolerability will be further assessed through the expansion of a few dose levels to determine an optimal recommended Phase 2 Dose (RP2D) and administration schedule of NC525, based on the PK/PD profiles. Taken together, preclinical data suggest that targeting LAIR-1 in AML and other myeloid malignancies may be an effective therapy for these diseases with the advantage of specifically eradicating leukemic stem cells and sparing normal HSCs.

The value of undenatured collagen for the normalization of the function of the cartilaginous tissue of the joints

Among the proteins of the human body, collagen accounts for at least 25–45% collagen; it is an essential structural component of skeletal tissues, connective tissue of internal organs (heart, intestines, lungs, liver, kidneys), as well as blood vessels. The variety of types of collagen is due to their role and function. The article presents information about the structure and synthesis of collagen, a high concentration of glycine, proline and hydroxyproline in the alpha chains of the collagen helix. Attention is drawn to the fact that cross-links and orientation of collagen fibers in the direction of the vector of external forces and loads provide protection to tissues and organs. The clinical significance of type II collagen is considered on the example of the cartilaginous tissue of the joints and tendons. It is emphasized that an imbalance in the structure of nutrition, genetic mutations, dysfunction of the endocrine and immune systems, especially in old age, are associated with the occurrence of one of the most common joint diseases – osteoarthritis (ОА). It has been established that the degradation or reduction of type II collagen in the cartilage matrix is accompanied by the progression of this disease. Due to the increasing prevalence of OA, signs of metabolic disorders and post-traumatic joint injuries, there is a growing interest in non-pharmacological and pharmacological interventions for the prevention and treatment of osteoarthritis. In recent years, convincing evidence has emerged of the successful use of drugs (biologically active food supplements) of collagen in osteoarthritis. The article presents the results of experimental and clinical studies, meta-analysis and systematic review, confirming the possibility of using these drugs (products) as part of the complex treatment of OA. The possibility of using compositions based on undenatured (native) collagen type II, with the inclusion of ascorbic acid, vitamin D, methylsulfonylmethane and boswellic acids promotes the synergy of these substances, slows down the rate of cartilage destruction, reduces the manifestation of pain and inflammation in the joints, improves functional joint and spinal conditions, promotes the synthesis of endogenous collagen.

The collagen type I alpha 1 chain gene is an alternative safe harbor locus in the porcine genome

Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors. Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects. However, relatively few safe harbor loci are available in pigs, a fact which has impeded the development of multi-transgenic pig research. We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain (COL1A1) gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. After the knock-in of a 2A peptide-green fluorescence protein (2A-GFP) transgene in the last codon of COL1A1 in multiple porcine cells, including porcine kidney epithelial (PK15), porcine embryonic fibroblast (PEF) and porcine intestinal epithelial (IPI-2I) cells, quantitative PCR (qPCR), Western blotting, RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus. The qPCR results showed that the GFP knock-in had no effect (P=0.29, P=0.66 and P=0.20 for PK15, PEF and IPI-2I cells, respectively) on the mRNA expression of COL1A1 gene. Similarly, no significant differences (P=0.64, P=0.48 and P=0.80 for PK15, PEF and IPI-2I cells, respectively) were found between the GFP knock-in and wild type cells by Western blotting. RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation (P<2.2e–16) with that of the wild type cells, indicating that the GFP knock-in did not alter the global expression of endogenous genes. Furthermore, the CCK8 assay showed that the GFP knock-in events had no adverse effects (P24h=0.31, P48h=0.96, P72h=0.24, P96h=0.17, and P120h=0.38) on cell proliferation of PK15 cells. These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment.

Soft Tissue and Biomolecular Preservation in Vertebrate Fossils from Glauconitic, Shallow Marine Sediments of the Hornerstown Formation, Edelman Fossil Park, New Jersey.

Simple SummaryOriginal organics and soft tissues are known to persist in the fossil record. To date, these discoveries derive from a limited number of ancient environments, (e.g., rivers, floodplains), and fossils from rarer environments remain largely unexplored. We studied Cretaceous–Paleogene fossils from a peculiar marine environment (glauconitic greensand) from Jean and Ric Edelman Fossil Park in Mantua Township, NJ. Twelve samples were demineralized in acid to remove the mineral component of bone. This treatment frequently yielded products that are visually consistent with bone cells, blood vessels, and bone matrix from modern animals. Fossil specimens that are dark in color exhibit excellent microscopic bone preservation and yielded a greater recovery of original soft tissues, whereas light-colored specimens exhibit poor microscopic preservation and yielded few to no soft tissues. Additionally, a well-preserved femur of a marine crocodilian was found to retain original bone protein by reactions with antibodies. Our results: (1) corroborate previous findings that original soft tissue and proteins can be recovered from fossils preserved in marine environments, and (2) expand the range of ancient environments documented to preserve original organics and soft tissues. This broadens the suite of fossils that may be fruitful to examine in future paleomolecular studies.Endogenous biomolecules and soft tissues are known to persist in the fossil record. To date, these discoveries derive from a limited number of preservational environments, (e.g., fluvial channels and floodplains), and fossils from less common depositional environments have been largely unexplored. We conducted paleomolecular analyses of shallow marine vertebrate fossils from the Cretaceous–Paleogene Hornerstown Formation, an 80–90% glauconitic greensand from Jean and Ric Edelman Fossil Park in Mantua Township, NJ. Twelve samples were demineralized and found to yield products morphologically consistent with vertebrate osteocytes, blood vessels, and bone matrix. Specimens from these deposits that are dark in color exhibit excellent histological preservation and yielded a greater recovery of cells and soft tissues, whereas lighter-colored specimens exhibit poor histology and few to no cells/soft tissues. Additionally, a well-preserved femur of the marine crocodilian Thoracosaurus was found to have retained endogenous collagen I by immunofluorescence and enzyme-linked immunosorbent assays. Our results thus not only corroborate previous findings that soft tissue and biomolecular recovery from fossils preserved in marine environments are possible but also expand the range of depositional environments documented to preserve endogenous biomolecules, thus broadening the suite of geologic strata that may be fruitful to examine in future paleomolecular studies.

Hidroxiapatita de Cálcio em harmonização orofacial na região frontal - revisão da literatura

Aging signs begin to appear due to decrease of collagen production, causing the skin to lose support, mechanical strength and elasticity, generating expression lines and static wrinkles. The frontal region that is responsible for the main part of the facial expression, is a highly innervated area and it is irrigated by very important vessels being susceptible to the formation of horizontal and vertical static lines, and also formation of depressions due to frontal bone resorption. This area is highly enervated and with important arteries, and also has a thin subcutaneous layer. This area is difficult to treat as subcutaneous tissue is thick and it is a region irrigated by important arteries prone to necrosis. The search for collagen stimulation with Calcium Hydroxyapatite, in order to delay the changes related to aging due to decrease of type I collagen production, stimulating the formation of endogenous collagen by fibroblasts and more long-lasting results is increasing. Calcium Hydroxyapatite is used for filling and promotes skin collagen type I formation, decreasing aging signs and presenting long-lasting results. So, the objective of the present paper is a literature review of the Calcium Hydroxyapatite use for collagen biostimulation and volumization of the frontal region. For the search of articles of this literature review in PubMed and SciELO databases the key words used were Calcium Hydroxyapatite, Collagen Biostimulators, Orofacial Harmonization. It was concluded that Calcium Hydroxyapatite use in the frontal region is indicated for collagen stimulation and volumization, but it requires a qualified operator that knows all the structures of the region where the product is injected and knows how to deal with the complications arising from this procedure.

A Novel Lair-1 Antibody Selectively Targets Acute Myeloid Leukemia (AML) Stem Cells

Extensive research has led to recent approval of novel therapies such as mylotarg, venetoclax, glasdegib and CC486, and small molecule inhibitors against actionable mutations such as ivosidenib (IDH1), enasidenib (IDH2), gliteritinib and midostaurin (FLT3) in AML. However, the mainstay of treatment in AML remains unchanged since the 1970s. There is a significant unmet need for AML patients that fail to respond to or relapse after standard-of-care (SOC) treatments including allogeneic stem cell transplantation and targeting actionable mutations. In addition, a large fraction of SOC patients invariably relapse due to persistence of chemotherapy-resistant leukemia stem cells (LSCs) or immune evasion. Therefore, identification of unique therapies that preferentially target elusive LSCs and promote immune responses to AML to prevent relapse are highly sought after. Unlike, targeting acute lymphoblastic leukemia (ALL) with CD19 or CD22 with various modalities, when developing AML therapies, it is of paramount importance to differentiate LSCs from hematopoietic stem cells (HSCs) to lessen or abolish unavoidable cytopenias.Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) is an immune checkpoint receptor on T cells and myeloid cells that delimits immune cell activation through binding to endogenous collagen ligands. In addition, LAIR-1 is universally expressed on AML blasts and may sustain AML survival signals. We demonstrated using multi-color flow cytometry that LAIR-1 is highly expressed in AML blasts (n=9 of 9) and that LAIR-1 expression in LSCs (markers: CD34 +CD38 -CD90 -CD45RA +/- or CD34 -CD117 +CD244 +/-) is high compared with negligible expression of LAIR-1 in HSCs (markers: CD34 +CD38 -CD90 +CD99 -) (n=3) (Figure 1). Based on these findings, we hypothesized that a LAIR-1 monoclonal antibody (mAb) would disrupt LAIR-1 mediated survival signaling and preferentially target LAIR-1 expressing AML LSCs and blast cells but not HSCs. To test this, we developed a novel LAIR-1 targeting mAb with a functional human IgG 1 isotype that blocks LAIR-1 binding to its ligands (including collagens, complement component C1q, MBL and SP-D)To characterize the anti-leukemic effect of the LAIR-1 mAb we performed an in vitro antibody dependent cell cytotoxicity (ADCC) assay with LAIR-1 expressing AML cells (MOLT4 and MV-4-11). Compared with isotype control, the LAIR-1 mAb significantly increased leukemia cell death (MV411 = 17% above isotype, and MOLT4 = 29.24% above isotype at 15 µg/ml), suggesting that the LAIR-1 mAb confers ADCC activity against LAIR1 + AML cells (Figure 2). To elucidate if the LAIR-1 mAb has a direct signaling effect on LAIR-1 + AML cells, a colony forming unit assay using primary AML cells was carried out. Interestingly, the LAIR-1 mAb inhibited colony formation by AML CD34 + cells (40-60% decreased compared with isotype control, N=4), but not normal CD34 + cells. These data suggests that our LAIR-1 mAb stimulated LAIR-1 signaling that inhibits LSC self-renewal. We then tested the in vivo anti-leukemia effect of the mAb in cell line derived xenograft (CDX) models (immune deficient mice transplanted with MV-4-11 expressing luciferase). In vivo bioluminescence imaging indicated that the LAIR-1 mAb significantly inhibited in vivo AML growth (91% reduction of total flux)(Figure 3). A significant increase in cell death was observed in the presence of the mAb in the blood (47%), spleen (89.4%) and bone marrow (27.6%). Similar to the anti-leukemic effect in CDX AML models, the LAIR-1 mAb significantly suppressed in vivo growth of AML patient derived xenografts (5 different primary AML donors) (10-90% human CD33 + AML cells in isotype control treatment vs 0.5-5% CD33 + AML cells in anti-LAIR-1 treatment, N=3) (Figure 4), while minimally impacting normal immune cells.Taken together, our studies suggest that the LAIR-1 mAb we generated is a novel AML immunomedicine that preferentially eradicates AML LSCs and blasts while preserving healthy HSCs through disruption of AML survival signals and clearance of AML through ADCP and ADCC. Additional studies are currently evaluating if this novel LAIR-1 mAb has other mechanisms of action that contribute to overall in vivo activity, including reduction of AML niche implantation, regulation of bone marrow homing and regulation of anti-tumor immunity. [Display omitted] DisclosuresTian: NextCure: Ended employment in the past 24 months. Paucarmayta: NextCure: Current Employment. Lovewell: NextCure: Current Employment. Maloveste: NextCure: Current Employment. Copeland: NextCure: Current Employment. O'Neill: NextCure: Current Employment. Patel: NextCure: Current Employment. Liu: NextCure: Current Employment, Current holder of stock options in a privately-held company. Myint: NextCure: Current Employment, Current holder of stock options in a privately-held company. Langermann: NextCure: Current Employment, Current holder of stock options in a privately-held company. Flies: NextCure: Current Employment, Current holder of stock options in a privately-held company. Kim: Nextcure: Research Funding.

Heat-induced structural changes in fish muscle collagen related to texture development in fish balls: Using eel ball as a study model.

In order to elucidate the substantial effect and underlying mechanism of endogenous collagen on the texture development of fish balls, the structural and gelling properties of eel muscle collagen (EMC) under different heat treatments, as well as their effects on texture of eel ball, were investigated. EMC resulted in significant improvement of eel ball texture via gelling ability, filler effect, and interaction with starch. Under mild heating below 90°C for 30 min, the structural and physicochemical changes of EMC varied gradually, resulting in improved storage modulus of starch‐containing myofibrillar gel, a mimic of eel ball. However, overheating (100°C, 30 min) induced EMC degradation and significantly decreased the gel formation and the improvements in textural properties. Supplementation of EMC to eel balls significantly improved its gel strength, springiness, cohesiveness, and chewiness, as well as uniformity and tightness of the microstructure. These results suggest the texture development of eel ball can be regulated by heat‐induced structural changes, as well as structure–function relationship of collagen, compared with previous studies on myofibrillar proteins and exogenous gelatin; and they may provide texture‐related insights to the quality control of fish balls and diverse heat‐treated products of surimi containing collagen.

Improved quality control criteria for stable carbon and nitrogen isotope measurements of ancient bone collagen

The carbon (δ13C) and nitrogen (δ15N) isotopic compositions of bone and tooth collagen provide a powerful tool for studying past biological, environmental, and cultural phenomena. Collagen has a well-understood chemical composition that has enabled the development of invaluable quality control (QC) criteria for isotopic data – something that is extremely rare among biogeochemical research on ancient biomolecules as a whole. The most important of these collagen QC criteria is atomic C:N ratio (ratio of carbon to nitrogen atoms), which provides an indicator of the extent to which the amount of carbon and nitrogen present in a sample matches the known composition of pure collagen, thereby indicating whether contamination or diagenesis may be influencing a sample's isotopic compositions. We present a model describing the relationship between the carbon and nitrogen isotopic and elemental compositions that accounts for the isotopic composition of the endogenous collagen and exogenous contaminants as well as taxon-specific information about the collagen's amino acid composition. In some cases the traditional C:N QC parameters are applicable, while in others they can result in the inclusion of unreliable (altered) isotopic data primarily due to contamination from humic substances. Using new and previously published data on taxa commonly encountered in ancient studies, we further illustrate how using traditional C:N QC parameters may lead to the inclusion of altered isotopic compositions in real archaeological and paleontological scenarios. We argue that the traditional ‘one size fits all’ approach to the C:N QC criterion should be avoided and we outline new collagen QC criteria specific to certain taxa and environments on the basis of the results of our model. These revised criteria will help to improve the interpretation of isotopic data by more accurately identifying samples with isotopic compositions altered by contamination.

The dipeptide prolyl-hydroxyproline promotes cellular homeostasis and lamellipodia-driven motility via active β1-integrin in adult tendon cells

Collagen-derived hydroxyproline (Hyp)-containing peptides have a variety of biological effects on cells. These bioactive collagen peptides are locally generated by the degradation of endogenous collagen in response to injury. However, no comprehensive study has yet explored the functional links between Hyp-containing peptides and cellular behavior. Here, we show that the dipeptide prolyl-4-hydroxyproline (Pro-Hyp) exhibits pronounced effects on mouse tendon cells. Pro-Hyp promotes differentiation/maturation of tendon cells with modulation of lineage-specific factors and induces significant chemotactic activity in vitro. In addition, Pro-Hyp has profound effects on cell proliferation, with significantly upregulated extracellular signal–regulated kinase phosphorylation and extracellular matrix production and increased type I collagen network organization. Using proteomics, we have predicted molecular transport, cellular assembly and organization, and cellular movement as potential linked-network pathways that could be altered in response to Pro-Hyp. Mechanistically, cells treated with Pro-Hyp demonstrate increased directional persistence and significantly increased directed motility and migration velocity. They are accompanied by elongated lamellipodial protrusions with increased levels of active β1-integrin–containing focal contacts, as well as reorganization of thicker peripheral F-actin fibrils. Pro-Hyp–mediated chemotactic activity is significantly reduced (p < 0.001) in cells treated with the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or the α5β1-integrin antagonist ATN-161. Furthermore, ATN-161 significantly inhibits uptake of Pro-Hyp into adult tenocytes. Thus, our findings document the molecular basis of the functional benefits of the Pro-Hyp dipeptide in cellular behavior. These dynamic properties of collagen-derived Pro-Hyp dipeptide could lead the way to its application in translational medicine.

Meat quality, post-mortem proteolytic enzymes, and myosin heavy chain isoforms of different Thai native cattle muscles.

ObjectiveThis study investigated the meat quality characteristics, endogenous proteolytic enzymes, collagen content, and myosin heavy chain (MyHC) isoforms of different muscles of Thai native cattle (TNC).MethodsInfraspinatus (IF), Longissimus thoracis (LT), and Supraspinatus (SS) muscles were obtained from two TNC breeds, Kho-Lan (KL, n = 7) and Kho-Isaan (KI, n = 7). The muscle and meat characteristics of TNC breeds and their relationship with MyHC expression were examined.ResultsThree MyHC isoforms namely MyHC I, MyHC IIa, and MyHC IIx were detected in the muscles. The KL had higher (p<0.05) MyHC IIx than the KI. The IF muscle had higher (p<0.05) MyHC I compared to other muscles. The LT muscle had the least MyHC I. The LT had higher (p<0.05) MyHC IIx than the IF and SS muscles. The IF presented the least MyHC IIx. The KL had higher (p<0.05) lightness and moisture content and lower crude protein, redness, cooking loss, shear force, and calpastatin than the KI. The glycogen, total collagen, soluble collagen, crude protein, ash contents, and troponin T degradation product of IF and SS were lower (p<0.05) than that of LT. Ether extract in LT was lower (p<0.05) than that of IF and SS. The percentage of MyHC I, MyHC IIa, and MyHC IIx were significantly correlated with muscle and meat characteristics of TNC.ConclusionThese results suggest that the differences in the MyHC isoforms may partly account for the variation in meat quality between breeds and among muscles of TNC.

Comprehensive collagen crosslinking comparison of microfluidic wet-extruded microfibers for bioactive surgical suture development

Collagen microfiber-based constructs have garnered considerable attention for ligament, tendon, and other soft tissue repairs, yet with limited clinical translation due to strength, biocompatibility, scalable manufacturing, and other challenges. Crosslinking collagen fibers improves mechanical properties; however, questions remain regarding optimal crosslinking chemistries, biocompatibility, biodegradation, long-term stability, and potential for biotextile assemble at scale, limiting their clinical usefulness. Here, we assessed over 50 different crosslinking chemistries on microfluidic wet-extruded collagen microfibers made with clinically relevant collagen to optimize collagen fibers as a biotextile yarn for suture or other medical device manufacture. The endogenous collagen crosslinker, glyoxal, provides extraordinary fiber ultimate tensile strength near 300MPa, and Young's modulus of over 3GPa while retaining 50% of the initial load-bearing capacity through 6 months as hydrated. Glyoxal crosslinked collagen fibers further proved cytocompatible and biocompatible per ISO 10993-based testing, and further elicits a predominantly M2 macrophage response. Remarkably these strong collagen fibers are amenable to industrial braiding to form strong collagen fiber sutures. Collagen microfluidic wet extrusion with glyoxal crosslinking thus progress bioengineered, strong, and stable collagen microfibers significantly towards clinical use for potentially promoting efficient healing compared to existing suture materials. Statement of SignificanceTowards improving clinical outcomes for over 1 million ligament and tendon surgeries performed annually, we report an advanced microfluidic extrusion process for type I collagen microfiber manufacturing for biological suture and other biotextile manufacturing. This manuscript reports the most extensive wet-extruded collagen fiber crosslinking compendium published to date, providing a tremendous recourse to the field. Collagen fibers made with clinical-grade collagen and crosslinked with glyoxal, exhibit tensile strength and stability that surpasses all prior reports. This is the first report demonstrating that glyoxal, a native tissue crosslinker, has the extraordinary ability to produce strong, cytocompatible, and biocompatible collagen microfibers. These collagen microfibers are ideal for advanced research and clinical use as surgical suture or other tissue-engineered medical products for sports medicine, orthopedics, and other surgical indications.

Autofluorescence spectroscopy and multivariate analysis for predicting the induced damages to other organs due to liver fibrosis

When our liver does not work well, it can induce damage to other organs causing their dysfunction. With this background, we aim to study the effect of liver fibrosis on other organs such as heart, lungs, kidney and spleen by assessing the variations in the inherent emission property of the tissue, using fluorescence spectroscopy. Fluorescence emission spectra from excised organs of liver fibrosis induced rats were collected at excitation wavelengths 320 and 410 nm. Optical redox ratio derived from the spectral data supported by multivariate statistical analysis, principal component analysis followed by linear discriminant analysis (PCA-LDA) distinguished between control and fibrosis induced groups. The two different excitation wavelength provided variations in the endogenous flurophores collagen, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), lipopigments and porphyrins. Additionally, evaluation of redox ratio provided variations in tissue metabolic activity of different organs. The PCA–LDA modelling yielded a sensitivity of 85 to 97% and specificity of 80 to 96% on 320 nm excitation and a sensitivity of 72 to 100% and specificity of 59 to 100% on 410 nm excitation. Fluorescence emission spectral study along with multivariate analysis paved way to identify the biochemical alterations caused to other organs due to the development of liver fibrosis, which could lead to their damage and dysfunction.