Mature Fibers Research Articles

Canonical ligand-dependent and non-canonical ligand-independent EphA2 signaling in the eye lens of wild-type, knockout, and aging mice.

Disruption of Eph-ephrin bidirectional signaling leads to human congenital and age-related cataracts, but the mechanisms for these opacities in the eye lens remain unclear. Eph receptors bind to ephrin ligands on neighboring cells to induce canonical ligand-mediated signaling. The EphA2 receptor also signals non-canonically without ligand binding in cancerous cells, leading to epithelial-to-mesenchymal transition (EMT). We have previously shown that the receptor EphA2 and the ligand ephrin-A5 have diverse functions in maintaining lens transparency in mice. Loss of ephrin-A5 leads to anterior cataracts due to EMT. Surprisingly, both canonical and non-canonical EphA2 activation are present in normal wild-type lenses and in the ephrin-A5 knockout lenses. Canonical EphA2 signaling is localized exclusively to lens epithelial cells and does not change with age. Non-canonical EphA2 signaling is in both epithelial and fiber cells and increases significantly with age. We hypothesize that canonical ligand-dependent EphA2 signaling is required for the morphogenesis and organization of hexagonal equatorial epithelial cells while non-canonical ligand-independent EphA2 signaling is needed for complex membrane interdigitations that change during fiber cell differentiation and maturation. This is the first demonstration of non-canonical EphA2 activation in a non-cancerous tissue or cell and suggests a possible physiological function for ligand-independent EphA2 signaling.

Proteomic Evidence for Amyloidogenic Cross-Seeding in Fibrinaloid Microclots

In classical amyloidoses, amyloid fibres form through the nucleation and accretion of protein monomers, with protofibrils and fibrils exhibiting a cross-β motif of parallel or antiparallel β-sheets oriented perpendicular to the fibre direction. These protofibrils and fibrils can intertwine to form mature amyloid fibres. Similar phenomena can occur in blood from individuals with circulating inflammatory molecules (and also some originating from viruses and bacteria). Such pathological clotting can result in an anomalous amyloid form termed fibrinaloid microclots. Previous proteomic analyses of these microclots have shown the presence of non-fibrin(ogen) proteins, suggesting a more complex mechanism than simple entrapment. We thus provide evidence against such a simple entrapment model, noting that clot pores are too large and centrifugation would have removed weakly bound proteins. Instead, we explore whether co-aggregation into amyloid fibres may involve axial (multiple proteins within the same fibril), lateral (single-protein fibrils contributing to a fibre), or both types of integration. Our analysis of proteomic data from fibrinaloid microclots in different diseases shows no significant quantitative overlap with the normal plasma proteome and no correlation between plasma protein abundance and their presence in fibrinaloid microclots. Notably, abundant plasma proteins like α-2-macroglobulin, fibronectin, and transthyretin are absent from microclots, while less abundant proteins such as adiponectin, periostin, and von Willebrand factor are well represented. Using bioinformatic tools, including AmyloGram and AnuPP, we found that proteins entrapped in fibrinaloid microclots exhibit high amyloidogenic tendencies, suggesting their integration as cross-β elements into amyloid structures. This integration likely contributes to the microclots’ resistance to proteolysis. Our findings underscore the role of cross-seeding in fibrinaloid microclot formation and highlight the need for further investigation into their structural properties and implications in thrombotic and amyloid diseases. These insights provide a foundation for developing novel diagnostic and therapeutic strategies targeting amyloidogenic cross-seeding in blood clotting disorders.

9291 Hypothyroidism impairs skeletal muscle regeneration after injury

Abstract Disclosure: P. Aguiari: None. V. Villani: None. K.Y. Liu: None. G.A. Brent: None. L. Perin: None. A. Milanesi: None. Thyroid hormone (TH) signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury. Disruption of TH signaling results in an abnormal skeletal muscle phenotype, impaired regeneration, and sarcopenia with aging, reflecting the myopathic changes described in hypothyroid patients. Muscle stem cells (MuSCs) are the mediators of post-natal skeletal muscle plasticity. Normally quiescent, in response to injury they enter the cell cycle (myoblasts) and proliferate. Myoblasts then exit the cell cycle and differentiate into myocytes that will fuse with existing myofibers to restore tissue architecture and function. Via TH receptor alpha, TH regulates all the stages of the regeneration program and regulates the expression of multiple myogenic genes. Despite all this evidence, to date there are no studies investigating MuSCs behavior in response to injury during hypothyroidism. We characterized injury-induced regeneration of the tibialis anterior muscle (TAM) in euthyroid and hypothyroid mice, fed a low iodine + 0.15% propylthiouracil diet. To investigate the cell cycle dynamics of MuSCs, we generated Pax7-Fucci mice carrying the fluorescent ubiquitination-based cell-cycle indicator (Fucci) system under the Pax7 promoter. Muscle injury was induced via cardiotoxin injection in the TAM of 3-month-old Pax7-Fucci mice. Hypothyroid mice present signs of impaired regeneration compared to euthyroid mice. From histological analysis, at different time points, we observed smaller fiber diameters, myofibers with central nuclei, and a significantly reduced number of fast glycolytic type IIb fibers, the prevalent muscle fiber type in the TA muscle. ScRNAseq analysis shows that at a late phase of regeneration (14 days after injury) hypothyroid MuSCs and myoblasts are still in the activation state and overexpress genes related to DNA replication and cell proliferation, while genes related to myogenic differentiation and cell cycle exit are downregulated. Differentiated myocytes in the hypothyroid fibers present an immature phenotype and are characterized by overexpression of embryonic/temporary and slow myosin genes and downregulation of mature fast myosins. Cell cycle analysis of the Fucci signal through Flow Cytometry confirmed a higher number of activated hypothyroid MuSCs at 4, 7, and 28 days after injury. These cells accumulate in the S phase and are unable to exit the cell cycle and differentiate towards myocytes. These data demonstrate that hypothyroidism impairs muscle tissue regeneration halting MuSCs progression through the cell cycle, myoblast cell cycle exit, and fiber maturation. Investigating muscle stem cell behavior and response to injury during hypothyroidism is crucial to understanding the mechanism behind thyroid hormone-induced myopathies and identifying possible therapeutical targets. Presentation: 6/2/2024

8670 Hypothyroidism impairs skeletal muscle regeneration after injury

Abstract Disclosure: P. Aguiari: None. V. Villani: None. K.Y. Liu: None. G.A. Brent: None. L. Perin: None. A. Milanesi: None. Thyroid hormone (TH) signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury. Disruption of TH signaling results in an abnormal skeletal muscle phenotype, impaired regeneration, and sarcopenia with aging, reflecting the myopathic changes described in hypothyroid patients. Muscle stem cells (MuSCs) are the mediators of post-natal skeletal muscle plasticity. Normally quiescent, in response to injury they enter the cell cycle (myoblasts) and proliferate. Myoblasts then exit the cell cycle and differentiate into myocytes that will fuse with existing myofibers to restore tissue architecture and function. Via TH receptor alpha, TH regulates all the stages of the regeneration program and regulates the expression of multiple myogenic genes. Despite all this evidence, to date there are no studies investigating MuSCs behavior in response to injury during hypothyroidism. We characterized injury-induced regeneration of the tibialis anterior muscle (TAM) in euthyroid and hypothyroid mice, fed a low iodine + 0.15% propylthiouracil diet. To investigate the cell cycle dynamics of MuSCs, we generated Pax7-Fucci mice carrying the fluorescent ubiquitination-based cell-cycle indicator (Fucci) system under the Pax7 promoter. Muscle injury was induced via cardiotoxin injection in the TAM of 3-month-old Pax7-Fucci mice. Hypothyroid mice present signs of impaired regeneration compared to euthyroid mice. From histological analysis, at different time points, we observed smaller fiber diameters, myofibers with central nuclei, and a significantly reduced number of fast glycolytic type IIb fibers, the prevalent muscle fiber type in the TA muscle. ScRNAseq analysis shows that at a late phase of regeneration (14 days after injury) hypothyroid MuSCs and myoblasts are still in the activation state and overexpress genes related to DNA replication and cell proliferation, while genes related to myogenic differentiation and cell cycle exit are downregulated. Differentiated myocytes in the hypothyroid fibers present an immature phenotype and are characterized by overexpression of embryonic/temporary and slow myosin genes and downregulation of mature fast myosins. Cell cycle analysis of the Fucci signal through Flow Cytometry confirmed a higher number of activated hypothyroid MuSCs at 4, 7, and 28 days after injury. These cells accumulate in the S phase and are unable to exit the cell cycle and differentiate towards myocytes. These data demonstrate that hypothyroidism impairs muscle tissue regeneration halting MuSCs progression through the cell cycle, myoblast cell cycle exit, and fiber maturation. Investigating muscle stem cell behavior and response to injury during hypothyroidism is crucial to understanding the mechanism behind thyroid hormone-induced myopathies and identifying possible therapeutical targets. Presentation: 6/2/2024

Assessment of land configuration techniques on in-situ soil moisture conservation and productivity of rainfed Bt. Cotton in semi-arid production systems

Rainfed agriculture is proving to be a significant contributor to cotton production. However, recent climate change characterizes a substantial threat to current rainfed agricultural production, and consequently to farmers’ livelihoods in semi-arid regions. Typical features of these areas are erratic rainfall to frequent dry spells that lead to frequent crop failure and lasting poverty. In-situ soil moisture conservation is an important climate resilience agricultural practice to enhance the productivity of crops in these regions including rainfed Bt cotton. It comprises agronomical land configuration techniques with several local modifications. The experiments were conducted in a field at Okkarai village (11o 22’ N latitude and 78o 53’ E longitudes), Thuraiyur Block, Tiruchirapallai District in the Northwestern Agro-climatic Zone of Tamil Nadu. It was undertaken during the Rabi season (North-East monsoon) of 2022 and 2023. The experiments were laid out in randomized block design (RBD) with 7 treatments with three replications. The treatments consist of different land configuration techniques viz., flat-bed, ridges and furrows, broad bed and furrows (BBF), raised bed, tied ridges, tied ridges (alternate rows) and tied broad beds (TBB). An analysis of pooled mean data of two years showed significant differences between the treatments. Among the land configuration techniques tested, tied ridges (alternate rows) registered the maximum soil moisture content during flowering and boll formation (90 DAS), boll development (120 DAS) and boll and fibre maturation (140 DAS) stages, and the increase was 34.4%, 31.8% and 15.74%, respectively over flat-bed method. Likewise, tied ridges (alternate rows) significantly enhanced the cotton growth characters viz., plant height (128.92 cm), leaf area index (4.52), dry matter production (5.19 t/ha), and yield attributes viz., number of squares (44.50/plant), number of bolls (43.73/plant), boll weight (4.76 g) and seed cotton yield (20.86 q/ha) and cost-benefit ratio (3.07). Tied ridges in alternative row served as low-cost micro catchment approach for trapping and holding water and its practice in semi-arid rainfed region has beneficial effects of reducing runoff and soil loss, increasing soil moisture and augment crop yield.

GhCASPL1 regulates secondary cell wall thickening in cotton fibers by stabilizing the cellulose synthase complex on the plasma membrane.

Cotton (Gossypium hirsutum) fibers are elongated single cells that rapidly accumulate cellulose during secondary cell wall (SCW) thickening, which requires cellulose synthase complex (CSC) activity. Here, we describe the CSC-interacting factor CASPARIAN STRIP MEMBRANE DOMAIN-LIKE1 (GhCASPL1), which contributes to SCW thickening by influencing CSC stability on the plasma membrane. GhCASPL1 is preferentially expressed in fiber cells during SCW biosynthesis and encodes a MARVEL domain protein. The ghcaspl1 ghcaspl2 mutant exhibited reduced plant height and produced mature fibers with fewer natural twists, lower tensile strength, and a thinner SCW compared to the wild type. Similarly, the Arabidopsis (Arabidopsis thaliana) caspl1 caspl2 double mutant showed a lower cellulose content and thinner cell walls in the stem vasculature than the wild type but normal plant morphology. Introducing the cotton gene GhCASPL1 successfully restored the reduced cellulose content of the Arabidopsis caspl1 caspl2 mutant. Detergent treatments, ultracentrifugation assays, and enzymatic assays showed that the CSC in the ghcaspl1 ghcaspl2 double mutant showed reduced membrane binding and decreased enzyme activity compared to the wild type. GhCASPL1 binds strongly to phosphatidic acid (PA), which is present in much higher amounts in thickening fiber cells compared to ovules and leaves. Mutating the PA-binding site in GhCASPL1 resulted in the loss of its colocalization with GhCesA8, and it failed to localize to the plasma membrane. PA may alter membrane structure to facilitate protein-protein interactions, suggesting that GhCASPL1 and PA collaboratively stabilize the CSC. Our findings shed light on CASPL functions and the molecular machinery behind SCW biosynthesis in cotton fibers.

Bifunctional Inhibitor Lentinan Inhibits Fibrillogenesis of Amyloid-β Protein and α-Synuclein and Alleviates Their Cytotoxicity: In Vitro and In Vivo Studies.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases in the world. Misfolding of β-amyloid (Aβ) and α-synuclein (α-syn) and subsequent fibril formation are closely associated with the pathogenesis of AD and PD, respectively. Lentinan is a natural product commonly used in medicine and dietary supplements. It has potential antitumor, anti-inflammatory, and antiviral effects, but the underlying mechanism of its action on AD and PD remains unclear. In this study, lentinan inhibited the formation of Aβ and α-syn fibers in a dose-dependent manner and disrupted their mature fibers. Lentinan inhibited the conversion of Aβ and α-syn conformations to β-sheet-rich conformations. Additionally, lentinan protected Caenorhabditis elegans against damage caused by the accumulation of Aβ and α-syn aggregation and prolonged their lifespan. Notably, the beneficial effects of lentinan in AD and PD mice were also demonstrated, including ameliorating the cognitive and memory impairments in AD mice and behavioral deficits in PD mice. Finally, molecular interactions between lentinan and Aβ/α-syn pentamers were also explored using molecular docking.

A Review on Dermal Wound Repair and Regeneration Using Antimicrobial-Loaded Microspheres Incorporated into Bovine Collagen Scaffold

In this paper, the developed microsphere-incorporated collagen scaffold presents a promising solution for delivering drugs over wound surfaces in a uniform and sustained manner. The biomaterials utilized in this system are of natural origin, minimizing potential toxicity from core materials, and collagen and gelatin exhibit active wound-healing properties. The collagen scaffold used for the delivery device offers several advantages, including minimizing dressing frequency, facilitating easier examination, and providing added aesthetic value. Furthermore, the protein-based biomaterial scaffold acts as a template for the extracellular matrix in the skin, enhancing the maturation of wounds and enabling the transition to the accelerated production of mature and aligned collagen fibers. The controlled release of antimicrobial drugs through denatured collagens and gelatin microspheres, as well as the efficient healing observed in in vivo studies, demonstrate the potential of this system for effective wound care. Overall, the incorporation of ciprofloxacin-loaded microspheres in porous collagen scaffold offers sustained releases of ciprofloxacin in infected environments, as supported by histological examination showing well-formed epidermis and dermis in the connective tissue of the skin. The system’s ability to heal full-thickness wounds within a significantly shorter time frame compared to antibiotic-incorporated collagen sponges further emphasizes its efficacy in wound healing.

Crosstalk among canonical Wnt and Hippo pathway members in skeletal muscle and at the neuromuscular junction.

Skeletal muscles are essential for locomotion, posture, and metabolic regulation. To understand physiological processes, exercise adaptation, and muscle-related disorders, it is critical to understand the molecular pathways that underlie skeletal muscle function. The process of muscle contraction, orchestrated by a complex interplay of molecular events, is at the core of skeletal muscle function. Muscle contraction is initiated by an action potential and neuromuscular transmission requiring a neuromuscular junction. Within muscle fibers, calcium ions play a critical role in mediating the interaction between actin and myosin filaments that generate force. Regulation of calcium release from the sarcoplasmic reticulum plays a key role in excitation-contraction coupling. The development and growth of skeletal muscle are regulated by a network of molecular pathways collectively known as myogenesis. Myogenic regulators coordinate the differentiation of myoblasts into mature muscle fibers. Signaling pathways regulate muscle protein synthesis and hypertrophy in response to mechanical stimuli and nutrient availability. Several muscle-related diseases, including congenital myasthenic disorders, sarcopenia, muscular dystrophies, and metabolic myopathies, are underpinned by dysregulated molecular pathways in skeletal muscle. Therapeutic interventions aimed at preserving muscle mass and function, enhancing regeneration, and improving metabolic health hold promise by targeting specific molecular pathways. Other molecular signaling pathways in skeletal muscle include the canonical Wnt signaling pathway, a critical regulator of myogenesis, muscle regeneration, and metabolic function, and the Hippo signaling pathway. In recent years, more details have been uncovered about the role of these two pathways during myogenesis and in developing and adult skeletal muscle fibers, and at the neuromuscular junction. In fact, research in the last few years now suggests that these two signaling pathways are interconnected and that they jointly control physiological and pathophysiological processes in muscle fibers. In this review, we will summarize and discuss the data on these two pathways, focusing on their concerted action next to their contribution to skeletal muscle biology. However, an in-depth discussion of the non-canonical Wnt pathway, the fibro/adipogenic precursors, or the mechanosensory aspects of these pathways is not the focus of this review.

R2R3 MYB Transcription Factor GhMYB201 Promotes Cotton Fiber Elongation via Cell Wall Loosening and Very-Long-Chain Fatty Acid Synthesis.

Cotton fiber is the leading natural textile material, and fiber elongation plays an essential role in the formation of cotton yield and quality. Although a number of components in the molecular network controlling cotton fiber elongation have been reported, a lot of players still need to be functionally dissected to understand the regulatory mechanism of fiber elongation comprehensively. In the present study, an R2R3-MYB transcription factor gene, GhMYB201, was characterized and functionally verified via CRISPR/Cas9-mediated gene editing. GhMYB201 was homologous to Arabidopsis AtMYB60, and both coding genes (GhMYB201At and GhMYB201Dt) were preferentially expressed in elongating cotton fibers. Knocking-out of GhMYB201 significantly reduced the rate and duration of fiber elongation, resulting in shorter and coarser mature fibers. It was found that GhMYB201 could bind and activate the transcription of cell wall loosening genes (GhRDLs) and also β-ketoacyl-CoA synthase genes (GhKCSs) to enhance very-long-chain fatty acid (VLCFA) levels in elongating fibers. Taken together, our data demonstrated that the transcription factor GhMYB201s plays an essential role in promoting fiber elongation via activating genes related to cell wall loosening and VLCFA biosynthesis.

Spider Silk: Rapid, Bottom‐Up Self‐Assembly of MaSp1 into Hierarchically Structured Fibers Through Biomimetic Processing

AbstractSpider dragline silk's exceptional mechanical performance, coupled with its benign production pathway, have inspired the design of diverse smart materials. Remarkably, relatively little is known about the self‐assembly process of major ampullate spidroin 1 (MaSp1) – the main protein constituent of dragline fiber – during its rapid conversion from soluble precursor protein into hierarchically structured mature silk fibers. Herein, the biomimetic potential of MaSp1 is explored using a new recombinant platform with full domain representation. MaSp1 is found to undergo efficient liquid‐liquid phase separation (LLPS) in response to kosmotropic ions, with notably higher propensity compared to MaSp2, and with MaSp1 exhibiting a more complex solubility profile relative to levels of sodium chloride. To further the understanding of silk spinning, the rapid assembly of mesoscale structures is monitored in real‐time in response to ion and pH gradients, revealing a progression from LLPS droplets toward aligned hierarchical fibers. Furthermore, using this biomimetic system, insoluble macroscopic MaSp1 fibers can be readily generated, wherein the application of mechanical deformation triggers the emergence of β‐sheet secondary structures. The insights gained from this study can have broader application in efforts to mimic the formation of biomaterials with spatially organized features across multiple length scales.

Novel Identification of Ankyrin-R in Cardiac Fibroblasts and a Potential Role in Heart Failure.

Altered ankyrin-R (AnkR; encoded by ANK1) expression is associated with diastolic function, left ventricular remodeling, and heart failure with preserved ejection fraction (HFpEF). First identified in erythrocytes, the role of AnkR in other tissues, particularly the heart, is less studied. Here, we identified the expression of both canonical and small isoforms of AnkR in the mouse myocardium. We demonstrate that cardiac myocytes primarily express small AnkR (sAnkR), whereas cardiac fibroblasts predominantly express canonical AnkR. As canonical AnkR expression in cardiac fibroblasts is unstudied, we focused on expression and localization in these cells. AnkR is expressed in both the perinuclear and cytoplasmic regions of fibroblasts with considerable overlap with the trans-Golgi network protein 38, TGN38, suggesting a potential role in trafficking. To study the role of AnkR in fibroblasts, we generated mice lacking AnkR in activated fibroblasts (Ank1-ifKO mice). Notably, Ank1-ifKO mice fibroblasts displayed reduced collagen compaction, supportive of a novel role of AnkR in normal fibroblast function. At the whole animal level, in response to a heart failure model, Ank1-ifKO mice displayed an increase in fibrosis and T-wave inversion compared with littermate controls, while preserving cardiac ejection fraction. Collagen type I fibers were decreased in the Ank1-ifKO mice, suggesting a novel function of AnkR in the maturation of collagen fibers. In summary, our findings illustrate the novel expression of AnkR in cardiac fibroblasts and a potential role in cardiac function in response to stress.

The DUF579 proteins GhIRX15s regulate cotton fiber development by interacting with proteins involved in xylan synthesis

Cotton provides the most abundant natural fiber for the textile industry. The mature cotton fiber largely consists of secondary cell walls with the highest proportion of cellulose and a small amount of hemicellulose and lignin. To dissect the roles of hemicellulosic polysaccharides during fiber development, four IRREGULAR XYLEM 15 (IRX15) genes, GhIRX15-1/-2/-3/-4, were functionally characterized in cotton. These genes encode DUF579 domain-containing proteins, which are homologs of AtIRX15 involved in xylan biosynthesis. The four GhIRX15 genes were predominantly expressed during fiber secondary wall thickening, and the encoded proteins were localized to the Golgi apparatus. Each GhIRX15 gene could restore the xylan deficient phenotype in the Arabidopsis irx15irx15l double mutant. Silencing of GhIRX15s in cotton resulted in shorter mature fibers with a thinner cell wall and reduced cellulose content as compared to the wild type. Intriguingly, GhIRX15-2 and GhIRX15-4 formed homodimers and heterodimers. In addition, the GhIRX15s showed physical interaction with glycosyltransferases GhGT43C, GhGT47A and GhGT47B, which are responsible for synthesis of the xylan backbone and reducing end sequence. Moreover, the GhIRX15s can form heterocomplexes with enzymes involved in xylan modification and side chain synthesis, such as GhGUX1/2, GhGXM1/2 and GhTBL1. These findings suggest that GhIRX15s participate in fiber xylan biosynthesis and modulate fiber development via forming large multiprotein complexes.

Influence of melatonin supplementation on tissue response of endodontic sealers in Wistar rats.

This study evaluated the influence of melatonin supplementation on tissue's response of endodontic sealers in Wistar rats. Forty-eight rats received subcutaneous implants of four polyethylene tubes: one empty (control) and three filled with endodontic sealers (AH Plus, Endofill and Sealapex). Half of the animals were supplemented with melatonin (ME) and the remaining treated with water (WA) for 15days before the implantation until euthanasia, forming the groups: control-WA, AH Plus-WA, Endofill-WA, Sealapex-WA, Control-ME, AH Plus-ME, Endofill-ME and Sealapex-ME. After 5, 15 and 30days, (n = 8) tubes were removed and evaluated in H&E., immunohistochemistry, PSR, VK and POL. The results were statistically analyzed (p < 0.05). In animals treated with water, Endofill-WA evoked more intense inflammatory infiltrate compared to AH Plus-WA and Control-WA in a 30-day period (p < 0.05). In animals supplemented with melatonin, there was any difference among endodontic sealers' response in any period of analysis (p > 0.05). Comparing the individual response of each sealer, over a 30-day period, Endofill-ME and Sealapex-ME showed less inflammatory infiltrate compared to Endofill-WA and Sealapex-WA, respectively (p < 0.05). Immunostaining for IL-6 and TNF-α was less intense for all groups in animals supplemented with melatonin, in most periods, except for the Endofill sealer (p < 0.05). Furthermore, Endofill-ME at 5days and AH-Plus-ME at 30days showed a higher percentage of mature collagen fibers compared to the Endofill-WA and AH Plus-WA, respectively (p < 0.05). Positive structures for von Kossa staining and birefringent to polarized light were observed only for Sealapex-WA and Sealapex-ME in all periods. It can be concluded that melatonin influences the tissue response to endodontic sealers, modulating the inflammatory and reparative process.

Intermittent cyclic stretch of engineered ligaments drives hierarchical collagen fiber maturation in a dose- and organizational-dependent manner

Hierarchical collagen fibers are the primary source of strength in tendons and ligaments; however, these fibers largely do not regenerate after injury or with repair, resulting in limited treatment options. We previously developed a static culture system that guides ACL fibroblasts to produce native-sized fibers and early fascicles by 6 weeks. These constructs are promising ligament replacements, but further maturation is needed. Mechanical cues are critical for development in vivo and in engineered tissues; however, the effect on larger fiber and fascicle formation is largely unknown. Our objective was to investigate whether intermittent cyclic stretch, mimicking rapid muscle activity, drives further maturation in our system to create stronger engineered replacements and to explore whether cyclic loading has differential effects on cells at different degrees of collagen organization to better inform engineered tissue maturation protocols. Constructs were loaded with an established intermittent cyclic loading regime at 5 or 10 % strain for up to 6 weeks and compared to static controls. Cyclic loading drove cells to increase hierarchical collagen organization, collagen crimp, and tissue tensile properties, ultimately producing constructs that matched or exceeded immature ACL properties. Further, the effect of loading on cells varied depending on degree of organization. Specifically, 10 % load drove early improvements in tensile properties and composition, while 5 % load was more beneficial later in culture, suggesting a shift in mechanotransduction. This study provides new insight into how cyclic loading affects cell-driven hierarchical fiber formation and maturation, which will help to develop better rehabilitation protocols and engineer stronger replacements. Statement of significanceCollagen fibers are the primary source of strength and function in tendons and ligaments throughout the body. These fibers have limited regenerate after injury, with repair, and in engineered replacements, reducing treatment options. Cyclic load has been shown to improve fibril level alignment, but its effect at the larger fiber and fascicle length-scale is largely unknown. Here, we demonstrate intermittent cyclic loading increases cell-driven hierarchical fiber formation and tissue mechanics, producing engineered replacements with similar organization and mechanics as immature ACLs. This study provides new insight into how cyclic loading affects cell-driven fiber maturation. A better understanding of how mechanical cues regulate fiber formation will help to develop better engineered replacements and rehabilitation protocols to drive repair after injury.

Clinicopathological and molecular genetic features of neuromuscular choristoma-associated desmoid type fibromatosis

Objective: To investigate the clinicopathological and genetic characteristics of neuromuscular choristoma-associated desmoid type fibromatosis (NMC-DF). Methods: The clinical morphological and immunohistochemical features of 7 NMC-DF cases diagnosed from January 2013 to January 2023 in Beijing Jishuitan Hospital were retrospectively analyzed. A series of neuromuscular choristoma and neuromuscular choristoma-associated desmoid type fibromatosis were evaluated for CTNNB1 mutations, and hotspot mutations for CTNNB1 were tested in 4 NMC-DF cases using Sanger sequencing. Results: The tumors were collected from 3 females and 4 males, aged 1 to 22 years (mean 7.1 years), involving the sciatic nerve (n=4), brachial plexus (n=2) or multiple nerves (n=1). The course of the disease spanned from 3 months to 10 years. Two cases were recurrent tumors. All the 7 NMC cases showed endoneurial intercalation of mature skeletal muscle fibers among the peripheral nerve fascicles, and the histologic features of the NMC-DF were strikingly similar to the conventional desmoid-type fibromatosis. By immunohistochemistry, all NMC and NMC-DF cases showed aberrant nuclear staining of β-catenin (7/7), the muscle cells in NMC were intensely immunoreactive for desmin, and the admixed nerve fibers were highlighted by NF and S-100 (7/7). Four NMC and NMC-DF had CTNNB1 mutations, 3 c.121A>G (p.T41A) and 1 c.134C>T (p.S45F). Follow-up of the 7 cases, ranging from 22 to 78 months, showed tumor recurrence in 2 patients at 3 and 8 months respectively after the first surgical resection, of which 1 patient underwent above-knee amputation. No recurrence occurred in other cases with tumor excision and neurological reconstruction surgery. There was no metastasis occurred in the 7 cases. Conclusions: NMC is a rare congenital lesion with differentiated mature skeletal muscle tissue found in peripheral nerve fascicles, and approximately 80% of patients with NMC develop a soft tissue fibromatosis. CTNNB1 mutation in the Wnt signaling pathway may be involved in the pathogenesis of NMC and NMC-DF, and S45F mutations seems to have a higher risk of disease progression.

P-380 Improvement of follicle growth and health during dynamic culture of bovine ovarian cortical tissue (BOCT) is associated to a higher remodeling of the extracellular matrix

Abstract Study question Is improvement of follicle growth and health during dynamic culture in Perifusion Bioreactor (PB) associated to a healthier condition and remodeling of extracellular matrix? Summary answer The dynamic culture in PB allows the extracellular matrix (ECM) network rearrangement improving follicle growth and viability compared to static culture What is known already Over the past two decades, in vitro folliculogenesis has achieved moderate success in large mammals, emerging as a distinctive and reliable method for obtaining secondary follicles. Recently, although dynamic culture in bioreactors has been demonstrated to enhance follicle progression, quality, and viability compared to static culture, less emphasis has been devoted to ECM remodeling. Ovarian tissue ECM plays a crucial role in cell–cell, and cell-ECM interactions involved in follicle activation and growth. The comprehension of ECM function deepens our understanding of follicular development and holds significant promise for future reproductive technologies Study design, size, duration Bovine ovaries (age 8-24 months) were collected at slaughterhouse. BOCT strips (1x1x0.5mm) from the same ovary were cultured in groups of 10 for 14 days in PB (dynamic culture) and conventional dishes (CD, static culture). At the end of culture, collagen thickness and packing density was assessed through PicroSirius red (PSR) staining, follicle stages and quality through histology, and follicle viability through live-dead far-red and propidium iodide labeling under confocal microscopy Participants/materials, setting, methods Neosynthesis and remodeling of collagen fibers was analyzed on PSR-stained samples under polarized light measuring the color of each pixel. A color threshold was set to isolate the four colors seen in PSR-stained samples under polarized light: green, thin fibers (neosynthesized); yellow, mid-sized fibers (low assembly); orange, mid-sized fibers (medium assembly); red, mature thick fibers (high assembly). The color threshold was set as follows: red 2–9, yellow 39-51, orange 10–38, green 52–128 Main results and the role of chance In fresh tissue, analysis of PSR-stained samples showed the following values: Red 1.35; Orange 4.93; Yellow 0.18; Green 0.15. At Day 14, compared to the static culture, culture in PB, exhibits a significantly higher production of green (PB 0.22 vs CD 0.02 P &amp;lt; 0.01) and yellow (PB 0.56 vs CD 0.26 P &amp;lt; 0.05) fibers indicating an improved neosynthesis and remodeling of collagen and ECM health condition. Such higher ECM remodeling after dynamic culture is associated to a significantly higher percentage of secondary follicles (PB 15.4– CD 4.1%, P &amp;lt; 0.01 ), superior follicle quality (grade I/II, PB 70,5 - CD 28.4%, P &amp;lt; 0.01) and viability (PB 70 – CD 41.1%, P &amp;lt; 0.01). PSR analysis suggests that the continuous nutrients and oxygen supply, catabolites removal, and biomechanical stimulation during dynamic PB culture, stimulates the deposition of newly synthesized collagen which in turn, promotes follicle growth Limitations, reasons for caution Although the bovine is a suitable reproductive model for human, such findings should be replicated on human ovarian tissue Wider implications of the findings The aim of ovarian tissue culture is to promote the growth of healthy secondary follicles that can be isolated for further culture to generate MII oocytes. The present findings suggest that adoption of dynamic ovarian tissue culture could enhance the outcome of folliculogenesis in vitro Trial registration number not applicable

Exploring the plasmodesmata callose-binding protein gene family in upland cotton: unraveling insights for enhancing fiber length.

Plasmodesmata are transmembrane channels embedded within the cell wall that can facilitate the intercellular communication in plants. Plasmodesmata callose-binding (PDCB) protein that associates with the plasmodesmata contributes to cell wall extension. Given that the elongation of cotton fiber cells correlates with the dynamics of the cell wall, this protein can be related to the cotton fiber elongation. This study sought to identify PDCB family members within the Gossypium. hirsutum genome and to elucidate their expression profiles. A total of 45 distinct family members were observed through the identification and screening processes. The analysis of their physicochemical properties revealed the similarity in the amino acid composition and molecular weight across most members. The phylogenetic analysis facilitated the construction of an evolutionary tree, categorizing these members into five groups mainly distributed on 20 chromosomes. The fine mapping results facilitated a tissue-specific examination of group V, revealing that the expression level of GhPDCB9 peaked five days after flowering. The VIGS experiments resulted in a marked decrease in the gene expression level and a significant reduction in the mature fiber length, averaging a shortening of 1.43-4.77 mm. The results indicated that GhPDCB9 played a pivotal role in the cotton fiber development and served as a candidate for enhancing cotton yield.

GhATL68b regulates cotton fiber cell development by ubiquitinating the enzyme required for β-oxidation of polyunsaturated fatty acids

E3 ligases are key enzymes required for protein degradation. Here, we identified a C3H2C3 RING domain-containing E3 ubiquitin ligase gene named GhATL68b. It is preferentially and highly expressed in developing cotton fiber cells and shows greater conservation in plants than in animals or archaea. The four orthologous copies of this gene in various diploid cottons and eight in the allotetraploid G. hirsutum were found to have originated from a single common ancestor that can be traced back to Chlamydomonas reinhardtii at about 992 million years ago. Structural variations in the GhATL68b promoter regions of G. hirsutum, G. herbaceum, G. arboreum, and G. raimondii are correlated with significantly different methylation patterns. Homozygous CRISPR-Cas9 knockout cotton lines exhibit significant reductions in fiber quality traits, including upper-half mean length, elongation at break, uniformity, and mature fiber weight. In vitro ubiquitination and cell-free protein degradation assays revealed that GhATL68b modulates the homeostasis of 2,4-dienoyl-CoA reductase, a rate-limiting enzyme for the β-oxidation of polyunsaturated fatty acids (PUFAs), via the ubiquitin proteasome pathway. Fiber cells harvested from these knockout mutants contain significantly lower levels of PUFAs important for production of glycerophospholipids and regulation of plasma membrane fluidity. The fiber growth defects of the mutant can be fully rescued by the addition of linolenic acid (C18:3), the most abundant type of PUFA, to the ovule culture medium. This experimentally characterized C3H2C3 type E3 ubiquitin ligase involved in regulating fiber cell elongation may provide us with a new genetic target for improved cotton lint production.

Improving Irrigation Management of Cotton with Small Unmanned Aerial Vehicle (UAV) in Texas High Plains

The rapid decline in water availability for irrigation on the Texas High Plains (THP) is a significant problem affecting crop production and the viability of a large regional economy worth approximately USD 7 billion annually. This region is the largest continuous cotton-producing area in the United States, and the timely delivery and efficient use of irrigation water are critical to the sustainability and profitability of cotton production in this region. Current irrigation scheduling must be improved to reduce water consumption without compromising crop production. Presently, irrigation scheduling based on reference evapotranspiration (ETo) is limited due to the lack of reliable and readily available in-field weather data and updated crop coefficients. Additionally, in-field variability in crop water demand is often overlooked, leading to lower irrigation efficiency. To address these challenges, we explored the potential use of an unmanned aerial vehicle (UAV)-based crop monitoring system to support irrigation management decisions. This study was conducted in Lubbock, Texas, in 2022, where high temporal and spatial resolution images were acquired using a UAV from a cotton field experiment with four irrigation levels. Soil moisture and canopy temperature sensors were deployed to monitor crop response to irrigation and rainfall. The results indicated a significant effect of water stress on crop growth (revealed by UAV-based canopy cover (CC) measurements), yield, and fiber quality. Strong correlations between multi-temporal CC and lint yield (R2 = 0.68 to 0.88) emphasized a clear trend: rainfed treatments with lower yields exhibited reduced CC, while irrigated plots with higher CC displayed increased yields. Furthermore, irrigated plots produced more mature and uniform fibers. This study also explored various evapotranspiration calculation approaches indicating that site-specific CC measurements obtained from a UAV could significantly reduce irrigation application. A regression model linking evapotranspiration to canopy cover demonstrated promising potential for estimating water demand in crops with an R2 as high as 0.68. The findings highlight the efficacy of UAV-based canopy features in assessing drought effects and managing irrigation water in water-limited production regions like the THP.