Rescuing error control in crosslinking mass spectrometry.

Crosslinking mass spectrometry is a powerful tool to study protein-protein interactions under native or near-native conditions in complex mixtures. Through novel search controls, we show how biassing results towards likely correct proteins can subtly undermine error estimation of crosslinks, with significant consequences. Without adjustments to address this issue, we have misidentified an average of 260 interspecies protein-protein interactions across 16 analyses in which we synthetically mixed data of different species, misleadingly suggesting profound biological connections that do not exist. We also demonstrate how data analysis procedures can be tested and refined to restore the integrity of the decoy-false positive relationship, a crucial element for reliably identifying protein-protein interactions.

Loss of the long form of Plod2 phenocopies contractures of Bruck syndrome - osteogenesis imperfecta.

Bruck syndrome is an autosomal recessive form of osteogenesis imperfecta (OI) caused by biallelic variants in PLOD2 or FKBP10 and is characterized by joint contractures, bone fragility, short stature, and scoliosis. PLOD2 encodes LH2, which hydroxylates type I collagen telopeptide lysines, a critical step for collagen crosslinking. The Plod2 global knockout mouse model is limited by early embryonic lethality, thus the role of PLOD2 in skeletogenesis is not well understood. We generated a novel Plod2 mouse line modeling a variant identified in two unrelated individuals with Bruck syndrome: PLOD2 c.1559dupC, predicting a frameshift and loss of the long isoform LH2b. In the mouse, the duplication led to loss of LH2b mRNA as well as significantly reduced total LH2 protein. This model, Plod2fs/fs, survived up to E18.5 although in non-Mendelian genotype frequencies. The homozygous frameshift model recapitulated the joint contractures seen in Bruck syndrome and had indications of absent type I collagen telopeptide lysine hydroxylation in bone. Genetically labeling tendons with Scleraxis-GFP in Plod2fs/fs mice revealed the loss of extensor tendons in the forelimb by E18.5 and developmental studies showed extensor tendons developed through E14.5 but were absent starting at E16.5. Second harmonic generation showed abnormal tendon type I collagen fiber organization, suggesting structurally abnormal tendons. Characterization of the skeleton by µCT and Raman spectroscopy showed normal bone mineralization levels. This work highlights the importance of properly crosslinked type I collagen in tendon and bone, providing a promising new mouse model to further our understanding of Bruck syndrome.

Bruck syndrome is a rare disease where individuals have brittle bone as well as contracted or stiff joints. Mutations in two genes are associated with Bruck syndrome and, in this work, we focus on PLOD2. Mice without Plod2 die at an early embryonic stage, before they have a chance to fully develop. In this work, we created a mouse with a PLOD2 mutation seen in people with Bruck syndrome. Some of these new Bruck syndrome model mice survived to a later gestational age, but all died at birth. The Bruck syndrome mice were small and had contracted joints. We found they were missing tendons in their arms and had structurally abnormal tendons in their knees. Bone mineralization was normal, but there were indications that the modifications needed for normal type I collagen structure were absent. Overall, this is an advantageous new mouse model of Bruck syndrome that can be used to study this rare disease and highlights the importance of Plod2 in tendon.

Structurally decoupled hyaluronic acid hydrogels for studying matrix metalloproteinase-mediated invasion of metastatic breast cancer cells.

In recent years, polymeric hydrogels have been employed to investigate cancer cell-extracellular matrix (ECM) interactions in vitro. In the context of breast cancer, cancer cells are known to degrade the ECM using matrix-metalloproteinases (MMPs) to support invasion resulting in disease progression. Polymeric hydrogels incorporating MMP-cleavable peptides have been employed to study cancer cell invasion, however, the approaches employed to incorporate these peptides often change other hydrogel properties. This underscores the need for decoupling hydrogel properties while incorporating MMP-cleavable peptides. Herein, we report structurally decoupled hyaluronic acid (HA) hydrogels formulated using varying ratios of a biologically sensitive MMP-cleavable peptide and an insensitive counterpart (Dithiothreitol (DTT) or polyethylene glycol dithiol (PEGDT)) to study MMP-mediated metastatic breast cancer cell invasion. Rheological, swelling ratio, estimated mesh size, and permeability measurements showed similar mechanical and physical properties for hydrogels crosslinked with different DTT (or PEGDT)/MMP ratios. However, their degradation rate in the presence of collagenase correlated with the ratio of MMP-cleavable peptide. Encapsulated metastatic breast cancer spheroids in HA hydrogels with MMP sensitivity exhibited increased invasiveness compared to those without MMP sensitivity after 14 days of culture. Overall, such structurally decoupled HA hydrogels provide a platform to study MMP-mediated breast cancer cell invasion in vitro.

Quantitative characterization of the crosslinking degree of hydroxypropyl guar gum fracturing fluid by low-field NMR.

As a widely used water-based fracturing fluid, the performance of hydroxypropyl guar gum fracturing fluid is closely related to the degree of crosslinking, the quantitative characterization of which can reveal a detailed crosslinking mechanism and guide the preparation of fracturing fluid gels with an excellent performance. However, the commonly used high-temperature rheology method for evaluating the performance of fracturing fluids only qualitatively reflects the degree of crosslinking. In this study, low-field nuclear magnetic resonance (LF-NMR) was used to characterize the degree of crosslinking in guar gum fracturing fluid gels. The spin-spin relaxation time of the H proton in guar gum was molecularly analyzed using LF-NMR. The viscoelastic properties met the requirements when the crosslinking degree of the gel was 88-94 %. The transformation of the linear structure into a membrane structure during the crosslinking process of the guar gum fracturing fluid was confirmed by freeze-drying and scanning electron microscopy (SEM) from a microscopic perspective. The changing trend of the microstructure and viscoelastic properties of the fracturing fluid gel under different crosslinker dosages was consistent with changes in the degree of crosslinking. The LF-NMR test process is non-destructive to the gel structure, and the test results demonstrate good accuracy and repeatability.

Rapid and Efficient Isolation of Total RNA-Bound Proteomes by Liquid Emulsion-Assisted Purification of RNA-Bound Protein (LEAP-RBP).

The critical roles of RNA-binding proteins (RBPs) in all aspects of RNA biology fostered the development of methods utilizing ultraviolet (UV) crosslinking and method-specific RNA enrichment steps for proteome-wide identification and assessment of RBP function. Despite the substantial contributions of these UV-based RNA-centric methods to our understanding of RNA-protein interaction networks, their utility is constrained by biases in RBP recovery and significant noise contributions, which can confound meaningful interpretation. To overcome these issues, we recently developed a method termed Liquid Emulsion-Assisted Purification of RNA-Bound Protein (LEAP-RBP) and introduced quantitative signal-to-noise (S:N)-based metrics for the proteome-wide identification of RNA interactomes and accurate assessment of global RBP occupancy dynamics. Compared to existing methodologies, LEAP-RBP provides significant advantages in speed, cost, efficiency, and selectivity for RNA-bound proteins. In this work, we provide a step-by-step guide for the successful application of the LEAP-RBP method for both small- and large-scale investigations of RNA-bound proteomes. Key features • Unbiased and efficient isolation of total RNA-bound protein, RNA, and protein from biological samples. • Cost-effective identification of proteome-wide RNA interactomes and validation of direct RNA-binding protein functionality. • Robust and accurate assessment of context- and/or condition-dependent RBP occupancy state dynamics.

Protein-Protein Interface Identification by Site-Specific Photo-Cross-linking/Cleavage in Mammalian Cells.

Identification of protein-protein interfaces is necessary for understanding and regulating biological events. Genetic code expansion enables site-specific photo-cross-linking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the cross-linked region still remains challenging. Our new protocol enables its identification by pre-installing a site-specific cleavage site, an α-hydroxy acid (Nε-allyloxycarbonyl-α-hydroxyl-L-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located on within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. This combination of site-specific crosslinking and cleavage promises to be useful for revealing binding interfaces and protein complex geometries. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Search for crosslinkable sites Basic Protocol 2: Site-specific photo-cross-linking/cleavage.

Bone turnover markers in the preoperative assessment of bone quality - A prospective investigation of bone microstructure and advanced glycation endproducts in lumbar fusion patients.

INTRODUCTION: Effective tools to evaluate bone quality preoperatively are scarce and the standard method to determine bone quality requires an invasive biopsy. A non-invasive, and preoperatively available method for bone quality assessment would be of clinical value. The purpose of this study is to investigate the associations of bone formation marker, serum bone alkaline phosphatase (BAP), and bone resorption marker, urine collagen cross-linked N-telopeptide (uNTX) to volumetric bone mineral density (vBMD), fluorescent advanced glycation endproducts (fAGEs) and bone microstructure. MATERIALS AND METHODS: A cross-secional analysis using prospective data of patients undergoing lumbar spinal fusion was performed. BAP and uNTX were preoperatively collected. Quantitative computed tomography (QCT) was performed at the lumbar spine (vBMD ≤ 120 mg/cm3 osteopenic/osteoporotic). Bone biopsies from the posterior superior iliac spine were obtained and evaluated with multiphoton fluorescence microscopy for fAGEs and microcomputed tomography (µCT) for bone microarchitecture. Correlations between BAP/uNTX to vBMD, fAGEs and µCT parameters were assessed with Spearman's ρ. Receiver operating characteristic (ROC) analysis evaluated BAP and uNTX as predictors for osteopenia/osteoporosis. Multivariable linear regression models adjusting for age, sex, BMI, race and diabetes mellitus determined associations between BAP/uNTX and fAGEs. RESULTS: 127 prospectively enrolled patients (50.4% female, 62.5 years, BMI 28.7 kg/m2) were analyzed. uNTX (ρ=-0.331,p < 0.005) and BAP (ρ=-0.245,p < 0.025) decreased with cortical fAGEs, and uNTX (ρ=-0.380,p < 0.001) decreased with trabecular fAGEs. BAP and uNTX revealed no significant correlation with vBMD. ROC analysis for BAP and uNTX discriminated osteopenia/osteoporosis with AUC of 0.477 and 0.561, respectively. In the multivariable analysis, uNTX decreased with increasing trabecular fAGEs after adjusting for covariates (ß = 0.923;p = 0.031). CONCLUSION: This study demonstrated an inverse association of bone turnover markers and fAGEs. Both uNTX and BAP could not predict osteopenia/osteoporosis in the spine. uNTX reflects collagen characteristics and might have a complementary role to vBMD, as a non-invasive tool for bone quality assessment in spine surgery.

The Structural Logic of Dynamic Signaling in the Escherichia coli Serine Chemoreceptor.

The experimental challenges posed by integral membrane proteins hinder molecular understanding of transmembrane signaling mechanisms. Here, we exploited protein crosslinking assays in living cells to follow conformational and dynamic stimulus signals in Tsr, the Escherichia coli serine chemoreceptor. Tsr mediates serine chemotaxis by integrating transmembrane serine-binding inputs with adaptational modifications of a methylation helix bundle to regulate a signaling kinase at the cytoplasmic tip of the receptor molecule. We created a series of cysteine replacements at Tsr residues adjacent to hydrophobic packing faces of the bundle helices and crosslinked them with a cell-permeable, bifunctional thiol-reagent. We identified an extensively crosslinked dynamic junction midway through the methylation helix bundle that seemed uniquely poised to respond to serine signals. We explored its role in mediating signaling shifts between different packing arrangements of the bundle helices by measuring crosslinking in receptor molecules with apposed pairs of cysteine reporters in each subunit and assessing their signaling behaviors with an in vivo kinase assay. In the absence of serine, the bundle helices evinced compact kinase-ON packing arrangements; in the presence of serine, the dynamic junction destabilized adjacent bundle segments and shifted the bundle to an expanded, less stable kinase-OFF helix-packing arrangement. An AlphaFold 3 model of kinase-active Tsr showed a prominent bulge and kink at the dynamic junction that might antagonize stable structure at the receptor tip. Serine stimuli probably inhibit kinase activity by shifting the bundle to a less stably-packed conformation that relaxes structural strain at the receptor tip, thereby freezing kinase activity.

Could Automated Objective Measurements Acquired at the Preoperative Stage Estimate the Corrected Distance Visual Acuity after Corneal Cross-Linking in Keratoconus?

INTRODUCTION: Objective markers describing corneal optical density (COD), thinnest corneal thickness (TCT), and anterior (ARC) and posterior (PRC) surface radii over the 3 mm thinnest region of the cornea were investigated to provide a model for estimating corrected distance visual acuity (CDVA) after corneal cross-linking (CXL) in keratoconus. METHODS: CDVA, COD, TCT, ARC, and PRC were monitored (using Pentacam™) over 1 year in patients with (1) keratoconus treated with routine CXL (2) relatively stable untreated keratoconus, and (3) age/gender-matched controls. RESULTS: In group 1 (n = 77), the median logMAR CDVA (mode, interquartile range) improved significantly (p < 0.01) from 0.26 (0.22, 0.12-0.65) to 0.07 (0.00, 0.02-0.21). The mean (± standard deviation, 95% confidence interval) COD (in 0-100 grey scale units) in the 0-2 mm central anterior corneal region (0-2 ant), TCT (µm), ARC (mm), and PRC (mm) changed significantly (p < 0.01), from 21.2 (± 3.70, 20.4-22.0), 454 (± 40.0, 446-462), 6.49 (± 0.71, 6.33-6.65), and 4.81 (± 0.65, 4.66-4.96) to 31.5 (± 9.19, 29.5-33.6), 423 (± 49.3, 412-434), 6.78 (± 0.80, 6.60-6.98), and 4.74 (± 0.64, 4.59-4.88), respectively, but remained stable in groups 2 (n = 23) and 3 (n = 24). Significant relationships (p < 0.01) were uncovered between postop CDVA and preop values of COD, TCT, ARC, and PRC. Multilinear regression revealed significant correlations between CDVA at 1 year and preop COD, TCT, ARC, and PRC (r2 = 0.533, r20-2ant = 0.126, r2TCT = 0.321, r2ARC = 0.506, r2PRC = 0.467). Including preop CDVA further enhanced this correlation (r2 = 0.637, r2LogMAR CDVApreop = 0.566). CONCLUSION: CXL improved CDVA, increased COD and ARC, and reduced TCT and PRC. The chance of correctly estimating the CDVA at 1 year after CXL using preoperative markers of COD, TCT, ARC, and PRC is 53%, improving to 64% with the inclusion of preoperative CDVA. Objective measurements taken at the preoperative screening stage may be useful to estimate the likely postoperative CDVA when preoperative CDVA measures are unreliable or unobtainable. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT06522789.

Chitosan Extracted from the Biomass of Tenebrio molitor Larvae as a Sustainable Packaging Film.

Waste from non-degradable packaging materials poses a serious environmental risk and has led to interest in developing sustainable bio-based packaging materials. Sustainable packaging materials have been made from diverse naturally derived materials such as bamboo, sugarcane, and corn starch. In this study, we made a sustainable packaging film using chitosan extracted from the biomass of yellow mealworm (Tenebrio molitor) shell waste. The extracted chitosan was used to create films, cross-linked with citric acid (CA) and with the addition of glycerol to impart flexibility, using the solvent casting method. The successful cross-linking was evaluated using Fourier-Transform Infrared (FTIR) analysis. The CA cross-linked mealworm chitosan (CAMC) films exhibited improved water resistance with moisture content reduced from 19.9 to 14.5%. Improved barrier properties were also noted, with a 28.7% and 10.2% decrease in vapor permeability and vapor transmission rate, respectively. Bananas were selected for food preservation, and significant changes were observed over a duration of 10 days. Compared to the control sample, bananas packaged in CAMC pouches exhibited a lesser loss in weight because of excellent barrier properties against water vapor. Moreover, the quality and texture of bananas packaged in CAMC pouch remained intact over the duration of the experiment. This indicates that adding citric acid and glycerol to the chitosan structure holds promise for effective food wrapping and contributes to the enhancement of banana shelf life. Through this study, we concluded that chitosan film derived from mealworm biomass has potential as a valuable resource for sustainable packaging solutions, promoting the adoption of environmentally friendly practices in the food industry.

Comparative Study of the Foaming Behavior of Ethylene-Vinyl Acetate Copolymer Foams Fabricated Using Chemical and Physical Foaming Processes.

Ethylene-vinyl acetate copolymer (EVA), a crucial elastomeric resin, finds extensive application in the footwear industry. Conventional chemical foaming agents, including azodicarbonamide and 4,4'-oxybis(benzenesulfonyl hydrazide), have been identified as environmentally problematic. Hence, this study explores the potential of physical foaming of EVA using supercritical nitrogen as a sustainable alternative, garnering considerable interest in both academia and industry. The EVA formulations and processing parameters were optimized and EVA foams with densities between 0.15 and 0.25 g/cm3 were produced. Key findings demonstrate that physical foaming not only reduces environmental impact but also enhances product quality by a uniform cell structure with small cell size (50-100 µm), a wide foaming temperature window (120-180 °C), and lower energy consumption. The research further elucidates the mechanisms of cell nucleation and growth within the crosslinked EVA network, highlighting the critical role of blowing agent dispersion and localized crosslinking around nucleated cells in defining the foam's cellular morphology. These findings offer valuable insights for producing EVA foams with a more controllable cellular structure, utilizing physical foaming techniques.

Revealing the Impact of Viscoelastic Characteristics on Performance Parameters of UV-Crosslinked Hotmelt Pressure-Sensitive Adhesives: Insights from Time-Temperature Superposition Analysis.

This study emphasizes the influential role of rheology in decoding the viscoelastic properties of pressure-sensitive adhesives (PSAs) vital to predicting key application features such as shear, tack, and peel, depending on the flow characteristics of PSAs during bonding and debonding processes. By applying the principle of time-temperature superposition (TTS), we extend the scope of our frequency analysis, surpassing the technical constraints of the available apparatus. Our exploration aims to uncover the general correlations between PSAs' viscoelastic properties and their performance in end-use applications. Initially, the adhesive performance and viscoelastic properties of a UV-crosslinkable styrene-butadiene-styrene (SBS) model adhesive prior and subsequent to UV irradiation were examined. The subsequent crosslinking reaction increased cohesive strength and heat resistance, although tack and peel strength observed a substantial decline. We successfully demonstrated these effects by logging the viscoelastic properties, specifically the storage modulus G' at lower frequencies, which mirrors the shear strength at higher temperatures and the shift in the tan δ peak to represent each PSA's tack. These correlations were partially reflected in three commercial UV crosslinkable acrylic PSA products, although the effect of UV irradiation was less distinctive. This study also revealed the challenges in predicting tack and peel strength, which result from a complex interplay of bonding and debonding processes. Our findings reinforce the necessity for more sophisticated analysis techniques and models that can accurately predict the end-use performance of PSAs across different physical structures and chemical compositions. Further research is needed to develop these predictive models, which may reduce the need for labor-intensive testing under real-life conditions.

Crosslinking and Swelling Properties of pH-Responsive Poly(Ethylene Glycol)/Poly(Acrylic Acid) Interpenetrating Polymer Network Hydrogels.

This study investigates the crosslinking dynamics and swelling properties of pH-responsive poly(ethylene glycol) (PEG)/poly(acrylic acid) (PAA) interpenetrating polymer network (IPN) hydrogels. These hydrogels feature denser crosslinked networks compared to PEG single network (SN) hydrogels. Fabrication involved a two-step UV curing process: First, forming PEG-SN hydrogels using poly(ethylene glycol) diacrylate (PEGDA) through UV-induced free radical polymerization and crosslinking reactions, then immersing them in PAA solutions with two different molar ratios of acrylic acid (AA) monomer and poly(ethylene glycol) dimethacrylate (PEGDMA) crosslinker. A subsequent UV curing step created PAA networks within the pre-fabricated PEG hydrogels. The incorporation of AA with ionizable functional groups imparted pH sensitivity to the hydrogels, allowing the swelling ratio to respond to environmental pH changes. Rheological analysis showed that PEG/PAA IPN hydrogels had a higher storage modulus (G') than PEG-SN hydrogels, with PEG/PAA-IPN5 exhibiting the highest modulus. Thermal analysis via thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) indicated increased thermal stability for PEG/PAA-IPN5 compared to PEG/PAA-IPN1, due to higher crosslinking density from increased PEGDMA content. Consistent with the storage modulus trend, PEG/PAA-IPN hydrogels demonstrated superior mechanical properties compared to PEG-SN hydrogels. The tighter network structure led to reduced water uptake and a higher gel modulus in swollen IPN hydrogels, attributed to the increased density of active network strands. Below the pKa (4.3) of acrylic acid, hydrogen bonds between PEG and PAA chains caused the IPN hydrogels to contract. Above the pKa, ionization of PAA chains induced electrostatic repulsion and osmotic forces, increasing water absorption. Adjusting the crosslinking density of the PAA network enabled fine-tuning of the IPN hydrogels' properties, allowing comprehensive comparison of single network and IPN characteristics.

Nanogels: Recent Advances in Synthesis and Biomedical Applications.

In the context of advanced nanomaterials research, nanogels (NGs) have recently gained broad attention for their versatility and promising biomedical applications. To date, a significant number of NGs have been developed to meet the growing demands in various fields of biomedical research. Summarizing preparation methods, physicochemical and biological properties, and recent applications of NGs may be useful to help explore new directions for their development. This article presents a comprehensive overview of the latest NG synthesis methodologies, highlighting advances in formulation with different types of hydrophilic or amphiphilic polymers. It also underlines recent biomedical applications of NGs in drug delivery and imaging, with a short section dedicated to biosafety considerations of these innovative nanomaterials. In conclusion, this article summarizes recent innovations in NG synthesis and their numerous applications, highlighting their considerable potential in the biomedical field.

Tumor-Suppressive Cross-Linking of Anti-T. cruzi Antibodies in Acute Lymphoblastic Leukemia.

Parasites have been associated with possible anticancer activity, including Trypanosoma cruzi, which has been linked to inhibiting the growth of solid tumors. To better understand this antitumor effect, we investigated the association of anti-T. cruzi antibodies with B cells of the acute lymphoblastic leukemia (ALL) SUPB15 cell line. The antibodies were generated in rabbits. IgGs were purified by affinity chromatography. Two procedures (flow cytometry (CF) and Western blot(WB)) were employed to recognize anti-T. cruzi antibodies on SUPB15 cells. We also used CF to determine whether the anti-T. cruzi antibodies could suppress SUPB15 cells. The anti-T. cruzi antibodies recognized 35.5% of the surface antigens of SUPB15. The complement-dependent cytotoxicity (CDC) results demonstrate the cross-suppression of anti-T. cruzi antibodies on up to 8.4% of SUPB15 cells. For the WB analysis, a band at 100 kDa with high intensity was sequenced using mass spectrometry, identifying the protein as nucleolin. This protein may play a role in the antitumor effect on T. cruzi. The anti-T. cruzi antibodies represent promising polyclonal antibodies that have the effect of tumor-suppressive cross-linking on cancer cells, which should be further investigated.

Site-specific photo-crosslinking of Hsc70 with the KFERQ pentapeptide motif in a chaperone-mediated autophagy and microautophagy substrate in mammalian cells.

Heat shock cognate protein 70 (Hsc70/HSPA8) belongs to the Hsp70 family of molecular chaperones. The fundamental functions of Hsp70 family molecular chaperones depend on ATP-dependent allosteric regulation of binding and release of hydrophobic polypeptide substrates. Hsc70 is also involved in various other cellular functions including selective pathways of protein degradation: chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI), in which Hsc70 recruits substrate proteins containing a KFERQ-like pentapeptide motif from the cytosol to lysosomes and late endosomes, respectively. However, whether the interaction between Hsc70 and the pentapeptide motif is direct or mediated by other molecules has remained unknown. In the present study, we introduced a photo-crosslinker near the KFERQ motif in a CMA/eMI model substrate and successfully detected its crosslinking with Hsc70, revealing the direct interaction between Hsc70 and the KFERQ motif for the first time. In addition, we demonstrated that the loss of the Hsc70 ATPase activity by the D10 N mutation appreciably reduced the crosslinking efficiency. Our present results suggested that the ATP allostery of Hsc70 is involved in the direct interaction of Hsc70 with the KFERQ-like pentapeptide.

An enzymatically crosslinked collagen type II/hyaluronic acid hybrid hydrogel: A biomimetic cell delivery system for cartilage tissue engineering.

This study presents new injectable hydrogels based on hyaluronic acid and collagen type II that mimic the polysaccharide-protein structure of natural cartilage. After collagen isolation from chicken sternal cartilage, tyramine-grafted hyaluronic acid and collagen type II (HA-Tyr and COL-II-Tyr) were synthesized. Hybrid hydrogels were prepared with different ratios of HA-Tyr/COL-II-Tyr using horseradish peroxidase and noncytotoxic concentrations of hydrogen peroxide to encapsulate human bone marrow-derived mesenchymal stromal cells (hBM-MSCs). The findings showed that a higher HA-Tyr content resulted in a higher storage modulus and a lower hydrogel shrinkage, resulting in hydrogel swelling. Incorporating COL-II-Tyr into HA-Tyr hydrogels induced a more favorable microenvironment for hBM-MSCs chondrogenic differentiation. Compared to HA-Tyr alone, the hybrid HA-Tyr/COL-II-Tyr hydrogel promoted enhanced chondrocyte adhesion, spreading, proliferation, and upregulation of cartilage-related gene expression. These results highlight the promising potential of injectable HA-Tyr/COL-II-Tyr hybrid hydrogels to deliver cells for cartilage regeneration.

The Formation Mechanism of Carbonized Polymer Dots: Crosslinking-Induced Nucleation and Carbonization.

Carbon dots (CDs), as a kind of zero-dimensional nanomaterials, have been widely synthesized by bottom-up methods from various precursors. However, the formation mechanism is still unclear and controversial, which also brings difficulty to the regulation of structures and properties. Only some tentative formation processes were postulated by analyzing the products obtained at different reaction times and temperatures. Here, the effect of crosslinking on the formation of carbonized polymer dots (CPDs) is explored. Crosslinking-induced nucleation and carbonization (CINC) is proposed as the driving force for the formation of CPDs. Under hydrothermal synthesis, the precursors are initiated to polymerize and crosslink. The crosslinking brings higher hydrophobicity to generate the hydrophilic/hydrophobic microphase separation, which promotes dehydration and carbonization resulting in the formation of CPDs. Based on the principle of CINC, the influence factors of size are also revealed. Moreover, the dissipative particle dynamics (DPD) simulation is employed to support this formation mechanism. This concept of CINC will bring light to the formation process of CPDs, as well as facilitate the regulation of CPDs' size and photoluminescence.

Comparative Evaluation of Anterior Segment Optical Coherence Tomography Findings Following Accelerated Corneal Crosslinking Protocols Using Different Riboflavin Formulations and Soaking Durations.

PURPOSE: To comparatively evaluate the influence of different riboflavin formulations and soaking durations on the anterior segment optical coherence tomography (AS-OCT) findings following accelerated corneal crosslinking (ACXL) at 9 mW/cm2 for in progressive keratoconus. METHODS: In this prospective study, consecutive patients with progressive keratoconus were randomized into 4 groups. Group 1: hydroxypropyl methylcellulose (HPMC)-based riboflavin for 10 min; Group 2: HPMC-based riboflavin for 20 min; Group 3: dextran-based riboflavin (0.1%) for 30 min. Riboflavin soaking was followed by ultraviolet-A irradiation at 9 mW/cm2 for 10 min in all three groups. Group 4 underwent conventional CXL (CCXL) using Dresden protocol. The AS-OCT features of the crosslinked cornea were evaluated at postoperative month 1 and correlated to the clinical outcomes at postoperative month 12. RESULTS: The study enrolled 26 eyes of 26 patients in each group. In groups 1 and 2, the AS-OCT findings were similar (p > .05) and the demarcation lines depth (DLD) were deep as obtained following CCXL. The DLD was significantly shallower in group 3 compared to the other groups (p < .01). There were no between-group differences in regards to the visual, refractive, keratometric, and tomographic outcomes at postoperative month 12. No significant endothelial cell loss or any other clinically significant adverse event was encountered in any patient's eye at 12 months follow-up. CONCLUSION: Although structural variations were noted in the crosslinked cornea, DLDs observed following ACXL (9 mW/cm2) using HPMC-based solution for 10 or 20 min were similar to those observed following CCXL. Whereas, ACXL (9 mW/cm2) using dextran-based solution for 30 min resulted in the shallowest DLD. Despite these remodeling differences, the visual, refractive and tomographic outcomes of all groups were comparable at postoperative 1-year follow-up. Studies with a greater number of patients and longer follow-ups are required to establish any relation between AS-OCT characteristics of crosslinked cornea and ACXL efficacy.

Coupling enterotoxigenic Escherichia coli heat-stable peptide toxin with 8-arm PEG enhances immunogenicity.

Enterotoxigenic Escherichia coli (ETEC) strains, which produce the heat-stable enterotoxin (ST) either alone or in combination with the heat-labile enterotoxin, contribute to the bulk of the burden of child diarrheal disease in resource-limited countries and are associated with mortality. Developing an effective vaccine targeting ST presents challenges due to its potent enterotoxicity, non-immunogenicity, and the risk of autoimmune reaction stemming from its structural similarity to the human endogenous ligands, guanylin, and uroguanylin. This study aimed to assess a novel synthetic vaccine carrier platform employing a single chemical coupling step for making human ST (STh) immunogenic. Specifically, the method involved cross-linking STh to an 8-arm N-hydroxysuccinimide (NHS) ester-activated PEG cross-linker. A conjugate of STh with 8-arm structure was prepared, and its formation was confirmed through immunoblotting analysis. The impact of conjugation on STh epitopes was assessed using ELISAs with polyclonal and monoclonal antibodies targeting various epitopes of STh. Immunization of mice with the conjugate induced the production of anti-STh antibodies, exhibiting neutralizing activity against STh.