Functional Scaffolds Research Articles

Catalyst-free synthesis of highly functionalized triazole hexahydroquinoline carbohydrazide scaffolds via four-component cyclocondensation reaction.

A new class of multi-functional triazole hexahydroquinoline carbohydrazide named 2-amino-7,7-dimethyl-5-oxo-4-phenyl-1-(4H-1,2,4-triazol-3-yl)-1,4,5,6,7,8-hexahydroquinoline-3-carbohydrazide has been synthesized by a novel multi-component process involving the reaction of dimedone, 3-amino-1,2,4-triazole, various benzaldehyde with cyanoacetohydrazide under mild conditions in the stoichiometric melt and chloroform in sequence. The simple one-pot process, straight product isolation without applying tedious purification procedures, progression of the reaction without using any catalyst, the application of diverse aldehydes causing a high molecular diversity, the existence of several nitrogen atoms in the product structure, and the possibility of creating multiple hydrogen bonding in the final compound are attractive specifications of the present strategy.

Strontium-doped mesoporous bioactive glass microspheres developed for drug delivering and enhancing the bioactivity of polylactic acid scaffolds

Strontium-doped mesoporous bioactive glass microspheres (Sr-MBGMs) were prepared by sol–gel method for delivering loxoprofen sodium (LX) drug. The loading capacity of the prepared Sr-MBGMs for LX drug was affected by the initial concentration of the drug solution and adsorption time. The LX-loaded Sr-MBGMs obtained under optimum conditions were coated with polydopamine (LX/Sr-MBGMs@PDA) and incorporated into the aminated modified poly-l-lactic acid nanofibrous matrix to develop a functional composite scaffold for bone tissue engineering applications. The in vitro studies revealed that the incorporation of the LX/Sr-MBGMs@PDA could promote cell proliferation and biomineralization, indicating that the as-developed composite scaffolds possessed an improved cell viability and osteogenic ability.

Cryo-EM structure of the Mre11-Rad50-Nbs1 complex reveals the molecular mechanism of scaffolding functions

The DNA double-strand break repair complex Mre11-Rad50-Nbs1 (MRN) detects and nucleolytically processes DNA ends, activates the ATM kinase, and tethers DNA at break sites. How MRN can act both as nuclease and scaffold protein is not well understood. The cryo-EM structure of MRN from Chaetomium thermophilum reveals a 2:2:1 complex with a single Nbs1 wrapping around the autoinhibited Mre11 nuclease dimer. MRN has two DNA-binding modes, one ATP-dependent mode for loading onto DNA ends and one ATP-independent mode through Mre11's C terminus, suggesting how it may interact with DSBs and intact DNA. MRNs two 60-nm-long coiled-coil domains form a linear rod structure, the apex of which is assembled by the two joined zinc-hook motifs. Apices from two MRN complexes can further dimerize, forming 120-nm spanning MRN-MRN structures. Our results illustrate the architecture of MRN and suggest how it mechanistically integrates catalytic and tethering functions.

Development of potent and selective degraders of PI5P4Kγ

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks), a family of three members in mammals (α, β and γ), have emerged as potential therapeutic targets due to their role in regulating many important cellular signaling pathways. In comparison to the PI5P4Kα and PI5P4Kβ, which usually have similar expression profiles across cancer cells, PI5P4Kγ exhibits distinct expression patterns, and pathological functions for PI5P4Kγ have been proposed in the context of cancer and neurodegenerative diseases. PI5P4Kγ has very low kinase activity and has been proposed to inhibit the PI4P5Ks through scaffolding function, providing a rationale for developing a selective PI5P4Kγ degrader. Here, we report the development and characterization of JWZ-1-80, a first-in-class PI5P4Kγ degrader. JWZ-1-80 potently degrades PI5P4Kγ via the ubiquitin-proteasome system and exhibits proteome-wide selectivity and is therefore a useful tool compound for further dissecting the biological functions of PI5P4Kγ.

ATX-101, a cell-penetrating protein targeting PCNA, can be safely administered as intravenous infusion in patients and shows clinical activity in a Phase 1 study

Proliferating Cell Nuclear Antigen (PCNA) is a highly conserved protein essential for DNA replication, repair and scaffold functions in the cytosol. Specific inhibition of PCNA in cancer cells is an attractive anti-cancer strategy. ATX-101 is a first-in-class drug targeting PCNA, primarily in cellular stress regulation. Multiple in vivo and in vitro investigations demonstrated anti-cancer activity of ATX-101 in many tumor types and a potentiating effect on the activity of anti-cancer therapies. Healthy cells were less affected. Based on preclinical data, a clinical phase 1 study was initiated. Twenty-five patients with progressive, late-stage solid tumors were treated with weekly ATX-101 infusions at four dose levels (20, 30, 45, 60 mg/m2). ATX-101 showed a favorable safety profile supporting that vital cellular functions are not compromised in healthy cells. Mild and moderate infusion-related reactions were observed in 64% of patients. ATX-101 was quickly cleared from blood with elimination half-lives of less than 30 min at all dose levels, probably due to both, a quick cell penetration and peptide digestion in serum, as demonstrated in vivo. No tumor responses were observed but stable disease was seen in 70% of the efficacy population (n = 20). Further studies have been initiated to provide evidence of efficacy. Trial registration numbers: ANZCTR 375262 and ANZCTR 375319.

Incorporation of a molybdenum atom in a Rubredoxin-type Centre of a de novo-designed α3DIV-L21C three-helical bundle peptide

The rational design and functionalization of small, simple, and stable peptides scaffolds is an attractive avenue to mimic catalytic metal-centres of complex proteins, relevant for the design of metalloenzymes with environmental, biotechnological and health impacts. The de novo designed α3DIV-L21C framework has a rubredoxin-like metal binding site and was used in this work to incorporate a Mo-atom. Thermostability studies using differential scanning calorimetry showed an increase of 4 °C in the melting temperature of the Mo-α3DIV-L21C when compared to the apo-α3DIV-L21C. Circular dichroism in the visible and far-UV regions corroborated these results showing that Mo incorporation provides stability to the peptide even though there were almost no differences observed in the secondary structure. A formal reduction potential of ∼ −408 mV vs. NHE, pH 7.6 was determined. Combining electrochemical results, EPR and UV–visible data we discuss the oxidation state of the molybdenum centre in Mo-α3DIV-L21C and propose that is mainly in a Mo (VI) oxidation state.

Nitrosothiol-functionalized silicate bioceramic scaffolds with enhanced antibacterial property for bone repair

Bacterial infection is a challenge in the field of bone tissue engineering. Antibiotics are often used to combat infection, but the abuse of antibiotics causes nonselective cytotoxicity and induces bacterial drug resistance. Therefore, it is of great clinical significance to develop bone tissue engineering scaffolds with antibacterial properties for bone repair. In this study, polymer-derived β-Ca2SiO4 (C2S) scaffolds were functionalized with nitric oxide (NO) precursors to repair infected bone defects due to their synergistic antibacterial properties by combining a photothermal effect and NO gas therapy. The results showed that the functionalization of C2S scaffolds with NO precursors not only maintained the initial macropore structure and photothermal effect of the scaffolds, but also endowed the scaffolds with sustained NO release ability. Importantly, the NO precursor-functionalized C2S (C2S–SNO) scaffolds improved the proliferation and alkaline phosphatase (ALP) activity of bone marrow mesenchymal stem cells (rBMSCs), as well as the expression of related osteogenic genes. Furthermore, the photothermal effect of the C2S–SNO scaffolds and the NO release induced by near-infrared (NIR) laser irradiation contributed to enhanced antibacterial efficacy, and the inhibition efficiencies of the C2S–SNO scaffolds against Staphylococcus aureus and Escherichia coli were estimated to be approximately 83.3% and 87.5%, respectively. Therefore, the C2S–SNO scaffolds are promising for repairing infected bone defects.

High expression of GSKIP is associated with poor prognosis in meningioma

Meningiomas are the most common extra-axial primary central nervous system tumors. There is no effective treatment or targeted therapy for meningioma except excision and radiotherapy. glycogen synthesis kinase 3β interaction protein (GSKIP) is an A-kinase anchor protein that has cytosolic scaffolding function and binds to a protein kinase A and glycogen synthesis kinase 3β to modulate different biological processes and malignant tumorigenesis through the Wnt pathway. The purpose of this study was to investigate the relationship between GSKIP expression and the clinico-pathological parameters in meningioma using immunohistochemical staining. We collected samples from 74 patients, from 2008 to 2012, in the Kaohsiung Medical University Hospital that had data on the staging and prognosis of the meningioma pathological section. Chi-square, Kaplan-Meier method, and cox regression were used to analyze the correlation between clinical parameters and immunohistochemistry staining for GSKIP. Following our immunohistochemical score, we found that higher expression of GSKIP was associated with high World Health Organization grading, recurrence, malignant transformation, and reduced overall survival time and recurrence-free survival time in meningioma. GSKIP may be a biomarker of poor prognosis and a target protein for therapy in meningioma.

Hyperdry Human Amniotic Membrane as a Protective Dressing for Open Wounds With Exposed Bowel in Mice

IntroductionAn enteroatmospheric fistula forms when the exposed bowel is perforated with chronic enteric fistula formation. Currently, there is no established preventative method for this condition. Hyperdry (HD) amniotic membrane (AM) can promote early granulation tissue formation on the exposed viscera and is suitable for dressing intractable wounds as it possesses anti-inflammatory, antibacterial, and immunomodulatory properties. This study investigated whether HD-AM promotes early formation of blood vessel–containing granulation tissue for enteroatmospheric fistula treatment. MethodsAn experimental animal model of an open wound with exposed bowel was developed. A 15 × 20 mm wound was prepared on the abdomen of Institute of Cancer Research mice, and the HD-AM was placed. The mice were assigned to one of the following groups: HD-AM group, in which the stromal layer of the HD-AM was placed in contact with the exposed bowel; HD-AM UD group, in which the epithelial layer of the HD-AM was placed in contact with the exposed bowel; and the HD-AM (-) or control group, in which the HD-AM was not used. ResultsOn postoperative days 7 and 14, granulation tissue thickness significantly increased in the HD-AM and HD-AM UD groups compared with that in the HD-AM (-) group. Macrophages accumulated in the HD-AM epithelium only in the HD-AM group. During HD-AM contact, a subset of invading macrophages switched from M1 to M2 phenotype. ConclusionsHD-AM is a practical wound dressing with its scaffolding function, regulation of TGF β-1 and C-X-C motif chemokine 5 (CXCL-5), and ability to induce M1-to-M2 macrophage conversion.

Fabrication and detection of a novel hybrid conductive scaffold based on alginate/gelatin/carboxylated carbon nanotubes (Alg/Gel/mMWCNTs) for neural tissue engineering

Carbon nanotubes (CNTs), as kinds of conductive carbon nanomaterials, were widely applied in neural tissue engineering due to their excellent electrical conductivity and good biocompatibility. In this study, the carboxyl-modified multi-walled carbon nanotubes (mMWCNTs) were introduced into sodium alginate/gelatin (Alg/Gel) scaffolds to optimize the function of the hybrid scaffolds. The Alg/Gel/mMWCNTs conductive scaffolds with mMWCNTs content of 1%, 3%, and 5% were prepared by freeze-drying, respectively. Following this, the physicochemical properties and biocompatibility of the hybrid scaffolds at different magnetic field intensities were evaluated. The conductive scaffolds were characterized by Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). In general, the mMWCNTs addition improved the hydrophilic, electrical conductivity and mechanical properties of the composite scaffold, and PC12 cells showed a trend of gradual increase over culture time. Particularly, the Alg/Gel-1%C scaffold exhibited the best cell proliferation behavior. Briefly, the surface contact angle decreased from 74 ± 1° to 60 ± 3°, the electrical conductivity and compressive modulus increased to 1.32 × 10-3 ± 2.1 × 10-4 S/cm and 1.40 ± 0.076 Mpa, the G1 phase from 55.67 ± 1.86% to 59.77 ± 0.94% and the G2 phase from 10.32 ± 0.35% to 13.93 ± 1.26%，respectively. In the SEM images, PC12 cells were well-shaped and densely distributed. Therefore, the Alg/Gel/mMWCNTs conductive scaffold has potential as a tissue engineering scaffold in nerve regeneration.

Biomechanical behavior of PMMA 3D printed biomimetic scaffolds: Effects of physiologically relevant environment

Functional cellular structures with controllable mechanical and morphological properties are of great interest for applications including tissue engineering, energy storage, and aerospace. Additive manufacturing (AM), also referred to as 3D printing, has enabled the potential for fabrication of functional porous scaffolds (i.e., meta-biomaterials) with controlled geometrical, morphological, and mechanical properties. Understanding the biomechanical behavior of 3D printed porous scaffolds under physiologically relevant loading and environmental conditions is crucial in accurately predicting the in vivo performance. This study was aimed to investigate the environmental dependency of the mechanical responses of 3D printed porous scaffolds of poly(methyl methacrylate) (PMMA) Class IIa biomaterial that was based on triply periodic minimal surfaces – TPMS (i.e., Primitive and Schoen-IWP). The 3D printed scaffolds (n = 5/study group) were tested under compressive loading in both ambient and fluidic (distilled water with pH = 7.4) environments according to ASTM D1621 standards. Outcomes of this study showed that compressive properties of the developed scaffolds are significantly lower in the fluidic condition than the ambient environment for the same topological and morphological group (p≤0.023). Additionally, compressive properties and flexural stiffness of the studied scaffolds were within the range of trabecular bone's properties, for both topological classes. Relationships between predicted mechanical responses and morphological properties (i.e., porosity) were evaluated for each topological class. Quantitative correlation analysis indicated that mechanical behavior of the developed 3D printed scaffolds can be controlled based on both topology and morphology.

The scaffolding function of LSD1/KDM1A reinforces a negative feedback loop to repress stem cell gene expression during primitive hematopoiesis

Lsd1/Kdm1a functions both as a histone demethylase enzyme and as a scaffold for assembling chromatin modifier and transcription factor complexes to regulate gene expression. The relative contributions of Lsd1's demethylase and scaffolding functions during embryogenesis are not known. Here, we analyze two independent zebrafish lsd1/kdm1a mutant lines and show Lsd1 is required to repress primitive hematopoietic stem cell gene expression. Lsd1 rescue constructs containing point mutations that selectively abrogate its demethylase or scaffolding capacity demonstrate the scaffolding function of Lsd1, not its demethylase activity, is required for repression of gene expression invivo. Lsd1's SNAG-binding domain mediates its scaffolding function and reinforces a negative feedback loop to repress the expression of SNAG-domain-containing genes during embryogenesis, including gfi1 and snai1/2. Our findings reveal a model in which the SNAG-binding and scaffolding function of Lsd1, and its associated negative feedback loop, provide transient and reversible regulation of gene expression during hematopoietic development.

The Syp1/FCHo2 protein induces septin filament bundling through its intrinsically disordered domain.

The septin collar of budding yeast is an ordered array of septin filaments that serves a scaffolding function for the cytokinetic machinery at the bud neck and compartmentalizes the membrane between mother and daughter cell. How septin architecture is aided by septin-binding proteins is largely unknown. Syp1 is an endocytic protein that was implicated in the timely recruitment of septins to the newly forming collar through an unknown mechanism. Using advanced microscopy and invitro reconstitution assays, we show that Syp1 is able to align laterally and tightly pack septin filaments, thereby forming flat bundles or sheets. This property is shared by the Syp1 mammalian counterpart FCHo2, thus emphasizing conserved protein functions. Interestingly, the septin-bundling activity of Syp1 resides mainly in its intrinsically disordered region. Our data uncover the mechanism through which Syp1 promotes septin collar assembly and offer another example of functional diversity of unstructured protein domains.

Role of filamin A in the pathogenesis of neuroendocrine tumors and adrenal cancer.

Cell cytoskeleton proteins are involved in tumor pathogenesis, progression and pharmacological resistance. Filamin A (FLNA) is a large actin-binding protein with both structural and scaffold functions implicated in a variety of cellular processes, including migration, cell adhesion, differentiation, proliferation and transcription. The role of FLNA in cancers has been studied in multiple types of tumors. FLNA plays a dual role in tumors, depending on its subcellular localization, post-translational modification (as phosphorylation at Ser2125) and interaction with binding partners. This review summarizes the experimental evidence showing the critical involvement of FLNA in the complex biology of endocrine tumors. Particularly, the role of FLNA in regulating expression and signaling of the main pharmacological targets in pituitary neuroendocrine tumors, pancreatic neuroendocrine tumors, pulmonary neuroendocrine tumors and adrenocortical carcinomas, with implications on responsiveness to currently used drugs in the treatment of these tumors, will be discussed.

Fabrication of functional and biodegradable scaffolds using nucleated poly(4-hydroxybutyrate) via 3D printing for bone tissue engineering

Poly (4-hydroxybutyrate) (P4HB) was a new generation of biosourced and biodegradable semi-crystalline polymer. However, due to the low crystallization temperature and slow crystallization rate of P4HB, there were problems such as insufficient cooling and product collapse during the 3D printing. The additive of biocompatible nucleating agents is one of the most effective means to promote the crystallization. Therefore, it was studied that the crystallization behavior and morphology of biodegradable P4HB were influenced by the zinc salt of d-phenylalanine (Zn(D-Phe)2) and cyanuric acid (CA) as efficient nucleating agents respectively. The addition of 2% Zn(D-Phe)2 and 0.6% CA could effectively increase the crystallization temperature, crystallization rate and nucleation density of P4HB spherulites, without damaging the crystal structure. Then, the bone tissue engineering scaffolds were prepared through 3D printing by the nucleated P4HB. The tensile strengths of P4HB/Zn(D-Phe)2 and P4HB/CA samples prepared by 3D printing were 33.4 MPa and 27.2 MPa, with the elongation at break of 449% and 370%, respectively, which fully met the requirements of bone tissue engineering on the mechanical properties of scaffolds. The results of biological property test showed that the P4HB were ideal scaffolds materials with good biological activity and biocompatibility.

LASP1 in Cellular Signaling and Gene Expression: More than Just a Cytoskeletal Regulator

LIM and SH3 protein 1 was originally identified as a structural cytoskeletal protein with scaffolding function. However, recent data suggest additional roles in cell signaling and gene expression, especially in tumor cells. These novel functions are primarily regulated by the site-specific phosphorylation of LASP1. This review will focus on specific phosphorylation-dependent interaction between LASP1 and cellular proteins that orchestrate primary tumor progression and metastasis. More specifically, we will describe the role of LASP1 in chemokine receptor, and PI3K/AKT signaling. We outline the nuclear role for LASP1 in terms of epigenetics and transcriptional regulation and modulation of oncogenic mRNA translation. Finally, newly identified roles for the cytoskeletal function of LASP1 next to its known canonical F-actin binding properties are included.

The XPA Protein—Life under Precise Control

Nucleotide excision repair (NER) is a central DNA repair pathway responsible for removing a wide variety of DNA-distorting lesions from the genome. The highly choreographed cascade of core NER reactions requires more than 30 polypeptides. The xeroderma pigmentosum group A (XPA) protein plays an essential role in the NER process. XPA interacts with almost all NER participants and organizes the correct NER repair complex. In the absence of XPA's scaffolding function, no repair process occurs. In this review, we briefly summarize our current knowledge about the XPA protein structure and analyze the formation of contact with its protein partners during NER complex assembling. We focus on different ways of regulation of the XPA protein's activity and expression and pay special attention to the network of post-translational modifications. We also discuss the data that is not in line with the currently accepted hypothesis about the functioning of the XPA protein.

ZBTB18 inhibits SREBP-dependent lipid synthesis by halting CTBPs and LSD1 activity in glioblastoma.

Enhanced fatty acid synthesis is a hallmark of tumors, including glioblastoma. SREBF1/2 regulate the expression of enzymes involved in fatty acid and cholesterol synthesis. Yet, little is known about the precise mechanism regulating SREBP gene expression in glioblastoma. Here, we show that a novel interaction between the co-activator/co-repressor CTBP and the tumor suppressor ZBTB18 regulates the expression of SREBP genes. In line with our findings, metabolic assays and glucose tracing analysis confirm the reduction in several phospholipid species upon ZBTB18 expression. Our study identifies CTBP1/2 and LSD1 as co-activators of SREBP genes and indicates that the functional activity of the CTBP-LSD1 complex is altered by ZBTB18. ZBTB18 binding to the SREBP gene promoters is associated with reduced LSD1 demethylase activity of H3K4me2 and H3K9me2 marks. Concomitantly, the interaction between LSD1, CTBP, and ZNF217 is increased, suggesting that ZBTB18 promotes LSD1 scaffolding function. Our results outline a new epigenetic mechanism enrolled by ZBTB18 and its co-factors to regulate fatty acid synthesis that could be targeted to treat glioblastoma patients.

Surface Functionalization of Hydroxyapatite Scaffolds with MgAlEu-LDH Nanosheets for High-Performance Bone Regeneration.

Although artificial bone repair scaffolds, such as titanium alloy, bioactive glass, and hydroxyapatite (HAp), have been widely used for treatment of large-size bone defects or serious bone destruction, they normally exhibit unsatisfied bone repair efficiency because of their weak osteogenic and angiogenesis performance as well as poor cell crawling and adhesion properties. Herein, the surface functionalization of MgAlEu-layered double hydroxide (MAE-LDH) nanosheets on porous HAp scaffolds is reported as a simple and effective strategy to prepare HAp/MAE-LDH scaffolds for enhanced bone regeneration. The surface functionalization of MAE-LDHs on the porous HAp scaffold can significantly improve its surface roughness, specific surface, and hydrophilicity, thus effectively boosting the cells adhesion and osteogenic differentiation. Importantly, the MAE-LDHs grown on HAp scaffolds enable the sustained release of Mg2+ and Eu3+ ions for efficient bone repair and vascular regeneration. In vitro experiments suggest that the HAp/MAE-LDH scaffold presents much enhanced osteogenesis and angiogenesis properties in comparison with the pristine HAp scaffold. In vivo assays further reveal that the new bone mass and mineral density of HAp/MAE-LDH scaffold increased by 3.18- and 2.21-fold, respectively, than that of pristine HAp scaffold. The transcriptome sequencing analysis reveals that the HAp/MAE-LDH scaffold can activate the Wnt/β-catenin signaling pathway to promote the osteogenic and angiogenic abilities.

Braided suture-reinforced fibrous yarn bundles as a scaffold for tendon tissue engineering in extensor digitorum tendon repair

In this study, we intend to develop an effective tendon tissue engineering scaffold that can provide mechanical stability and tendon regeneration ability. Using a novel electrospinning process, a biodegradable suture was continuously covered with aligned polycaprolactone fibers in core/sheath structure to produce a single yarn. The single yarn was braided together to fabricate a multi yarn (MY) scaffold, which can be surface modified with oxygen plasma and conjugated with heparin. The fibroblast growth factor 2 (FGF2) was bound to MY through bioaffinity between heparin and FGF2 to generate a functional scaffold (MY-FGF2) suitable for extensor digitorum tendon (EDT) repair. The physico-chemical properties of the scaffolds were characterized throughout the modification steps using microscopy, spectroscopy and mechanical testing. In vitro static culture using rabbit tendon-derived fibroblasts (rTFs) indicates combined effects of FGF2 and fiber alignment can enhance cell proliferation and extracellular matrix synthesis rates, as well as fasten tendon maturation. The cytoskeleton staining further endorses aligned morphology of fibers direct cell growth and collagen fiber deposition along the fiber axial direction, mimicking native tendon features. The dynamic culture in a bioreactor under uniaxial cyclic tensile loading authenticates 5 % mechanical stimulation can further increase cell proliferation and tenogenic differentiation rates compared to static culture. After mechanical stimulation for 7 days in vitro, the MY-FGF2/rTFs construct was used for repair of EDT defects in rabbits. The retrieved MY-FGF2/rTFs sample 6-week post-implantation shows superior mechanical properties and tendon regeneration abilities over acellular MY-FGF2. Taken together, we demonstrate a combinatory approach with MY-FGF2 where chemical and physical cues provided by FGF2, fiber alignment and dynamic stimulation contribute to tendon regeneration with a specific focus on EDT repair.