Soluble Form Research Articles

Structure-Activity Relationship of Fluorinated Benzenesulfonamides as Inhibitors of Amyloid-β Aggregation.

Amyloid-beta aggregation is considered one of the factors influencing the onset of the Alzheimer's disease. Early prevention of such aggregation should alleviate disease condition by applying small molecule compounds that shift the aggregation equilibrium toward the soluble form of the peptide or slow down the process. We have discovered that fluorinated benzenesulfonamides of particular structure slowed the amyloid-beta peptide aggregation process by more than three-fold. We synthesized a series of ortho-para and meta-para double-substituted fluorinated benzenesulfonamides that inhibited the aggregation process to a variable extent yielding a detailed picture of the structure-activity relationship. Analysis of compound chemical structure effect on aggregation in artificial cerebrospinal fluid showed the necessity to arrange the benzenesulfonamide, hydrophobic substituent, and benzoic acid in a particular way. The amyloid beta peptide aggregate fibril structures varied in cross-sectional height depending on the applied inhibitor indicating the formation of a complex with the compound. Application of selected inhibitors increased the survivability of cells affected by the amyloid beta peptide. Such compounds may be developed as drugs against Alzheimer's disease.

Biochemical characterization of paralyzed flagellum proteins A (PflA) and B (PflB) from Helicobacter pylori flagellar motor.

Motility by means of flagella plays an important role in the persistent colonization of Helicobacter pylori in the human stomach. The H. pylori flagellar motor has a complex structure that includes a periplasmic scaffold, the components of which are still being identified. Here, we report the isolation and characterization of the soluble forms of two putative essential H. pylori motor scaffold components, proteins PflA and PflB. We developed an on-column refolding procedure, overcoming the challenge of inclusion body formation in E. coli. We employed mild detergent sarkosyl to enhance protein recovery and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO)-containing buffers to achieve optimal solubility and monodispersity. In addition, we showed that PflA lacking the β-rich N-terminal domain is expressed in a soluble form, and behaves as a monodisperse monomer in solution. The methods for producing the soluble, folded forms of H. pylori PflA and PflB established in this work will facilitate future biophysical and structural studies aimed at deciphering their location and their function within the flagellar motor.

Importance of the (Pro)renin Receptor in Activating the Renin-Angiotensin System During Normotensive and Preeclamptic Pregnancies.

For a healthy pregnancy to occur, a controlled interplay between the maternal circulating renin-angiotensin-aldosterone system (RAAS), placental renin-angiotensin system (RAS) and intrarenal renin-angiotensin system (iRAS) is necessary. Functionally, both the RAAS and iRAS interact to maintain blood pressure and cardiac output, as well as fluid and electrolyte balance. The placental RAS is important for placental development while also influencing the maternal circulating RAAS and iRAS. This narrative review concentrates on the (pro)renin receptor ((P)RR) and its soluble form (s(P)RR) in the context of the hypertensive pregnancy pathology, preeclampsia. The (P)RR and the s(P)RR have become of particular interest as not only can they activate prorenin and renin, thus influencing levels of angiotensin II (Ang II), but s(P)RR has now been shown to directly interact with and stimulate the Angiotensin II type 1 receptor (AT1R). Levels of both placental (P)RR and maternal circulating s(P)RR are elevated in patients with preeclampsia. Furthermore, s(P)RR has been shown to increase blood pressure in non-pregnant and pregnant rats and mice. In preeclamptic pregnancies, which are characterised by maternal hypertension and impaired placental development and function, we propose that there is enhanced secretion of s(P)RR from the placenta into the maternal circulation. Due to its ability to both activate prorenin and act as an AT1R agonist, excess maternal circulating s(P)RR can act on both the maternal vasculature, and the kidney, leading to RAS over-activation. This results in dysregulation of the maternal circulating RAAS and overactivation of the iRAS, contributing to maternal hypertension, renal damage, and secondary changes to neurohumoral regulation of fluid and electrolyte balance, ultimately contributing to the pathophysiology of preeclampsia.

Oxidative degradation and detoxification of multiple mycotoxins using a dye-decolorizing peroxidase from the white-rot fungus Bjerkandera adusta

Mycotoxins are widely present from cereal crops to food products, posing a significant risk to food safety and causing serious health and economic losses. Therefore, it is imperative to develop a sustainable and efficient detoxification strategy to address mycotoxin contamination. Herein, a dye-decolorizing peroxidase, BaDyP, from the white-rot fungus Bjerkandera adusta was successfully expressed as a soluble form in Escherichia coli with the help of molecular chaperones. Purified recombinant BaDyP could efficiently degrade multiple mycotoxins including aflatoxin B1, zearalenone, and deoxynivalenol in the presence of mediators such as Mn2+ or 1-HBT. Meanwhile, their degradation products were identified using UPLC-MS/MS, including AFB1-diol, AFQ1, 15-OH-ZEN, HZEN, and C15H18O8. Furthermore, cell survival tests exhibited a significant reduction in the biological toxicity of these degradation products. These findings indicate that the use of dye-decolorizing peroxidase could be a promising approach for the effective removal of multiple mycotoxins in cereal crops and food products.

Soluble E-Cadherin Contributes to Airway Inflammation in Severe Asthma.

Impaired airway epithelial barrier and decreased expression of E-cadherin are key features of severe asthma. As a gatekeeper of the mucosa, E-cadherin can be cleaved from the cell surface and released into the apical lumen as a soluble form (sE-cadherin).This study was aimed to investigate the role of sE-cadherin in severe asthma.Induced sputum was obtained from healthy subjects and patients with asthma. Two murine models of severe asthma were established using either TDI (toluene diisocyanate) or OVA (ovalbumin)/CFA (complete Freund's adjuvants). The role of sE-cadherin in severe asthma was evaluated by intraperitoneal injection of DECMA-1, a neutralizing antibody against sE-cadherin. Mice or THP-1-derived macrophages were treated with recombinant sE-cadherin to explore the pro-inflammatory mechanism of sE-cadherin.Severe asthma patients had a significantly higher sputum sE-cadherin level than the health subjects with mild to moderate asthma, which were positively correlated with sputum HMGB1 level and glucocorticoid dosage required for daily control. Allergen exposure markedly increased sE-cadherin level in the bronchoalveolar lavage fluid in mice. Treatment of DECMA-1 significantly attenuated allergen-induced airway inflammation and hyperresponsivenes in both models of severe asthma. While exposure to recombinant sE-cadherin dramatically up-regulated VEGF expression in THP-1-derived macrophages, and increased neutophlil and eosinophil infiltration into the airway as well as the release of VEGF and IL-6 in mice, both of which can be suppressed by pharmacological inhibition of ERK signaling.Taken together, our data indicated that sE-cadherin contributed to the airway inflammation of severe asthma in an ERK-depedent pathway.

Assessment of contamination, mobility and application of selected technology-critical elements as indicators of anthropogenic pollution of bottom sediments.

The study investigates the potential of technology-critical elements (TCEs) in the bottom sediments of the Biała Przemsza River as indicators of anthropogenic activities. The mass fractions of TCEs: Ge, Ga, In, Tl, Sb and Te (and other elements) in the sediment were analysed by inductively coupled plasma mass spectrometry with the maximum mass fractions: 2.46, 25.6, 0.528, 27.7, 12.5 and 0.293mg/kg, respectively. Distribution and identification of TCE sources were supported by statistical analysis (principal component analysis coupled with varimax rotation and hierarchical cluster analysis). Assessments of TCE contamination using the geoaccumulation index, pollution index, contamination factor, enrichment factor and the antimony-to-arsenic ratio highlighted the high contamination of bottom sediments by Sb, Ga, Tl, Cd, As, Zn, Pb and moderate contamination by Co, In and V. Distinct behaviour patterns were observed among TCEs, revealing Sb and Tl as potential indicators of Zn-Pb ore mining activities. Co, V, Ge and, to a lesser extent, Te emerged as promising indicators of coal and coal fly ash effluents. Sequential chemical extraction of TCEs showed that Sb, In and Tl had the highest mobility from sediments. The Risk Assessment Code calculations suggest, that in the Biała Przemsza River bottom sediments, there is an average risk of contamination by As, Tl and Mn. Soluble forms of Tl, Ge, Sb, Te and In were identified in descending order, indicating their bioavailability.

Shifts in receptors during submergence of an encephalitic arbovirus.

Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s1-3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity(known as viral submergence3) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors4. However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2-E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.

Fractalkine in Health and Disease

CX3CL1 is one of the 50 up-to-date identified and characterized chemokines. While other chemokines are produced as small, secreted proteins, CX3CL1 (fractalkine) is synthetized as a transmembrane protein which also leads to a soluble form produced as a result of proteolytic cleavage. The membrane-bound protein and the soluble forms exhibit different biological functions. While the role of the fractalkine/CX3CR1 signaling axis was described in the nervous system and was also related to the migration of leukocytes to sites of inflammation, its actions are controversial in cancer progression and anti-tumor immunity. In the present review, we first describe the known biology of fractalkine concerning its action through its cognate receptor, but also its role in the activation of different integrins. The second part of this review is dedicated to its role in cancer where we discuss its role in anti-cancer or procarcinogenic activities.

Simulation of Soluble and Bound VEGF-stimulated in vitro Capillary-like Network Formation on Deformed Substrate.

Capillary plexus cultivation is crucial in tissue engineering and regenerative medicine. Theoretical simulations have been conducted to supplement the expensive experimental works. However, the mechanisms connecting mechanical and chemical stimuli remained undefined, and the functions of the different VEGF forms in the culture environment were still unclear. In this paper, we developed a hybrid model for simulating short-term in vitro capillary incubations. We used the Cellular Potts model to predict individual cell migration, morphology change, and continuum mechanics to quantify biogel deformation and VEGF transport dynamics. By bridging the mechanical regulation and chemical stimulation in the model, the results showed good agreement between the predicted network topology and experiments, in which elongated cells connected, forming the network cords and round cells gathered, creating cobblestone-like aggregates. The results revealed that the capillary-like networks could develop in high integrity only when the mechanical and chemical couplings worked adequately, with the cell morphology and haptotaxis driven by the soluble and bound forms of VEGF, respectively, functioning simultaneously.

Recombinant Expression and Characterization of a Novel Thermo-Alkaline Lipase with Increased Solvent Stability from the Antarctic Thermophilic Bacterium Geobacillus sp. ID17.

Lipases are enzymes that hydrolyze long-chain carboxylic esters, and in the presence of organic solvents, they catalyze organic synthesis reactions. However, the use of solvents in these processes often results in enzyme denaturation, leading to a reduction in enzymatic activity. Consequently, there is significant interest in identifying new lipases that are resistant to denaturing conditions, with extremozymes emerging as promising candidates for this purpose. Lip7, a lipase from Geobacillus sp. ID17, a thermophilic microorganism isolated from Deception Island, Antarctica, was recombinantly expressed in E. coli C41 (DE3) in functional soluble form. Its purification was achieved with 96% purity and 23% yield. Enzymatic characterization revealed Lip7 to be a thermo-alkaline enzyme, reaching a maximum rate of 3350 U mg-1 at 50 °C and pH 11.0, using p-nitrophenyl laurate substrate. Notably, its kinetics displayed a sigmoidal behavior, with a higher kinetic efficiency (kcat/Km) for substrates of 12-carbon atom chain. In terms of thermal stability, Lip7 demonstrates stability up to 60 °C at pH 8.0 and up to 50 °C at pH 11.0. Remarkably, it showed high stability in the presence of organic solvents, and under certain conditions even exhibited enzymatic activation, reaching up to 2.5-fold and 1.35-fold after incubation in 50% v/v ethanol and 70% v/v isopropanol, respectively. Lip7 represents one of the first lipases from the bacterial subfamily I.5 and genus Geobacillus with activity and stability at pH 11.0. Its compatibility with organic solvents makes it a compelling candidate for future research in biocatalysis and various biotechnological applications.

Three-Dimensional DNA Hydrogel Mediated Dual-Mode Sensing Method for Quantification of Epithelial Cell Adhesion Molecule in Biological Fluid Samples.

Monitoring changes in the expression of marker proteins in biological fluids is essential for biomarker-based disease diagnosis. Epithelial cell adhesion molecule (EpCAM) has been identified as a broad-spectrum biomarker for various chronic diseases and as a therapeutic target. However, the development of simple and reliable methods for quantifying EpCAM changes in biological fluids faces challenges due to the variability of its expression across different diseases, the presence of soluble forms, and matrix effects. In this paper, a surface-enhanced Raman scattering (SERS)-fluorescence (FL) dual-mode sensing method was established for quantification of trace EpCAM in biological fluids based on bimetallic Au@Ag nanoparticles and nitrogen-doped quantum dots encapsulated DNA hydrogel hybrid with graphene oxide (Au@Ag-NQDs/GO). The DNA hydrogel was constructed based on three-dimensional (3D) structure DNA-mediated strategy using an aptamer DNA (AptDNA) linker. The interaction of the AptDNA with EpCAM triggered the disassembly of the DNA hydrogel. Consequently, the release of Au@Ag nanoparticles induced an "on-off" switch in the SERS signal while the weakened FL quenching effect in Au@Ag-NQDs/GO system achieved "off-on" switch of FL signal, enabling the simultaneous SERS-FL quantification of EpCAM. The established dual-mode method exhibited outstanding sensitivity and stability in quantifying EpCAM in the range of 0.5-60.0 pg/mL, with the limits of detection (LODs) of SERS and FL as 0.17 and 0.35 pg/mL, respectively. When applied for real sample analysis, the method showed satisfactory specificity and recoveries in cancer cells lysate, serum, and urine samples with RSDs of 2.8-6.3%, 4.0-6.3%, and 2.8-5.7%, respectively. The developed SERS-FL sensing method offered a sensitive, reliable, and practical quantification strategy for trace EpCAM in diverse biological fluid samples, which would benefit the early diagnosis of disease and further health management.

Inter domain linker region affects properties of CBM6 in GH5_34 arabinoxylanases and alters oligosaccharide product profile.

Understanding the relation between enzyme domain structure and catalytic activity is crucial for optimal engineering of novel enzymes for lignocellulose bioconversion. Xylanases with varying specificities are commonly used to valorise the hemicellulose arabinoxylan (AX), yet characterization of specific arabinoxylanases remain limited. Two homologous GH5_34 arabinoxylanases, HhXyn5A and CtXyn5A, in which the two domains are connected by a 40-residue linker, exhibit distinct activity on AX, yielding different reaction product patterns, despite high sequence identity, conserved active sites and similar domain composition. In this study, the carbohydrate binding module 6 (CBM6), or the inter domain linker together with CBM6, were swapped to investigate their influence on hydrolytic activity and oligosaccharide product pattern on cereal AXs. The variants, with only CBM6 swapped, displayed reduced activity on commercial wheat and rye AX, as well as on extracted oat fibre, compared to the original enzymes. Additionally, exchange of both linker and CBM6 resulted in a reduced ratio of enzyme produced in soluble form in Escherichia coli cultivations, causing loss of activity of both HhXyn5A and CtXyn5A variants. Analysis of oligosaccharide product patterns applying HPAEC-PAD revealed a decreased number of reaction products for CtXyn5A with swapped CBM6, which resembled the product pattern of HhXyn5A. These findings emphasize the importance of the CBM6 interactions with the linker and the catalytic domain for enzyme activity and specificity, and underlines the role of the linker in enzyme structure organisation and product formation, where alterations in linker interactions with the catalytic and/or CBM6 domains, influence enzyme-substrate association and specificity.

Characterizing soluble immune checkpoint molecules and TGF-β1,2,3 in pleural effusion of malignant pleural mesothelioma

The clinical impact of soluble molecules in pleural effusion (PE) is unclear in patients with malignant pleural mesothelioma (MPM). In this single-center, retrospective, observational study, we assessed soluble forms of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and PD-1 ligand 1 (PD-L1) using enzyme-linked immunosorbent assays; three TGF-β isoforms were measured via multiplex assay in PE of patients with fibrinous pleuritis (FP) or MPM, to assess relationships between the levels of six molecules, clinicopathological characteristics, and efficacy of immune checkpoint inhibitors. Soluble forms of CTLA-4, PD-L1, PD-1, TGF-β1, TGF-β2, and TGF-β3 were variably produced in PE of FP (n = 34) and MPM (n = 79); we found significant relationships between the six molecules and clinicopathological features. Although none of the three soluble immune checkpoint molecules showed diagnostic or prognostic effects in patients with MPM, TGF-β2 level in PE is a useful differential diagnostic marker between FP and MPM. Both TGF-β1 and TGF-β3 levels are promising prognostic markers for MPM. Moreover, we found that higher baseline levels of PD-1 soluble forms predicted the response to anti-PD1 monotherapy. Our findings identify novel diagnostic, prognostic, and predictive biomarkers for anti-PD1 therapy in patients with MPM.

Characterizing soluble immune checkpoint molecules and TGF-β1,2,3 in pleural effusion of malignant pleural mesothelioma

The clinical impact of soluble molecules in pleural effusion (PE) is unclear in patients with malignant pleural mesothelioma (MPM). In this single-center, retrospective, observational study, we assessed soluble forms of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and PD-1 ligand 1 (PD-L1) using enzyme-linked immunosorbent assays; three TGF-β isoforms were measured via multiplex assay in PE of patients with fibrinous pleuritis (FP) or MPM, to assess relationships between the levels of six molecules, clinicopathological characteristics, and efficacy of immune checkpoint inhibitors. Soluble forms of CTLA-4, PD-L1, PD-1, TGF-β1, TGF-β2, and TGF-β3 were variably produced in PE of FP (n = 34) and MPM (n = 79); we found significant relationships between the six molecules and clinicopathological features. Although none of the three soluble immune checkpoint molecules showed diagnostic or prognostic effects in patients with MPM, TGF-β2 level in PE is a useful differential diagnostic marker between FP and MPM. Both TGF-β1 and TGF-β3 levels are promising prognostic markers for MPM. Moreover, we found that higher baseline levels of PD-1 soluble forms predicted the response to anti-PD1 monotherapy. Our findings identify novel diagnostic, prognostic, and predictive biomarkers for anti-PD1 therapy in patients with MPM.

Cleavage site-directed antibodies reveal the prion protein in humans is shed by ADAM10 at Y226 and associates with misfolded protein deposits in neurodegenerative diseases

Proteolytic cell surface release (‘shedding’) of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

Palmitoylation at a conserved cysteine residue facilitates gasdermin D-mediated pyroptosis and cytokine release

Gasdermin D (GSDMD)-mediated pyroptotic cell death drives inflammatory cytokine release and downstream immune responses upon inflammasome activation, which play important roles in host defense and inflammatory disorders. Upon activation by proteases, the GSDMD N-terminal domain (NTD) undergoes oligomerization and membrane translocation in the presence of lipids to assemble pores. Despite intensive studies, the molecular events underlying the transition of GSDMD from an autoinhibited soluble form to an oligomeric pore form inserted into the membrane remain incompletely understood. Previous work characterized S-palmitoylation for gasdermins from bacteria, fungi, invertebrates, as well as mammalian gasdermin E (GSDME). Here, we report that a conserved residue Cys191 in human GSDMD was S-palmitoylated, which promoted GSDMD-mediated pyroptosis and cytokine release. Mutation of Cys191 or treatment with palmitoyltransferase inhibitors cyano-myracrylamide (CMA) or 2-bromopalmitate (2BP) suppressed GSDMD palmitoylation, its localization to the membrane and dampened pyroptosis or IL-1β secretion. Furthermore, Gsdmd-dependent inflammatory responses were alleviated by inhibition of palmitoylation in vivo. By contrast, coexpression of GSDMD with palmitoyltransferases enhanced pyroptotic cell death, while introduction of exogenous palmitoylation sequences fully restored pyroptotic activities to the C191A mutant, suggesting that palmitoylation-mediated membrane localization may be distinct from other molecular events such as GSDMD conformational change during pore assembly. Collectively, our study suggests that S-palmitoylation may be a shared regulatory mechanism for GSDMD and other gasdermins, which points to potential avenues for therapeutically targeting S-palmitoylation of gasdermins in inflammatory disorders.

Palmitoylation at a conserved cysteine residue facilitates gasdermin D-mediated pyroptosis and cytokine release

Gasdermin D (GSDMD)-mediated pyroptotic cell death drives inflammatory cytokine release and downstream immune responses upon inflammasome activation, which play important roles in host defense and inflammatory disorders. Upon activation by proteases, the GSDMD N-terminal domain (NTD) undergoes oligomerization and membrane translocation in the presence of lipids to assemble pores. Despite intensive studies, the molecular events underlying the transition of GSDMD from an autoinhibited soluble form to an oligomeric pore form inserted into the membrane remain incompletely understood. Previous work characterized S-palmitoylation for gasdermins from bacteria, fungi, invertebrates, as well as mammalian gasdermin E (GSDME). Here, we report that a conserved residue Cys191 in human GSDMD was S-palmitoylated, which promoted GSDMD-mediated pyroptosis and cytokine release. Mutation of Cys191 or treatment with palmitoyltransferase inhibitors cyano-myracrylamide (CMA) or 2-bromopalmitate (2BP) suppressed GSDMD palmitoylation, its localization to the membrane and dampened pyroptosis or IL-1β secretion. Furthermore, Gsdmd-dependent inflammatory responses were alleviated by inhibition of palmitoylation in vivo. By contrast, coexpression of GSDMD with palmitoyltransferases enhanced pyroptotic cell death, while introduction of exogenous palmitoylation sequences fully restored pyroptotic activities to the C191A mutant, suggesting that palmitoylation-mediated membrane localization may be distinct from other molecular events such as GSDMD conformational change during pore assembly. Collectively, our study suggests that S-palmitoylation may be a shared regulatory mechanism for GSDMD and other gasdermins, which points to potential avenues for therapeutically targeting S-palmitoylation of gasdermins in inflammatory disorders.

Issues in the design of tissue-engineered collagen constructs and some approaches to their solution: A review

This review article analyzes modern literature sources on the design of bioinks and tissue-engineered constructs on the basis of soluble forms of collagen, including gelatin. The choice of soluble forms of collagen as a biopolymer basis for bioinks and this type of constructs is determined by their unique biocompatibility, bioresorbability, as well as the presence of adhesive sites (motifs) for binding cells with their subsequent proliferation and organ or tissue maturation. However, the poor mechanical properties of products derived from soluble collagens, rapid biodegradation, tendency to lose the solubility of highly viscous solutions when stored or with pH increase limit their application in tissue engineering. The use of more stable low-viscosity collagen solutions does not enable the creation of dimensionally stable tissue-engineered constructs. It is shown that the introduction of various water-soluble biocompatible polymeric additives into hydrogels on the basis of soluble collagens allows the above-mentioned problems to be solved, as well as providing a means to customize the required characteristics of bioinks and tissue-engineered constructs. The additives that improve their characteristics include biopolymers: silk sericin and fibroin, as well as alginates and fibrinogen, which can form cross-links in the presence of Ca2+. This type of crosslinking is shown to further improve the performance of these constructs. All of these biopolymers are commercially available. The article comparatively analyzes approaches to stabilizing the shape, improving the mechanical properties, and adjusting the bioresorption time of 3D printed tissue-engineered constructs during organ or tissue maturation.

Comparing the Soluble Form of Recombinant Human Insulin-like Growth Factor-1 (rhIGF-1) in Escherichia coli Using Thioredoxin as Fused and Co-expressed Protein.

Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide with various physiological functions. Escherichia coli is one of the most desirable hosts for recombinant protein production, especially for human proteins whose post-translation modifications are not essential for their bioactivity, such as hIGF-1. In this study, bacterial thioredoxin (Trx) was studied as a fused and non-fused protein to convert the insoluble form of recombinant human IGF-1 (rhIGF-1) to its soluble form in E. coli. The rhIGF-1 was expressed in the E. coli Origami strain in the form of fused-Trx. It was co-expressed with Trx and then purified and quantified. In the next step, the biological activity of rhIGF-1 was evaluated by alkaline phosphatase (ALP) activity assay in human adipose-derived stem cells (hASCs) regarding the differentiation enhancement effect of IGF-1 through the osteogenic process. Results showed that Trx in both the fused and non-fused forms had a positive effect on the production of the soluble form of rhIGF-1. A significant increase in ALP activity in hASCs after rhIGF-1 treatment was observed, confirming protein bioactivity. It was strongly suggested that the overproduction of Trx could increase the solubility of co-expressed recombinant proteins by changing the redox state in E. coli cells.

Serum Vascular Adhesion Protein-1 and Endothelial Dysfunction in Hepatic Cirrhosis: Searching for New Prognostic Markers.

Endothelial dysfunction plays a key role in the development of liver cirrhosis. Among the biomarkers of endothelial dysfunction, the soluble form of Vascular Adhesion Protein-1 (sVAP-1) is an unconventional and less known adhesion molecule endowed also with amine oxidase activity. The aim of this study was to explore and correlate the behavior of sVAP-1 with that of the soluble vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1) and with the severity of liver cirrhosis. A cross-sectional study was carried out by enrolling 28 controls, 59 cirrhotic patients without hepatocellular carcinoma, and 56 patients with hepatocellular carcinoma (HCC), mainly caused by alcohol abuse. The levels of adhesion molecules and of the pro-inflammatory cytokines (IL-6 and TNF-αα) were determined by immunoassay and the enzymatic activity of sVAP-1 by a fluorometric assay. In non-diabetic patients without HCC, a specific behavior of sVAP-1 was highlighted. Differently from sVCAM-1, sICAM-1, and cytokines, the sVAP-1 level was significantly increased only in the early stage of disease, and then, it decreased in the last stage (866 ± 390 ng/mL vs. 545 ± 316 ng/mL, in Child-Pugh class A vs. C, respectively, p < 0.05). Bivariate analysis correlates sVAP-1 to sVCAM-1, in the absence of HCC (Spearman's rho = 0.403, p < 0.01). Multiple linear regression analysis revealed that sVCAM-1 appears to be a predictor of sVAP-1 (β coefficient = 0.374, p = 0.021). In conclusion, in non-diabetic and non-HCC cirrhotic patients, sVAP-1 may be a potential prognostic biomarker that, together with sVCAM-1 and pro-inflammatory cytokines, may provide information on the progression of sinusoidal liver endothelium damage.