Application of an NMR/Crystallography Fragment Screening Platform for the Assessment and Rapid Discovery of New HIV-CA Binding Fragments.

Identification and assessment of novel targets is essential to combat drug resistance in the treatment of HIV/AIDS. HIV Capsid (HIV-CA), the protein playing a major role in both the early and late stages of the viral life cycle, has emerged as an important target. We have applied an NMR fragment screening platform and identified molecules that bind to the N-terminal domain (NTD) of HIV-CA at a site close to the interface with the C-terminal domain (CTD). Using X-ray crystallography, we have been able to obtain crystal structures to identify the binding mode of these compounds. This allowed for rapid progression of the initial, weak binding, fragment starting points to compounds 37 and 38, which have 19F-pKi values of 5.3 and 5.4 respectively.

Structural insights into hydrolytic defluorination of difluoroacetate by microbial fluoroacetate dehalogenases.

Fluorine forms the strongest single bond to carbon with the highest bond dissociation energy among natural products. However, fluoroacetate dehalogenases (FADs) have been shown to hydrolyze this bond in fluoroacetate under mild reaction conditions. Furthermore, two recent studies demonstrated that the FAD RPA1163 from Rhodopseudomonas palustris can also accept bulkier substrates. In this study, we explored the substrate promiscuity of microbial FADs and their ability to defluorinate polyfluorinated organic acids. Enzymatic screening of eight purified dehalogenases with reported fluoroacetate defluorination activity revealed significant hydrolytic activity against difluoroacetate in three proteins. Product analysis using liquid chromatography-mass spectrometry identified glyoxylic acid as the final product of enzymatic DFA defluorination. The crystal structures of DAR3835 from Dechloromonas aromatica and NOS0089 from Nostoc sp. were determined in the apo-state along with the DAR3835 H274N glycolyl intermediate. Structure-based site-directed mutagenesis of DAR3835 demonstrated a key role for the catalytic triad and other active site residues in the defluorination of both fluoroacetate and difluoroacetate. Computational analysis of the dimer structures of DAR3835, NOS0089, and RPA1163 indicated the presence of one substrate access tunnel in each protomer. Moreover, protein-ligand docking simulations suggested similar catalytic mechanisms for the defluorination of both fluoroacetate and difluoroacetate, with difluoroacetate being defluorinated via two consecutive defluorination reactions producing glyoxylate as the final product. Thus, our findings provide molecular insights into substrate promiscuity and catalytic mechanism of FADs, which are promising biocatalysts for applications in synthetic chemistry and bioremediation of fluorochemicals.

Defluorination Capability of l-2-Haloacid Dehalogenases in the HAD-Like Hydrolase Superfamily Correlates with Active Site Compactness.

l-2-Haloacid dehalogenases, industrially and environmentally important enzymes that catalyse cleavage of the carbon-halogen bond in S-2-halocarboxylic acids, were known to hydrolyse chlorinated, brominated and iodinated substrates but no activity towards fluorinated compounds had been reported. A screen for novel dehalogenase activities revealed four l-2-haloacid dehalogenases capable of defluorination. We now report crystal structures for two of these enzymes, Bpro0530 and Rha0230, as well as for the related proteins PA0810 and RSc1362, which hydrolyse chloroacetate but not fluoroacetate, all at ∼2.2 Šresolution. Overall structure and active sites of these enzymes are highly similar. In molecular dynamics (MD) calculations, only the defluorinating enzymes sample more compact conformations, which in turn allow more effective interactions with the small fluorine atom. Structural constraints, based on X-ray structures and MD calculations, correctly predict the defluorination activity of the homologous enzyme ST2570.

Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5' end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3'-5' exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.

Processing of NMR and MS metabolomics data using chemometrics methods: a global tool for fungi biotransformation reactions monitoring.

INTRODUCTION: Biotransformation constitutes an important aspect of the drug discovery process, to mimic human metabolism of active principal ingredient but also to generate new chemical entities. Several microorganisms such as fungi are well adapted to transform drug, whether at the stage of screening or for large-scale production. OBJECTIVES: Due to the high chemical complexity of the biotransformation media, it seems attractive to develop new analytical strategies in order to guarantee an adequate monitoring and optimize the production of targeted metabolites or drug candidates. METHODS: The model designed for this purpose concerns the biotransformation of a potential histamine H3 antagonist (S38093) in order to produce phase I metabolites. MS, NMR and chemometrics tools were used to monitor biotransformation reactions. RESULTS: First, a screening of eleven filamentous fungi was carried out by UHPLC-UV-MS and principal component analysis to select the best candidates. Subsequently, MS (tR, m/z) and NMR (1H, JRES) fingerprints associated with Consensus OPLS-DA multiblock approach were used to better understand the bioreaction mechanisms in terms of nutrient consumption and hydroxylated metabolites production. Then an experimental design was set up to optimize the production conditions (pH, kinetic) of these target metabolites. CONCLUSION: This study demonstrates how NMR and MS acquisitions combined with chemometric methods offer an innovative analytical strategy to have a grasp of functionalization mechanisms, and identify metabolites and other compounds (amino acids, nutrients, etc.) in complex biotransformation mixtures.

A Deeper Investigation of Drug Degradation Mixtures Using a Combination of MS and NMR Data: Application to Indapamide.

A global approach that is based on a combination of mass spectrometry (MS) and nuclear magnetic resonance (NMR) data has been developed for a complete and rapid understanding of drug degradation mixtures. We proposed a workflow based on a sample preparation protocol that is compatible to MS and NMR, the selection of the most appropriate experiments for each technique, and the implementation of prediction software and multivariable analysis method for a better interpretation and correlation of MS and NMR spectra. We have demonstrated the efficient quantification of the remaining active pharmaceutical ingredient (API). The unambiguous characterization of degradation products (DPs) was reached while using the potential of ion mobility-mass spectrometry (IM-MS) for fragment ions filtering (HDMSE) and the implementation of two-dimensional (2D) NMR experiments with the non-uniform sampling (NUS) method. We have demonstrated the potential of quantitative NMR (qNMR) for the estimation of low level DPs. Finally, in order to simultaneously monitor multi-samples, the contribution of partial least squares (PLS) regression was evaluated. Our methodology was tested on three indapamide forced degradation conditions (acidic, basic, and oxidative) and it could be easily transposed in the drug development field to assist in the interpretation of complex mixtures (stability studies, impurities profiling, and biotransformation screening).

A structural study of the complex between neuroepithelial cell transforming gene 1 (Net1) and RhoA reveals a potential anticancer drug hot spot.

The GTPase RhoA is a major player in many different regulatory pathways. RhoA catalyzes GTP hydrolysis, and its catalysis is accelerated when RhoA forms heterodimers with proteins of the guanine nucleotide exchange factor (GEF) family. Neuroepithelial cell transforming gene 1 (Net1) is a RhoA-interacting GEF implicated in cancer, but the structural features supporting the RhoA/Net1 interaction are unknown. Taking advantage of a simple production and purification process, here we solved the structure of a RhoA/Net1 heterodimer with X-ray crystallography at 2-Å resolution. Using a panel of several techniques, including molecular dynamics simulations, we characterized the RhoA/Net1 interface. Moreover, deploying an extremely simple peptide-based scanning approach, we found that short peptides (penta- to nonapeptides) derived from the protein/protein interaction region of RhoA could disrupt the RhoA/Net1 interaction and thereby diminish the rate of nucleotide exchange. The most inhibitory peptide, EVKHF, spanning residues 102-106 in the RhoA sequence, displayed an IC50 of ∼100 µm without further modifications. The peptides identified here could be useful in further investigations of the RhoA/Net1 interaction region. We propose that our structural and functional insights might inform chemical approaches for transforming the pentapeptide into an optimized pseudopeptide that antagonizes Net1-mediated RhoA activation with therapeutic anticancer potential.

Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM.

Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation.

Magnetic resonance imaging of painful metal-on-metal total hip replacement.

Consecutive patients that had primary metal-on-metal (MoM) or metal-on-polyethylene (MoP) hip arthroplasty were prospectively enrolled to this study. All operated hips were evaluated with MRI by one radiologist who was blinded to the radiographic findings and clinical symptoms. Three groups of patients were formed: (1) thirteen MoM THRs in 13 patients with groin pain (Group 1), (2) ten MoM THRs in 10 patients with no pain (Group 2), (3) five MoP THRs in 4 patients without pain (control group). Abnormal MRI findings were distributed in all groups in a balanced way, irrespective of the patients' symptoms, prostheses, or metal ion levels. Two patients from Group 1 and one patient of Group 3 (control group) were diagnosed with a large periprosthetic mass (pseudotumor).

Pyridine to aniline: an exceptional biologically driven rearrangement.

During the course of our study on the innovative ligand for nicotinic acetylcholinergic receptors, LNAChR, and in order to assess activity and toxicity profiles of the drug's metabolites, synthesis of the main metabolites was undertaken. This synthesis work was done in parallel by organic chemistry and by biotransformation of LNAChR. Filamentous fungus Aspergillus alliaceus (NRRL 315) neatly afforded three of the main metabolites, one of which arose from a very unexpected and very uncommon rearrangement.

Presence of cobalt and chromium ions in the seminal fluid of young patients with metal-on-metal total hip arthroplasty.

We aimed to investigate the effect of metal ions on the semen of males of child fathering age with metal-on-metal (MM) total hip arthroplasty (THA). Semen was collected form 11 patients with MM THA and 5 control of comparable age. Cobalt and chromium concentrations were measured in both the seminal plasma and in the blood of patients. Results showed that cobalt level was higher in the seminal plasma of MM THA patients (2.89 µg/L) compared to control patients (1.12 µg/L) (P = .011). The ejaculate volume, the sperm density, the total sperm count, the pH, and the percentage of cells with normal morphology were in the range of the World Health Organization criteria for fertile population.

The structural basis for the interaction between the CAF1 nuclease and the NOT1 scaffold of the human CCR4-NOT deadenylase complex.

The CCR4-NOT complex plays a crucial role in post-transcriptional mRNA regulation in eukaryotic cells. It catalyzes the removal of mRNA poly(A) tails, thereby repressing translation and committing mRNAs to decay. The conserved core of the complex consists of a catalytic module comprising two deadenylases (CAF1/POP2 and CCR4a/b) and the NOT module, which contains at least NOT1, NOT2 and NOT3. NOT1 bridges the interaction between the two modules and therefore, acts as a scaffold protein for the assembly of the complex. Here, we present the crystal structures of the CAF1-binding domain of human NOT1 alone and in complex with CAF1. The NOT1 domain comprises five helical hairpins that adopt an MIF4G (middle portion of eIF4G) fold. This NOT1 MIF4G domain binds CAF1 through a pre-formed interface and leaves the CAF1 catalytic site fully accessible to RNA substrates. The conservation of critical structural and interface residues suggests that the NOT1 MIF4G domain adopts a similar fold and interacts with CAF1 in a similar manner in all eukaryotes. Our findings shed light on the assembly of the CCR4-NOT complex and provide the basis for dissecting the role of the NOT module in mRNA deadenylation.

Long-term follow-up and metal ion trend of patients with metal-on-metal total hip arthroplasty.

PURPOSE: Long-term studies are required to support the use of metal-on-metal (MoM) bearings in total hip arthroplasty (THA) given the concern about systemic metal ion release and reports of adverse local soft tissue reactions. The purpose of this study was to report the seven to 13-year clinical, radiographic, and metal ion results in patients following MoM THA. METHODS: We studied 163 prostheses after second-generation MoM THA between July 1997 and November 2003. Cobalt and chromium metal ions were collected using whole and analysed by inductively-coupled plasma-mass spectrometry. RESULTS: The mean follow-up was 8.87 years (range, 7-13 years). Four hips (2.5 %) were revised. The Kaplan-Meier survivorship was 91.3 % for revision for all causes, and 97.5 % when excluding the hips revised for a manufacturer's defect. Median whole blood cobalt levels peaked at a value of 2.87 µg/L at four years (p < 0.0001 vs. pre-operative) and subsequently decreased to 2.0 µg/L after nine years (p = 0.002 vs. four years). Median chromium levels maximally increased up to 0.75 µg/L after five years (p < 0.0001 vs. pre-operative) and tended to decrease thereafter to values of 0.56 µg/L after seven years. CONCLUSIONS: This seven to 13-year follow-up study indicates that the clinical and radiological results following MoM THA are satisfactory with low revision rates. Cobalt and chromium ion levels peaked at four and five years, respectively, and gradually decreased thereafter.

The effect of operative factors on outlier ion levels in patients with metal-on-metal hip arthroplasties.

UNLABELLED: Metal-on-metal (MoM) hip arthroplasties comprised of cobalt and chromium alloys continue to be successful alternatives to conventional bearings in younger patients with osteoarthritis (OA). A small proportion of patients have unusually high levels of cobalt and chromium ions postoperatively. Given the increasing prevalence of MoM bearings and the potential for cellular toxicity, the purpose of our study was to determine whether patient or surgical factors could account for abnormally elevated ion levels following MoM hip arthroplasty. MATERIALS AND METHODS: Cobalt and chromium levels were analyzed from whole blood in 761 patients with MoM hip arthroplasties. Patient outliers were defined as those who had ion levels greater than or equal to three-fold the median value. Thirty-four patients (4.5%) met this criteria. This included 20 patients who underwent standard total hip arthroplasty (THA) and hip resurfacing, respectively. Patients were followed prospectively with the Harris hip scores (HHS) and the University of California Los Angeles (UCLA) activity scores. Serial radiographs and ion levels were analyzed at regular intervals. RESULTS: At a mean follow-up of 2 years, the median values for outlier cobalt and chromium ions were 13.7 µg/L and 6.0 µg/L, respectively. Postoperative HHS and UCLA activity scores improved significantly when compared to pre-operative values. There was no statistical correlation between outlier ion levels, patient demographics, and HHS and UCLA activity scores. Acetabular inclination correlated with chromium values. Outlier cobalt levels were higher in patients after THA. CONCLUSIONS: We identified that various patient and surgical factors could account for some of the abnormally high metal ion levels in outliers. Further studies are necessary to better understand the effect of abnormal elevations in metal ions, given the recent concerns of adverse local soft tissue reactions following MoM hip implants.

Metal-on-metal total hip arthroplasty - five- to 11-year follow-up.

Metal-on-metal (MoM) total hip arthroplasty (THA) has been introduced in an attempt to reduce the wear rate and the consequent osteolysis around implants. The aim of this study was to present the intermediate to long-term clinical and radiological outcomes and to investigate the metal ion levels in the blood of patients who had undergone primary uncemented MoM THA in our institution. Between July 1997 and November 2003, 166 patients (193 hips), with a mean age of 50 years (range, 18-65 years), underwent primary MoM THA. Clinical data, radiographs, and blood samples were obtained at regular follow-up visits. Cobalt (Co), chromium (Cr), and molybdenum (Mo) ions were measured by inductively coupled plasma-mass spectrometry (ICP-MS) from the patient's whole blood. All patients were prospectively followed for a minimum of 5 years (mean, 7 years; range, 5-11 years). The mean Harris hip score (HHS) and the University of California at Los Angeles (UCLA) activity score at the latest follow-up was 88 ± 11 and 7 ± 1.8 points, respectively. Thirteen hips have been revised. Ten acetabular components had early failure, due to factory manufacturing problems. All other implants have been found stable, with no signs of aseptic loosening. The probability of survival at 11 years, if the hips that were revised due to manufacturing problems were excluded, was 98.4%. The Co and Cr metal ion levels, after increasing significantly during the first 4 to 5 years post-surgery, remained stable, with a tendency to decrease thereafter, but not significantly. During the same follow-up period, Mo ion levels remained stable. In this 5-to-11 year follow-up study of MoM THA patients, excellent survivorship, with low complications rates, was found. Results of longer follow-up studies are necessary to clarify the possible long-term effects of metal ion release.

Titania-hydroxyapatite nanocomposite coatings support human mesenchymal stem cells osteogenic differentiation.

In addition to mechanical and chemical stability, the third design goal of the ideal bone-implant coating is the ability to support osteogenic differentiation of mesenchymal stem cells (MSCs). Plasma-sprayed TiO(2)-based bone-implant coatings exhibit excellent long-term mechanical properties, but their applications in bone implants are limited by their bioinertness. We have successfully produced a TiO(2) nanostructured (grain size <50 nm) based coating charged with 10% wt hydroxyapatite (TiO(2)-HA) sprayed by high-velocity oxy-fuel. On Ti64 substrates, the novel TiO(2)-HA coating bond 153× stronger and has a cohesive strength 4× higher than HA coatings. The HA micro- and nano-sized particles covering the TiO(2)-HA coating surface are chemically bound to the TiO(2) coating matrix, producing chemically stable coatings under high mechanical solicitations. In this study, we elucidated the TiO(2)-HA nanocomposite coating surface chemistry, and in vitro osteoinductive potential by culturing human MSCs (hMSCs) in basal and in osteogenic medium (hMSC-ob). We assessed the following hMSCs and hMSC-ob parameters over a 3-week period: (i) proliferation; (ii) cytoskeleton organization and cell-substrate adhesion; (iii) coating-cellular interaction morphology and growth; and (iv) cellular mineralization. The TiO(2) -HA nanocomposite coatings demonstrated 3× higher hydrophilicity than HA coatings, a TiO(2)-nanostructured surface in addition to the chemically bound HA micron- and nano-sized rod to the surface. hMSCs and hMSC-ob demonstrated increased proliferation and osteoblastic differentiation on the nanostructured TiO(2)-HA coatings, suggesting the TiO(2)-HA coatings nanostructure surface properties induce osteogenic differentiation of hMSC and support hMSC-ob osteogenic potential better than our current golden standard HA coating.

Stem cells, nitrogen-rich plasma-polymerized culture surfaces, and type X collagen suppression.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into chondrocytes, osteoblasts, myocytes, adipocytes, and a variety of other cell types. Several studies have been directed toward using MSCs from patients with osteoarthritis (OA) for cartilage repair, not only because these are the ones that will require a source of autologous stem cells if biological repair of cartilage lesions is to be a therapeutic option, but also to further an understanding of stem cell differentiation. Previous studies have shown that a major drawback of current cartilage and intervertebral disc tissue repair is that human MSCs from OA patients express type X collagen (COL X). COL X, a marker of late-stage chondrocyte hypertrophy, is implicated in endochondral ossification. However, those studies also revealed that a novel plasma-polymerized thin film material, named nitrogen-rich plasma-polymerized ethylene (PPE:N), was able to inhibit COL X expression in committed MSCs. The specific aim of this present study was to determine if the suppression of COL X by PPE:N is maintained when MSCs are transferred to pellet cultures in serum-free media. Our results confirmed the potential of two different types of PPE:N surfaces (low-pressure-PPE:N [L-PPE:N] and high-pressure-PPE:N [H-PPE:N]) in suppressing COL X expression, more so on the latter. Interestingly, when MSCs were transferred to pellet cultures, the expression level of COL X was further decreased by preincubation on H-PPE:N, suggesting that these kinds of coatings show promise for tissue engineering of cartilage and disc tissues. Further studies are needed to assess the relative importance of surface-chemistry versus surface-morphology in the mechanism of COL X suppression.

Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes.

BACKGROUND: Recent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.% ) of N. METHODS: In the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes) cultured on PPE:N. We also studied the effect of PPE:N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13) and molecules implicated in cell division (cyclin B2). Two other culture surfaces, "hydrophilic" polystyrene (PS, regular culture dishes) and nitrogen-containing cation polystyrene (Primaria®), were also investigated for comparison. RESULTS: Results showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE:N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels. CONCLUSION: Hypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such "intelligent surfaces" over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving.

Effect of synthetic link N peptide on the expression of type I and type II collagens in human intervertebral disc cells.

Intervertebral disc (IVD) degeneration is associated with proteolytic degradation of proteoglycan aggregates present within the extracellular matrix of the disc. Link N peptide (DHLSDNYTLDHDRAIH) is the N-terminal peptide of link protein, which stabilizes the proteoglycan aggregates. It is generated in vivo by proteolytic degradation during tissue turnover. It has been previously shown that this peptide can stimulate the synthesis of collagens by articular cartilage and bovine IVD cells in vitro. Being a synthetic peptide, Link N has considerable financial benefits for clinical use over recombinant growth factors because it is extremely cheap to produce. The purpose of the present study was to determine the effect of Link N on the expression of types I and II collagen and investigate the cellular mechanisms of Link N signal transduction in human IVD cells. The present results suggest that Link N stimulates the expression of types I and II collagen in human IVD cells. More specifically, Link N stimulated the expression of type I in nucleus pulposus (NP) cells, but not in annulus fibrosus cells. As Link N also decreased the phosphorylation of p38 in NP cells only, results suggest that p38 is a mediator of the effect of Link N on type I collagen expression. p38 is a member of the mitogen-activated protein kinase family highlighted by three major cascades: p38, c-Jun amino-terminal kinase, and extracellular signal-regulated kinase pathways. Link N showed no effect on the latter two pathways, suggesting a specific effect of Link N on the p38 cascade. On the other hand, Link N stimulated the expression of type II collagen in both NP and annulus fibrosus, suggesting that other mechanisms are implicated in the control of type II collagen expression in disc cells, without excluding p38 for the NP. In conclusion, the present study showed that Link N can modulate the expression of collagen in human IVD cells.

Effect of sterilization on non-woven polyethylene terephthalate fiber structures for vascular grafts.

Non-woven polyethylene terephthalate (PET) fibers produced via melt blowing and compounded into a 6 mm diameter 3D tubular scaffold were developed with artery matching mechanical properties. This work compares the effects of ethylene oxide (EtO) and low temperature plasma (LTP) sterilization on PET surface chemistry and biocompatibility. As seen through X-ray photoelectron spectroscopy (XPS) analysis, LTP sterilization led to an increase in overall oxygen content and the creation of new hydroxyl groups. EtO sterilization induced alkylation of the PET polymer. The in vitro cytotoxicity showed similar fibroblastic viability on LTP- and EtO-treated PET fibers. However, TNF-α release levels, indicative of macrophage activation, were significantly higher when macrophages were incubated on EtO-treated PET fibers. Subcutaneous mice implantation revealed an inflammatory response with foreign body reaction to PET grafts independent of the sterilization procedure.