Differences in the Synovial Fluid Proteome of Septic and Aseptic Implant Failure.

Implant loosening is a severe complication after total joint replacement. Here, differential diagnosis between septic and aseptic cases is crucial for further surgical treatment, but low-grade periprosthetic joint infections (PJIs) in particular remain a challenge. In this study, we analyzed the synovial fluid proteome of 21 patients undergoing revision surgery for septic (eight cases) or aseptic (thirteen cases) implant failure using LC-MS/MS to identify potential new biomarkers as future diagnostic tools. Staphylococci were found in four cases, Streptococci in two cases, Serratia marcescens and Cutibacterium acnes in one case. Proteomic analysis of the synovial fluid resulted in the identification of 515 different proteins based on at least two peptides. A statistical comparison revealed 37 differentially abundant proteins (p < 0.05), of which 17 proteins (46%) showed a higher abundance in the septic group. The proteins with the highest fold change included the known marker proteins c-reactive protein (7.57-fold) and the calprotectin components protein S100-A8 (4.41-fold) and protein S100-A9 (3.1-fold). However, the protein with the highest fold change was leucine-rich alpha-2-glycoprotein 1 (LRG1) (9.07-fold), a currently discussed new biomarker for inflammatory diseases. Elevated LRG1 levels could facilitate the diagnosis of PJI in the future, but their significance needs to be further investigated.

Surgical Site-Released Tissue Is Potent to Generate Bone onto TCP and PCL-TCP Scaffolds In Vitro.

There is evidence that surgical site tissue (SSRT) released during orthopedic surgery has a strong mesenchymal regenerative potential. Some data also suggest that this tissue may activate synthetic or natural bone substitute materials and can thus upgrade its osteopromoting properties. In this comparative in vitro study, we investigate the composition of SSRT during total hip replacement (n = 20) harvested using a surgical suction handle. In addition, the osteopromoting effect of the cells isolated from SSRT is elucidated when incubated with porous beta-tricalcium phosphate (ß-TCP) or 80% medical-grade poly-ε-caprolactone (PCL)/20% TCP composite material. We identified multiple growth factors and cytokines with significantly higher levels of PDGF and VEGF in SSRT compared to peripheral blood. The overall number of MSC was 0.09 ± 0.12‱ per gram of SSRT. A three-lineage specific differentiation was possible in all cases. PCL-TCP cultures showed a higher cell density and cell viability compared to TCP after 6 weeks in vitro. Moreover, PCL-TCP cultures showed a higher osteocalcin expression but no significant differences in osteopontin and collagen I synthesis. We could demonstrate the high regenerative potential from SSRT harvested under vacuum in a PMMA filter device. The in vitro data suggest advantages in cytocompatibility for the PCL-TCP composite compared to TCP alone.

The Implant Proteome-The Right Surgical Glue to Fix Titanium Implants In Situ.

Titanium implants are frequently applied to the bone in orthopedic and trauma surgery. Although these biomaterials are characterized by excellent implant survivorship and clinical outcomes, there are almost no data available on the initial protein layer binding to the implant surface in situ. This study aims to investigate the composition of the initial protein layer on endoprosthetic surfaces as a key initiating step in osseointegration. In patients qualified for total hip arthroplasty, the implants are inserted into the femoral canal, fixed and subsequently explanted after 2 and 5 min. The proteins adsorbed to the surface (the implant proteome) are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A statistical analysis of the proteins' alteration with longer incubation times reveals a slight change in their abundance according to the Vroman effect. The pathways involved in the extracellular matrix organization of bone, sterile inflammation and the beginning of an immunogenic response governed by neutrophils are significantly enriched based on the analysis of the implant proteome. Those are generally not changed with longer incubation times. In summary, proteins relevant for osseointegration are already adsorbed within 2 min in situ. A deeper understanding of the in situ protein-implant interactions in patients may contribute to optimizing implant surfaces in orthopedic and trauma surgery.

Supramolecular Enhancement of a Natural 14-3-3 Protein Ligand.

Rational design of protein-protein interaction (PPI) inhibitors is challenging. Connecting a general supramolecular protein binder with a specific peptidic ligand provides a novel conceptual approach. Thus, lysine-specific molecular tweezers were conjugated to a peptide-based 14-3-3 ligand and produced a strong PPI inhibitor with 100-fold elevated protein affinity. X-ray crystal structure elucidation of this supramolecular directed assembly provides unique molecular insight into the binding mode and fully aligns with Molecular Dynamics (MD) simulations. This new supramolecular chemical biology concept opens the path to novel chemical tools for studying PPIs.

Functionalization of Synthetic Bone Substitutes.

Bone substitutes have been applied to treat osseous defects for a long time. To prevent implant related infection (IRI) and enhance bone healing functionalized biomaterials, antibiotics and osteoinductive substances have been introduced. This study gives an overview of the current available surface-coated bone substitutes and provides an outlook for future perspectives.

Supramolecular Mechanism of Viral Envelope Disruption by Molecular Tweezers.

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the "phosphate tweezer" CLR01, a "carboxylate tweezer" CLR05, and a "phosphate clip" PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.

Intrasurgical Protein Layer on Titanium Arthroplasty Explants: From the Big Twelve to the Implant Proteome.

PURPOSE: Aseptic loosening in total joint replacement due to insufficient osteointegration is an unsolved problem in orthopaedics. The purpose of the study is to obtain a picture of the initial protein adsorption layer on femoral endoprosthetic surfaces as the key to the initiation of osseointegration. EXPERIMENTAL DESIGN: The paper describes the first study of femoral stem explants from patients for proteome analysis of the primary protein layer. After 2 min in situ, the stems are explanted and frozen in liquid nitrogen. Proteins are eluted under reducing conditions and analyzed by LC-MS/MS. RESULTS: After exclusion of proteins identified by a single peptide, the implant proteome is found to consist of 2802 unique proteins. Of these, 77% are of intracellular origin, 9% are derived from the plasma proteome, 8% from the bone proteome, and four proteins with highest specificity score could be assigned to the bone marrow proteome (transcriptome). The most abundant protein in the adsorbed total protein layer is hemoglobin (8-11%) followed by serum albumin (3.6-6%). CONCLUSIONS: A detailed knowledge of the initial protein film deposited onto the implants, as demonstrated here for the first time, may help to understand and predict the response of the osseous microenvironment to implant surfaces.

Molecular Tweezers with Additional Recognition Sites.

A new synthetic access to molecular tweezers with one or two aliphatic phosphate ester groups in the central benzene spacer-unit is presented. Alkynyl ester groups offer the prospect to attach additional functional units by click chemistry and greatly broaden the scope of these tools for chemical biology. We present two alternative strategies: the trichloroacetonitrile method involves activation of only one OH group of each phosphoric acid substituent by way of trichloroacetimidate intermediates and subsequent introduction of an aliphatic ester alcohol moiety. The method is versatile, robust and combines simple workup with high yields. Mono- and disubstituted novel host structures are thus accessible in a convenient way. Alternatively, the phosphoramidite strategy activates the hydroquinone precursor by way of phosphoramidite intermediates and couples the desired ester alcohols followed by mild oxidation to the desired phosphate esters. Each step of the synthesis is carried out at very mild conditions and allows to combine sensitive host candidates and recognition elements. After neutralization of the phosphoric acids to water-soluble tri- and tetra-anions the cavities of the new tweezer derivatives are open to bind lysine and arginine as well as peptidic guests. The concept of introducing clickable alkynyl phosphates to free OH groups may be transferred to other major macrocyclic host classes to introduce additional recognition elements, biomolecules or fluorescence labels.

The molecular tweezer CLR01 inhibits Ebola and Zika virus infection.

Ebola (EBOV) and Zika viruses (ZIKV) are responsible for recent global health threats. As no preventive vaccines or antiviral drugs against these two re-emerging pathogens are available, we evaluated whether the molecular tweezer CLR01 may inhibit EBOV and ZIKV infection. This small molecule has previously been shown to inactivate HIV-1 and herpes viruses through a selective interaction with lipid-raft-rich regions in the viral envelope, which results in membrane disruption and loss of infectivity. We found that CLR01 indeed blocked infection of EBOV and ZIKV in a dose-dependent manner. The tweezer inhibited infection of epidemic ZIKV strains in cells derived from the anogenital tract and the central nervous system, and remained antivirally active in the presence of semen, saliva, urine and cerebrospinal fluid. Our findings show that CLR01 is a broad-spectrum inhibitor of enveloped viruses with prospects as a preventative microbicide or antiviral agent.

The Molecular Tweezer CLR01 Stabilizes a Disordered Protein-Protein Interface.

Protein regions that are involved in protein-protein interactions (PPIs) very often display a high degree of intrinsic disorder, which is reduced during the recognition process. A prime example is binding of the rigid 14-3-3 adapter proteins to their numerous partner proteins, whose recognition motifs undergo an extensive disorder-to-order transition. In this context, it is highly desirable to control this entropy-costly process using tailored stabilizing agents. This study reveals how the molecular tweezer CLR01 tunes the 14-3-3/Cdc25CpS216 protein-protein interaction. Protein crystallography, biophysical affinity determination and biomolecular simulations unanimously deliver a remarkable finding: a supramolecular "Janus" ligand can bind simultaneously to a flexible peptidic PPI recognition motif and to a well-structured adapter protein. This binding fills a gap in the protein-protein interface, "freezes" one of the conformational states of the intrinsically disordered Cdc25C protein partner and enhances the apparent affinity of the interaction. This is the first structural and functional proof of a supramolecular ligand targeting a PPI interface and stabilizing the binding of an intrinsically disordered recognition motif to a rigid partner protein.

An NMR Method To Pinpoint Supramolecular Ligand Binding to Basic Residues on Proteins.

Targeting protein surfaces involved in protein-protein interactions by using supramolecular chemistry is a rapidly growing field. NMR spectroscopy is the method of choice to map ligand-binding sites with single-residue resolution by amide chemical shift perturbation and line broadening. However, large aromatic ligands affect NMR signals over a greater distance, and the binding site cannot be determined unambiguously by relying on backbone signals only. We herein employed Lys- and Arg-specific H2(C)N NMR experiments to directly observe the side-chain atoms in close contact with the ligand, for which the largest changes in the NMR signals are expected. The binding of Lys- and Arg-specific supramolecular tweezers and a calixarene to two model proteins was studied. The H2(C)N spectra track the terminal CH2 groups of all Lys and Arg residues, revealing significant differences in their binding kinetics and chemical shift perturbation, and can be used to clearly pinpoint the order of ligand binding.

A molecular tweezer antagonizes seminal amyloids and HIV infection.

Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion-amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses.