Staphylococcal Protein Research Articles

Human Plasma Significantly Reduces Bacteriophage Infectivity Against Staphylococcus aureus Clinical Isolates.

Bacteriophage therapy has been regaining interest as a potential therapeutic in treating a wide range of infections. However, there is a paucity of knowledge regarding numerous aspects of bacteriophage therapy, thereby hindering the development of proper treatment protocols and effective clinical trials. In this report, the activities of three bacteriophages are evaluated against clinical bacterial isolates in the presence and absence of human plasma (HP). The bacteriophages used in this experiment were residual therapeutic doses from the United States Food and Drug Administration (FDA) approved compassionate use cases to treat recalcitrant prosthetic joint infections (PJIs). Herein we demonstrate that in the presence of HP, the infectivity of these Staphylococcal bacteriophages was significantly reduced compared to the infectivity in the absence of HP. Inhibition of infectivity ranged from 48% to 81% for two methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates independently infected with the same bacteriophage and 98% for a third MRSA clinical isolate infected with a different bacteriophage. In contrast, bacteriophage infectivity of an Enterococcus faecalis clinical isolate was not affected by the presence of HP. We hypothesize that the inhibition is correlated with plasma proteins binding to Staphylococcal surface proteins masking the receptors associated with bacteriophage attachment, thereby reducing infectivity. This has clinical ramifications for bacteriophage therapy use in treating Staphylococcal bacteremia and periprosthetic joint infections.

Mitochondrion-Localized SND1 Promotes Mitophagy and Liver Cancer Progression Through PGAM5.

Staphylococcal nuclease domain-containing protein 1 (SND1) is an evolutionarily conserved multifunctional protein that functions mainly in the nucleus and cytoplasm. However, whether SND1 regulates cellular activity through mitochondrial-related functions remains unclear. Herein, we demonstrate that SND1 is localized to mitochondria to promote phosphoglycerate mutase 5 (PGAM5)-mediated mitophagy. We find that SND1 is present in mitochondria based on mass spectrometry data and verified this phenomenon in different liver cancer cell types by performing organelle subcellular isolation. Specifically, The N-terminal amino acids 1-63 of SND1 serve as a mitochondrial targeting sequence (MTS), and the translocase of outer membrane 70 (TOM 70) promotes the import of SND1 into mitochondria. By immunoprecipitation-mass spectrometry (IP-MS), we find that SND1 interacts with PGAM5 in mitochondria and is crucial for the binding of PGAM5 to dynamin-related protein 1 (DRP1). Importantly, we demonstrate that PGAM5 and SND1-MTS are required for SND1-mediated mitophagy under FCCP and glucose deprivation treatment as well as for SND1-mediated cell proliferation and tumor growth both in vitro and in vivo. Aberrant expression of SND1 and PGAM5 predicts poor outcomes in hepatocellular carcinoma (HCC) patients. Taken together, these findings establish a previously unappreciated role of SND1 and the association of mitochondrion-localized SND1 with PGAM5 in mitophagy and tumor progression.

Staphylococcal Protein A with Engineered Cysteine: Comparison of Monomeric Content as a Critical Quality Attribute during Intracellular and Extracellular Expression

Background: The introduction of engineered cysteine in staphylococcal protein A (SPA-cys) for site-specific conjugation results in a substantial amount of dimerized SPA due to spontaneous oxidation during its production, leading to inaccessibility and thus rendering it unusable. Monomers are usually recovered from dimers by using reducing agents before conjugation in subsequent steps. However, this leads to low conjugation efficiency and increases overall cost and production time. This study aims to systematically compare and quantify the monomeric and dimeric content of SPA when produced through intracellular and extracellular routes in E. coli. Methods: Purified SPAs with and without cysteine from both intracellular and extracellular processes are compared for their monomeric content and efficiency to conjugate on solid support matrix with and without an additional pre-step of reduction. Results: The monomeric form of SPA-cys, which is a desired key quality attribute, is less than 50% when produced extracellularly. SPA-cys produced through the intracellular production process has high monomeric content (≥85%) and shows higher binding to solid support. Conclusion: The study demonstrates that the intracellular route for production of SPA-cys should be the preferred method, and the release assays for SPA-cys products should include the amount of monomeric content as one of the quality attributes. The abundance of monomeric content enhances the site-specific conjugation efficiency and density of SPA on the resin matrix.

Determination of Abrin by a Magnetic Affinity Immunoassay (MAIA) Based on the Signal Amplification of the Aptamer and Staphylococcal Protein A (SPA) Functionalized Gold Magnetic Microparticles (GMPs)

A new magnetic affinity immunoassay (MAIA) was developed for the determination of abrin based on signal amplification of staphylococcal protein A (SPA) functionalized gold magnetic microparticles (GMPs) and the aptamer. This assay consisted of a sandwich format in which SPA-coated GMPs coupled anti-abrin monoclonal antibodies (mcAb) were used as the magnetic capture probe and the enzyme-labeled abrin aptamer was used as the signal probe. Abrin was successfully determined by the proposed strategy. This method exhibited a linear response to abrin from 0.031 to 31.25 μg/L with a detection limit of 0.031 μg/L. The staphylococcal protein A, gold magnetic microparticles, and aptamer increased the sensitivity by 4 times, 2 times and 2 times, respectively The integrated amplification produced by these parameters increased the sensitivity by 16-fold compared to the traditional double-antibody sandwich enzyme linked immunosorbent assay (ELISA). This method possesses a low detection limit, acceptable reproducibility, and high specificity, which demonstrates that the MAIA shows promise in trace toxin determination.

Multiple Domains of Staphylococcal Superantigen-like Protein 11 (SSL11) Contribute to Neutrophil Inhibition.

Staphylococcus aureus is an opportunistic pathogen producing many immune evasion molecules targeting various components of the host immune defense, including the Staphylococcal superantigen-like protein (SSL 1-14) family. Despite sharing similar structures with the powerful superantigens (SAgs), which cause massive T cell activation, SSLs interfere with a wide range of innate immune defenses. SSLs are divided into two subgroups, SSLs that contain a conserved carbohydrate Sialyl Lewis X [Neu5Acα2-3Galβ1-4(Fucα1-3) GlcNAcβ, SLeX] binding site and SSLs that lack the SLeX binding site. SSL2-6 and SSL11 possess the SLeX binding site. Our previous studies showed that SSL11 arrests cell motility by inducing cell adhesion in differentiated HL60 (dHL60) cells, while SSL7 did not bind dHL60 cells. SSL7-based chimeras were engineered by exchanging the SSL7 sequence with the corresponding SSL11 sequence and assaying for a gain of SSL11 function, namely, the induction of cell spreading and motility arrest. In addition to the SLeX-binding site, we observed that three beta-strands β6, β7, and β9 and the N-terminal residues, Y16 and Y17, transitioned SSL7 to gain SSL11 activities. These studies define the structure-function properties of SSL11 that may allow SSL11 to inhibit S. aureus clearance by the host innate immune system, allowing S. aureus to maintain a carrier state in humans, an understudied aspect of S. aureus pathogenesis.

Natural Human Immunity Against Staphylococcal Protein A Relies on Effector Functions Triggered by IgG3.

Staphylococcal protein A (SpA) is a multifunctional, highly conserved virulence factor of Staphylococcus aureus. By binding the Fc portion of all human IgG subclasses apart from IgG3, SpA interferes with antibody and complement deposition on the bacterial surface, impairing staphylococcal clearance by phagocytosis. Because of its anti-opsonic properties, SpA is not investigated as a surface antigen to mediate bacterial phagocytosis. Herein we investigate human sera for the presence of SpA-opsonizing antibodies. The screening revealed that sera containing IgG3 against SpA were able to correctly opsonize the target and drive Fcγ receptor-mediated interactions and phagocytosis. We demonstrated that IgG3 Fc is significantly more efficient in inducing phagocytosis of SpA-expressing S. aureus as compared to IgG1 Fc in an assay resembling physiological conditions. Furthermore, we show that the capacity of SpA antibodies to induce phagocytosis depends on the specific epitope recognized by the IgGs on SpA molecules. Overall, our results suggest that anti-SpA IgG3 antibodies could favor the anti-staphylococcal response in humans, paving the way towards the identification of a correlate of protection against staphylococcal infections.

A peptide from the staphylococcal protein Efb binds P‐selectin and inhibits the interaction of platelets with leukocytes

AimsP‐selectin is a key surface adhesion molecule for the interaction of platelets with leukocytes. We have shown previously that the N‐terminal domain of Staphylococcus aureus extracellular fibrinogen‐binding protein (Efb) binds to P‐selectin and interferes with platelet‐leukocyte aggregate formation. Here, we aimed to identify the minimal Efb motif required for binding platelets and to characterize its ability to interfering with the formation of platelet‐leukocyte aggregates. Methods and ResultsUsing a library of synthetic peptides, we mapped the platelet‐binding site to a continuous 20 amino acid stretch. The peptide Efb68‐87 was able to bind to resting and, to a greater extent, thrombin‐stimulated platelets in the absence of fibrinogen. Dot blots, pull‐down assays and P‐selectin glycoprotein ligand‐1 (PSGL‐1) competitive binding experiments identified P‐selectin as the cellular docking site mediating Efb68‐87 platelet binding. Accordingly, Efb68‐87 did not bind to other blood cells and captured platelets from human whole blood under low shear stress conditions. Efb68‐87 did not affect platelet activation as tested by aggregometry, flow cytometry and immunoblotting, but inhibited the formation of platelet‐leukocyte aggregates (PLAs). Efb68‐87 also interfered with the platelet‐dependent stimulation of neutrophil extracellular traps (NETs) formation in vitro. ConclusionsWe have identified Efb68‐87 as a novel selective platelet‐binding peptide. Efb68‐87 binds directly to P‐selectin and inhibits interactions of platelets with leukocytes that lead to PLA and NET formation. As PLAs and NETs play a key role in thromboinflammation, Efb68‐87 is an exciting candidate for the development of novel selective inhibitors of the proinflammatory activity of platelets.

Bacterial ligands as flexible and sensitive detectors in rapid tests for antibodies to SARS-CoV-2

Lateral flow immunoassay (LFIA) is widely employed as point-of-care tests (POCT) for the diagnosis of infectious diseases. The accuracy of LFIA largely depends on the quality of the immunoreagents used. Typical LFIAs to reveal the immune response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) employ anti-human immunoglobulin (hIG) antibodies and recombinant viral antigens, which usually are unstable and poorly soluble. Broad selective bacterial proteins, such as Staphylococcal protein A (SpA) and Streptococcal protein G (SpG) can be considered alternatives to anti-hIG to increase versatility and sensitivity of serological LFIAs because of their high binding capacity, interspecies reactivity, and robustness. We developed two colorimetric LFA devices including SpA and SpG linked to gold nanoparticles (GNP) as detectors and explored the use of a specific, stable, and soluble immunodominant fraction of the nucleocapsid protein from SARS-CoV-2 as the capturing agent. The optimal amount of SpA-GNP and SpG-GNP conjugates and the protein-to-GNP ratios were defined through a full factorial experimental design to maximize the diagnostic sensitivity of the LFIAs. The new LFA devices were applied to analyze 105 human serum samples (69 positive and 36 negatives according to reference molecular diagnostic methods). The results showed higher sensitivity (89.9%, 95% CI 82.7–97.0) and selectivity (91.7%, 82.6–100) for the SpA-based compared to the SpG-based LFA. In addition, 18 serum samples from cats and dogs living with COVID-19 patients were analyzed and 14 showed detectable levels of anti-SARS-CoV-2 antibodies, thus illustrating the flexibility of the SpA- and SpG-based LFAs.Graphical abstract

More Than Meets the Kappa for Antibody Superantigen Protein L (PpL).

Immunoglobulin superantigens play an important role in affinity purification of antibodies and the microbiota-immune axis at mucosal areas. Based on current understanding, Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG) and Finegoldia Protein L (PpL) are thought to only bind specific regions of human antibodies, allowing for selective purification of antibody isotypes and chains. Clinically, these superantigens are often classified as toxins and increase the virulence of the producing pathogen through unspecific interactions with immune proteins. To perform an in-depth interaction study of these three superantigens with antibodies, bio-layer interferometry (BLI) measurements of their interactions with a permutation panel of 63 IgG1 variants of Pertuzumab and Trastuzumab CDRs grafted to the six human Vκ and seven human VH region families were tested. Through this holistic and systemic analysis of IgG1 variants with various antibody regions modified, comparisons revealed novel PpL–antibody interactions influenced by other non-canonical antibody known light-chain framework regions, whereas SpA and SpG showed relatively consistent interactions. These findings have implications on PpL-based affinity antibody purification and design that can guide the engineering and understanding of PpL-based microbiota-immune effects.

SxsA, a novel surface protein mediating cell aggregation and adhesive biofilm formation of Staphylococcus xylosus.

Biofilm formation of staphylococci has been an emerging field of research for many years. However, the underlying molecular mechanisms are still not fully understood and vary widely between species and strains. The aim of this study was to identify new effectors impacting biofilm formation of two Staphylococcus xylosus strains. We identified a novel surface protein conferring cell aggregation, adherence to abiotic surfaces, and biofilm formation. The S. xylosus surface protein A (SxsA) is a large protein occurring in variable sizes. It lacks sequence similarity to other staphylococcal surface proteins but shows similar structural domain organization and functional features. Upon deletion of sxsA, adherence of S. xylosus strain TMW 2.1523 to abiotic surfaces was completely abolished and significantly reduced in TMW 2.1023. Macro- and microscopic aggregation assays further showed that TMW 2.1523 sxsA mutants exhibit reduced cell aggregation compared with the wildtype. Comparative genomic analysis revealed that sxsA is part of the core genome of S. xylosus, Staphylococcus paraxylosus, and Staphylococcus nepalensis and additionally encoded in a small group of Staphylococcus cohnii and Staphylococcus saprophyticus strains. This study provides insights into protein-mediated biofilm formation of S. xylosus and identifies a new cell wall-associated protein influencing cell aggregation and biofilm formation.

Ti3C2-MXene-assisted signal amplification for sensitive and selective surface plasmon resonance biosensing of biomarker

Surface plasmon resonance (SPR) technology has become one of the powerful tools for real-time bioassay of various targets without needing labels. However, SPR biosensing faces great challenge with the extension of application fields due to the limited sensitivity. Herein, we report an ultrasensitive SPR biosensing strategy for detecting carcinoembryonic antigen (CEA) with the introduction of amino-functionalized Ti3C2-MXene (N-Ti3C2-MXene) nanosheets. The ultrathin Ti3C2-MXene nanosheets were prepared by hydrochloric acid (HF) etching and further modified by (3-aminopropyl) triethoxysilane (APTES) to obtain amino terminals for conjugating polyclonal anti-CEA antibody (Ab2) via covalent bonding. Staphylococcal protein A (SPA) decorated Au film was utilized as sensing platform to orientedly immobilize monoclonal anti-CEA antibody (Ab1) via its Fc region. The N-Ti3C2-MXene-Ab2 nanoconjugates were introduced to the sensing system after capture of CEA, forming sandwiched immunocomplexes on the SPR chip. A signal amplification strategy of Ti3C2-MXene-induced spontaneous gold reductive deposition on Ti3C2-MXene nanosheets was employed to further enhance sensitivity. The proposed biosensor exhibits a wide linear range for CEA determination of 10−11 to 10−6 g mL−1 with a detection limit of 1.7 pg mL−1 (S/N=3). Moreover, good reproducibility and high specificity were demonstrated by serum samples analysis, which indicates the present method holds potential in evaluating CEA in human serum for early diagnosis and monitoring of cancer.

Mechanism for Bioactive Nanomaterial circ0024831 Regulation of Staphylococcal Nuclease Domain Containing 1 via RNA Methylation Recognition in Osteosarcoma

Bioactive nanomaterial circular RNA (circRNA) is an important non-coding RNA with a strong specificity, stable structure and high expression abundance. It can affect many diseases and physiological processes and may become a new way of disease diagnosis and targeted therapy. Recent studies have shown that Staphylococcal Nuclease Domain-Containing Protein 1 (SND1) can recognize N6-methyladenine (M6A) modified mRNA and regulate target mRNA stability. It can then control the expression of a series of downstream genes. However, whether SND1 can directly combine with circRNA and regulate its stability and function are new issues to be discussed. Results showed bioactive nanomaterial circ0024831 could directly bind to the Tudor domain of SND1 in the cytoplasm to block the recognition of SND1 to M6A modified RNA thus reducing the stability of downstream target gene mRNA and inhibiting the expression of downstream regulatory proteins. The down-regulation of circ0024831 expression in osteosarcoma cells relieved inhibition of SND1 which lead to change of tumor-related gene expression profile, promoting the occurrence and development of osteosarcoma.

Extracellular fibrinogen-binding protein (Efb), a key immune evasion protein of Staphylococcus aureus and a potential therapeutic target

Due to the continued growth of antibiotic-resistant Staphylococcus aureus strains, it is necessary to explore alternative targets for future therapeutic applications. For this reason, is important to understand the staphylococcal immune evasion mechanisms with a special focus on extracellular fibrinogen-binding protein (Efb) and Efb related proteins. Therefore, it was conducted a literature review to compile relevant information on this protein. It was found that Efb has three binding sites with biological relevance that could be used as therapeutic targets with specificity for fibrinogen, platelets, and complements. First, the fibrinogen-binding motifs also found in coagulase block neutrophil αMβ2 adherence to fibrinogen and attract fibrinogen to the bacterial surface, forming capsule-like structures that block phagocytosis. Second, Efb is a potent anti-thrombotic agent, probably related to its P-selectin binding capacity. Efb P-selectin binding blocks the interaction of P-selectin with the PSGL-1 receptor, thereby impairs the mechanisms of platelet-mediated leukocyte recruitment to the site of vascular injury. Third, the Efb complement binding domain, also found in other staphylococcal complement inhibitory proteins like Ecb, Sbi, and SCIN, is responsible for the evasion of the complement-mediated immune response. Efb reduces the formation of C3 convertase and the interaction with neutrophils, affect B-cells activation, and maturation. Efb binding sites have a clear implication on the virulence of Staphylococcus aureus in mastitis, wound infection, staphylococcal pneumonia, and infections related to implanted devices, and contributes to staphylococcal persistence in host tissues and abscess formation in the kidneys. Given the biological relevance of Efb binding sites in staphylococcal infections, they are promising vaccine targets. Additionally, due to the inhibitory effect of Efb on platelets and complements, Efb can be a potential therapeutic agent to treat diseases associated with thrombosis and abnormal complement activity.

Engineered human antibodies for the opsonization and killing of Staphylococcus aureus

Gram-positive organisms with their thick envelope cannot be lysed by complement alone. Nonetheless, antibody-binding on the surface can recruit complement and mark these invaders for uptake and killing by phagocytes, a process known as opsonophagocytosis. The crystallizable fragment of immunoglobulins (Fcγ) is key for complement recruitment. The cell surface of S. aureus is coated with Staphylococcal protein A (SpA). SpA captures the Fcγ domain of IgG and interferes with opsonization by anti-S. aureus antibodies. In principle, the Fcγ domain of therapeutic antibodies could be engineered to avoid the inhibitory activity of SpA. However, the SpA-binding site on Fcγ overlaps with that of the neonatal Fc receptor (FcRn), an interaction that is critical for prolonging the half-life of serum IgG. This evolutionary adaptation poses a challenge for the exploration of Fcγ mutants that can both weaken SpA-IgG interactions and retain stability. Here, we use both wild-type and transgenic human FcRn mice to identify antibodies with enhanced half-life and increased opsonophagocytic killing in models of S. aureus infection and demonstrate that antibody-based immunotherapy can be improved by modifying Fcγ. Our experiments also show that by competing for FcRn-binding, staphylococci effectively reduce the half-life of antibodies during infection. These observations may have profound impact in treating cancer, autoimmune, and asthma patients colonized or infected with S. aureus and undergoing monoclonal antibody treatment.

Fingerprints of Element Concentrations in Infective Endocarditis Obtained by Mass Spectrometric Imaging and t-Distributed Stochastic Neighbor Embedding.

Staphylococcus aureus-induced infective endocarditis (IE) is a life-threatening disease. Differences in virulence between distinct S. aureus strains, which are partly based on the molecular mechanisms during bacterial adhesion, are not fully understood. Yet, distinct molecular or elemental patterns, occurring during specific steps in the adhesion process, may help to identify novel targets for accelerated diagnosis or improved treatment. Here, we use laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) of post-mortem tissue slices of an established mouse model of IE to obtain fingerprints of element distributions in infected aortic valve tissue. Three S. aureus strains with different virulence due to deficiency in distinct adhesion molecules (fibronectin-binding protein A and staphylococcal protein A) were used to assess strain-specific patterns. Data analysis was performed by t-distributed stochastic neighbor embedding (t-SNE) of mass spectrometry imaging data, using manual reference tissue classification in histological specimens. This procedure allowed for obtaining distinct element patterns in infected tissue for all three bacterial strains and for comparing those to patterns observed in healthy mice or after sterile inflammation of the valve. In tissue from infected mice, increased concentrations of calcium, zinc, and magnesium were observed compared to noninfected mice. Between S. aureus strains, pronounced variations were observed for manganese. The presented approach is sensitive for detection of S. aureus infection. For strain-specific tissue characterization, however, further improvements such as establishing a database with elemental fingerprints may be required.

Сomparative analysis of cytokine determination in neutrophil supernatants stimulated by S. aureus supernatants with and without the SPA gene

This study presents the results of the determination of cytokines by multiplex analysis in the blood neutrophil supernatants of donors stimulated by Staphylococcus aureus supernatants, depending on the presence or absence of the staphylococcal protein A (spa) gene in bacteria. Cytokine indication was carried out by multiplex analysis using BioRad kits (USA) for the determination of 17 human cytokines (G-CSF, GM-CSF, INF-γ, TNF-alfa, MCP-1, MIP-1beta, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12p70, IL-13, IL-17A) after 1 hour incubation of staphylococcal supernatants with neutrophils. It was shown that S. aureus, which had the staphylococcal protein A (spa) gene, significantly stimulated the secretion of neutrophils G-CSF (growth factor), IL-12p70, IL-17A, IFN-gamma, IL-13, IL-1b, IL-4, IL-6, IL-7, TNF-alfa (proinflammatory cytokines), MCP-1 (chemokine). But at the same time, they significantly reduced the secretion of IL-10 (anti-inflammatory cytokine), IL-8, MIP-1beta (chemokines). S. aureus supernatants, which did not have spa, also acted almost identically. IFN-gamma increased less significantly under their action, and IL-1b, on the contrary, more significantly compared to S. aureus supernatants having the staphylococcal protein A gene. It was found that the supernatants of all studied clinical isolates of S. aureus with a pronounced ability to produce cytokine-like substances, regardless of the presence/absence of the spa gene, affect the ability of neutrophils to secrete cytokines. Thus, the presence of cytokine-like activity in S. aureus affects the response of neutrophils to secrete a pool of cytokines upon contact of phagocytic cells with staphylococcal supernatants. In particular, pro-inflammatory interleukins, some chemokines and growth factors. This indicates that the interaction of neutrophils with extracellular cytokine-like products of S. aureus can significantly modulate the inflammatory process.

Composition and Dynamics of Protein Complexes Measured by Quantitative Mass Spectrometry of Affinity-Purified Samples.

Multiple protein complexes are fundamental parts of living systems. Identification of the components of these complexes and characterization of the molecular mechanisms that allow their formation, function, and regulation can be done by affinity purification of proteins and associated factors followed by mass spectrometry of peptides. Speed and specificity for the isolation of complexes from whole cell extracts improved over time, together with the reliable identification and quantification of proteins by mass spectrometry. Relative quantification of proteins in such samples can now be done to characterize even relatively nonabundant complexes. We describe here our experience with proteins fused with the Z domain, derived from staphylococcal protein A, and IgG affinity purification for the analysis of protein complexes involved in RNA metabolism in the budding yeast Saccharomyces cerevisiae. We illustrate the use of enrichment calculations for proteins in purified samples as a way to robust identification of protein partners. While the protocols presented here are specific for yeast, their principles can be applied to the study of protein complexes in any other organism.

SYNTHESIS AND APPLICATION OF NON-SPHERICAL NANOPARTICLES AS LABELS IN IMMUNOCHROMATOGRAPHY OF SURFACE-ACTIVE SUBSTANCES

Series of preparations of nanoparticles of various shapes, compositions, and surface patterns have been obtained. All preparations are conjugated with staphylococcal protein A by physical adsorption and tested in an indirect scheme for the immunochromatographic determination of nonylphenol, a non-ionic surfactant, a significant technogenic toxic environmental contaminant.

Biological activity of Cucurbita maxima and Momordica charantia seed extracts against the biofilm-associated protein of Staphylococcus aureus: An in vitro and in silico studies

Nowadays, it is necessary to find new and effective antimicrobial drugs because of rising drug-resistance microbes. Through in vitro analysis, two selected plants' seed (Cucurbita maxima, and Momordica charantia) extracts were evaluated to determine their antibacterial activity, and an in silico screening was used to identify potential compounds of the two selected plants' seed extracts that could be used in developing new drugs against Staphylococcus aureus. Despite an inhibitory zone being seen against all bacteria in the antibacterial activity test, the highest inhibition zone of the seed extracts from the selected plants was identified against Staphylococcus aureus at the dose of 200 μg/disc. Particular plant seed extracts also demonstrated significant antioxidant content and low risk of negative impacts. The two selected plant seed extracts exhibited remarkable biofilm inhibition ability. The binding interaction was investigated using molecular docking to identify bioactive components against Staphylococcal BAP protein (7C7U) that are responsible for biofilm formation. The binding energies of the D1 (CID- 620905), D2 (CID- 66760004), and D3 (CID- 5379220) compound from Cucurbita maxima had binding energy of −8.6 kcal/mol, −7.5 kcal/mol, and −7.1 kcal/mol, respectively. The D1 (CID- 123409), D2 (CID- 5280435), and D3 (CID- 541568) compound from Momordica charantia had an energy of −6.1 kcal/mol, −6.0 kcal/mol, and −5.9 kcal/mol, respectively. Thus, two selected plants’ seed extracts could be utilized to combat antibiotic-resistant bacteria as a natural source of drugs.

BioAdhere: tailor-made bioadhesives for epiretinal visual prostheses.

Introduction: Visual prostheses, i.e. epiretinal stimulating arrays, are a promising therapy in treating retinal dystrophies and degenerations. In the wake of a new generation of devices, an innovative method for epiretinal fixation of stimulator arrays is required. We present the development of tailor-made bioadhesive peptides (peptesives) for fixating epiretinal stimulating arrays omitting the use of traumatic retinal tacks. Materials and methods: Binding motifs on the stimulating array (poly[chloro-p-xylylene] (Parylene C)) and in the extracellular matrix of the retinal surface (collagens I and IV, laminin, fibronectin) were identified. The anchor peptides cecropin A (CecA), KH1, KH2 (author's initials) and osteopontin (OPN) were genetically fused to reporter proteins to assess their binding behavior to coated microtiter plates via fluorescence-based assays. Domain Z (DZ) of staphylococcal protein A was used as a separator to generate a bioadhesive peptide. Following ISO 10993 "biological evaluation of medical materials", direct and non-direct cytotoxicity testing (L-929 and R28 retinal progenitor cells) was performed. Lastly, the fixating capabilities of the peptesives were tested in proof-of-principle experiments. Results: The generation of the bioadhesive peptide required evaluation of the N- and C-anchoring of investigated APs. The YmPh-CecA construct showed the highest activity on Parylene C in comparison with the wildtype phytase without the anchor peptide. eGFP-OPN was binding to all four investigated ECM proteins (collagen I, laminin > collagen IV, fibronectin). The strongest binding to collagen I was observed for eGFP-KH1, while the strongest binding to fibronectin was observed for eGFP-KH2. The selectivity of binding was checked by incubating eGFP-CecA and eGFP-OPN on ECM proteins and on Parylene C, respectively. Direct and non-direct cytotoxicity testing of the peptide cecropin-A-DZ-OPN using L-929 and R28 cells showed good biocompatibility properties. Proof-of-concept experiments in post-mortem rabbit eyes suggested an increased adhesion of CecA-DZ-OPN-coated stimulating arrays. Conclusion: This is the first study to prove the applicability and biocompatibility of peptesives for the fixation of macroscopic objects.