Peter Panizzi

Staphylocoagulase (SC), a crucial virulence protein secreted by several strains of the major pathogen Staphylococcus aureus , functions as a potent inducer of blood clotting. SC binds its zymogen target, prothrombin (ProT) with subnanomolar affinity, allosterically inducing a thrombin-like active site in the bound zymogen. The resulting SC·(Pro)T* active complexes effectively convert fibrinogen (Fbg) into clot-forming fibrin (Fbn). We previously reported crystal structures of the N-terminal SC domains, responsible for ProT binding and activation, and characterized the mechanism of cofactor-induced zymogen activation in detail. However, the lack of three-dimensional (3D) structures for the C-terminal SC region has hampered a complete understanding of its role in Fbg recognition and cleavage. Here, we present and discuss 3D models of the Fbg-binding SC region. Our results suggest that the major C-terminal domain of SC folds into a single-layer β-sheet structurally similar to the membrane occupation and recognition nexus (MORN) family of tandem repeats. We further propose a folding pathway for this C-terminal SC region that is contingent upon the presence of its substrate, Fbg. These structural and mechanistic insights are critical for understanding SC-mediated fibrin generation, which is highly relevant to heart valve vegetations and biofilm formation during S. aureus infection.

Background: Although manipulation of host hemostasis is a central mechanism in Staphylococcus aureus virulence, clinically relevant phenotypes of S aureus coagulase activity and the variability thereof in the context of bloodstream infections are unknown. Methods: We created a high-throughput staphylothrombin activity and growth fitness assay to characterize fibrin formation and S aureus replication by adding bacterial culture supernatant to human blood plasma. Supernatant is rich in bacterial proteins, including coagulases secreted during growth, but it contains relatively few viable cells. As a result, staphylothrombin activity driven by secreted coagulases can initially be observed and a contribution from bacterial replication can also be captured. Results: Some S aureus isolates showed slow or absent staphylothrombin activity in a subset of plasma specimens despite robust and rapid fibrin polymerization in other plasmas, suggesting that a host-pathogen match or mismatch determines the observed phenotype. Characterization of S aureus transposon mutant behavior, host plasma coagulation factors, pharmacologic direct thrombin inhibition, and across-host patterns provides clues to complex determinants of coagulase-mediated virulence. Conclusions: Observations of variable staphylothrombin phenotypes may have implications for novel therapeutic approaches and clinical risk stratification in S aureus bloodstream infections.

Implant failures in orthopedic surgeries can occur due to inflammation, poor osteointegration, and biofilm formation. The local delivery of anti-inflammatory drugs is one of the potential solutions to address orthopedic implant failures caused by extensive inflammation, poor osteointegration, and biofilm formation. This study utilized a simple spray coating technique to deposit multilayer coatings of the anti-inflammatory drug meloxicam and PLGA on 3D printed 316L stainless steel implants. The implants were evaluated for their drug content, drug-polymer miscibility, static contact angle, corrosion resistance, in vitro release, cell adhesion, cell proliferation, and bacterial adhesion studies. The coatings aimed to improve osteointegration and reduce biofilm formation. The drug release studies demonstrated an initial burst release accompanied by sustained release for more than 30 days. The meloxicam-PLGA coated implants showed higher resistance to pitting corrosion, improved adhesion, and proliferation of human U2OS cells in comparison to uncoated implants. In addition, results from cell cytotoxicity studies confirmed that all the coatings are biocompatible and safe for in vivo application. The meloxicam-PLGA coated implants were also found to have significantly lower bacterial adhesion and biofilm formation compared to uncoated implants for both S. aureus ( p < 0.01) and S. pseudintermedius ( p < 0.01). The multilayer coatings developed in this study have the potential to improve adhesion at the implant-bone interface and thus might prevent implant rejections in patients undergoing orthopedic surgery. Moreover, this method can be applied to other drugs and polymers to address the issues related to implant failure, pain management, and surgical site infections. Graphical Abstract

Background/Objectives: Personalized 3D-printed (3DP) metallic implants delivery systems are being explored to repair bone fractures, allowing the customization of medical implants that respond to individual patient needs, making it potentially more effective and of greater quality than mass-produced devices. However, challenges associated with postsurgical infections caused by bacterial adhesion remain a clinical issue. To address this, local antibiotic therapies are receiving extensive attention to minimize the risk of implant-related infections. This study investigated the use of amikacin (AMK), a broad-spectrum aminoglycoside antibiotic, incorporated onto 3D-printed 316L stainless steel implants using biodegradable polymer coatings of chitosan and poly lactic-co-glycolic acid (PLGA). Methods: This research examined different approaches to coat 3DP implants with amikacin. Various polymer-based coatings were studied to determine the optimal formulation based on the characteristics and release profile. The optimal formulation was performed on the antibacterial activity studies. Results: AMK-chitosan with PLGA coating implants controlled the rate of drug release for up to one month. The 3DP drug-loaded substrates demonstrated effective, concentration-dependent antibacterial activity against common infective pathogens. AMK-loaded substrates showed antimicrobial effectiveness for one week and inhibited bacteria significantly compared to the uncoated controls. Conclusions: This study demonstrated that 3DP metal surfaces coated with amikacin can provide customizable drug release profiles while effectively inhibiting bacterial growth. These findings highlight the potential of combining 3D printing with localized delivery strategies to prevent implant-associated infections and advance the development of personalized therapies.

A surgically implantable device is an inevitable treatment option for millions of people worldwide suffering from diseases arising from orthopedic injuries. A global paradigm shift is currently underway to tailor and personalize replacement or reconstructive joints. Additive manufacturing (AM) has provided dynamic outflow to the customized fabrication of orthopedic implants by enabling need-based design and surface modification possibilities. Surgical grade 316L Stainless Steel (316L SS) is promising with its cost, strength, composition, and corrosion resistance to fabricate 3D implants. This work investigates the possibilities of application of the laser powder bed fusion (L-PBF) technique to fabricate 3D-printed (3DP) implants, which are functionalized with a multilayered antimicrobial coating to treat potential complications arising due to postsurgical infections (PSIs). Postsurgical implant-associated infection is a primary reason for implantation failure and is complicated mainly by bacterial colonization and biofilm formation at the installation site. PLGA (poly-d,l-lactide-co-glycolide), a biodegradable polymer, was utilized to impart multiple layers of coating using the airbrush spray technique on 3DP implant surfaces loaded with gentamicin (GEN). Various PLGA-based polymers were tested to optimize the ideal lactic acid: glycolic acid ratio and molecular weight suited for our investigation. 3D-Printed PLGA-GEN substrates sustained the release of gentamicin from the surface for approximately 6 weeks. The 3DP surface modification with PLGA-GEN facilitated cell adhesion and proliferation compared to control surfaces. The cell viability studies showed that the implants were safe for application. The 3DP PLGA-GEN substrates showed good concentration-dependent antibacterial efficacy against the common PSI pathogen Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). The GEN-loaded substrates demonstrated antimicrobial longevity and showed significant biofilm growth inhibition compared to control. The substrates offered great versatility regarding the in vitro release rates, antimicrobial properties, and biocompatibility studies. These results radiate great potential in future human and veterinary clinical applications pertinent to complications arising from PSIs, focusing on personalized sustained antibiotic delivery.

BAPCs are PEG free nanoparticles with potential to deliver therapeutic mRNA in vivo. Association of mRNA with BAPCs surface enhanced localization in lung by avoiding liver and spleen. Therefore, potentiating their application in lung therapy.

Background: Computed tomography (CT) generates a three‐dimensional rendering that can be used to interrogate a given region or desired structure from any orientation. However, in preclinical research, its deployment remains limited due to relatively high upfront costs. Existing integrated imaging systems that provide merged planar X‐ray also dwarfs CT popularity in small laboratories due to their added versatility.Purpose: In this paper, we sought to generate CT‐like data using an existing small‐animal X‐ray imager with a specialized specimen rotation system, or MiSpinner. This setup conforms to the cone‐beam CT (CBCT) geometry, which demands high spatial calibration accuracy. Therefore, a simple but robust geometry calibration algorithm is necessary to ensure that the entire imaging system works properly and accurately.Methods: Because the rotation system is not permanently affixed, we propose a structure tensor‐based two‐step online (ST‐TSO) geometry calibration algorithm. Specifically, two datasets are needed, namely, calibration and actual measurements. A calibration measurement detects the background of the system forward X‐ray projections. A study on the background image reveals the characteristics of the X‐ray photon distribution, and thus, provides a reliable estimate of the imaging geometry origin. Actual measurements consisted of an X‐ray of the intended object, including possible geometry errors. A comprehensive image processing technique helps to detect spatial misalignment information. Accordingly, the first processing step employs a modified projection matrix‐based calibration algorithm to estimate the relevant geometric parameters. Predicted parameters are then fine‐tuned in a second processing step by an iterative strategy based on the symmetry property of the sum of projections. Virtual projections calculated from the parameters after two‐step processing compensate for the scanning errors and are used for CT reconstruction. Experiments on phantom and mouse imaging data were performed to validate the calibration algorithm.Results: Once system correction was conducted, CBCT of a CT bar phantom and a cohort of euthanized mice were analyzed. No obvious structure error or spatial artifacts were observed, validating the accuracy of the proposed geometry calibration method. Digital phantom simulation indicated that compared with the preset spatial values, errors in the final estimated parameters could be reduced to 0.05° difference in dominant angle and 0.5‐pixel difference in dominant axis bias. The in‐plane resolution view of the CT‐bar phantom revealed that the resolution approaches 150 m.Conclusions: A constrained two‐step online geometry calibration algorithm has been developed to calibrate an integrated X‐ray imaging system, defined by a first‐step analytical estimation and a second‐step iterative fine‐tuning. Test results have validated its accuracy in system correction, thus demonstrating the potential of the described system to be modified and adapted for preclinical research.

Streptococcus pyogenes (S. pyogenes) can thrive in its host during an infection, and, as a result, it must be able to respond to external stimuli and available carbon sources. The preclinical use of engineered pathogens capable of constitutive light production may provide real-time information on microbial-specific metabolic processes. In this study, we mapped the central metabolism of a luxABCDE-modified S. pyogenes Xen20 (Strep. Xen20) to its de novo synthesis of luciferase substrates as assessed by the rate of light production in response to different environmental triggers. Previous characterization predicted that the lux operon was under the myo-inositol iolE promotor. In this study, we revealed that supplementation with myo-inositol generated increased Strep. Xen20 luminescence. Surprisingly, when supplemented with infection-relevant carbon sources, such as glucose or glycine, light production was diminished. This was presumably due to the scavenging of pyruvate by L-lactate dehydrogenase (LDH). Inhibition of LDH by its inhibitor, oxamate, partially restored luminescent signal in the presence of glucose, presumably by allowing the resulting pyruvate to proceed to acetyl-coenzyme A (CoA). This phenomenon appeared specific to the lactic acid bacterial metabolism as glucose or glycine did not reduce signal in an analogous luxABCDE-modified Gram-positive pathogen, Staph. Xen29. The Strep. Xen20 cells produced light in a concentration-dependent manner, inversely related to the amount of glucose present. Taken together, our measures of microbial response could provide new information regarding the responsiveness of S. pyogenes metabolism to acute changes in its local environments and cellular health.

Interactions between the immune and central nervous systems strongly influence brain health. Although the blood–brain barrier restricts this crosstalk, we now know that meningeal gateways through brain border tissues facilitate intersystem communication. Cerebrospinal fluid (CSF), which interfaces with the glymphatic system and thereby drains the brain’s interstitial and perivascular spaces, facilitates outward signaling beyond the blood–brain barrier. In the present study, we report that CSF can exit into the skull bone marrow. Fluorescent tracers injected into the cisterna magna of mice migrate along perivascular spaces of dural blood vessels and then travel through hundreds of sub-millimeter skull channels into the calvarial marrow. During meningitis, bacteria hijack this route to invade the skull’s hematopoietic niches and initiate cranial hematopoiesis ahead of remote tibial sites. As skull channels also directly provide leukocytes to meninges, the privileged sampling of brain-derived danger signals in CSF by regional marrow may have broad implications for inflammatory neurological disorders.