Engineering antimicrobial supramolecular polymer assemblies

Antibacterial resistance against conventional antibiotics has emerged as a global health problem. To address this problem, antimicrobial peptides (AMPs) have been recognized as alternatives due to their fast-killing activity and less propensity to induce resistance. Here, the AMPs are engineered via a supramolecular fashion to control and increase their biological performance. The AMPs are modified with ureido-pyrimidinone (UPy) to obtain UPy-AMP monomers, followed by modular self-assembling to realize antibacterial UPy-AMP supramolecular polymers. These positively charged assemblies are illustrated as stable, short fibrous or rod-like UPy-AMP nanostructures with enhanced antibacterial activity and modulable cytotoxicity. Moreover, these antibacterial UPy-AMP assemblies can be internalized by both THP-1 derived macrophages and human kidney cells, which would be an effective potential therapy to deliver the AMPs into mammalian cells to address intracellular infections. Overall, the results present here demonstrate that supramolecular engineering of AMPs provides a powerful tool to enhance the antibacterial activity, modulate cytotoxicity and accelerate the clinical application of AMPs.</p

Engineering antimicrobial supramolecular polymer assemblies

Antibacterial resistance against conventional antibiotics has emerged as a global health problem. To address this problem, antimicrobial peptides (AMPs) have been recognized as alternatives due to their fast-killing activity and less propensity to induce resistance. Here, the AMPs are engineered via a supramolecular fashion to control and increase their biological performance. The AMPs are modified with ureido-pyrimidinone (UPy) to obtain UPy-AMP monomers, followed by modular self-assembling to realize antibacterial UPy-AMP supramolecular polymers. These positively charged assemblies are illustrated as stable, short fibrous or rod-like UPy-AMP nanostructures with enhanced antibacterial activity and modulable cytotoxicity. Moreover, these antibacterial UPy-AMP assemblies can be internalized by both THP-1 derived macrophages and human kidney cells, which would be an effective potential therapy to deliver the AMPs into mammalian cells to address intracellular infections. Overall, the results present here demonstrate that supramolecular engineering of AMPs provides a powerful tool to enhance the antibacterial activity, modulate cytotoxicity and accelerate the clinical application of AMPs.</p

Development of an Antimicrobial Peptide SAAP-148-Functionalized Supramolecular Coating on Titanium to Prevent Biomaterial-Associated Infections

Titanium implants are widely used in medicine but have a risk of biomaterial-associated infection (BAI), of which traditional antibiotic-based treatment is affected by resistance. Antimicrobial peptides (AMPs) are used to successfully kill antibiotic-resistant bacteria. Herein, a supramolecular coating for titanium implants is developed which presents the synthetic antimicrobial and antibiofilm peptide SAAP-148 via supramolecular interactions using ureido-pyrimidinone supramolecular units (UPy-SAAP-148GG). Material characterization of dropcast coatings shows the presence of UPy-SAAP-148GG at the surface. The supramolecular immobilized peptide remains antimicrobially active in dropcast polymer films and can successfully kill (antibiotic-resistant) Staphylococcus aureus, Acinetobacter baumannii, and Escherichia coli. Minor toxicity for human dermal fibroblasts is observed, with a reduced cell attachment after 24 h. Subsequently, a dipcoat coating on titanium implants is developed and tested in vivo in a subcutaneous implant infection mouse model with S. aureus administered locally on the implant before implantation to mimic contamination during surgery. The supramolecular coating containing 5 mol% of UPy-SAAP-148GG significantly prevents colonization of the implant surface as well as of the surrounding tissue, with no signs of toxicity. This shows that supramolecular AMP coatings on titanium are eminently suitable to prevent BAI.</p

Heparin-guided binding of vascular endothelial growth factor to supramolecular biomaterial surfaces

Growth factors can steer the biological response to a biomaterial post implantation. Heparin is a growth factor binding molecule that can coordinate growth factor presentation to cells and therefore is able to regulate many biological processes. One way to functionalize biomaterials with heparin and growth factors is via a supramolecular approach. Here, we show a proof-of-concept study in which a supramolecular approach based on ureido-pyrimidinone (UPy) was used, which allows for modular functionalization. PCLdiUPy was functionalized with a UPy-modified heparin binding peptide (UPy-HBP) to facilitates binding of heparin, which in turn can bind vascular endothelial growth factor (VEGF) via its heparin binding domain. The adsorption of both heparin and VEGF were studied in two different functionalization approaches (pre-complex and two-step) and at different molecular ratios. Quartz crystal microbalance with dissipation energy adsorption data showed that VEGF and pre-complexed heparin:VEGF adsorbed non-specifically, with no distinguish between non-specific adsorption and heparin guided-adsorption. On the biological side, heparin guided-adsorption of Heparin:VEGF enhanced HUVECs surface coverage as compared to non-specific adsorption. These results provide a detailed insight on the molecular sandwich which is useful for new design strategies of supramolecular biomaterials with well-controlled immobilization of different growth factors.</p

Clan Structure Analysis and Rapidity Gap Probability

Clan structure analysis in rapidity intervals is generalized from negative binomial multiplicity distribution to the wide class of compound Poisson distributions. The link of generalized clan structure analysis with correlation functions is also established. These theoretical results are then applied to minimum bias events and evidentiate new interesting features, which can be inspiring and useful in order to discuss data on rapidity gap probability at TEVATRON and HERA.Comment: (14 pages in Plain TeX plus 5 Postscript Figures, all compressed via uufiles) DFTT 28/9

False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance

A variant in the GBA1 gene is one of the most common genetic risk factors to develop Parkinson's disease (PD). Here the serendipitous finding is reported of a polymerase dependent allelic imbalance when using next generation sequencing, potentially resulting in false-negative results when the allele frequency falls below the variant calling threshold (by default commonly at 30%). The full GBA1 gene was sequenced using next generation sequencing on saliva derived DNA from PD patients. Four polymerase chain reaction conditions were varied in twelve samples, to investigate the effect on allelic imbalance: (1) the primers (n=4); (2) the polymerase enzymes (n=2); (3) the primer annealing temperature (T-a) specified for the used polymerase; and (4) the amount of DNA input. Initially, 1295 samples were sequenced using Q5 High-Fidelity DNA Polymerase. 112 samples (8.6%) had an exonic variant and an additional 104 samples (8.0%) had an exonic variant that did not pass the variant frequency calling threshold of 30%. After changing the polymerase to TaKaRa LA Taq DNA Polymerase Hot-Start Version: RR042B, all samples had an allele frequency passing the calling threshold. Allele frequency was unaffected by a change in primer, annealing temperature or amount of DNA input. Sequencing of the GBA1 gene using next generation sequencing might be susceptible to a polymerase specific allelic imbalance, which can result in a large amount of flase-negative results. This was resolved in our case by changing the polymerase. Regions displaying low variant calling frequencies in GBA1 sequencing output in previous and future studies might warrant additional scrutiny.Perioperative Medicine: Efficacy, Safety and Outcome (Anesthesiology/Intensive Care

The unusual kinetics of lactate dehydrogenase of Schistosoma mansoni and their role in the rapid metabolic switch after penetration of the mammalian host

Lactate dehydrogenase (LDH) from Schistosoma mansoni has peculiar properties for a eukaryotic LDH. Schistosomal LDH (SmLDH) isolated from schistosomes, and the recombinantly expressed protein, are strongly inhibited by ATP, which is neutralized by fructose-1,6-bisphosphate (FBP). In the conserved FBP/anion binding site we identified two residues in SmLDH (Val187 and Tyr190) that differ from the conserved residues in LDHs of other eukaryotes, but are identical to conserved residues in FBP-sensitive prokaryotic LDHs. Three-dimensional (3D) models were generated to compare the structure of SmLDH with other LDHs. These models indicated that residues Val187, and especially Tyr190, play a crucial role in the interaction of FBP with the anion pocket of SmLDH. These 3D models of SmLDH are also consistent with a competitive model of SmLDH inhibition in which ATP (inhibitor) and FBP (activator) compete for binding in a well-defined anion pocket. The model of bound ATP predicts a distortion of the nearby key catalytic residue His195, resulting in enzyme inhibition. To investigate a possible physiological role of this allosteric regulation of LDH in schistosomes we made a kinetic model in which the allosteric regulation of the glycolytic enzymes can be varied. The model showed that inhibition of LDH by ATP prevents fermentation to lactate in the free-living stages in water and ensures complete oxidation via the Krebs cycle of the endogenous glycogen reserves. This mechanism of allosteric inhibition by ATP prevents the untimely depletion of these glycogen reserves, the only fuel of the free-living cercariae. Neutralization by FBP of this ATP inhibition of LDH prevents accumulation of glycolytic intermediates when S. mansoni schistosomula are confronted with the sudden large increase in glucose availability upon penetration of the final host. It appears that the LDH of S. mansoni is special and well suited to deal with the variations in glucose availability the parasite encounters during its life cycle.</p

Rapid analysis of local data to inform off-label tocilizumab use early in the COVID-19 pandemic

The interleukin-6 receptor antagonist tocilizumab became widely used early in the coronavirus disease 2019 (COVID-19) pandemic based on small observational studies that suggested clinical benefit in COVID-19 patients with a hyperinflammatory state. To inform our local treatment algorithms in the absence of randomized clinical trial results, we performed a rapid analysis of the first 11 hospitalized COVID-19 patients treated with tocilizumab at our academic medical center. We report their early clinical outcomes and describe the process by which we assembled a team of diverse trainees and stakeholders to extract, analyze, and disseminate data during a time of clinical uncertainty