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SUMMARY Fragment screening is widely used to identify attractive starting points for drug design. However, its potential and limitations to assess the tractability of often challenging protein:protein interfaces have been underexplored. Here, we address this question by means of a systematic deconstruction of lead-like inhibitors of the pVHL:HIF-1α interaction into their component fragments. Using biophysical techniques commonly employed for screening, we could only detect binding of fragments that violate the Rule of Three, are more complex than those typically screened against classical druggable targets, and occupy two adjacent binding subsites at the interface rather than just one. Analyses based on ligand and group lipophilicity efficiency of anchored fragments were applied to dissect the individual subsites and probe for binding hot spots. The implications of our findings for targeting protein interfaces by fragment-based approaches are discussed.

Coagulase-negative staphylococci (CNS; n = 417) were isolated from bovine milk and identified by matrix-assisted laser desorption/ionization time-offlight mass spectrometry. Nineteen different species were identified, and Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus haemolyticus, and Staphylococcus sciuri were the most prevalent species. Resistance to oxacillin (47.0% of the isolates), fusidic acid (33.8%), tiamulin (31.9%), penicillin (23.3%), tetracycline (15.8%), streptomycin (9.6%), erythromycin (7.0%), sulfonamides (5%), trimethoprim (4.3%), clindamycin (3.4%), kanamycin (2.4%), and gentamicin (2.4%) was detected. Resistance to oxacillin was attributed to the mecA gene in 9.7% of the oxacillin-resistant isolates. The remaining oxacillin-resistant CNS did not contain the mecC gene or mecA1 promoter mutations. The mecA gene was detected in Staphylococcus fleurettii, Staphylococcus epidermidis, Staph. haemolyticus, and Staph. xylosus. Resistance to tetracycline was attributed to the presence of tet(K) and tet(L), penicillin resistance to blaZ, streptomycin resistance to str and ant(6)-Ia, and erythromycin resistance to erm(C), erm(B), and msr. Resistance to tiamulin and fusidic acid could not be attributed to an acquired resistance gene. In total, 15.1% of the CNS isolates were multidrug resistant (i.e., resistant to 2 or more antimicrobials). The remaining CNS isolates were susceptible to antimicrobials commonly used in mastitis treatment. Methicillin-resistant CNS isolates were diverse, as determined by mecA gene sequence analysis, staphylococcal cassette chromosome mec typing, and pulsed-field gel electrophoresis. Arginine catabolic mobile element types 1 and 3 were detected in both methicillin-resistant and methicillin-susceptible Staph. epidermidis and were associated with sequence types ST59 and ST111. Because this study revealed the presence of multidrugresistant CNS in a heterogeneous CNS population, we recommend antibiogram analysis of CNS in persistent infections before treatment with antimicrobials.

ReviewTargeting the immune system Dendritic cells (DCs) play crucial roles in promoting and regulating immune defenses, providing important targets for prophylactic and therapeutic approaches (FiguRe 1). Particulate formulations, including liposomes and other nanoparticles, have been employed as delivery vehicles. Despite the large number of reports, current in-depth knowledge on the cellular processes involved remains relatively limited, particularly for nucleic acid delivery. Certain structures in the nucleic acid may also signal the cell in the sense of an adjuvant or immunomodulatory activity. Importantly, the optimized characteristics for delivery of an antigen or therapeutic agent may be distinct from those for nucleic acid delivery, especially RNA species. Moreover, RNA delivery for interference therapy will not necessarily require the same delivery routes as mRNA delivery wherein RNA translation is the main requisite.The efficacy of delivery can be further improved by targeting the appropriate cells of the immune system, an area in which nanoparticle-based delivery platforms have found an important niche [1]. Advances with prophylactic applications can also prove valuable for therapeutic applications and vice versa, as seen with RNA delivery. This review will consider how growing knowledge on delivery mechanisms has found application with nucleic acids, in both prophylaxis and therapy, focusing on interaction with DCs.The initial components of the DC endocytic pathways are critically important in determining how the cell handles the delivered material; thereafter, correct cytosolic delivery is a critical element for a number of desired outcomes, including delivery of nucleic acids. Advances made with protein delivery -in particular, vaccines -are of value to highlight the potential of a particular mode of application or the high risk for nucleic acid integrity. Particularly pertinent is the application of cationic elements in the delivery mechanisms and how application of ligands for cell receptors influence intracellular compartmentalization.It is not the aim of the present review to retrace all the fine details of work contributing to our current knowledge. Accordingly, review articles covering areas that have already received considerable attention will be employed and assimilated to provide a more elaborate picture of the current situation. This will allow a focusing on more recent advances, along with problems and pitfalls therein. While advances on protein and DNA delivery will be presented, these will be used to highlight how our current knowledge can be applied to RNA delivery. There are numerous reviews on protein delivery to DC, wherein many of the procedures employed are not relevant for RNA delivery, which must escape into the cytosol undamaged. With DNA delivery, there is also the question of whether the DNA will activate the DC or reach the nucleus for transcription; this latter area has most often Functional RNA delivery targeted to dendritic cells by synthetic nanoparticles Dendritic cell...

Because of the frequency of multiple antibiotic resistance, Staphylococcus species often represent a challenge in incisional infections of horses undergoing colic surgery. To investigate the evolution of antibiotic resistance patterns before and after preventative peri-and postoperative penicillin treatment, staphylococci were isolated from skin and wound samples at different times during hospitalization. Most staphylococci were normal skin commensals and belonged to the common coagulase-negative group. In some cases they turned out to be opportunistic pathogens present in wound infections. MICs were determined for 12 antibiotics, and antibiotic resistance genes were detected by microarray. At hospital admission, horses harbored staphylococci that were susceptible to antibiotics or resistant to one group of drugs, mainly due to the presence of new variants of the methicillin and macrolide resistance genes mecA and mph(C), respectively. After 3 days, the percentage of Staphylococcus isolates displaying antibiotic resistance, as well as the number of resistance genes per isolate, increased moderately in hospitalized horses without surgery or penicillin treatment but dramatically in hospitalized horses after colic surgery as well as penicillin treatment. Staphylococcus species displaying multiple resistance were found to harbor mainly genes conferring resistance to ␤-lactams (mecA and blaZ), aminoglycosides [str and aac(6)-Ie-aph(2)-Ia], and trimethoprim [dfr(A) and dfr(D)]. Additional genes conferring resistance to macrolides [mph(C), erm(C), and erm(B)], tetracycline [tet(K) and tet(M)], chloramphenicol [cat(pC221) and cat(pC223)], and streptothricin (sat4) appeared in several strains. Hospitalization and preventive penicillin use were shown to act as selection agents for multidrug-resistant commensal staphylococcal flora.Postoperative incisional infection is a major complication after colic surgery in horses and can lead to intensive postoperative care, prolonged hospitalization, and increased costs (15). All equine patients undergoing colic surgery at the equine clinic of the Vetsuisse Faculty of the University of Berne receive preventative treatment of 30,000 IU penicillin G per kg body weight once preoperatively and then daily for at least 3 days postoperatively. Despite antimicrobial prevention, the rates of postoperative wound infection after colic surgery

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During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic. However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro, it provides a real competitive advantage in vivo, particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.

Self-amplifying replicon RNA (RepRNA) possesses high potential for increasing antigen load within dendritic cells (DCs). The major aim of the present work was to define how RepRNA delivered by biodegradable, chitosan-based nanoparticulate delivery vehicles (nanogel-alginate (NGA)) interacts with DCs, and whether this could lead to translation of the RepRNA in the DCs. Although studies employed virus replicon particles (VRPs), there are no reports on biodegradable, nanoparticulate vehicle delivery of RepRNA. VRP studies employed cytopathogenic agents, contrary to DC requirements—slow processing and antigen retention. We employed noncytopathogenic RepRNA with NGA, demonstrating for the first time the efficiency of RepRNA association with nanoparticles, NGA delivery to DCs, and RepRNA internalization by DCs. RepRNA accumulated in vesicular structures, with patterns typifying cytosolic release. This promoted RepRNA translation, in vitro and in vivo. Delivery and translation were RepRNA concentration-dependent, occurring in a kinetic manner. Including cationic lipids with chitosan during nanoparticle formation enhanced delivery and translation kinetics, but was not required for translation of immunogenic levels in vivo. This work describes for the first time the characteristics associated with chitosan-nanoparticle delivery of self-amplifying RepRNA to DCs, leading to translation of encoded foreign genes, namely influenza virus hemagglutinin and nucleoprotein.