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We studied the effects of glucosylation of RhoA, Rac1, and Cdc42 at threonine-35 and -37 by Clostridium difficile toxin B on nucleotide binding, GTPase activity, and effector coupling and compared these results with the ADP ribosylation of RhoA at asparagine-41 catalyzed by Clostridium botulinum C3 transferase. Whereas glucosylation and ADP ribosylation had no major effects on GDP release from RhoA, Rac1, and Cdc42, the rate of GTPgammaS release from Rho proteins was increased 3-6-fold by glucosylation. ADP ribosylation decreased the rate of GTPgammaS release by about 50%. Glucosylation reduced the intrinsic activities of the GTPases by 3-7-fold and completely blocked GTPase stimulation by Rho-GAP. In contrast, ADP ribosylation slightly increased GTPase activity ( approximately 2-fold) and had no major effect on GAP stimulation of GTPase. Whereas ADP ribosylation did not affect the interaction of RhoA with the binding domain of protein kinase N, glucosylation inhibited this interaction. Glucosylation of Rac1 markedly diminished its ability to support the activation of the superoxide-generating NADPH oxidase of phagocytes. Glucosylated Rac1 did not interfere with NADPH oxidase activation by unmodified Rac1, even when present in marked molar excess, indicating that it was incapable of competing for a common effector. The data indicate that the functional inactivation of small GTPases by glucosylation is mainly caused by inhibition of GTPase-effector protein interaction.

BACKGROUNDFollowing the emergence of the Omicron variant of concern, we investigated immunogenicity, efficacy and safety of BNT162b2 or mRNA1273 fourth dose in an open-label, clinical intervention trial.METHODSPrimary end-points were safety and immunogenicity and secondary end-points were vaccine efficacy in preventing SARS-CoV-2 infections and COVID-19 symptomatic disease. The two intervention arms were compared to a matched control group. Eligible participants were healthcare-workers (HCW) vaccinated with three BNT162b2 doses, and whose IgG antibody levels were ≤700 BAU (40-percentile). IgG and neutralizing titers, direct neutralization of live VOCs, and T-cell activation were assessed. All participants were actively screened for SARS-CoV-2 infections on a weekly basis.RESULTSOf 1050 eligible HCW, 154 and 120 were enrolled to receive BNT162b2 and mRNA1273, respectively, and compared to 426 age-matched controls. Recipients of both vaccine types had a ∼9-10-fold increase in IgG and neutralizing titers within 2 weeks of vaccination and an 8-fold increase in live Omicron VOC neutralization, restoring titers to those measured after the third vaccine dose. Breakthrough infections were common, mostly very mild, yet, with high viral loads. Vaccine efficacy against infection was 30% (95%CI:-9% to 55%) and 11% (95%CI:-43% to +43%) for BNT162b2 and mRNA1273, respectively. Local and systemic adverse reactions were reported in 80% and 40%, respectively.CONCLUSIONSThe fourth COVID-19 mRNA dose restores antibody titers to peak post-third dose titers. Low efficacy in preventing mild or asymptomatic Omicron infections and the infectious potential of breakthrough cases raise the urgency of next generation vaccine development.Trial registration numberclicaltrials.gov: NCT05231005, NCT05230953

The in vitro susceptibilities of 86 recent clinical isolates of Brucella melitensis to minocycline, streptomycin, co-trimoxazole, rifampin, and six fluoroquinolones were determined. Minocycline exhibited the lowest MIC and was followed by rifampin and streptomycin. Among the quinolones, WIN 57273 and ciprofloxacin were the most active agents. No antibiotic combination of these agents exhibited synergy against 15 selected isolates. In killing rate experiments, streptomycin exhibited the most rapid kill (<12 h), while a complete kill with minocycline, rifampin, and ciprofloxacin was delayed up to 48 h. The combinations of streptomycin with each of minocycline, rifampin, or ciprofloxacin exhibited the fastest kills (within 2 h), while with the other combinations, a complete kill was delayed up to 96 h. These results demonstrate the discrepancy between the results of various in vitro methods in evaluating the antibiotic susceptibility of B. melitensis.Human brucellosis is an important ongoing medical problem. The Mediterranean basin is one of the most heavily afflicted regions (7). Despite the availability of many antibacterial agents, the complete cure of the infection with prevention of the frequent relapses is still an unattainable goal (7). The new fluoroquinolone antibacterial agents, because of their broad spectrum of bactericidal activity against a variety of gram-negative bacteria and because of their favorable intracellular penetration, could potentially be candidates for the therapy of brucellosis (13, 19). We therefore tested the in vitro activities of several fluoroquinolones (pefloxacin, ofloxacin, ciprofloxacin, fleroxacin, sparfloxacin, and WIN 57273) and compared them with the activities of the conventional anti-Brucella antibiotics: tetracycline, streptomycin, rifampin, and sulfamethoxazole-trimethoprim (co-trimoxazole), recommended for the therapy of brucellosis. In addition, we also studied the possible synergistic effects of several combinations of these antibiotics.Bacteria. Eighty-six Brucella melitensis strains isolated from patients at three different geographical districts in Israel were used. The identities of all B. melitensis strains were confirmed (by Menachem Banai) in the National Brucella Reference Laboratory by the Stamp stain appropriate biochemical reactions and by phage typing (2). Stock strains were kept frozen in small aliquots at -80°C and were kept between experiments on agar slants.Antibiotics. Antibiotics were obtained from their manufacturers as laboratory powders and were reconstituted in their recommended diluents to yield stock solutions of 1,000 ,u.g/ml that were kept at -70°C. Minocycline (Lederle, Pearl River, N.Y.) was used as a representative of the tetracycline family. Streptomycin sulfate (Teva, Jerusalem, Israel), sulfamethoxazole-trimethoprim (Wellcome, Beckenham, United Kingdom), rifampin (Ciba-Geigy, Basel, Switzerland), ciprofloxacin (Bayer AG, Leverkusen, Germany), ofloxacin (Hoechst AG, Frankfurt, Germany), pefloxacin and sparfloxacin (Rhone-Poulenc-Rorer, ...