Chloramphenicol (Cam) is a broad-spectrum antibiotic used to combat bacterial infections in humans and animals. Cam export from bacterial cells is one of the mechanisms by which pathogens resist Cam's antibacterial effects, and several different proteins are known to facilitate this process. However, to date no report exists on any specific transport protein that facilitates Cam uptake. The proton-coupled oligopeptide transporter (POT) YdgR from is a prototypical member of the POT family, functioning in proton-coupled uptake of di- and tripeptides. By following bacterial growth and conducting LC-MS-based assays we show here that YdgR facilitates Cam uptake. Some YdgR variants displaying reduced peptide uptake also exhibited reduced Cam uptake, indicating that peptides and Cam bind YdgR at similar regions. Homology modeling of YdgR, Cam docking, and mutational studies suggested a binding mode that resembles that of Cam binding to the multidrug resistance transporter MdfA. To our knowledge, this is the first report of Cam uptake into bacterial cells mediated by a specific transporter protein. Our findings suggest a specific bacterial transporter for drug uptake that might be targeted to promote greater antibiotic influx to increase cytoplasmic antibiotic concentration for enhanced cytotoxicity.

Background The impact of body size, fat-free mass (FFM) and fat mass (FM) on cardiorespiratory fitness in pediatric renal transplant recipients (TX) has not been established. Study objectives were to assess maximal oxygen consumption (VO2max) in TX and controls, adjusted for body composition, and to identify risk factors for reduced fitness in TX. Methods Cycle ergometry and DXA were obtained in 50 TX and 70 controls, ages 8 to 21 yr. Control recruitment was targeted to include obese subjects with body mass index (BMI) Z-scores comparable to TX. Allometric regression models were utilized. Results TX had significantly lower height Z-scores (p < 0.001) and comparable BMI Z-scores. VO2max per body weight (ml/kg/min) and per FFM (ml/kgFFM/min) did not differ between groups. However, VO2max was 13% lower (95% CI 18, 8; p < 0.001) in TX, compared with controls, adjusted for FM, FFM, sex and race. Greater FFM, lower FM, non-black race, and male sex were independently associated with greater VO2max. Within TX, hemoglobin levels were positively associated with VO2max (p = 0.04) and sirolimus use was associated with lower VO2max (p < 0.01). Conclusions TX had significant VO2max deficits that were not captured by conventional measures (ml/kg/min). Greater FM was an independent risk factor for low VO2max. Lower fitness in TX may be related to sirolimus effects on skeletal muscle.

Objective: Links between pain and joint degradation are poorly understood. We investigated the role of activation of Toll-like receptors (TLR) by cartilage metabolites in initiating and maintaining the inflammatory loop in OA causing joint destruction. Methods: Synovial membrane explants (SMEs) were prepared from OA patients' synovial biopsies. SMEs were cultured for 10 days under following conditions: culture medium alone, OSM þ TNFa, TLR2 agonist -Pam2CSK4, Pam3CSK4 or synthetic aggrecan 32-mer, TLR4 agonist -Lipid A. Release of pro-inflammatory and degradation biomarkers (acMMP3 and C3M) were measured by ELISA in conditioned media along with IL-6. Additionally, human cartilage was digested with ADAMTS-5, with or without the ADAMTS-5 inhibiting nanobody -M6495. Digested cartilage solution (DCS) and synthetic 32-mer were tested for TLR activation in SEAP based TLR reporter assay. Results: Western blotting confirmed TLR2 and TLR4 in untreated OA synovial biopsies. TLR agonists showed an increase in release of biomarkers -acMMP3 and C3M in SME. Synthetic 32-mer showed no activation in the TLR reporter assay. ADAMTS-5 degraded cartilage fragments activated TLR2 in vitro. Adding M6495 e an anti-ADAMTS-5 inhibiting nanobody®, blocked ADAMTS-5-mediated DCS TLR2 activation. Conclusion: TLR2 is expressed in synovium of OA patients and their activation by synthetic ligands causes increased tissue turnover. ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments which activates the TLR2 receptor in vitro. M6495 suppressed cartilage degradation by ADAMTS-5, limiting the activation of TLR2. In conclusion, pain and joint destruction may be linked to generation of ADAMTS-5 cartilage metabolites.

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