Endosomal trafficking ensures the proper distribution of lipids and proteins to various cellular compartments, facilitating intracellular communication, nutrient transport, waste disposal, and the ...maintenance of cell structure. Retromer, a peripheral membrane protein complex, plays an important role in this process by recruiting the associated actin‐polymerizing WASH complex to establish distinct sorting domains. The WASH complex is recruited through the interaction of the VPS35 subunit of retromer with the WASH complex subunit FAM21. Here, we report the identification of two separate fragments of FAM21 that interact with VPS35, along with a third fragment that binds to the VPS29 subunit of retromer. The crystal structure of VPS29 bound to a peptide derived from FAM21 shows a distinctive sharp bend that inserts into a conserved hydrophobic pocket with a binding mode similar to that adopted by other VPS29 effectors. Interestingly, despite the network of interactions between FAM21 and retromer occurring near the Parkinson's disease‐linked mutation (D620N) in VPS35, this mutation does not significantly impair the direct association with FAM21 in vitro.
Nowadays, the meat industry requires non-destructive, sustainable, and rapid methods that can provide objective and accurate quality assessment with little human intervention. Therefore, the present ...research aimed to create a model that can classify beef samples from longissimus thoracis muscle according to their tenderness degree based on hyperspectral imaging (HSI). In order to obtain different textures, two main strategies were used: (a) aging type (wet and dry aging with or without starters) and (b) aging times (0, 7, 13, 21, and 27 days). Categorization into two groups was carried out for further chemometric analysis, encompassing group 1 (ngroup1 = 30) with samples with WBSF ˂ 53 N whereas group 2 (ngroup2 = 28) comprised samples with WBSF values ≥ 53 N. Then, classification models were created by applying the partial least squares discriminant analysis (PLS-DA) method. The best results were achieved by combining the following pre-processing algorithms: 1st derivative + mean center, reaching 70.83% of correctly classified (CC) samples and 67.14% for cross validation (CV) and prediction, respectively. In general, it can be concluded that HSI technology combined with chemometrics has the potential to differentiate and classify meat samples according to their textural characteristics.
Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen Legionella pneumophila encodes RidL to hijack the host scaffold protein VPS29, ...a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of L. pneumophila RidL in complex with the human VPS29–VPS35 retromer subcomplex. A hairpin loop protruding from RidL inserts into a conserved pocket on VPS29 that is also used by cellular ligands, such as Tre-2/Bub2/Cdc16 domain family member 5 (TBC1D5) and VPS9-ankyrin repeat protein for VPS29 binding. Consistent with the idea of molecular mimicry in protein interactions, RidL outcompeted TBC1D5 for binding to VPS29. Furthermore, the interaction of RidL with retromer did not interfere with retromer dimerization but was essential for association of RidL with retromer-coated vacuolar and tubular endosomes. Our work thus provides structural and mechanistic evidence into how RidL is targeted to endosomal membranes.
Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen Legionella pneumophila encodes RidL to hijack the host scaffold protein VPS29, ...a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of L. pneumophila RidL in complex with the human VPS29-VPS35 retromer subcomplex. A hairpin loop protruding from RidL inserts into a conserved pocket on VPS29 that is also used by cellular ligands, such as Tre-2/Bub2/Cdc16 domain family member 5 (TBC1D5) and VPS9-ankyrin repeat protein for VPS29 binding. Consistent with the idea of molecular mimicry in protein interactions, RidL outcompeted TBC1D5 for binding to VPS29. Furthermore, the interaction of RidL with retromer did not interfere with retromer dimerization but was essential for association of RidL with retromer-coated vacuolar and tubular endosomes. Our work thus provides structural and mechanistic evidence into how RidL is targeted to endosomal membranes.
Human DNA polymerase δ is essential for DNA replication and acts in conjunction with the processivity factor proliferating cell nuclear antigen (PCNA). In addition to its catalytic subunit (p125), ...pol δ comprises three regulatory subunits (p50, p68, and p12). PCNA interacts with all of these subunits, but only the interaction with p68 has been structurally characterized. Here, we report solution NMR–, isothermal calorimetry–, and X-ray crystallography–based analyses of the p12–PCNA interaction, which takes part in the modulation of the rate and fidelity of DNA synthesis by pol δ. We show that p12 binds with micromolar affinity to the classical PIP-binding pocket of PCNA via a highly atypical PIP box located at the p12 N terminus. Unlike the canonical PIP box of p68, the PIP box of p12 lacks the conserved glutamine; binds through a 2-fork plug made of an isoleucine and a tyrosine residue at +3 and +8 positions, respectively; and is stabilized by an aspartate at +6 position, which creates a network of intramolecular hydrogen bonds. These findings add to growing evidence that PCNA can bind a diverse range of protein sequences that may be broadly grouped as PIP-like motifs as has been previously suggested.
Abstract
p15PAF is an oncogenic intrinsically disordered protein that regulates DNA replication and lesion bypass by interacting with the human sliding clamp PCNA. In the absence of DNA, p15PAF ...traverses the PCNA ring via an extended PIP-box that contacts the sliding surface. Here, we probed the atomic-scale structure of p15PAF-PCNA-DNA ternary complexes. Crystallography and MD simulations show that, when p15PAF occupies two subunits of the PCNA homotrimer, DNA within the ring channel binds the unoccupied subunit. The structure of PCNA-bound p15PAF in the absence and presence of DNA is invariant, and solution NMR confirms that DNA does not displace p15PAF from the ring wall. Thus, p15PAF reduces the available sliding surfaces of PCNA, and may function as a belt that fastens the DNA to the clamp during synthesis by the replicative polymerase (pol δ). This constraint, however, may need to be released for efficient DNA lesion bypass by the translesion synthesis polymerase (pol η). Accordingly, our biochemical data show that p15PAF impairs primer synthesis by pol η-PCNA holoenzyme against both damaged and normal DNA templates. In light of our findings, we discuss the possible mechanistic roles of p15PAF in DNA replication and suppression of DNA lesion bypass.
Nowadays, the meat industry requires non-destructive, sustainable, and rapid methods that can provide objective and accurate quality assessment with little human intervention. Therefore, the present ...research aimed to create a model that can classify beef samples from longissimus thoracis muscle according to their tenderness degree based on hyperspectral imaging (HSI). In order to obtain different textures, two main strategies were used: (a) aging type (wet and dry aging with or without starters) and (b) aging times (0, 7, 13, 21, and 27 days). Categorization into two groups was carried out for further chemometric analysis, encompassing group 1 (nsub.group1 = 30) with samples with WBSF ˂ 53 N whereas group 2 (nsub.group2 = 28) comprised samples with WBSF values ≥ 53 N. Then, classification models were created by applying the partial least squares discriminant analysis (PLS-DA) method. The best results were achieved by combining the following pre-processing algorithms: 1st derivative + mean center, reaching 70.83% of correctly classified (CC) samples and 67.14% for cross validation (CV) and prediction, respectively. In general, it can be concluded that HSI technology combined with chemometrics has the potential to differentiate and classify meat samples according to their textural characteristics.
Pathophysiological changes involved in drug disposition in critically ill patients should be considered in order to optimize the dosing of vancomycin administered by continuous infusion, and certain ...strategies must be applied to reach therapeutic targets on the first day of treatment. The aim of this study was to develop a population pharmacokinetic model of vancomycin to determine clinical covariates, including mechanical ventilation, that influence the wide variability of this antimicrobial. Plasma vancomycin concentrations from 54 critically ill patients were analyzed simultaneously by a population pharmacokinetic approach. A nomogram for dosing recommendations was developed and was internally evaluated through stochastic simulations. The plasma vancomycin concentration-versus-time data were best described by a one-compartment open model with exponential interindividual variability associated with vancomycin clearance and the volume of distribution. Residual error followed a homoscedastic trend. Creatinine clearance and body weight significantly dropped the objective function value, showing their influence on vancomycin clearance and the volume of distribution, respectively. Characterization based on the presence of mechanical ventilation demonstrated a 20% decrease in vancomycin clearance. External validation (
= 18) was performed to evaluate the predictive ability of the model; median bias and precision values were 0.7 mg/liter (95% confidence interval CI, -0.4, 1.7) and 5.9 mg/liter (95% CI, 5.4, 6.4), respectively. A population pharmacokinetic model was developed for the administration of vancomycin by continuous infusion to critically ill patients, demonstrating the influence of creatinine clearance and mechanical ventilation on vancomycin clearance, as well as the implications for targeting dosing rates to reach the therapeutic range (20 to 30 mg/liter).
•Amikacin pharmacokinetics is described by a two-compartment open model in elderly•Amikacin clearance in elderly patients is better related to Crockoft-Gault equation•Initial amikacin dose should be ...chosen based on body mass index in elderly patients•Therapeutic drug monitoring could be performed with final pharmacokinetic model
The aim of this study was to characterize the population pharmacokinetics of amikacin in elderly patients by means of nonlinear mixed effects modelling and to propose initial dosing schemes to optimize therapy based on PK/PD targets.
A total of 137 elderly patients from 65 to 94 years receiving intravenous amikacin and routine therapeutic drug monitoring at Hospital Universitario Severo Ochoa were included. Concentration–time data and clinical information were retrospectively collected; initial doses of amikacin ranged from 5.7 to 22.5 mg/kg/day and each patient provided between 1 and 10 samples.
Amikacin pharmacokinetics were best described by a two-compartment open model; creatinine clearance (CrCL) was related to drug clearance (2.75 L/h/80 mL/min) and it was augmented 28% when non-steroidal anti-inflammatory drugs were concomitantly administered. Body mass index (BMI) influenced the central volume of distribution (17.4 L/25 kg/m2). Relative absolute prediction error was reduced from 33.2% (base model) to 17.9% (final model) when predictive performance was evaluated with a different group of elderly patients. A nomogram for initial amikacin dosage was developed and evaluated based on stochastic simulations considering final model to achieve PK/PD targets (Cmax/MIC>10 and AUC/MIC>75) and to avoid toxic threshold (Cmin<2.5 mg/L).
Initial dosing approach for amikacin was designed for elderly patients based on nonlinear mixed effects modeling to maximize the probability to attain efficacy and safety targets considering individual BMI and CrCL.
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