Search

Musculoskeletal and pain sequelae of COVID-19 are common in both the acute infection and patients experiencing longer term symptoms associated with recovery, known as postacute sequelae of COVID-19 (PASC). Patients with PASC may experience multiple manifestations of pain and other concurrent symptoms that complicate their experience of pain. In this review, the authors explore what is currently known about PASC-related pain and its pathophysiology as well as strategies for diagnosis and management.

Inactivation of Cytochrome P450 (CYP450) enzymes can lead to significant increases in exposure of comedicants. The majority of reported in vitro to in vivo extrapolation (IVIVE) data have historically focused on CYP3A, leaving the assessment of other CYP isoforms insubstantial. To this end, the utility of human hepatocytes (HHEP) and human liver microsomes (HLM) to predict clinically relevant drug-drug interactions was investigated with a focus on CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6. Evaluation of IVIVE for CYP2B6 was limited to only weak inhibition. A search of the University of Washington Drug-Drug Interaction Database was conducted to identify a clinically relevant weak, moderate, and strong inhibitor for selective substrates of CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6, resulting in 18 inhibitors for in vitro characterization against 119 clinical interaction studies. Pooled human hepatocytes and HLM were preincubated with increasing concentrations of inhibitors for designated timepoints. Time dependent inhibition was detected in HLM for four moderate/strong inhibitors, suggesting that some optimization of incubation conditions (i.e., lower protein concentrations) is needed to capture weak inhibition. Clinical risk assessment was conducted by incorporating the in vitro derived kinetic parameters maximal rate of enzyme inactivation (min−1) (kinact) and concentration of inhibitor resulting in 50% of the maximum enzyme inactivation (KI) into static equations recommended by regulatory authorities. Significant overprediction was observed when applying the basic models recommended by regulatory agencies. Mechanistic static models, which consider the fraction of metabolism through the impacted enzyme, using the unbound hepatic inlet concentration lead to the best overall prediction accuracy with 92% and 85% of data from HHEPs and HLM, respectively, within twofold of the observed value.SIGNIFICANCE STATEMENTCoupling time-dependent inactivation parameters derived from pooled human hepatocytes and human liver microsomes (HLM) with a mechanistic static model provides an easy and quantitatively accurate means to determine clinical drug-drug interaction risk from in vitro data. Optimization is needed to evaluate time-dependent inhibition (TDI) for weak and moderate inhibitors using HLM. Recommendations are made with respect to input parameters for in vitro to in vivo extrapolation (IVIVE) of TDI with non-CYP3A enzymes using available data from HLM and human hepatocytes.

Aldehyde oxidase (AOX) is a soluble, cytosolic enzyme that metabolizes various N-heterocyclic compounds and organic aldehydes. It has wide tissue distribution with highest levels found in liver, kidney, and lung. Human clearance projections of AOX substrates by in vitro assessments in isolated liver fractions (cytosol, S9) and even hepatocytes have been largely underpredictive of clinical outcomes. Various hypotheses have been suggested as to why this is the case. One explanation is that extrahepatic AOX expression contributes measurably to AOX clearance and is at least partially responsible for the often observed underpredictions. Although AOX expression has been confirmed in several extrahepatic tissues, activities therein and potential contribution to overall human clearance have not been thoroughly studied. In this work, the AOX enzyme activity using the S9 fractions of select extrahepatic human tissues (kidney, lung, vasculature, and intestine) were measured using carbazeran as a probe substrate. Measured activities were scaled to a whole-body clearance using best-available parameters and compared with liver S9 fractions. Here, the combined scaled AOX clearance obtained from the kidney, lung, vasculature, and intestine is very low and amounted to <1% of liver. This work suggests that AOX metabolism from extrahepatic sources plays little role in the underprediction of activity in human. One of the notable outcomes of this work has been the first direct demonstration of AOX activity in human vasculature.

Digital protein assays have great potential to advance immunodiagnostics because of their single-molecule sensitivity, high precision, and robust measurements. However, translating digital protein assays to acute clinical care has been challenging because it requires deployment of these assays with a rapid turnaround. Herein, we present a technology platform for ultrafast digital protein biomarker detection by using single-molecule counting of immune-complex formation events at an early, pre-equilibrium state. This method, which we term “pre-equilibrium digital enzyme-linked immunosorbent assay” (PEdELISA), can quantify a multiplexed panel of protein biomarkers in 10 µL of serum within an unprecedented assay incubation time of 15 to 300 seconds over a 104 dynamic range. PEdELISA allowed us to perform rapid monitoring of protein biomarkers in patients manifesting post-chimeric antigen receptor T-cell therapy cytokine release syndrome, with ∼30-minute sample-to-answer time and a sub–picograms per mL limit of detection. The rapid, sensitive, and low-input volume biomarker quantification enabled by PEdELISA is broadly applicable to timely monitoring of acute disease, potentially enabling more personalized treatment.

This study examined the incidence and trends of surfing-related and mild traumatic brain injuries that presented to United States emergency departments between 2001 and 2016. Subjects with surging-related head injuries were retrieved from the National Electronic Injury Surveillance System. A weighted total of 34,337 surfing-related head injuries were identified. The annual incidence of surfing-related head injuries insignificantly decreased from 2001 to 2016 (R2= .119; p = .19). Most common injuries included lacerations (50.4%), blunt head injuries (25.7%), and mild traumatic brain injuries (16.1%). Mild traumatic brain injury incidence and annual percentage increased significantly during the study period (R2= .251; p = .05 and R2= .346; p = .02, respectively).

How Rho GTPases are directed to effector pathways is an important question. We show here that BEM-type adaptors play unique roles in sequentially directing Cdc42 to an effector MAPK pathway. Our study may provide insight into Rho GTPase specification in other systems.

Polarized secretion facilitates polarized cell growth. For a secretory vesicle to dock at the plasma membrane, it must mature with a progressive association or dissociation of molecules that are, respectively, necessary or inhibitory to vesicle docking, including an exchange of Rab GTPases. In current models, oxysterol binding protein homologue 4 (Osh4p) establishes a PI4P gradient along the secretory trafficking pathway such that vesicles have higher PI4P levels after budding from the trans-Golgi relative to when vesicles arrive at the plasma membrane. In this study, using the lipid binding domain P4M and live cell imaging, we show that secretory vesicle-associated PI4P levels remain constant when vesicles traffic from the trans-Golgi to the plasma membrane. We also show that deletion of OSH4does not alter vesicle-associated PI4P levels, though loss of any individual member of the OSHfamily or complete loss of OSHfamily function alters the intracellular distribution of PI4P. We propose a model in which the Rab GTPases Ypt32p and Sec4p, remain associated with a secretory vesicle during trafficking, independent of PI4P levels and Osh4p. Together these data indicate the necessity of experiments revealing the location and timing of events required for vesicle maturation.Movie S1Movie S1In WT cells, GFP‐Ypt32p‐marked vesicles localize to sites of polarized growth (KKY1403). The arrowhead indicates an area where GFP‐Ypt32p‐marked vesicles can be seen moving from the mother cell to the bud neck. 3 frames/sec. Scale bar, 4μm.Movie S2Movie S2In WT cells, GFP‐Sec4p‐marked vesicles localize to sites of polarized growth (KKY1408). The arrowhead indicates an area where GFP‐Sec4p‐marked vesicles can be seen moving from the mother cell to the bud neck. 3 frames/sec. Scale bar, 4μm.Movie S3Movie S3In WT cells, mCherry2b‐P4M localizes to GFP‐Ypt32p‐marked vesicles as vesicles traffic from the mother cell to the bud neck (KKY1400). The same cells shown in Figure 3A. The arrowhead indicates an area where dual‐labeled vesicles can be seen moving from the mother cell to the bud neck. 3 frames/sec. Scale bar, 4μm.Movie S4Movie S4In WT cells, GFP‐Sec4p localizes to sites of polarized growth, but mCherry2b‐P4M does not strongly co‐localize with GFP‐Sec4p‐marked vesicles in the mother cell (KKY1405). The arrowhead indicates an area where a GFP‐Sec4p‐marked vesicle can be seen moving from the mother cell to the bud neck. 3 frames/sec. Scale bar, 4μm.

•BRCA1 binds directly to DNA double-strand ends.•Interactions with the DNA major grove is not found sufficient for complex stabilization.•Integrative NMR and MD simulations can reveal functions of fuzzy complexes.

New scientific frontiers and emerging technologies within the life sciences pose many global challenges to society. Big Data is a premier example, especially with respect to individual, national, and international security. Here a Special Agent of the Federal Bureau of Investigation discusses the security implications of Big Data and the need for security in the life sciences.