Digital Biomarker Applications Across the Spectrum of Opioid Use Disorder.

Opioid use disorder (OUD) is one of the most pressing public health problems of the past decade, with over eighty thousand overdose related deaths in 2021 alone. Digital technologies to measure and respond to disease states encompass both on- and off-body sensors. Such devices can be used to detect and monitor end-user physiologic or behavioral measurements (i.e. digital biomarkers) that correlate with events of interest, health, or pathology. Recent work has demonstrated the potential of digital biomarkers to be used as a tools in the prevention, risk mitigation, and treatment of opioid use disorder (OUD). Multiple physiologic adaptations occur over the course of opioid use, and represent potential targets for digital biomarker based monitoring strategies. This review explores the current evidence (and potential) for digital biomarkers monitoring across the spectrum of opioid use. Technologies to detect opioid administration, withdrawal, hyperalgesia and overdose will be reviewed. Driven by empirically derived algorithms, these technologies have important implications for supporting the safe prescribing of opioids, reducing harm in active opioid users, and supporting those in recovery from OUD.

Analysis of AKAP7γ Dimerization.

A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that contribute to spatiotemporal regulation of PKA-mediated phosphorylation events. In particular, AKAP7 is a family of alternatively spliced proteins that participates in cardiac calcium dynamics. Here, we demonstrate via pull-down from transfected cells and by direct protein-protein association that AKAP7γ self-associates. Self-association appears to be an isoform specific phenomenon, as AKAP7α did not associate with itself or with AKAP7γ. However, AKAP7γ did associate with AKAP7δ, suggesting the long isoforms of the AKAP can form heterodimers. Surface plasmon resonance found that the AKAP7γ self-association occurs via two high affinity binding sites with K D values in the low nanomolar range. Mapping of the binding sites by peptide array reveals that AKAP7γ interacts with itself through multiple regions. Photon counting histogram analysis (PCH) of AKAP7γ-EGFP expressed in HEK-293 cells confirmed that AKAP7γ-EGFP self-associates in a cellular context. Lastly, computational modeling of PKA dynamics within AKAP7γ complexes suggests that oligomerization may augment phosphorylation of scaffolded PKA substrates. In conclusion, our study reveals that AKAP7γ forms both homo- and heterodimers with the long isoforms of the AKAP and that this phenomenon could be an important step in mediating effective substrate phosphorylation in cellular microdomains.

Phosphorylation state-dependent interaction between AKAP7δ/γ and phospholamban increases phospholamban phosphorylation.

Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca(2+) cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca(2+) re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3]. Here, we further these findings by showing that phosphorylation of PLB requires interaction with AKAP7δ/γ and that this interaction occurs only when PLB is unphosphorylated. Additionally, we find that two mutants of PLB (R9C and Δ14), which are associated with dilated cardiomyopathy in humans, prevent association with AKAP7δ/γ and display reduced phosphorylation in vitro. This finding implicates the AKAP7δ/γ-PLB interaction in the pathology of the disease phenotype. Further exploration of the AKAP7δ/γ-PLB association demonstrated a phosphorylation state-dependence of the interaction. Computational modeling revealed that this mode of interaction allows for small amounts of AKAP and PKA (100-200nM) to regulate the phosphorylation of large quantities of PLB (50µM). Our results confirm that AKAP7γ/δ binding to PLB is important for phosphorylation of PLB, and describe a novel phosphorylation state-dependent binding mechanism that explains how phosphorylation of highly abundant PKA substrates can be regulated by AKAPs present at ~100-200 fold lower concentrations.

Pharmacological inhibition of Mdm2 triggers growth arrest and promotes DNA breakage in mouse colon tumors and human colon cancer cells.

The p53 tumor suppressor protein performs a number of cellular functions, ranging from the induction of cell cycle arrest and apoptosis to effects on DNA repair. Modulating p53 activity with Mdm2 inhibitors is a promising approach for treating cancer; however, it is presently unclear how the in vivo application of Mdm2 inhibitors impact the myriad processes orchestrated by p53. Since approximately half of all colon cancers (predominately cancers with microsatellite instability) are p53-normal, we assessed the anticancer activity of the Mdm2 inhibitor Nutlin-3 in the mouse azoxymethane (AOM) colon cancer model, in which p53 remains wild type. Using a cell line derived from an AOM-induced tumor, we found that four daily exposures to Nutlin-3 induced persistent p53 stabilization and cell cycle arrest without significant apoptosis. A 4-day dosing schedule in vivo generated a similar response in colon tumors; growth arrest without significantly increased apoptosis. In adjacent normal colon tissue, Nutlin-3 treatment reduced both cell proliferation and apoptosis. Surprisingly, Nutlin-3 induced a transient DNA damage response in tumors but not in adjacent normal tissue. Nutlin-3 likewise induced a transient DNA damage response in human colon cancer cells in a p53-dependent manner, and enhanced DNA strand breakage and cell death induced by doxorubicin. Our findings indicate that Mdm2 inhibitors not only trigger growth arrest, but may also stimulate p53's reported ability to slow homologous recombination repair. The potential impact of Nutlin-3 on DNA repair in tumors suggests that Mdm2 inhibitors may significantly accentuate the tumoricidal actions of certain therapeutic modalities.

DNA damage response to the Mdm2 inhibitor nutlin-3.

Mdm2 inhibitors represent a promising class of p53 activating compounds that may be useful in cancer treatment and prevention. However, the consequences of pharmacological p53 activation are not entirely clear. We observed that Nutlin-3 triggered a DNA damage response in azoxymethane-induced mouse AJ02-NM(0) colon cancer cells, characterized by the phosphorylation of H2AX (at Ser-139) and p53 (at Ser-15). The DNA damage response was highest in cells showing robust p53 stabilization, it could be triggered by the active but not the inactive Nutlin-3 enantiomer, and it was also activated by another pharmacological Mdm2 inhibitor (Caylin-1). Quantification of gamma H2AX-positive cells following Nutlin-3 exposure showed that approximately 17% of cells in late S and G2/M were mounting a DNA damage response (compared to a approximately 50% response to 5-fluorouracil). Nutlin-3 treatment caused the formation of double-strand DNA strand breaks, promoted the formation of micronuclei, accentuated strand breakage induced by doxorubicin and sensitized the mouse colon cancer cells to DNA break-inducing topoisomerase II inhibitors. Although the HCT116 colon cancer cells did not mount a significant DNA damage response following Nutlin-3 treatment, Nutlin-3 enhanced the DNA damage response to the nucleotide synthesis inhibitor hydroxyurea in a p53-dependent manner. Finally, p21 deletion also sensitized HCT116 cells to the Nutlin-3-induced DNA damage response, suggesting that cell cycle checkpoint abnormalities may promote this response. We propose that p53 activation by Mdm2 inhibitors can result in the slowing of double-stranded DNA repair. Although this effect may suppress illegitimate homologous recombination repair, it may also increase the risk of clastogenic events.