Extracting regulatory active chromatin footprint from cell-free DNA.

Cell-free DNA (cfDNA) has emerged as a pivotal player in precision medicine, revolutionizing the diagnostic and therapeutic landscape. While its clinical applications have significantly increased in recent years, current cfDNA assays have limited ability to identify the active transcriptional programs that govern complex disease phenotypes and capture the heterogeneity of the disease. To address these limitations, we have developed a non-invasive platform to enrich and examine the active chromatin fragments (cfDNAac) in peripheral blood. The deconvolution of the cfDNAac signal from traditional nucleosomal chromatin fragments (cfDNAnuc) yields a catalog of features linking these circulating chromatin signals in blood to specific regulatory elements across the genome, including enhancers, promoters, and highly transcribed genes, mirroring the epigenetic data from the ENCODE project. Notably, these cfDNAac counts correlate strongly with RNA polymerase II activity and exhibit distinct expression patterns for known circadian genes. Additionally, cfDNAac signals across gene bodies and promoters show strong correlations with whole blood gene expression levels defined by GTEx. This study illustrates the utility of cfDNAac analysis for investigating epigenomics and gene expression, underscoring its potential for a wide range of clinical applications in precision medicine.

Pain sensitivity differs between dog breeds but not in the way veterinarians believe.

Background: Veterinarians hold distinct breed-specific pain sensitivity beliefs that differ from the general public but are highly consistent with one another. This is remarkable as there is no current scientific evidence for biological differences in pain sensitivity across dog breeds. Therefore, the present study evaluated whether pain sensitivity thresholds differ across a set of dog breeds and, if so, whether veterinarians' pain sensitivity ratings explain these differences or whether these ratings are attributed to behavioral characteristics. Methods: Pain sensitivity thresholds [using quantitative sensory testing (QST) methods] and canine behaviors (using owner questionnaires and emotional reactivity tests) were prospectively measured across selected dog breeds. Adult, healthy dogs from 10 dog breeds/breed types were recruited, representing breeds subjectively rated by veterinarians as high (chihuahua, German shepherd, Maltese, Siberian husky), average (border collie, Boston terrier, Jack Russell terrier), or low (golden retriever, pitbull, Labrador retriever) pain sensitivity. A final sample of 149 dogs was included in statistical analyses. Results: Veterinarians' pain sensitivity ratings provided a minimal explanation for pain sensitivity thresholds measured using QST in dogs; however, dog breeds did differ in their pain sensitivity thresholds across the QST methods evaluated. Breed differences were observed for some aspects of emotional reactivity tests; however, these behavioral differences did not explain the differences in pain sensitivity thresholds found. Veterinarians' pain sensitivity ratings were positively associated with dog approach scores for the disgruntled stranger test suggesting that the way dogs greet strangers may be a factor influencing veterinarians' ratings of pain sensitivity across dog breeds. Conclusions and clinical relevance: Overall, these findings highlight a need to investigate biological mechanisms that may explain breed differences in pain sensitivity because this may inform pain management recommendations. Further, future research should focus on when and how these breed-specific pain sensitivity beliefs developed in veterinarians, as veterinarians' beliefs could impact the recognition and treatment of pain for canine patients.

Evaluation of a nutritional supplement for the alleviation of pain associated with feline degenerative joint disease: a prospective, randomized, stratified, double-blind, placebo-controlled clinical trial.

OBJECTIVES: The purpose of this study was to evaluate the pain-alleviating and activity-enhancing effects of glucosamine/chondroitin sulfate (Dasuquin) in cats that had degenerative joint disease (DJD) and owner-noted mobility/activity impairment. We hypothesized that the nutritional supplement would produce pain-relieving and activity-enhancing effects in cats with painful DJD. METHODS: In this prospective, randomized, stratified, double-blind, placebo-controlled clinical trial, 59 cats with DJD pain were assigned to receive a placebo (n = 30) or supplement (n = 29) for 6 weeks after 2 weeks of placebo. Outcome measures (at-home accelerometry and client-specific outcome measures [feline (CSOMf); Feline Musculoskeletal Pain Index (FMPI); quality of life (QoL)]; and veterinarian examination) were collected at days 14, 28, 42 and 56. RESULTS: Twenty-seven cats in the treatment group and 30 in the placebo group completed the trial. Within the first 2 weeks (placebo administration to all cats), 78% of all cats had an improvement in CSOMf scores. Both groups showed significant improvement at most time points in CSOMf, FMPI, QoL and pain scores, with the placebo group showing greater improvement than the supplement group (significant for CSOMf [P = 0.01]). Overall, no differences in activity were seen between the groups. Cumulative distribution function analysis indicated that for most levels of activity, the placebo-treated cats were more active; however, the least active cats were more active on the supplement (P = 0.013). CONCLUSIONS AND RELEVANCE: This study showed a strong placebo effect. The glucosamine/chondroitin sulfate supplement did not show pain-relieving effects when compared with placebo.

Assessment of Sensory Thresholds in Dogs using Mechanical and Hot Thermal Quantitative Sensory Testing.

Quantitative sensory testing (QST) is used to evaluate the function of the somatosensory system in dogs by assessing the response to applied mechanical and thermal stimuli. QST is used to determine normal dogs' sensory thresholds and evaluate alterations in peripheral and central sensory pathways caused by various disease states, including osteoarthritis, spinal cord injury, and cranial cruciate ligament rupture. Mechanical sensory thresholds are measured by electronic von Frey anesthesiometers and pressure algometers. They are determined as the force at which the dog exhibits a response indicating conscious stimulus perception. Hot thermal sensory thresholds are the latency to respond to a fixed or ramped temperature stimulus applied by a contact thermode. Following a consistent protocol for performing QST and paying attention to details of the testing environment, procedure, and individual study subjects are critical for obtaining accurate QST results for dogs. Protocols for the standardized collection of QST data in dogs have not been described in detail. QST should be performed in a quiet, distraction-free environment that is comfortable for the dog, the QST operator, and the handler. Ensuring that the dog is calm, relaxed, and properly positioned for each measurement helps produce reliable, consistent responses to the stimuli and makes the testing process more manageable. The QST operator and handler should be familiar and comfortable with handling dogs and interpreting dogs' behavioral responses to potentially painful stimuli to determine the endpoint of testing, reduce stress, and maintain safety during the testing process.

Nucleolar Stress Induction by Oxaliplatin and Derivatives.

Platinum(II) compounds are a critical class of chemotherapeutic agents. Recent studies have highlighted the ability of a subset of Pt(II) compounds, including oxaliplatin but not cisplatin, to induce cytotoxicity via nucleolar stress rather than a canonical DNA damage response. In this study, influential properties of Pt(II) compounds were investigated using redistribution of nucleophosmin (NPM1) as a marker of nucleolar stress. NPM1 assays were coupled to calculated and measured properties such as compound size and hydrophobicity. The oxalate leaving group of oxaliplatin is not required for NPM1 redistribution. Interestingly, although changes in diaminocyclohexane (DACH) ligand ring size and aromaticity can be tolerated, ring orientation appears important for stress induction. The specificity of ligand requirements provides insight into the striking ability of only certain Pt(II) compounds to activate nucleolar processes.

Pt-induced crosslinks promote target enrichment and protection from serum nucleases.

Identifying the interactions of small molecules with biomolecules in complex cellular environments is a significant challenge. As one important example, despite being widely used for decades, much is still not understood regarding the cellular targets of Pt(II)-based anticancer drugs. In this study we introduce a novel method for isolation of Pt(II)-bound biomolecules using a DNA hybridization pull-down approach. Using a modified Pt reagent, click-ligation of a DNA oligonucleotide to both a Pt(II)-bound DNA hairpin and bovine serum albumin (BSA) are demonstrated. Subsequent hybridization to a biotin-labeled oligonucleotide allows for efficient isolation of Pt(II)-bound species by streptavidin pulldown. We also find that platinated bovine serum albumin readily crosslinks to DNA in the absence of click ligation, and that a fraction of BSA-bound Pt(II) can transfer to DNA over time. Interestingly, in in vitro studies, fragmented mammalian DNA that is crosslinked to BSA through Pt(II) exhibits significantly increased protection from degradation by serum nucleases.

Platinum Binds Proteins in the Endoplasmic Reticulum of S. cerevisiae and Induces Endoplasmic Reticulum Stress.

Pt(II)-based anticancer drugs are widely used in the treatment of a variety of cancers, but their clinical efficacy is hindered by undesirable side effects and resistance. While much research has focused on Pt(II) drug interactions with DNA, there is increasing interest in proteins as alternative targets and contributors to cytotoxic and resistance mechanisms. Here, we describe a chemical proteomic method for isolation and identification of cellular protein targets of platinum compounds using Pt(II) reagents that have been modified for participation in the 1,3 dipolar cycloaddition "click" reaction. Using this method to visualize and enrich for targets, we identified 152 proteins in Pt(II)-treated Saccharomyces cerevisiae. Of interest was the identification of multiple proteins involved in the endoplasmic reticulum (ER) stress response, which has been proposed to be an important cytoplasmic mediator of apoptosis in response to cisplatin treatment. Consistent with possible direct targeting of this pathway, the ER stress response was confirmed to be induced in Pt(II)-treated yeast along with in vitro Pt(II)-inhibition of one of the identified proteins, protein disulfide isomerase.

Convenient detection of metal-DNA, metal-RNA, and metal-protein adducts with a click-modified Pt(II) complex.

cis-[Pt(2-azido-1,3-propanediamine)Cl2] is a reagent for high-yield post-treatment fluorescent labelling of Pt(II) biomolecular targets using click chemistry and exhibits a bias in conformational isomers in the context of duplex DNA. Pt-protein adducts are detected using BSA as a model. Following in vivo treatment, long-lived Pt-RNA adducts are detected on ribosomal RNA.