Evaluation of shelter dog activity levels before and during COVID-19 using automated analysis.

Animal shelters have been found to represent stressful environments for pet dogs, both affecting behavior and influencing welfare. The current COVID-19 pandemic has brought to light new uncertainties in animal sheltering practices which may affect shelter dog behavior in unexpected ways. To evaluate this, we analyzed changes in dog activity levels before COVID-19 and during COVID-19 using an automated video analysis within a large, open-admission animal shelter in New York City, USA. Shelter dog activity was analyzed during two two-week long time periods: (i) just before COVID-19 safety measures were put in place (Feb 26-Mar 17, 2020) and (ii) during the COVID-19 quarantine (July 10-23, 2020). During these two periods, video clips of 15.3 second, on average, were taken of participating kennels every hour from approximately 8 am to 8 pm. Using a two-step filtering approach, a matched sample (based on the number of days of observation) of 34 dogs was defined, consisting of 17 dogs in each group (N1/N2 = 17). An automated video analysis of active/non-active behaviors was conducted and compared to manual coding of activity. The automated analysis validated by comparison to manual coding reaching above 79% accuracy. Significant differences in the patterns of shelter dog activity were observed: less activity was observed in the afternoons before COVID-19 restrictions, while during COVID-19, activity remained at a constant average. Together, these findings suggest that 1) COVID-19 lockdown altered shelter dog in-kennel activity, likely due to changes in the shelter environment and 2) automated analysis can be used as a hands-off tool to monitor activity. While this method of analysis presents immense opportunity for future research, we discuss the limitations of automated analysis and guidelines in the context of shelter dogs that can increase accuracy of detection, as well as reflect on policy changes that might be helpful in mediating canine stress in changing shelter environments.

Amiodarone-Induced Hypothyroidism Related to Pericardial Effusion With Tamponade Physiology.

Hypothyroidism is a commonly encountered clinical diagnosis, particularly in the elderly population. While management of this disorder is rather simple with thyroxine replacement, healthcare providers may occasionally encounter patient non-adherence, which may lead to life-threatening complications. In this report, we present a case of a 74-year-old veteran with a long-standing history of amiodarone-induced asymptomatic hypothyroidism, who was non-adherent to thyroxine replacement therapy and presented to the hospital after a mechanical fall. His chest X-ray showed a globular heart with an enlarged cardiac silhouette, and transthoracic echocardiography (TTE) subsequently confirmed a large pericardial effusion with tamponade physiology. Physicians should be aware of and patients should be counseled about the potentially serious consequences of untreated hypothyroidism that could be avoided with proper patient education and adherence to the therapeutic plan.

Functional genomics screen identifies proteostasis targets that modulate prion protein (PrP) stability.

Prion protein (PrP) adopts either a helical conformation (PrPC) or an alternative, beta sheet-rich, misfolded conformation (PrPSc). The PrPSc form has the ability to "infect" PrPC and force it into the misfolded state. Accumulation of PrPSc is associated with a number of lethal neurodegenerative disorders, including Creutzfeldt-Jacob disease (CJD). Knockout of PrPC protects cells and animals from PrPSc infection; thus, there is interest in identifying factors that regulate PrPC stability, with the therapeutic goal of reducing PrPC levels and limiting infection by PrPSc. Here, we assembled a short-hairpin RNA (shRNA) library composed of 25+ shRNA sequences for each of 133 protein homeostasis (aka proteostasis) factors, such as molecular chaperones and co-chaperones. This Proteostasis shRNA Library was used to identify regulators of PrPC stability in HEK293 Hu129M cells. Strikingly, the screen identified a number of Hsp70 family members and their co-chaperones as putative targets. Indeed, a chemical pan-inhibitor of Hsp70s reduced PrPC levels and limited conversion to PrPSc in N2a cells. These results implicate specific proteostasis sub-networks, especially the Hsp70 system, as potential new targets for the treatment of CJD. More broadly, the Proteostasis shRNA Library might be a useful tool for asking which proteostasis factors are important for a given protein.

Protein-Protein Interactions in the Molecular Chaperone Network.

Molecular chaperones play a central role in protein homeostasis (a.k.a. proteostasis) by balancing protein folding, quality control, and turnover. To perform these diverse tasks, chaperones need the malleability to bind nearly any "client" protein and the fidelity to detect when it is misfolded. Remarkably, these activities are carried out by only ∼180 dedicated chaperones in humans. How do a relatively small number of chaperones maintain cellular and organismal proteostasis for an entire proteome? Furthermore, once a chaperone binds a client, how does it "decide" what to do with it? One clue comes from observations that individual chaperones engage in protein-protein interactions (PPIs)-both with each other and with their clients. These physical links coordinate multiple chaperones into organized, functional complexes and facilitate the "handoff" of clients between them. PPIs also link chaperones and their clients to other cellular pathways, such as those that mediate trafficking (e.g., cytoskeleton) and degradation (e.g., proteasome). The PPIs of the chaperone network have a wide range of affinity values (nanomolar to micromolar) and involve many distinct types of domain modules, such as J domains, zinc fingers, and tetratricopeptide repeats. Many of these motifs have the same binding surfaces on shared partners, such that members of one chaperone class often compete for the same interactions. Somehow, this collection of PPIs draws together chaperone families and creates multiprotein subnetworks that are able to make the "decisions" of protein quality control. The key to understanding chaperone-mediated proteostasis might be to understand how PPIs are regulated. This Account will discuss the efforts of our group and others to map, measure, and chemically perturb the PPIs within the molecular chaperone network. Structural biology methods, including X-ray crystallography, NMR spectroscopy, and electron microscopy, have all played important roles in visualizing the chaperone PPIs. Guided by these efforts and -omics approaches to measure PPIs, new advances in high-throughput chemical screening that are specially designed to account for the challenges of this system have emerged. Indeed, chemical biology has played a particularly important role in this effort, as molecules that either promote or inhibit specific PPIs have proven to be invaluable research probes in cells and animals. In addition, these molecules have provided leads for the potential treatment of protein misfolding diseases. One of the major products of this research field has been the identification of putative PPI drug targets within the chaperone network, which might be used to change chaperone "decisions" and rebalance proteostasis.

Hierarchical functional specificity of cytosolic heat shock protein 70 (Hsp70) nucleotide exchange factors in yeast.

Heat shock protein 70 (Hsp70) molecular chaperones play critical roles in protein homeostasis. In the budding yeast Saccharomyces cerevisiae, cytosolic Hsp70 interacts with up to three types of nucleotide exchange factors (NEFs) homologous to human counterparts: Sse1/Sse2 (Heat shock protein 110 (Hsp110)), Fes1 (HspBP1), and Snl1 (Bag-1). All three NEFs stimulate ADP release; however, it is unclear why multiple distinct families have been maintained throughout eukaryotic evolution. In this study we investigate NEF roles in Hsp70 cell biology using an isogenic combinatorial collection of NEF deletion mutants. Utilizing well characterized model substrates, we find that Sse1 participates in most Hsp70-mediated processes and is of particular importance in protein biogenesis and degradation, whereas Fes1 contributes to a minimal extent. Surprisingly, disaggregation and resolubilization of thermally denatured firefly luciferase occurred independently of NEF activity. Simultaneous deletion of SSE1 and FES1 resulted in constitutive activation of heat shock protein expression mediated by the transcription factor Hsf1, suggesting that these two factors are important for modulating stress response. Fes1 was found to interact in vivo preferentially with the Ssa family of cytosolic Hsp70 and not the co-translational Ssb homolog, consistent with the lack of cold sensitivity and protein biogenesis phenotypes for fes1Δ cells. No significant consequence could be attributed to deletion of the minor Hsp110 SSE2 or the Bag homolog SNL1. Together, these lines of investigation provide a comparative analysis of NEF function in yeast that implies Hsp110 is the principal NEF for cytosolic Hsp70, making it an ideal candidate for therapeutic intervention in human protein folding disorders.

Coupled assays for monitoring protein refolding in Saccharomyces cerevisiae.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences (1). External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases (10). It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme (12). Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 (13), known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system (5), to protein refolding to validate this approach.

Biology of the heat shock response and protein chaperones: budding yeast (Saccharomyces cerevisiae) as a model system.

The eukaryotic heat shock response is an ancient and highly conserved transcriptional program that results in the immediate synthesis of a battery of cytoprotective genes in the presence of thermal and other environmental stresses. Many of these genes encode molecular chaperones, powerful protein remodelers with the capacity to shield, fold, or unfold substrates in a context-dependent manner. The budding yeast Saccharomyces cerevisiae continues to be an invaluable model for driving the discovery of regulatory features of this fundamental stress response. In addition, budding yeast has been an outstanding model system to elucidate the cell biology of protein chaperones and their organization into functional networks. In this review, we evaluate our understanding of the multifaceted response to heat shock. In addition, the chaperone complement of the cytosol is compared to those of mitochondria and the endoplasmic reticulum, organelles with their own unique protein homeostasis milieus. Finally, we examine recent advances in the understanding of the roles of protein chaperones and the heat shock response in pathogenic fungi, which is being accelerated by the wealth of information gained for budding yeast.

How does participation of youth with spina bifida vary by age?

BACKGROUND: Youth with disabilities are at risk for decreased participation in community activities. However, little is known about participation at different developmental periods of childhood and adolescence among youth with spina bifida (SB) or whether child, family, and SB-associated factors influence participation. QUESTIONS/PURPOSES: Our cross-sectional study examined participation among youth with SB and assessed how participation differs between youth ages 2-5, 6-12, and 13-18; how participation relates to child (gender) and family (caregiver marital status, education, and employment) characteristics; and how participation relates to SB-related factors (motor level, hydrocephalus, ambulation, medical issues, and bladder/bowel needs). PATIENTS AND METHODS: Sixty-three youth ages 2-18 years and/or their caregivers completed age-appropriate measures of participation for youth with disabilities. The patients had an average age of 9.52 years (SD = 5.22), 83% had a shunt, 34% had a motor level of L2 or higher, and 66% L3 or lower. RESULTS: A comparison of youth ages 2-5 (n = 19), 6-12 (n = 21), and 13-18 (n = 23) revealed older youth participated less in recreational, physical, and skill-based activities. Caregiver employment facilitated participation in social activities. Youth who did not have a shunt participated more often in physical and skill-based activities. Youth without recent major medical issues participated more often in physical and social activities. More caregivers reported bladder and bowel needs as barriers to participation for youth ages 6-12 than those ages 2-5 or 13-18. CONCLUSIONS: Participation of youth with SB varies by age and across child and caregiver factors and should be understood in a developmental and situational context.

Physical activity promotion: a local and state health department perspective.

Local and state health departments are well-positioned to serve as catalysts for the institutional and community changes needed to increase physical activity across the population. Efforts should focus on evidence-based strategies, including promotion of high-quality physical education in schools, social support networks and structured programs for physical activity in communities, and organizational practices, policies, and programs that promote physical activity in the workplace. Health departments must also focus on land use and transportation practices and policies in communities where the built environment creates major impediments to physical activity, particularly in economically disadvantaged communities disproportionately burdened by chronic disease.