Winning the race with aging: age-related changes in gait speed and its association with cognitive performance in dogs.

Introduction: In humans, gait speed is a crucial component in geriatric evaluation since decreasing speed can be a harbinger of cognitive decline and dementia. Aging companion dogs can suffer from age-related mobility impairment, cognitive decline and dementia known as canine cognitive dysfunction syndrome. We hypothesized that there would be an association between gait speed and cognition in aging dogs. Methods: We measured gait speed on and off leash in 46 adult and 49 senior dogs. Cognitive performance in senior dogs was assessed by means of the Canine Dementia Scale and a battery of cognitive tests. Results: We demonstrated that dogs' food-motivated gait speed off leash is correlated with fractional lifespan and cognitive performance in dogs, particularly in the domains of attention and working memory. Discussion: Food-motivated gait speed off leash represents a relatively easy variable to measure in clinical settings. Moreover, it proves to be a more effective indicator of age-related deterioration and cognitive decline than gait speed on leash.

Flow behavior of protein solutions in a lab-scale chromatographic system.

A fluid dynamics model has been developed to describe flow behavior in a lab-scale chromatographic system dedicated for protein processing. The case study included a detailed analysis of elution pattern of a protein, which was a monoclonal antibody, glycerol, and their mixtures in aqueous solutions. Glycerol solutions mimicked viscous environment of the concentrated protein solutions. The model accounted for concentration dependences of solution viscosity and density, and dispersion anisotropy in the packed bed. It was implemented into a commercial computational fluid dynamics software using user-defined functions. The prediction efficiency was successfully verified by comparing the model simulations in the form of the concentration profiles and their variances with the corresponding experimental data. The contribution of the individual elements of the chromatographic system to protein band broadening was evaluated for different configurations: for the extra-column volumes in the absence of the chromatographic column, for the zero-length column without the packed bed and for the column containing the packed bed. The influence of the operating variables, including: the mobile phase flowrate, the type of the injection system, i.e., the injection loop capillary or the superloop, the injection volume and the length of the packed bed, on band broadening of the protein was determined under nonadsorbing conditions. For protein solutions having viscosity comparable with the mobile phase, the flow behavior either in the column hardware or in the injection system made major contributions to band broadening, which depended on the type of the injection system. For highly viscous protein solution, the flow behavior in the packed bed exerted a dominant influence on band broadening.

Age-associated changes in electroretinography measures in companion dogs.

PURPOSE: To determine the association between age and retinal full-field electroretinographic (ERG) measures in companion (pet) dogs, an important translational model species for human neurologic aging. METHODS: Healthy adult dogs with no significant ophthalmic abnormalities were included. Unilateral full-field light- and dark-adapted electroretinography was performed using a handheld device, with mydriasis and topical anaesthesia. Partial least squares effect screening analysis was performed to determine the effect of age, sex, body weight and use of anxiolytic medication on log-transformed ERG peak times and amplitudes; age and anxiolytic usage had significant effects on multiple ERG outcomes. Mixed model analysis was performed on data from dogs not receiving anxiolytic medications. RESULTS: In dogs not receiving anxiolytics, median age was 118 months (interquartile range 72-140 months, n = 77, 44 purebred, 33 mixed breed dogs). Age was significantly associated with prolonged peak times of a-waves (dark-adapted 3 and 10 cds/m2 flash p < 0.0001) and b-waves (cone flicker p = 0.03, dark-adapted 0.01 cds/m2 flash p = 0.001). Age was also significantly associated with reduced amplitudes of a-waves (dark-adapted 3 cds/m2 flash p < 0.0001, 10 cds/m2 flash p = 0.005) and b-waves (light-adapted 3 cds/m2 flash p < 0.0001, dark-adapted 0.01 cds/m2 flash p = 0.0004, 3 cds/m2 flash p < 0.0001, 10 cds/m2 flash p = 0.007) and flicker (light-adapted 30 Hz 3 cds/m2 p = 0.0004). Within the Golden Retriever breed, these trends were matched in a cross-sectional analysis of 6 individuals that received no anxiolytic medication. CONCLUSIONS: Aged companion dogs have slower and reduced amplitude responses in both rod- and cone-mediated ERG. Consideration of anxiolytic medication use should be made when conducting ERG studies in dogs.

HLA class I signal peptide polymorphism determines the level of CD94/NKG2-HLA-E-mediated regulation of effector cell responses.

Human leukocyte antigen (HLA)-E binds epitopes derived from HLA-A, HLA-B, HLA-C and HLA-G signal peptides (SPs) and serves as a ligand for CD94/NKG2A and CD94/NKG2C receptors expressed on natural killer and T cell subsets. We show that among 16 common classical HLA class I SP variants, only 6 can be efficiently processed to generate epitopes that enable CD94/NKG2 engagement, which we term 'functional SPs'. The single functional HLA-B SP, known as HLA-B/-21M, induced high HLA-E expression, but conferred the lowest receptor recognition. Consequently, HLA-B/-21M SP competes with other SPs for providing epitope to HLA-E and reduces overall recognition of target cells by CD94/NKG2A, calling for reassessment of previous disease models involving HLA-B/-21M. Genetic population data indicate a positive correlation between frequencies of functional SPs in humans and corresponding cytomegalovirus mimics, suggesting a means for viral escape from host responses. The systematic, quantitative approach described herein will facilitate development of prediction algorithms for accurately measuring the impact of CD94/NKG2-HLA-E interactions in disease resistance/susceptibility.

Lip Squamous Cell Carcinoma With Spontaneous Eruption and Drainage.

A 69-year-old woman with a newly diagnosed squamous cell carcinoma of the lower lip mucosa presented 3 days after initiating neoadjuvant immune checkpoint blockade immunotherapy with redness and swelling of the tumor site. What is your diagnosis?

Virtual Screening of Hepatitis B Virus Pre-Genomic RNA as a Novel Therapeutic Target.

The global burden imposed by hepatitis B virus (HBV) infection necessitates the discovery and design of novel antiviral drugs to complement existing treatments. One attractive and underexploited therapeutic target is ε, an ~85-nucleotide (nt) cis-acting regulatory stem-loop RNA located at the 3'- and 5'-ends of the pre-genomic RNA (pgRNA). Binding of the 5'-end ε to the viral polymerase protein (P) triggers two early events in HBV replication: pgRNA and P packaging and reverse transcription. Our recent solution nuclear magnetic resonance spectroscopy structure of ε permits structure-informed drug discovery efforts that are currently lacking for P. Here, we employ a virtual screen against ε using a Food and Drug Administration (FDA)-approved compound library, followed by in vitro binding assays. This approach revealed that the anti-hepatitis C virus drug Daclatasvir is a selective ε-targeting ligand. Additional molecular dynamics simulations demonstrated that Daclatasvir targets ε at its flexible 6-nt priming loop (PL) bulge and modulates its dynamics. Given the functional importance of the PL, our work supports the notion that targeting ε dynamics may be an effective anti-HBV therapeutic strategy.

Salt-sensitive hypertension after reversal of unilateral ureteral obstruction.

The incidence of ureter obstruction is increasing and patients recovering from this kidney injury often progress to chronic kidney injury. There is evidence that a long-term consequence of recovery from ureter obstruction is an increased risk for salt-sensitive hypertension. A reversal unilateral ureteral obstruction (RUUO) model was used to study long-term kidney injury and salt-sensitive hypertension. In this model, we removed the ureteral obstruction at day 10 in mice. Mice were divided into four groups: (1) normal salt diet, (2) high salt diet, (3) RUUO normal salt diet, and (4) RUUO high salt diet. At day 10, the mice were fed a normal or high salt diet for 4 weeks. Blood pressure was measured, and urine and kidney tissue collected. There was a progressive increase in blood pressure in the RUUO high salt diet group. RUUO high salt group had decreased sodium excretion and glomerular injury. Renal epithelial cell injury was evident in RUUO normal and high salt mice as assessed by neutrophil gelatinase-associated lipocalin (NGAL). Kidney inflammation in the RUUO high salt group involved an increase in F4/80 positive macrophages; however, CD3+ positive T cells were not changed. Importantly, RUUO normal and high salt mice had decreased vascular density. RUUO was also associated with renal fibrosis that was further elevated in RUUO mice fed a high salt diet. Overall, these findings demonstrate long-term renal tubular injury, inflammation, decreased vascular density, and renal fibrosis following reversal of unilateral ureter obstruction that could contribute to impaired sodium excretion and salt-sensitive hypertension.

Management of Multiple Head and Neck Paragangliomas With Assistance of a 3-D Model.

INTRODUCTION: Extirpation of multiple head and neck paragangliomas carries challenge due to close anatomic relationships with critical neurovascular bundles. OBJECTIVES: This study aims to assess whether the application of 3-D models can assist with surgical planning and treatment of these paragangliomas, decrease surgically related morbidity and mortality. METHODS: Fourteen patients undergoing surgical resection of multiple head and neck paragangliomas were enrolled in this study. A preoperative 3-D model was created based on radiologic data, and relevant critical anatomic relationships were preoperatively assessed and intraoperatively validated. RESULTS: All 14 patients presented with multiple head and neck paragangliomas, including bilateral carotid body tumors (CBT, n = 9), concurrent CBT with glomus jugulare tumors (GJT, n = 4), and multiple vagal paragangliomas (n = 1). Ten patients underwent genomic analysis and all harbored succinate dehydrogenase complex subunit D (SDHD) mutations. Under guidance of the 3-D model, the internal carotid artery (ICA) was circumferentially encased by tumor on 5 of the operated sides, in 4 (80%) of which the tumor was successfully dissected out from the ICA, whereas ICA reconstruction was required on one side (20%). Following removal of CBT, anterior rerouting of the facial nerve was avoided in 3 (75%) of 4 patients during the extirpation of GJT with assistance of a 3-D model. Two patients developed permanent postoperative vocal cord paralysis. There was no vessel rupture or mortality in this study cohort. CONCLUSION: The 3-D model is beneficial for establishment of a preoperative strategy, as well as planning and guiding the intraoperative procedure for resection of multiple head and neck paragangliomas.

Resection of Carotid Body Tumors in Patients of Advanced Age: Experience From a Single Center.

INTRODUCTION: Resection of carotid body tumor (CBT) in patients of advanced ages has not been appreciated. OBJECTIVES: This study aims to assess the clinical characteristics and perioperative comorbidities for CBT resection in patients of advanced age and to validate the application of an "isolated island" technique for extirpation of CBT. METHODS: Eight patients of advanced age (≥60 years) who underwent CBT resection were enrolled as the study group (SG). Another 29 patients of younger age (<45 years old) underwent CBT extirpation were assigned as the control group (CG). The perioperative issues were compared between these 2 groups. RESULTS: The "isolated island" technique was successfully applied for resection of CBT in all 37 patients. The prevalence of Shamblin classification I, II, and III tumors in the SG was 12.5%, 62.5%, and 25%; whereas in the CG was 10.3%, 55.2%, and 34.5%, respectively. Bilateral CBT was observed in 7 patients of the CG and none in the SG. Vascular reconstruction was required for 1 (12.5%) patient in the SG, while it was required for 8 (27.6%) patients in the CG. Postoperative vocal cord palsy occurred in 37.5% of patients in SG, whereas the vocal cord palsy (34.5%) and dysphagia (6.9%) were commonly encountered in CG. In addition to postoperative length of stay (P = .004), no significant difference for operative time, intraoperative blood loss, or mortality were observed between these 2 groups (P > .05). CONCLUSION: Extirpation of CBT in patients of advanced age is rationale in appropriately selected patients. The "isolated island" technique is safe for CBT resection with seemingly low complication rates.

Application of Molecular Dynamics to Expand Docking Program's Exploratory Capabilities and to Evaluate Its Predictions.

Recognition of the growing importance of RNA as a target for therapeutic or diagnostic ligands brings the importance of computational predictions of docking poses to such receptors to the forefront. Most docking programs have been optimized for protein targets, based on a relatively rich pool of known docked protein structures. Unfortunately, despite progress, numbers of known docked RNA complexes are low and the accuracy of the computational predictions trained on those inadequate samples lags behind that achieved for proteins. Compared to proteins, RNA structures generally have fewer docking pockets, have less diverse electrostatic surfaces, and are more flexible, raising the possibility of producing only transiently available good docking targets. We are presenting a docking prediction protocol that adds molecular dynamics simulations before and after the actual docking in order to explore the conformational space of the target RNA and then to reevaluate the stability of the predicted RNA-ligand complex. In this way we are attempting to overcome important limitations of the docking programs: the rigid (fully or mostly) target structure and imperfect nature of the docking scoring functions.

Relationship between hearing, cognitive function, and quality of life in aging companion dogs.

BACKGROUND: Elderly people with presbycusis are at higher risk for dementia and depression than the general population. There is no information regarding consequences of presbycusis in dogs. OBJECTIVE: Evaluate the relationship between cognitive function, quality of life, and hearing loss in aging companion dogs. ANIMALS: Thirty-nine elderly companion dogs. METHODS: Prospective study. Hearing was evaluated using brainstem auditory evoked response (BAER) testing. Dogs were grouped by hearing ability. Owners completed the canine dementia scale (CADES) and canine owner-reported quality of life (CORQ) questionnaire. Cognitive testing was performed, and cognitive testing outcomes, CADES and CORQ scores and age were compared between hearing groups. RESULTS: Nineteen dogs could hear at 50 dB, 12 at 70 dB, and 8 at 90 dB with mean ages (months) of 141 ± 14, 160 ± 16, and 172 ± 15 for each group respectively (P = .0002). Vitality and companionship CORQ scores were significantly lower as hearing deteriorated (6.6-5.4, 50-90 dB group, P = .03 and 6.9-6.2, 50-90 dB group, P = .02, respectively). Cognitive classification by CADES was abnormal in all 90 dB group dogs and normal in 3/12 70 dB group and 11/19 50 dB group dogs (P = .0004). Performance on inhibitory control, detour and sustained gaze tasks decreased significantly with hearing loss (P = .001, P = .008, P = .002, respectively). In multivariate analysis, higher CADES score was associated with worse hearing (P = .01). CONCLUSIONS AND CLINICAL IMPORTANCE: Presbycusis negatively alters owner-pet interactions and is associated with poor executive performance and owner-assessed dementia severity.

Conformational Dynamics of the Hepatitis B Virus Pre-genomic RNA on Multiple Time Scales: Implications for Viral Replication.

Human hepatitis B virus (HBV) replication is initiated by the binding of the viral polymerase (P) to epsilon (ε), an ≈85-nucleotide (nt) cis-acting regulatory stem-loop RNA located at the 5'-end of the pre-genomic RNA (pgRNA). This interaction triggers P and pgRNA packaging and protein-primed reverse transcription and is therefore an attractive therapeutic target. Our recent nuclear magnetic resonance (NMR) structure of ε provides a useful starting point toward a detailed understanding of HBV replication, and hints at the functional importance of ε dynamics. Here, we present a detailed description of ε motions on the ps to ns and µs to ms time scales by NMR spin relaxation and relaxation dispersion, respectively. We also carried out molecular dynamics simulations to provide additional insight into ε conformational dynamics. These data outline a series of complex motions on multiple time scales within ε. Moreover, these motions occur in mostly conserved nucleotides from structural regions (i.e., priming loop, pseudo-triloop, and U43 bulge) that biochemical and mutational studies have shown to be essential for P binding, P-pgRNA packaging, protein-priming, and DNA synthesis. Taken together, our work implicates RNA dynamics as an integral feature that governs HBV replication.

Use of Cognitive Testing, Questionnaires, and Plasma Biomarkers to Quantify Cognitive Impairment in an Aging Pet Dog Population.

BACKGROUND: Aging dogs may suffer from canine cognitive dysfunction syndrome (CCDS), a condition in which cognitive decline is associated with amyloid pathology and cortical atrophy. Presumptive diagnosis is made through physical examination, exclusion of systemic/metabolic conditions, and completion of screening questionnaires by owners. OBJECTIVE: This study aimed to determine whether cognitive function could be quantified in aging pet dogs, and to correlate cognitive testing with validated questionnaires and plasma neurofilament light chain (pNfL) concentration. METHODS: Thirty-nine dogs from fifteen breeds were recruited (9.3 to 15.3 years). Owners completed the Canine Dementia Scale (CADES) and Canine Cognitive Dysfunction Rating scale (CCDR). Executive control and social cues were tested, and pNfL was measured with single molecule array assay. Comparisons were made between cognitive testing scores, CADES, CCDR scores, and pNfL. RESULTS: CADES scoring classified five dogs as severe CCDS, six as moderate, ten as mild, and eighteen as normal. CCDR identified seven dogs at risk of CCDS and thirty-two as normal. Cognitive testing was possible in the majority of dogs, although severely affected dogs were unable to learn tasks. CADES score correlated with sustained attention duration (r = -0.47, p = 0.002), inhibitory control (r = -0.51, p = 0.002), detour (r = -0.43, p = 0.001), and pNfL (r = 0.41, p = 0.025). Concentration of pNfL correlated with inhibitory control (r = -0.7, p≤0.001). The CCDR scale correlated with performance on inhibitory control (r = -0.46, p = 0.005). CONCLUSION: Our findings suggest that a multi-dimensional approach using a combination of questionnaires, specific cognitive tests, and pNfL concentration can be used to quantify cognitive decline in aging pet dogs.

Cure of syngeneic carcinomas with targeted IL-12 through obligate reprogramming of lymphoid and myeloid immunity.

Therapeutic IL-12 has demonstrated the ability to reduce local immune suppression in preclinical models, but clinical development has been limited by severe inflammation-related adverse events with systemic administration. Here, we show that potent immunologic tumor control of established syngeneic carcinomas can be achieved by i.t. administration of a tumor-targeted IL-12 antibody fusion protein (NHS-rmIL-12) using sufficiently low doses to avoid systemic toxicity. Single-cell transcriptomic analysis and ex vivo functional assays of NHS-rmIL-12-treated tumors revealed reinvigoration and enhanced proliferation of exhausted CD8+ T lymphocytes, induction of Th1 immunity, and a decrease in Treg number and suppressive capacity. Similarly, myeloid cells transitioned toward inflammatory phenotypes and displayed reduced suppressive capacity. Cell type-specific IL-12 receptor-KO BM chimera studies revealed that therapeutic modulation of both lymphoid and myeloid cells is required for maximum treatment effect and tumor cure. Study of single-cell data sets from human head and neck carcinomas revealed IL-12 receptor expression patterns similar to those observed in murine tumors. These results describing the diverse mechanisms underlying tumor-directed IL-12-induced antitumor immunity provide the preclinical rationale for the clinical study of i.t. NHS-IL-12.

Prevalence of human papillomavirus in head and neck cancers at tertiary care centers in the United States over time.

BACKGROUND: Human papillomavirus (HPV) is responsible for a growing proportion of oropharyngeal squamous cell carcinomas (OPSCCs) among men and White individuals. Whether similar trends apply to women, non-Whites, and non-oropharyngeal squamous cell carcinomas (non-OPSCCs) is unknown. METHODS: This is a cross-sectional analysis combining 2 multi-institutional case series of incident head and neck squamous cell carcinoma (HNSCC) cases. Incident HNSCCs from 1995 to 2012 were enrolled retrospectively using banked tumor samples and medical record abstraction. Incident HNSCCs from 2013 to 2019 were enrolled prospectively. The prevalence of tumor HPV biomarkers was tested over 3 time periods (1995-2003, 2004-2012, and 2013-2019). Centralized testing was done for p16 immunohistochemistry (p16) and oncogenic HPV in situ hybridization (ISH). RESULTS: A total of 1209 incident cases of HNSCC were included. Prevalence of p16- and ISH-positive tumors increased significantly for oropharynx cancers over time. The majority were positive after 2013 for White patients (p16, 92%; P < .001; ISH 94%; P < .001), Black patients (p16, 72%; P = .021; ISH 67%; P = .011), and Hispanic patients (p16, 100%; P = .04; ISH 100%; P = .013). For women with OPSCC, the prevalence of p16- and ISH-positive tumors increased significantly to 82% (P < .001) and 78% (P = .004), respectively. For non-OPSCCs, there was increased p16 and ISH positivity overall with 24% p16 and 16% ISH positivity in the most recent time period (P < .001 for both). CONCLUSIONS: The majority of OPSCCs in US tertiary care centers are now p16 and ISH positive for all sex and race groups. In some populations in the United States, 91% of OPSCCs are now caused by HPV. Few non-OPSCCs are p16 and ISH positive.

Structural characterization of a new subclass of panicum mosaic virus-like 3' cap-independent translation enhancer.

Canonical eukaryotic mRNA translation requires 5'cap recognition by initiation factor 4E (eIF4E). In contrast, many positive-strand RNA virus genomes lack a 5'cap and promote translation by non-canonical mechanisms. Among plant viruses, PTEs are a major class of cap-independent translation enhancers located in/near the 3'UTR that recruit eIF4E to greatly enhance viral translation. Previous work proposed a single form of PTE characterized by a Y-shaped secondary structure with two terminal stem-loops (SL1 and SL2) atop a supporting stem containing a large, G-rich asymmetric loop that forms an essential pseudoknot (PK) involving C/U residues located between SL1 and SL2. We found that PTEs with less than three consecutive cytidylates available for PK formation have an upstream stem-loop that forms a kissing loop interaction with the apical loop of SL2, important for formation/stabilization of PK. PKs found in both subclasses of PTE assume a specific conformation with a hyperreactive guanylate (G*) in SHAPE structure probing, previously found critical for binding eIF4E. While PTE PKs were proposed to be formed by Watson-Crick base-pairing, alternative chemical probing and 3D modeling indicate that the Watson-Crick faces of G* and an adjacent guanylate have high solvent accessibilities. Thus, PTE PKs are likely composed primarily of non-canonical interactions.

Structural insights of the conserved "priming loop" of hepatitis B virus pre-genomic RNA.

Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral polymerase with a cis-acting regulatory signal, designated epsilon (ε), located at the 5'-end of its pre-genomic RNA (pgRNA). Binding of polymerase to ε is also necessary for pgRNA encapsidation. While the mechanistic basis of this interaction remains elusive, mutagenesis studies suggest its internal 6-nt "priming loop" provides an important structural contribution. ε might therefore be considered a promising target for small molecule interventions to complement current nucleoside-analog based anti-HBV therapies. An ideal prerequisite to any RNA-directed small molecule strategy would be a detailed structural description of this important element. Herein, we present a solution NMR structure for HBV ε which, in combination with molecular dynamics and docking simulations, reports on a flexible ligand "pocket", reminiscent of those observed in proteins. We also demonstrate the binding of the selective estrogen receptor modulators (SERMs) Raloxifene, Bazedoxifene, and a de novo derivative to the priming loop.Communicated by Ramaswamy H. Sarma.

Clinician's commentary: Atypia in salivary gland fine needle aspiration.

Due to the heterogenicity and morphological overlap among the broad spectrum of benign and malignant salivary gland lesions, cytopathology result interpretations can be challenging and variable even among the most experienced head and neck pathologist. There was no standardization of cytopathology result reporting until the recently proposed "Milan system for reporting salivary gland cytopathology" (MSRSGC). MSRSGC may offer more clarity and help minimize ambiguity, but surgeons, as part of multidisciplinary teams, do not only rely on the tiered stratification and risk of malignancy assessment. With only the MSRSGC reported, there may be critical information missing from the overall diagnostic evaluation of salivary gland masses. Cytopathologist evaluation, description of findings, and expert interpretation of the fine-needle aspiration cytology along with differential diagnosis can be critical pieces of information, that is, utilized in management discussions with patients and their families. This information needs to be included in every cytopathology interpretation in addition to the MSRSGC classification. In clinical practice, decisions concerning salivary gland tumor management are not based on single examinations but incorporate information from multiple sources including patient histories, clinical symptoms and signs, physical examinations, imaging studies, and when available, cytopathology. Additional cytopathology information will likely help to improve the utility and predictive power of MSRSGC, similar to Bethesda Classification and the predictive importance of nuclear atypia in indeterminate thyroid biopsy material for thyroid neoplasms.