Neuronal activation in the geomagnetic responsive region Cluster N covaries with nocturnal migratory restlessness in white-throated sparrows (Zonotrichia albicollis).

Cluster N is a region of the visual forebrain of nocturnally migrating songbirds that supports the geomagnetic compass of nocturnal migrants. Cluster N expresses immediate-early genes (ZENK), indicating neuronal activation. This neuronal activity has only been recorded at night during the migratory season. Night-to-night variation in Cluster N activity in relation to migratory behaviour has not been previously examined. We tested whether Cluster N is activated only when birds are motivated to migrate and presumably engage their magnetic compass. We measured immediate-early gene activation in Cluster N of white-throated sparrows (Zonotrichia albicollis) in three conditions: daytime, nighttime migratory restless and nighttime resting. Birds in the nighttime migratory restlessness group had significantly greater numbers of ZENK-labelled cells in Cluster N compared to both the daytime and the nighttime resting groups. Additionally, the degree of migratory restlessness was positively correlated with the number of ZENK-labelled cells in the nighttime migratory restless group. Our study adds to the number of species observed to have neural activation in Cluster N and demonstrates for the first time that immediate early gene activation in Cluster N is correlated with the amount of active migratory behaviour displayed across sampled individuals. We conclude that Cluster N is facultatively regulated by the motivation to migrate, together with nocturnal activity, rather than obligatorily active during the migration season.

Population-based surveillance for cervical cancer precursors in three central cancer registries, United States 2009.

PURPOSE: The USA has a well-established network of central cancer registries (CCRs) that collect data using standardized definitions and protocols to provide population-based estimates of cancer incidence. The addition of cervical cancer precursors in select CCR operations would facilitate future studies measuring the population-level impact of human papillomavirus (HPV) vaccine. To assess the feasibility of collecting data on cervical cancer precursors, we conducted a multi-site surveillance study in three state-wide CCRs, to obtain annual case counts and compare rates of precursor lesions to those for invasive cervical cancer. METHODS: We developed standardized methods for case identification, data collection and transmission, training and quality assurance, while allowing for registry-specific strategies to accomplish surveillance objectives. We then conducted population-based surveillance for precancerous cervical lesions in three states using the protocols. RESULTS: We identified 5,718 cases of cervical cancer precursors during 2009. Age-adjusted incidence of cervical cancer precursors was 77 (Kentucky), 60 (Michigan), and 54 (Louisiana) per 100,000 women. Highest rates were observed in those aged 20-29 years: 274 (Kentucky), 202 (Michigan), and 196 (Louisiana) per 100,000. The variable with the most missing data was race/ethnicity, which was missing for 13 % of cases in Kentucky, 18 % in Michigan, and 1 % in Louisiana. Overall rates of cervical cancer precursors were over sixfold higher than invasive cervical cancer rates [rate ratios: 8.6 (Kentucky), 8.3 (Michigan), and 6.2 (Louisiana)]. CONCLUSIONS: Incorporating surveillance of cervical cancer precursors using existing CCR infrastructure is feasible and results in collection of population-based incidence data. Standardized collection of these data in high-quality registry systems will be useful in future activities monitoring the impact of HPV vaccination across states. As a result of this study, ongoing surveillance of these lesions has now been conducted in four CCRs since 2010.

Non-pathogenic microbiota accelerate age-related CpG Island methylation in colonic mucosa.

DNA methylation is an epigenetic process altered in cancer and ageing. Age-related methylation drift can be used to estimate lifespan and can be influenced by extrinsic factors such as diet. Here, we report that non-pathogenic microbiota accelerate age-related methylation drift in the colon when compared with germ-free mice. DNA methylation analyses showed that microbiota and IL10KO were associated with changes in 5% and 4.1% of CpG sites, while mice with both factors had 18% alterations. Microbiota, IL10KO, and their combination altered 0.4%, 0.4%, and 4% of CpG island methylation, respectively. These are comparable to what is seen in colon cancer. Ageing changes were accelerated in the IL10KO mice with microbiota, and the affected genes were more likely to be altered in colon cancer. Thus, the microbiota affect DNA methylation of the colon in patterns reminiscent of what is observed in ageing and colorectal cancer.

EASI Transformation Protocol: An Agrobacterium-Mediated Transient Transformation Protocol for Catharanthus roseus Seedlings.

Catharanthus roseus produces medicinal terpenoid indole alkaloids, including the critical anti-cancer compounds vinblastine and vincristine in its leaves. Recently, we developed a highly efficient transient expression method relying on Agrobacterium-mediated transformation of seedlings to facilitate rapid and high-throughput studies on the regulation of terpenoid indole alkaloid biosynthesis in C. roseus . We detail our optimized protocol known as efficient Agrobacterium-mediated seedling infiltration method (EASI), including the development of constructs used in EASI and an example experimental design that includes appropriate controls. We applied our EASI method to rapidly screen and evaluate transcriptional activators and repressors and promoter activity. Our EASI method can be used for promoter transactivation studies or transgene overexpression paired with downstream analyses like quantitative PCR or metabolite analysis. Our protocol takes about 16 days from sowing seeds to obtaining the results of the experiment.

Relationship Between Cognition and Gait at 2- and 12-Months Post-Traumatic Brain Injury.

Background: A common and debilitating challenge experienced by people with TBI is gait-associated mobility impairment and persisting cognitive impairments. Cognitive and physical impairments are often addressed independently during rehabilitation, however, increasing evidence links cognitive and motor processes more closely. Objectives: (1) To determine if correlations exist between measures of cognitive and gait recovery, post-TBI. (2) To investigate the predictive power of cognition at 2-months on gait outcomes at 12-months post-TBI. Methods: In this secondary, longitudinal study of cognitive and neural recovery, data from 93 participants admitted to an inpatient neurorehabilitation program were analyzed. Spatiotemporal gait variables [velocity, step time variability (STV), step length variability (SLV)] were collected along with cognitive variables [Trail Making Test-B (TMT-B), Digit Span-Forward (DS-F)]. Spearman's correlation coefficients were calculated between gait and cognitive variables. Multilinear and step wise regression analyses were calculated to determine predictive value of cognitive variables at 2-months on gait performance at 12-months-post TBI. Results: At 2-months post-injury, TMT-B was significantly correlated with gait velocity and STV; and DS-F was significantly correlated with velocity. At 12-months post-injury, TMT-B and DS-F was still significant correlated with velocity. TMT-B at 2-months was correlated with SLV and STV at 12-months; and DS-F correlated significantly with velocity. Regression models showed TMT-B at 2-months predicting STV, SLV, and velocity at 12-months. Conclusions: Significant associations and predictions between physical and cognitive recovery post-TBI were observed in this study. Future directions may consider a "neural internetwork" model as a salient rehabilitation approach in TBI that integrates physical and cognitive functions.

Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells.

Chromatin accessibility of a promoter is fundamental in regulating transcriptional activity. The histone variant H2A.Z has been shown to contribute to this regulation, but its role has remained poorly understood. Here, we prepare high-depth maps of the position and accessibility of H2A.Z-containing nucleosomes for all human Pol II promoters in epithelial, mesenchymal and isogenic cancer cell lines. We find that, in contrast to the prevailing model, many different types of active and inactive promoter structures are observed that differ in their nucleosome organization and sensitivity to MNase digestion. Key aspects of an active chromatin structure include positioned H2A.Z MNase resistant nucleosomes upstream or downstream of the TSS, and a MNase sensitive nucleosome at the TSS. Furthermore, the loss of H2A.Z leads to a dramatic increase in the accessibility of transcription factor binding sites. Collectively, these results suggest that H2A.Z has multiple and distinct roles in regulating gene expression dependent upon its location in a promoter.

Neurologic Music Therapy via Telehealth: A Survey of Clinician Experiences, Trends, and Recommendations During the COVID-19 Pandemic.

This cross-sectional survey investigated the transition of Neurologic Music Therapy (NMT) services from in-person (pre-COVID-19) to telehealth (since COVID-19) to (1) determine whether the use of an NMT paradigm contributes to the successful transition of therapy services to telehealth, (2) identify which NMT domains and techniques are transferable from in-person to telehealth, (3) identify whether there are differences in the transition of NMT services across different employment settings, and (4) evaluate the potential benefits and challenges of telehealth NMT. An online survey comprised of 49 closed and open-ended questions was distributed by the Academy of Neurologic Music Therapy to 2,778 NMT affiliates worldwide. The survey sought information on demographics, telehealth perceptions, technology, assessment, clinical practice, safety, and caregiver involvement. Quantitative and qualitative analyses were applied. Eighty-one participants answered the survey and the 69 who completed the survey in its entirety were included in the analysis. Results indicated that the frequency of NMT technique usage had no impact on the overall number of clinical hours retained over telehealth. Correlation analysis revealed an association between more frequent NMT usage and perceived likelihood of using telehealth in the future (i.e., once COVID-19 is no longer a major threat), as well as with fewer group sessions lost over telehealth. All NMT domains transferred to telehealth, although within the sensorimotor domain, fewer therapists implemented rhythmic auditory stimulation for telehealth sessions compared to in-person. Overall, NMTs had fewer hours for telehealth compared to in-person regardless of employment setting. Technological challenges were notable drawbacks, while major benefits included the ability to continue providing NMT when in-person sessions were not possible, increased accessibility for remote clients, and positive outcomes related to increased caregiver involvement. Based on the results, our recommendations for implementing telehealth in Neurologic Music Therapy include integrating telehealth into routine care, mitigating safety concerns, identifying those who could benefit most from remote delivery, involving caregivers, and developing/sharing resources for telehealth NMT.

Probable early-onset Alzheimer's disease in an apolipoprotein E2 homozygote.

OBJECTIVE: To describe a case of early-onset Alzheimer's disease (AD) in an apolipoprotein (Apo) ε2/ε2 homozygote. BACKGROUND: Apo ε2/ε2 is the rarest of the ApoE genotypes, representing only 1.4% of the population. Cognitive decline in ApoE ε2 homozygotes has rarely been reported. CASE REPORT/METHODS: We report a 58-year-old Apo ε2/ε2 female who meets clinical criteria for probable AD as confirmed by neuropsychological testing, positron emission/computed tomography scan, CSF analysis and genetic screening for known mutations. RESULTS: The clinical course is typical of AD, with progressive cognitive and functional decline. CONCLUSION: Clinically confirmed early-onset AD is atypical in ApoE2 homozygotes but can occur.

MNase Profiling of Promoter Chromatin in Salmonella typhimurium-Stimulated GM12878 Cells Reveals Dynamic and Response-Specific Nucleosome Architecture.

The nucleosome is the primary unit of chromatin structure and commonly imputed as a regulator of nuclear events, although the exact mechanisms remain unclear. Recent studies have shown that certain nucleosomes can have different sensitivities to micrococcal nuclease (MNase) digestion, resulting in the release of populations of nucleosomes dependent on the concentration of MNase. Mapping MNase sensitivity of nucleosomes at transcription start sites genome-wide reveals an important functional nucleosome organization that correlates with gene expression levels and transcription factor binding. In order to understand nucleosome distribution and sensitivity dynamics during a robust genome response, we mapped nucleosome position and sensitivity using multiple concentrations of MNase. We used the innate immune response as a model system to understand chromatin-mediated regulation. Herein we demonstrate that stimulation of a human lymphoblastoid cell line (GM12878) with heat-killed Salmonella typhimurium (HKST) results in changes in nucleosome sensitivity to MNase. We show that the HKST response alters the sensitivity of -1 nucleosomes at highly expressed promoters. Finally, we correlate the increased sensitivity with response-specific transcription factor binding. These results indicate that nucleosome sensitivity dynamics reflect the cellular response to HKST and pave the way for further studies that will deepen our understanding of the specificity of genome response.

Flexible heteroarotinoid (Flex-Het) SHetA2 inhibits angiogenesis in vitro and in vivo.

Flexible heteroarotinoids (Flex-Hets) compounds regulate growth, differentiation and apoptosis in cancer cells. The hypothesis of this study was that the lead Flex-Het, SHetA2, inhibits angiogenesis by blocking cytokine release from cancer cells. SHetA2 altered secretion of thrombospondin-4 (TSP-4), vascular endothelial growth factor A (VEGF) and fibroblast growth factor (bFGF) proteins from normal and cancerous ovarian and renal cultures. Thymidine phosphorylase (TP) expression was inhibited in cancer, but not normal cultures. Endothelial tube formation was stimulated by conditioned media from cancer but not normal cultures, and SHetA2 reduced secretion of this angiogenic activity. SHetA2 directly inhibited endothelial cell tube formation and proliferation through G1 cell cycle arrest, but not apoptosis. Recombinant TP reversed SHetA2 anti-angiogenic activity. SHetA2 inhibition of in vivo angiogenesis was observed in Caki-1 renal cancer xenografts. In conclusion, SHetA2 inhibits angiogenesis through alteration of angiogenic factor secretion by cancer cells and through direct effects on endothelial cells.

EASI Transformation: An Efficient Transient Expression Method for Analyzing Gene Function in Catharanthus roseus Seedlings.

The Catharanthus roseus plant is the exclusive source of the valuable anticancer terpenoid indole alkaloids, vinblastine (VB) and vincristine (VC). The recent availability of transcriptome and genome resources for C. roseus necessitates a fast and reliable method for studying gene function. In this study, we developed an Agrobacterium-mediated transient expression method to enable the functional study of genes rapidly in planta, conserving the compartmentalization observed in the VB and VC pathway. We focused on (1) improving the transformation method (syringe versus vacuum agroinfiltration) and cultivation conditions (seedling age, Agrobacterium density, and time point of maximum transgene expression), (2) improving transformation efficiency through the constitutive expression of the virulence genes and suppressing RNA silencing mechanisms, and (3) improving the vector design by incorporating introns, quantitative and qualitative reporter genes (luciferase and GUS genes), and accounting for transformation heterogeneity across the tissue using an internal control. Of all the parameters tested, vacuum infiltration of young seedlings (10-day-old, harvested 3 days post-infection) resulted in the strongest increase in transgene expression, at 18 - 57 fold higher than either vacuum or syringe infiltration of other seedling ages. Endowing the A. tumefaciens strain with the mutated VirGN54D or silencing suppressors within the same plasmid as the reporter gene further increased expression by 2 - 10 fold. For accurate measurement of promoter transactivation or activity, we included an internal control to normalize the differences in plant mass and transformation efficiency. Including the normalization gene (Renilla luciferase) on the same plasmid as the reporter gene (firefly luciferase) consistently yielded a high signal and a high correlation between RLUC and FLUC. As proof of principle, we applied this approach to investigate the regulation of the CroSTR1 promoter with the well-known activator ORCA3 and repressor ZCT1. Our method demonstrated the quantitative assessment of both the activation and repression of promoter activity in C. roseus. Our efficient Agrobacterium-mediated seedling infiltration (EASI) protocol allows highly efficient, reproducible, and homogenous transformation of C. roseus cotyledons and provides a timely tool for the community to rapidly assess the function of genes in planta, particularly for investigating how transcription factors regulate terpenoid indole alkaloid biosynthesis.

The regulation of ZCT1, a transcriptional repressor of monoterpenoid indole alkaloid biosynthetic genes in Catharanthus roseus.

Cys2/His2-type (C2H2) zinc finger proteins, such as ZCT1, are an important class of transcription factors involved in growth, development, and stress responses in plants. In the medicinal plant Catharanthus roseus, the zinc finger Catharanthus transcription factor (ZCT) family represses monoterpenoid indole alkaloid (MIA) biosynthetic gene expression. Here, we report the analysis of the ZCT1 promoter, which contains several hormone-responsive elements. ZCT1 is responsive to not only jasmonate, as was previously known, but is also induced by the synthetic auxin, 1-naphthalene acetic acid (1-NAA). Through promoter deletion analysis, we show that an activation sequence-1-like (as-1-like)-motif and other motifs contribute significantly to ZCT1 expression in seedlings. We also show that the activator ORCA3 does not transactivate the expression of ZCT1 in seedlings, but ZCT1 represses its own promoter, suggesting a feedback mechanism by which the expression of ZCT1 can be limited.

Rapid reversal of colonic pneumatosis with restoration of mesenteric arterial supply.

Pneumatosis cystoides intestinalis (PCI) is characterized by gas-filled cystic lesions within the wall of the large intestine and presents along a spectrum of clinical severity ranging from benign to life threatening. Etiopathogenesis is multifactorial and postulated to result from either mechanical or bacterial causes. In this report, we present a patient with chronic abdominal pain evaluated with colonoscopy revealing segmental PCI isolated to the distal colon. Further investigation revealed an abdominal aortic aneurysm (AAA) compromising the inferior mesenteric artery takeoff. Endovascular repair of the AAA resulted in clinical resolution of abdominal pain and endoscopic resolution of PCI. To our knowledge, this is the first report to document endoscopic resolution of PCI with restoration of mesenteric arterial supply, highlighting vascular insufficiency as a predisposing and reversible pathogenic mechanism.

Immune cell subset differentiation and tissue inflammation.

Immune cells were traditionally considered as major pro-inflammatory contributors. Recent advances in molecular immunology prove that immune cell lineages are composed of different subsets capable of a vast array of specialized functions. These immune cell subsets share distinct duties in regulating innate and adaptive immune functions and contribute to both immune activation and immune suppression responses in peripheral tissue. Here, we summarized current understanding of the different subsets of major immune cells, including T cells, B cells, dendritic cells, monocytes, and macrophages. We highlighted molecular characterization, frequency, and tissue distribution of these immune cell subsets in human and mice. In addition, we described specific cytokine production, molecular signaling, biological functions, and tissue population changes of these immune cell subsets in both cardiovascular diseases and cancers. Finally, we presented a working model of the differentiation of inflammatory mononuclear cells, their interaction with endothelial cells, and their contribution to tissue inflammation. In summary, this review offers an updated and comprehensive guideline for immune cell development and subset differentiation, including subset characterization, signaling, modulation, and disease associations. We propose that immune cell subset differentiation and its complex interaction within the internal biological milieu compose a "pathophysiological network," an interactive cross-talking complex, which plays a critical role in the development of inflammatory diseases and cancers.

Lysophospholipid Receptors, as Novel Conditional Danger Receptors and Homeostatic Receptors Modulate Inflammation-Novel Paradigm and Therapeutic Potential.

There are limitations in the current classification of danger-associated molecular patterns (DAMP) receptors. To overcome these limitations, we propose a new paradigm by using endogenous metabolites lysophospholipids (LPLs) as a prototype. By utilizing a data mining method we pioneered, we made the following findings: (1) endogenous metabolites such as LPLs at basal level have physiological functions; (2) under sterile inflammation, expression of some LPLs is elevated. These LPLs act as conditional DAMPs or anti-inflammatory homeostasis-associated molecular pattern molecules (HAMPs) for regulating the progression of inflammation or inhibition of inflammation, respectively; (3) receptors for conditional DAMPs and HAMPs are differentially expressed in human and mouse tissues; and (4) complex signaling mechanism exists between pro-inflammatory mediators and classical DAMPs that regulate the expression of conditional DAMPs and HAMPs. This novel insight will facilitate identification of novel conditional DAMPs and HAMPs, thus promote development of new therapeutic targets to treat inflammatory disorders.

Sensitivity of primary fibroblasts in culture to atmospheric oxygen does not correlate with species lifespan.

Differences in the way human and mouse fibroblasts experience senescence in culture had long puzzled researchers. While senescence of human cells is mediated by telomere shortening, Parrinello et al. demonstrated that senescence of mouse cells is caused by extreme oxygen sensitivity. It was hypothesized that the striking difference in oxygen sensitivity between mouse and human cells explains their different rates of aging. To test if this hypothesis is broadly applicable, we cultured cells from 16 rodent species with diverse lifespans in 3% and 21% oxygen and compared their growth rates. Unexpectedly, fibroblasts derived from laboratory mouse strains were the only cells demonstrating extreme sensitivity to oxygen. Cells from hamster, muskrat, woodchuck, capybara, blind mole rat, paca, squirrel, beaver, naked mole rat and wild-caught mice were mildly sensitive to oxygen, while cells from rat, gerbil, deer mouse, chipmunk, guinea pig and chinchilla showed no difference in the growth rate between 3% and 21% oxygen. We conclude that, although the growth of primary fibroblasts is generally improved by maintaining cells in 3% oxygen, the extreme oxygen sensitivity is a peculiarity of laboratory mouse strains, possibly related to their very long telomeres, and fibroblast oxygen sensitivity does not directly correlate with species' lifespan.

Examining the incidence of human papillomavirus-associated head and neck cancers by race and ethnicity in the U.S., 1995-2005.

BACKGROUND: Head and neck cancer (HNC) incidence, mortality and survival rates vary by sex and race, with men and African Americans disproportionately affected. Risk factors for HNC include tobacco and alcohol exposure, with a recent implication of human papillomavirus (HPV) in the pathogenesis of HNC. This study describes the epidemiology of HNC in the United States, examining variation of rates by age, sex, race/ethnicity and potential HPV-association. METHODS: We used the North American Association of Central Cancer Registries (NAACCR) Cancer in North America (CINA) Deluxe Analytic Data to analyze HNC incidence for 1995-2005 from forty population-based cancer registries. We calculated age-adjusted incidence rates and incidence trends using annual percent change by age, sex, race/ethnicity and HPV-association. RESULTS: Males and Non-Hispanic Blacks experienced greater HNC incidence compared to women and other race/ethnicity groupings. A significant overall increase in HNC incidence was observed among HPV-associated sites during 1995-2005, while non HPV-associated sites experienced a significant decline in HNC incidence. Overall, younger age groups, Non-Hispanic Whites and Hispanics experienced greater increases in incidence for HPV-associated sites, while HNC incidence declined for Non-Hispanic Blacks independent of HPV-association. In particular, for HPV-associated sites, HNC incidence for Non-Hispanic White males aged 45-54 increased at the greatest rate, with an APC of 6.28% (p<0.05). Among non HPV-associated sites, Non-Hispanic Black males aged 0-44 years experienced the greatest reduction in incidence (APC, -8.17%, p<0.05), while a greater decline among the older, 55-64 year age group (APC, -5.44%, p<0.05) occurred in females. CONCLUSIONS: This study provides evidence that HPV-associated tumors are disproportionately affecting certain age, sex and race/ethnicity groups, representing a different disease process for HPV-associated tumors compared to non HPV-associated tumors. Our study suggests that HPV tumor status should be incorporated into treatment decisions for HNC patients to improve prognosis and survival.

Positron emission tomography and neuropathologic estimates of fibrillar amyloid-ß in a patient with Down syndrome and Alzheimer disease.

BACKGROUND: Down syndrome appears to be associated with a virtually certain risk of fibrillar amyloid-ß (Aß) pathology by the age of 40 and a very high risk of dementia at older ages. The positron emission tomography (PET) ligand florbetapir F18 has been shown to characterize fibrillar Aß in the living human brain and to provide a close correlation with subsequent Aß neuropathology in individuals proximate to and after the end of life. The extent to which the most frequently used PET ligands can be used to detect fibrillar Aß in patients with Down syndrome remains to be determined. OBJECTIVES: To characterize PET estimates of fibrillar Aß burden in a Down syndrome patient very close to the end of life and to compare them with neuropathologic assessment made after his death. Design/ METHODS: With the family's informed consent, florbetapir PET was used to study a 55-year-old Down syndrome patient with Alzheimer disease near the end of life; his brain was donated for neuropathologic assessment when he died 14 days later. Visual ratings of cerebral florbetapir uptake were performed by trained readers who were masked to the patient's diagnosis as part of a larger study, and an automated algorithm was used to characterize regional-to-cerebellar standard uptake value ratios in 6 cerebral regions of interest. Neuropathologic assessments were performed masked to the patient's diagnosis or PET measurements. RESULTS: Visual ratings and automated analyses of the PET image revealed a heavy fibrillar Aß burden in cortical, striatal, and thalamic regions, similar to that reported for patients with late-onset Alzheimer disease. This matched neuropathologic findings of frequent neuritic and diffuse plaques, as well as frequent amyloid angiopathy, except for neuropathologically demonstrated frequent cerebellar diffuse plaques and amyloid angiopathy that were not detected by the PET scan. CONCLUSIONS: Florbetapir PET can be used to detect increased cerebral-to-cerebellar fibrillar Aß burden in a Down syndrome patient with Alzheimer disease, even in the presence of frequent amyloid angiopathy and diffuse plaques in the cerebellum. Additional studies are needed to determine the extent to which PET could be used to detect and to track fibrillar Aß and to evaluate investigational Aß-modifying treatments in the presymptomatic and symptomatic stages of Alzheimer disease.