Combining Isoprenoid Probes with Antibody Markers for Mass Cytometric Analysis of Prenylation in Single Cells.

Protein prenylation is an essential post-translational modification that plays a key role in facilitating protein localization. Aberrations in protein prenylation have been indicated in multiple disease pathologies including progeria, some forms of cancer, and Alzheimer's disease. While there are single-cell methods to study prenylation, these methods cannot simultaneously assess prenylation and other cellular changes in the complex cell environment. Here, we report a novel method to monitor, at the single-cell level, prenylation and expression of autophagy markers. An isoprenoid analogue containing a terminal alkyne, substrate of prenylation enzymes, was metabolically incorporated into cells in culture. Treatment with a terbium reporter containing an azide functional group, followed by copper-catalyzed azide-alkyne cycloaddition, covalently attached terbium ions to prenylated proteins within cells. In addition, simultaneous treatment with a holmium-containing analogue of the reporter, without an azide functional group, was used to correct for non-specific retention at the single-cell level. This procedure was compatible with other mass cytometric sample preparation steps that use metal-tagged antibodies. We demonstrate that this method reports changes in levels of prenylation in competitive and inhibitor assays, while tracking autophagy molecular markers with metal-tagged antibodies. The method reported here makes it possible to track prenylation along with other molecular pathways in single cells of complex systems, which is essential to elucidate the role of this post-translational modification in disease, cell response to pharmacological treatments, and aging.

Nonspecific Binding Correction for Single-Cell Mass Cytometric Analysis of Autophagy and Myoblast Differentiation.

Satellite cells provide regenerative capacity to the skeletal muscle after injury. In this process, termed myogenesis, satellite cells get activated, proliferate, and differentiate. Myogenesis is recapitulated in the tissue culture of myoblasts that differentiate by fusion and then by the formation of myotubes. Autophagy plays an important role in myogenesis, but the asynchronous and unique trajectory of differentiation of each myoblast along the myogenic lineage complicates teasing apart at what stages of differentiation autophagy plays a critical role. In this report, we describe a mass cytometric, multidimensional, individual cell analysis of differentiating myoblasts that characterizes autophagy flux (i.e., autophagy rate) at separate myogenesis stages. Because mass cytometry uses a set of lanthanide-tagged antibodies, each being specific for a desired molecular target, quantification of each molecular target could be exaggerated by nonspecific binding of its respective antibody to other nontarget cellular regions. In this report, we used lanthanide-tagged isotypes, which allowed for correction for nonspecific binding at the single-cell level. Using this approach, myoblasts were phenotypically identified by their position in the myogenic lineage, simultaneously with the quantification of autophagic flux in each identified subset. We found that generally autophagy flux is upregulated specifically during myoblast fusion and declines in myotubes. We also observed that mitophagy (i.e., selective autophagic degradation of mitochondria) is also active after myotube formation. The ability to track different types of autophagy is another feature of this methodology, which could be key to expand the current understanding of autophagy regulation in regenerating the skeletal muscle.

Using health technology assessment for better healthcare decisions.

Canadian health leaders can benefit from greater involvement in the design and production of Health Technology Assessment (HTA) through enhanced working relationships with HTA producers. The HTA producers and health leaders have a shared interest in the appropriate use of pharmaceuticals, healthcare devices, and procedures which benefit patients and support sustainable health systems. This article highlights the shared responsibility of HTA producers and decision makers in the appropriate use of these healthcare interventions through an examination of a HTA-informed policy and practice change in the management diabetes in elderly residents in long-term care settings. Consideration is given to the role of the co-responsibility model and LEADS in a Caring Environment framework (LEADS) in helping to facilitate partnerships between decision-makers and HTA producers in the realization of HTA-informed policy and practice.

Maturation in auditory event-related potentials explains variation in language ability in children.

Processing of auditory information in the cortex continues to develop into later childhood and adolescence. Recent research has indicated that intraclass correlation (ICC) is the best method for capturing maturation in auditory event-related potentials (AEPs) of school-age children. However, the sensitivity of the ICC approach in discerning AEP changes in children has not been consistently demonstrated and positive results have not been replicated. We attempted this replication and further explored whether AEP maturation estimated using the ICC approach predicts cognitive and linguistic abilities in addition to chronological age. We measured AEPs in response to simple tones in groups of 7-, 8-, 9- and 10-year olds with typical development (N = 67) and used ICC to estimate the age equivalent of each child's AEP (AEP-age). Results indicated that ICC differentiated 7- to 8-year-old children from 9- to 10-year-old children and that AEP-age predicted both chronological age and significant, unique variance in language ability, but not in nonverbal IQ. Our findings support the view that auditory organization in children reflects both general developmental maturation and more specific development of language skills, and support the future use of AEP-age to identify and understand individual differences in brain maturation in typically developing and clinical populations.

Quantifying Heterogeneity of Individual Organelles in Mixed Populations via Mass Cytometry.

Macroautophagy is a complex degradative intracellular process by which long-lived proteins and damaged organelles are cleared. Common methods for the analysis of autophagy are bulk measurements which mask organelle heterogeneity and complicate the analysis of interorganelle association and trafficking. Thus, methods for individual organelle quantification are needed to address these deficiencies. Current techniques for quantifying individual autophagy organelles are either low through-put or are dimensionally limited. We make use of the multiparametric capability of mass cytometry to investigate phenotypic heterogeneity in autophagy-related organelle types that have been isolated from murine brain, liver, and skeletal muscle. Detection and phenotypic classification of individual organelles were accomplished through the use of a lanthanide-chelating membrane stain and organelle-specific antibodies. Posthoc sample matrix background correction and nonspecific antibody binding corrections provide measures of interorganelle associations and heterogeneity. This is the first demonstration of multiparametric individual organelle analysis via mass cytometry. The method described here illustrates the potential for further investigation of the inherently complex interorganelle associations, trafficking, and heterogeneity present in most eukaryotic biological systems.

Being able to be myself: Understanding autonomy and autonomy-support from the perspectives of autistic adults with intellectual disabilities.

LAY ABSTRACT: Autistic young adults with intellectual disabilities want to be autonomous but are less autonomous than other people. However, they can be autonomous with appropriate support. We wanted to learn how we can support autistic adults with intellectual disabilities to be more autonomous. We designed our study with help from five autistic community partners to make sure the research was relevant to autistic people and would improve their lives. We talked with eight autistic young adults with intellectual disabilities about autonomy. We defined "talk" as verbal language, as well as non-verbal cues such as body language, facial expressions, vocalizations, and laughter. We did art projects and played games while we talked. We met in small groups over multiple sessions. Our participants told us that being autonomous meant being able to be themselves. They told us three main ways to support their autonomy: (1) having choice and control, (2) being able to communicate in their own way, and (3) being in a safe environment. Families, support staff, and caregivers can use this information to help autistic young adults with intellectual disabilities to be autonomous.

Chlamydia trachomatis infection causes mitotic spindle pole defects independently from its effects on centrosome amplification.

Chlamydiae are Gram negative, obligate intracellular bacteria, and Chlamydia trachomatis is the etiologic agent of the most commonly reported sexually transmitted disease in the United States. Chlamydiae undergo a biphasic life cycle that takes place inside a parasitophorous vacuole termed an inclusion. Chlamydial infections have been epidemiologically linked to cervical cancer in patients previously infected by human papillomavirus (HPV). The inclusion associates very closely with host cell centrosomes, and this association is dependent upon the host motor protein dynein. We have previously reported that this interaction induces supernumerary centrosomes in infected cells, leading to multipolar mitotic spindles and inhibiting accurate chromosome segregation. Our findings demonstrate that chlamydial infection causes mitotic spindle defects independently of its effects on centrosome amplification. We show that chlamydial infection increases centrosome spread and inhibits the spindle assembly checkpoint delay to disrupt centrosome clustering. These data suggest that chlamydial infection exacerbates the consequences of centrosome amplification by inhibiting the cells' ability to suppress the effects of these defects on mitotic spindle organization. We hypothesize that these combined effects on mitotic spindle architecture identifies a possible mechanism for Chlamydia as a cofactor in cervical cancer formation.

Chlamydial infection induces host cytokinesis failure at abscission.

Chlamydia trachomatis is an obligate intracellular bacteria and the infectious agent responsible for the sexually transmitted disease Chlamydia. Infection with Chlamydia can lead to serious health sequelae such as pelvic inflammatory disease and reproductive tract scarring contributing to infertility and ectopic pregnancies. Additionally, chlamydial infections have been epidemiologically linked to cervical cancer in patients with a prior human papilomavirus (HPV) infection. Chlamydial infection of cultured cells causes multinucleation, a potential pathway for chromosomal instability. Two mechanisms that are known to initiate multinucleation are cell fusion and cytokinesis failure. This study demonstrates that multinucleation of the host cell by Chlamydia is entirely due to cytokinesis failure. Moreover, cytokinesis failure is due in part to the chlamydial effector CPAF acting as an anaphase promoting complex mimic causing cells to exit mitosis with unaligned and unattached chromosomes. These lagging and missegregated chromosomes inhibit cytokinesis by blocking abscission, the final stage of cytokinesis.

Toward understanding and enhancing self-determination: a qualitative exploration with autistic adults without co-occurring intellectual disability.

Introduction: Self-determination is a fundamental human right positively related to quality of life. However, Autistic people are reported to be less self-determined than non-autistic people. We aimed to (1) understand what self-determination means to Autistic people from their perspective, (2) explore their perceptions of current barriers to being self-determined, and (3) learn from Autistic people about how they would like to be supported to be self-determined. Methods: Semi-structured interviews were done with 19 Autistic adults without co-occurring intellectual disability. Data were analyzed by three Autistic and two non-autistic researchers through an iterative process of data familiarization, coding, and theme development, informed by reflexive thematic analysis. Autistic Community Partners (ACP) were also engaged throughout the study, and provided substantive feedback on all methods and results. Results: Self-determination held the same meaning for Autistic people as non-autistic people. More specifically, participants discussed having the opportunity and support to make choices and decisions in life without unnecessary control from others. Experiences of self-determination were centered around: (1) lack of opportunity, influenced by ableist expectations and discrimination, and (2) executive processing differences that interfered with choice and decision-making. Desired areas of support related to providing opportunities to (1) make choices and exert autonomy, (2) be supported to unmask and be valued as one's authentic Autistic self, and (3) offering pragmatic support for executive processing differences. Conclusion: Autistic adults desire to be self-determined and can flourish with support, as they determine to be appropriate, which might look different from support commonly offered or sought by non-autistic people. Although individualized support was discussed, the ideal desired support was for an inclusive society that values and respects their neurodivergence, rather than imposing ableist expectations. An inclusive society is only achievable through reduced (or eliminated) stigma and prejudice against Autistic people.

Improving autism identification and support for individuals assigned female at birth: clinical suggestions and research priorities.

Emerging evidence suggests that the higher prevalence of autism in individuals who are assigned male than assigned female at birth results from both biological factors and identification biases. Autistic individuals who are assigned female at birth (AFAB) and those who are gender diverse experience health disparities and clinical inequity, including late or missed diagnosis and inadequate support. In this Viewpoint, an international panel of clinicians, scientists, and community members with lived experiences of autism reviewed the challenges in identifying autism in individuals who are AFAB and proposed clinical and research directions to promote the health, development, and wellbeing of autistic AFAB individuals. The recognition challenges stem from the interplay between cognitive differences and nuanced or different presentations of autism in some AFAB individuals; expectancy, gender-related, and autism-related biases held by clinicians; and social determinants. We recommend that professional development for clinicians be supported by health-care systems, professional societies, and governing bodies to improve equitable access to assessment and earlier identification of autism in AFAB individuals. Autistic AFAB individuals should receive tailored support in education, identity development, health care, and social and professional sense of belonging.

The Autism Intervention Research Network on Physical Health (AIR-P) Research Agenda.

OBJECTIVES: In the United States, autistic individuals experience disproportionate physical and mental health challenges relative to non-autistic individuals, including higher rates of co-occurring and chronic conditions and lower physical, social, and psychological health-related quality of life. The Autism Intervention Research Network on Physical Health (AIR-P) is an interdisciplinary, multicenter research network for scientific collaboration and infrastructure that aims to increase the life expectancy and quality of life for autistic individuals, with a focus on underserved or vulnerable populations. The current paper describes the development of the AIR-P Research Agenda. METHODS: Development of the research agenda involved an iterative and collaborative process between the AIR-P Advisory Board, Steering Committee, and Autistic Researcher Review Board. The methodology consisted of 3 phases: (1) ideation and design, (2) literature review and synthesis; and (3) network engagement. RESULTS: Six core research priorities related to the health of autistic individuals were identified: (1) primary care services and quality, (2) community-based lifestyle interventions, (3) health systems and services, (4) gender, sexuality, and reproductive health, (5) neurology, and (6) genetics. Specific topics within each of these priorities were identified. Four cross-cutting research priorities were also identified: (1) neurodiversity-oriented care, (2) facilitating developmental transitions, (3) methodologically rigorous intervention studies, and (4) addressing health disparities. CONCLUSIONS: The AIR-P Research Agenda represents an important step forward for enacting large-scale health-promotion efforts for autistic individuals across the lifespan. This agenda will catalyze autism research in historically underrepresented topic areas while adopting a neurodiversity-oriented approach to health-promotion.

C. elegans RPM-1 regulates axon termination and synaptogenesis through the Rab GEF GLO-4 and the Rab GTPase GLO-1.

C. elegans RPM-1 (for Regulator of Presynaptic Morphology) is a member of a conserved protein family that includes Drosophila Highwire and mammalian Pam and Phr1. These are large proteins recently shown to regulate synaptogenesis through E3 ubiquitin ligase activities. Here, we report the identification of an RCC1-like guanine nucleotide exchange factor, GLO-4, from mass spectrometry analysis of RPM-1-associated proteins. GLO-4 colocalizes with RPM-1 at presynaptic terminals. Loss of function in glo-4 or in its target Rab GTPase, glo-1, causes neuronal defects resembling those in rpm-1 mutants. We show that the glo pathway functions downstream of rpm-1 and acts in parallel to fsn-1, a partner of RPM-1 E3 ligase function. We find that late endosomes are specifically disorganized at the presynaptic terminals of glo-4 mutants. Our data suggest that RPM-1 positively regulates a Rab GTPase pathway to promote vesicular trafficking via late endosomes.

The Caenorhabditis elegans UNC-14 RUN domain protein binds to the kinesin-1 and UNC-16 complex and regulates synaptic vesicle localization.

Kinesin-1 is a heterotetramer composed of kinesin heavy chain (KHC) and kinesin light chain (KLC). The Caenorhabditis elegans genome has a single KHC, encoded by the unc-116 gene, and two KLCs, encoded by the klc-1 and klc-2 genes. We show here that UNC-116/KHC and KLC-2 form a complex orthologous to conventional kinesin-1. KLC-2 also binds UNC-16, the C. elegans JIP3/JSAP1 JNK-signaling scaffold protein, and the UNC-14 RUN domain protein. The localization of UNC-16 and UNC-14 depends on kinesin-1 (UNC-116 and KLC-2). Furthermore, mutations in unc-16, klc-2, unc-116, and unc-14 all alter the localization of cargos containing synaptic vesicle markers. Double mutant analysis is consistent with these four genes functioning in the same pathway. Our data support a model whereby UNC-16 and UNC-14 function together as kinesin-1 cargos and regulators for the transport or localization of synaptic vesicle components.

Curricular Activities that Promote Metacognitive Skills Impact Lower-Performing Students in an Introductory Biology Course.

This study explores the impacts of repeated curricular activities designed to promote metacognitive skills development and academic achievement on students in an introductory biology course. Prior to this study, the course curriculum was enhanced with pre-assignments containing comprehension monitoring and self-evaluation questions, exam review assignments with reflective questions related to study habits, and an optional opportunity for students to explore metacognition and deep versus surface learning. We used a mixed-methods study design and collected data over two semesters. Self-evaluation, a component of metacognition, was measured via exam score postdictions, in which students estimated their exam scores after completing their exam. Metacognitive awareness was assessed using the Metacognitive Awareness Inventory (MAI) and a reflective essay designed to gauge students' perceptions of their metacognitive skills and study habits. In both semesters, more students over-predicted their Exam 1 scores than under-predicted, and statistical tests revealed significantly lower mean exam scores for the over-predictors. By Exam 3, under-predictors still scored significantly higher on the exam, but they outnumbered the over-predictors. Lower-performing students also displayed a significant increase in exam postdiction accuracy by Exam 3. While there was no significant difference in students' MAI scores from the beginning to the end of the semester, qualitative analysis of reflective essays indicated that students benefitted from the assignments and could articulate clear action plans to improve their learning and performance. Our findings suggest that assignments designed to promote metacognition can have an impact on students over the course of one semester and may provide the greatest benefits to lower-performing students.

Pilot study of the Olympia oyster Ostrea conchaphila in the San Francisco Bay estuary: description and distribution of diseases.

Olympia oysters Ostrea conchaphila have declined markedly during the last century and are a focus of restoration in many embayments, including the San Francisco Bay (SFB) estuary. Oysters were collected from 17 sites in this estuary and nearby Tomales Bay in an effort to characterize diseases that may impact recovery of this species and captive rearing programs. Three diseases/disease agents including a Mikrocytos-like protist (microcell), a haplosporidian and hemic neoplasia were observed from several sites along the western margins of the SFB estuary suggesting a geographic localization of disease presence. Based on fluoresecent in situ hybridization (FISH) assays, the microcell is distinct from M. mackini and Bonamia spp. These data highlight the need for further elucidation of the haplosporidian and for careful health management of a declining species destined for captive rearing and supplementation.

Cytology of secondary vulvar Paget's disease of urothelial origin: a case report.

BACKGROUND: Primary cutaneous Paget's disease of the vulva is an intraepithelial adenocarcinoma most likely arising from a cutaneous stem cell with sweat gland epithelial differentiation or can be of sweat gland origin. Primary vulvar Paget's disease, however, can be mimicked by an internal noncutaneous neoplasm htat has extended to secondarily involve the vulva. Most commonly, this is due to an anal or rectal adenocarcinoma or a urothelial carcinoma. These malignancies may be detected in a vaginal or vulvar cytologic smear. CASE: An 81-year-old woman with a past history of urothelial carcinoma in situ of the bladder presented severalyears subsequent to treatment for bladder cancer with extensive vulvar and vaginal disease, clinically interpreted as primary vulvar Paget's disease involving the vagina. Vaginal cytology showed a high grade malignancy. The patient subsequently underwent radical (total deep) vulvectomy and vaginal excision. Subsequent investigation of her bladder showed recurrent urothelial carcinoma in situ with extensive spread to the vagina and vulva, simulating primary cutaneous vulvar Paget's disease. CONCLUSION: It is important to recognize secondary vulvar Paget's disease, although uncommon, because of the difference in therapy for primary and secondary vulvar Paget's disease. Certain cytologic characteristics in a vaginal or vulvar smear in a patient with suspected vulvar Paget's disease may aid in distinguishing them.