The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM.

SNAREs provide a large part of the specificity and energy needed for membrane fusion and, to do so, must be localized to their correct membranes. Here, we show that the R-SNAREs VAMP8, VAMP3, and VAMP2, which cycle between the plasma membrane and endosomes, bind directly to the ubiquitously expressed, PtdIns4,5P(2)-binding, endocytic clathrin adaptor CALM/PICALM. X-ray crystallography shows that the N-terminal halves of their SNARE motifs bind the CALM(ANTH) domain as helices in a manner that mimics SNARE complex formation. Mutation of residues in the CALM:SNARE interface inhibits binding in vitro and prevents R-SNARE endocytosis in vivo. Thus, CALM:R-SNARE interactions ensure that R-SNAREs, required for the fusion of endocytic clathrin-coated vesicles with endosomes and also for subsequent postendosomal trafficking, are sorted into endocytic vesicles. CALM's role in directing the endocytosis of small R-SNAREs may provide insight into the association of CALM/PICALM mutations with growth retardation, cognitive defects, and Alzheimer's disease.

The One Hour Human Proteome.

We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS1 scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach.

Youth Participatory Action Communication Research: A Model for Developing Youth-Driven Health Campaigns.

Youth offer valuable insight on health communication needs and solutions in their communities. We propose youth participatory action communication research (YPACR) as a model for health campaign development that engages youth perspectives in applying systematic theory-informed communication research to addressing youth-identified health priorities. YPACR informed a series of paid high school internship programs in West Philadelphia, in which youth interns identified mental health help-seeking communication as a need among peers. In Phase 1, guided by the reasoned action approach and Hornik & Woolf method, youth interns conducted a survey measuring behavioral beliefs, normative beliefs, and control beliefs associated with mental health help-seeking, as well as trusted sources of mental health information, among local high school students. Survey results suggested control (self-efficacy) was an important message target and peers were trusted mental health information sources. In Phase 2, youth interns developed TikTok-style messages focused on strengthening control beliefs and promoting a youth-selected mental health support resource. Youth interns distributed an online survey experiment to test whether youth-created messages shown alongside resource information increased help-seeking self-efficacy compared to an information-only control. The YPACR framework contributed to youth-relevant campaign goals, study measurements, recruitment approaches, data interpretation, and message design. We discuss the benefits and challenges of this youth-driven health campaign development model and recommendations for future research.

The impact of a stress management intervention including cultural components on stress biomarker levels and mental health indicators among indigenous women.

We examined the effectiveness of a 26-week culture-inclusive intervention on reducing salivary stress biomarker levels, and perceived stress, depressive, and post-traumatic stress disorder (PTSD) symptoms measured using scales in 53 Indigenous women in Ontario, Canada. Statistical analyses compared the average biomarker levels, and the area under the curve (AUC) of biomarkers. Differences in biomarkers and mental health scale scores pre- and post-intervention were compared using mixed models with a random intercept. Interaction terms were included between the intervention and age, education, disability, and HIV status, individually, to test for sub-group differences. Cortisol AUC post-intervention was decreased compared to pre-intervention (ß -1.29 µg/dL; 95%CI -2.35, -0.23). There was a slight decrease in perceived stress levels (aOR: -2.80; 95%CI -5.09, -0.50). The associations were stronger among women of younger age, higher education, and no disabilities. These interventions can be effective, but future interventions should target Indigenous population sub-groups to address individual needs.

Adaptor protein complex 4 deficiency: a paradigm of childhood-onset hereditary spastic paraplegia caused by defective protein trafficking.

Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3-5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.

A highly multiplexed quantitative phosphosite assay for biology and preclinical studies.

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho-signaling in drug-treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.

A Quantitative Intersectional Exploration of Sexual Violence and Mental Health among Bi + People: Looking within and across Race and Gender.

Young bisexual people report disparities related to mental health and sexual violence compared to their heterosexual and gay/lesbian peers. However, the majority of research in these areas does not employ an intersectional design, despite evidence that health outcomes vary by race and gender within bi + populations. The goal of this paper is to provide an intersectionally-informed exploration of the prevalence of sexual violence among a diverse sample of 112 bi + people age 18-26, as well as descriptive data on stigma, mental health, and social support. Most (82%) of participants reported at least once experience of sexual violence since the age of 16. Sexual violence was positively associated with sexual stigma, anxiety, depression, and suicidality. Nonbinary participants reported greater prevalence of violence, exposure to stigma, and worse mental health outcomes relative to cisgender participants. Nonbinary BIPOC participants reported higher levels of anxiety and depression than cisgender BIPOC participants.

Fast and cloning-free CRISPR/Cas9-mediated genomic editing in mammalian cells.

CHoP-In (CRISPR/Cas9-mediated Homology-independent PCR-product integration) is a fast, non-homologous end-joining based, strategy for genomic editing in mammalian cells. There is no requirement for cloning in generation of the integration donor, instead the desired integration donor is produced as a polymerase chain reaction (PCR) product, flanked by the Cas9 recognition sequences of the target locus. When co-transfected with the cognate Cas9 and guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon-homologous end joining. The approach is versatile, allowing N-terminal, C-terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a selection of well characterised membrane trafficking proteins. The lack of donor vectors offers advantages over existing methods in terms of both speed and hands-on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems.

Adaptor protein complexes and disease at a glance.

Adaptor protein (AP) complexes are heterotetramers that select cargo for inclusion into transport vesicles. Five AP complexes (AP-1 to AP-5) have been described, each with a distinct localisation and function. Furthermore, patients with a range of disorders, particularly involving the nervous system, have now been identified with mutations in each of the AP complexes. In many cases this has been correlated with aberrantly localised membrane proteins. In this Cell Science at a Glance article and the accompanying poster, we summarize what is known about the five AP complexes and discuss how this helps to explain the clinical features of the different genetic disorders.

Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval.

The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR), GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5-associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders.

Poverty in Lesbian, Gay, Bisexual, Transgender, Queer, Two-Spirit, and Other Sexual and Gender Minority (LGBTQ2S+) Communities in Canada: Implications for Social Work Practice.

In this article, we draw on a recent review of the Canadian literature on poverty in lesbian, gay, bisexual, transgender, queer, two-spirit, and other sexual and gender minority (LGBTQ2S+) communities to conceptualize social work interventions that may be used to address material inequities among these groups. Our literature review, which was based on a total of 39 works, revealed distinctive expressions of poverty among younger and older LGBTQ2S+ groups, as well as racialized, newcomer, and Indigenous sexual and gender minorities. Drawing on these insights, together with theoretical frameworks grounded in intersectionality and relational poverty analysis, we conceptualize these expressions of material inequity as salient sites of social work practice and propose interventions targeting these manifestations of LGBTQ2S+ poverty at various levels. Given the centrality of anti-poverty work as part of the social work profession's commitment to social justice, and the dearth of social work literature on LGBTQ2S+ poverty, this article promises to make significant contributions to social work scholarship and professional practice.

Evidence of Paracrystalline Cation Order in the Ruddlesden-Popper Phase LaSr3NiRuO8 through Neutron Total Scattering Techniques.

Cation ordering in perovskite-derived phases can lead to a wealth of tunable physical properties. Ordering is typically driven by a large difference between the cation size and charge, but many Ruddlesden-Popper phases An+1BnO3n+1 appear to lack such B-site ordering, even when these differences are present. One such example is the "double" Ruddlesden-Popper n = 1 composition LaSr3NiRuO8. In this material, a lack of B-site ordering is observed through traditional crystallographic techniques, but antiferromagnetic ordering in the magnetism data suggests that B-site cation ordering is indeed present. Neutron total scattering, particularly analysis of the neutron pair distribution function, reveals that the structure is locally B-site-ordered below 6 Å but becomes slightly disordered in the midrange structure around 12 Å. This provides evidence for paracrystalline order in this material: cation ordering within a single perovskite sheet that lacks perfect registry within the three-dimensional stack of sheets. This work highlights the importance of employing a structural technique that can probe both the local and midrange order in addition to the crystallographic structure and provides a structural origin to the observed magnetic properties of LaSr3NiRuO8. Further, it is proposed that paracrystalline order is likely to be common among these layered-type oxides.

Prioritizing Primary Care Patients for a Communication Intervention Using the "Surprise Question": a Prospective Cohort Study.

BACKGROUND: Communication about priorities and goals improves the value of care for patients with serious illnesses. Resource constraints necessitate targeting interventions to patients who need them most. OBJECTIVE: To evaluate the effectiveness of a clinician screening tool to identify patients for a communication intervention. DESIGN: Prospective cohort study. SETTING: Primary care clinics in Boston, MA. PARTICIPANTS: Primary care physicians (PCPs) and nurse care coordinators (RNCCs) identified patients at high risk of dying by answering the Surprise Question (SQ): "Would you be surprised if this patient died in the next 2 years?" MEASUREMENTS: Performance of the SQ for predicting mortality, measured by the area under receiver operating curve (AUC), sensitivity, specificity, and likelihood ratios. RESULTS: Sensitivity of PCP response to the SQ at 2 years was 79.4% and specificity 68.6%; for RNCCs, sensitivity was 52.6% and specificity 80.6%. In univariate regression, the odds of 2-year mortality for patients identified as high risk by PCPs were 8.4 times higher than those predicted to be at low risk (95% CI 5.7-12.4, AUC 0.74) and 4.6 for RNCCs (3.4-6.2, AUC 0.67). In multivariate analysis, both PCP and RNCC prediction of high risk of death remained associated with the odds of 2-year mortality. LIMITATIONS: This study was conducted in the context of a high-risk care management program, including an initial screening process and training, both of which affect the generalizability of the results. CONCLUSION: When used in combination with a high-risk algorithm, the 2-year version of the SQ captured the majority of patients who died, demonstrating better than expected performance as a screening tool for a serious illness communication intervention in a heterogeneous primary care population.

Bisexual Women's Experience of Microaggressions and Microaffirmations: A Community-Based, Mixed-Methods Scale Development Project.

In order to better serve bisexual women, clinicians and researchers need tools that accurately reflect and capture bisexual women's experiences of stigma and affirmation. These tools are essential as research indicates that bisexual women experience poorer mental health than either heterosexual or lesbian women. Our community-based study developed and psychometrically evaluated the Bisexual Microaggression and Microaffirmation Scales for Women (BMMS-W). We held focus groups and advisory committee meetings with bisexual women to identify common experiences of microaggressions and microaffirmations and drafted over 200 potential survey items. Exploratory factor analysis of data from 382 participants across Canada and the U.S. yielded five microaggression factors (dismissal; mistrust; sexualization; social exclusion; and denial of complexity) and four microaffirmation factors (acceptance; social support; recognition of bisexuality and biphobia; and emotional support). Confirmatory factor analysis of data from a separate sample of 323 participants across Canada and the U.S. tested the model. The development of the BMMS-W responds to calls to examine the distinctiveness of bisexual women's experience and gives mental health service providers and researchers a tool to better understand their experiences.

Molecular Basis for the Interaction Between AP4 ß4 and its Accessory Protein, Tepsin.

The adaptor protein 4 (AP4) complex (ϵ/ß4/µ4/σ4 subunits) forms a non-clathrin coat on vesicles departing the trans-Golgi network. AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin adaptors AP1 and AP2. AP4 is important for human health because mutations in any AP4 subunit cause severe neurological problems, including intellectual disability and progressive spastic para- or tetraplegias. We have used a range of structural, biochemical and biophysical approaches to determine the molecular basis for how the AP4 ß4 C-terminal appendage domain interacts with tepsin, the only known AP4 accessory protein. We show that tepsin harbors a hydrophobic sequence, LFxG[M/L]x[L/V], in its unstructured C-terminus, which binds directly and specifically to the C-terminal ß4 appendage domain. Using nuclear magnetic resonance chemical shift mapping, we define the binding site on the ß4 appendage by identifying residues on the surface whose signals are perturbed upon titration with tepsin. Point mutations in either the tepsin LFxG[M/L]x[L/V] sequence or in its cognate binding site on ß4 abolish in vitro binding. In cells, the same point mutations greatly reduce the amount of tepsin that interacts with AP4. However, they do not abolish the binding between tepsin and AP4 completely, suggesting the existence of additional interaction sites between AP4 and tepsin. These data provide one of the first detailed mechanistic glimpses at AP4 coat assembly and should provide an entry point for probing the role of AP4-coated vesicles in cell biology, and especially in neuronal function.

Optically Detected Magnetic Resonance for Selective Imaging of Diamond Nanoparticles.

While there is great interest in understanding the fate and transport of nanomaterials in the environment and in biological systems, the detection of nanomaterials in complex matrices by fluorescence methods is complicated by photodegradation, blinking, and the presence of natural organic material and other fluorescent background signals that hamper detection of fluorescent nanomaterials of interest. Optically detected magnetic resonance (ODMR) of nitrogen-vacancy (NV) centers in diamond nanoparticles provides a pathway toward background-free fluorescence measurements, as the application of a resonant microwave field can selectively modulate the intensity from NV centers in nanodiamonds of various diameters in complex materials systems using on-resonance and off-resonance microwave fields. This work represents the first investigation showing how nanoparticle diameter impacts the NV center lifetime and thereby directly impacts the accessible contrast and signal-to-noise ratio when using ODMR to achieve background-free imaging of NV-nanodiamonds in the presence of interfering fluorophores. These results provide new insights that will guide the choice of optimum nanoparticle size and methodology for background-free imaging and sensing applications, while also providing a model system to explore the fate and transport of nanomaterials in the environment.

Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation.

Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide and is a major risk factor for liver failure and hepatocellular carcinoma. Current antiviral therapy inhibits cytoplasmic HBV genomic replication, but is not curative because it does not directly affect nuclear HBV closed circular DNA (cccDNA), the genomic form that templates viral transcription and sustains viral persistence. Novel approaches that directly target cccDNA regulation would therefore be highly desirable. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications (PTMs). Here, using a new cccDNA ChIP-Seq approach, we report, to our knowledge, the first genome-wide maps of PTMs in cccDNA-containing chromatin from de novo infected HepG2 cells, primary human hepatocytes, and from HBV-infected liver tissue. We find high levels of PTMs associated with active transcription enriched at specific sites within the HBV genome and, surprisingly, very low levels of PTMs linked to transcriptional repression even at silent HBV promoters. We show that transcription and active PTMs in HBV chromatin are reduced by the activation of an innate immunity pathway, and that this effect can be recapitulated with a small molecule epigenetic modifying agent, opening the possibility that chromatin-based regulation of cccDNA transcription could be a new therapeutic approach to chronic HBV infection.

Forty Years of Clathrin-coated Vesicles.

The purification of coated vesicles and the discovery of clathrin by Barbara Pearse in 1975 was a landmark in cell biology. Over the past 40 years, work from many labs has uncovered the molecular details of clathrin and its associated proteins, including how they assemble into a coated vesicle and how they select cargo. Unexpected connections have been found with signalling, development, neuronal transmission, infection, immunity and genetic disorders. But there are still a number of unanswered questions, including how clathrin-mediated trafficking is regulated and how the machinery evolved.

Loss of AP-5 results in accumulation of aberrant endolysosomes: defining a new type of lysosomal storage disease.

Adaptor proteins (AP 1-5) are heterotetrameric complexes that facilitate specialized cargo sorting in vesicular-mediated trafficking. Mutations in AP5Z1, encoding a subunit of the AP-5 complex, have been reported to cause hereditary spastic paraplegia (HSP), although their impact at the cellular level has not been assessed. Here we characterize three independent fibroblast lines derived from skin biopsies of patients harbouring nonsense mutations in AP5Z1 and presenting with spastic paraplegia accompanied by neuropathy, parkinsonism and/or cognitive impairment. In all three patient-derived lines, we show that there is complete loss of AP-5 ζ protein and a reduction in the associated AP-5 µ5 protein. Using ultrastructural analysis, we show that these patient-derived lines consistently exhibit abundant multilamellar structures that are positive for markers of endolysosomes and are filled with aberrant storage material organized as exaggerated multilamellar whorls, striated belts and 'fingerprint bodies'. This phenotype can be replicated in a HeLa cell culture model by siRNA knockdown of AP-5 ζ. The cellular phenotype bears striking resemblance to features described in a number of lysosomal storage diseases (LSDs). Collectively, these findings reveal an emerging picture of the role of AP-5 in endosomal and lysosomal homeostasis, illuminates a potential pathomechanism that is relevant to the role of AP-5 in neurons and expands the understanding of recessive HSPs. Moreover, the resulting accumulation of storage material in endolysosomes leads us to propose that AP-5 deficiency represents a new type of LSDs.

Religion, Alcohol Use and Risk Drinking Among Canadian Adults Living in Ontario.

This research examines (1) the association between risk drinking and religious affiliation and (2) differences between religions for risk drinking among adults living in Ontario, Canada, for Christians, Buddhists, Sikhs, Muslims, Hindus, Jews, other religious groups and the non-religious. Data are based on telephone interviews with 16,596 respondents and are derived from multiple cycles (2005-2011) of the Centre for Addiction and Mental Health's (CAMH) Monitor survey, an ongoing cross-sectional survey of adults in Ontario, Canada, aged 18 years and older. Data were analysed using bivariate cross-tabulations, Mann-Whitney U nonparametric test and logistic regression. Alcohol use and risk drinking occur among members of all religious groups; however, the rate of drinking ranges widely. Risk drinking is significantly associated with religion. When compared to the No religion/Atheist group, several religious groups (Baptist, Christian, Hindu, Jehovah's Witness, Jewish, Muslim/Islam, Non-denominational, Pentecostal, Sikh and Other religion) in our sample have significantly lower odds of risk drinking. Risk drinkers also attended significantly fewer services among several religions. Results suggest that there are differences in the risk drinking rates among Canadian adults, living in Ontario, by religion. It appears that religious traditions of prohibition and abstention do hold sway among Canadian adults for some religious groups.