Effectiveness of Operation K9 Assistance Dogs on Suicidality in Australian Veterans with PTSD: A 12-Month Mixed-Methods Follow-Up Study.

Post-traumatic stress disorder (PTSD) is a pervasive disorder among both current and ex-serving Australian Defence Force (ADF) members. Studies have shown current psychological and pharmacological treatments for PTSD are suboptimal in veterans, with high dropout rates and poor adherence to treatment protocols. Therefore, evaluating complementary interventions, such as assistance dogs, is needed for veterans who may not receive the ultimate benefit from traditional therapies. The present longitudinal mixed-method study examined the effectiveness of Operation K9 assistance dogs among sixteen veterans with PTSD, specifically, their effects on suicidality, PTSD, depression, and anxiety from baseline to 12 months post-matching. Self-reported measures were completed prior to receiving their dog (baseline) and at three time points (3, 6, and 12 months) following matching. The Clinician-Administered PTSD Scale for DSM-5 was used to assess the severity of every PTSD case. Veterans participated in a semi-structured interview 3 months post-matching. Whilst there was a reduction in the proportion of veterans reporting any suicidality, there was no significant change in the probability of veterans reporting suicidality between time points. There was a significant effect of time on PTSD, depression, and anxiety symptoms. Three major themes emerged from qualitative data analysis: life changer, constant companion, and social engagement. Qualitative data suggest assistance dogs can have a positive impact on important areas of daily life and support veterans in achieving some of the prerequisites for health, including access to services, transport, education, employment, and development of new and diverse social and community connections. Connections were key in improving health and wellbeing. This study exemplifies the power of human-animal relationships and adds emphasis to the need to take these seriously and create supportive healthy environments for veterans with PTSD. Our findings could be used to inform public health policy and service delivery, in line with the Ottawa Charter action areas and indicate that for veterans with PTSD, assistance dogs may be a feasible adjunct intervention.

High-Resolution Ion-Mobility-Enabled Peptide Mapping for High-Throughput Critical Quality Attribute Monitoring.

Characterization and monitoring of post-translational modifications (PTMs) by peptide mapping is a ubiquitous assay in biopharmaceutical characterization. Often, this assay is coupled to reversed-phase liquid chromatographic (LC) separations that require long gradients to identify all components of the protein digest and resolve critical modifications for relative quantitation. Incorporating ion mobility (IM) as an orthogonal separation that relies on peptide structure can supplement the LC separation by providing an additional differentiation filter to resolve isobaric peptides, potentially reducing ambiguity in identification through mobility-aligned fragmentation and helping to reduce the run time of peptide mapping assays. A next-generation high-resolution ion mobility (HRIM) technique, based on structures for lossless ion manipulations (SLIM) technology with a 13 m ion path, provides peak capacities and higher resolving power that rivals traditional chromatographic separations and, owing to its ability to resolve isobaric peptides that coelute in faster chromatographic methods, allows for up to 3× shorter run times than conventional peptide mapping methods. In this study, the NIST monoclonal antibody IgG1κ (NIST RM 8671, NISTmAb) was characterized by LC-HRIM-MS and LC-HRIM-MS with collision-induced dissociation (HRIM-CID-MS) using a 20 min analytical method. This approach delivered a sequence coverage of 96.5%. LC-HRIM-CID-MS experiments provided additional confidence in sequence determination. HRIM-MS resolved critical oxidations, deamidations, and isomerizations that coelute with their native counterparts in the chromatographic dimension. Finally, quantitative measurements of % modification were made using only the m/z-extracted HRIM arrival time distributions, showing good agreement with the reference liquid-phase separation. This study shows, for the first time, the analytical capability of HRIM using SLIM technology for enhancing peptide mapping workflows relevant to biopharmaceutical characterization.

High-defined quantitative snapshots of the ganglioside lipidome using high resolution ion mobility SLIM assisted shotgun lipidomics.

Defects in sphingolipid metabolism have emerged as a common link across neurodegenerative disorders, and a deeper understanding of the lipid content in preclinical models and patient specimens offers opportunities for development of new therapeutic targets and biomarkers. Sphingolipid metabolic pathways include the formation of glycosphingolipid species that branch into staggeringly complex structural heterogeneity within the globoside and ganglioside sub-lipidomes. Characterization of these sub-lipidomes has typically relied on liquid chromatography-mass spectrometry-based (LC-MS) approaches, but such assays are challenging and resource intensive due to the close structural heterogeneity, the presence of isobaric and isomeric species, and broad dynamic range of endogenous glycosphingolipids. Here, we apply Structures for Lossless Ion Manipulations (SLIM)-based High Resolution Ion Mobility (HRIM)-MS to enable rapid, repeatable, quantitative assays with deep structural information sufficient to resolve endogenous brain gangliosides at the level of individual molecular species. Analyses were performed using a prototype SLIM-MS instrument equipped with a 13-m serpentine path which enabled resolution of closely related isomeric analytes such as GD1a d36:1 and GD1b d36:1 based on recorded mass-to-charge (m/z) and arrival times. To demonstrate the power of our methodology, brain extracts derived from wild-type mice hemi-brains were analyzed by HRIM-MS using flow injection analyses (FIA) without the need for additional separation by liquid chromatography. Endogenous ganglioside species were readily resolved, identified, and quantified by FIA-SLIM-MS analyses within 2 min per sample. Thus, the FIA-SLIM-MS platform enables robust quantification across a broad range of lipid species in biological specimens in a standardized assay format that is readily scalable to support studies with large sample numbers.

Test Administration Methods and Cognitive Test Scores in Older Adults with Hearing Loss.

In light of the high prevalence of hearing loss and cognitive impairment in the aging population, it is important to know how cognitive tests should be administered for older adults with hearing loss. The purpose of the present study is to examine this question with a cognitive screening test and a working memory test. Specifically, we asked the following questions in 2 experiments. First, does a controlled amplification method affect cognitive test scores? Second, does test modality (visual vs. auditory) impact cognitive test scores? Three test administration conditions were created for both Montreal Cognitive Assessment (MoCA) and working memory test (a word recognition and recall test): auditory amplified, auditory unamplified, and visual. The auditory administration was implemented through a computer program to control for presentation level while the visual condition was implemented through timed computer slides. Data were collected from older individuals with mild-to-moderate sensorineural hearing loss. We did not find any effect of amplification or test modality on the total score of the cognitive screening test (i.e., MoCA). Amplification improved working memory performance as measured by word recall performance, but test modality (auditory vs. visual) did not. These results are consistent with literature in demonstrating a downstream effect of audibility on working memory performance. From a clinical perspective, these findings are informative for developing clinical administration protocols of these tests for older individuals with hearing loss.

An Amyloid Core Sequence in the Major Candida albicans Adhesin Als1p Mediates Cell-Cell Adhesion.

The human fungal commensal Candida albicans can become a serious opportunistic pathogen in immunocompromised hosts. The C. albicans cell adhesion protein Als1p is a highly expressed member of a large family of paralogous adhesins. Als1p can mediate binding to epithelial and endothelial cells, is upregulated in infections, and is important for biofilm formation. Als1p includes an amyloid-forming sequence at amino acids 325 to 331, identical to the sequence in the paralogs Als5p and Als3p. Therefore, we mutated Val326 to test whether this sequence is important for activity. Wild-type Als1p (Als1pWT) and Als1p with the V326N mutation (Als1pV326N) were expressed at similar levels in a Saccharomyces cerevisiae surface display model. Als1pV326N cells adhered to bovine serum albumin (BSA)-coated beads similarly to Als1pWT cells. However, cells displaying Als1pV326N showed visibly smaller aggregates and did not fluoresce in the presence of the amyloid-binding dye Thioflavin-T. A new analysis tool for single-molecule force spectroscopy-derived surface mapping showed that statistically significant force-dependent Als1p clustering occurred in Als1pWT cells but was absent in Als1pV326N cells. In single-cell force spectroscopy experiments, strong cell-cell adhesion was dependent on an intact amyloid core sequence on both interacting cells. Thus, the major adhesin Als1p interacts through amyloid-like ß-aggregation to cluster adhesin molecules in cis on the cell surface as well as in trans to form cell-cell bonds.IMPORTANCE Microbial cell surface adhesins control essential processes such as adhesion, colonization, and biofilm formation. In the opportunistic fungal pathogen Candida albicans, the agglutinin-like sequence (ALS) gene family encodes eight cell surface glycoproteins that mediate adherence to biotic and abiotic surfaces and cell-cell aggregation. Als proteins are critical for commensalism and virulence. Their activities include attachment and invasion of endothelial and epithelial cells, morphogenesis, and formation of biofilms on host tissue and indwelling medical catheters. At the molecular level, Als5p-mediated cell-cell aggregation is dependent on the formation of amyloid-like nanodomains between Als5p-expressing cells. A single-site mutation to valine 326 abolishes cellular aggregation and amyloid formation. Our results show that the binding characteristics of Als1p follow a mechanistic model similar to Als5p, despite its differential expression and biological roles.

Fluidic Force Microscopy Demonstrates That Homophilic Adhesion by Candida albicans Als Proteins Is Mediated by Amyloid Bonds between Cells.

The fungal pathogen Candida albicans frequently forms drug-resistant biofilms in hospital settings and in chronic disease patients. Cell adhesion and biofilm formation involve a family of cell surface Als (agglutinin-like sequence) proteins. It is now well documented that amyloid-like clusters of laterally arranged Als proteins activate cell-cell adhesion under mechanical stress, but whether amyloid-like bonds form between aggregating cells is not known. To address this issue, we measure the forces driving Als5-mediated intercellular adhesion using an innovative fluidic force microscopy platform. Strong cell-cell adhesion is dependent on expression of amyloid-forming Als5 at high cell surface density and is inhibited by a short antiamyloid peptide. Furthermore, there is greatly attenuated binding between cells expressing amyloid-forming Als5 and cells with a nonamyloid form of Als5. Thus, homophilic bonding between Als5 proteins on adhering cells is the major mode of fungal aggregation, rather than protein-ligand interactions. These results point to a model whereby amyloid-like ß-sheet interactions play a dual role in cell-cell adhesion, that is, in formation of adhesin nanoclusters ( cis-interactions) and in homophilic bonding between amyloid sequences on opposing cells ( trans-interactions). Because potential amyloid-forming sequences are found in many microbial adhesins, we speculate that this novel mechanism of amyloid-based homophilic adhesion might be widespread and could represent an interesting target for treating biofilm-associated infections.

Development and validation of Portable Automated Rapid Testing (PART) measures for auditory research.

The current state of consumer-grade electronics means that researchers, clinicians, students, and members of the general public across the globe can create high-quality auditory stimuli using tablet computers, built-in sound hardware, and calibrated consumer-grade headphones. Our laboratories have created a free application that supports this work: PART (Portable Automated Rapid Testing). PART has implemented a range of psychoacoustical tasks including: spatial release from speech-on-speech masking, binaural sensitivity, gap discrimination, temporal modulation, spectral modulation, and spectrotemporal modulation (STM). Here, data from the spatial release and STM tasks are presented. Data were collected across the globe on tablet computers using applications available for free download, built-in sound hardware, and calibrated consumer-grade headphones. Spatial release results were as good or better than those obtained with standard laboratory methods. Spectrotemporal modulation thresholds were obtained rapidly and, for younger normal hearing listeners, were also as good or better than those in the literature. For older hearing impaired listeners, rapid testing resulted in similar thresholds to those reported in the literature. Listeners at five different testing sites produced very similar STM thresholds, despite a variety of testing conditions and calibration routines. Download Spatial Release, PART, and Listen: An Auditory Training Experience for free at https://bgc.ucr.edu/games/.

Amyloid-Like ß-Aggregates as Force-Sensitive Switches in Fungal Biofilms and Infections.

Cellular aggregation is an essential step in the formation of biofilms, which promote fungal survival and persistence in hosts. In many of the known yeast cell adhesion proteins, there are amino acid sequences predicted to form amyloid-like ß-aggregates. These sequences mediate amyloid formation in vitro. In vivo, these sequences mediate a phase transition from a disordered state to a partially ordered state to create patches of adhesins on the cell surface. These ß-aggregated protein patches are called adhesin nanodomains, and their presence greatly increases and strengthens cell-cell interactions in fungal cell aggregation. Nanodomain formation is slow (with molecular response in minutes and the consequences being evident for hours), and strong interactions lead to enhanced biofilm formation. Unique among functional amyloids, fungal adhesin ß-aggregation can be triggered by the application of physical shear force, leading to cellular responses to flow-induced stress and the formation of robust biofilms that persist under flow. Bioinformatics analysis suggests that this phenomenon may be widespread. Analysis of fungal abscesses shows the presence of surface amyloids in situ, a finding which supports the idea that phase changes to an amyloid-like state occur in vivo. The amyloid-coated fungi bind the damage-associated molecular pattern receptor serum amyloid P component, and there may be a consequential modulation of innate immune responses to the fungi. Structural data now suggest mechanisms for the force-mediated induction of the phase change. We summarize and discuss evidence that the sequences function as triggers for protein aggregation and subsequent cellular aggregation, both in vitro and in vivo.

Serum Amyloid P Component and Systemic Fungal Infection: Does It Protect the Host or Is It a Trojan Horse?

It is a striking observation that tissue of patients invaded by the deep mycoses often lacks evidence of an inflammatory response. This lack of host response is often attributed to neutropenia secondary to chemotherapy. However, systematic studies do not support this simplistic explanation. However, invasive fungal lesions are characterized by abundant fungal functional amyloid, which in turn is bound by serum amyloid P component (SAP). We postulate that SAP is important in the local immune response in invasive fungal infections. The interaction between fungal functional amyloid, SAP, and the immune response in deep mycoses is discussed.

A unique biofilm in human deep mycoses: fungal amyloid is bound by host serum amyloid P component.

BACKGROUND/OBJECTIVES: We have demonstrated the presence of Candida cell surface amyloids that are important in aggregation of fungi and adherence to tissue. Fungal amyloid was present in invasive human candidal infections and host serum amyloid P component (SAP) bound to the fungal amyloid. SAP is a protease-resistant glycoprotein that binds avidly to amyloid and interferes with host defence, especially against bacterial pathogens for which neutrophils are important. In this study, we investigated whether biofilm of fungal amyloid and SAP was a feature of other disseminated fungal infections. METHODS: Tissue specimens from 15 autopsies were systematically evaluated with multiple histochemical stains including thioflavin T and Congo red (dyes that stain amyloid), as well as antibody to SAP. We studied specimens with disseminated aspergillosis, mucormycosis and coccidioidomycosis. The structure of the lesions, host inflammatory cells and the presence of fungal amyloid and SAP were determined. RESULTS: The structure of the lesions was characteristic in aspergillosis ('starburst') and mucormycosis (closely apposed bundles of hyphae). Host inflammatory cells were absent or few in number within these lesions. In Coccidioides lesions, host inflammation was sparse as well. Fungal amyloid was a prominent feature of all lesions along with abundant SAP bound to hyphae and spherules. Fungal amyloid and SAP perhaps contributed to persistence in caseous necrosis lesions. SAP also bound to Aspergillus and Mucorales amyloid in vitro. CONCLUSIONS: A biofilm including amyloid and SAP is present in invasive fungal infections. This biofilm may dampen host defence leading to the characteristic sparse inflammatory reaction found in these infections.

Between Amyloids and Aggregation Lies a Connection with Strength and Adhesion.

We tell of a journey that led to discovery of amyloids formed by yeast cell adhesins and their importance in biofilms and host immunity. We begin with the identification of the adhesin functional amyloid-forming sequences that mediate fiber formation in vitro. Atomic force microscopy and confocal microscopy show 2-dimensional amyloid "nanodomains" on the surface of cells that are activated for adhesion. These nanodomains are arrays of adhesin molecules that bind multivalent ligands with high avidity. Nanodomains form when adhesin molecules are stretched in the AFM or under laminar flow. Treatment with antiamyloid perturbants or mutation of the amyloid sequence prevents adhesion nanodomain formation and activation. We are now discovering biological consequences. Adhesin nanodomains promote formation and maintenance of biofilms, which are microbial communities. Also, in abscesses within candidiasis patients, we find adhesin amyloids on the surface of the fungi. In both human infection and a Caenorhabditis elegans infection model, the presence of fungal surface amyloids elicits anti-inflammatory responses. Thus, this is a story of how fungal adhesins respond to extension forces through formation of cell surface amyloid nanodomains, with key consequences for biofilm formation and host responses.

Peptide detection of fungal functional amyloids in infected tissue.

Many fungal cell adhesion proteins form functional amyloid patches on the surface of adhering cells. The Candida albicans Agglutinin-like sequence (Als) adhesins are exemplars for this phenomenon, and have amyloid forming sequences that are conserved between family members. The Als5p amyloid sequence mediates amyloid fibril formation and is critical for cell adhesion and biofilm formation, and is also present in the related adhesins Als1p and Als3p. We have developed a fluorescent peptide probe containing the conserved Als amyloid-forming sequence. This peptide bound specifically to yeast expressing Als5p, but not to cells lacking the adhesin. The probe bound to both yeast and hyphal forms of C. albicans. Δals1/Δals3 single and double deletion strains exhibited reduced fluorescence, indicating that probe binding required expression of these proteins. Additionally, the Als peptide specifically stained fungal cells in abscesses in autopsy sections. Counterstaining with calcofluor white showed colocalization with the amyloid peptide. In addition, fungi in autopsy sections derived from the gastrointestinal tract showed colocalization of the amyloid-specific dye thioflavin T and the fluorescent peptide. Collectively, our data demonstrate that we can exploit amyloid sequence specificity for detection of functional amyloids in situ.

Improving influenza vaccination rates in pregnant women.

OBJECTIVE: To determine whether a provider-focused reminder improved vaccination rates as compared with no reminder. STUDY DESIGN: This was a retrospective cohort study comparing vaccination rates among pregnant patients seen in October and November 2003 (no reminder) and October and November 2005 (provider-focused reminder). Charts of all patients presenting for prenatal care during those months were reviewed for vaccination order. Vaccination rates were calculated and compared by year, provider, age, race, education, primary language, insurance type, and presence or absence of medical risk factors. RESULTS: A total of 1,367 records were reviewed: 504 from 2003 and 863 from 2005. Overall vaccination rate increased from 15-52% with a provider-focused reminder in place. In our study population medical risk factors were identified in 396 patients (29%). Vaccination rates for patients with medical risk factors increased from 18-47%. All provider groups demonstrated significant increases in the rates of vaccination with a reminder, however, there were no differences in age, race, education, primary language, or insurance. CONCLUSION: Although a low-cost, provider-focused reminder improved vaccination rates to 52%, additional measures including patient and provider education, dedicated vaccination clinics, and standing orders will be needed to achieve the ACOG goal of 100% vaccination.