Treatment of dissociative identity disorder: leveraging neurobiology to optimize success.

INTRODUCTION: Dissociative identity disorder (DID) is a treatable mental health condition that is associated with a range of psychobiological manifestations. However, historical controversy, modern day misunderstanding, and lack of professional education have prevented accurate treatment information from reaching most clinicians and patients. These obstacles also have slowed empirical efforts to improve treatment outcomes for people with DID. Emerging neurobiological findings in DID provide essential information that can be used to improve treatment outcomes. AREAS COVERED: In this narrative review, the authors discuss symptom characteristics of DID, including dissociative self-states. Current treatment approaches are described, focusing on empirically supported psychotherapeutic interventions for DID and pharmacological agents targeting dissociative symptoms in other conditions. Neurobiological correlates of DID are reviewed, including recent research aimed at identifying a neural signature of DID. EXPERT OPINION: Now is the time to move beyond historical controversy and focus on improving DID treatment availability and efficacy. Neurobiological findings could optimize treatment by reducing shame, aiding assessment, providing novel interventional brain targets and guiding novel pharmacologic and psychotherapeutic interventions. The inclusion of those with lived experience in the design, planning and interpretation of research investigations is another powerful way to improve health outcomes for those with DID.

Global Indigenous gender concepts, gender-based violence and resilience: A scoping review.

BACKGROUND & OBJECTIVE: The legacy of colonialism includes ongoing trauma and disruption of traditional teachings on relationality, which has contributed to Indigenous populations being disproportionately exposed to gender-based violence (GBV). GBV in Indigenous populations is explored to consider gender-specific findings and points of resilience in relational networks. PARTICIPANTS & SETTING: Included articles sampled Indigenous groups in Canada, US, Mexico, Guatemala, and Israel. All participants self-identified as Indigenous, and were either GBV survivors or service providers working in GBV contexts. METHODS: A scoping review was conducted in OVID Medline, Embase, APA Psycinfo, and Informit Indigenous Collection, using keywords for Indigenous peoples, gender concepts, and GBV. Articles were screened and extracted by two reviewers; a third reviewer resolved conflicts. RESULTS: Our search yielded one mixed-method study and seven qualitative studies, all published since 2016. North American studies identified colonial, patriarchal disruptions (e.g. residential schools) to positive pre-contact gender norms (e.g. non-hierarchical roles) that contribute to emerging GBV. Studies conducted in Guatemala and Israel also described local patriarchal cultures contributing to GBV. Lack of understanding of the Two-Spirit identity (i.e. supra-binary gender identity used by Indigenous persons) led to harmful attitudes and stigma. Interpersonal support and return to traditional matriarchal practices were identified as key resilience processes. CONCLUSIONS: There is limited literature on Indigenous gender concepts and GBV, particularly regarding GBV against males and Two-Spirit persons. Colonization-related violence and/or patriarchal gender norms were identified as precursors for GBV. Decolonization processes should be further explored to address GBV in Indigenous populations.

Electrosynthetic C-F bond cleavage.

Fluorinated organic compounds are common among pharmaceuticals, agrochemicals and materials. The significant strength of the C-F bond results in chemical inertness that, depending on the context, is beneficial, problematic or simply a formidable synthetic challenge. Electrosynthesis is a rapidly expanding methodology that can enable new reactivity and selectivity for cleavage and formation of chemical bonds. Here, a comprehensive overview of synthetically relevant electrochemically driven protocols for C-F bond activation and functionalization is presented, including photoelectrochemical strategies.

Characterization of cerebrospinal fluid biomarkers associated with neurodegenerative diseases in healthy cynomolgus and rhesus macaque monkeys.

Monkeys are becoming important translational models of neurodegenerative disease. To facilitate model development, we measured cerebrospinal fluid (CSF) concentrations of key biomarkers in healthy male and female cynomolgus and rhesus macaques. Amyloid beta (Aß40, Aß42), tau (total tau [t-tau], phosphorylated tau [pThr181]), and neurofilament light (NfL) concentrations were measured in CSF of 82 laboratory-housed, experimentally naïve cynomolgus (n = 33) and rhesus (n = 49) macaques. Aß40 and Aß42 were significantly higher in rhesus, and female rhesus were higher than males. NfL and t-tau were higher in males, and NfL was higher in rhesus macaques. p-tau was not affected by species or sex. We also examined whether sample location (lumbar or cisterna puncture) affected concentrations. Sample acquisition site only affected NfL, which was higher in CSF from lumbar puncture compared to cisterna magna puncture. Establishing normative biomarker values for laboratory-housed macaque monkeys provides an important resource by which to compare to monkey models of neurodegenerative diseases.

Osteoarthritic and non-osteoarthritic patients show comparable coronal knee joint line orientations in a cross-sectional study based on 3D reconstructed CT images.

PURPOSE: Recently introduced total knee arthroplasty (TKA) alignment strategies aim to restore the pre-arthritic alignment of an individual patient. The native alignment of a patient can only be restored with detailed knowledge about the native and osteoarthritic alignment as well as differences between them. The first aim of this study was to assess the alignment of a large series of osteoarthritic (OA) knees and investigate whether femoral and tibial joint lines vary within patients with the same overall lower limb alignment. The secondary aim was to compare the alignment of OA patients to the previously published data of non-OA patients. This information could be useful for surgeons considering implementing one of the new alignment concepts. MATERIAL: Coronal alignment parameters of 2692 knee OA patients were measured based on 3D reconstructed CT data using a validated planning software (Knee-PLAN®, Symbios, Yverdon les Bains, Switzerland). Based on these measurements, patients' coronal alignment was phenotyped according to the functional knee phenotype concept. These phenotypes represent an alignment variation of either the overall alignment, the femoral joint line orientation or the tibial joint line orientation. Each phenotype is defined by a specific mean and covers a range of ± 1.5° from this mean. Mean values and distribution among the phenotypes are presented and compared between two populations (OA patients of this study and non-OA patients of a previously published study) as well as between HKA subgroups (varus, valgus and neutral) using t tests and Chi-square tests (p < 0.05). RESULTS: Femoral and tibial joint lines varied within patients with the same overall lower limb alignment. A total of 162 functional knee phenotypes were found (119 males, 136 females and 94 mutual phenotypes). Mean values differed between the OA and non-OA population, but differences were small (< 2°) except for the overall alignment (e.g. HKA). The distribution of OA and non-OA patients among the phenotypes differed significantly, especially among the limb phenotypes. CONCLUSION: Differences between OA and non-OA knees are small regarding coronal femoral and tibial joint line orientation. Femoral and tibial joint line orientation of osteoarthritic patients can, therefore, be used to estimate their native coronal alignment and plan an individualized knee alignment. LEVEL OF CLINICAL EVIDENCE: III.

Change of tibial tuberosity-trochlear groove (TT-TG) distance during total knee arthroplasty had no influence on clinical outcome and anterior knee pain.

PURPOSE: The main purpose of this study was to determine whether there is a correlation between the change of tibial tuberosity-trochlear groove (TT-TG) distance and clinical outcomes after total knee arthroplasty (TKA). METHODS: A total of 52 knees undergoing TKA due to primary osteoarthritis were included in this retrospective study. All patients had pre- and postoperative CT scans. TT-TG distance was measured by two independent observers and the following alignment parameters were measured: hip-knee ankle angle (HKA), femoral mechanical angle (FMA), tibial mechanical angle (TMA), and posterior condylar angle (PCA). Clinical outcome was assessed using Knee Society Score (KSS) pre- and post-operatively and at a minimum of 12-month follow-up. Evidence of AKP was noted from follow-up reports. Pre- and postoperative scores were compared using a paired Student t-test. Pearson correlations were calculated to assess the influence of TT-TG on clinical outcome and of alignment parameters on the change in TT-TG. TT-TG between patients with and without AKP was compared using unpaired Student's t-test (p < 0.05). RESULTS: Neither the absolute postoperative TT-TG nor the amount of change in TT-TG correlated with the post-operative KSS or the change in KSS. Post-operative TT-TG and change in TT-TG did not differ significantly between patients with and patients without AKP. Only the change in FMA showed a correlation with the change in TT-TG (p = 0.01, r = 0.36). CONCLUSION: Despite a missing correlation between outcomes and TT-TG distance in this study, excessive TT-TG distance should be avoided. Furthermore, surgeons need to be aware that changes in femoral joint line orientation might affect TT-TG distance.

A comparison of femoral component rotation after total knee arthroplasty in Kanekasu radiographs, axial CT slices and 3D reconstructed images.

OBJECTIVE: To compare the posterior condylar angle measured with Kanekasu radiograph and 2D-CT with the gold standard 3D-CT following primary total knee arthroplasty (TKA). METHODS: Eighty-two knees with pain following TKA were included in this retrospective study. Two independent raters measured the anatomical and surgical posterior condylar angles twice on each Kanekasu radiograph and 2D-CT. These measurements were compared against the 3D-CT measurement. The intra- and interrater reliability of the Kanekasu radiograph and 2D-CT and the correlation with 3D-CT were calculated. RESULTS: The intra- and interrater reliability for measurements of the anatomical posterior condyle angle for the Kanekasu radiograph and the 2D-CT were excellent for both raters (0.85-0.92). For the less experienced rater 1, the intrarater reliability was significantly better for 2D-CT than Kanekasu radiograph for measuring both the surgical (p < 0.01) and anatomical posterior condyle angles (p < 0.05). For the experienced rater 2, the intrarater reliability was significantly better for Kanekasu radiograph than 2D-CT for measurement of the surgical posterior condyle angle (p < 0.05). The correlation with 3D-CT is higher in 2D-CT than in Kanekasu radiograph (p < 0.01). While the Kanekasu radiograph predicts the 3D-CT angle with 65.9%, 2D-CT can measure the true angle with 82.9% certainty. CONCLUSION: Measurements using the anatomical transepicondylar axis are easier to replicate compared to the surgical transepicondylar axis. In comparison with the gold standard 3D-CT, 2D-CT showed a significantly higher correlation with 3D-CT than the Kanekasu measurements. If 3D-CT is available, it should be preferred over 2D-CT and Kanekasu view radiograph for femoral component rotation measurements.

Typical MRI-pattern suggests peak maturation of the ACI graft 2 years after third-generation ACI: a systematic review.

PURPOSE: The purpose of the present article was (1) to systematically review the current literature and (2) to collect data regarding the postoperative magnetic resonance imaging (MRI) appearance of third-generation autologous chondrocyte implantation (ACI) grafts and (3) to provide an overview of imaging findings at various postoperative time points. METHODS: A systematic review of the literature in Medline (Pubmed) and Embase was performed using the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Articles which reported the post-operative MRI morphological outcomes following the use of third-generation ACI for treatment of knee cartilage lesions were included. All MRI results were allocated to six different time intervals: ≤ 3 months, > 3-6 months, > 6 months-1 year, > 1 year-2 years, > 2-5 years and > 5 years after surgery. RESULTS: A total of 22 studies were included and the study populations ranged from 13 to 180 patients adding up to a total of 951 patients. Parameters such as defect fill, border integration, surface contour, graft morphology and integrity of the subchondral lamina all improve gradually with a peak two years following surgery suggesting complete graft maturation at this time point. After this peak, a statistically insignificant decline is noted for most of the parameters. Signal intensity was found to gradually shift from hyperintense to isointense in the first 36 months and to hypointense later on. Contrarily, subchondral bone edema is not only a postoperative feature of the procedure but also can reappear or persist up to ten years after surgery. As graft failures can appear after two years, consequently, the MRI composite score is also affected. CONCLUSION: Recurring patterns in postoperative MRI appearance were observed in certain parameters including defect filling, graft signal intensity and structure, border integration of the graft while parameters like subchondral bone tend to be unpredictable. Given the heterogenous findings in terms of clinical correlation, and relating that aspect to the patterns found in this review, an MRI is justified at three months, one year, two years and five years after surgery, unless the clinical symptomatology and individual patient needs dictate otherwise. LEVEL OF EVIDENCE: IV.

The effect of lumbar puncture on the neurodegeneration biomarker neurofilament light in macaque monkeys.

INTRODUCTION: Neurofilament light (NFL) in cerebrospinal fluid (CSF) is elevated in neurodegenerative disease patients, and may track disease progression and treatment. Macaque monkeys are emerging as important translational models of neurodegeneration, and NFL may be a useful biomarker. METHODS: To determine the influence of a previous lumbar puncture (LP) on NFL, we collected CSF at multiple time points in macaque monkeys via LP or cisterna magna puncture. NFL, amyloid beta (Aß40, Aß42), and tau (tTau, pTau) in CSF were measured by standard enzyme-linked immunosorbent assay and multiplex. RESULTS: NFL was significantly elevated at 14 to 23 days after an LP (median increase: 162%). Aß and tau biomarkers remained stable. NFL peaked and decayed over 1 to 2 months after LP. NFL was not elevated after cisterna magna puncture. DISCUSSION: Results suggest damage of the cauda equina during LP may increase NFL. Caution should be taken in interpreting NFL concentration in studies in which repeat LPs are performed.

Prescribers' experiences of, and attitudes to, use of morphine for palliative care at a tertiary hospital in Zambia.

OBJECTIVE: To explore medical doctors' experiences of, and attitudes to, use of morphine for palliative care at a tertiary hospital in Zambia. METHODS: A qualitative, exploratory case study was undertaken. Semi-structured interviews were used to collect data from 14 medical doctors working in the fields of oncology, pediatrics, and internal medicine at a tertiary hospital in Lusaka, Zambia, regarding their experiences and attitudes to prescribing morphine for palliative care. Thematic analysis of interview transcripts was carried out to establish common themes in the data. The study was approved by BSMS and UNZA research ethics committees. RESULTS: All participants agreed that doctors were becoming more comfortable with the prescribing of morphine, although experiences were notably different for doctors working in oncology, compared to other departments. Themes of difficulty discussing end-of-life, poor recognition of pain, and fear of patient addiction, were more prominent in the responses of non-cancer doctors. Morphine use was generally restricted to cancer and sickle cell disease patients, with most non-cancer doctors stating that they rarely prescribe morphine for outpatient use. Training in pain management and the presence of a palliative care team were perceived to be facilitators to morphine prescribing. CONCLUSIONS: Although there is an increased willingness to prescribe morphine, limited knowledge of pain management, especially for nonmalignant disease, underlies many of the findings in this study. Opportunity exists for professional development in pain management to further improve the acceptance and use of opioids in palliative care, especially for out-patients.

Drivers of Public Attitudes towards Small Wind Turbines in the UK.

Small Wind Turbines (SWTs) are a growing micro-generation industry with over 870,000 installed units worldwide. No research has focussed on public attitudes towards SWTs, despite evidence the perception of such attitudes are key to planning outcomes and can be a barrier to installations. Here we present the results of a UK wide mail survey investigating public attitudes towards SWTs. Just over half of our respondents, who were predominantly older, white males, felt that SWTs were acceptable across a range of settings, with those on road signs being most accepted and least accepted in hedgerows and gardens. Concern about climate change positively influenced how respondents felt about SWTs. Respondent comments highlight visual impacts and perceptions of the efficiency of this technology are particularly important to this sector of the UK public. Taking this into careful consideration, alongside avoiding locating SWTs in contentious settings such as hedgerows and gardens where possible, may help to minimise public opposition to proposed installations.

Genotypic Diversity Is Associated with Clinical Outcome and Phenotype in Cryptococcal Meningitis across Southern Africa.

Cryptococcal meningitis is a major cause of mortality throughout the developing world, yet little is known about the genetic markers underlying Cryptococcal virulence and patient outcome. We studied a cohort of 230 Cryptococcus neoformans (Cn) isolates from HIV-positive South African clinical trial patients with detailed clinical follow-up using multi-locus sequence typing and in vitro phenotypic virulence assays, correlating these data with clinical and fungal markers of disease in the patient. South African Cn displayed high levels of genetic diversity and locus variability compared to globally distributed types, and we identified 50 sequence types grouped within the main molecular types VNI, VNII and VNB, with 72% of isolates typed into one of seven 'high frequency' sequence types. Spatial analysis of patients' cryptococcal genotype was not shown to be clustered geographically, which might argue against recent local acquisition and in favour of reactivation of latent infection. Through comparison of MLST genotyping data with clinical parameters, we found a relationship between genetic lineage and clinical outcome, with patients infected with the VNB lineage having significantly worse survival (n=8, HR 3.35, CI 1.51-7.20, p=0.003), and this was maintained even after adjustment for known prognostic indicators and treatment regimen. Comparison of fungal genotype with in vitro phenotype (phagocytosis, laccase activity and CSF survival) performed on a subset of 89 isolates revealed evidence of lineage-associated virulence phenotype, with the VNII lineage displaying increased laccase activity (p=0.001) and ex vivo CSF survival (p=0.0001). These findings show that Cryptococcus neoformans is a phenotypically heterogeneous pathogen, and that lineage plays an important role in cryptococcal virulence during human infection. Furthermore, a detailed understanding of the genetic diversity in Southern Africa will support further investigation into how genetic diversity is structured across African environments, allowing assessment of the risks different ecotypes pose to infection.

Microrheology with optical tweezers: measuring the relative viscosity of solutions 'at a glance'.

We present a straightforward method for measuring the relative viscosity of fluids via a simple graphical analysis of the normalised position autocorrelation function of an optically trapped bead, without the need of embarking on laborious calculations. The advantages of the proposed microrheology method are evident when it is adopted for measurements of materials whose availability is limited, such as those involved in biological studies. The method has been validated by direct comparison with conventional bulk rheology methods, and has been applied both to characterise synthetic linear polyelectrolytes solutions and to study biomedical samples.

Efficient phagocytosis and laccase activity affect the outcome of HIV-associated cryptococcosis.

BACKGROUND: Cryptococcal meningitis (CM) is a leading cause of HIV-associated mortality globally. High fungal burden in cerebrospinal fluid (CSF) at diagnosis and poor fungal clearance during treatment are recognized adverse prognostic markers; however, the underlying pathogenic factors that drive these clinical manifestations are incompletely understood. We profiled a large set of clinical isolates for established cryptococcal virulence traits to evaluate the contribution of C. neoformans phenotypic diversity to clinical presentation and outcome in human cryptococcosis. METHODS: Sixty-five C. neoformans isolates from clinical trial patients with matched clinical data were assayed in vitro to determine murine macrophage uptake, intracellular proliferation rate (IPR), capsule induction, and laccase activity. Analysis of the correlation between prognostic clinical and host immune parameters and fungal phenotypes was performed using Spearman's r, while the fungal-dependent impact on long-term survival was determined by Cox regression analysis. RESULTS: High levels of fungal uptake by macrophages in vitro, but not the IPR, were associated with CSF fungal burden (r = 0.38, P = 0.002) and long-term patient survival (hazard ratio [HR] 2.6, 95% CI 1.2-5.5, P = 0.012). High-uptake strains were hypocapsular (r = -0.28, P = 0.05) and exhibited enhanced laccase activity (r = 0.36, P = 0.003). Fungal isolates with greater laccase activity exhibited heightened survival ex vivo in purified CSF (r = 0.49, P < 0.0001) and resistance to clearance following patient antifungal treatment (r = 0.39, P = 0.003). CONCLUSION: These findings underscore the contribution of cryptococcal-phagocyte interactions and laccase-dependent melanin pathways to human clinical presentation and outcome. Furthermore, characterization of fungal-specific pathways that drive clinical manifestation provide potential targets for the development of therapeutics and the management of CM. FUNDING: This work was made possible by funding from the Wellcome Trust (WT088148MF), the Medical Research Council (MR/J008176/1), the NIHR Surgical Reconstruction and Microbiology Research Centre and the Lister Institute for Preventive Medicine (to R.C. May), and a Wellcome Trust Intermediate fellowship (089966, to T. Bicanic). The C. neoformans isolates were collected within clinical trials funded by the British Infection Society (fellowship to T. Bicanic), the Wellcome Trust (research training fellowships WT069991, to A.E. Brouwer and WT081794, to J.N. Jarvis), and the Medical Research Council, United Kingdom (76201). The funding sources had no role in the design or conduct of this study, nor in preparation of the manuscript.

Cryptococcus neoformans ex vivo capsule size is associated with intracranial pressure and host immune response in HIV-associated cryptococcal meningitis.

BACKGROUND: The Cryptococcus neoformans polysaccharide capsule is a well-characterized virulence factor with immunomodulatory properties. The organism and/or shed capsule is postulated to raise intracranial pressure (ICP) in cryptococcal meningitis (CM) by mechanical obstruction of cerebrospinal fluid (CSF) outflow. Little is known regarding capsule phenotype in human cryptococcosis. We investigated the relationship of ex vivo CSF capsular phenotype with ICP and CSF immune response, as well as in vitro phenotype. METHODS: In total, 134 human immunodeficiency virus (HIV)-infected Ugandan adults with CM had serial lumbar punctures with measurement of CSF opening pressures, quantitative cultures, ex vivo capsule size and shedding, viscosity, and CSF cytokines; 108 had complete data. Induced capsular size and shedding were measured in vitro for 48 C. neoformans isolates. RESULTS: Cryptococcal strains producing larger ex vivo capsules in the baseline (pretreatment) CSF correlated with higher ICP (P = .02), slower rate of fungal clearance (P = .02), and paucity of CSF inflammation, including decreased CSF white blood cell (WBC) count (P < .001), interleukin (IL)-4 (P = .02), IL-6 (P = .01), IL-7 (P = .04), IL-8 (P = .03), and interferon γ (P = .03). CSF capsule shedding did not correlate with ICP. On multivariable analysis, capsule size remained independently associated with ICP. Ex vivo capsular size and shedding did not correlate with that of the same isolates grown in vitro. CONCLUSIONS: Cryptococcal capsule size ex vivo is an important contributor to virulence in human cryptococcal meningitis.

EDTA inhibits biofilm formation, extracellular vesicular secretion, and shedding of the capsular polysaccharide glucuronoxylomannan by Cryptococcus neoformans.

The fungal pathogen Cryptococcus neoformans can grow as a biofilm on a range of synthetic and prosthetic materials. Cryptococcal biofilm formation can complicate the placement of shunts used to relieve increased intracranial pressure in cryptococcal meningitis and can serve as a nidus for chronic infection. Biofilms are generally advantageous to pathogens in vivo, as they can confer resistance to antimicrobial compounds, including fluconazole and voriconazole in the case of C. neoformans. EDTA can inhibit biofilm formation by several microbes and enhances the susceptibility of biofilms to antifungal drugs. In this study, we evaluated the effect of sublethal concentrations of EDTA on the growth of cryptococcal biofilms. EDTA inhibited biofilm growth by C. neoformans, and the inhibition could be reversed by the addition of magnesium or calcium, implying that the inhibitory effect was by divalent cation starvation. EDTA also reduced the amount of the capsular polysaccharide glucuronoxylomannan shed into the biofilm matrix and decreased vesicular secretion from the cell, thus providing a potential mechanism for the inhibitory effect of this cation-chelating compound. Our data imply that the growth of C. neoformans biofilms requires the presence of divalent metals in the growth medium and suggest that cations are required for the export of materials needed for biofilm formation, possibly including extracellular vesicles.

Nonlytic exocytosis of Cryptococcus neoformans from macrophages occurs in vivo and is influenced by phagosomal pH.

UNLABELLED: A unique aspect of the interaction of the fungus Cryptococcus neoformans with macrophages is the phenomenon of nonlytic exocytosis, also referred to as "vomocytosis" or phagosome extrusion/expulsion, which involves the escape of fungal cells from the phagocyte with the survival of both cell types. This phenomenon has been observed only in vitro using subjective and time-consuming microscopic techniques. In spite of recent advances in our knowledge about its mechanisms, a major question still remaining is whether this phenomenon also occurs in vivo. In this study, we describe a novel flow cytometric method that resulted in a substantial gain in throughput for studying phagocytosis and nonlytic exocytosis in vitro and used it to explore the occurrence of this phenomenon in a mouse model of infection. Furthermore, we tested the hypothesis that host cell phagosomal pH affected nonlytic exocytosis. The addition of the weak bases ammonium chloride and chloroquine resulted in a significant increase of nonlytic exocytosis events, whereas the vacuolar ATPase inhibitor bafilomycin A1 had the opposite effect. Although all three agents are known to neutralize phagosomal acidity, their disparate effects suggest that phagosomal pH is an important and complex variable in this process. Our experiments established that nonlytic exocytosis occurred in vivo with a frequency that is possibly much higher than that observed in vitro. These results in turn suggest that nonlytic exocytosis has a potential role in the pathogenesis of cryptococcosis. IMPORTANCE: Cryptococcus neoformans causes disease in people with immune deficiencies such as AIDS. Upon infection, C. neoformans cells are ingested by macrophage immune cells, which provide a niche for survival and replication. After ingestion, macrophages can expel the fungi without causing harm to either cell type, a process named nonlytic exocytosis. To dissect this phenomenon, we evaluated its dependence on the pH inside the macrophage and addressed its occurrence during infection of mice. We developed new techniques using flow cytometry to measure C. neoformans internalization by and nonlytic exocytosis from macrophages. Neutralizing the phagosome acidity changed the rate of nonlytic exocytosis: activity increased with the weak bases chloroquine and ammonium chloride, whereas the vacuolar ATPase inhibitor bafilomycin A1 caused it to decrease. Experiments in mice suggested that nonlytic exocytosis occurred during infection with C. neoformans. These results shed new light on the interaction between C. neoformans and host macrophages.

The putative RxLR effector protein SpHtp1 from the fish pathogenic oomycete Saprolegnia parasitica is translocated into fish cells.

The fish pathogenic oomycete Saprolegnia parasitica causes the disease Saprolegniosis in salmonids and other freshwater fish, resulting in considerable economic losses in aquaculture. Very little is known about the molecular and cellular mechanisms underlying the infection process of fish pathogenic oomycetes. In order to investigate the interaction in detail, an in vitro infection assay using an Oncorhynchus mykiss (rainbow trout) cell line (RTG-2) was developed. In a zoospore/cyst cDNA library, we identified the ORF SpHtp1, which encodes a secreted protein containing an RxLR motif. Detailed expression analysis indicated that SpHtp1 is highly expressed in zoospores/cysts from S. parasitica and in the very early stages of infection on RTG-2 cells, when compared with in vitro-grown mycelium. Moreover, the protein, SpHtp1, was found to translocate into the RTG-2 trout cells, during the interaction with S. parasitica, and also when the RTG-2 cells were treated with recombinant SpHtp1 fused to a C-terminal His-tag. These findings suggest that protein translocation could play an important role in Saprolegniosis.