A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction.

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis. Detection of these contact sites at nanometer scale over time in living cells is challenging. Here, we developed a tool kit for detecting contact sites based on Fluorogen-Activated Bimolecular complementation at CONtact sites, FABCON, using a reversible, low affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic switch.

Female Sex but Not Oestrogen Receptor Expression Predicts Survival in Advanced Gastroesophageal Adenocarcinoma-A Post-hoc Analysis of the GO2 Trial.

Gastroesophageal adenocarcinoma is a disease of older adults that is associated with a very poor prognosis. It is less common and has better outcomes in females. The reason for this is unknown but may relate to signalling via the main oestrogen receptors (ER) α and ß. In this study, we sought to investigate this using the GO2 clinical trial patient cohort. GO2 recruited older and/or frail patients with advanced gastroesophageal cancer. Immunohistochemistry was performed on tumour samples from 194 patients. The median age of the population was 76 years (range 52-90), and 25.3% were female. Only one (0.5%) tumour sample was positive for ERα, compared to 70.6% for ERß expression. There was no survival impact according to ERß expression level. Female sex and younger age were associated with lower ERß expression. Female sex was also associated with improved overall survival. To our knowledge, this is the largest study worldwide of ER expression in a cohort of patients with advanced gastroesophageal adenocarcinoma. It is also unique, given the age of the population. We have demonstrated that female sex is associated with better survival outcomes with palliative chemotherapy but that this does not appear to be related to ER IHC expression. The differing ER expression according to age supports the concept of a different disease biology with age.

A Qualitative Study to Develop and Evaluate the Content Validity of the Vitiligo Patient Priority Outcome (ViPPO) Measures.

INTRODUCTION: Vitiligo can be associated with a psychological burden, stigmatization and impaired quality of life. Tools to assess the impact of vitiligo exist; however, none were developed in line with the FDA's patient-reported outcome (PRO) Guidance for Industry. This study aimed to explore the content validity of two newly developed PRO measures to assess the impact of facial and total body vitiligo on how patients feel and function. METHODS: Draft PRO measures were developed from existing literature and input from PRO experts, a patient advocate and a clinical expert. Qualitative interviews were conducted with US participants living with vitiligo and international dermatologists with vitiligo expertise. Concept elicitation methodology explored the relevance of concepts in the draft PRO, while cognitive debriefing assessed conceptual relevance and understanding/interpretation. Items were iteratively amended/added throughout the interview study. RESULTS: The 60 participants included adults (n = 48, 63% female, 18-62 years old) and adolescents (n = 12, 67% female, 12-17 years old) with Fitzpatrick Skin Types I-VI. Expert dermatologists from the US (n = 8), EU (n = 4), India (n = 1) and Egypt (n = 1) participated. Concept elicitation was utilized to confirm the signs/symptoms of vitiligo and the associated impact on emotional/psychological wellbeing, social functioning, daily life and work/school. Conceptual saturation was achieved. Most participants reported impacts on their emotional/psychological wellbeing (n = 57, 95%), e.g. feeling self-conscious (n = 35, 58%). Participants reported impacts on social functioning (n = 53, 88%), e.g. vitiligo being noticed by others (n = 42, 70%). There was general consensus between participants and expert dermatologists. Cognitive debriefing confirmed that the items were well understood. Most items were conceptually relevant; feeling self-conscious and feeling frustrated were highly endorsed. Items were removed based on low conceptual relevance (feeling abandoned, skin roughness) and expected redundancy (four items), resulting in two measures with three proposed domain scores: Emotional/Psychological Wellbeing; Social Functioning; and Physical Sensation. No comprehension concerns were observed in relation to the 7-day recall period or the item response scale/options. Eight dermatologists reviewed the PRO measures, confirming comprehensiveness and relevance. CONCLUSION: The draft Vitiligo Patient Priority Outcomes (ViPPO) measures evaluate the impact of facial (ViPPO-F) and total body (ViPPO-T) vitiligo on emotional/psychological and social functioning. The ViPPO measures are well understood, comprehensive and content valid for adults and adolescents with vitiligo.

Management of CLN1 Disease: International Clinical Consensus.

BACKGROUND: CLN1 disease (neuronal ceroid lipofuscinosis type 1) is a rare, genetic, neurodegenerative lysosomal storage disorder caused by palmitoyl-protein thioesterase 1 (PPT1) enzyme deficiency. Clinical features include developmental delay, psychomotor regression, seizures, ataxia, movement disorders, visual impairment, and early death. In general, the later the age at symptom onset, the more protracted the disease course. We sought to evaluate current evidence and to develop expert practice consensus to support clinicians who have not previously encountered patients with this rare disease. METHODS: We searched the literature for guidelines and evidence to support clinical practice recommendations. We surveyed CLN1 disease experts and caregivers regarding their experiences and recommendations, and a meeting of experts was conducted to ascertain points of consensus and clinical practice differences. RESULTS: We found a limited evidence base for treatment and no clinical management guidelines specific to CLN1 disease. Fifteen CLN1 disease experts and 39 caregivers responded to the surveys, and 14 experts met to develop consensus-based recommendations. The resulting management recommendations are uniquely informed by family perspectives, due to the inclusion of caregiver and advocate perspectives. A family-centered approach is supported, and individualized, multidisciplinary care is emphasized in the recommendations. Ascertainment of the specific CLN1 disease phenotype (infantile-, late infantile-, juvenile-, or adult-onset) is of key importance in informing the anticipated clinical course, prognosis, and care needs. Goals and strategies should be periodically reevaluated and adapted to patients' current needs, with a primary aim of optimizing patient and family quality of life.

Serosurvey for Brucella spp. and Coxiella burnetii in animals on Caribbean islands.

To determine the prevalence of antibodies to Brucella melitensis, Brucella abortus and Coxiella burnetii in animals on Caribbean islands we obtained sera from convenience samples of cattle (C), sheep (S), goats (G) and cats (F) from Dominica (C, S, G), Grenada (C, S, G), Montserrat (C, S, G), Puerto Rico (C), Nevis (C, S, G), St Kitts (C, S, G, F) and St Lucia (C, G). The sera were tested for antibodies against the Brucella spp. using commercial ELISA kits. Some sera were also tested at 1/80 for antibodies to C. burnetii using an indirect fluorescent antibody test. Positive sera were also tested at 1/640. None of 599 cattle, 462 sheep or 434 goats were positive in the Brucella ELISAs. None of 230 cattle had antibodies against C. burnetii, but one of 299 sheep was positive at 1/80 (Dominica - 1/54, 2%, 95% CI (0%-5.6%)), as were two of 314 goats, at 1/80 (Grenada - 1/53, 2%, 95% CI (0%-7.5%)) and 1/640 (St Kitts - 1/18, 5.6%, 95% CI (0%-16.7%)), and one of 34 cats, at 1/80 (St Kitts - 1/34; 3%, 95% CI (0%-8.8%)). Our data suggests that there is a very low prevalence or absence of B. melitensis and B. abortus on Caribbean islands. Coxiella burnetii, however, is present but it appears to be present on only some islands and then only at low levels. Overall, there appears to be a low threat to human and animal health from these organisms in the Caribbean.

DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome.

The cellular stress response has a vital role in regulating homeostasis by modulating cell survival and death. Stress granules are cytoplasmic compartments that enable cells to survive various stressors. Defects in the assembly and disassembly of stress granules are linked to neurodegenerative diseases, aberrant antiviral responses and cancer1-5. Inflammasomes are multi-protein heteromeric complexes that sense molecular patterns that are associated with damage or intracellular pathogens, and assemble into cytosolic compartments known as ASC specks to facilitate the activation of caspase-1. Activation of inflammasomes induces the secretion of interleukin (IL)-1ß and IL-18 and drives cell fate towards pyroptosis-a form of programmed inflammatory cell death that has major roles in health and disease6-12. Although both stress granules and inflammasomes can be triggered by the sensing of cellular stress, they drive contrasting cell-fate decisions. The crosstalk between stress granules and inflammasomes and how this informs cell fate has not been well-studied. Here we show that the induction of stress granules specifically inhibits NLRP3 inflammasome activation, ASC speck formation and pyroptosis. The stress granule protein DDX3X interacts with NLRP3 to drive inflammasome activation. Assembly of stress granules leads to the sequestration of DDX3X, and thereby the inhibition of NLRP3 inflammasome activation. Stress granules and the NLRP3 inflammasome compete for DDX3X molecules to coordinate the activation of innate responses and subsequent cell-fate decisions under stress conditions. Induction of stress granules or loss of DDX3X in the myeloid compartment leads to a decrease in the production of inflammasome-dependent cytokines in vivo. Our findings suggest that macrophages use the availability of DDX3X to interpret stress signals and choose between pro-survival stress granules and pyroptotic ASC specks. Together, our data demonstrate the role of DDX3X in driving NLRP3 inflammasome and stress granule assembly, and suggest a rheostat-like mechanistic paradigm for regulating live-or-die cell-fate decisions under stress conditions.

Performance evaluation of extended depth of field microscopy in the presence of spherical aberration and noise.

Effectiveness of extended depth of field microscopy (EDFM) implementation with wavefront encoding methods is reduced by depth-induced spherical aberration (SA) due to reliance of this approach on a defined point spread function (PSF). Evaluation of the engineered PSF's robustness to SA, when a specific phase mask design is used, is presented in terms of the final restored image quality. Synthetic intermediate images were generated using selected generalized cubic and cubic phase mask designs. Experimental intermediate images were acquired using the same phase mask designs projected from a liquid crystal spatial light modulator. Intermediate images were restored using the penalized space-invariant expectation maximization and the regularized linear least squares algorithms. In the presence of depth-induced SA, systems characterized by radially symmetric PSFs, coupled with model-based computational methods, achieve microscope imaging performance with fewer deviations in structural fidelity (e.g., artifacts) in simulation and experiment and 50% more accurate positioning of 1-µm beads at 10-µm depth in simulation than those with radially asymmetric PSFs. Despite a drop in the signal-to-noise ratio after processing, EDFM is shown to achieve the conventional resolution limit when a model-based reconstruction algorithm with appropriate regularization is used. These trends are also found in images of fixed fluorescently labeled brine shrimp, not adjacent to the coverslip, and fluorescently labeled mitochondria in live cells.

Reducing effects of aberration in 3D fluorescence imaging using wavefront coding with a radially symmetric phase mask.

In this work, a wavefront encoded (WFE) imaging system built using a squared cubic phase mask, designed to reduce the sensitivity of the imaging system to spherical aberration, is investigated. The proposed system allows the use of a space-invariant image restoration algorithm, which uses a single PSF, to restore intensity distribution in images suffering aberration, such as sample-induced aberration in thick tissue. This provides a computational advantage over depth-variant image restoration algorithms developed previously to address this aberration. Simulated PSFs of the proposed system are shown to change up to 25% compared to the 0 µm depth PSF (quantified by the structural similarity index) over a 100 µm depth range, while the conventional system PSFs change up to 84%. Results from experimental test-sample images show that restoration error is reduced by 29% when the proposed WFE system is used instead of the conventional system over a 30 µm depth range.

Obstructive Sleep Apnea as an Independent Stroke Risk Factor: A Review of the Evidence, Stroke Prevention Guidelines, and Implications for Neuroscience Nursing Practice.

BACKGROUND: Stroke is a leading cause of death and disability affecting nearly 800,000 people in the United States every year. Obstructive sleep apnea (OSA) is found in over 60% of patients with stroke/transient ischemic attack (TIA) and identified as an independent stroke risk factor in large epidemiology studies and Canadian Stroke Prevention Guidelines (SPG) but not in the United States. The 2014 Secondary SPG recommend OSA screening and treatment as a consideration only, not a requirement. The twofold purpose of this article is, first, to present the evidence supporting OSA as an independent stroke risk factor in national SPG with mandatory recommendations and, second, to engage neuroscience nurses to incorporate OSA assessment and interventions into the nursing process and thereby promote excellence in stroke/TIA patient care. METHODS: A systematic literature search was conducted in Medline, CINAHL, and PubMed to identify research from 2003 through 2013 on the independent risk, mortality, and prevalence relationship between OSA and stroke/TIA including recurrence and recovery outcomes with continuous positive airway pressure (CPAP) therapy. RESULTS: Twenty-eight research articles were reviewed: 14 observational cohorts, five case-control studies, four cross-sectional studies, and four randomized control trials representing 12 countries and 10,671 subjects. DISCUSSION: OSA is highly prevalent in patients with stroke/TIA independently increasing stroke risk. CPAP studies revealed reduced stroke recurrence and improved recovery with feasible initiation in stroke units. Patients with stroke/TIA have less OSA-associated daytime sleepiness and obesity, making the usual screening tools insufficient and CPAP adherence challenging. Treating OSA decreases stroke prevalence and mortality. OSA initiatives empower neuroscience nurses to integrate this OSA evidence into clinical practice and improve stroke/TIA patient outcomes.

Spatial light modulator phase mask implementation of wavefront encoded 3D computational-optical microscopy.

Spatial light modulator (SLM) implementation of wavefront encoding enables various types of engineered point-spread functions (PSFs), including the generalized-cubic and squared-cubic phase mask wavefront encoded (WFE) PSFs, shown to reduce the impact of sample-induced spherical aberration in fluorescence microscopy. This investigation validates dynamic experimental parameter variation of these WFE-PSFs. We find that particular design parameter bounds exist, within which the divergence of computed and experimental WFE-PSFs is of the same order of magnitude as that of computed and experimental conventional PSFs, such that model-based approaches for solving the inverse imaging problem can be applied to a wide range of SLM-WFE systems. Interferometric measurements were obtained to evaluate the SLM implementation of the numeric mask. Agreement between experiment and theory in terms of a wrapped phase, 0-2π, validates the phase mask implementation and allows characterization of the SLM response. These measurements substantiate experimental practice of computational-optical microscope imaging with an SLM-engineered PSF.

Evidence for aldosterone-dependent growth of renal cell carcinoma.

The aim if this study was to investigate the hypothesis that K-RAS 4A is upregulated in a mineralocorticoid-dependent manner in renal cell carcinoma and that this supports the proliferation and survival of some renal cancers. Expression of the K-RAS in renal tumour tissues and cell lines was examined by real-time PCR and Western blot and mineralocorticoid receptor, and its gatekeeper enzyme 11ß-hydroxysteroid dehydrogenase-2 was examined by immunocytochemistry on a tissue microarray of 27 cases of renal cell carcinoma. Renal cancer cells lines 04A018 (RCC4 plus VHL) and 04A019 (RCC4 plus vector alone) were examined for the expression of K-RAS4A and for the effect on K-RAS expression of spironolactone blockade of the mineralocorticoid receptor. K-RAS4A was suppressed by siRNA, and the effect on cell survival, proliferation and activation of the Akt and Raf signalling pathways was investigated in vitro. K-RAS4A was expressed in RCC tissue and in the renal cancer cell lines but K-RAS was downregulated by spironolactone and upregulated by aldosterone. Spironolactone treatment and K-RAS suppression both led to a reduction in cell number in vitro. Both Akt and Raf pathways showed activation which was dependent on K-RAS expression. K-RAS expression in renal cell carcinoma is at least partially induced by aldosterone. Aldosterone supports the survival and proliferation of RCC cells by upregulation of K-RAS acting through the Akt and Raf pathways.

Immunohistochemical detection of Polo-like kinase-1 (PLK1) in primary breast cancer is associated with TP53 mutation and poor clinical outcom.

INTRODUCTION: Polo-like kinase-1 (PLK1) is a crucial driver of cell cycle progression and its down-regulation plays an important checkpoint role in response to DNA damage. Mechanistically, this is mediated by p53 which represses PLK1 expression through chromatin remodelling. Consistent with this model, cultured cells lacking p53 fail to repress PLK1 expression. This study examined PLK1 expression, p53 mutation and clinical outcome in breast cancer. METHODS: Immunohistochemistry was performed using antibodies to PLK1, MDM2 and Ki67 on Tissue Micro-Array (TMA) slides of a cohort of 215 primary breast cancers. The TP53 gene (encoding p53) was sequenced in all tumour samples. Protein expression scored using the "Quickscore" method was compared with clinical and pathological data, including survival. RESULTS: Staining of PLK1 was observed in 11% of primary breast tumours and was significantly associated with the presence of TP53 mutation (P = 0.0063). Moreover, patients with both PLK1 expression and TP53 mutation showed a significantly worse survival than those with either PLK1 expression or TP53 mutation alone. There was also a close association of elevated PLK1 with triple negative tumours, considered to be poor prognosis breast cancers that generally harbour TP53 mutation. Further association was observed between elevated PLK1 levels and the major p53 negative regulator, MDM2. CONCLUSIONS: The significant association between elevated PLK1 and TP53 mutation in women with breast cancer is consistent with escape from repression of PLK1 expression by mutant p53. Tumours expressing elevated PLK1, but lacking functional p53, may be potential targets for novel anti-PLK1-targeted drugs.

International seroepidemiology of adenovirus serotypes 5, 26, 35, and 48 in pediatric and adult populations.

Recombinant adenovirus serotype 5 (rAd5) vaccine vectors for HIV-1 and other pathogens have been shown to be limited by high titers of Ad5 neutralizing antibodies (NAbs) in the developing world. Alternative serotype rAd vectors have therefore been constructed. Here we report Ad5, Ad26, Ad35, and Ad48 NAb titers in 4381 individuals from North America, South America, sub-Saharan Africa, and Southeast Asia. As expected, Ad5 NAb titers were both frequent and high magnitude in sub-Saharan Africa and Southeast Asia. In contrast, Ad35 NAb titers proved infrequent and low in all regions studied, and Ad48 NAbs were rare in all regions except East Africa. Ad26 NAbs were moderately common in adults in sub-Saharan Africa and Southeast Asia, but Ad26 NAb titers proved markedly lower than Ad5 NAb titers in all regions, and these relatively low Ad26 NAb titers did not detectably suppress the immunogenicity of 4×10(10)vp of a rAd26-Gag/Pol/Env/Nef vaccine in rhesus monkeys. These data inform the clinical development of alternative serotype rAd vaccine vectors in the developing world.

p53-dependent repression of polo-like kinase-1 (PLK1).

PLK1 is a critical mediator of G2/M cell cycle transition that is inactivated and depleted as part of the DNA damage-induced G2/M checkpoint. Here we show that downregulation of PLK1 expression occurs through a transcriptional repression mechanism and that p53 is both necessary and sufficient to mediate this effect. Repression of PLK1 by p53 occurs independently of p21 and of arrest at G1/S where PLK1 levels are normally repressed in a cell cycle-dependent manner through a CDE/CHR element. Chromatin immunoprecipitation analysis indicates that p53 is present on the PLK1 promoter at two distinct sites termed p53RE1 and p53RE2. Recruitment of p53 to p53RE2, but not to p53RE1, is stimulated in response to DNA damage and/or p53 activation and is coincident with repression-associated changes in the chromatin. Downregulation of PLK1 expression by p53 is relieved by the histone deacetylase inhibitor, trichostatin A, and involves recruitment of histone deacetylase to the vicinity of p53RE2, further supporting a transcriptional repression mechanism. Additionally, wild type, but not mutant, p53 represses expression of the PLK1 promoter when fused upstream of a reporter gene. Silencing of PLK1 expression by RNAi interferes with cell cycle progression consistent with a role in the p53-mediated checkpoint. These data establish PLK1 as a direct transcriptional target of p53, independently of p21, that is required for efficient G2/M arrest.

Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys.

The worldwide diversity of HIV-1 presents an unprecedented challenge for vaccine development. Antigens derived from natural HIV-1 sequences have elicited only a limited breadth of cellular immune responses in nonhuman primate studies and clinical trials to date. Polyvalent 'mosaic' antigens, in contrast, are designed to optimize cellular immunologic coverage of global HIV-1 sequence diversity. Here we show that mosaic HIV-1 Gag, Pol and Env antigens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors markedly augmented both the breadth and depth without compromising the magnitude of antigen-specific T lymphocyte responses as compared with consensus or natural sequence HIV-1 antigens in rhesus monkeys. Polyvalent mosaic antigens therefore represent a promising strategy to expand cellular immunologic vaccine coverage for genetically diverse pathogens such as HIV-1.

Pathways to Assisted Living: The Influence of Race and Class.

This article examines how race and class influence decisions to move to assisted living facilities. Qualitative methods were used to study moving decisions of residents in 10 assisted living facilities varying in size and location, as well as race and socioeconomic status of residents. Data were derived from in-depth interviews with 60 residents, 43 family members and friends, and 12 administrators. Grounded theory analysis identified three types of residents based on their decision-making control: proactive, compliant, and passive/resistant. Only proactive residents (less than a quarter of residents) had primary control. Findings show that control of decision making for elders who are moving to assisted living is influenced by class, though not directly by race. The impact of class primarily related to assisted-living placement options and strategies available to forestall moves. Factors influencing the decision-making process were similar for Black and White elders of comparable socioeconomic status.

Adenovirus-specific immunity after immunization with an Ad5 HIV-1 vaccine candidate in humans.

The immunologic basis for the potential enhanced HIV-1 acquisition in adenovirus serotype 5 (Ad5)-seropositive individuals who received the Merck recombinant Ad5 HIV-1 vaccine in the STEP study remains unclear. Here we show that baseline Ad5-specific neutralizing antibodies are not correlated with Ad5-specific T lymphocyte responses and that Ad5-seropositive subjects do not develop higher vector-specific cellular immune responses as compared with Ad5-seronegative subjects after vaccination. These findings challenge the hypothesis that activated Ad5-specific T lymphocytes were the cause of the potential enhanced HIV-1 susceptibility in the STEP study.