Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake

Adenovirus type 2 (Ad2) binds the coxsackie B virus Ad receptor and is endocytosed upon activation of the alphav integrin coreceptors. Here, we demonstrate that expression of dominant negative clathrin hub, eps15, or K44A-dynamin (dyn) inhibited Ad2 uptake into epithelial cells, indicating clathrin-dependent viral endocytosis. Surprisingly, Ad strongly stimulated the endocytic uptake of fluid phase tracers, coincident with virus internalization but without affecting receptor-mediated transferrin uptake. A large amount of the stimulated endocytic activity was macropinocytosis. Macropinocytosis depended on alphav integrins, PKC, F-actin, and the amiloride-sensitive Na+/H+ exchanger, which are all required for Ad escape from endosomes and infection. Macropinocytosis stimulation was not a consequence of viral escape, since it occurred in K44A-dyn-expressing cells. Surprisingly, 30-50% of the endosomal contents were released into the cytosol of control and also K44A-dyn-expressing cells, and the number of fluid phase-positive endosomes dropped below the levels of noninfected cells, indicating macropinosomal lysis. The release of macropinosomal contents was Ad dose dependent, but the presence of Ad particles on macropinosomal membranes was not sufficient for contents release. We conclude that Ad signaling from the cell surface controls the induction of macropinosome formation and leakage, and this correlates with viral exit to the cytosol and infection.     

Anti-mouse GPI-PLD antisera highlight structural differences between murine and bovine GPI-PLDs

Despite its well characterised biochemistry, the physiological role of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is unknown. Most of the previous studies investigating the distribution of GPI-PLD have focused on the human and bovine forms of the enzyme. Studies on mouse GPI-PLD are rare, partly due to the lack of a specific anti-mouse GPI-PLD antibody, but also due to the apparent low reactivity of existing antibodies to rodent GPI-PLDs. Here we describe the isolation of a mouse liver cDNA, the construction and expression of a recombinant enzyme and the generation of an affinity-purified rabbit anti-mouse GPI-PLD antiserum. The antibody shows good reactivity to partially purified murine and purified bovine GPI-PLD. In contrast, a rat anti-bovine GPI-PLD antibody shows no reactivity with the mouse enzyme and the two antibodies recognise different proteolytic fragments of the bovine enzyme. Comparison between the rodent, bovine and human enzymes indicates that small changes in the amino acid sequence of a short peptide in the mouse and bovine GPI-PLDs may contribute to the different reactivities of the two antisera. We discuss the implications of these results and stress the importance of antibody selection while investigating GPI-PLD in the mouse.     

A gimbal-mounted pressurization chamber for macroscopic and microscopic assessment of ocular tissues

The biomechanical model of glaucoma considers intraocular pressure-related stress and resultant strain on load bearing connective tissues of the optic nerve and surrounding peripapillary sclera as one major causative influence that effects cellular, vascular, and axonal components of the optic nerve. By this reasoning, the quantification of variations in the microstructural architecture and macromechanical response of scleral shells in glaucomatous compared to healthy populations provides an insight into any variations that exist between patient populations. While scleral shells have been tested mechanically in planar and pressure-inflation scenarios the link between the macroscopic biomechanical response and the underlying microstructure has not been determined to date. A potential roadblock to determining how the microstructure changes based on pressure is the ability to mount the spherical scleral shells in a method that does not induce unwanted stresses to the samples (for instance, in the flattening of the spherical specimens), and then capturing macroscopic and microscopic changes under pressure. Often what is done is a macroscopic test followed by sample fixation and then imaging to determine microstructural organization. We introduce a novel device and method, which allows spherical samples to be pressurized and macroscopic and microstructural behavior quantified on fully hydrated ocular specimens. The samples are pressurized and a series of markers on the surface of the sclera imaged from several different perspectives and reconstructed between pressure points to allow for mapping of nonhomogenous strain. Pictures are taken from different perspectives through the use of mounting the pressurization scheme in a gimbal that allows for positioning the sample in several different spherical coordinate system configurations. This ability to move the sclera in space about the center of the globe, coupled with an upright multiphoton microscope, allows for collecting collagen, and elastin signal in a rapid automated fashion so the entire globe can be imaged.     

Acute psychiatric care: approaches to increasing the range of services and improving access and quality of care

Acute services for mental health crises are very important to service users and their supporters, and consume a substantial share of mental health resources in many countries. However, acute care is often unpopular and sometimes coercive, and the evidence on which models are best for patient experience and outcomes remains surprisingly limited, in part reflecting challenges in conducting studies with people in crisis. Evidence on best ap­proaches to initial assessment and immediate management is particularly lacking, but some innovative models involving extended assessment, brief interventions, and diversifying settings and strategies for providing support are potentially helpful. Acute wards continue to be central in the intensive treatment phase following a crisis, but new approaches need to be developed, evaluated and implemented to reducing coercion, addressing trauma, diversifying treatments and the inpatient workforce, and making decision‐making and care collaborative. Intensive home treatment services, acute day units, and community crisis services have supporting evidence in diverting some service users from hospital admission: a greater understanding of how best to implement them in a wide range of contexts and what works best for which service users would be valuable. Approaches to crisis management in the voluntary sector are more flexible and informal: such services have potential to complement and provide valuable learning for statutory sector services, especially for groups who tend to be underserved or disengaged. Such approaches often involve staff with personal experience of mental health crises, who have important potential roles in improving quality of acute care across sectors. Large gaps exist in many low‐ and middle‐income countries, fuelled by poor access to quality mental health care. Responses need to build on a foundation of existing community responses and contextually relevant evidence. The necessity of moving outside formal systems in low‐resource settings may lead to wider learning from locally embedded strategies.     

Immune Senescence: Relative Contributions of Age and Cytomegalovirus Infection

Immune senescence, defined as the age-associated dysregulation and dysfunction of the immune system, is characterised by impaired protective immunity and decreased efficacy of vaccines. Recent clinical, epidemiological and immunological studies suggest that Cytomegalovirus (CMV) infection may be associated with accelerated immune senescence, possibly by restricting the naïve T cell repertoire. However, direct evidence whether and how CMV-infection is implicated in immune senescence is still lacking. In this study, we have investigated whether latent mouse CMV (MCMV) infection with or without thymectomy (Tx) alters antiviral immunity of young and aged mice. After infection with lymphocytic choriomeningitis virus (LCMV) or Vaccinia virus, specific antiviral T cell responses were significantly reduced in old, old MCMV-infected and/or Tx mice compared to young mice. Importantly, control of LCMV replication was more profoundly impaired in aged MCMV-infected mice compared to age-matched MCMV-naïve or young mice. In addition, latent MCMV infection was associated with slightly reduced vaccination efficacy in old Tx mice. In contrast to the prevailing hypothesis of a CMV-mediated restriction of the naïve T cell repertoire, we found similar naïve T cell numbers in MCMV-infected and non-infected mice, whereas ageing and Tx clearly reduced the naïve T cell pool. Instead, MCMV-infection expanded the total CD8⁺ T cell pool by a massive accumulation of effector memory T cells. Based on these results, we propose a new model of increased competition between CMV-specific memory T cells and any ‘de novo’ immune response in aged individuals. In summary, our results directly demonstrate in a mouse model that latent CMV-infection impairs immunity in old age and propagates immune senescence.     

Nutritional regulation of hepatic heme biosynthesis and porphyria through PGC-1alpha

Inducible hepatic porphyrias are inherited genetic disorders of enzymes of heme biosynthesis. The main clinical manifestations are acute attacks of neuropsychiatric symptoms frequently precipitated by drugs, hormones, or fasting, associated with increased urinary excretion of delta-aminolevulinic acid (ALA). Acute attacks are treated by heme infusion and glucose administration, but the mechanisms underlying the precipitating effects of fasting and the beneficial effects of glucose are unknown. We show that the rate-limiting enzyme in hepatic heme biosynthesis, 5-aminolevulinate synthase (ALAS-1), is regulated by the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha). Elevation of PGC-1alpha in mice via adenoviral vectors increases the levels of heme precursors in vivo as observed in acute attacks. The induction of ALAS-1 by fasting is lost in liver-specific PGC-1alpha knockout animals, as is the ability of porphyrogenic drugs to dysregulate heme biosynthesis. These data show that PGC-1alpha links nutritional status to heme biosynthesis and acute hepatic porphyria.     

The indris of Anjanaharibe-Sud,northeastern Madagascar

During a short field trip to the Special Reserve of Anjanaharibe-Sud in northeastern Madagascar, data concerning pelage coloration, behavior (especially vocalization), and ecology of indris were collected. Anjanaharibe-Sud is the northernmost locality of indri distribution. In comparison to the better-known indris from the southern part of their distribution, the indris in this region show different pelage coloration. Several types of loud vocalizations are analyzed, based on a small sample of tape recordings. Their song structure is more complicated than previously reported, containing distinct sequences of duetting. Data on behavior and ecology were collected by interviewing guides and local inhabitants. Some information contrasts with reports on the more southern indri populations. The conservation status of indris in Anjanaharibe-Sud and the future of the reserve are outlined.