Capsule independent uptake of the fungal pathogen Cryptococcus neoformans into brain microvascular endothelial cells.

Cryptococcosis is a life-threatening fungal disease with a high rate of mortality among HIV/AIDS patients across the world. The ability to penetrate the blood-brain barrier (BBB) is central to the pathogenesis of cryptococcosis, but the way in which this occurs remains unclear. Here we use both mouse and human brain derived endothelial cells (bEnd3 and hCMEC/D3) to accurately quantify fungal uptake and survival within brain endothelial cells. Our data indicate that the adherence and internalisation of cryptococci by brain microvascular endothelial cells is an infrequent event involving small numbers of cryptococcal yeast cells. Interestingly, this process requires neither active signalling from the fungus nor the presence of the fungal capsule. Thus entry into brain microvascular endothelial cells is most likely a passive event that occurs following ‘trapping’ within capillary beds of the BBB

Bariatric surgery in morbidly obese insulin resistant humans normalises insulin signalling but not insulin-stimulated glucose disposal.

This is the final published version. Available from PLoS via the DOI in this record.All relevant data are available from Figshare, under the DOI http://dx.doi. org/10.6084/m9.figshare.1292883.AIMS: Weight-loss after bariatric surgery improves insulin sensitivity, but the underlying molecular mechanism is not clear. To ascertain the effect of bariatric surgery on insulin signalling, we examined glucose disposal and Akt activation in morbidly obese volunteers before and after Roux-en-Y gastric bypass surgery (RYGB), and compared this to lean volunteers. MATERIALS AND METHODS: The hyperinsulinaemic euglycaemic clamp, at five infusion rates, was used to determine glucose disposal rates (GDR) in eight morbidly obese (body mass index, BMI=47.3 ± 2.2 kg/m(2)) patients, before and after RYGB, and in eight lean volunteers (BMI=20.7 ± 0.7 kg/m2). Biopsies of brachioradialis muscle, taken at fasting and insulin concentrations that induced half-maximal (GDR50) and maximal (GDR100) GDR in each subject, were used to examine the phosphorylation of Akt-Thr308, Akt-473, and pras40, in vivo biomarkers for Akt activity. RESULTS: Pre-operatively, insulin-stimulated GDR was lower in the obese compared to the lean individuals (P<0.001). Weight-loss of 29.9 ± 4 kg after surgery significantly improved GDR50 (P=0.004) but not GDR100 (P=0.3). These subjects still remained significantly more insulin resistant than the lean individuals (p<0.001). Weight loss increased insulin-stimulated skeletal muscle Akt-Thr308 and Akt-Ser473 phosphorylation, P=0.02 and P=0.03 respectively (MANCOVA), and Akt activity towards the substrate PRAS40 (P=0.003, MANCOVA), and in contrast to GDR, were fully normalised after the surgery (obese vs lean, P=0.6, P=0.35, P=0.46, respectively). CONCLUSIONS: Our data show that although Akt activity substantially improved after surgery, it did not lead to a full restoration of insulin-stimulated glucose disposal. This suggests that a major defect downstream of, or parallel to, Akt signalling remains after significant weight-loss.Diabetes Research and Wellness FoundatioMedical Research Council (MRC)Astra Zenec

The Human Fungal Pathogen Cryptococcus neoformans Escapes Macrophages by a Phagosome Emptying Mechanism That Is Inhibited by Arp2/3 Complex-Mediated Actin Polymerisation

The lysis of infected cells by disease-causing microorganisms is an efficient but risky strategy for disseminated infection, as it exposes the pathogen to the full repertoire of the host's immune system. Cryptococcus neoformans is a widespread fungal pathogen that causes a fatal meningitis in HIV and other immunocompromised patients. Following intracellular growth, cryptococci are able to escape their host cells by a non-lytic expulsive mechanism that may contribute to the invasion of the central nervous system. Non-lytic escape is also exhibited by some bacterial pathogens and is likely to facilitate long-term avoidance of the host immune system during latency. Here we show that phagosomes containing intracellular cryptococci undergo repeated cycles of actin polymerisation. These actin ‘flashes’ occur in both murine and human macrophages and are dependent on classical WASP-Arp2/3 complex mediated actin filament nucleation. Three dimensional confocal imaging time lapse revealed that such flashes are highly dynamic actin cages that form around the phagosome. Using fluorescent dextran as a phagosome membrane integrity probe, we find that the non-lytic expulsion of Cryptococcus occurs through fusion of the phagosome and plasma membranes and that, prior to expulsion, 95% of phagosomes become permeabilised, an event that is immediately followed by an actin flash. By using pharmacological agents to modulate both actin dynamics and upstream signalling events, we show that flash occurrence is inversely related to cryptococcal expulsion, suggesting that flashes may act to temporarily inhibit expulsion from infected phagocytes. In conclusion, our data reveal the existence of a novel actin-dependent process on phagosomes containing cryptococci that acts as a potential block to expulsion of Cryptococcus and may have significant implications for the dissemination of, and CNS invasion by, this organism.\ud \u

Two Planets, One Species: Does a Mission to Mars Alter the Balance in Favour of Human Enhancement?

In this chapter we examine the implications of a crewed mission to Mars, possible colonisation of the planet, and the wider implications this may have on genetic enhancement in both a terrestrial and space context. We consider the usage of both somatic and germ-line genetic engineering, and its potential impact on the evolution of Homo sapiens. We acknowledge that a mission to Mars may require the usage of such technologies if it is to be successful. Our investigation suggests that the use of such technologies might ultimately be linked with the transformation of our own species. We also consider projected timescales for the development of these genetic enhancements and the ethical questions raised by the possibility of speciation. Cooperation among spacefaring nations in this context and the development of norms for the use of such technologies is desirable

Central modulation in cluster headache patients treated with occipital nerve stimulation: an FDG-PET study

Peer reviewe

The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis.

Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype

Invasion speeds for structured populations in fluctuating environments

We live in a time where climate models predict future increases in environmental variability and biological invasions are becoming increasingly frequent. A key to developing effective responses to biological invasions in increasingly variable environments will be estimates of their rates of spatial spread and the associated uncertainty of these estimates. Using stochastic, stage-structured, integro-difference equation models, we show analytically that invasion speeds are asymptotically normally distributed with a variance that decreases in time. We apply our methods to a simple juvenile-adult model with stochastic variation in reproduction and an illustrative example with published data for the perennial herb, \emph{Calathea ovandensis}. These examples buttressed by additional analysis reveal that increased variability in vital rates simultaneously slow down invasions yet generate greater uncertainty about rates of spatial spread. Moreover, while temporal autocorrelations in vital rates inflate variability in invasion speeds, the effect of these autocorrelations on the average invasion speed can be positive or negative depending on life history traits and how well vital rates ``remember'' the past

Uptake of home-based voluntary HIV testing in sub-Saharan Africa: a systematic review and meta-analysis

Improving access to HIV testing is a key priority in scaling up HIV treatment and prevention services. Home-based voluntary counselling and testing (HBT) as an approach to delivering wide-scale HIV testing is explored here

Coxiella burnetii Phagocytosis Is Regulated by GTPases of the Rho Family and the RhoA Effectors mDia1 and ROCK

The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that processFil: Salinas Ojeda, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ortiz Flores, Rodolfo Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Distel, Jesús Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Aguilera, Milton Osmar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Beron, Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Heterogeneities in leishmania infantum infection : using skin parasite burdens to identify highly infectious dogs

Background: The relationships between heterogeneities in host infection and infectiousness (transmission to arthropod vectors) can provide important insights for disease management. Here, we quantify heterogeneities in Leishmania infantum parasite numbers in reservoir and non-reservoir host populations, and relate this to their infectiousness during natural infection. Tissue parasite number was evaluated as a potential surrogate marker of host transmission potential. Methods: Parasite numbers were measured by qPCR in bone marrow and ear skin biopsies of 82 dogs and 34 crab-eating foxes collected during a longitudinal study in Amazon Brazil, for which previous data was available on infectiousness (by xenodiagnosis) and severity of infection. Results: Parasite numbers were highly aggregated both between samples and between individuals. In dogs, total parasite abundance and relative numbers in ear skin compared to bone marrow increased with the duration and severity of infection. Infectiousness to the sandfly vector was associated with high parasite numbers; parasite number in skin was the best predictor of being infectious. Crab-eating foxes, which typically present asymptomatic infection and are non-infectious, had parasite numbers comparable to those of non-infectious dogs. Conclusions: Skin parasite number provides an indirect marker of infectiousness, and could allow targeted control particularly of highly infectious dogs