Vacuolar ATPase depletion contributes to dysregulation of endocytosis in bloodstream forms of Trypanosoma brucei

BACKGROUND Vacuolar H-ATPase (V-ATPase) is a highly conserved protein complex which hydrolyzes ATP and pumps protons to acidify vacuolar vesicles. Beyond its role in pH maintenance, the involvement of V-ATPase in endocytosis is well documented in mammals and plants but is less clear in Trypanosoma brucei. METHODS In this study, the subcellular localization of V-ATPase subunit B (TbVAB) of T. brucei was assessed via in situ N-terminal YFP-tagging and immunofluorescence assays. Transgenic bloodstream forms (BSF) of T. brucei were generated which comprised either a V-ATPase subunit B (TbVAB) conditional knockout or a V-ATPase subunit A (TbVAA) knockdown. Acridine orange and BCECF-AM were employed to assess the roles of V-ATPase in the pH regulation of BSF T. brucei. The endocytic activities of three markers were also characterized by flow cytometry analyses. Furthermore, trypanosomes were counted from trypanolysis treatment groups (either containing 1% or 5% NHS) and endocytosed trypanosome lytic factor (TLF) was also analyzed by an immunoblotting assay. RESULTS TbVAB was found to localize to acidocalcisomes, lysosomes and probably also to endosomes of BSF of T. brucei and was demonstrated to be essential for cell growth. TbVAB depletion neutralized acidic organelles at 24 hours post-tetracycline depletion (hpd), meanwhile the steady state intracellular pH increased from 7.016 ± 0.013 to 7.422 ± 0.058. Trypanosomes with TbVAB depletion at 24 hpd were found to take up more transferrin (2.068 ± 0.277 fold) but less tomato lectin (49.31 ± 22.57%) by endocytosis, while no significant change was detected in dextran uptake. Similar endocytic dysregulated phenotypes were also observed in TbVAA knockdown cells. In addition, TbVAB depleted trypanosomes showed a low uptake of TLF and exhibited less sensitive to lysis in both 1% and 5% NHS treatments. CONCLUSIONS TbVAB is a key component of V-ATPase and was found to play a key function in endocytosis as well as exhibiting different effects in a receptor/cargo dependent manner in BSF of T. brucei. Besides vacuolar alkalinization, the dysregulation of endocytosis in TbVAB depleted T. brucei is considered to contribute to the reduced sensitivity to lysis by normal human serum

Evidence for high levels of vertical transmission in Toxoplasma gondii

Toxoplasma gondii is a highly ubiquitous and prevalent parasite. Despite the cat being the only definitive host, it is found in almost all geographical areas and warm blooded animals. Three routes of transmission are recognised: ingestion of oocysts shed by the cat, carnivory and congenital transmission. In natural populations, it is difficult to establish the relative importance of these routes. This paper reviews recent work in our laboratory which suggests that congenital transmission may be much more important than previously thought. Using PCR detection of the parasite, studies in sheep show that congenital transmission may occur in as many as 66% of pregnancies. Furthermore, in families of sheep on the same farm, exposed to the same sources of oocysts, significant divergent prevalences of Toxoplasma infection and abortion are found between different families. The data suggest that breeding from infected ewes increases the risk of subsequent abortion and infection in lambs. Congenital transmission rates in a natural population of mice were found to be 75%. Interestingly, congenital transmission rates in humans were measured at 19.8%. The results presented in these studies differ from those of other published studies and suggest that vertical transmission may be much more important than previously thought

Human African trypanosomiasis : the current situation in endemic regions and the risks for non-endemic regions from imported cases

Human African trypanosomiasis (HAT) is caused by Trypanosoma brucei gambiense and T. b. rhodesiense and caused devastating epidemics during the 20th century. Due to effective control programs implemented in the last two decades, the number of reported cases has fallen to a historically low level. Although fewer than 977 cases were reported in 2018 in endemic countries, HAT is still a public health problem in endemic regions until it is completely eliminated. In addition, almost 150 confirmed HAT cases were reported in non-endemic countries in the last three decades. The majority of non-endemic HAT cases were reported in Europe, United States and South Africa, due to historical alliances, economic links or geographic proximity to disease endemic countries. Furthermore, with the implementation of the “Belt and Road” project, sporadic imported HAT cases have been reported in China as a warning sign of tropical diseases prevention. In this paper, we explore and interpret the data on HAT incidence and find no positive correlation between the number of HAT cases from endemic and non-endemic countries.This data will provide useful information for better understanding the imported cases of HAT globally in the post-elimination phase

Divergent LIN28-mRNA associations result in translational suppression upon the initiation of differentiation

LIN28 function is fundamental to the activity and behavior of human embryonic stem cells (hESCs) and induced pluripotent stem cells. Its main roles in these cell types are the regulation of translational efficiency and let-7 miRNA maturation. However, LIN28-associated mRNA cargo shifting and resultant regulation of translational efficiency upon the initiation of differentiation remain unknown. An RNA-immunoprecipitation and microarray analysis protocol, eRIP, that has high specificity and sensitivity was developed to test endogenous LIN28-associated mRNA cargo shifting. A combined eRIP and polysome analysis of early stage differentiation of hESCs with two distinct differentiation cues revealed close similarities between the dynamics of LIN28 association and translational modulation of genes involved in the Wnt signaling, cell cycle, RNA metabolism and proteasomal pathways. Our data demonstrate that change in translational efficiency is a major contributor to early stages of differentiation of hESCs, in which LIN28 plays a central role. This implies that eRIP analysis of LIN28-associated RNA cargoes may be used for rapid functional quality control of pluripotent stem cells under manufacture for therapeutic applications

Baroclinic, Kelvin and inertia-gravity waves in the barostrat instability experiment

International audienceThe differentially heated rotating annulus is a laboratory experiment historically designed for modelling large-scale features of the mid-latitude atmosphere. In the present study, we investigate a modified version of the classic baroclinic experiment in which a juxtaposition of convective and motionless stratified layers is created by introducing a vertical salt stratification. The thermal convective motions are suppressed in a central region at mid-depth of the rotating tank, therefore double-diffusive convection rolls can develop only in thin layers located at top and bottom, where the salt stratification is weakest. For high enough rotation rates, the baroclinic instability destabilises the flow in the top and the bottom shallow convective layers, generating cyclonic and anticyclonic eddies separated by the stable stratified layer. Thanks to this alternation of layers resembling the convective and radiative layers of stars, the planetary's atmospheric troposphere and stratosphere or turbulent layers at the sea surface above stratified waters, this new laboratory setup is of interest for both astrophysics and geophysical sciences. More specifically, it allows to study the exchange of momentum and energy between the layers, primarly by the propagation of internal gravity waves (IGW). PIV velocity maps are used to describe the wavy flow pattern at different heights. Using a co-rotating laser and camera, the wave field is well resolved and different wave types can be found: baroclinic waves, Kelvin, and Poincaré type waves. The signature of small-scale IGW can also be observed attached to the baroclinic jet. The baroclinic waves occur at the thin convectively active layer at the surface and the bottom of the tank, though decoupled they show different manifestation of nonlinear interactions. The inertial Kelvin and Poincaré waves seem to be mechanically forced. The small-scale wave trains attached to the meandering jet point to an imbalance of the large-scale flow. For the first time, the simultaneous occurrence of differentwave types is reported in detail for a differentially heated rotating annulus experiment

The effect of normal human serum on the mouse trypanosome Trypanosoma musculi in vitro and in vivo

Trypanosoma musculi, a common blood flagellate found in mice, is similar in morphology and life cycle to the rat trypanosome T. lewisi. Both species belong to the subgenus Herpetosoma, and as T. lewisi has recently been shown to be a zoonotic pathogen, there is concern that T. musculi could also be potentially infective to humans. To test this hypothesis, a well-established method, the normal human serum (NHS) incubation test, was carried out which distinguishes human and non-human infective trypanosomes. We found that T. musculi could grow in 0.31% NHS in vitro, and even kept their infectivity to mice after incubation with 10% NHS for 24 h. In in vivo experiments, T. musculi were only slightly affected by NHS injection, confirming that it was less sensitive to the NHS than T. b. brucei, but more sensitive than T. lewisi. This resistance probably does not rely on a restricted uptake of ApoL-1. Due to this partial resistance, we cannot definitively confirm that T. musculi has the potential for infection to humans. As resistance is less than that of T. lewisi, our data suggest that it is unlikely to be a zoonotic pathogen although we would advise caution in the case of immunocompromised people such as AIDS and cancer patients

Topographic Core-Mantle Coupling and Fluctuations in the Earth's Rotation

Astronomically-determined irregular fluctuations in the Earth's rotation vector on decadal time scales can be used to estimate the fluctuating torque on the lower surface of the Earth's mantle produced by magnetohydrodynamic flow in the underlying liquid metallic core. A method has been proposed for testing the hypothesis that the torque is due primarily to fluctuating dynamic pressure forces acting on irregular topographic features of the core-mantle boundary and also on the equatorial bulge. The method exploits (a) geostrophically-constrained models of fluid motions in the upper reaches of the core based on geomagnetic secular variation data, and (b) patterns of the topography of the CMB based on the mantle flow models constrained by data from seismic tomography, determinations of long wave-length anomalies of the Earth's gravitational field and other geophysical and geodetic data. According to the present study, the magnitude of the axial component of the torque implied by determinations of irregular changes in the length of the day is compatible with models of the Earth's deep interior characterized by the presence of irregular CMB topography of effective “height” no more than about 0.5 km (about 6% of the equatorial bulge) and strong horizontal variations in the properties of the D″ layer at the base of the mantle. The investigation is now being extended to cover a wider range of epochs and also the case of polar motion on decadal time scales produced by fluctuations in the equatorial components of the torque

Prediction of infrared light emission from pi-conjugated polymers: a diagrammatic exciton basis valence bond theory

There is currently a great need for solid state lasers that emit in the infrared, as this is the operating wavelength regime for applications in telecommunications. Existing $\pi$--conjugated polymers all emit in the visible or ultraviolet, and whether or not $\pi$--conjugated polymers that emit in the infrared can be designed is an interesting challenge. On the one hand, the excited state ordering in trans-polyacetylene, the $\pi$--conjugated polymer with relatively small optical gap, is not conducive to light emission because of electron-electron interaction effects. On the other hand, excited state ordering opposite to that in trans-polyacetylene is usually obtained by chemical modification that increases the effective bond-alternation, which in turn increases the optical gap. We develop a theory of electron correlation effects in a model $\pi$-conjugated polymer that is obtained by replacing the hydrogen atoms of trans-polyacetylene with transverse conjugated groups, and show that the effective on-site correlation in this system is smaller than the bare correlation in the unsubstituted system. An optical gap in the infrared as well as excited state ordering conducive to light emission is thereby predicted upon similar structural modifications.Comment: 15 pages, 15 figures, 1 tabl

Mesoscopic Cooperative Emission From a Disordered System

We study theoretically the cooperative light emission from a system of $N\gg 1$ classical oscillators confined within a volume with spatial scale, $L$, much smaller than the radiation wavelength, $\lambda_0=2\pi c/\omega_0$. We assume that the oscillators frequencies are randomly distributed around a central frequency, $\omega_0$, with some characteristic width, $\Omega\ll\omega_0$. In the absence of disorder, that is $\Omega=0$, the cooperative emission spectrum is composed of a narrow subradiant peak superimposed on a wide superradiant band. When $\Omega\neq 0$, we demonstrate that if $N$ is large enough, the subradiant peak is not simply broadened by the disorder but rather splits into a system of random narrow peaks. We estimate the spectral width of these peaks as a function of $N, L, \Omega$, and $\lambda_0$. We also estimate the amplitude of this mesoscopic structure in the emission spectrum.Comment: 25 pages including 6 figure