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

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

The secondary Bjerknes force between two oscillating bubbles in Kelvin-Voigt-type viscoelastic fluids driven by harmonic ultrasonic pressure

The interaction between two small bubbles experiencing transient cavitation in a nonlinear Kelvin-Voigt fluid is investigated. The time-delay effect in the interaction is incorporated in the coupled Keller-Miksis model. The refined model predicts that bubbles with radii smaller than 2μm will be repelled by large bubbles, in contrast to predictions from previous models. The matching pressure needed to obtain same level of transient cavitation in different Kelvin-Voigt fluids is shown to depend mainly on the shear modulus and is insensitive to other parameters, which makes it a useful parameter to correlate the results. When the radii of the bubbles fall between 4μm and 6μm, the secondary Bjerknes force obtained with matching pressures shows only weak dependence on the shear modulus. For the pressure amplitudes investigated, equilibrium distances can be found between two bubbles when the equilibrium radius of one of the bubbles is in a narrow range around 2 μm. The equilibrium distance decreases when the shear modulus is increased. A simple relation between the two quantities is established

Surface response of spherical core-shell structured nanoparticle by optically induced elastic oscillations of soft shell against hard core

The optically induced oscillatory response of a spherical two-component, shell-core structured, nanoparticle by nodeless elastic vibrations of soft peripheral shell against hard and dynamically immobile inner core is considered. The eigenfrequencies of the even-parity, spheroidal and odd-parity torsional vibrational modes trapped in the finite-depth shell are obtained which are of practical interest for modal specification of individual resonances in spectra of resonant scattering of long wavelength electromagnetic waves by ultrafine particles.Comment: Surface Review and Letters (World Scientific) Year: 2009 Vol: 16 Issue: 1 (February 2009) Page: 5 - 1

Cyclin-dependent Kinase 5 (Cdk5)-dependent Phosphorylation of p70 Ribosomal S6 Kinase 1 (S6K) Is Required for Dendritic Spine Morphogenesis.

The maturation and maintenance of dendritic spines depends on neuronal activity and protein synthesis. One potential mechanism involves mammalian target of rapamycin, which promotes protein synthesis through phosphorylation of eIF4E-binding protein and p70 ribosomal S6 kinase 1 (S6K). Upon extracellular stimulation, mammalian target of rapamycin phosphorylates S6K at Thr-389. S6K also undergoes phosphorylation at other sites, including four serine residues in the autoinhibitory domain. Despite extensive biochemical studies, the importance of phosphorylation in the autoinhibitory domain in S6K function remains unresolved, and its role has not been explored in the cellular context. Here we demonstrated that S6K in neuron was phosphorylated at Ser-411 within the autoinhibitory domain by cyclin-dependent kinase 5. Ser-411 phosphorylation was regulated by neuronal activity and brain-derived neurotrophic factor (BDNF). Knockdown of S6K in hippocampal neurons by RNAi led to loss of dendritic spines, an effect that mimics neuronal activity blockade by tetrodotoxin. Notably, coexpression of wild type S6K, but not the phospho-deficient S411A mutant, could rescue the spine defects. These findings reveal the importance of cyclin-dependent kinase 5-mediated phosphorylation of S6K at Ser-411 in spine morphogenesis driven by BDNF and neuronal activity.published_or_final_versio

Dose-dependent effects of luteinizing hormone and follicle stimulating hormone on in vitro maturation, apoptosis, secretion function and expression of follicle stimulating hormone receptor and luteinizing hormone receptor of sheep oocytes

TThe present study compared the effects of follicle stimulating hormone (FSH) and luteinizing hormone (LH) on in vitro maturation (IVM), apoptosis, and secretion function in sheep oocytes, as well as gene expressions of the receptors (FSHR, LHR, and GnRHR) in cumulus-oocyte complexes (COCs). The COCs were recovered from sheep ovaries and pooled in groups. The COCs were cultured for 24 hours in IVM medium supplemented with various concentrations of LH (5–30 μg/mL) and FSH (5–30 IU/mL). They were allocated to LH-1 (5 µg/mL), LH-2 (10 µg/mL), LH-3 (20 µg/mL), and LH-4 (30 µg/mL) groups, and FSH-1 (5 IU/mL), FSH-2 (10 IU/mL), FSH-3 (20 IU/mL), and FSH-4 (30IU/mL) groups. The apoptosis of COCs was assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The maturation rates of oocytes improved gradually as LH and FSH concentration increased from 0 to 10 μg/mL(IU/mL), reaching a peak value of 44.1% of LH-2 and 48.5% of FSH-2 group. Oocyte apoptosis rates of LH-2 and FSH-2 groups were the lowest among LH- and FSH-treated groups, respectively. The germinal vesicle breakdown (GVBD) rate of the FSH-2 group was higher than that of the control group (CG) and FSH-4 groups. The GVBD rate of LH-2 group also increased in comparison with the CG group. FSH concentration of the FSH-2 group was greater than that of CG. Expression levels of FSHR, LHR, and GnRHR mRNAs of FSH-2, LH-3, and LH-3 group, respectively, were higher than CG. Levels of FSHR proteins in FSH-2 and FSH-3 groups were greater than CG. Levels of GnRHR proteins were increased with a maximum increment of FSH-4. The FSH and LH supplemented into the IVM medium could promote the maturation rate, reduce the apoptosis rate of sheep oocytes, and increase FSH concentrations in IVM medium fluid. Additionally, FSH and LH enhanced expression levels of FSHR, LHR, and GnRHR mRNAs of sheep COCs.Keywords: Apoptosis, cumulus-oocyte complexes, germinal vesicle breakdown, protein expression, recepto

Growth Models And The Question Of Universality Classes

In the past many papers have appeared which simulated surface growth with different growth models. The results showed that, if models differed only slightly in their `growth' rules, the resulting surfaces may belong to different universality classes, i.e. they are described by different differential equations. In the present paper we describe a mapping of ``growth rules'' to differential operators and give plausibility arguments for this mapping. We illustrate the validity of our theory by applying it to published results

Intermittent emission of particles from three coupled condensates in a one-dimensional lattice

We investigate particle emission, driven by periodically modulating the interaction strength, from three coupled Bose-Einstein condensates in a one-dimensional lattice. Within perturbative analyses, which lead to the regimes of instabilities for different modes, we not only obtain two main frequencies, under which the system can emit a large particle jet, but also find that the emission is distinctly intermittent rather than continuous. The time evolution of the trapped particles exhibits a stair-like decay, and a larger drive induces a more significant intermittency. We further shed light on the dynamics of the stimulating process, and demonstrate that instead of a real suspension, the intermittency represents a build-up stage of the particles. The theoretical framework might be generalized, to the explorations on other multiple-condensate systems with analogous configurations and couplings.Comment: 7 pages, 10 figure

Engineering d-band center of FeN<inf>4</inf> moieties for efficient oxygen reduction reaction electrocatalysts

Atomically-dispersed FeN4 moieties are emerging as low-cost electrocatalysts for oxygen reduction reaction (ORR), which can be applied in fuel cells and metal-air batteries. Whereas, the unsatisfactory position of the d-band center from the metal sites offered by FeN4 affects the adsorption-desorption behaviors of oxygenated intermediates, further impeding the improvement of their ORR performances. Herein, we report a well-designed diatomic Fe/Zn-CNHC catalyst on a microporous hollow support. This strategy drives the position of the d-band center of Fe upward, thus making FeN4 active sites more favorable and stable during the ORR kinetic processes. The material exhibits an excellent ORR activity with a half-wave potential of 0.91 V and excellent stability (insignificant attenuation after 5,000 cycles), surpassing commercial Pt/C and many other single-atom catalysts. DFT calculations further indicate that the tuning effect of Zn on the d-orbital electron distribution of Fe facilitates the stretching and cleavage of Fe-O, thus accelerating the rate-determining step. This work presents a simple strategy to fabricate well-defined diatomic coordination in single-atom ORR electrocatalysts and inspires future research on developing new syntheses to control the coordination of single-atom electrocatalysts