Flagellar targeting of an arginine kinase requires a conserved lipidated protein intraflagellar transport (LIFT) pathway in Trypanosoma brucei.

Both intraflagellar transport (IFT) and lipidated protein intraflagellar transport (LIFT) pathways are essential for cilia/flagella biogenesis, motility, and sensory functions. In the LIFT pathway, lipidated cargoes are transported into the cilia through the coordinated actions of cargo carrier proteins such as Unc119 or PDE6δ, as well as small GTPases Arl13b and Arl3 in the cilium. Our previous studies have revealed a single Arl13b ortholog in the evolutionarily divergent Trypanosoma brucei, the causative agent of African sleeping sickness. TbArl13 catalyzes two TbArl3 homologs, TbArl3A and TbArl3C, suggesting the presence of a conserved LIFT pathway in these protozoan parasites. Only a single homolog to the cargo carrier protein Unc119 has been identified in T. brucei genome, but its function in lipidated protein transport has not been characterized. In this study, we exploited the proximity-based biotinylation approach to identify binding partners of TbUnc119. We showed that TbUnc119 binds to a flagellar arginine kinase TbAK3 in a myristoylation-dependent manner and is responsible for its targeting to and enrichment in the flagellum. Interestingly, only TbArl3A, but not TbArl3C interacted with TbUnc119 in a GTP-dependent manner, suggesting functional specialization of Arl3-GTPases in T. brucei These results establish the function of TbUnc119 as a myristoylated cargo carrier and support the presence of a conserved LIFT pathway in T. brucei.

The unusual flagellar-targeting mechanism and functions of the trypanosome ortholog of the ciliary GTPase Arl13b.

The small GTPase Arl13b is one of the most conserved and ancient ciliary proteins. In human and animals, Arl13b is primarily associated with the ciliary membrane, where it acts as a guanine-nucleotide-exchange factor (GEF) for Arl3 and is implicated in a variety of ciliary and cellular functions. We have identified and characterized Trypanosoma brucei (Tb)Arl13, the sole Arl13b homolog in this evolutionarily divergent, protozoan parasite. TbArl13 has conserved flagellar functions and exhibits catalytic activity towards two different TbArl3 homologs. However, TbArl13 is distinctly associated with the axoneme through a dimerization/docking (D/D) domain. Replacing the D/D domain with a sequence encoding a flagellar membrane protein created a viable alternative to the wild-type TbArl13 in our RNA interference (RNAi)-based rescue assay. Therefore, flagellar enrichment is crucial for TbArl13, but mechanisms to achieve this could be flexible. Our findings thus extend the understanding of the roles of Arl13b and Arl13b-Arl3 pathway in a divergent flagellate of medical importance.This article has an associated First Person interview with the first author of the paper.

Adolescent exposure to environmental level of PCBs (Aroclor 1254) induces non-alcoholic fatty liver disease in male mice.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants found in various environmental media, and there is growing evidence that PCBs may contribute to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). The purposes of this study were to investigate whether environmental level of Aroclor 1254 (a commercial mixture of PCBs) exposure to adolescent male mice could induce the development of NAFLD and the mechanisms involved. Twenty-one-day-old male C57BL/6 mice were exposed to Aroclor 1254 (0.5-500 µg/kg body weight) by oral gavage once every third day for 60 days. The results showed that exposure to Aroclor 1254 increased body weight and decreased the liver-somatic index in a dose-dependent manner. Aroclor 1254 administration increased lipid accumulation in the liver and induced the mRNA expression of genes associated with lipogenesis, including acetyl-CoA carboxylase 1 (Acc1), acetyl-CoA carboxylase 2 (Acc2) and fatty acid synthase (Fasn). Moreover, Aroclor 1254 decreased peroxisome proliferator-activated receptor alpha (PPARα) signaling and lipid oxidation. In addition, we found that Aroclor 1254 administration induced oxidative stress in mouse liver and elevated the protein level of cyclooxygenase 2 (COX-2), an inflammatory molecule, possibly via the endoplasmic reticulum (ER) stress inositol-requiring enzyme 1α-X-box-binding protein-1 (IRE1α-XBP1) pathway, but not the nuclear factor-κB (NF-κB) pathway. In summary, adolescent exposure to environmental level of PCBs stimulated oxidative stress, ER stress and the inflammatory response and caused NAFLD in male mice. This work provides new insight into the idea that adolescent exposure to environmental level of PCBs might induce the development of NAFLD under the regulation of ER stress in males.

ARL3 GTPases facilitate ODA16 unloading from IFT in motile cilia.

Eukaryotic cilia and flagella are essential for cell motility and sensory functions. Their biogenesis and maintenance rely on the intraflagellar transport (IFT). Several cargo adapters have been identified to aid IFT cargo transport, but how ciliary cargos are discharged from the IFT remains largely unknown. During our explorations of small GTPases ARL13 and ARL3 in Trypanosoma brucei, we found that ODA16, a known IFT cargo adapter present exclusively in motile cilia, is a specific effector of ARL3. In the cilia, active ARL3 GTPases bind to ODA16 and dissociate ODA16 from the IFT complex. Depletion of ARL3 GTPases stabilizes ODA16 interaction with the IFT, leading to ODA16 accumulation in cilia and defects in axonemal assembly. The interactions between human ODA16 homolog HsDAW1 and ARL GTPases are conserved, and these interactions are altered in HsDAW1 disease variants. These findings revealed a conserved function of ARL GTPases in IFT transport of motile ciliary components, and a mechanism of cargo unloading from the IFT.

Puerarin protects against H2O2-induced apoptosis of HTR-8/SVneo cells by regulating the miR-20a-5p/VEGFA/Akt axis.

INTRODUCTION: Preeclampsia seriously affects the health of pregnant women and fetuses. It has been reported that puerarin has a positive therapeutic effect on the treatment of preeclampsia. In this study, oxidative stress-induced trophoblast cell injury was established to explore the potential interaction between puerarin and preeclampsia. METHODS: A CCK-8 assay was performed to investigate the effect of puerarin on the viability of HTR-8/SVneo cells. To mimic oxidative stress-induced trophoblast cell injury, human villous trophoblasts (HTR-8/SVneo) were treated with H2O2. Then, the relationships among MMP2, VEGFA and miR-20a-5p in HTR-8/SVneo cells were confirmed using a dual-luciferase reporter assay. Finally, Western blot assays were performed to measure the expression levels of MMP2, VEGFA, p-Akt, Akt, Bcl-2 and cleaved caspase 3. RESULTS: In this study, puerarin eliminated H2O2-induced cytotoxicity of HTR-8/SVneo cells. In addition, puerarin was able to reverse H2O2-induced apoptosis and metastasis inhibition in cells. Meanwhile, puerarin significantly abrogated H2O2-induced mitochondrial membrane potential (MMP) decline in HTR-8/SVneo cells. And, MMP2 and VEGFA were identified as direct targets of miR-20a-5p. Furthermore, puerarin reversed H2O2-induced growth inhibition in HTR-8/SVneo cells by regulating the miR-20a-5p/VEGFA/Akt axis. DISCUSSION: All these data indicated that puerarin could abolish H2O2-induced growth inhibition in HTR-8/SVneo cells by regulating the miR-20a-5p/VEGFA/AKT axis.

Neuroprotective effects of low-intensity transcranial ultrasound stimulation combined with Baicalin intervention on traumatic brain injury in animals.

Low-intensity transcranial ultrasound stimulation (LITUS) can improve the inflammatory reaction after traumatic brain injury (TBI), and Baicalin also has a good protective effect on TBI. The purpose of this study was to observe the neuroprotective effect of LITUS combined with Baicalin intervention in the TBI rats. Sprague Dawley (SD) rats were randomly divided into 5 groups (n = 15) which were Sham control group, TBI group, LITUS group, Baicalin group, LITUS combined with Baicalin group (LB group). The rats were scanned with 3.0 T magnetic resonance imager, and the apparent diffusion coefficient (ADC) and the fractional anisotropy (FA) of the brain injury cortical area were determined at 3 h, 1, 3, 7 and 10 d after TBI. The ADC value, FA value, neurological function score and Nissl staining were used to assess the level of brain damage of rats. The results showed that on the 10th day after TBI, the ADC values of the TBI group, the LITUS group and the Baicalin group were remarkable greater than that of the L-B group (all adjusted P < 0.05), FA values were remarkable smaller than that of the L-B group (all adjusted P < 0.05), neurological function scores were remarkable greater than that of the L-B group (all adjusted P < 0.05), and Nissl body loss rates were remarkable greater than that of the L-B group (all adjusted P < 0.001). This study indicated that compared with the LITUS group and the Baicalin group, the L-B group can more effectively reduce level of brain damage after TBI, and the method of LITUS combined with Baicalin intervention was a more effective neuroprotection for brain injury.

Redistribution of intracellular and extracellular free & adsorbed antibiotic resistance genes through a wastewater treatment plant by an enhanced extracellular DNA extraction method with magnetic beads.

Due to the limitations of current extraction methods, extracellular DNA (eDNA) is rarely discerned from intracellular DNA (iDNA) despite having unique contributions to antibiotic resistance genes (ARGs) propagation. Furthermore, eDNA may be free (f-eDNA) or adsorbed to or suspended solids, including cells (a-eDNA), which affects ARG persistence and transmissivity. We developed a novel method using magnetic beads to separate iDNA, a-eDNA, and f-eDNA to assess how these physical states of ARGs change across a wastewater treatment plant. This method efficiently extracted eDNA (>85.3%) with higher recovery than current methods such as alcohol precipitation, CTAB-based extraction, and DNA extraction kits (<10%). Biological treatment and UV disinfection decreased the concentration of intracellular ARGs (iARGs) and adsorbed extracellular ARGs (a-eARGs), causing an increase of released free extracellular ARGs (f-eARGs). More ARGs were discharged through the wasted biosolids than in the effluent; iARGs and a-eARGs are prevalent in wasted biosolids ((73.9 ±â€¯22.5) % and (23.4 ±â€¯15.3) % of total ARGs respectively), while f-eARGs were prevalent in the effluent ((90.3 ±â€¯16.5) %). Bacterial community analysis showed significant correlations between specific genera and ARGs (e.g., Aeromonas, Pseudomonas and Acinetobacter were strongly correlated with multidrug-resistance gene blaTEM). This treatment system decreased the discharge of iARGs to receiving environments, however, increased eARG concentrations were present in the effluent, which may contribute to the environmental resistome.

Fast Multidimensional Ellipsoid-Specific Fitting by Alternating Direction Method of Multipliers.

Many problems in computer vision can be formulated as multidimensional ellipsoid-specific fitting, which is to minimize the residual error such that the underlying quadratic surface is a multidimensional ellipsoid. In this paper, we present a fast and robust algorithm for solving ellipsoid-specific fitting directly. Our method is based on the alternating direction method of multipliers, which does not introduce extra positive semi-definiteness constraints. The computation complexity is thus significantly lower than those of semi-definite programming (SDP) based methods. More specifically, to fit n data points into a p dimensional ellipsoid, our complexity is O(p(6) + np(4))+O(p(3)), where the former O results from preprocessing data once, while that of the state-of-the-art SDP method is O(p(6) + np(4) + n(3/2)p(2)) for each iteration. The storage complexity of our algorithm is about 1/2np(2), which is at most 1/4 of those of SDP methods. Extensive experiments testify to the great speed and accuracy advantages of our method over the state-of-the-art approaches. The implementation of our method is also much simpler than SDP based methods.