Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.     

Different fermentation strategies by Schizochytrium mangrovei strain pq6 to produce feedstock for exploitation of squalene and omega‐3 fatty acids

Schizochytrium mangrovei strain PQ 6 was investigated for coproduction of docosahexaenoic acid (C22: 6ω‐3, DHA ) and squalene using a 30‐L bioreactor with a working volume of 15 L under various batch and fed‐batch fermentation process regimes. The fed‐batch process was a more efficient cultivation strategy for achieving higher biomass production rich in DHA and squalene. The final biomass, total lipid, unsaponifiable lipid content, and DHA productivity were 105.25 g · L^?1, 43.40% of dry cell weight, 8.58% total lipid, and 61.66 mg · g^?1 · L^?1, respectively, after a 96 h fed‐batch fermentation. The squalene content was highest at 48 h after feeding glucose (98.07 mg · g^?1 of lipid). Differences in lipid accumulation during fermentation were correlated with changes in ultrastructure using transmission electron microscopy and Nile Red staining of cells. The results may be of relevance to industrial‐scale coproduction of DHA and squalene in heterotrophic marine microalgae such as Schizochytrium .     

Expanding the DNA alphabet in the fruit fly

DNA integrity is under the control of multiple pathways of nucleotide metabolism and DNA damage recognition and repair. Unusual sets of protein factors involved in these control mechanisms may result in tolerance and accumulation of non-canonical bases within the DNA. We investigate the presence of uracil in genomic DNA of Drosophila melanogaster. Results indicate a developmental pattern and strong correlations between uracil-DNA levels, dUTPase expression and developmental fate of different tissues. The intriguing lack of the catalytically most efficient uracil-DNA glycosylase in Drosophila melanogaster may be a general attribute of Holometabola and is suggested to be involved in the specific characteristics of uracil-DNA metabolism in these insects.     

Quinidine as an ABCB1 probe for testing drug interactions at the blood-brain barrier: an in vitro in vivo correlation study

This study provides evidence that quinidine can be used as a probe substrate for ABCB1 in multiple experimental systems both in vitro and in vivo relevant to the blood-brain barrier (BBB). The combination of quinidine and PSC-833 (valspodar) is an effective tool to assess investigational drugs for interactions on ABCB1. Effects of quinidine and substrate-inhibitor interactions were tested in a membrane assay and in monolayer assays. The authors compared quinidine and digoxin as ABCB1 probes in the in vitro assays and found that quinidine was more potent and at least as specific as digoxin in ATPase and monolayer efflux assays employing MDCKII-MDR1 and the rat brain microcapillary endothelial cell system. Brain exposure to quinidine was tested in dual-/triple-probe microdialysis experiments in rats by assessing levels of quinidine in blood and brain. Comparing quinidine levels in dialysate samples from valspodar-treated and control animals, it is evident that systemic/local administration of the inhibitor diminishes the pumping function of ABCB1 at the BBB, resulting in an increased brain penetration of quinidine. In sum, quinidine is a good probe to study ABCB1 function at the BBB. Moreover, quinidine/PSC-833 is an ABCB1-specific substrate/inhibitor combination applicable to many assay systems both in vitro and in vivo.     

Kinetics of viremia and NS1 antigenemia are shaped by immune status and virus serotype in adults with dengue

Background

Dengue is a major public health problem in tropical and subtropical countries. Exploring the relationships between virological features of infection with patient immune status and outcome may help to identify predictors of disease severity and enable rational therapeutic strategies.

Methods

Clinical features, antibody responses and virological markers were characterized in Vietnamese adults participating in a randomised controlled treatment trial of chloroquine.

Results

Of the 248 patients with laboratory-confirmed dengue and defined serological and clinical classifications 29 (11.7%) had primary DF, 150 (60.5%) had secondary DF, 4 (1.6%) had primary DHF and 65 (26.2%) had secondary DHF. DENV-1 was the commonest serotype (57.3%), then DENV-2 (20.6%), DENV-3 (15.7%) and DENV-4 (2.8%). DHF was associated with secondary infection (Odds ratio = 3.13, 95% CI 1.04–12.75). DENV-1 infections resulted in significantly higher viremia levels than DENV-2 infections. Early viremia levels were higher in DENV-1 patients with DHF than with DF, even if the peak viremia level was often not observed because it occurred prior to enrolment. Peak viremias were significantly less often observed during secondary infections than primary for all disease severity grades (P = 0.001). The clearance of DENV viremia and NS1 antigenemia occurs earlier and faster in patients with secondary dengue (P<0.0001). The maximum daily rate of viremia clearance was significantly higher in patients with secondary infections than primary (P<0.00001).

Conclusions

Collectively, our findings suggest that the early magnitude of viremia is positively associated with disease severity. The clearance of DENV is associated with immune status, and there are serotype dependent differences in infection kinetics. These findings are relevant for the rational design of randomized controlled trials of therapeutic interventions, especially antivirals.     

Heterogeneity of populations of the mollusc Bithynia inflata according to the degree of trematode infestation in opisthorchiasis foci

Results of studies of differences in the infection rate of natural populations of Bithynia inflata with trematodes (including Opisthorchis felineus) in the nidi of opisthorchiasis in the Sumsk and Chernigov Districts of the Ukrainian SSR and Tomsk District of the RSFSR are given. It has been shown that in the Ukrain over 1/3 of all populations of the specific intermediate host of opisthorchis does not take part in the circulation of the agent due to fundamentally different reasons: resistence to the infection and hyperinfection with parthenites of trematodes.     

Role of Insulin Resistance in the Alzheimer's Disease Progression

Recent studies continue to find evidence linking Type 2 diabetes (T2D) with Alzheimer's disease (AD), the most common cause of dementia, a general term for memory loss and other cognitive abilities serious enough to interfere with daily life. Insulin resistance or dysfunction of insulin signaling is a universal feature of T2D, the main culprit for altered glucose metabolism and its interdependence on cell death pathways, forming the basis of linking T2D with AD as it may exacerbate Aβ accumulation, tau hyperphosphorylation and devastates glucose transportation, energy metabolism, hippocampal framework and promulgate inflammatory pathways. The current work demonstrates the basic mechanisms of the insulin resistance mediates dysregulation of bioenergetics and progress to AD as a mechanistic link between diabetes mellitus and AD. This work also aimed to provide a potential and feasible zone to succeed in the development of therapies in AD by enhanced hypometabolism and altered insulin signaling.