The ciliary machinery is repurposed for T cell immune synapse trafficking of LCK

Upon engagement of the T cell receptor with an antigen-presenting cell, LCK initiates TCR signaling by phosphorylating its activation motifs. However, the mechanism of LCK activation specifically at the immune synapse is a major question. We show that phosphorylation of the LCK activating Y394, despite modestly increasing its catalytic rate, dramatically focuses LCK localization to the immune synapse. We describe a trafficking mechanism whereby UNC119A extracts membrane-bound LCK by sequestering the hydrophobic myristoyl group, followed by release at the target membrane under the control of the ciliary ARL3/ARL13B. The UNC119A N terminus acts as a “regulatory arm” by binding the LCK kinase domain, an interaction inhibited by LCK Y394 phosphorylation, thus together with the ARL3/ARL13B machinery ensuring immune synapse focusing of active LCK. We propose that the ciliary machinery has been repurposed by T cells to generate and maintain polarized segregation of signals such as activated LCK at the immune synapse

Palladium nanoparticles from desulfovibrio alaskensis G20 catalyze biocompatible sonogashira and biohydrogenation cascades

[Image: see text] Transition-metal nanoparticles produced by living bacteria are emerging as novel catalysts for sustainable synthesis. However, the scope of their catalytic activity and their ability to be integrated within metabolic pathways for the bioproduction of non-natural small molecules has been underexplored. Herein we report that Pd nanoparticles synthesized by the sulfate-reducing bacterium Desulfovibrio alaskensis G20 (DaPdNPs) catalyze the Sonogashira coupling of phenyl acetylenes and aryl iodides, and the subsequent one-pot hydrogenation to bibenzyl derivatives using hydrogen gas generated from d-glucose by engineered Escherichia coli DD-2. The formal hydroarylation reaction is biocompatible, occurs in aqueous media at ambient temperature, and affords products in 70–99% overall yield. This is the first reported microbial nanoparticle to catalyze the Sonogashira reaction and the first demonstration that these biogenic catalysts can be interfaced with the products of engineered metabolism for small molecule synthesis

Complex I dysfunction underlies the glycolytic switch in pulmonary hypertensive smooth muscle cells.

ATP is essential for cellular function and is usually produced through oxidative phosphorylation. However, mitochondrial dysfunction is now being recognized as an important contributing factor in the development cardiovascular diseases, such as pulmonary hypertension (PH). In PH there is a metabolic change from oxidative phosphorylation to mainly glycolysis for energy production. However, the mechanisms underlying this glycolytic switch are only poorly understood. In particular the role of the respiratory Complexes in the mitochondrial dysfunction associated with PH is unresolved and was the focus of our investigations. We report that smooth muscle cells isolated from the pulmonary vessels of rats with PH (PH-PASMC), induced by a single injection of monocrotaline, have attenuated mitochondrial function and enhanced glycolysis. Further, utilizing a novel live cell assay, we were able to demonstrate that the mitochondrial dysfunction in PH-PASMC correlates with deficiencies in the activities of Complexes I-III. Further, we observed that there was an increase in mitochondrial reactive oxygen species generation and mitochondrial membrane potential in the PASMC isolated from rats with PH. We further found that the defect in Complex I activity was due to a loss of Complex I assembly, although the assembly of Complexes II and III were both maintained. Thus, we conclude that loss of Complex I assembly may be involved in the switch of energy metabolism in smooth muscle cells to glycolysis and that maintaining Complex I activity may be a potential therapeutic target for the treatment of PH

A Single-Visit Field Test of Critical Speed

Purpose: To compare critical speed (CS) measured from a single-visit field test of the distance–time relationship with the “traditional” treadmill time-to-exhaustion multivisit protocol. Methods: Ten male distance runners completed treadmill and field tests to calculate CS and the maximum distance performed above CS (D′). The field test involved 3 runs on a single visit to an outdoor athletics track over 3600, 2400, and 1200 m. Two field-test protocols were evaluated using either a 30-min recovery or a 60-min recovery between runs. The treadmill test involved runs to exhaustion at 100%, 105%, and 110% of velocity at VO2max, with 24 h recovery between runs. Results: There was no difference in CS measured with the treadmill and 30-min- and 60-minrecovery field tests (P .05). A typical error of the estimate of 0.14 m/s (95% confidence limits 0.09–0.26 m/s) was seen for CS and 88 m (95% confidence limits 60–169 m) for D′. A coefficient of variation of 0.4% (95% confidence limits: 0.3–0.8%) was found for repeat tests of CS and 13% (95% confidence limits 10–27%) for D′. Conclusion: The single-visit method provides a useful alternative for assessing CS in the field

Mild hypohydration increases the frequency of driver errors during a prolonged, monotonous driving task

The aim of the present study was to examine the effect of mild hypohydration on performance during a prolonged, monotonous driving task. Methods: Eleven healthy males (age 22 ± 4 y) were instructed to consume a volume of fluid in line with published guidelines (HYD trial) or 25% of this intake (FR trial) in a crossover manner. Participants came to the laboratory the following morning after an overnight fast. One hour following a standard breakfast, a 120 min driving simulation task began. Driver errors, including instances of lane drifting or late breaking, EEG and heart rate were recorded throughout the driving task. Results: Pre-trial bodymass (P=0.692), urine osmolality (P=0.838) and serumosmolality (P=0.574)were the same on both trials. FR resulted in a 1.1±0.7% reduction in bodymass, compared to−0.1±0.6% in the HYD trial (P = 0.002). Urine and serum osmolality were both increased following FR (P b 0.05). There was a progressive increase in the total number of driver errors observed during both the HYD and FR trials, but significantly more incidents were recorded throughout the FR trial (HYD 47 ± 44, FR 101 ± 84; ES = 0.81; P = 0.006). Conclusions: The results of the present study suggest that mild hypohydration, produced a significant increase in minor driving errors during a prolonged, monotonous drive, compared to that observed while performing the same task in a hydrated condition. The magnitude of decrement reported,was similar to that observed following the ingestion of an alcoholic beverage resulting in a blood alcohol content of approximately 0.08% (the current UK legal driving limit), or while sleep deprived

ARL3 mutations cause Joubert syndrome by disrupting ciliary protein composition

Joubert syndrome (JBTS) is a genetically heterogeneous autosomal recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTSrelated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997-109106128 (hg 19)), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP Ribosylation Factor-Like GTPase 3, ARL3, is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localisation in cilia. We propose that ARL3 provides a potential hub in the network of encoded ciliopathy genes, whereby perturbation of ARL3 results in the mislocalisation of multiple ciliary proteins due to abnormal displacement of lipidated protein cargo

Development of a regional glycerol dialkyl glycerol tetraether (GDGT)-temperature calibration for Antarctic and sub-Antarctic lakes

A regional network of quantitative reconstructions of past climate variability is required to test climate models. In recent studies, temperature calibration models based on the relative abundances of sedimentary glycerol dialkyl glycerol tetraethers (GDGTs) have enabled past temperature reconstructions in both marine and terrestrial environments. Nevertheless, to date these methods have not been widely applied in high latitude environments due to poor performance of the GDGT–temperature calibrations at lower temperatures. To address this we studied 32 lakes from Antarctica, the sub-Antarctic Islands and Southern Chile to: 1) quantify their GDGT composition and investigate the environmental controls on GDGT composition; and 2) develop a GDGT–temperature calibration model for inferring past temperatures from Antarctic and sub-Antarctic lakes. GDGTs were found in all 32 lakes studied and in 31 lakes branched GDGTs (brGDGTs) were the dominant compounds. Statistical analyses of brGDGT composition in relation to temperature, pH, conductivity and water depth showed that the composition of brGDGTs is strongly correlated with mean summer air temperature (MSAT). This enabled the development of the first regional brGDGT–temperature calibration for use in Antarctic and sub-Antarctic lakes using four brGDGT compounds (GDGT-Ib, GDGT-II, GDGT-III and GDGT-IIIb). A key discovery was that GDGT-IIIb is of particular importance in cold lacustrine environments. The addition of this compound significantly improved the model's performance from r2=0.67r2=0.67, RMSEP-LOO (leave-one-out) = 2.23 °C, RMSEP-H (h-block) = 2.37 °C when applying the re-calibrated global GDGT–temperature calibration to our Antarctic dataset to r2=0.83r2=0.83, RMSEP-LOO = 1.68 °C, RMSEP-H = 1.65 °C for our new Antarctic calibration. This shows that Antarctic and sub-Antarctic, and possibly other high latitude, palaeotemperature reconstructions should be based on a regional GDGT–temperature calibration where specific compounds can be identified and included to improve model performance. Finally, downcore temperature reconstructions using the new Antarctic brGDGT–temperature calibration were tested in sub-Antarctic Fan Lake from South Georgia providing a proof of concept for the new calibration model in the Southern Hemisphere