An alternate proton acceptor for excited-state proton transfer in green fluorescent protein: Rewiring GFP

The neutral form of the chromophore in wild-type green fluorescent protein (wtGFP) undergoes excited-state proton transfer (ESPT) upon excitation, resulting in characteristic green (508 nm) fluorescence. This ESPT reaction involves a proton relay from the phenol hydroxyl of the chromophore to the ionized side chain of E222, and results in formation of the anionic chromophore in a protein environment optimized for the neutral species (the I* state). Reorientation or replacement of E222, as occurs in the S65T and E222Q GFP mutants, disables the ESPT reaction and results in loss of green emission following excitation of the neutral chromophore. Previously, it has been shown that the introduction of a second mutation (H148D) into S65T GFP allows the recovery of green emission, implying that ESPT is again possible. A similar recovery of green fluorescence is also observed for the E222Q/H148D mutant, suggesting that D148 is the proton acceptor for the ESPT reaction in both double mutants. The mechanism of fluorescence emission following excitation of the neutral chromophore in S65T/H148D and E222Q/H148D has been explored through the use of steady state and ultrafast time-resolved fluorescence and vibrational spectroscopy. The data are contrasted with those of the single mutant S65T GFP. Time-resolved fluorescence studies indicate very rapid (<1 ps) formation of I* in the double mutants, followed by vibrational cooling on the picosecond time scale. The time-resolved IR difference spectra are markedly different to those of wtGFP or its anionic mutants. In particular, no spectral signatures are apparent in the picosecond IR difference spectra that would correspond to alteration in the ionization state of D148, leading to the proposal that a low-barrier hydrogen bond (LBHB) is present between the phenol hydroxyl of the chromophore and the side chain of D148, with different potential energy surfaces for the ground and excited states. This model is consistent with recent high-resolution structural data in which the distance between the donor and acceptor oxygen atoms is =2.4 Å. Importantly, these studies indicate that the hydrogen-bond network in wtGFP can be replaced by a single residue, an observation which, when fully explored, will add to our understanding of the various requirements for proton-transfer reactions within proteins

Complete Proton Transfer Cycle in GFP and Its T203V and S205V Mutants

Proton transfer is critical in many important biochemical reactions. The unique three‐step excited‐state proton transfer in avGFP allows observations of protein proton transport in real‐time. In this work we exploit femtosecond to microsecond transient IR spectroscopy to record, in D2O, the complete proton transfer photocycle of avGFP, and two mutants (T203V and S205V) which modify the structure of the proton wire. Striking differences and similarities are observed among the three mutants yielding novel information on proton transfer mechanism, rates, isotope effects, H‐bond strength and proton wire stability. These data provide a detailed picture of the dynamics of long‐range proton transfer in a protein against which calculations may be compared

Social Interaction and Communities of Practice in Formative Period NW Argentina: A Multi-analytical Study of Ceramics

This is the author accepted manuscript. The final version is available from University of New Mexico Press via the link in this recordThe study of long-distance exchange of goods and resources has long been central to the understanding of socio-political and cultural complexity in the south-central Andes. Traditional studies have emphasized typological similarities to reconstruct regional networks, proposing the dominance of different centres through time. While these approaches were informative on the general direction of contacts, the nature and scale of interaction has remained speculative. This chapter summarises the latest results of our ongoing research project on long-distance circulation of archaeological materials in northwestern Argentina during part of the Formative Period (ca. 1500 BC-AD 1000). The study applied a multianalytical methodological strategy integrating archaeological analysis with archaeometric techniques, including thin section petrography, instrumental neutron activation analysis (INAA) and laser ablation inductively coupled massspectometry (LA-ICP-MS) in order to contribute an evidence-based holistic view of preHispanic exchange networks. The study examined materials traditionally studied separately, including 542 ceramic samples and 113 obsidian and volcanic rock artifacts, from seven sectors in the semi-arid valleys area. We summarize here the results of the ceramic analysis, showing the 19-2 following trends: (1) inter-valley heterogeneity of clay and fabrics for ordinary wares; (2) intervalley homogeneity of clay and fabrics for a wide range of decorated wares; (3) selective circulation of two distinct polychrome wares. These trends reflect the complex inter-community relationships experienced in small-scale societies. The study offers a new platform to model ancient exchange, and circulation and interaction more broadly, based on actual material transfers. The results call for the re-examination of the centralized models of exchange and interaction that are often drawn upon to account for emergent cultural complexity in the past, both in the Andes and beyond.British AcademyArts and Humanities Research Council (AHRC)Argentinean National Agency for Science and Technology (ANPCyT)Raíces ProgramArgentinian National Council for Scientific and Technical Research (CONICET

Poor sleep quality, antepartum depression and suicidal ideation among pregnant women.

To evaluate the independent and combined associations of maternal self-reported poor sleep quality and antepartum depression with suicidal ideation during the third trimester METHODS: A cross-sectional study was conducted among 1298 pregnant women (between 24 and 28 gestational weeks) attending prenatal clinics in Lima, Peru. Antepartum depression and suicidal ideation were assessed using the Patient Health Questionnaire-9 (PHQ-9). The Pittsburgh Sleep Quality Index (PSQI) questionnaire was used to assess sleep quality. Multivariate logistical regression procedures were used to estimate odds ratios (OR) and 95% confidence intervals (95% CI) after adjusting for putative confounders. Approximately, 17% of women were classified as having poor sleep quality (defined using the recommended criteria of PSQI global score of &gt;5 vs. ≤5). Further, the prevalence of antepartum depression and suicidal ideation were 10.3% and 8.5%, respectively in this cohort. After adjusting for confounders including depression, poor sleep quality was associated with a 2.81-fold increased odds of suicidal ideation (OR=2.81; 95% CI 1.78-4.45). When assessed as a continuous variable, each 1-unit increase in the global PSQI score resulted in a 28% increase in odds for suicidal ideation, even after adjusting for depression (OR=1.28; 95% CI 1.15-1.41). The odds of suicidal ideation was particularly high among depressed women with poor sleep quality (OR=13.56 95% CI 7.53-24.41) as compared with women without either risk factor. This cross-sectional study utilized self-reported data. Causality cannot be inferred, and results may not be fully generalizable. Poor sleep quality, even after adjusting for depression, is associated with antepartum suicidal ideation. Our findings support the need to explore sleep-focused interventions for pregnant women

Quantitative Microscopy of Hepatic Changes Induced by Phenethyl Isothiocyanate in Fischer-344 Rats Fed Either a Cereal-Based Diet or a Purified Diet

Hepatic changes induced by phenethyl isothiocyanate (PEITC) in the liver of rats were determined by quantitative microscopy. Groups of male Fischer-344 rats were fed either a standard, cereal-based diet (Wayne rodent meal) or a purified diet (AIN-76A) containing PEITC at concentrations of 0.75 and 6.0 mmol/kg for 13 wk. Severe hepatic lipidosis was observed in control rats fed the purified diet. Addition of PEITC to the purified diet significantly reduced lipid content in hepatocytes. In contrast, lipid content in the liver of the rats fed the cereal-based diet containing PEITC was greater than in control rats maintained on the same diet. In addition, dose-related reductions in hepatocyte, lipid droplet, peroxisome, and mitochondrial volumes were observed in PEITC-treated rats fed the cereal-based diet. These results indicate that PEITC exerts differential effects on the liver of rats fed either the cereal-based or purified diet.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/68493/2/10.1177_019262339502300602.pd

Photoacid behaviour in a fluorinated green fluorescent protein chromophore:Ultrafast formation of anion and zwitterion states

The photophysics of the chromophore of the green fluorescent protein in Aequorea victoria (avGFP) are dominated by an excited state proton transfer reaction. In contrast the photophysics of the same chromophore in solution are dominated by radiationless decay, and photoacid behaviour is not observed. Here we show that modification of the pKa of the chromophore by fluorination leads to an excited state proton transfer on an extremely fast (50 fs) time scale. Such a fast rate suggests a barrierless proton transfer and the existence of a pre-formed acceptor site in the aqueous solution, which is supported by solvent and deuterium isotope effects. In addition, at lower pH, photochemical formation of the elusive zwitterion of the GFP chromophore is observed by means of an equally fast excited state proton transfer from the cation. The significance of these results for understanding and modifying the properties of fluorescent proteins are discusse

Leg Fidgeting Improves Executive Function following Prolonged Sitting with a Typical Western Meal: A Randomized, Controlled Cross-Over Trial.

Prolonged uninterrupted sitting and a typical Western meal, high in fat and refined sugar, can additively impair cognitive and cerebrovascular functions. However, it is unknown whether interrupting these behaviours, with a simple desk-based activity, can attenuate the impairment. The aim of this study was to determine whether regular leg fidgeting can off-set the detrimental effects of prolonged sitting following the consumption of a typical Western meal, on executive and cerebrovascular function. Using a randomized cross-over design, 13 healthy males consumed a Western meal and completed 180-min of prolonged sitting with leg fidgeting of 1 min on/4 min off (intervention [INT]) and without (control [CON]). Cognitive function was assessed pre and post sitting using the Trail Maker Test (TMT) parts A and B. Common carotid artery (CCA) blood flow, as an index of brain flow, was measured pre and post, and cerebral (FP1) perfusion was measured continuously. For TMT B the CON trial signifi-cantly increased (worsened) completion time (mean difference [MD]=5.2s, d= 0.38), the number of errors (MD=3.33, d= 0.68) and cognitive fatigue (MD=0.73, d= 0.92). Compared to CON, the INT trial significantly improved completion time (MD=2.3s, d= 0.97), and prevented declines in cognitive fatigue and a reduction in the number of errors. No significant changes in cerebral perfusion or CCA blood flow were found. Leg fidgeting for 1-min on/4-min off following a meal high in fats and refined sugars attenuated the impairment in executive function. This attenuation in executive function may not be caused by al-terations in CCA blood flow or cerebral perfusion

Relationship between Spatial Working Memory Performance and Diet Specialization in Two Sympatric Nectar Bats

Behavioural ecologists increasingly recognise spatial memory as one the most influential cognitive traits involved in evolutionary processes. In particular, spatial working memory (SWM), i.e. the ability of animals to store temporarily useful information for current foraging tasks, determines the foraging efficiency of individuals. As a consequence, SWM also has the potential to influence competitive abilities and to affect patterns of sympatric occurrence among closely related species. The present study aims at comparing the efficiency of SWM between generalist (Glossophaga soricina) and specialist (Leptonycteris yerbabuenae) nectarivorous bats at flowering patches. The two species differ in diet – the generalist diet including seasonally fruits and insects with nectar and pollen while the specialist diet is dominated by nectar and pollen yearlong – and in some morphological traits – the specialist being heavier and with proportionally longer rostrum than the generalist. These bats are found sympatrically within part of their range in the Neotropics. We habituated captive individuals to feed on artificial flower patches and we used infrared video recordings to monitor their ability to remember and avoid the spatial location of flowers they emptied in previous visits in the course of 15-min foraging sequences. Experiments revealed that both species rely on SWM as their foraging success attained significantly greater values than random expectations. However, the nectar specialist L. yerbabuenae was significantly more efficient at extracting nectar (+28% in foraging success), and sustained longer foraging bouts (+27% in length of efficient foraging sequences) than the generalist G. soricina. These contrasting SWM performances are discussed in relation to diet specialization and other life history traits

Integral Relaxation Time of Single-Domain Ferromagnetic Particles

The integral relaxation time \tau_{int} of thermoactivating noninteracting single-domain ferromagnetic particles is calculated analytically in the geometry with a magnetic field H applied parallel to the easy axis. It is shown that the drastic deviation of \tau_{int}^{-1} from the lowest eigenvalue of the Fokker-Planck equation \Lambda_1 at low temperatures, starting from some critical value of H, is the consequence of the depletion of the upper potential well. In these conditions the integral relaxation time consists of two competing contributions corresponding to the overbarrier and intrawell relaxation processes.Comment: 8 pages, 3 figure