Mapping uterine calcium dynamics during the ovulatory cycle in live mice.

Uterine contraction patterns vary during the ovulatory cycle and throughout pregnancy but prior measurements have produced limited and conflicting information on these patterns. We combined a virally delivered genetically encoded calcium reporter (GCaMP8m) and ultra-widefield imaging in live nonpregnant mice to characterize uterine calcium dynamics at organ scale throughout the estrous cycle. Prior to ovulation (proestrus and estrus) uterine excitations primarily initiated in a region near the oviduct, but after ovulation (metestrus and diestrus), excitations initiated at loci homogeneously distributed throughout the organ. The frequency of excitation events was lowest in proestrus and estrus, higher in metestrus and highest in diestrus. These results establish a platform for mapping uterine activity, and show that the question of whether there is an anatomically localized trigger for uterine excitations depends on the estrous cycle phase.

Non-Destructive Monitoring of Foliar Fungicide Efficacy with Hyperspectral Sensing in Grapevine.

Frequent fungicide applications are required to manage grapevine powdery mildew (Erysiphe necator). However, this practice is costly and has led to widespread fungicide resistance. A method of monitoring in-field fungicide efficacy could help growers maximize spray-interval length, thereby reducing costs and the rate of fungicide resistance emergence. The goal of this study was to evaluate if hyperspectral sensing in the visible to shortwave infrared range (400 to 2,400 nm) can quantify foliar fungicide efficacy on grape leaves. Commercial formulations of metrafenone, Bacillus mycoides isolate J (BmJ), and sulfur were applied on Chardonnay grapevines in vineyard or greenhouse settings. Foliar reflectance was measured with handheld hyperspectral spectroradiometers at multiple days post-application. Fungicide efficacy was estimated as a proxy for fungicide residue and disease control measured with the Blackbird microscopy imaging robot. Treatments could be differentiated from the untreated control with an accuracy of 73.06% for metrafenone, 67.76% for BmJ, and 94.10% for sulfur. The change in spectral reflectance was moderately correlated with the cube root of the area under the disease progress curve for metrafenone- and sulfur-treated samples (R2 = 0.38 and 0.36, respectively) and with sulfur residue (R2 = 0.42). BmJ treatment impacted foliar physiology by enhancing the leaf mass/area and reducing the nitrogen and total phenolic content as estimated from spectral reflectance. The results suggest that hyperspectral sensing can be used to monitor in-situ fungicide efficacy, and the prediction accuracy depends on the fungicide and the time point measured. The ability to monitor in-situ fungicide efficacy could facilitate more strategic fungicide applications and promote sustainable grapevine protection. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Identification of genetic loci associated with five agronomic traits in alfalfa using multi-environment trials.

KEY MESSAGE: The use of multi-environment trials to test yield-related traits in a diverse alfalfa panel allowed to find multiple molecular markers associated with complex agronomic traits. Yield is one of the most important target traits in alfalfa breeding; however, yield is a complex trait affected by genetic and environmental factors. In this study, we used multi-environment trials to test yield-related traits in a diverse panel composed of 200 alfalfa accessions and varieties. Phenotypic data of maturity stage measured as mean stage by count (MSC), dry matter content, plant height (PH), biomass yield (Yi), and fall dormancy (FD) were collected in three locations in Idaho, Oregon, and Washington from 2018 to 2020. Single-trial and stagewise analyses were used to obtain estimated trait means of entries by environment. The plants were genotyped using a genotyping by sequencing approach and obtained a genotypic matrix with 97,345 single nucleotide polymorphisms. Genome-wide association studies identified a total of 84 markers associated with the traits analyzed. Of those, 29 markers were in noncoding regions and 55 markers were in coding regions. Ten significant SNPs at the same locus were associated with FD and they were linked to a gene annotated as a nuclear fusion defective 4-like (NFD4). Additional SNPs associated with MSC, PH, and Yi were annotated as transcription factors such as Cysteine3Histidine (C3H), Hap3/NF-YB family, and serine/threonine-protein phosphatase 7 proteins, respectively. Our results provide insight into the genetic factors that influence alfalfa maturity, yield, and dormancy, which is helpful to speed up the genetic gain toward alfalfa yield improvement.

Effects of Nighttime Applications of Germicidal Ultraviolet Light Upon Powdery Mildew (Erysiphe necator), Downy Mildew (Plasmopara viticola), and Sour Rot of Grapevine.

Nighttime applications of germicidal ultraviolet were evaluated as a means to suppress three diseases of grapevine. In laboratory studies, UV-C light (peak 254 nm, FWHM 5 nm) applied during darkness strongly inhibited the germination of conidia of Erysiphe necator, and at a dose of 200 J/m2, germination was zero. Reciprocity of irradiance and duration of exposure with respect to conidial germination was confirmed for UV-C doses between 0 and 200 J/m2 applied at 4 or 400 s. When detached grapevine leaves were exposed during darkness to UV-C at 100 J/m2 up to 7 days before they were inoculated with zoospores of Plasmopara viticola, infection and subsequent sporulation was reduced by over 70% compared to untreated control leaves, indicating an indirect suppression of the pathogen exerted through the host. A hemicylindrical array of low-pressure discharge UV-C lamps configured for trellised grapevines was designed and fitted to both a tractor-drawn carriage and a fully autonomous robotic carriage for vineyard applications. In 2019, in a Chardonnay research vineyard with a history of high inoculum and severe disease, weekly nighttime applications of UV-C suppressed E. necator on leaves and fruit at doses of 100 and 200 J/m2. In the same vineyard in 2020, UV-C was applied once or twice weekly at doses of 70, 100, or 200 J/m2, and severity of E. necator on both leaves and fruit was significantly reduced compared to untreated controls; twice-weekly applications at 200 J/m2 provided suppression equivalent to a standard fungicide program. None of the foregoing UV-C treatments significantly reduced the severity of P. viticola on Chardonnay vines compared to the untreated control in 2020. However, twice-weekly applications of UV-C at 200 J/m2 to the more downy mildew-resistant Vitis interspecific hybrid cultivar Vignoles in 2021 significantly suppressed foliar disease severity. In commercial Chardonnay vineyards with histories of excellent disease control in Dresden, NY, E. necator remained at trace levels on foliage and was zero on fruit following weekly nighttime applications of UV-C at 200 J/m2 in 2020 and after weekly or twice-weekly application of UV-C at 100 or 200 J/m2 in 2021. In 2019, weekly nighttime applications of UV-C at 200 J/m2 also significantly reduced the severity of sour rot, a decay syndrome of complex etiology, on fruit of 'Vignoles' but not the severity of bunch rot caused by Botrytis cinerea. A similar level of suppression of sour rot was observed on 'Vignoles' vines treated twice-weekly with UV-C at 200 J/m2 in 2021. Nighttime UV-C applications did not produce detectable indications of metabolic abnormalities, phytotoxicity, growth reduction, or reductions of fruit yield or quality parameters, even at the highest doses and most frequent intervals employed.

Deep semantic segmentation for the quantification of grape foliar diseases in the vineyard.

Plant disease evaluation is crucial to pathogen management and plant breeding. Human field scouting has been widely used to monitor disease progress and provide qualitative and quantitative evaluation, which is costly, laborious, subjective, and often imprecise. To improve disease evaluation accuracy, throughput, and objectiveness, an image-based approach with a deep learning-based analysis pipeline was developed to calculate infection severity of grape foliar diseases. The image-based approach used a ground imaging system for field data acquisition, consisting of a custom stereo camera with strobe light for consistent illumination and real time kinematic (RTK) GPS for accurate localization. The deep learning-based pipeline used the hierarchical multiscale attention semantic segmentation (HMASS) model for disease infection segmentation, color filtering for grapevine canopy segmentation, and depth and location information for effective region masking. The resultant infection, canopy, and effective region masks were used to calculate the severity rate of disease infections in an image sequence collected in a given unit (e.g., grapevine panel). Fungicide trials for grape downy mildew (DM) and powdery mildew (PM) were used as case studies to evaluate the developed approach and pipeline. Experimental results showed that the HMASS model achieved acceptable to good segmentation accuracy of DM (mIoU > 0.84) and PM (mIoU > 0.74) infections in testing images, demonstrating the model capability for symptomatic disease segmentation. With the consistent image quality and multimodal metadata provided by the imaging system, the color filter and overlapping region removal could accurately and reliably segment grapevine canopies and identify repeatedly imaged regions between consecutive image frames, leading to critical information for infection severity calculation. Image-derived severity rates were highly correlated (r > 0.95) with human-assessed values, and had comparable statistical power in differentiating fungicide treatment efficacy in both case studies. Therefore, the developed approach and pipeline can be used as an effective and efficient tool to quantify the severity of foliar disease infections, enabling objective, high-throughput disease evaluation for fungicide trial evaluation, genetic mapping, and breeding programs.

Alfalfa biotypes with putative enhanced cell wall digestibility and effects on performance of growing beef steers.

Three alfalfa biotypes were chosen based on the presumption that they would be sources of alfalfa herbage that differed in lignin concentration and therefore cell wall digestibility. The hypothesis was that a lesser lignin concentration would result in greater alfalfa neutral detergent fiber (NDF) digestibility and greater beef steer growth performance. The three alfalfa biotypes were HarvXtra (Forage Genetics International), Hi-Gest 360 (Alforex Seeds), and a control alfalfa, LegenDairy XHD (Winfield Solutions LLC). High-moisture wrapped bales were prepared from second-harvest, d 30 crops. Digestibility of NDF was determined using in vitro incubations and a steer digestibility trial. Alfalfa baleage and trace mineral salt were fed to Angus steers (300 kg initial body weight, 4 pens/treatment) in an 83-day growing-phase trial. Alfalfa acid detergent lignin concentrations were 75.6, 71.8, and 63.0 g/kg dry matter (P = 0.34) for LegenDairy, Hi-Gest and HarvXtra, respectively. Based on in vitro total-tract NDF digestibility coefficients, HarvXtra tended (P ≥ 0.09) to have the highest NDF digestibility. Alfalfa biotype affected in vivo apparent total tract digestibility of NDF (P < 0.001) and there was a trend for an effect on acid detergent fiber digestibility (P = 0.051). Hi-Gest and HarvXtra had similar in vivo apparent NDF digestibilities, which were greater than for LegenDairy (P < 0.05). There was no alfalfa biotype effect on daily alfalfa dry matter intake (DMI; P = 0.51) or average daily gain (P = 0.25) by growing steers. The absence of an effect by the novel alfalfa biotypes on DMI by growing steers suggests that the compositional and digestibility differences of the novel alfalfa biotypes compared to LegenDairy were not sufficient to alleviate the limitation of physical fill (if evident) on DMI. If more disparity in cell wall composition and NDF digestibility were to exist between control and reduced-lignin biotypes, then perhaps an advantage in cattle growth performance for a reduced-lignin alfalfa biotype would be detectable.

Reduced Infestation by Xylosandrus germanus (Coleoptera: Curculionidae: Scolytinae) in Apple Trees Treated with Host Plant Defense Compounds.

The ambrosia beetle Xylosandrus germanus (Blandford) is an invasive pest that has caused tree decline and death in numerous NY dwarf apple orchards during the past ten years, despite efforts to control them using trunk sprays of chlorpyrifos or pyrethroids, either alone or combined with the repellent verbenone. From 2017 to 2019, we tested trunk applications of different repellents and plant defense compounds for protection against X. germanus in potted apple trees adjacent to infested orchards. Treatments included topical formulations of verbenone and methyl salicylate (MeSa), alone and in combination, at different rates and timings. Additional treatments evaluated included the systemic acquired resistance activators acibenzolar-S-methyl, Reynoutria sachalinensis extract, and salicylic acid. The combination verbenone+MeSa treatments had the lowest incidences of attack sites and galleries containing adults or brood, although results varied among years. In a separate trial, we found no significant difference in numbers of adults caught in ethanol-baited traps placed 5-20 m from an apple bolt treated with the verbenone+MeSa repellent, suggesting that the repellent's effect did not extend to those distances from the treated target. Cross-sectional discs of trunk tissue sampled in August were analyzed for levels of phytohormones. Quantities of ergosterol, abscissic acid, salicylic acid, jasmonic acid, methyl salicylate, methyl jasmonate, trans-cinnamic acid, and indole-3-cinnamic acid did not significantly vary across treatments; however, trees with greater beetle damage contained higher levels of jasmonic and salicylic acid, which are key molecules in plant defense pathways.

Genome-Wide Association Studies Identifying Multiple Loci Associated With Alfalfa Forage Quality.

Autotetraploid alfalfa is a major hay crop planted all over the world due to its adaptation in different environments and high quality for animal feed. However, the genetic basis of alfalfa quality is not fully understood. In this study, a diverse panel of 200 alfalfa accessions were planted in field trials using augmented experimental design at three locations in 2018 and 2019. Thirty-four quality traits were evaluated by Near Infrared Reflectance Spectroscopy (NIRS). The plants were genotyped using a genotyping by sequencing (GBS) approach and over 46,000 single nucleotide polymorphisms (SNPs) were obtained after variant calling and filtering. Genome-wide association studies (GWAS) identified 28 SNP markers associated with 16 quality traits. Among them, most of the markers were associated with fiber digestibility and protein content. Phenotypic variations were analyzed from three locations and different sets of markers were identified by GWAS when using phenotypic data from different locations, indicating that alfalfa quality traits were also affected by environmental factors. Among different sets of markers identified by location, two markers were associated with nine traits of fiber digestibility. One marker associated with lignin content was identified consistently in multiple environments. Putative candidate genes underlying fiber-related loci were identified and they are involved in the lignin and cell wall biosynthesis. The DNA markers and associated genes identified in this study will be useful for the genetic improvement of forage quality in alfalfa after the validation of the markers.

Validation of algorithms to identify adverse perinatal outcomes in the Medicaid Analytic Extract database.

BACKGROUND: The Medicaid Analytic eXtract (MAX) is a health care utilization database from publicly insured individuals that has been used for studies of drug safety in pregnancy. Claims-based algorithms for defining many important maternal and neonatal outcomes have not been validated. OBJECTIVE: To validate claims-based algorithms for identifying selected pregnancy outcomes in MAX using hospital medical records. METHODS: The medical records of mothers who delivered between 2000 and 2010 within a single large healthcare system were linked to their claims in MAX. Claims-based algorithms for placental abruption, preeclampsia, postpartum hemorrhage, small for gestational age, and noncardiac congenital malformation were defined. Fifty randomly sampled cases for each outcome identified using these algorithms were selected, and their medical records were independently reviewed by two physicians to confirm the presence of the diagnosis of interest; disagreements were resolved by a third physician reviewer. Positive predictive values (PPVs) and 95% confidence intervals (CIs) of the claims-based algorithms were calculated using medical records as the gold standard. RESULTS: The linked cohort included 10,899 live-birth pregnancies. The PPV was 92% (95% CI, 82%-97%) for placental abruption, 82% (95% CI, 70%-91%) for preeclampsia, 74% (95% CI, 61%-85%) for postpartum hemorrhage, 92% (95% CI, 82%-97%) for small for gestational age, and 86% (95% CI, 74%-94%) for noncardiac congenital malformation. CONCLUSIONS: Across the perinatal outcomes considered, PPVs ranged between 74% and 92%. These PPVs can inform bias analyses that correct for outcome misclassification.

Effect of milk feeding strategy and lactic acid probiotics on growth and behavior of dairy calves fed using an automated feeding system1.

Automated milk feeders offer flexibility to feed calves high milk allowances, to change the daily quantity of milk offered, and also to dispense additives like probiotics on an individual basis. Our objectives were to test the effects of 2 milk feeding protocols and a lactic acid bacterium probiotic on performance and behavior in calves. Heifer dairy calves (n = 96) were enrolled at birth in a 2 × 2 factorial study design comparing feeding (1) 2 milk feeding protocols and (2) a lactic acid bacterium-based probiotic program, or a placebo, using automated milk feeders. The early milk feeding strategy (EM) offered a maximum of 11 L/d on day 1 and a peak maximum allowance of 15 L/d on day 21. The late milk feeding strategy (LM) offered a maximum of 7 L/d on day 1 and increased slowly to its peak at 13 L/d on day 28. Both feeding strategies gradually weaned the calves after peak milk allowance until complete weaning at day 53, offering a total of 543 liters of milk. Probiotics or placebo were fed orally in a gel once after colostrum, and twice daily in the milk until weaning. Water and calf starter were provided ad libitum. The experimental period was divided into 3 periods: from day 1 on the automated feeder to day 28 (Period 1), from day 29 to day 53 (Period 2), and the week post-weaning (Period 3). For Period 1, the average daily gain (ADG) of the probiotic group was greater than that of the placebo group (0.84 ± 0.10 kg/d vs. 0.74 ± 0.10 kg/d, respectively), but was not different between milk feeding strategies. For Period 2, ADG was not affected by probiotic or milk feeding strategies. For Period 3, ADG was greater for EM compared to LM (1.27 ± 0.10 kg/d vs. 1.02 ± 0.10 kg/d, respectively), but not between probiotic and placebo groups. During the whole experimental period, LM calves consumed significantly more milk than the EM calves (431.84 ± 33.0 liters vs. 378.64 ± 34.2 liters, respectively). During Period 3, probiotics affected the frequency of visits to the calf starter feed bunk (37.72 ± 2.8 vs. 23.27 ± 2.8 visits per day for probiotic and placebo groups, respectively), but did not affect total time spent at the feed bunk. The supplementation of a lactic acid-based probiotic improved ADG during early life and altered some aspects of the feeding behavior of dairy calves. Calves receiving an early accelerated milk allowance had improved growth during post-weaning and consumed less milk in total, which may indicate better use of solid feed.

Xylosandrus germanus (Coleoptera: Curculionidae: Scolytinae) Occurrence, Fungal Associations, and Management Trials in New York Apple Orchards.

Xylosandrus germanus (Blandford) has caused increasing damage in high-density New York apple orchards since 2013, resulting in tree decline and death. We documented their occurrence and timing in > 50 orchards using ethanol-baited traps from 2014 to 2016. First captures ranged from 48 to 83 degree days (base 10 °C) from 1 January. Captures were numerically higher at the orchard-woods interface than within the orchard interior, but differences were not significant in locations with lower populations. Control using insecticide trunk sprays was tested in potted, waterlogged apple trees placed in orchards and nurseries, and inside wooded areas adjacent to orchards. A verbenone repellent was used in combination with trunk sprays to improve control. Overall, insecticide sprays were inconsistent and marginal in preventing new infestations. Chlorpyrifos significantly reduced infestations versus lambda-cyhalothrin and untreated trees at one location in the 2015 orchard trials, and versus untreated trees at one location in the 2016 nursery trials, but otherwise performed no better than other treatments. The addition of verbenone to either the check or permethrin treatments resulted in significantly fewer attack sites containing brood at one orchard site in 2016. Chlorpyrifos, lambda-cyhalothrin, and permethrin significantly reduced the number of attack sites containing adults compared with untreated trees at one nursery trial location in 2016, but were otherwise ineffective in reducing numbers of trees in other locations and infestation categories. We found several fungal and bacterial species associated with X. germanus and its infestation of apples. These microbes likely play a minimal role in apple decline.

Inclusion of Specialist and Generalist Stimuli in Attract-and-Kill Programs: Their Relative Efficacy in Apple Maggot Fly (Diptera: Tephritidae) Pest Management.

Investigating the chemical ecology of agricultural systems continues to be a salient part of integrated pest management programs. Apple maggot fly, a key pest of apple in eastern North America, is a visual specialist with attraction to host fruit-mimicking cues. These cues have been incorporated into red spherical traps used for both monitoring and behaviorally based management. Incorporating generalist or specialist olfactory cues can potentially increase the overall success of this management system. The primary aim of this study was to evaluate the attractiveness of a generalist olfactory cue, ammonium carbonate, and the specialist olfactory cue, a five-component apple volatile blend, when included as a component of a red attracticidal sphere system. Secondly, we assessed how critical it was to maintain minimal deviation from the optimal, full-round specialist visual stimulus provided by red spheres. Finally, attracticidal spheres were deployed with specialist olfactory cues in commercial apple orchards to evaluate their potential for effective management of apple maggot. Ammonium carbonate did not increase residency, feeding time, or mortality in the laboratory-based trials. Field deployment of specialist olfactory cues increased apple maggot captures on red spheres, while the generalist cue did not. Apple maggot tolerated some deviation from the optimal visual stimulus without reducing captures on red spheres. Attracticidal spheres hung in perimeter trees in orchards resulted in acceptable and statistically identical levels of control compared with standard insecticide programs used by growers. Overall, our study contributes valuable information for developing a reliable attract-and-kill system for apple maggot.

Sphingomyelinase D inhibits store-operated Ca2+ entry in T lymphocytes by suppressing ORAI current.

Infections caused by certain bacteria including Mycobacterium tuberculosis and Corynebacterium pseudotuberculosis provoke inflammatory responses characterized by the formation of granulomas with necrotic foci-so-called caseous necrosis. The granulomas of infected animals show prominent infiltration by T lymphocytes, and T cell depletion increases host mortality. Notorious zoonotic C. pseudotuberculosis secretes sphingomyelinase (SMase) D, a phospholipase that cleaves off the choline moiety of sphingomyelin, a phospholipid found primarily in the outer leaflet of host cell plasma membranes. Experimental C. pseudotuberculosis strains that lack SMase D are markedly less infectious and unable to spread in hosts, indicating that this enzyme is a crucial virulence factor for sustaining the caseous lymphadenitis infections caused by this microbe. However, the molecular mechanism by which SMase D helps bacteria evade the host's immune response remains unknown. Here, we find that SMase D inhibits store-operated Ca(2+) entry (SOCE) in human T cells and lowers the production of the SOCE-dependent cytokines interleukin-2, which is critical for T cell growth, proliferation, and differentiation, and tumor necrosis factor α, which is crucial for the formation and maintenance of granulomas in microbial infections. SMase D inhibits SOCE through a previously unknown mechanism, namely, suppression of Orai1 current, rather than through altering gating of voltage-gated K(+) channels. This finding suggests that, whereas certain genetic mutations abolish Orai1 activity causing severe combined immunodeficiency (SCID), bacteria have the ability to suppress Orai1 activity with SMase D to create an acquired, chronic SCID-like condition that allows persistent infection. Thus, in an example of how virulence factors can disrupt key membrane protein function by targeting phospholipids in host cell membranes, our study has uncovered a novel molecular mechanism that bacteria can use to thwart host immunity.

Who is most vulnerable to social rejection? The toxic combination of low self-esteem and lack of negative emotion differentiation on neural responses to rejection.

People have a fundamental need to belong that, when satisfied, is associated with mental and physical well-being. The current investigation examined what happens when the need to belong is thwarted-and how individual differences in self-esteem and emotion differentiation modulate neural responses to social rejection. We hypothesized that low self-esteem would predict heightened activation in distress-related neural responses during a social rejection manipulation, but that this relationship would be moderated by negative emotion differentiation-defined as adeptness at using discrete negative emotion categories to capture one's felt experience. Combining daily diary and neuroimaging methodologies, the current study showed that low self-esteem and low negative emotion differentiation represented a toxic combination that was associated with stronger activation during social rejection (versus social inclusion) in the dorsal anterior cingulate cortex and anterior insula-two regions previously shown to index social distress. In contrast, individuals with greater negative emotion differentiation did not show stronger activation in these regions, regardless of their level of self-esteem; fitting with prior evidence that negative emotion differentiation confers equanimity in emotionally upsetting situations.

Justice for the average Joe: the role of envy and the mentalizing network in the deservingness of others' misfortunes.

The misfortunes of enviable individuals are met by observers with pleasure whereas those of "average", non-enviable individuals elicit pain. These responses are mirrored in deservingness judgments, as enviable individuals' misfortunes are perceived as deserved and those of non-enviable individuals perceived as undeserved. However, the neural underpinnings of these deservingness disparities remain unknown. To explore this phenomenon, we utilized fMRI to test the hypotheses that (A) non-enviable targets' misfortunes would be associated with activation of brain regions that mediate empathic responding (pain matrix, mentalizing network) and not for enviable targets and (B) that activation of those regions would predict decreases in deservingness judgments. Supporting our first hypothesis, the misfortunes of non-enviable targets (as opposed to good fortunes) were associated with activation of the mentalizing network: medial prefrontal cortex, posterior cingulate cortex, temporal-parietal junction, and anterior temporal lobes. Supporting our second hypothesis, dorsomedial prefrontal cortex activation from this contrast was negatively correlated with subsequent reports of how much the non-enviable target deserved his/her misfortune. These findings suggest that non-enviable individuals' misfortunes are perceived as unjust due, in part, to the recruitment of the mentalizing network.

Tuning voltage-gated channel activity and cellular excitability with a sphingomyelinase.

Voltage-gated ion channels generate action potentials in excitable cells and help set the resting membrane potential in nonexcitable cells like lymphocytes. It has been difficult to investigate what kinds of phospholipids interact with these membrane proteins in their native environments and what functional impacts such interactions create. This problem might be circumvented if we could modify specific lipid types in situ. Using certain voltage-gated K(+) (KV) channels heterologously expressed in Xenopus laevis oocytes as a model, our group has shown previously that sphingomyelinase (SMase) D may serve this purpose. SMase D is known to remove the choline group from sphingomyelin, a phospholipid primarily present in the outer leaflet of plasma membranes. This SMase D action lowers the energy required for voltage sensors of a KV channel to enter the activated state, causing a hyperpolarizing shift of the Q-V and G-V curves and thus activating them at more hyperpolarized potentials. Here, we find that this SMase D effect vanishes after removing most of the voltage-sensor paddle sequence, a finding supporting the notion that SMase D modification of sphingomyelin molecules alters these lipids' interactions with voltage sensors. Then, using SMase D to probe lipid-channel interactions, we find that SMase D not only similarly stimulates voltage-gated Na(+) (Na(V)) and Ca(2+) channels but also markedly slows Na(V) channel inactivation. However, the latter effect is not observed in tested mammalian cells, an observation highlighting the profound impact of the membrane environment on channel function. Finally, we directly demonstrate that SMase D stimulates both native K(V)1.3 in nonexcitable human T lymphocytes at their typical resting membrane potential and native Na(V) channels in excitable cells, such that it shifts the action potential threshold in the hyperpolarized direction. These proof-of-concept studies illustrate that the voltage-gated channel activity in both excitable and nonexcitable cells can be tuned by enzymatically modifying lipid head groups.

Do neural responses to rejection depend on attachment style? An fMRI study.

Social bonds fulfill the basic human need to belong. Being rejected thwarts this basic need, putting bonds with others at risk. Attachment theory suggests that people satisfy their need to belong through different means. Whereas anxious attachment is associated with craving acceptance and showing vigilance to cues that signal possible rejection, avoidant attachment is associated with discomfort with closeness and using avoidant strategies to regulate one's relationships. Given these different styles by which people satisfy their need to belong (that can operate simultaneously within the same individual), responses to social rejection may differ according to these individual differences in attachment anxiety and avoidance. To test this hypothesis, we used neuroimaging techniques to examine how the degree to which people display each of the two attachment dimensions (anxiety and avoidance) uniquely correlated with their neural activity during a simulated experience of social exclusion. Anxious attachment related to heightened activity in the dorsal anterior cingulate cortex (dACC) and anterior insula, regions previously associated with rejection-related distress. In contrast, avoidant attachment related to less activity in these regions. Findings are discussed in terms of the strategies that individuals with varying attachment styles might use to promote maintenance of social bonds.

Post-translational regulation and nuclear entry of TIMELESS and PERIOD are affected in new timeless mutant.

The molecular circadian clock consists of a feedback loop in which canonical clock proteins negatively regulate transcription of their own genes. Timed nuclear entry of these proteins is critical, but regulation of this event is poorly understood. In Drosophila melanogaster, the idea that nuclear entry of PERIOD (PER) is controlled by its partner protein TIMELESS (TIM) has been challenged by several studies. We identify here a novel mutation in the tim gene that eliminates behavioral rhythms while allowing robust expression of TIM and PER. Mutant TIM can bind to and stabilize PER. However, neither protein is expressed cyclically, and phosphorylation of both is reduced. In addition, TIM and PER are localized in the cytoplasm at all times of day, and mutant TIM attenuates transcriptional feedback by PER in cultured cells, suggesting that it holds PER in the cytoplasm. In fact, much of the reduced phosphorylation of PER in the new tim mutant appears to result from the cytoplasmic localization of PER. Interestingly, mutating a threonine near the original mutation produces similar phenotypes, raising the possibility that defective phosphorylation is the basis of TIM dysfunction in the novel tim mutant. We also show that a stable form of PER is cytoplasmic in tim-null flies. These studies establish an essential role of TIM in the timed nuclear entry of PER.

Acetaminophen reduces social pain: behavioral and neural evidence.

Pain, whether caused by physical injury or social rejection, is an inevitable part of life. These two types of pain-physical and social-may rely on some of the same behavioral and neural mechanisms that register pain-related affect. To the extent that these pain processes overlap, acetaminophen, a physical pain suppressant that acts through central (rather than peripheral) neural mechanisms, may also reduce behavioral and neural responses to social rejection. In two experiments, participants took acetaminophen or placebo daily for 3 weeks. Doses of acetaminophen reduced reports of social pain on a daily basis (Experiment 1). We used functional magnetic resonance imaging to measure participants' brain activity (Experiment 2), and found that acetaminophen reduced neural responses to social rejection in brain regions previously associated with distress caused by social pain and the affective component of physical pain (dorsal anterior cingulate cortex, anterior insula). Thus, acetaminophen reduces behavioral and neural responses associated with the pain of social rejection, demonstrating substantial overlap between social and physical pain.

Preactivated carboxyl linker for the rapid conjugation of alkylamines to oligonucleotides on solid support.

A C10 linker phosphoramidite reagent terminated with a succinimidyl-activated carboxyl group was prepared and used to couple to the 5'-end of an oligonucleotide synthesized on a solid support. The succinimidyl-activated carboxyl functionality can be used for rapid conjugation of amines to oligonucleotides on solid support or it can be hydrolyzed to form a carboxylic acid functionality. The activated linker was successfully used for conjugation of several primary and secondary aliphatic amine derivatives (including biotin and fluorescein cadaverine) onto a solid support-bound 12-mer DNA oligonucleotide at scales ranging from 0.15 to 1.0 micromol. The overall yields of the conjugation products after AMA deprotection and cleavage from the solid support ranged from 43 to 75% of the total oligonucleotide product. This value is significant, as it includes oligonucleotide synthesis, coupling of the linker, and conjugation of the amine. In addition, the entire process of oligonucleotide synthesis, linker coupling, amine conjugation, deprotection, and cleavage of the oligonucleotide from solid support can be accomplished in 1 day.