Response to: “Response to Different measures of 'genome-wide' DNA methylation exhibit unique properties in placental and somatic tissues

We are responding to a Letter to the Editor addressing the Method section of our paper “Different measures of ‘genome-wide’ DNA methylation exhibit unique properties in placental and somatic tissues.” The letter raised concerns that the protocol for Epigentek’s MethylFlash kit was followed incorrectly based on the wording of an online publication of our article. We admittedly made an error in the language used to describe the MethylFlash protocol in our initial submission and thus this was corrected as soon as it was brought to our attention. However, the error was only in language and not procedure. We are confident that the protocol was followed as stated in the insert provided with the MethylFlash^TM Methylated DNA Quantification kit (Colorimetric).We are responding to a Letter to the Editor addressing the Method section of our paper “Different measures of ‘genome-wide’ DNA methylation exhibit unique properties in placental and somatic tissues” (Price ME, Cotton AM, PeÒaherrera MS, McFadden DE, Kobor MS, Robinson WP. Different measures of “genome-wide” DNA methylation exhibit unique properties in placental and somatic tissues. Epigenetics 2012; 7: 652–63; PMID: 22531475; 10.4161/epi.20221). The letter raised concerns that the protocol for Epigentek’s MethylFlash kit was followed incorrectly based on the wording of an online publication of our article. We admittedly made an error in the language used to describe the MethylFlash protocol in our initial submission and thus this was corrected as soon as it was brought to our attention. However, the error was only in language and not procedure. We are confident that the protocol was followed as stated in the insert provided with the MethylFlash^TM Methylated DNA Quantification kit (Colorimetric).     

Counting fluorescently labeled proteins in tissues in the spinning–disk microscope using single–molecule calibrations

Quantification of molecular numbers and concentrations in living cells is critical for testing models of complex biological phenomena. Counting molecules in cells requires estimation of the fluorescence intensity of single molecules, which is generally limited to imaging near cell surfaces, in isolated cells, or where motions are diffusive. To circumvent this difficulty, we have devised a calibration technique for spinning–disk confocal microscopy, commonly used for imaging in tissues, that uses single–step bleaching kinetics to estimate the single–fluorophore intensity. To cross–check our calibrations, we compared the brightness of fluorophores in the SDC microscope to those in the total internal reflection and epifluorescence microscopes. We applied this calibration method to quantify the number of end–binding protein 1 (EB1)–eGFP in the comets of growing microtubule ends and to measure the cytoplasmic concentration of EB1–eGFP in sensory neurons in fly larvae. These measurements allowed us to estimate the dissociation constant of EB1–eGFP from the microtubules as well as the GTP–tubulin cap size. Our results show the unexplored potential of single–molecule imaging using spinning–disk confocal microscopy and provide a straightforward method to count the absolute number of fluorophores in tissues that can be applied to a wide range of biological systems and imaging techniques.     

The Functional Repertoire of Survivin's Tails

Survivin is a multitasking protein that can inhibit cell death and that is essential for mitosis. Due to these prosurvival activities and the correlation of its expression with tumor resistance to conventional cancer treatments, survivin has received much attention as a potential oncotherapeutic target. Nevertheless, many questions regarding its exact role at the molecular level remain to be elucidated. In this study we ask whether the extreme C- and NH₂ termini of survivin are required for it to carry out its cytoprotective and mitotic duties. When assayed for their ability to act as a cytoprotectant, both survivin_1–120 and survivin_11–142 were able to protect cells against TRAIL-mediated apoptosis, but when challenged with irradiation cells expressing survivin_11–142 had no survival advantage. During mitosis, however, removing the NH₂ terminal 10 amino acids (survivin_11–142) had no apparent effect but truncating 22 amino acids from the C-terminus (survivin_1–120) prevented survivin from transferring to the midzone microtubules during anaphase. Collectively the data herein presented suggest that the C-terminus is required for cell division, and that the NH₂ terminus is dispensable for apoptosis and mitosis but required for protection from irradiation.     

Do I Have My Attention? Speed of Processing Advantages for the Self-Face Are Not Driven by Automatic Attention Capture

We respond more quickly to our own face than to other faces, but there is debate over whether this is connected to attention-grabbing properties of the self-face. In two experiments, we investigate whether the self-face selectively captures attention, and the attentional conditions under which this might occur. In both experiments, we examined whether different types of face (self, friend, stranger) provide differential levels of distraction when processing self, friend and stranger names. In Experiment 1, an image of a distractor face appeared centrally – inside the focus of attention – behind a target name, with the faces either upright or inverted. In Experiment 2, distractor faces appeared peripherally – outside the focus of attention – in the left or right visual field, or bilaterally. In both experiments, self-name recognition was faster than other name recognition, suggesting a self-referential processing advantage. The presence of the self-face did not cause more distraction in the naming task compared to other types of face, either when presented inside (Experiment 1) or outside (Experiment 2) the focus of attention. Distractor faces had different effects across the two experiments: when presented inside the focus of attention (Experiment 1), self and friend images facilitated self and friend naming, respectively. This was not true for stranger stimuli, suggesting that faces must be robustly represented to facilitate name recognition. When presented outside the focus of attention (Experiment 2), no facilitation occurred. Instead, we report an interesting distraction effect caused by friend faces when processing strangers’ names. We interpret this as a “social importance” effect, whereby we may be tuned to pick out and pay attention to familiar friend faces in a crowd. We conclude that any speed of processing advantages observed in the self-face processing literature are not driven by automatic attention capture.     

From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Cα deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein.     

Semi-Automated Detection of Cerebral Microbleeds on 3.0 T MR Images

Cerebral microbleeds are associated with vascular disease and dementia. They can be detected on MRI and receive increasing attention. Visual rating is the current standard for microbleed detection, but is rater dependent, has limited reproducibility, modest sensitivity, and can be time-consuming. The goal of the current study is to present a tool for semi-automated detection of microbleeds that can assist human raters in the rating procedure. The radial symmetry transform is originally a technique to highlight circular-shaped objects in two-dimensional images. In the current study, the three-dimensional radial symmetry transform was adapted to detect spherical microbleeds in a series of 72 patients from our hospital, for whom a ground truth visual rating was made by four raters. Potential microbleeds were automatically identified on T2*-weighted 3.0 T MRI scans and the results were visually checked to identify microbleeds. Final ratings of the radial symmetry transform were compared to human ratings. After implementing and optimizing the radial symmetry transform, the method achieved a high sensitivity, while maintaining a modest number of false positives. Depending on the settings, sensitivities ranged from 65%–84% compared to the ground truth rating. Rating of the processed images required 1–2 minutes per participant, in which 20–96 false positive locations per participant were censored. Sensitivities of individual raters ranged from 39%–86% compared to the ground truth and required 5–10 minutes per participant per rater. The sensitivities that were achieved by the radial symmetry transform are similar to those of individual experienced human raters, demonstrating its feasibility and usefulness for semi-automated microbleed detection.     

Patients Covertly Recording Clinical Encounters: Threat or Opportunity? A Qualitative Analysis of Online Texts

Background

The phenomenon of patients covertly recording clinical encounters has generated controversial media reports. This study aims to examine the phenomenon and analyze the underlying issues.

Methods and Findings

We conducted a qualitative analysis of online posts, articles, blogs, and forums (texts) discussing patients covertly recording clinical encounters. Using Google and Google Blog search engines, we identified and analyzed 62 eligible texts published in multiple countries between 2006 and 2013. Thematic analysis revealed four key themes: 1) a new behavior that elicits strong reactions, both positive and negative, 2) an erosion of trust, 3) shifting patient-clinician roles and relationships, and 4) the existence of confused and conflicting responses. When patients covertly record clinical encounters – a behavior made possible by various digital recording technologies – strong reactions are evoked among a range of stakeholders. The behavior represents one consequence of an erosion of trust between patients and clinicians, and when discovered, leads to further deterioration of trust. Confused and conflicting responses to the phenomenon by patients and clinicians highlight the need for policy guidance.

Conclusions

This study describes strong reactions, both positive and negative, to the phenomenon of patients covertly recording clinical encounters. The availability of smartphones capable of digital recording, and shifting attitudes to patient-clinician relationships, seems to have led to this behavior, mostly viewed as a threat by clinicians but as a welcome and helpful innovation by some patients, possibly indicating a perception of subordination and a lack of empowerment. Further examination of this tension and its implications is needed.     

The normalized segment classification model: A new tool to compare spectral reflectance curves

1. Color patterns are complex traits under selective pressures from conspecifics, mutualists, and antagonists. To evaluate the salience of a pattern or the similarity between colors, several visual models are available. Color discrimination models estimate the perceptual difference between any two colors. Their application to a diversity of taxonomic groups has become common in the literature to answer behavioral, ecological, and evolutionary questions. To use these models, we need information about the visual system of our beholder species. However, many color patterns are simultaneously subject to selective pressures from different species, often from different taxonomic groups, with different visual systems. Furthermore, we lack information about the visual system of many species, leading ecologists to use surrogate values or theoretical estimates for model parameters.
2. Here, we present a modification of the segment classification method proposed by Endler (Biological Journal of the Linnean Society, 1990 41, 315–352): the normalized segment classification model (NSC). We explain its logic and use, exploring how NSC differs from other visual models. We also compare its predictions with available experimental data.
3. Even though the NSC model includes no information about the visual system of the receiver species, it performed better than traditional color discrimination models when predicting the output of some behavioral tasks. Although vision scientists define color as independent of stimulus brightness, a likely explanation for the goodness of fit of the NSC model is that its distance measure depends on brightness differences, and achromatic information can influence the decision‐making process of animals when chromatic information is missing.
4. Species‐specific models may be insufficient for the study of color patterns in a community context. The NSC model offers a species‐independent solution for color analyses, allowing us to calculate color differences when we ignore the intended viewer of a signal or when different species impose selective pressures on the signal.

     

Influence of correct secondary and tertiary RNA folding on the binding of cellular factors to the HCV IRES

Structural integrity of the hepatitus C virus (HCV) 5′ UTR region that includes the internal ribosome entry site (IRES) element is known to be essential for efficient protein synthesis. The functional explanation for this observation has been provided by the recent evidence that binding of several cellular factors to the HCV IRES is dependent on the conservation of its secondary structure. In order to better define the relationship between IRES activity, protein binding and RNA folding of the HCV IRES, we have focused our attention on its major stem–loop region (domain III) and the binding of several cellular factors: two subunits of eukaryotic initiation factor eIF3 and ribosomal protein S9. Our results show that binding of eIF3 p170 and p116/p110 subunits is dependent on the ability of the domain III apical stem–loop region to fold in the correct secondary structure whilst secondary structure of hairpin IIId is important for the binding of S9 ribosomal protein. In addition, we show that binding of S9 ribosomal protein also depends on the disposition of domain III on the HCV 5′ UTR, indicating the presence of necessary interdomain interactions required for the binding of this protein (thus providing the first direct evidence that tertiary folding of the HCV RNA does affect protein binding).     

Evaluating Letter Recognition,Flicker Fusion,and the Talbot-Plateau Law using Microsecond-Duration Flashes

Four experiments examined the ability of respondents to identify letters that were displayed on an LED array with flashes lasting little more than a microsecond. The first experiment displayed each letter with a single, simultaneous flash of all the dots forming the letter and established the relation of flash intensity to the probability of letter identification. The second experiment displayed the letters with multiple flashes at different frequencies to determine the probability that the sequence of flashes would be perceived as fused. The third experiment displayed the letters at a frequency that was above the flicker-fusion frequency, varying flash intensity to establish the amount needed to elicit a given probability of letter identification. The fourth experiment displayed each letter twice, once at a frequency where no flicker was perceived and also with steady light emission. The intensity of each flash was fixed and the steady intensity was varied; respondents were asked to judge whether the fused-flicker display and the steady display appeared to be the same brightness. Steady intensity was about double the average flash intensity where the two conditions were perceived as being equal in brightness. This is at odds with Talbot-Plateau law, which predicts that these two values should be equal. The law was formulated relative to a flash lasting half of each period, so it is surprising that it comes this close to being correct where the flash occupies only a millionth of the total period.     

Predator-induced flow disturbances alert prey,from the onset of an attack

Many prey species, from soil arthropods to fish, perceive the approach of predators, allowing them to escape just in time. Thus, prey capture is as important to predators as prey finding. We extend an existing framework for understanding the conjoint trajectories of predator and prey after encounters, by estimating the ratio of predator attack and prey danger perception distances, and apply it to wolf spiders attacking wood crickets. Disturbances to air flow upstream from running spiders, which are sensed by crickets, were assessed by computational fluid dynamics with the finite-elements method for a much simplified spider model: body size, speed and ground effect were all required to obtain a faithful representation of the aerodynamic signature of the spider, with the legs making only a minor contribution. The relationship between attack speed and the maximal distance at which the cricket can perceive the danger is parabolic; it splits the space defined by these two variables into regions differing in their values for this ratio. For this biological interaction, the ratio is no greater than one, implying immediate perception of the danger, from the onset of attack. Particular attention should be paid to the ecomechanical aspects of interactions with such small ratio, because of the high degree of bidirectional coupling of the behaviour of the two protagonists. This conclusion applies to several other predator–prey systems with sensory ecologies based on flow sensing, in air and water.     

Recognition of Handwriting from Electromyography

Handwriting – one of the most important developments in human culture – is also a methodological tool in several scientific disciplines, most importantly handwriting recognition methods, graphology and medical diagnostics. Previous studies have relied largely on the analyses of handwritten traces or kinematic analysis of handwriting; whereas electromyographic (EMG) signals associated with handwriting have received little attention. Here we show for the first time, a method in which EMG signals generated by hand and forearm muscles during handwriting activity are reliably translated into both algorithm-generated handwriting traces and font characters using decoding algorithms. Our results demonstrate the feasibility of recreating handwriting solely from EMG signals – the finding that can be utilized in computer peripherals and myoelectric prosthetic devices. Moreover, this approach may provide a rapid and sensitive method for diagnosing a variety of neurogenerative diseases before other symptoms become clear.     

Invitation Choice Structure Has No Impact on Attendance in a Female Business Training Program in Kenya

Business training programs are a common form of support to small businesses, but organizations providing this training often struggle to get business owners to attend. We evaluate the role of invitation choice structure in determining agreement to participate and actual attendance. A field experiment randomly assigned female small business owners in Kenya (N = 1172) to one of three invitation types: a standard opt-in invitation; an active choice invitation where business owners had to explicitly say yes or no to the invitation; and an enhanced active choice invitation which highlighted the costs of saying no. We find no statistically significant effect of these alternative choice structures on willingness to participate in training, attending at least one day, and completing the course. The 95 percent confidence interval for the active treatment effect on attendance is [−1.9%, +9.5%], while for the enhanced active choice treatment it is [−4.1%, +7.7%]. The effect sizes consistent with our data are smaller than impacts measured in health and retirement savings studies in the United States. We examine several potential explanations for the lack of effect in a developing country setting. We find evidence consistent with two potential reasons being limited decision-making power amongst some women, and lower levels of cognition making the enhanced active choice wording less effective.     

Applying an Attentional Set to Perceived and Remembered Features

Previous research has examined our ability to attend selectively to particular features of perceptual objects, as well as our ability to switch from attending to one type of feature to another. This is usually done in the context of anticipatory attentional-set control, comparing the neural mechanisms involved as participants prepare to attend to the same stimulus feature as on the previous trial (“task-stay” trials) with those required as participants prepare to attend to a different stimulus feature to that previously attended (“task-switch” trials). We wanted to establish how participants maintain or switch attentional set retrospectively, as they attend to features of objects held in visual short-term memory (VSTM). We found that switching, relative to maintaining attentional set retrospectively, was associated with a performance cost, which can be reduced over time. This control process was mirrored by a large parietal and frontal amplitude difference in the event-related brain potentials (ERPs) and significant differences in global field power (GFP) between switch and stay trials. However, when taking into account the switch/stay GFP differences, thereby controlling for this difference in amplitude, we could not distinguish these trial types topographically. By contrast, we found clear topographic differences between preparing an anticipatory feature-based attentional set versus applying it retrospectively within VSTM. These complementary topographical and amplitude analyses suggested that anticipatory and retrospective set control recruited a qualitatively different configuration of underlying neural generators. In contrast, switch/stay differences were largely quantitative, with them differing primarily in terms of amplitude rather than topography.     

Negative Example Selection for Protein Function Prediction: The NoGO Database

Negative examples – genes that are known not to carry out a given protein function – are rarely recorded in genome and proteome annotation databases, such as the Gene Ontology database. Negative examples are required, however, for several of the most powerful machine learning methods for integrative protein function prediction. Most protein function prediction efforts have relied on a variety of heuristics for the choice of negative examples. Determining the accuracy of methods for negative example prediction is itself a non-trivial task, given that the Open World Assumption as applied to gene annotations rules out many traditional validation metrics. We present a rigorous comparison of these heuristics, utilizing a temporal holdout, and a novel evaluation strategy for negative examples. We add to this comparison several algorithms adapted from Positive-Unlabeled learning scenarios in text-classification, which are the current state of the art methods for generating negative examples in low-density annotation contexts. Lastly, we present two novel algorithms of our own construction, one based on empirical conditional probability, and the other using topic modeling applied to genes and annotations. We demonstrate that our algorithms achieve significantly fewer incorrect negative example predictions than the current state of the art, using multiple benchmarks covering multiple organisms. Our methods may be applied to generate negative examples for any type of method that deals with protein function, and to this end we provide a database of negative examples in several well-studied organisms, for general use (The NoGO database, available at: bonneaulab.bio.nyu.edu/nogo.html).     

After 2 years of a pandemic,students are struggling to find strong reference letters

Writing and receiving reference letters in the time of COVID. Subject Categories: Careers

“People influence people. Nothing influences people more than a recommendation from a trusted friend. A trusted referral influences people more than the best broadcast message.” —Mark Zuckerberg.

I regularly teach undergraduate courses in genetics and genomics. Sure enough, at the end of each semester, after the final marks have been submitted, my inbox is bombarded with reference letter requests. “Dear Dr. Smith, I was a student in your Advanced Genetics course this past term and would be forever grateful if you would write me a reference for medical school…” I understand how hard it can be to find references, but I have a general rule that I will only write letters of support for individuals that I have interacted with face‐to‐face on at least a few occasions. This could include, for example, research volunteers in my laboratory, honors thesis students that I have supervised, and students who have gone out of their way to attend office hours and/or been regularly engaged in class discussions. I am selective about who I will write references for, not because I am unkind or lazy, but because I know from experience that a strong letter should include concrete examples of my professional interactions with the individual and should speak to their character and their academic abilities. In today''s highly competitive educational system, a letter that merely states that a student did well on the midterm and final exams will not suffice to get into medical or graduate school.However, over the past 2 years many, if not most, students have been attending university remotely with little opportunity to foster meaningful relationships with their instructors, peers, and mentors, especially for those in programs with large enrollments. Indeed, during the peak of Covid‐19, I stopped taking on undergraduate volunteers and greatly reduced the number of honors students in my laboratory. Similarly, my undergraduate lectures have been predominantly delivered online via Zoom, meaning I did not see or speak with most of the students in my courses. It did not help that nearly all of them kept their cameras and microphones turned off and rarely attended online office hours. Consequently, students are desperately struggling to identify individuals who can write them strong letters of reference. In fact, this past spring, I have had more requests for reference letters than ever before, and the same is true for many of my colleagues. Some of the emails I have received have been heartfelt and underscore how taxing the pandemic has been on young adults. With permission, I have included an excerpt from a message I received in early May:Hi Dr. Smith. You may not remember me, but I was in Genome Evolution this year. I enjoyed the class despite being absent for most of your live Zoom lectures because of the poor internet connection where I live. Believe it or not, my mark from your course was the highest of all my classes this term! Last summer, I moved back home to rural Northern Ontario to be closer to my family. My mom is a frontline worker and so I''ve been helping care for my elderly grandmother who has dementia as well as working part‐time as a tutor at the local high school to help pay tuition. All of this means that I''ve not paid as much attention to my studies as I should have. I''m hoping to go to graduate school this coming fall, but I have yet to find a professor who will write a reference for me. Would you please, please consider writing me a letter?I am sympathetic to the challenges students faced and continue to face during Covid‐19 and, therefore, I have gone out of my way to provide as many as I can with letters of support. But, it is no easy feat writing a good reference for someone you only know via an empty Zoom box and a few online assignments. My strategy has been to focus on their scholarly achievements in my courses, providing clear, tangible examples from examinations and essays, and to highlight the notable aspects of their CVs. I also make a point to stress how hard online learning can be for students (and instructors), reiterating some of the themes touched upon above. This may sound unethical to some readers but, in certain circumstances, I have allowed students to draft their own reference letters, which I can then vet, edit, and rewrite as I see fit.But it is not just undergraduates. After months and months of lockdowns and social distancing, many graduate students, postdocs, and professors are also struggling to find suitable references. In April, I submitted my application for promotion to Full Professor, which included the names of 20 potential reviewers. Normally, I would have selected at least some of these names from individuals I met at recent conferences and invited to university seminars, except I have not been to a conference in over 30 months. Moreover, all my recent invited talks have been on Zoom and did not include any one‐on‐one meetings with faculty or students. Thus, I had to include the names of scientists that I met over 3 years ago, hoping that my research made a lasting impression on them. I have heard similar anecdotes from many of my peers both at home and at other universities. Given all of this, I would encourage academics to be more forthcoming than they may have traditionally been when students or colleagues approach them for letters of support. Moreover, I think we could all be a little more forgiving and understanding when assessing our students and peers, be it for admissions into graduate school, promotion, or grant evaluations.Although it seems like life on university campuses is returning to a certain degree of normality, many scholars are still learning and working remotely, and who knows what the future may hold with regard to lockdowns. With this uncertainty, we need to do all we can to engage with and have constructive and enduring relationships with our university communities. For undergraduate and graduate students, this could mean regularly attending online office hours, even if it is only to introduce yourself, as well as actively participating in class discussions, whether they are in‐person, over Zoom, or on digital message boards. Also, do not disregard the potential and possibilities of remote volunteer research positions, especially those related to bioinformatics. Nearly, every laboratory in my department has some aspect of their research that can be carried out from a laptop computer with an Internet connection. Although not necessarily as enticing as working at the bench or in the field, computer‐based projects can be rewarding and an excellent path to a reference letter.If you are actively soliciting references, try and make it as easy as possible on your potential letter writers. Clearly and succinctly outline why you want this person to be a reference, what the letter writing/application process entails, and the deadline. Think months ahead, giving your references ample time to complete the letter, and do not be shy about sending gentle reminders. It is great to attach a CV, but also briefly highlight your most significant achievements in bullet points in your email (e.g., Dean''s Honours List 2021–22). This will save time for your references as they will not have to sift through many pages of a CV. No matter the eventual result of the application or award, be sure to follow up with your letter writers. There is nothing worse than spending time crafting a quality support letter and never learning the ultimate outcome of that effort. And, do not be embarrassed if you are unsuccessful and need to reach out again for another round of references—as Winston Churchill said, “Success is stumbling from failure to failure with no loss of enthusiasm.”     

Individual Differences in Temporal Selective Attention as Reflected in Pupil Dilation

Background

Attention is restricted for the second of two targets when it is presented within 200–500 ms of the first target. This attentional blink (AB) phenomenon allows one to study the dynamics of temporal selective attention by varying the interval between the two targets (T1 and T2). Whereas the AB has long been considered as a robust and universal cognitive limitation, several studies have demonstrated that AB task performance greatly differs between individuals, with some individuals showing no AB whatsoever.

Methodology/Principal Findings

Here, we studied these individual differences in AB task performance in relation to differences in attentional timing. Furthermore, we investigated whether AB magnitude is predictive for the amount of attention allocated to T1. For both these purposes pupil dilation was measured, and analyzed with our recently developed deconvolution method. We found that the dynamics of temporal attention in small versus large blinkers differ in a number of ways. Individuals with a relatively small AB magnitude seem better able to preserve temporal order information. In addition, they are quicker to allocate attention to both T1 and T2 than large blinkers. Although a popular explanation of the AB is that it is caused by an unnecessary overinvestment of attention allocated to T1, a more complex picture emerged from our data, suggesting that this may depend on whether one is a small or a large blinker.

Conclusion

The use of pupil dilation deconvolution seems to be a powerful approach to study the temporal dynamics of attention, bringing us a step closer to understanding the elusive nature of the AB. We conclude that the timing of attention to targets may be more important than the amount of allocated attention in accounting for individual differences.     

Ltc1 is an ER-localized sterol transporter and a component of ER–mitochondria and ER–vacuole contacts

Organelle contact sites perform fundamental functions in cells, including lipid and ion homeostasis, membrane dynamics, and signaling. Using a forward proteomics approach in yeast, we identified new ER–mitochondria and ER–vacuole contacts specified by an uncharacterized protein, Ylr072w. Ylr072w is a conserved protein with GRAM and VASt domains that selectively transports sterols and is thus termed Ltc1, for Lipid transfer at contact site 1. Ltc1 localized to ER–mitochondria and ER–vacuole contacts via the mitochondrial import receptors Tom70/71 and the vacuolar protein Vac8, respectively. At mitochondria, Ltc1 was required for cell viability in the absence of Mdm34, a subunit of the ER–mitochondria encounter structure. At vacuoles, Ltc1 was required for sterol-enriched membrane domain formation in response to stress. Increasing the proportion of Ltc1 at vacuoles was sufficient to induce sterol-enriched vacuolar domains without stress. Thus, our data support a model in which Ltc1 is a sterol-dependent regulator of organelle and cellular homeostasis via its dual localization to ER–mitochondria and ER–vacuole contact sites.     

The centromeric nucleosome-like CENP–T–W–S–X complex induces positive supercoils into DNA

The centromere is a specific genomic region upon which the kinetochore is formed to attach to spindle microtubules for faithful chromosome segregation. To distinguish this chromosomal region from other genomic loci, the centromere contains a specific chromatin structure including specialized nucleosomes containing the histone H3 variant CENP–A. In addition to CENP–A nucleosomes, we have found that centromeres contain a nucleosome-like structure comprised of the histone-fold CENP–T–W–S–X complex. However, it is unclear how the CENP–T–W–S–X complex associates with centromere chromatin. Here, we demonstrate that the CENP–T–W–S–X complex binds preferentially to ∼100 bp of linker DNA rather than nucleosome-bound DNA. In addition, we find that the CENP–T–W–S–X complex primarily binds to DNA as a (CENP–T–W–S–X)₂ structure. Interestingly, in contrast to canonical nucleosomes that negatively supercoil DNA, the CENP–T–W–S–X complex induces positive DNA supercoils. We found that the DNA-binding regions in CENP–T or CENP–W, but not CENP–S or CENP–X, are required for this positive supercoiling activity and the kinetochore targeting of the CENP–T–W–S–X complex. In summary, our work reveals the structural features and properties of the CENP–T–W–S–X complex for its localization to centromeres.