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The preferred specimen orientation problem limits accuracy and resolution in structure determination by cryo-EM. Collecting data at defined sample tilts yielded near-atomic-resolution structures for the influenza hemagglutinin trimer and ribosomal biogenesis intermediates. We present a strategy for tackling preferred specimen orientation in single-particle cryogenic electron microscopy by employing tilts during data collection. We also describe a tool to quantify the resulting directional resolution using 3D Fourier shell correlation volumes. We applied these methods to determine the structures at near-atomic resolution of the influenza hemagglutinin trimer, which adopts a highly preferred specimen orientation, and of ribosomal biogenesis intermediates, which adopt moderately preferred orientations.

The habenula is a small, bilateral brain structure located at the dorsal end of the diencephalon. This structure sends projections to the dopaminergic striatum and receives inputs from the limbic forebrain, making the habenula a unique modulator of cross-talk between these brain regions. Despite strong interest in the habenula during the seventies and eighties (Herkenham and Nauta, 1977; Beckstead, 1979; Beckstead et al., 1979; Herkenham and Nauta, 1979; Caldecott-Hazard et al., 1988), interest waned due to lack of a clearly identifiable functional role. Following Matsumoto and Hikosaka's seminal work on the lateral habenula as a predictor of negative reward in monkeys, the habenula has undergone a resurgence of scientific interest. Matsumoto and Hikosaka demonstrated an increase in habenular neuron firing when monkeys did not receive an expected juice reward (Matsumoto and Hikosaka, 2007). Studies have shown that increased habenular activity inactivates dopaminergic cells in the Rostromedial Tegmental Nucleus (RMTg) through GABAergic mechanisms (Jhou et al., 2009a,b). Additional studies link habenular activity to the regulation of serotonin and norepinephrine, suggesting the habenula modulates multiple brain systems (Strecker and Rosengren, 1989; Amat et al., 2001). These discoveries ushered in a series of new studies that have refocused attention on the lateral habenula and the importance of this small brain structure (Bianco and Wilson, 2009; Jhou et al., 2009a; Matsumoto and Hikosaka, 2009; Sartorius et al., 2010; Savitz et al., 2011). Recently, Geisler and Trimble reviewed this renewed interest in: The Lateral Habenula: No Longer Neglected (Geisler and Trimble, 2008). While the lateral habenula (LHb) has been extensively studied, the anatomically and histochemically distinct medial habenula (MHb) remains largely understudied. This short review argues that the MHb is functionally important and should be studied more aggressively.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting “preferred orientations” on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps. Here, the authors quantify the effect of cryo-EM data acquisition with stage-tilt on the global resolution of reconstructions and present a tool for predicting an optimal stage-tilt angle to ameliorate the effects of preferred specimen orientation.

Little is known about how prior beliefs impact biophysically described processes in the presence of neuroactive drugs, which presents a profound challenge to the understanding of the mechanisms and treatments of addiction. We engineered smokers’ prior beliefs about the presence of nicotine in a cigarette smoked before a functional magnetic resonance imaging session where subjects carried out a sequential choice task. Using a model-based approach, we show that smokers’ beliefs about nicotine specifically modulated learning signals (value and reward prediction error) defined by a computational model of mesolimbic dopamine systems. Belief of “no nicotine in cigarette” (compared with “nicotine in cigarette”) strongly diminished neural responses in the striatum to value and reward prediction errors and reduced the impact of both on smokers’ choices. These effects of belief could not be explained by global changes in visual attention and were specific to value and reward prediction errors. Thus, by modulating the expression of computationally explicit signals important for valuation and choice, beliefs can override the physical presence of a potent neuroactive compound like nicotine. These selective effects of belief demonstrate that belief can modulate model-based parameters important for learning. The implications of these findings may be far ranging because belief-dependent effects on learning signals could impact a host of other behaviors in addiction as well as in other mental health problems. Significance Nicotine is the primary addictive substance in tobacco, which stimulates neural pathways mediating reward processing. However, pure biochemical explanations are not sufficient to account for the difficulty in quitting and remaining smoke-free among smokers, and in fact cognitive factors are now considered to contribute critically to addiction. Using model-based functional neuroimaging, we show that smokers’ prior beliefs about nicotine specifically impact learning signals defined by principled computational models of mesolimbic dopamine systems. We further demonstrate that these specific changes in neural signaling are accompanied by measurable changes in smokers’ choice behavior. Our findings suggest that subjective beliefs can override the physical presence of a powerful drug like nicotine by modulating learning signals processed in the brain’s reward system.

Calcium-phosphate (CaP) is a ubiquitous inorganic compound that plays an important structural role in healthy bone and teeth formation, but its pathologic buildup can occur in dyshomeostatic calcium disorders like Alzheimer’s disease and Leigh syndrome. The nexus of pathologic extracellular CaP in the nervous system is not well understood, but prior evidence suggests mitochondria could be a source. We have observed mitochondria-sized sheet-like CaP aggregates within functional wild type cortical neuron cultures at 1 and 20 days in vitro. Neurons were extracted from embryonic day 18 (E18) rat embryos following standard protocols to study neuronal structure and function. We have used a combination of cryo-ET, cryo-CLEM, and LDSAED to demonstrate that these aggregates are octacalcium phosphate-like, are associated with mitochondria, and that at least a portion are extruded via migrasomes. Visually similar aggregates were previously observed in Huntington’s disease model neurons, but in that study they were not observed in WT controls. These findings show that this CaP aggregation process occurs routinely in WT neurons and may reveal an important link for how mitochondria may participate in calcification, highlighting them as potential therapeutic targets in neurological disorders characterized by pathological calcification, such as Alzheimer’s disease.

Background Nearly 6 million deaths and over a half trillion dollars in healthcare costs worldwide are attributed to tobacco smoking each year. Extensive research efforts have been pursued to elucidate the molecular underpinnings of smoking addiction and facilitate cessation. In this study, we genotyped and obtained both resting state and task-based functional magnetic resonance imaging from 64 non-smokers and 42 smokers. Smokers were imaged after having smoked normally (“sated”) and after having not smoked for at least 12 h (“abstinent”). Results While abstinent smokers did not differ from non-smokers with respect to pairwise resting state functional connectivities (RSFCs) between 12 brain regions of interest, RSFCs involving the caudate and putamen of sated smokers significantly differed from those of non-smokers ( P  < 0.01). Further analyses of caudate and putamen activity during elicited experiences of reward and disappointment show that caudate activity during reward (CR) correlated with smoking status ( P  = 0.015). Moreover, abstinent smokers with lower CR experienced greater withdrawal symptoms ( P  = 0.024), which suggests CR may be related to smoking urges. Associations between genetic variants and CR, adjusted for smoking status, were identified by genome-wide association study (GWAS). Genes containing or exhibiting caudate-specific expression regulation by these variants were enriched within Gene Ontology terms that describe cytoskeleton functions, synaptic organization, and injury response ( P  < 0.001, FDR < 0.05). Conclusions By integrating genomic and imaging data, novel insights into potential mechanisms of caudate activation and homeostasis are revealed that may guide new directions of research toward improving our understanding of addiction pathology.

Although positive reward prediction error, a key element in learning that is signaled by dopamine cells, has been extensively studied, little is known about negative reward prediction errors in humans. Detailed animal electrophysiology shows that the habenula, an integrative region involved in many processes including learning, reproduction, and stress responses, also encodes negative reward-related events such as negative reward prediction error signals. In humans, however, the habenula's extremely small size has prevented direct assessments of its function. We developed a method to functionally locate and study the habenula in humans using fMRI, based on the expected reward-dependent response phenomenology of habenula and striatum and, we provide conclusive evidence for activation in human habenula to negative reward prediction errors.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of bacterial infections, but treatment options are limited due to MRSA multi-drug resistance. The anti-hypertensive drug Candesartan cilexetil (CC) exhibits potent anti-MRSA activity. It permeabilizes the membrane of MRSA cells and potentiates the activity of aminoglycoside antibiotics. Here, we used a variety of methods to elucidate the mechanism by which CC disrupts membrane homeostasis. We show that CC binds to bilayer lipid molecules, decreases membrane fluidity, down-regulates cell membrane and cell wall related genes and related metabolites, and decreases C20 fatty acids (C20:0). Decreasing C20:0 fatty acids confers CC- resistance, which can be reversed by C20:0 supplementation. Structural activity relationship analysis shows that the tetrazole ring and ester carbonic acid of CC are critical for antibacterial activity. Finally, CC reduces MRSA-MW2 replication in a murine MRSA abscess model, supporting a potential role of CC as a lead antimicrobial compound/potentiator against MRSA. In this work, authors show that candesartan cilexetil acts as a potent antibiotic potentiator, disrupting Staphylococcus aureus membranes and markedly enhancing the activity of clinically used antibiotics against drug-resistant infections.

Electronic cigarettes (e-cigarettes) are nicotine-delivery devices that are increasingly used, especially by young people. Because e-cigarettes lack many of the substances found in regular tobacco, they are often perceived as a safer smoking alternative, especially in high-risk situations such as pregnancy. However, studies suggest that it is exposure to nicotine that is most detrimental to prenatal development. The authors studied perceptions of tobacco and e-cigarette health risks using a multiple-choice survey. To study the perceived safety of e-cigarettes versus tobacco cigarettes, 184 modified Global Health Youth Surveys (WHO, http://www.who.int/tobacco/surveillance/gyts/en/ ) were completed electronically or on paper. Age range, smoking status, and perceptions about tobacco cigarettes and e-cigarettes were studied. The results verified that younger people use e-cigarettes more than older people. Tobacco cigarettes were perceived as more harmful than e-cigarettes to health in general, including lung cancer and pregnancy. Although more research is necessary, the authors postulate that the perception that e-cigarettes are safer during pregnancy may induce pregnant women to use these devices more freely. Given that nicotine is known to cause fetal harm, pregnant mothers who smoke e-cigarettes could cause even greater harm to the fetus because e-cigarettes are perceived as being safer than tobacco cigarettes. Until more data about the effects of nicotine during pregnancy are available, the authors advocate for labeling of e-cigarettes as potentially harmful, at least during pregnancy.