Working memory enables us to bridge past sensory information to upcoming future behaviour. Accordingly, by its very nature, working memory is concerned with two components: the past and the future. Yet, in conventional laboratory tasks, these two components are often conflated, such as when sensory information in working memory is encoded and tested at the same location. We developed a task in which we dissociated the past (encoded location) and future (to-be-tested location) attributes of visual contents in working memory. This enabled us to independently track the utilisation of past and future memory attributes through gaze, as observed during mnemonic selection. Our results reveal the joint consideration of past and future locations. This was prevalent even at the single-trial level of individual saccades that were jointly biased to the past and future. This uncovers the rich nature of working memory representations, whereby both past and future memory attributes are retained and can be accessed together when memory contents become relevant for behaviour.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

A protein's genetic architecture - the set of causal rules by which its sequence produces its functions - also determines its possible evolutionary trajectories. Prior research has proposed that the genetic architecture of proteins is very complex, with pervasive epistatic interactions that constrain evolution and make function difficult to predict from sequence. Most of this work has analyzed only the direct paths between two proteins of interest - excluding the vast majority of possible genotypes and evolutionary trajectories - and has considered only a single protein function, leaving unaddressed the genetic architecture of functional specificity and its impact on the evolution of new functions. Here, we develop a new method based on ordinal logistic regression to directly characterize the global genetic determinants of multiple protein functions from 20-state combinatorial deep mutational scanning (DMS) experiments. We use it to dissect the genetic architecture and evolution of a transcription factor's specificity for DNA, using data from a combinatorial DMS of an ancient steroid hormone receptor's capacity to activate transcription from two biologically relevant DNA elements. We show that the genetic architecture of DNA recognition consists of a dense set of main and pairwise effects that involve virtually every possible amino acid state in the protein-DNA interface, but higher-order epistasis plays only a tiny role. Pairwise interactions enlarge the set of functional sequences and are the primary determinants of specificity for different DNA elements. They also massively expand the number of opportunities for single-residue mutations to switch specificity from one DNA target to another. By bringing variants with different functions close together in sequence space, pairwise epistasis therefore facilitates rather than constrains the evolution of new functions.

Wound infections are highly prevalent and can lead to delayed or failed healing, causing significant morbidity and adverse economic impacts. These infections occur in various contexts, including diabetic foot ulcers, burns, and surgical sites. Enterococcus faecalis is often found in persistent non-healing wounds, but its contribution to chronic wounds remains understudied. To address this, we employed single-cell RNA sequencing (scRNA-seq) on infected wounds in comparison to uninfected wounds in a mouse model. Examining over 23,000 cells, we created a comprehensive single-cell atlas that captures the cellular and transcriptomic landscape of these wounds. Our analysis revealed unique transcriptional and metabolic alterations in infected wounds, elucidating the distinct molecular changes associated with bacterial infection compared to the normal wound healing process. We identified dysregulated keratinocyte and fibroblast transcriptomes in response to infection, jointly contributing to an anti-inflammatory environment. Notably, E. faecalis infection prompted a premature, incomplete epithelial-mesenchymal transition in keratinocytes. Additionally, E. faecalis infection modulated M2-like macrophage polarization by inhibiting pro-inflammatory resolution in vitro, in vivo, and in our scRNA-seq atlas. Furthermore, we discovered macrophage crosstalk with neutrophils, which regulates chemokine signaling pathways, while promoting anti-inflammatory interactions with endothelial cells. Overall, our findings offer new insights into the immunosuppressive role of E. faecalis in wound infections.

Osteoarthritis (OA) is a degenerative disease with a high prevalence in the elderly population, but our understanding of its mechanisms remains incomplete. Analysis of serum exosomal small RNA sequencing data from clinical patients and gene expression data from OA patient serum and cartilage obtained from the GEO database revealed a common dysregulated miRNA, miR-199b-5p. In vitro cell experiments demonstrated that miR-199b-5p inhibits chondrocyte vitality and promotes extracellular matrix degradation. Conversely, inhibition of miR-199b-5p under inflammatory conditions exhibited protective effects against damage. Local viral injection of miR-199b-5p into mice induced a decrease in pain threshold and OA-like changes. In an OA model, inhibition of miR-199b-5p alleviated the pathological progression of OA. Furthermore, bioinformatics analysis and experimental validation identified Gcnt2 and Fzd6 as potential target genes of MiR-199b-5p. Thus, these results indicated that MiR-199b-5p/Gcnt2 and Fzd6 axis might be a novel therapeutic target for the treatment of OA.

Rich data from large biobanks, coupled with increasingly accessible association statistics from genome-wide association studies (GWAS), provide great opportunities to dissect the complex relationships among human traits and diseases. We introduce BADGERS, a powerful method to perform polygenic score-based biobank-wide association scans. Compared to traditional approaches, BADGERS uses GWAS summary statistics as input and does not require multiple traits to be measured in the same cohort. We applied BADGERS to two independent datasets for late-onset Alzheimer's disease (AD; n=61,212). Among 1738 traits in the UK biobank, we identified 48 significant associations for AD. Family history, high cholesterol, and numerous traits related to intelligence and education showed strong and independent associations with AD. Furthermore, we identified 41 significant associations for a variety of AD endophenotypes. While family history and high cholesterol were strongly associated with AD subgroups and pathologies, only intelligence and education-related traits predicted pre-clinical cognitive phenotypes. These results provide novel insights into the distinct biological processes underlying various risk factors for AD.

Alterations in the function of K+ channels such as the voltage- and Ca2+-activated K+ channel of large conductance (BKCa) reportedly promote breast cancer (BC) development and progression. Underlying molecular mechanisms remain, however, elusive. Here, we provide electrophysiological evidence for a BKCa splice variant localized to the inner mitochondrial membrane of murine and human BC cells (mitoBKCa). Through a combination of genetic knockdown and knockout along with a cell permeable BKCa channel blocker, we show that mitoBKCa modulates overall cellular and mitochondrial energy production, and mediates the metabolic rewiring referred to as the 'Warburg effect', thereby promoting BC cell proliferation in the presence and absence of oxygen. Additionally, we detect mitoBKCa and BKCa transcripts in low or high abundance, respectively, in clinical BC specimens. Together, our results emphasize, that targeting mitoBKCa could represent a treatment strategy for selected BC patients in future.

Leptospirosis is an emerging infectious disease caused by pathogenic Leptospira spp. Humans and some mammals can develop severe forms of leptospirosis accompanied by a dysregulated inflammatory response, which often results in death. The gut microbiota has been increasingly recognized as a vital element in systemic health. However, the precise role of the gut microbiota in severe leptospirosis is still unknown. Here, we aimed to explore the function and potential mechanisms of the gut microbiota in a hamster model of severe leptospirosis. Our study showed that leptospires were able to multiply in the intestine, cause pathological injury, and induce intestinal and systemic inflammatory responses. 16S rRNA gene sequencing analysis revealed that Leptospira infection changed the composition of the gut microbiota of hamsters with an expansion of Proteobacteria. In addition, gut barrier permeability was increased after infection, as reflected by a decrease in the expression of tight junctions. Translocated Proteobacteria were found in the intestinal epithelium of moribund hamsters, as determined by fluorescence in situ hybridization, with elevated lipopolysaccharide (LPS) levels in the serum. Moreover, gut microbiota depletion reduced the survival time, increased the leptospiral load, and promoted the expression of proinflammatory cytokines after Leptospira infection. Intriguingly, fecal filtration and serum from moribund hamsters both increased the transcription of TNF-α, IL-1β, IL-10, and TLR4 in macrophages compared with those from uninfected hamsters. These stimulating activities were inhibited by LPS neutralization using polymyxin B. Based on our findings, we identified an LPS neutralization therapy that significantly improved the survival rates in severe leptospirosis when used in combination with antibiotic therapy or polyclonal antibody therapy. In conclusion, our study not only uncovers the role of the gut microbiota in severe leptospirosis but also provides a therapeutic strategy for severe leptospirosis.

Development of the mammalian oocyte requires physical contact with the surrounding granulosa cells of the follicle, which provide it with essential nutrients and regulatory signals. This contact is achieved through specialized filopodia, termed transzonal projections (TZPs), that extend from the granulosa cells to the oocyte surface. Transforming growth factor (TGFβ) family ligands produced by the oocyte increase the number of TZPs, but how they do so is unknown. Using an inducible Cre recombinase strategy together with expression of green fluorescent protein to verify Cre activity in individual cells, we examined the effect of depleting the canonical TGFβ mediator, SMAD4, in mouse granulosa cells. We observed a 20-50% decrease in the total number of TZPs in SMAD4-depleted granulosa cell-oocyte complexes, and a 50% decrease in the number of newly generated TZPs when the granulosa cells were reaggregated with wild-type oocytes. Three-dimensional image analysis revealed that TZPs of SMAD4-depleted cells were longer than controls and more frequently oriented towards the oocyte. Strikingly, the transmembrane proteins, N-cadherin and Notch2, were reduced by 50% in SMAD4-depleted cells. SMAD4 may thus modulate a network of cell adhesion proteins that stabilize the attachment of TZPs to the oocyte, thereby amplifying signalling between the two cell types.

Bacteria of the family Enterobacteriaceae are associated with gastrointestinal (GI) bleeding and bacteremia and are a leading cause of death, from sepsis, for individuals with inflammatory bowel diseases. The bacterial behaviors and mechanisms underlying why these bacteria are prone to bloodstream entry remain poorly understood. Herein, we report that clinical isolates of non-typhoidal Salmonella enterica serovars, Escherichia coli, and Citrobacter koseri are rapidly attracted toward sources of human serum. To simulate GI bleeding, we utilized an injection-based microfluidics device and found that femtoliter volumes of human serum are sufficient to induce bacterial attraction to the serum source. This response is orchestrated through chemotaxis and the chemoattractant L-serine, an amino acid abundant in serum that is recognized through direct binding by the chemoreceptor Tsr. We report the first crystal structures of Salmonella Typhimurium Tsr in complex with L-serine and identify a conserved amino acid recognition motif for L-serine shared among Tsr orthologues. We find Tsr to be widely conserved among Enterobacteriaceae and numerous World Health Organization priority pathogens associated with bloodstream infections. Lastly, we find that Enterobacteriaceae use human serum as a source of nutrients for growth and that chemotaxis and the chemoreceptor Tsr provide a competitive advantage for migration into enterohemorrhagic lesions. We define this bacterial behavior of taxis toward serum, colonization of hemorrhagic lesions, and the consumption of serum nutrients as 'bacterial vampirism', which may relate to the proclivity of Enterobacteriaceae for bloodstream infections.

Comorbidity with type 2 diabetes (T2D) results in worsening of cancer-specific and overall prognosis in colorectal cancer (CRC) patients. The treatment of CRC per se may be diabetogenic. We assessed the impact of different types of surgical cancer resections and oncological treatment on risk of T2D development in CRC patients.

Sex-biased genes offer insights into the evolution of sexual dimorphism. Sex-biased genes, especially those with male bias, show elevated evolutionary rates of protein sequences driven by positive selection and relaxed purifying selection in animals. Although rapid sequence evolution of sex-biased genes and evolutionary forces have been investigated in animals and brown algae, less is known about evolutionary forces in dioecious angiosperms. In this study, we separately compared the expression of sex-biased genes between female and male floral buds and between female and male flowers at anthesis in dioecious Trichosanthes pilosa (Cucurbitaceae). In floral buds, sex-biased gene expression was pervasive, and had significantly different roles in sexual dimorphism such as physiology. We observed higher rates of sequence evolution for male-biased genes in floral buds compared to female-biased and unbiased genes. Male-biased genes under positive selection were mainly associated with functions to abiotic stress and immune responses, suggesting that high evolutionary rates are driven by adaptive evolution. Additionally, relaxed purifying selection may contribute to accelerated evolution in male-biased genes generated by gene duplication. Our findings, for the first time in angiosperms, suggest evident rapid evolution of male-biased genes, advance our understanding of the patterns and forces driving the evolution of sexual dimorphism in dioecious plants.

Sexual bonds are central to the social lives of many species, including humans, and monogamous prairie voles have become the predominant model for investigating such attachments. We developed an automated whole-brain mapping pipeline to identify brain circuits underlying pair-bonding behavior. We identified bonding-related c-Fos induction in 68 brain regions clustered in seven major brain-wide neuronal circuits. These circuits include known regulators of bonding, such as the bed nucleus of the stria terminalis, paraventricular hypothalamus, ventral pallidum, and prefrontal cortex. They also include brain regions previously unknown to shape bonding, such as ventromedial hypothalamus, medial preoptic area, and the medial amygdala, but that play essential roles in bonding-relevant processes, such as sexual behavior, social reward, and territorial aggression. Contrary to some hypotheses, we found that circuits active during mating and bonding were largely sexually monomorphic. Moreover, c-Fos induction across regions was strikingly consistent between members of a pair, with activity best predicted by rates of ejaculation. A novel cluster of regions centered in the amygdala remained coordinated after bonds had formed, suggesting novel substrates for bond maintenance. Our tools and results provide an unprecedented resource for elucidating the networks that translate sexual experience into an enduring bond.

Eph receptor tyrosine kinases participate in a variety of normal and pathogenic processes during development and throughout adulthood. This versatility is likely facilitated by the ability of Eph receptors to signal through diverse cellular signalling pathways: primarily by controlling cytoskeletal dynamics, but also by regulating cellular growth, proliferation, and survival. Despite many proteins linked to these signalling pathways interacting with Eph receptors, the specific mechanisms behind such links and their coordination remain to be elucidated. In a proteomics screen for novel EPHB2 multi-effector proteins, we identified human MYC binding protein 2 (MYCBP2 or PAM or Phr1). MYCBP2 is a large signalling hub involved in diverse processes such as neuronal connectivity, synaptic growth, cell division, neuronal survival, and protein ubiquitination. Our biochemical experiments demonstrate that the formation of a complex containing EPHB2 and MYCBP2 is facilitated by FBXO45, a protein known to select substrates for MYCBP2 ubiquitin ligase activity. Formation of the MYCBP2-EPHB2 complex does not require EPHB2 tyrosine kinase activity and is destabilised by binding of ephrin-B ligands, suggesting that the MYCBP2-EPHB2 association is a prelude to EPHB2 signalling. Paradoxically, the loss of MYCBP2 results in increased ubiquitination of EPHB2 and a decrease of its protein levels suggesting that MYCBP2 stabilises EPHB2. Commensurate with this effect, our cellular experiments reveal that MYCBP2 is essential for efficient EPHB2 signalling responses in cell lines and primary neurons. Finally, our genetic studies in Caenorhabditis elegans provide in vivo evidence that the ephrin receptor VAB-1 displays genetic interactions with known MYCBP2 binding proteins. Together, our results align with the similarity of neurodevelopmental phenotypes caused by MYCBP2 and EPHB2 loss of function, and couple EPHB2 to a signalling effector that controls diverse cellular functions.

The ratio between κ and λ light chain (LC)-expressing B cells varies considerably between species. We recently identified Kinase D-interacting substrate of 220 kDa (Kidins220) as an interaction partner of the BCR. In vivo ablation of Kidins220 in B cells resulted in a marked reduction of λLC-expressing B cells. Kidins220 knockout B cells fail to open and recombine the genes of the Igl locus, even in genetic scenarios where the Igk genes cannot be rearranged or where the κLC confers autoreactivity. Igk gene recombination and expression in Kidins220-deficient B cells is normal. Kidins220 regulates the development of λLC B cells by enhancing the survival of developing B cells and thereby extending the time-window in which the Igl locus opens and the genes are rearranged and transcribed. Further, our data suggest that Kidins220 guarantees optimal pre-BCR and BCR signaling to induce Igl locus opening and gene recombination during B cell development and receptor editing.

Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria also produce sterols, but their function in bacteria is not known. Moreover, many more species, including pathogens and commensal microbes, acquire or modify sterols from eukaryotic hosts through poorly understood molecular mechanisms. The aerobic methanotroph Methylococcus capsulatus was the first bacterium shown to synthesize sterols, producing a mixture of C-4 methylated sterols that are distinct from those observed in eukaryotes. C-4 methylated sterols are synthesized in the cytosol and localized to the outer membrane, suggesting that a bacterial sterol transport machinery exists. Until now, the identity of such machinery remained a mystery. In this study, we identified three novel proteins that may be the first examples of transporters for bacterial sterol lipids. The proteins, which all belong to well-studied families of bacterial metabolite transporters, are predicted to reside in the inner membrane, periplasm, and outer membrane of M. capsulatus, and may work as a conduit to move modified sterols to the outer membrane. Quantitative analysis of ligand binding revealed their remarkable specificity for 4-methylsterols, and crystallographic structures coupled with docking and molecular dynamics simulations revealed the structural bases for substrate binding by two of the putative transporters. Their striking structural divergence from eukaryotic sterol transporters signals that they form a distinct sterol transport system within the bacterial domain. Finally, bioinformatics revealed the widespread presence of similar transporters in bacterial genomes, including in some pathogens that use host sterol lipids to construct their cell envelopes. The unique folds of these bacterial sterol binding proteins should now guide the discovery of other proteins that handle this essential metabolite.

Migraine headache is hypothesized to involve the activation and sensitization of trigeminal sensory afferents that innervate the cranial meninges. To better understand migraine pathophysiology and improve clinical translation, we used two-photon calcium imaging via a closed cranial window in awake mice to investigate changes in the responses of meningeal afferent fibers using a preclinical model of migraine involving cortical spreading depolarization (CSD). A single CSD episode caused a seconds-long wave of calcium activation that propagated across afferents and along the length of individual afferents. Surprisingly, unlike previous studies in anesthetized animals with exposed meninges, only a very small afferent population was persistently activated in our awake mouse preparation, questioning the relevance of this neuronal response to the onset of migraine pain. In contrast, we identified a larger subset of meningeal afferents that developed augmented responses to acute three-dimensional meningeal deformations that occur in response to locomotion bouts. We observed increased responsiveness in a subset of afferents that were already somewhat sensitive to meningeal deformation before CSD. Furthermore, another subset of previously insensitive afferents also became sensitive to meningeal deformation following CSD. Our data provides new insights into the mechanisms underlying migraine, including the emergence of enhanced meningeal afferent responses to movement-related meningeal deformations as a potential neural substrate underlying the worsening of migraine headache during physical activity.

Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury. Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked β-N-acetylglucosamine (O-GlcNAc) signaling, a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhance in vivo neuron regeneration. Here, we define the specific metabolic pathway by which O-GlcNAc transferase (ogt-1) loss of function mediates increased regenerative outgrowth. Performing in vivo laser axotomy and measuring subsequent regeneration of individual neurons in C. elegans, we find that glycolysis, serine synthesis pathway (SSP), one-carbon metabolism (OCM), and the downstream transsulfuration metabolic pathway (TSP) are all essential in this process. The regenerative effects of ogt-1 mutation are abrogated by genetic and/or pharmacological disruption of OCM and the SSP linking OCM to glycolysis. Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway. These results are further supported by RNA sequencing that reveals dramatic transcriptional changes by the ogt-1 mutation, in the genes involved in glycolysis, OCM, TSP, and ATP metabolism. Strikingly, the beneficial effects of the ogt-1 mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals. Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron in ogt-1 animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury.

Temporal binding has been understood as an illusion in timing judgment. When an action triggers an outcome (e.g. a sound) after a brief delay, the action is reported to occur later than if the outcome does not occur, and the outcome is reported to occur earlier than a similar outcome not caused by an action. We show here that an attention mechanism underlies the seeming illusion of timing judgment. In one method, participants watch a rotating clock hand and report event times by noting the clock hand position when the event occurs. We find that visual spatial attention is critically involved in shaping event time reports made in this way. This occurs because action and outcome events result in shifts of attention around the clock rim, thereby biasing the perceived location of the clock hand. Using a probe detection task to measure attention, we show a difference in the distribution of visual spatial attention between a single-event condition (sound only or action only) and a two-event agency condition (action plus sound). Participants accordingly report the timing of the same event (the sound or the action) differently in the two conditions: spatial attentional shifts masquerading as temporal binding. Furthermore, computational modeling based on the attention measure can reproduce the temporal binding effect. Studies that use time judgment as an implicit marker of voluntary agency should first discount the artefactual changes in event timing reports that actually reflect differences in spatial attention. The study also has important implications for related results in mental chronometry obtained with the clock-like method since Wundt, as attention may well be a critical confounding factor in the interpretation of these studies.

Orbitofrontal cortex (OFC) is classically linked to inhibitory control, emotion regulation, and reward processing. Recent perspectives propose that the OFC also generates predictions about perceptual events, actions, and their outcomes. We tested the role of the OFC in detecting violations of prediction at two levels of abstraction (i.e., hierarchical predictive processing) by studying the event-related potentials (ERPs) of patients with focal OFC lesions (n = 12) and healthy controls (n = 14) while they detected deviant sequences of tones in a local-global paradigm. The structural regularities of the tones were controlled at two hierarchical levels by rules defined at a local (i.e., between tones within sequences) and at a global (i.e., between sequences) level. In OFC patients, ERPs elicited by standard tones were unaffected at both local and global levels compared to controls. However, patients showed an attenuated mismatch negativity (MMN) and P3a to local prediction violation, as well as a diminished MMN followed by a delayed P3a to the combined local and global level prediction violation. The subsequent P3b component to conditions involving violations of prediction at the level of global rules was preserved in the OFC group. Comparable effects were absent in patients with lesions restricted to the lateral PFC, which lends a degree of anatomical specificity to the altered predictive processing resulting from OFC lesion. Overall, the altered magnitudes and time courses of MMN/P3a responses after lesions to the OFC indicate that the neural correlates of detection of auditory regularity violation are impacted at two hierarchical levels of rule abstraction.