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Abstract Hsp70 chaperones are central components of the cellular network that ensures the structural quality of proteins. Despite crucial roles in processes such as protein disaggregation and protein translocation into organelles, their physical mechanism of action has remained hotly debated. To the best of our knowledge, no experimental data has directly proven any of the models proposed to date (Power Stroke, Brownian Ratchet, or Entropic Pulling) due to a lack of suitable methods. Here, we use nanopores, a powerful single-molecule tool, to investigate the mechanism of Hsp70s. We demonstrate that Hsp70s extract trapped polypeptide substrates from the nanopore by generating strong forces (equivalent to 46 pN over distances of 1 nm), that rely on the size of Hsp70. The findings provide unambiguous evidence of the Entropic Pulling mechanism, thus solving a long-standing debate, and proposing a potentially universal principle governing diverse cellular processes. Additionally, these results highlight the utility of biological nanopores for protein studies.

Julien Ghelfi,1– 3,&ast; Zuzana Macek Jilkova,1,3,4,&ast; Christian Sengel,2 Bleuenn Brusset,4 Yann Teyssier,2 Charlotte Costentin,1,3,4 Marion Mercey-Ressejac,3,4 Lucile Dumolard,1,3 Marc Manceau,5 Eliott Mathieu,2 Ivan Bricault,1,2 Thomas Decaens1,3,4 1University of Grenoble-Alpes, Saint Martin d’Hères, France; 2Department of Radiology, Grenoble-Alpes University Hospital, Grenoble, France; 3Institute for Advanced Biosciences - INSERM U1209/CNRS UMR 5309/University of Grenoble-Alpes, La Tronche, France; 4Department of Hepatology and Gastrointestinal Medical Oncology, Grenoble-Alpes University Hospital, Grenoble, France; 5Clinical Pharmacology Unit, Inserm Clinical Research Center, Grenoble-Alpes University Hospital, Grenoble, France&ast;These authors contributed equally to this workCorrespondence: Julien Ghelfi, Department of Radiology, Grenoble-Alpes University Hospital, Boulevard de la Chantourne, Grenoble, 38000, France, Tel +33 476768909, Email jghelfi@chu-grenoble.fr Zuzana Macek Jilkova, Department of Hepato-Gastroenterology, Grenoble-Alpes University Hospital, Boulevard de la Chantourne, Grenoble, 38000, France, Tel +33 669048426, Email zmacekjilkova@chu-grenoble.frPurpose: Percutaneous thermal ablation (PTA) is a cornerstone in the management of early-stage hepatocellular carcinoma (HCC). However, intrahepatic distant recurrence (IDR) occurs in the majority of patients after PTA. The aim of this study was to evaluate the immune signature associated with very early IDR.Patients and Methods: Thirty-one patients (26 men, 5 women; mean age:72.4 ± 8.6; age range:57– 86 years) who underwent PTA for HCC were included in this study. After PTA for HCC, patients were followed and later divided into two groups: a “very early recurrence” group in case of IDR within 12 months after PTA, and a “prolonged recurrence-free” group in case of no recurrence before 12 months of follow-up. Freshly harvested intratumoral and nontumoral liver tissues and peripheral blood were obtained before PTA and explored by multiparametric flow cytometry.Results: The frequency of PD1+CD4+ T cells was higher in the early recurrence group than in the prolonged recurrence-free group in the peripheral blood (24.3%, IQR: 22.3– 36.5 vs 14.0%, IQR: 11.5– 16.4, p< 0.0001), in the nontumoral liver (37.9%, IQR: 36.0– 50.0 vs 22.5%, IQR: 18.0– 29.9, p=0.0004), and in the tumor (37.6%, IQR: 32.3– 39.3 vs 24.0%, IQR: 20.0– 30.3, p=0.0137). Similarly, the frequency of TIM+CD8+ T cells was higher in the very early recurrence group in the peripheral blood (p=0.0021), non-tumoral liver (p=0.0012), and tumor (p=0.0239).Conclusion: The expression of immune checkpoint molecules, such as PD1 and TIM3 on T cells identified HCC patients at risk of very early IDR after PTA who would likely benefit from adjuvant immunotherapy. Thus, our study contributes to a better understanding of the potential association of PTA with adjuvant immunotherapies.Keywords: immune checkpoint molecules, predictive factors, hepatocellular carcinoma, percutaneous thermal ablation, intrahepatic distant recurrence

Astrocyte morphology affects function, including the regulation of glutamatergic signaling. This morphology changes dynamically in response to the environment. However, how early life manipulations alter adult cortical astrocyte morphology is underexplored. Our lab uses brief postnatal resource scarcity, the limited bedding and nesting (LBN) manipulation, in rats. We previously found that LBN augments maternal behaviors and promotes later resilience to adult addiction-related behaviors, reducing impulsivity, risky decision-making, and morphine self-administration. These behaviors rely on glutamatergic transmission in the medial orbitofrontal (mOFC) and medial prefrontal (mPFC) cortex. Here we tested whether LBN changed astrocyte morphology in the mOFC and mPFC of adult rats using a novel viral approach that, unlike traditional markers, fully labels astrocytes. Prior exposure to LBN causes an increase in the surface area and volume of astrocytes in the mOFC and mPFC of adult males and females relative to control-raised rats. We next used bulk RNA sequencing of OFC tissue to assess transcriptional changes that could increase astrocyte size in LBN rats. LBN caused mainly sex-specific changes in differentially expressed genes. Pathway analysis revealed that OFC glutamatergic signaling is altered by LBN in males and females, but the gene changes in that pathway differed across sex. This may represent a convergent sex difference where glutamatergic signaling, which affects astrocyte morphology, is altered by LBN via sex-specific mechanisms. Collectively, these studies highlight that astrocytes may be an important cell type that mediates the effect of early resource scarcity on adult brain function.

Abstract Background Glutamate signaling within the nucleus accumbens underlies motivated behavior and is involved in psychiatric disease. Although behavioral sex differences in these processes are well-established, the neural mechanisms driving these differences are largely unexplored. In these studies, we examine potential sex differences in synaptic plasticity and excitatory transmission within the nucleus accumbens core. Further understanding of baseline sex differences in reward circuitry will shed light on potential mechanisms driving behavioral differences in motivated behavior and psychiatric disease. Methods Behaviorally naïve adult male and female Long-Evans rats, C57Bl/6J mice, and constitutive PKMζ knockout mice were killed and tissue containing the nucleus accumbens core was collected for ex vivo slice electrophysiology experiments. Electrophysiology recordings examined baseline sex differences in synaptic plasticity and transmission within this region and the potential role of PKMζ in long-term depression. Results Within the nucleus accumbens core, both female mice and rats exhibit higher AMPA/NMDA ratios compared to male animals. Further, female mice have a larger readily releasable pool of glutamate and lower release probability compared to male mice. No significant sex differences were detected in spontaneous excitatory postsynaptic current amplitude or frequency. Finally, the threshold for induction of long-term depression was lower for male animals than females, an effect that appears to be mediated, in part, by PKMζ. Conclusions We conclude that there are baseline sex differences in synaptic plasticity and excitatory transmission in the nucleus accumbens core. Our data suggest there are sex differences at multiple levels in this region that should be considered in the development of pharmacotherapies to treat psychiatric illnesses such as depression and substance use disorder.

Abstract Background Global warming is a major challenge for plant survival and growth. Understanding the molecular mechanisms by which higher plants sense and adapt to upsurges in the ambient temperature is essential for developing strategies to enhance plant tolerance to heat stress. Here, we designed a heat-responsive Arabidopsis thaliana reporter line that allows an in-depth investigation of the mechanisms underlying the accumulation of protective heat-shock proteins (HSPs) in response to high temperature. Methods A transgenic Arabidopsis thaliana reporter line named “Heat-Inducible Bioluminescence And Toxicity” (HIBAT) was designed to express from a conditional heat-inducible promoter, a fusion gene encoding for nanoluciferase and d-amino acid oxidase, whose expression is toxic in the presence of d-valine. HIBAT seedlings were exposed to different heat treatments in presence or absence of d-valine and analyzed for survival rate, bioluminescence and HSP gene expression. Results Whereas at 22 °C, HIBAT seedlings grew unaffected by d-valine, and all survived iterative heat treatments without d-valine, 98% died following heat treatments on d-valine. The HSP17.3B promoter was highly specific to heat as it remained unresponsive to various plant hormones, Flagellin, H2O2, osmotic stress and high salt. RNAseq analysis of heat-treated HIBAT seedlings showed a strong correlation with expression profiles of two wild type lines, confirming that HIBAT does not significantly differ from its Col-0 parent. Using HIBAT, a forward genetic screen revealed candidate loss-of-function mutants, apparently defective either at accumulating HSPs at high temperature or at repressing HSP accumulation at non-heat-shock temperatures. Conclusion HIBAT is a valuable candidate tool to identify Arabidopsis mutants defective in the response to high temperature stress. It opens new avenues for future research on the regulation of HSP expression and for understanding the mechanisms of plant acquired thermotolerance.

The regulation of gene expression underlies many forms of learning and behaviour in the mammalian brain. Carpenter et al. define a molecular mechanism whereby Nr4a1 activation leads to persistent changes in gene expression, chromatin and behaviour, in the context of cocaine abstinence.

Life is a non-equilibrium phenomenon. Owing to their high free energy content, the macromolecules of life tend to spontaneously react with ambient oxygen and water and turn into more stable inorganic molecules. A similar thermodynamic picture applies to the complex shapes of proteins: While a polypeptide is emerging unfolded from the ribosome, it may spontaneously acquire secondary structures and collapse into its functional native conformation. The spontaneity of this process is evidence that the free energy of the unstructured state is higher than that of the structured native state. Yet, under stress or because of mutations, complex polypeptides may fail to reach their native conformation and form instead thermodynamically stable aggregates devoid of biological activity. Cells have evolved molecular chaperones to actively counteract the misfolding of stress-labile proteins dictated by equilibrium thermodynamics. HSP60, HSP70 and HSP100 can inject energy from ATP hydrolysis into the forceful unfolding of stable misfolded structures in proteins and convert them into unstable intermediates that can collapse into the native state, even under conditions inauspicious for that state. Aggregates and misfolded proteins may also be forcefully unfolded and degraded by chaperone-gated endo-cellular proteases, and in eukaryotes also by chaperone-mediated autophagy, paving the way for their replacement by new, unaltered functional proteins. The greater energy cost of degrading and replacing a polypeptide, with respect to the cost of its chaperone-mediated repair represents a thermodynamic dilemma: some easily repairable proteins are better to be processed by chaperones, while it can be wasteful to uselessly try recover overly compromised molecules, which should instead be degraded and replaced. Evolution has solved this conundrum by creating a host of unfolding chaperones and degradation machines and by tuning their cellular amounts and activity rates.

Mortality in COVID-19 patients predominantly results from an acute respiratory distress syndrome (ARDS), in which lungs alveolar cells undergo programmed cell death. Mortality in a sepsis-induced ARDS rat model is reduced by adenovirus over-expression of the HSP70 chaperone. A natural rise of body temperature during mild fever can naturally accumulate high cellular levels of HSP70 that can arrest apoptosis and protect alveolar lung cells from inflammatory damages. However, beyond 1–2 h of fever, no HSP70 is being further produced and a decreased in body temperature required to the restore cell's ability to produce more HSP70 in a subsequent fever cycle. We suggest that antipyretics may be beneficial in COVID-19 patients subsequent to several hours of mild (

Stress can disrupt memory and contribute to cognitive impairments in psychiatric disorders, including schizophrenia and attention deficit hyperactivity disorder. These diseases are more common in men than in women, with men showing greater cognitive impairments. Mnemonic deficits induced by stress are mediated, in part, by corticotropin releasing factor (CRF). However, where CRF is acting to regulate memory, and sex differences therein, is understudied. Here we assessed whether CRF in the medial septum (MS), which projects to the hippocampus, affected memory formation in male and female rats. CRF in the MS did not alter hippocampal-independent object recognition memory, but impaired hippocampal-dependent object location memory in both sexes. Interestingly, males were more sensitive than females to the disruptive effect of a low dose of CRF in the MS. Female resistance was not due to circulating ovarian hormones. However, compared to males, females had higher MS expression of CRF binding protein, which reduces CRF bioavailability and thus may mitigate the effect of the low dose of CRF in females. In contrast, there was no sex difference in CRF1 expression in the MS. Consistent with this finding, CRF1 antagonism blocked the memory impairment caused by the high dose of CRF in the MS in both sexes. Collectively, these results suggest that males are more vulnerable than females to the memory impairments caused by CRF in the MS. In both sexes, CRF1 antagonists prevented MS-mediated memory deficits caused by high levels of CRF, and such levels can result from very stressful events. Thus, CRF1 antagonists may be a viable option for treating cognitive deficits in stressed individuals with psychiatric disorders. Keywords: Corticotropin releasing hormone, Medial septum, Memory, Sex differences, Ovarian hormones, Stress

Antoine D’Hollander,1–3 Evelien Mathieu,1,4 Hilde Jans,1 Greetje Vande Velde,2,3 Tim Stakenborg,1 Pol Van Dorpe,1,4 Uwe Himmelreich,2,3 Liesbet Lagae1,4 1Department of Life Science Technology, Imec, 2Department of Imaging and Pathology, Faculty of Medicine, Biomedical MRI Unit, 3Faculty of Medicine, Molecular Small Animal Imaging Center (MoSAIC), 4Department of Physics, Faculty of Sciences, Laboratory of Solid State Physics and Magnetism, KU Leuven, Leuven, Belgium Abstract: The need for sensitive imaging techniques to detect tumor cells is an important issue in cancer diagnosis and therapy. Surface-enhanced Raman scattering (SERS), realized by chemisorption of compounds suitable for Raman spectroscopy onto gold nanoparticles, is a new method for detecting a tumor. As a proof of concept, we studied the use of biocompatible gold nanostars as sensitive SERS contrast agents targeting an ovarian cancer cell line (SKOV3). Due to a high intracellular uptake of gold nanostars after 6 hours of exposure, they could be detected and located with SERS. Using these nanostars for passive targeting after systemic injection in a xenograft mouse model, a detectable signal was measured in the tumor and liver in vivo. These signals were confirmed by ex vivo SERS measurements and darkfield microscopy. In this study, we established SERS nanostars as a highly sensitive contrast agent for tumor detection, which opens the potential for their use as a theranostic agent against cancer. Keywords: SERS, gold nanostars, cancer imaging, Raman active

One of the most significant problems facing older individuals is difficulty staying asleep at night and awake during the day. Understanding the mechanisms by which the regulation of sleep/wake goes awry with age is a critical step in identifying novel therapeutic strategies to improve quality of life for the elderly. We measured wake, non-rapid eye movement (NREM) and rapid-eye movement (REM) sleep in young (2-4 months-old) and aged (22-24 months-old) C57BL6/NIA mice. We used both conventional measures (i.e., bout number and bout duration) and an innovative spike-and-slab statistical approach to characterize age-related fragmentation of sleep/wake. The short (spike) and long (slab) components of the spike-and-slab mixture model capture the distribution of bouts for each behavioral state in mice. Using this novel analytical approach, we found that aged animals are less able to sustain long episodes of wakefulness or NREM sleep. Additionally, spectral analysis of EEG recordings revealed that aging slows theta peak frequency, a correlate of arousal. These combined analyses provide a window into the mechanisms underlying the destabilization of long periods of sleep and wake and reduced vigilance that develop with aging.

This is an account of the research activities of our group during the first two years of its existence. First results from our work on proton-coupled electron transfer and long-range charge tunneling reactions are presented. This includes a hydrogen-bonded cation–anion pair in which a proton-coupled electron transfer process can be phototriggered and followed by simple optical spectroscopic means, as well as a series of rigid rod-like donor-bridge-acceptor molecules which we use to investigate physical phenomena associated with the tunneling of electrons or holes. A unifying feature of this research is the use of light (photons) to induce proton and/or electron transfer.

The sequencing of the full transcriptome (RNA-seq) has become the preferred choice for the measurement of genome-wide gene expression. Despite its widespread use, challenges remain in RNA-seq data analysis. One often-overlooked aspect is normalization. Despite the fact that a variety of factors or 'batch effects' can contribute unwanted variation to the data, commonly used RNA-seq normalization methods only correct for sequencing depth. The study of gene expression is particularly problematic when it is influenced simultaneously by a variety of biological factors in addition to the one of interest. Using examples from experimental neuroscience, we show that batch effects can dominate the signal of interest; and that the choice of normalization method affects the power and reproducibility of the results. While commonly used global normalization methods are not able to adequately normalize the data, more recently developed RNA-seq normalization can. We focus on one particular method, RUVSeq and show that it is able to increase power and biological insight of the results. Finally, we provide a tutorial outlining the implementation of RUVSeq normalization that is applicable to a broad range of studies as well as meta-analysis of publicly available data.