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Many animal species use olfaction to extract information about objects in their environment. Yet, the specific molecular signature that any given object emits varies due to various factors. Here, we detail why such variability makes chemosensory-mediated object recognition such a hard problem, and we propose that a major function of the elaborate chemosensory network is to overcome it. We describe previous work addressing different elements of the problem and outline future research directions that we consider essential for a full understanding of object-oriented olfaction. In particular, we call for extensive representation of olfactory object variability in chemical, behavioral, and electrophysiological analyses. While written with an emphasis on macrosmatic mammalian species, our arguments apply to all organisms that employ chemosensation to navigate complex environments., Competing Interests: Declaration of interests The authors declare no competing or financial interests related to this work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Downregulation of the urea cycle enzyme argininosuccinate synthase (ASS1) in multiple tumors is associated with a poor prognosis partly because of the metabolic diversion of cytosolic aspartate for pyrimidine synthesis, supporting proliferation and mutagenesis owing to nucleotide imbalance. Here, we find that prolonged loss of ASS1 promotes DNA damage in colon cancer cells and fibroblasts from subjects with citrullinemia type I. Following acute induction of DNA damage with doxorubicin, ASS1 expression is elevated in the cytosol and the nucleus with at least a partial dependency on p53; ASS1 metabolically restrains cell cycle progression in the cytosol by restricting nucleotide synthesis. In the nucleus, ASS1 and ASL generate fumarate for the succination of SMARCC1, destabilizing the chromatin-remodeling complex SMARCC1-SNF5 to decrease gene transcription, specifically in a subset of the p53-regulated cell cycle genes. Thus, following DNA damage, ASS1 is part of the p53 network that pauses cell cycle progression, enabling genome maintenance and survival. Loss of ASS1 contributes to DNA damage and promotes cell cycle progression, likely contributing to cancer mutagenesis and, hence, adaptability potential., (© 2024. The Author(s).)

In most mammals, conspecific chemosensory communication relies on semiochemical release within complex bodily secretions and subsequent stimulus detection by the vomeronasal organ (VNO). Urine, a rich source of ethologically relevant chemosignals, conveys detailed information about sex, social hierarchy, health, and reproductive state, which becomes accessible to a conspecific via vomeronasal sampling. So far, however, numerous aspects of social chemosignaling along the vomeronasal pathway remain unclear. Moreover, since virtually all research on vomeronasal physiology is based on secretions derived from inbred laboratory mice, it remains uncertain whether such stimuli provide a true representation of potentially more relevant cues found in the wild. Here, we combine a robust low-noise VNO activity assay with comparative molecular profiling of sex- and strain-specific mouse urine samples from two inbred laboratory strains as well as from wild mice. With comprehensive molecular portraits of these secretions, VNO activity analysis now enables us to (i) assess whether and, if so, how much sex/strain-selective 'raw' chemical information in urine is accessible via vomeronasal sampling; (ii) identify which chemicals exhibit sufficient discriminatory power to signal an animal's sex, strain, or both; (iii) determine the extent to which wild mouse secretions are unique; and (iv) analyze whether vomeronasal response profiles differ between strains. We report both sex- and, in particular, strain-selective VNO representations of chemical information. Within the urinary 'secretome', both volatile compounds and proteins exhibit sufficient discriminative power to provide sex- and strain-specific molecular fingerprints. While total protein amount is substantially enriched in male urine, females secrete a larger variety at overall comparatively low concentrations. Surprisingly, the molecular spectrum of wild mouse urine does not dramatically exceed that of inbred strains. Finally, vomeronasal response profiles differ between C57BL/6 and BALB/c animals, with particularly disparate representations of female semiochemicals., Competing Interests: MN, MN, RB, DF, AL, RS, PS, YB, MS No competing interests declared, (© 2023, Nagel et al.)

The physiological performance of any sensory organ is determined by its anatomy and physical properties. Consequently, complex sensory structures with elaborate features have evolved to optimize stimulus detection. Understanding these structures and their physical nature forms the basis for mechanistic insights into sensory function. Despite its crucial role as a sensor for pheromones and other behaviorally instructive chemical cues, the vomeronasal organ (VNO) remains a poorly characterized mammalian sensory structure. Fundamental principles of its physico-mechanical function, including basic aspects of stimulus sampling, remain poorly explored. Here, we revisit the classical vasomotor pump hypothesis of vomeronasal stimulus uptake. Using advanced anatomical, histological, and physiological methods, we demonstrate that large parts of the lateral mouse VNO are composed of smooth muscle. Vomeronasal smooth muscle tissue comprises two subsets of fibers with distinct topography, structure, excitation-contraction coupling, and, ultimately, contractile properties. Specifically, contractions of a large population of noradrenaline-sensitive cells mediate both transverse and longitudinal lumen expansion, whereas cholinergic stimulation targets an adluminal group of smooth muscle fibers. The latter run parallel to the VNO's rostro-caudal axis and are ideally situated to mediate antagonistic longitudinal constriction of the lumen. This newly discovered arrangement implies a novel mode of function. Single-cell transcriptomics and pharmacological profiling reveal the receptor subtypes involved. Finally, 2D/3D tomography provides non-invasive insight into the intact VNO's anatomy and mechanics, enables measurement of luminal fluid volume, and allows an assessment of relative volume change upon noradrenergic stimulation. Together, we propose a revised conceptual framework for mouse vomeronasal pumping and, thus, stimulus sampling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Social communication is crucial for the survival of many species. In most vertebrates, a dedicated chemosensory system, the vomeronasal system (VNS), evolved to process ethologically relevant chemosensory cues. The first central processing stage of the VNS is the accessory olfactory bulb (AOB), which sends information to downstream brain regions via AOB mitral cells (AMCs). Recent studies provided important insights about the functional properties of AMCs, but little is known about the principles that govern their coordinated activity. Here, we recorded local field potentials (LFPs) and single-unit activity in the AOB of adult male and female mice during presentation of natural stimuli. Our recordings reveal prominent LFP theta-band oscillatory episodes with a characteristic spatial pattern across the AOB. Throughout an experiment, the AOB network shows varying degrees of similarity to this pattern, in a manner that depends on the sensory stimulus. Analysis of LFP signal polarity and single-unit activity indicates that oscillatory episodes are generated locally within the AOB, likely representing a reciprocal interaction between AMCs and granule cells. Notably, spike times of many AMCs are constrained to the negative LFP oscillation phase in a manner that can drastically affect integration by downstream processing stages. Based on these observations, we propose that LFP oscillations may gate, bind, and organize outgoing signals from individual AOB neurons to downstream processing stages. Our findings suggest that, as in other neuronal systems and brain regions, population-level oscillations play a key role in organizing and enhancing transmission of socially relevant chemosensory information. SIGNIFICANCE STATEMENT The accessory olfactory bulb (AOB) is the first central stage of the vomeronasal system, a chemosensory system dedicated to processing cues from other organisms. Information from the AOB is conveyed to other brain regions via activity of its principal neurons, AOB mitral cells (AMCs). Here, we show that socially relevant sensory stimulation of the mouse vomeronasal system leads not only to changes in AMC activity, but also to distinct theta-band (∼5 Hz) oscillatory episodes in the local field potential. Notably AMCs favor the negative phase of these oscillatory events. Our findings suggest a novel mechanism for the temporal coordination of distributed patterns of neuronal activity, which can serve to efficiently activate downstream processing stages., (Copyright © 2023 the authors.)

The choroid plexus (CP) plays a key role in remotely controlling brain function in health, aging, and disease. Here, we report that CP epithelial cells express the brain-specific cholesterol 24-hydroxylase (CYP46A1) and that its levels are decreased under different mouse and human brain conditions, including amyloidosis, aging, and SARS-CoV-2 infection. Using primary mouse CP cell cultures, we demonstrate that the enzymatic product of CYP46A1, 24(S)-hydroxycholesterol, downregulates inflammatory transcriptomic signatures within the CP, found here to be elevated across multiple neurological conditions. In vitro, the pro-inflammatory cytokine tumor necrosis factor α (TNF-α) downregulates CYP46A1 expression, while overexpression of CYP46A1 or its pharmacological activation in mouse CP organ cultures increases resilience to TNF-α. In vivo, overexpression of CYP46A1 in the CP in transgenic mice with amyloidosis is associated with better cognitive performance and decreased brain inflammation. Our findings suggest that CYP46A1 expression in the CP impacts the role of this niche as a guardian of brain immune homeostasis., Competing Interests: Declaration of interests M.S. is an inventor of the intellectual property that forms the basis for the development of PD-L1 immunotherapy for AD., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells by binding to the angiotensin-converting enzyme 2 (hACE2) receptor. This process is aided by the transmembrane protease serine 2 (TMPRSS2), which enhances entry efficiency and infectiousness by cleaving the SARS-CoV-2 surface glycoprotein (Spike). The cleavage primes the Spike protein, promoting membrane fusion instead of receptor-mediated endocytosis. Despite the pivotal role played by TMPRSS2, our understanding of its non-protease distinct domains remains limited. In this report, we present evidence indicating the potential phosphorylation of a minimum of six tyrosine residues within the cytosolic tail (CT) of TMPRSS2. Via the use of TMPRSS2 CT phospho-mimetic mutants, we observed a reduction in TMPRSS2 protease activity, accompanied by a decrease in SARS-CoV-2 pseudovirus transduction, which was found to occur mainly via the endosomal pathway. We expanded our investigation beyond TMPRSS2 CT and discovered the involvement of other non-protease domains in regulating infection. Our co-immunoprecipitation experiments demonstrated a strong interaction between TMPRSS2 and Spike. We revealed a 21 amino acid long TMPRSS2-Spike-binding region (TSBR) within the TMPRSS2 scavenger receptor cysteine-rich (SRCR) domain that contributes to this interaction. Our study sheds light on novel functionalities associated with TMPRSS2's cytosolic tail and SRCR region. Both of these regions have the capability to regulate SARS-CoV-2 entry pathways. These findings contribute to a deeper understanding of the complex interplay between viral entry and host factors, opening new avenues for potential therapeutic interventions.

Proteasomes critically regulate proteostasis via protein degradation. Proteasomes are multi-subunit complexes composed of the 20S proteolytic core particle (20S CP) that, in association with one or two 19S regulatory particles (19S RPs), generates the 26S proteasome, which is the major proteasomal complex in cells. Native gel protocols are used to investigate the 26S/20S ratio. However, a simple method for detecting these proteasome complexes in cells is missing. To this end, using CRISPR technology, we YFP-tagged the endogenous PSMB6 (β1) gene, a 20S CP subunit, and co-tagged endogenous PSMD6 (Rpn7), a 19S RP subunit, with the mScarlet fluorescent protein. We observed the colocalization of the YFP and mScarlet fluorescent proteins in the cells, with higher nuclear accumulation. Nuclear proteasomal granules are formed under osmotic stress, and all were positive for YFP and mScarlet. Previously, we have reported that PSMD1 knockdown, one of the 19 RP subunits, gives rise to a high level of "free" 20S CPs. Intriguingly, under this condition, the 20S-YFP remained nuclear, whereas the PSMD6-mScarlet was mostly in cytoplasm, demonstrating the distinct subcellular distribution of uncapped 20S CPs. Lately, we have shown that the PSMA3 (α7) C-terminus, a 20S CP subunit, binds multiple intrinsically disordered proteins (IDPs). Remarkably, the truncation of the PSMA3 C-terminus is phenotypically reminiscent of PSMD1 knockdown. These data suggest that the PSMA3 C-terminal region is critical for 26S proteasome integrity.

Increasing evidence suggests that ribosome composition and modifications contribute to translation control. Whether direct mRNA binding by ribosomal proteins regulates the translation of specific mRNA and contributes to ribosome specialization has been poorly investigated. Here, we used CRISPR-Cas9 to mutate the RPS26 C-terminus (RPS26dC) predicted to bind AUG upstream nucleotides at the exit channel. RPS26 binding to positions -10 to -16 of short 5' untranslated region (5'UTR) mRNAs exerts positive and negative effects on translation directed by Kozak and Translation Initiator of Short 5'UTR (TISU), respectively. Consistent with that, shortening the 5'UTR from 16 to 10 nt diminished Kozak and enhanced TISU-driven translation. As TISU is resistant and Kozak is sensitive to energy stress, we examined stress responses and found that the RPS26dC mutation confers resistance to glucose starvation and mTOR inhibition. Furthermore, the basal mTOR activity is reduced while AMP-activated protein kinase is activated in RPS26dC cells, mirroring energy-deprived wild-type (WT) cells. Likewise, the translatome of RPS26dC cells is correlated to glucose-starved WT cells. Our findings uncover the central roles of RPS26 C-terminal RNA binding in energy metabolism, in the translation of mRNAs bearing specific features and in the translation tolerance of TISU genes to energy stress., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RPS3, a universal core component of the 40S ribosomal subunit, interacts with mRNA at the entry channel. Whether RPS3 mRNA-binding contributes to specific mRNA translation and ribosome specialization in mammalian cells is unknown. Here we mutated RPS3 mRNA-contacting residues R116, R146 and K148 and report their impact on cellular and viral translation. R116D weakened cap-proximal initiation and promoted leaky scanning, while R146D had the opposite effect. Additionally, R146D and K148D displayed contrasting effects on start-codon fidelity. Translatome analysis uncovered common differentially translated genes of which the downregulated set bears long 5'UTR and weak AUG context, suggesting a stabilizing role during scanning and AUG selection. We identified an RPS3-dependent regulatory sequence (RPS3RS) in the sub-genomic 5'UTR of SARS-CoV-2 consisting of a CUG initiation codon and a downstream element that is also the viral transcription regulatory sequence (TRS). Furthermore, RPS3 mRNA-binding residues are essential for SARS-CoV-2 NSP1-mediated inhibition of host translation and for its ribosomal binding. Intriguingly, NSP1-induced mRNA degradation was also reduced in R116D cells, indicating that mRNA decay occurs in the ribosome context. Thus, RPS3 mRNA-binding residues have multiple translation regulatory functions and are exploited by SARS-CoV-2 in various ways to influence host and viral mRNA translation and stability., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The COVID-19 pandemic resulted from the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since its first appearance in 2019, new SARS-CoV-2 variants of concern (VOCs) have emerged frequently, changing the infection's dynamic. SARS-CoV-2 infects cells via two distinct entry routes; receptor-mediated endocytosis or membrane fusion, depending on the absence or presence of transmembrane serine protease 2 (TMPRSS2), respectively. In laboratory conditions, the Omicron SARS-CoV-2 strain inefficiently infects cells predominantly via endocytosis and is phenotypically characterized by decreased syncytia formation compared to the earlier Delta variant. Thus, it is important to characterize Omicron's unique mutations and their phenotypic manifestations. Here, by utilizing SARS-CoV-2 pseudovirions, we report that the specific Omicron Spike F375 residue decreases infectivity, and its conversion to the Delta S375 sequence significantly increases Omicron infectivity. Further, we identified that residue Y655 decreases Omicron's TMPRSS2 dependency and entry via membrane fusion. The Y655H, K764N, K856N and K969N Omicron revertant mutations, bearing the Delta variant sequence, increased the cytopathic effect of cell-cell fusion, suggesting these Omicron-specific residues reduced the severity of SARS-CoV-2. This study of the correlation of the mutational profile with the phenotypic outcome should sensitize our alertness towards emerging VOCs.

Background: Proteasome inhibitors are in use in treating certain types of cancers. These drugs inhibit the catalytic activity of the 20S proteasome, shared by all the different proteasome complexes. Inhibitors of the 26S-associated deubiquitinating activity explicitly inhibit the 26S proteasomal degradation of ubiquitinylated substrates. We have previously reported an alternative strategy that is based on reducing the 26S/20S ratio by depleting PSMD1, 6, and 11, the subunits of the 19S proteasome regulatory complex. Given the addiction of the many cancer types to a high 26S/20S ratio, the depletion strategy is highly effective in killing many aggressive cancer cell lines but not mouse and human immortalized and normal cells., Methods: We used two aggressive cell lines, MDA-MB-231, a triple-negative breast tumor cell line, and OVCAR8, a high-grade ovary adenocarcinoma. Cell culture, mouse MDA-MB-231, OVCAR8 xenografts, and patient-derived ovarian cancer xenograft (PDX) models were transduced with lentivectors expressing PSMD1 shRNA. Tumor size was measured to follow treatment efficacy., Results: Using different experimental strategies of expressing shRNA, we found that PSMD1 depletion, either by expressing PSMD1 shRNA in an inducible manner or in a constitutive manner, robustly inhibited MDA-MB-231, and OVCAR8 xenograft tumor growth. Furthermore, the PSMD1 depletion strategy compromised the growth of the PDX of primary ovarian cancer., Conclusion: Our results suggest that reducing the 26S/20S ratio might be a valuable strategy for treating drug-resistant aggressive types of cancers., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

The degradation of intrinsically disordered proteins (IDPs) by a non-26S proteasome process does not require proteasomal targeting by polyubiquitin. However, whether and how IDPs are recognized by the non-26S proteasome, including the 20S complex, remains unknown. Analyses of protein interactome datasets revealed that the 20S proteasome subunit, PSMA3, preferentially interacts with many IDPs. In vivo and cell-free experiments revealed that the C-terminus of PSMA3, a 69-amino-acids-long fragment, is an IDP trapper. A recombinant trapper is sufficient to interact with many IDPs, and blocks IDP degradation in vitro by the 20S proteasome, possibly by competing with the native trapper. In addition, over a third of the PSMA3 trapper-binding proteins have previously been identified as 20S proteasome substrates and, based on published datasets, many of the trapper-binding proteins are associated with the intracellular proteasomes. The PSMA3-trapped IDPs that are proteasome substrates have the unique features previously recognized as characteristic 20S proteasome substrates in vitro. We propose a model whereby the PSMA3 C-terminal region traps a subset of IDPs to facilitate their proteasomal degradation.

CRISPR technology affords a simple and robust way to edit the genomes of cells, providing powerful tools for basic research and medicine. While using Cas9 to target a genomic site is very efficient, making a specific mutation at that site is much less so, as it depends on the endogenous DNA repair machinery. Various strategies have been developed to increase the efficiency of knock-in mutagenesis, but often the desired cells remain a small percentage of the total population. To improve efficiency, strategies to select edited cells have been developed. In some applications, a selectable foreign gene is linked directly to the gene of interest (GOI). Alternatively, co-editing, where the GOI is edited along with a selectable gene, enriches the desired cells since the cells that successfully edited the selectable gene are likely to have also edited the GOI. To minimize perturbations of the host genome, "scarless" selection strategies have been developed, where the modified cells are mutated solely in the GOI. In this review, we will discuss strategies employed to improve specific genome editing in mammalian cells, focusing on ways to select successfully edited cells.

In the Bruce effect, a mated female mouse becomes resistant to the pregnancy-blocking effect of the stud. Various lines of evidence suggest that this form of behavioral imprinting results from reduced sensitivity of the female's accessory olfactory bulb (AOB) to the stud's chemosignals. However, the AOB's combinatorial code implies that diminishing responses to one individual will distort representations of other stimuli. Here, we record extracellular responses of AOB neurons in mated and unmated female mice while presenting urine stimuli from the stud and from other sources. We find that, while initial sensory responses in the AOB (within a timescale required to guide social interactions) remain stable, responses to extended stimulation (as required for eliciting the pregnancy block) display selective attenuation of stud-responsive neurons. Such temporal disassociation could allow attenuation of slow-acting endocrine processes in a stimulus-specific manner without compromising ongoing representations that guide behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

We directly tested whether, when given the choice to ascend or descend, rodents would favor traveling downwards or upwards. The test incorporated different rodent species that dwell in different habitats and display different life and motor styles. Testing was performed in a three-dimensional Y-maze in which the basis was horizontal and, by rotating it, one arm of the maze could be pointing upwards at a certain angle and the other arm pointed downwards at the same angle. All the tested species displayed a general preference for descent, with rodents from complex habitats being less affected by inclination compared with rodents from flatlands. Unlike laboratory rats, wild species traveled greater distances along the lower compared to the upper maze arm. All the rodents initially tended to travel the entire length of the inclined maze arms, but such complete trips decreased with the increase in inclination. When introduced into the maze from top or bottom, flatland dwellers traveled mainly in the entry arm. Overall, when given the choice to ascend or descend, all the tested species displayed a preference to descend, perhaps as attraction to the ground, where they usually have their burrows.

The hepatitis B virus (HBV) is one of the smallest but most highly infectious human pathogens. With a DNA genome of only 3.2 kb and only four genes, HBV successfully completes its life cycle by using intricate processes to hijack the host machinery. HBV infects non-dividing liver cells in which dNTPs are limited. As a DNA virus, HBV requires dNTPs for its replication. HBV induces the ATR-mediated cellular DNA damage response pathway to overcome this constraint. This pathway upregulates R2 (RRM2) expression in generating an active RNR holoenzyme catalyzing de novo dNTP synthesis. Previously we reported that ERE, a small RNA fragment within the HBx ORF, is sufficient to induce R2 upregulation. Interestingly, there is high sequence similarity between ERE and a region within the R2 5'UTR that we named R2-box. Here, we established a mutant cell line in the R2-box region of the R2 gene using CRISPR-Cas9 technology to investigate the R2 regulation by ERE. This cell line expresses a much lower R2 level than the parental cell line. Interestingly, the HBV infection and life cycle were severely impaired. These cells became permissive to HBV infection upon ectopically R2 expression. These results validate the requirement of the R2 gene expression for HBV replication. Remarkably, the R2-box mutated cells became ERE refractory, suggesting that the homology region between ERE and R2 gene is critical for ERE-mediated R2 upregulation. Thus, along with the induction of the ATR pathway of the DNA damage response, ERE might also directly target the R2 gene via the R2-box.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal agent of the COVID-19 pandemic. More than 274 million individuals have suffered from COVID-19 and over five million people have died from this disease so far. Therefore, there is an urgent need for therapeutic drugs. Repurposing FDA approved drugs should be favored since evaluation of safety and efficacy of de-novo drug design are both costly and time consuming. We report that imatinib, an Abl tyrosine kinase inhibitor, robustly decreases SARS-CoV-2 infection and uncover a mechanism of action. We show that imatinib inhibits the infection of SARS-CoV-2 and its surrogate lentivector pseudotype. In latter, imatinib inhibited both routes of viral entry, endocytosis and membrane-fusion. We utilized a system to quantify in real-time cell-cell membrane fusion mediated by the SARS-CoV-2 surface protein, Spike, and its receptor, hACE2, to demonstrate that imatinib inhibits this process in an Abl1 and Abl2 independent manner. Furthermore, cellular thermal shift assay revealed a direct imatinib-Spike interaction that affects Spike susceptibility to trypsin digest. Collectively, our data suggest that imatinib inhibits Spike mediated viral entry by an off-target mechanism. These findings mark imatinib as a promising therapeutic drug in inhibiting the early steps of SARS-CoV-2 infection., (© 2022. The Author(s).)

DNA viruses require dNTPs for replication and have developed different strategies to increase intracellular dNTP pools. Hepatitis B virus (HBV) infects non-dividing cells in which dNTPs are scarce and the question is how viral replication takes place. Previously we reported that the virus induces the DNA damage response (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR holoenzyme, the key regulator of dNTP levels, leading to an increase in dNTPs. How the virus induces DDR and RNR-R2 upregulation is not completely known. The viral HBx open reading frame (ORF) was believed to trigger this pathway. Unexpectedly, however, we report here that the production of HBx protein is dispensable. We found that a small conserved region of 125 bases within the HBx ORF is sufficient to upregulate RNR-R2 expression in growth-arrested HepG2 cells and primary human hepatocytes. The observed HBV mRNA embedded regulatory element is named ERE. ERE in isolation is sufficient to activate the ATR-Chk1-E2F1-RNR-R2 DDR pathway. These findings demonstrate a non-coding function of HBV transcripts to support its propagation in non-cycling cells.

Background: For many animals, chemosensory cues are vital for social and defensive interactions and are primarily detected and processed by the vomeronasal system (VNS). These cues are often inherently associated with ethological meaning, leading to stereotyped behaviors. Thus, one would expect consistent representation of these stimuli across different individuals. However, individuals may express different arrays of vomeronasal sensory receptors and may vary in the pattern of connections between those receptors and projection neurons in the accessory olfactory bulb (AOB). In the first part of this study, we address the ability of individuals to form consistent representations despite these potential sources of variability. The second part of our study is motivated by the fact that the majority of research on VNS physiology involves the use of stimuli derived from inbred animals. Yet, it is unclear whether neuronal representations of inbred-derived stimuli are similar to those of more ethologically relevant wild-derived stimuli., Results: First, we compared sensory representations to inbred, wild-derived, and wild urine stimuli in the AOBs of males from two distinct inbred strains, using them as proxies for individuals. We found a remarkable similarity in stimulus representations across the two strains. Next, we compared AOB neuronal responses to inbred, wild-derived, and wild stimuli, again using male inbred mice as subjects. Employing various measures of neuronal activity, we show that wild-derived and wild stimuli elicit responses that are broadly similar to those from inbred stimuli: they are not considerably stronger or weaker, they show similar levels of sexual dimorphism, and when examining population-level activity, cluster with inbred mouse stimuli., Conclusions: Despite strain-specific differences and apparently random connectivity, the AOB can maintain stereotypic sensory representations for broad stimulus categories, providing a substrate for common stereotypical behaviors. In addition, despite many generations of inbreeding, AOB representations capture the key ethological features (i.e., species and sex) of wild-derived and wild counterparts. Beyond these broad similarities, representations of stimuli from wild mice are nevertheless distinct from those elicited by inbred mouse stimuli, suggesting that laboratory inbreeding has indeed resulted in marked modifications of urinary secretions.

Silent information regulator 2-related enzyme 1 (SIRT1) is an NAD + -dependent class III deacetylase and a key component of the cellular metabolic sensing pathway. The requirement of NAD + for SIRT1 activity led us to assume that NQO1, an NADH oxidoreductase producing NAD + , regulates SIRT1 activity. We show here that SIRT1 is capable of increasing NQO1 (NAD(P)H Dehydrogenase Quinone 1) transcription and protein levels. NQO1 physically interacts with SIRT1 but not with an enzymatically dead SIRT1 H363Y mutant. The interaction of NQO1 with SIRT1 is markedly increased under mitochondrial inhibition. Interestingly, under this condition the nuclear pool of NQO1 is elevated. Depletion of NQO1 compromises the role of SIRT1 in inducing transcription of several target genes and eliminates the protective role of SIRT1 following mitochondrial inhibition. Our results suggest that SIRT1 and NQO1 form a regulatory loop where SIRT1 regulates NQO1 expression and NQO1 binds and mediates the protective role of SIRT1 during mitochondrial stress. The interplay between an NADH oxidoreductase enzyme and an NAD + dependent deacetylase may act as a rheostat in sensing mitochondrial stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tsvetkov, Adler, Strobelt, Adamovich, Asher, Reuven and Shaul.)

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has revolutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology-directed repair (HDR), active in only a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, editing a gene of interest (GOI) concomitantly with rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using the repair of the ts mutation as a selectable marker, the selection is "scarless" since editing restores the wild-type (wt) sequence. As proof of principle, we used HEK293 and HeLa cells with a ts mutation in the essential TAF1 gene. CRISPR co-editing of TAF1ts and a GOI resulted in up to 90% of the temperature-resistant cells bearing the desired mutation in the GOI. We used this system to insert large cassettes encoded by plasmid donors and smaller changes encoded by single-stranded oligonucleotide donors (ssODN). Of note, among the genes we edited was the introduction of a T35A mutation in the proteasome subunit PSMB6, which eliminates its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating this system's utility in generating cell lines with biologically relevant mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for selecting genome-edited cells requiring HDR.

Monogenic disorders of the blood system have the potential to be treated by autologous stem cell transplantation of ex vivo genetically modified hematopoietic stem and progenitor cells (HSPCs). The sgRNA/Cas9 system allows for precise modification of the genome at single nucleotide resolution. However, the system is reliant on endogenous cellular DNA repair mechanisms to mend a Cas9-induced double stranded break (DSB), either by the non-homologous end joining (NHEJ) pathway or by the cell-cycle regulated homology-directed repair (HDR) pathway. Here, we describe a panel of ectopically expressed DNA repair factors and Cas9 variants assessed for their ability to promote gene correction by HDR or inhibit gene disruption by NHEJ at the HBB locus. Although transient global overexpression of DNA repair factors did not improve the frequency of gene correction in primary HSPCs, localization of factors to the DSB by fusion to the Cas9 protein did alter repair outcomes toward microhomology-mediated end joining (MMEJ) repair, an HDR event. This strategy may be useful when predictable gene editing outcomes are imperative for therapeutic success., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Benitez, Lomova Kaufman, Cervantes, Clark, Ayoub, Senadheera, Osborne, Sanchez, Crisostomo, Wang, Reuven, Shaul, Hollis, Romero and Kohn.)

The 26S proteasome is the endpoint of the ubiquitin- and ATP-dependent degradation pathway. Over the years, ATP was regarded as completely essential for 26S proteasome function due to its role in ubiquitin-signaling, substrate unfolding and ensuring its structural integrity. We have previously reported that physiological concentrations of NADH are efficient in replacing ATP to maintain the integrity of an enzymatically functional 26S PC. However, the substrate specificity of the NADH-stabilized 26S proteasome complex (26S PC) was never assessed. Here, we show that the binding of NADH to the 26S PC inhibits the ATP-dependent and ubiquitin-independent degradation of the structured ODC enzyme. Moreover, the NADH-stabilized 26S PC is efficient in degrading intrinsically disordered protein (IDP) substrates that might not require ATP-dependent unfolding, such as p27, Tau, c-Fos and more. In some cases, NADH-26S proteasomes were more efficient in processing IDPs than the ATP-26S PC. These results indicate that in vitro, physiological concentrations of NADH can alter the processivity of ATP-dependent 26S PC substrates such as ODC and, more importantly, the NADH-stabilized 26S PCs promote the efficient degradation of many IDPs. Thus, ATP-independent, NADH-dependent 26S proteasome activity exemplifies a new principle of how mitochondria might directly regulate 26S proteasome substrate specificity.

Stereotypic behavior (SB) is common in emotional stress-involved psychiatric disorders and is often attributed to glutamatergic impairments, but the underlying molecular mechanisms are unknown. Given the neuro-modulatory role of acetylcholine, we sought behavioral-transcriptomic links in SB using TgR transgenic mice with impaired cholinergic transmission due to over-expression of the stress-inducible soluble 'readthrough' acetylcholinesterase-R splice variant AChE-R. TgR mice showed impaired organization of behavior, performance errors in a serial maze test, escape-like locomotion, intensified reaction to pilocarpine and reduced rearing in unfamiliar situations. Small-RNA sequencing revealed 36 differentially expressed (DE) microRNAs in TgR mice hippocampi, 8 of which target more than 5 cholinergic transcripts. Moreover, compared to FVB/N mice, TgR prefrontal cortices displayed individually variable changes in over 400 DE mRNA transcripts, primarily acetylcholine and glutamate-related. Furthermore, TgR brains presented c-fos over-expression in motor behavior-regulating brain regions and immune-labeled AChE-R excess in the basal ganglia, limbic brain nuclei and the brain stem, indicating a link with the observed behavioral phenotypes. Our findings demonstrate association of stress-induced SB to previously unknown microRNA-mediated perturbations of cholinergic/glutamatergic networks and underscore new therapeutic strategies for correcting stereotypic behaviors.

The accessory olfactory system controls social and sexual behavior. In the mouse accessory olfactory bulb, the first central stage of information processing along the accessory olfactory pathway, projection neurons (mitral cells) display infra-slow oscillatory discharge with remarkable periodicity. The physiological mechanisms that underlie this default output state, however, remain controversial. Moreover, whether such rhythmic infra-slow activity patterns exist in awake behaving mice and whether such activity reflects the functional organization of the accessory olfactory bulb circuitry remain unclear. Here, we hypothesize that mitral cell ensembles form synchronized microcircuits that subdivide the accessory olfactory bulb into segregated functional clusters. We use a miniature microscope to image the Ca 2+ dynamics within the apical dendritic compartments of large mitral cell ensembles in vivo We show that infra-slow periodic patterns of concerted neural activity, indeed, reflect the idle state of accessory olfactory bulb output in awake male and female mice. Ca 2+ activity profiles are distinct and glomerulus-specific. Confocal time-lapse imaging in acute slices reveals that groups of mitral cells assemble into microcircuits that exhibit correlated Ca 2+ signals. Moreover, electrophysiological profiling of synaptic connectivity indicates functional coupling between mitral cells. Our results suggest that both intrinsically rhythmogenic neurons and neurons entrained by fast synaptic drive are key elements in organizing the accessory olfactory bulb into functional microcircuits, each characterized by a distinct default pattern of infra-slow rhythmicity. SIGNIFICANCE STATEMENT Information processing in the accessory olfactory bulb (AOB) plays a central role in conspecific chemosensory communication. Surprisingly, many basic physiological principles that underlie neuronal signaling in the AOB remain elusive. Here, we show that AOB projection neurons (mitral cells) form parallel synchronized ensembles both in vitro and in vivo Infra-slow synchronous oscillatory activity within AOB microcircuits thus adds a new dimension to chemosensory coding along the accessory olfactory pathway., (Copyright © 2020 Tsitoura et al.)

CRISPR/Cas9 is a powerful tool for genome editing in cells and organisms. Nevertheless, introducing directed templated changes by homology-directed repair (HDR) requires the cellular DNA repair machinery, such as the MRN complex (Mre11/Rad50/Nbs1). To improve the process, we tailored chimeric constructs of Cas9, in which SpCas9 was fused at its N- or C-terminus to a 126aa intrinsically disordered domain from HSV-1 alkaline nuclease (UL12) that recruits the MRN complex. The chimeric Cas9 constructs were two times more efficient in homology-directed editing of endogenous loci in tissue culture cells. This effect was dependent upon the MRN-recruiting activity of the domain and required lower amounts of the chimeric Cas9 in comparison with unmodified Cas9. The new constructs improved the yield of edited cells when making endogenous point mutations or inserting small tags encoded by oligonucleotide donor DNA (ssODN), and also with larger insertions encoded by plasmid DNA donor templates. Improved editing was achieved with both transfected plasmid-encoded Cas9 constructs as well as recombinant Cas9 protein transfected as ribonucleoprotein complexes. Our strategy was highly efficient in restoring a genetic defect in a cell line, exemplifying the possible implementation of our strategy in gene therapy. These constructs provide a simple approach to improve directed editing.

Behavioral responses can be classified as innate or learned and are often mediated by distinct neuronal pathways. In many animals, chemical cues are crucial for directing behaviors, and multiple chemosensory subsystems serve this purpose. The major subsystems in vertebrates are the main olfactory system (MOS) and the vomeronasal system (VNS). While the MOS has well-documented associative capabilities, the VNS is known for its role in mediating innate responses to sensory cues with clear ethological significance. However, it remains unknown whether the VNS can map arbitrary sensory activation to novel behavioral outputs. To address this question, we used several optogenetic strategies for selective vomeronasal activation and tested whether mice could associate stimulation patterns with particular reward locations. Our experiments indicate that mice can, indeed, exploit VNS activity to direct novel behavioral responses, implying that the VNS holds a substantial capacity for redirecting and adapting behavioral responses to given stimulation patterns., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

RFX proteins are a family of conserved DNA binding proteins involved in various, essential cellular and developmental processes. RFX1 is a ubiquitously expressed, dual-activity transcription factor capable of both activation and repression of target genes. The exact mechanism by which RFX1 regulates its target is not known yet. In this work, we show that the C-terminal repression domain of RFX1 interacts with the Serine/Threonine protein phosphatase PP1c, and that interaction with RFX1 can target PP1c to specific sites in the genome. Given that PP1c was shown to de-phosphorylate several transcription factors, as well as the regulatory C-terminal domain of RNA Polymerase II the recruitment of PP1c to promoters may be a mechanism by which RFX1 regulates the target genes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Short-chain fatty acids (SCFAs) have immunomodulatory effects, but the underlying mechanisms are not well understood. Here we show that pentanoate, a physiologically abundant SCFA, is a potent regulator of immunometabolism. Pentanoate induces IL-10 production in lymphocytes by reprogramming their metabolic activity towards elevated glucose oxidation. Mechanistically, this reprogramming is mediated by supplying additional pentanoate-originated acetyl-CoA for histone acetyltransferases, and by pentanoate-triggered enhancement of mTOR activity. In experimental mouse models of colitis and multiple sclerosis, pentanoate-induced regulatory B cells mediate protection from autoimmune pathology. Additionally, pentanoate shows a potent histone deacetylase-inhibitory activity in CD4 + T cells, thereby reducing their IL-17A production. In germ-free mice mono-colonized with segmented filamentous bacteria (SFB), pentanoate inhibits the generation of small-intestinal Th17 cells and ameliorates SFB-promoted inflammation in the central nervous system. Taken together, by enhancing IL-10 production and suppressing Th17 cells, the SCFA pentanoate might be of therapeutic relevance for inflammatory and autoimmune diseases.

The Hippo pathway utilizes a well-characterized Ser/Thr kinase cascade to control the downstream effectors, Yap and Taz. In addition, Yap/Taz and other Hippo pathway components are directly regulated by tyrosine kinases (TKs). The methodological strategies described here use the example of the c-Abl non-receptor TK and the Yap substrate to outline the steps used to identify and to validate tyrosine phosphorylation sites, including bioinformatic approaches, ectopic expression of proteins in transfected tissue culture cells, and mutagenesis of endogenous proteins by CRISPR-Cas9. These general strategies can be applied to investigate regulation of protein signaling moieties by tyrosine phosphorylation in the context of distinct TKs.

Proteasomal degradation is the main route of regulated proteostasis. The 20S proteasome is the core particle (CP) responsible for the catalytic activity of all proteasome complexes. Structural constraints mean that only unfolded, extended polypeptide chains may enter the catalytic core of the 20S proteasome. It has been previously shown that the 20S CP is active in degradation of certain intrinsically disordered proteins (IDP) lacking structural constrains. Here, a comprehensive analysis of the 20S CP substrates in vitro is conducted. It is revealed that the 20S CP substrates are highly disordered. However, not all the IDPs are 20S CP substrates. The group of the IDPs that are 20S CP substrates, termed 20S-IDPome are characterized by having significantly more protein binding partners, more posttranslational modification sites, and are highly enriched for RNA binding proteins. The vast majority of them are involved in splicing, mRNA processing, and translation. Remarkably, it is found that low complexity proteins with prion-like domain (PrLD), which interact with GR or PR di-peptide repeats, are the most preferential 20S CP substrates. The finding suggests roles of the 20S CP in gene transcription and formation of phase-separated granules., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In many mammalian species, the accessory olfactory system plays a central role in guiding behavioral and physiological responses to social and reproductive interactions. Because of its relatively compact structure and its direct access to amygdalar and hypothalamic nuclei, the accessory olfactory pathway provides an ideal system to study sensory control of complex mammalian behavior. During the last several years, many studies employing molecular, behavioral, and physiological approaches have significantly expanded and enhanced our understanding of this system. The purpose of the current review is to integrate older and newer studies to present an updated and comprehensive picture of vomeronasal signaling and coding with an emphasis on early accessory olfactory system processing stages. These include vomeronasal sensory neurons in the vomeronasal organ, and the circuitry of the accessory olfactory bulb. Because the overwhelming majority of studies on accessory olfactory system function employ rodents, this review is largely focused on this phylogenetic order, and on mice in particular. Taken together, the emerging view from both older literature and more recent studies is that the molecular, cellular, and circuit properties of chemosensory signaling along the accessory olfactory pathway are in many ways unique. Yet, it has also become evident that, like the main olfactory system, the accessory olfactory system also has the capacity for adaptive learning, experience, and state-dependent plasticity. In addition to describing what is currently known about accessory olfactory system function and physiology, we highlight what we believe are important gaps in our knowledge, which thus define exciting directions for future investigation.

Proteasomes are large intracellular complexes responsible for the degradation of cellular proteins. The altered protein homeostasis of cancer cells results in increased dependency on proteasome function. The cellular proteasome composition comprises the 20S catalytic complex that is frequently capped with the 19S regulatory particle in forming the 26S proteasome. Proteasome inhibitors target the catalytic barrel (20S) and thus this inhibition does not allow the deconvolution of the distinct roles of 20S versus 26S proteasomes in cancer progression. We examined the degree of dependency of cancer cells specifically to the level of the 26S proteasome complex. Oncogenic transformation of human and mouse immortalized cells with mutant Ras induced a strong posttranscriptional increase of the 26S proteasome subunits, giving rise to high 26S complex levels. Depletion of a single subunit of the 19S RP was sufficient to reduce the 26S proteasome level and lower the cellular 26S/20S ratio. Under this condition the viability of the Ras-transformed MCF10A cells was severely compromised. This observation led us to hypothesize that cancer cell survival is dependent on maximal utilization of its 26S proteasomes. We validated this possibility in a large number of cancer cell lines and found that partial reduction of the 26S proteasome level impairs viability in all cancer cells examined and was not correlated with cell doubling time or reduction efficiency. Interstingly, normal human fibroblasts are refractory to the same type of 26S proteasome reduction. The suppression of 26S proteasomes in cancer cells activated the UPR and caspase-3 and cells stained positive with Annexin V. In addition, suppression of the 26S proteasome resulted in cellular proteasome redistribution, cytoplasm shrinkage, and nuclear deformation, the hallmarks of apoptosis. The observed tumor cell-specific addiction to the 26S proteasome levels sets the stage for future strategies in exploiting this dependency in cancer therapy.

The vomeronasal system (VNS) is a major vertebrate chemosensory system that functions in parallel to the main olfactory system (MOS). Despite many similarities, the two systems dramatically differ in the temporal domain. While MOS responses are governed by breathing and follow a subsecond temporal scale, VNS responses are uncoupled from breathing and evolve over seconds. This suggests that the contribution of response dynamics to stimulus information will differ between these systems. While temporal dynamics in the MOS are widely investigated, similar analyses in the accessory olfactory bulb (AOB) are lacking. Here, we have addressed this issue using controlled stimulus delivery to the vomeronasal organ of male and female mice. We first analyzed the temporal properties of AOB projection neurons and demonstrated that neurons display prolonged, variable, and neuron-specific characteristics. We then analyzed various decoding schemes using AOB population responses. We showed that compared with the simplest scheme (i.e., integration of spike counts over the entire response period), the division of this period into smaller temporal bins actually yields poorer decoding accuracy. However, optimal classification accuracy can be achieved well before the end of the response period by integrating spike counts within temporally defined windows. Since VNS stimulus uptake is variable, we analyzed decoding using limited information about stimulus uptake time, and showed that with enough neurons, such time-invariant decoding is feasible. Finally, we conducted simulations that demonstrated that, unlike the main olfactory bulb, the temporal features of AOB neurons disfavor decoding with high temporal accuracy, and, rather, support decoding without precise knowledge of stimulus uptake time. SIGNIFICANCE STATEMENT A key goal in sensory system research is to identify which metrics of neuronal activity are relevant for decoding stimulus features. Here, we describe the first systematic analysis of temporal coding in the vomeronasal system (VNS), a chemosensory system devoted to socially relevant cues. Compared with the main olfactory system, timescales of VNS function are inherently slower and variable. Using various analyses of real and simulated data, we show that the consideration of response times relative to stimulus uptake can aid the decoding of stimulus information from neuronal activity. However, response properties of accessory olfactory bulb neurons favor decoding schemes that do not rely on the precise timing of stimulus uptake. Such schemes are consistent with the variable nature of VNS stimulus uptake., (Copyright © 2018 the authors 0270-6474/18/384957-20$15.00/0.)

Background: Achieving controlled stimulus delivery is a major challenge in the physiological analysis of the vomeronasal system (VNS)., New Method: We provide a comprehensive description of a setup allowing controlled stimulus delivery into the vomeronasal organ (VNO) of anesthetized mice. VNO suction is achieved via electrical stimulation of the sympathetic nerve trunk (SNT) using cuff electrodes, followed by flushing of the nasal cavity. Successful application of this methodology depends on several aspects including the surgical preparation, fabrication of cuff electrodes, experimental setup modifications, and the stimulus delivery and flushing. Here, we describe all these aspects in sufficient detail to allow other researchers to readily adopt it. We also present a custom written MATLAB based software with a graphical user interface that controls all aspects of the actual experiment, including trial sequencing, hardware control, and data logging., Results: The method allows measurement of stimulus evoked sensory responses in brain regions that receive vomeronasal inputs. An experienced investigator can complete the entire surgical procedure within thirty minutes., Comparison With Existing Methods: This is the only approach that allows repeated and controlled stimulus delivery to the intact VNO, employing the natural mode of stimulus uptake. The approach is economical with respect to stimuli, requiring stimulus volumes as low as 1-2μl., Conclusions: This comprehensive description will allow other investigators to adapt this setup to their own experimental needs and can thus promote our physiological understanding of this fascinating chemosensory system. With minor changes it can also be adapted for other rodent species., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Accurate determination of mouse positions from video data is crucial for various types of behavioral analyses. While detection of body positions is straightforward, the correct identification of nose positions, usually more informative, is far more challenging. The difficulty is largely due to variability in mouse postures across frames., Results: Here, we present OptiMouse, an extensively documented open-source MATLAB program providing comprehensive semiautomatic analysis of mouse position data. The emphasis in OptiMouse is placed on minimizing errors in position detection. This is achieved by allowing application of multiple detection algorithms to each video, including custom user-defined algorithms, by selection of the optimal algorithm for each frame, and by correction when needed using interpolation or manual specification of positions., Conclusions: At a basic level, OptiMouse is a simple and comprehensive solution for analysis of position data. At an advanced level, it provides an open-source and expandable environment for a detailed analysis of mouse position data.

In this issue of Neuron, Iurilli and Datta (2017) conduct a comprehensive analysis of odor responses in the posterolateral cortical amygdala, a brain region implicated in innate processing of olfactory cues. Unexpectedly, neuronal responses in this region do not reveal preferred representations of stimulus valence., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The polyomavirus middle T antigen (PyMT) oncogene activates the cellular nonreceptor tyrosine kinase c-Src and recruits the Hippo pathway effectors, Yap (yes-associated protein) and Taz (transcriptional coactivator with PDZ-binding motif), as key steps in oncogenesis. Yap and Taz are transcription coactivators shuttling from the cytoplasm to the nucleus. The Hippo pathway kinase Lats1/2 (large tumor suppressor homolog) reduces Yap/Taz nuclear localization and minimizes their cytoplasmic levels by facilitating their ubiquitination by the E3 ligase SCF(β-TrCP). In contrast, PyMT increases the cytoplasmic Taz level. Here we show that this unique PyMT behavior is mediated by Src. We demonstrate that PyMT-induced Src activation inhibits degradation of both wild-type and tyrosine-less Taz, ruling out Taz modification as a mechanism of escaping degradation. Instead, we found that Src attenuates the SCF(β-TrCP) E3-ligase activity in blunting Taz proteasomal degradation. The role of Src in rescuing Taz from TrCP-mediated degradation gives rise to higher cell proliferation under dense cell culture. Finally, IkB (NF-kappa-B inhibitor), a known substrate of β-TrCP, was rescued by Src, suggesting a wider effect of Src on β-TrCP substrates. These findings introduce the Src tyrosine kinase as a regulator of SCF(β-TrCP).

The accessory olfactory system controls social and sexual behavior. However, key aspects of sensory signaling along the accessory olfactory pathway remain largely unknown. Here, we investigate patterns of spontaneous neuronal activity in mouse accessory olfactory bulb mitral cells, the direct neural link between vomeronasal sensory input and limbic output. Both in vitro and in vivo, we identify a subpopulation of mitral cells that exhibit slow stereotypical rhythmic discharge. In intrinsically rhythmogenic neurons, these periodic activity patterns are maintained in absence of fast synaptic drive. The physiological mechanism underlying mitral cell autorhythmicity involves cyclic activation of three interdependent ionic conductances: subthreshold persistent Na(+) current, R-type Ca(2+) current, and Ca(2+)-activated big conductance K(+) current. Together, the interplay of these distinct conductances triggers infraslow intrinsic oscillations with remarkable periodicity, a default output state likely to affect sensory processing in limbic circuits., Significance Statement: We show for the first time that some rodent accessory olfactory bulb mitral cells-the direct link between vomeronasal sensory input and limbic output-are intrinsically rhythmogenic. Driven by ≥ 3 distinct interdependent ionic conductances, infraslow intrinsic oscillations show remarkable periodicity both in vitro and in vivo. As a novel default state, infraslow autorhythmicity is likely to affect limbic processing of pheromonal information., (Copyright © 2016 the authors 0270-6474/16/363127-18$15.00/0.)

For many animals, chemosensation is essential for guiding social behavior. However, because multiple factors can modulate levels of individual chemical cues, deriving information about other individuals via natural chemical stimuli involves considerable challenges. How social information is extracted despite these sources of variability is poorly understood. The vomeronasal system provides an excellent opportunity to study this topic due to its role in detecting socially relevant traits. Here, we focus on two such traits: a female mouse's strain and reproductive state. In particular, we measure stimulus-induced neuronal activity in the accessory olfactory bulb (AOB) in response to various dilutions of urine, vaginal secretions, and saliva, from estrus and non-estrus female mice from two different strains. We first show that all tested secretions provide information about a female's receptivity and genotype. Next, we investigate how these traits can be decoded from neuronal activity despite multiple sources of variability. We show that individual neurons are limited in their capacity to allow trait classification across multiple sources of variability. However, simple linear classifiers sampling neuronal activity from small neuronal ensembles can provide a substantial improvement over that attained with individual units. Furthermore, we show that some traits are more efficiently detected than others, and that particular secretions may be optimized for conveying information about specific traits. Across all tested stimulus sources, discrimination between strains is more accurate than discrimination of receptivity, and detection of receptivity is more accurate with vaginal secretions than with urine. Our findings highlight the challenges of chemosensory processing of natural stimuli, and suggest that downstream readout stages decode multiple behaviorally relevant traits by sampling information from distinct but overlapping populations of AOB neurons.

Small caliber synthetic vascular grafts are commonly used for bypass surgery and dialysis access sites but have high failure rates because of neointima formation and thrombosis. Seeding synthetic grafts with endothelial cells (ECs) provides a biocompatible surface that may prevent graft failure. However, EC detachment following exposure to blood flow still remains a major obstacle in the development of biosynthetic grafts. We tested the hypothesis that induced expression by the seeded EC, of vascular endothelial growth factor165 (VEGF165) and of fibulin-5, an extracellular matrix glycoprotein that has a crucial role in elastin fiber organization and increase EC adherence to surfaces, may improve long-term graft patency. Autologous ECs were isolated from venous segments, and were transduced with retroviral vectors expressing fibulin-5 and VEGF165. The modified cells were seeded on expanded polytetrafluoroethylene (ePTFE) grafts and implanted in a large animal model. Three months after transplantation, all grafts seeded with modified EC were patent on a selective angiography, whereas only a third of the control grafts were patent. Similar results were shown at 6 months. Thus, seeding ePTFE vascular grafts with genetically modified EC improved long-term small caliber graft patency. The biosynthetic grafts may provide a novel therapeutic modality for patients with peripheral vascular disease and patients requiring vascular access for hemodialysis.

Persistent activity has been reported in many brain areas and is hypothesized to mediate working memory and emotional brain states and to rely upon network or biophysical feedback. Here, we demonstrate a novel mechanism by which persistent neuronal activity can be generated without feedback, relying instead on the slow removal of Na+ from neurons following bursts of activity. We show that mitral cells in the accessory olfactory bulb (AOB), which plays a major role in mammalian social behavior, may respond to a brief sensory stimulation with persistent firing. By combining electrical recordings, Ca2+ and Na+ imaging, and realistic computational modeling, we explored the mechanisms underlying the persistent activity in AOB mitral cells. We found that the exceptionally slow inward current that underlies this activity is governed by prolonged dynamics of intracellular Na+ ([Na+]i), which affects neuronal electrical activity via several pathways. Specifically, elevated dendritic [Na+]i reverses the Na+-Ca2+ exchanger activity, thus modifying the [Ca2+]i set-point. This process, which relies on ubiquitous membrane mechanisms, is likely to play a role in other neuronal types in various brain regions.

Across all sensory modalities, stimuli can vary along multiple dimensions. Efficient extraction of information requires sensitivity to those stimulus dimensions that provide behaviorally relevant information. To derive social information from chemosensory cues, sensory systems must embed information about the relationships between behaviorally relevant traits of individuals and the distributions of the chemical cues that are informative about these traits. In simple cases, the mere presence of one particular compound is sufficient to guide appropriate behavior. However, more generally, chemosensory information is conveyed via relative levels of multiple chemical cues, in non-trivial ways. The computations and networks needed to derive information from multi-molecule stimuli are distinct from those required by single molecule cues. Our current knowledge about how socially relevant information is encoded by chemical blends, and how it is extracted by chemosensory systems is very limited. This manuscript explores several scenarios and the neuronal computations required to identify them.

Background & Aims: Hepatitis B virus (HBV) infects and replicates in quiescent hepatocytes, which are deficient in dNTPs, the critical precursors of HBV replication. Most tumor viruses promote dNTP production in host cells by inducing cell proliferation. Although HBV is known as a major cause of hepatocellular carcinoma, it does not lead to cellular proliferation. Instead, HBV acquires dNTPs by activating the expression of the R2 subunit of the Ribonucleotide Reductase (RNR) holoenzyme, the cell cycle gene that is rate-limiting for generation of dNTPs, without inducing the cell cycle. We wished to elucidate the molecular basis of HBV-dependent R2 expression in quiescent cells., Methods: Quiescent HepG2 cells were transduced with an HBV-containing lentiviral vector, and primary human hepatocytes were infected with HBV. DNA damage response and RNR-R2 gene expression were monitored under this condition., Results: We report here that HBV-induced R2 expression is mediated by the E2F1 transcription factor, and that HBV induces E2F1 accumulation, modification and binding to the R2 promoter. We found that Chk1, a known E2F1 kinase that functions in response to DNA damage, was activated by HBV. In cells where Chk1 was pharmacologically inhibited, or depleted by shRNA-mediated knockdown, HBV-mediated R2 expression was severely attenuated. Furthermore, we found that HBV attenuates DNA repair, thus reducing cellular dNTP consumption., Conclusions: Our findings demonstrate that HBV exploits the Chk1-E2F1 axis of the DNA damage response pathway to induce R2 expression in a cell cycle-independent manner. This suggests that inhibition of this pathway may have a therapeutic value for HBV carriers., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The polyomavirus middle T antigen (PyMT) is an oncogene that activates the non-receptor tyrosine kinase, c-Src, and physically interacts with Taz (WWTR1). Taz is a pro-oncogenic transcription coactivator of the Tead transcription factors. The Hippo tumor suppressor pathway activates the kinase Lats, which phosphorylates Taz, leading to its nuclear exclusion and blunting Tead coactivation. We found that Taz was required for transformation by PyMT, but counter-intuitively, Taz was exclusively cytoplasmic in the presence of PyMT. We demonstrate that in the presence of PyMT, wild-type Taz was phosphorylated by Lats, in a Src-dependent manner. Consistently, a Lats refractory Taz mutant did not undergo cytoplasmic retention by PyMT. We show that Yap, the Taz paralog, and Shp2 phosphatase were nuclear excluded as well. Our findings describe a noncanonical activation of Lats, and an unprecedented Tead-independent role for Taz and Yap in viral-mediated oncogenesis.

The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser(46) in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser(46), and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

YES-associated protein (YAP) is a central transcription coactivator that functions as an oncogene in a number of experimental systems. However, under DNA damage, YAP activates pro-apoptotic genes in conjunction with p73. This program switching is mediated by c-Abl (Abelson murine leukemia viral oncogene) via phosphorylation of YAP at the Y357 residue (pY357). YAP as an oncogene coactivates the TEAD (transcriptional enhancer activator domain) family transcription factors. Here we asked whether c-Abl regulates the YAP-TEAD functional module. We found that DNA damage, through c-Abl activation, specifically depressed YAP-TEAD-induced transcription. Remarkably, c-Abl counteracts YAP-induced transformation by interfering with the YAP-TEAD transcriptional program. c-Abl induced TEAD1 phosphorylation, but the YAP-TEAD complex remained unaffected. In contrast, TEAD coactivation was compromised by phosphomimetic YAP Y357E mutation but not Y357F, as demonstrated at the level of reporter genes and endogenous TEAD target genes. Furthermore, YAP Y357E also severely compromised the role of YAP in cell transformation, migration, anchorage-independent growth, and epithelial-to-mesenchymal transition (EMT) in human mammary MCF10A cells. These results suggest that YAP pY357 lost TEAD transcription activation function. Our results demonstrate that YAP pY357 inactivates YAP oncogenic function and establish a role for YAP Y357 phosphorylation in cell-fate decision.

The mouse vomeronasal organ (VNO) plays a critical role in semiochemical detection and social communication. Vomeronasal stimuli are typically secreted in various body fluids. Following direct contact with urine deposits or other secretions, a peristaltic vascular pump mediates fluid entry into the recipient's VNO. Therefore, while vomeronasal sensory neurons (VSNs) sample various stimulatory semiochemicals dissolved in the intraluminal mucus, they might also be affected by the general physicochemical properties of the "solvent." Here, we report cycle stage-correlated variations in urinary pH among female mice. Estrus-specific pH decline is observed exclusively in urine samples from sexually experienced females. Moreover, patch-clamp recordings in acute VNO slices reveal that mouse VSNs reliably detect extracellular acidosis. Acid-evoked responses share the biophysical and pharmacological hallmarks of the hyperpolarization-activated current Ih. Mechanistically, VSN acid sensitivity depends on a pH-induced shift in the voltage-dependence of Ih activation that causes the opening of HCN channels at rest, thereby increasing VSN excitability. Together, our results identify extracellular acidification as a potent activator of vomeronasal Ih and suggest HCN channel-dependent vomeronasal gain control of social chemosignaling. Our data thus reveal a potential mechanistic basis for stimulus pH detection in rodent chemosensory communication., (Copyright © 2015 the authors 0270-6474/15/354025-15$15.00/0.)