Parallel neural pathways control sodium consumption and taste valence.

The hedonic value of salt fundamentally changes depending on the internal state. High concentrations of salt induce innate aversion under sated states, whereas such aversive stimuli transform into appetitive ones under sodium depletion. Neural mechanisms underlying this state-dependent salt valence switch are poorly understood. Using transcriptomics state-to-cell-type mapping and neural manipulations, we show that positive and negative valences of salt are controlled by anatomically distinct neural circuits in the mammalian brain. The hindbrain interoceptive circuit regulates sodium-specific appetitive drive , whereas behavioral tolerance of aversive salts is encoded by a dedicated class of neurons in the forebrain lamina terminalis (LT) expressing prostaglandin E2 (PGE2) receptor, Ptger3. We show that these LT neurons regulate salt tolerance by selectively modulating aversive taste sensitivity, partly through a PGE2-Ptger3 axis. These results reveal the bimodal regulation of appetitive and tolerance signals toward salt, which together dictate the amount of sodium consumption under different internal states.

Neural Control and Modulation of Thirst, Sodium Appetite, and Hunger.

The function of central appetite neurons is instructing animals to ingest specific nutrient factors that the body needs. Emerging evidence suggests that individual appetite circuits for major nutrients-water, sodium, and food-operate on unique driving and quenching mechanisms. This review focuses on two aspects of appetite regulation. First, we describe the temporal relationship between appetite neuron activity and consumption behaviors. Second, we summarize ingestion-related satiation signals that differentially quench individual appetite circuits. We further discuss how distinct appetite and satiation systems for each factor may contribute to nutrient homeostasis from the functional and evolutional perspectives.

Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior.

The ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) contains ∼4,000 neurons that project to multiple targets and control innate social behaviors including aggression and mounting. However, the number of cell types in VMHvl and their relationship to connectivity and behavioral function are unknown. We performed single-cell RNA sequencing using two independent platforms-SMART-seq (∼4,500 neurons) and 10x (∼78,000 neurons)-and investigated correspondence between transcriptomic identity and axonal projections or behavioral activation, respectively. Canonical correlation analysis (CCA) identified 17 transcriptomic types (T-types), including several sexually dimorphic clusters, the majority of which were validated by seqFISH. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity.

Sensory representation and detection mechanisms of gut osmolality change.

Ingested food and water stimulate sensory systems in the oropharyngeal and gastrointestinal areas before absorption1,2. These sensory signals modulate brain appetite circuits in a feed-forward manner3-5. Emerging evidence suggests that osmolality sensing in the gut rapidly inhibits thirst neurons upon water intake. Nevertheless, it remains unclear how peripheral sensory neurons detect visceral osmolality changes, and how they modulate thirst. Here we use optical and electrical recording combined with genetic approaches to visualize osmolality responses from sensory ganglion neurons. Gut hypotonic stimuli activate a dedicated vagal population distinct from mechanical-, hypertonic- or nutrient-sensitive neurons. We demonstrate that hypotonic responses are mediated by vagal afferents innervating the hepatic portal area (HPA), through which most water and nutrients are absorbed. Eliminating sensory inputs from this area selectively abolished hypotonic but not mechanical responses in vagal neurons. Recording from forebrain thirst neurons and behavioural analyses show that HPA-derived osmolality signals are required for feed-forward thirst satiation and drinking termination. Notably, HPA-innervating vagal afferents do not sense osmolality itself. Instead, these responses are mediated partly by vasoactive intestinal peptide secreted after water ingestion. Together, our results reveal visceral hypoosmolality as an important vagal sensory modality, and that intestinal osmolality change is translated into hormonal signals to regulate thirst circuit activity through the HPA pathway.

Recovery of missing single-cell RNA-sequencing data with optimized transcriptomic references.

Single-cell RNA-sequencing (scRNA-seq) is an indispensable tool for characterizing cellular diversity and generating hypotheses throughout biology. Droplet-based scRNA-seq datasets often lack expression data for genes that can be detected with other methods. Here we show that the observed sensitivity deficits stem from three sources: (1) poor annotation of 3' gene ends; (2) issues with intronic read incorporation; and (3) gene overlap-derived read loss. We show that missing gene expression data can be recovered by optimizing the reference transcriptome for scRNA-seq through recovering false intergenic reads, implementing a hybrid pre-mRNA mapping strategy and resolving gene overlaps. We demonstrate, with a diverse collection of mouse and human tissue data, that reference optimization can substantially improve cellular profiling resolution and reveal missing cell types and marker genes. Our findings argue that transcriptomic references need to be optimized for scRNA-seq analysis and warrant a reanalysis of previously published datasets and cell atlases.

The cellular basis of distinct thirst modalities.

Fluid intake is an essential innate behaviour that is mainly caused by two distinct types of thirst1-3. Increased blood osmolality induces osmotic thirst that drives animals to consume pure water. Conversely, the loss of body fluid induces hypovolaemic thirst, in which animals seek both water and minerals (salts) to recover blood volume. Circumventricular organs in the lamina terminalis are critical sites for sensing both types of thirst-inducing stimulus4-6. However, how different thirst modalities are encoded in the brain remains unknown. Here we employed stimulus-to-cell-type mapping using single-cell RNA sequencing to identify the cellular substrates that underlie distinct types of thirst. These studies revealed diverse types of excitatory and inhibitory neuron in each circumventricular organ structure. We show that unique combinations of these neuron types are activated under osmotic and hypovolaemic stresses. These results elucidate the cellular logic that underlies distinct thirst modalities. Furthermore, optogenetic gain of function in thirst-modality-specific cell types recapitulated water-specific and non-specific fluid appetite caused by the two distinct dipsogenic stimuli. Together, these results show that thirst is a multimodal physiological state, and that different thirst states are mediated by specific neuron types in the mammalian brain.

Chondroitin 4-O-sulfation regulates hippocampal perineuronal nets and social memory.

Glycan alterations are associated with aging, neuropsychiatric, and neurodegenerative diseases, although the contributions of specific glycan structures to emotion and cognitive functions remain largely unknown. Here, we used a combination of chemistry and neurobiology to show that 4-O-sulfated chondroitin sulfate (CS) polysaccharides are critical regulators of perineuronal nets (PNNs) and synapse development in the mouse hippocampus, thereby affecting anxiety and cognitive abilities such as social memory. Brain-specific deletion of CS 4-O-sulfation in mice increased PNN densities in the area CA2 (cornu ammonis 2), leading to imbalanced excitatory-to-inhibitory synaptic ratios, reduced CREB activation, elevated anxiety, and social memory dysfunction. The impairments in PNN densities, CREB activity, and social memory were recapitulated by selective ablation of CS 4-O-sulfation in the CA2 region during adulthood. Notably, enzymatic pruning of the excess PNNs reduced anxiety levels and restored social memory, while chemical manipulation of CS 4-O-sulfation levels reversibly modulated PNN densities surrounding hippocampal neurons and the balance of excitatory and inhibitory synapses. These findings reveal key roles for CS 4-O-sulfation in adult brain plasticity, social memory, and anxiety regulation, and they suggest that targeting CS 4-O-sulfation may represent a strategy to address neuropsychiatric and neurodegenerative diseases associated with social cognitive dysfunction.

Chemosensory modulation of neural circuits for sodium appetite.

Sodium is the main cation in the extracellular fluid and it regulates various physiological functions. Depletion of sodium in the body increases the hedonic value of sodium taste, which drives animals towards sodium consumption1,2. By contrast, oral sodium detection rapidly quenches sodium appetite3,4, suggesting that taste signals have a central role in sodium appetite and its satiation. Nevertheless, the neural mechanisms of chemosensory-based appetite regulation remain poorly understood. Here we identify genetically defined neural circuits in mice that control sodium intake by integrating chemosensory and internal depletion signals. We show that a subset of excitatory neurons in the pre-locus coeruleus express prodynorphin, and that these neurons are a critical neural substrate for sodium-intake behaviour. Acute stimulation of this population triggered robust ingestion of sodium even from rock salt, while evoking aversive signals. Inhibition of the same neurons reduced sodium consumption selectively. We further demonstrate that the oral detection of sodium rapidly suppresses these sodium-appetite neurons. Simultaneous in vivo optical recording and gastric infusion revealed that sodium taste-but not sodium ingestion per se-is required for the acute modulation of neurons in the pre-locus coeruleus that express prodynorphin, and for satiation of sodium appetite. Moreover, retrograde-virus tracing showed that sensory modulation is in part mediated by specific GABA (γ-aminobutyric acid)-producing neurons in the bed nucleus of the stria terminalis. This inhibitory neural population is activated by sodium ingestion, and sends rapid inhibitory signals to sodium-appetite neurons. Together, this study reveals a neural architecture that integrates chemosensory signals and the internal need to maintain sodium balance.

Hierarchical neural architecture underlying thirst regulation.

Neural circuits for appetites are regulated by both homeostatic perturbations and ingestive behaviour. However, the circuit organization that integrates these internal and external stimuli is unclear. Here we show in mice that excitatory neural populations in the lamina terminalis form a hierarchical circuit architecture to regulate thirst. Among them, nitric oxide synthase-expressing neurons in the median preoptic nucleus (MnPO) are essential for the integration of signals from the thirst-driving neurons of the subfornical organ (SFO). Conversely, a distinct inhibitory circuit, involving MnPO GABAergic neurons that express glucagon-like peptide 1 receptor (GLP1R), is activated immediately upon drinking and monosynaptically inhibits SFO thirst neurons. These responses are induced by the ingestion of fluids but not solids, and are time-locked to the onset and offset of drinking. Furthermore, loss-of-function manipulations of GLP1R-expressing MnPO neurons lead to a polydipsic, overdrinking phenotype. These neurons therefore facilitate rapid satiety of thirst by monitoring real-time fluid ingestion. Our study reveals dynamic thirst circuits that integrate the homeostatic-instinctive requirement for fluids and the consequent drinking behaviour to maintain internal water balance.

[Malignant Melanoma of a Male Breast That Was Difficult to Differentiate from Breast Cancer-A Case Report].

INTRODUCTION: Malignant melanoma in the male breast is extremely rare. Here we report a case of malignant melanoma in which a small cystic lesion in the male breast gradually increased during follow-up and was difficult to distinguish from breast cancer. CASE: A 65-year-old male was diagnosed with a tumor in the right breast and was referred to our department for further examination. At 42 years of age, he underwent tumor resection of a malignant melanoma of the abdominal skin. Mammary ultrasonography showed a 0.6 cm cystic mass in his right breast. Eight months later, the right breast mass had increased to 1.4 cm, and a core needle biopsy suggested breast cancer. Total mastectomy with axillary lymph node dissection was performed. HE staining of the resected tumor showed intranuclear inclusion bodies and some large nucleoli. On the basis of various immunostaining methods, malignant melanoma was diagnosed instead of breast cancer. After surgery, adjuvant chemotherapy with molecularly targeted drugs was administered. DISCUSSION: This might have been a case of male breast metastasis of malignant melanoma with very late recurrence.

Multistate Structural Switching of [3]Catenanes with Cyclic Porphyrin Dimers by Complexation with Amine Ligands.

Catenanes with multistate switchable properties are promising components for next-generation molecular machines and supramolecular materials. Herein, we report a ligand-controlled switching method, a novel method for the multistate switching of catenanes controlled by complexation with added amine ligands. To verify this method, a [3]catenane comprising cyclic porphyrin dimers with a rigid π-system has been synthesized. Owing to the rigidity, the relative positions among the cyclic components of the [3]catenane can be precisely controlled by complexation with various amine ligands. Moreover, ligand-controlled multistate switching affects the optical properties of the [3]catenanes: the emission intensity can be tuned by modulating the sizes and coordination numbers of integrated amine ligands. This work shows the utility of using organic ligands for the structural switching of catenanes, and will contribute to the further development of multistate switchable mechanically interlocked molecules.

Thirst driving and suppressing signals encoded by distinct neural populations in the brain.

Thirst is the basic instinct to drink water. Previously, it was shown that neurons in several circumventricular organs of the hypothalamus are activated by thirst-inducing conditions. Here we identify two distinct, genetically separable neural populations in the subfornical organ that trigger or suppress thirst. We show that optogenetic activation of subfornical organ excitatory neurons, marked by the expression of the transcription factor ETV-1, evokes intense drinking behaviour, and does so even in fully water-satiated animals. The light-induced response is highly specific for water, immediate and strictly locked to the laser stimulus. In contrast, activation of a second population of subfornical organ neurons, marked by expression of the vesicular GABA transporter VGAT, drastically suppresses drinking, even in water-craving thirsty animals. These results reveal an innate brain circuit that can turn an animal's water-drinking behaviour on and off, and probably functions as a centre for thirst control in the mammalian brain.

Ty-2 and Ty-3a Conferred Resistance are Insufficient Against Tomato Yellow Leaf Curl Kanchanaburi Virus from Southeast Asia in Single or Mixed Infections of Tomato.

Tomato yellow leaf curl virus (TYLCV), a monopartite begomovirus that originated in the eastern Mediterranean, has spread worldwide, becoming a serious threat to tomato (Solanum lycopersicum L.) production. Southeast Asia is considered one of the hotspots for begomovirus diversity, and a wide variety of local begomovirus species distinct from TYLCV have been identified. In this study, the protection effect of introgressions of single TYLCV Ty resistance genes, Ty-2 and Ty-3a, in tomato was examined against inoculations of the bipartite begomoviruses Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV) and Pepper yellow leaf curl Indonesia virus (PepYLCIV) isolated from Indonesia. Our findings suggest that Ty-2 in the heterozygous state was found to be ineffective against PepYLCIV and TYLCKaV, whereas Ty-3a in the heterozygous state was effective against PepYLCIV and partially effective against TYLCKaV. Quantification of viral DNAs showed correlation between symptom expression and viral DNA accumulation. Moreover, mixed infections of TYLCKaV and PepYLCIV caused notably severe symptoms in tomato plants harboring Ty-3a. In cases of mixed infection, quantifying viral DNAs showed a relatively high accumulation of PepYLCIV, indicating that Ty-3a loses its effectiveness against PepYLCIV when TYLCKaV is also present. This study demonstrates the lack of effectiveness of Ty resistance genes against single and mixed infections of distinct local begomoviruses from Southeast Asia.

High salt recruits aversive taste pathways.

In the tongue, distinct classes of taste receptor cells detect the five basic tastes; sweet, sour, bitter, sodium salt and umami. Among these qualities, bitter and sour stimuli are innately aversive, whereas sweet and umami are appetitive and generally attractive to animals. By contrast, salty taste is unique in that increasing salt concentration fundamentally transforms an innately appetitive stimulus into a powerfully aversive one. This appetitive-aversive balance helps to maintain appropriate salt consumption, and represents an important part of fluid and electrolyte homeostasis. We have shown previously that the appetitive responses to NaCl are mediated by taste receptor cells expressing the epithelial sodium channel, ENaC, but the cellular substrate for salt aversion was unknown. Here we examine the cellular and molecular basis for the rejection of high concentrations of salts. We show that high salt recruits the two primary aversive taste pathways by activating the sour- and bitter-taste-sensing cells. We also demonstrate that genetic silencing of these pathways abolishes behavioural aversion to concentrated salt, without impairing salt attraction. Notably, mice devoid of salt-aversion pathways show unimpeded, continuous attraction even to very high concentrations of NaCl. We propose that the 'co-opting' of sour and bitter neural pathways evolved as a means to ensure that high levels of salt reliably trigger robust behavioural rejection, thus preventing its potentially detrimental effects on health.

The cells and peripheral representation of sodium taste in mice.

Salt taste in mammals can trigger two divergent behavioural responses. In general, concentrated saline solutions elicit robust behavioural aversion, whereas low concentrations of NaCl are typically attractive, particularly after sodium depletion. Notably, the attractive salt pathway is selectively responsive to sodium and inhibited by amiloride, whereas the aversive one functions as a non-selective detector for a wide range of salts. Because amiloride is a potent inhibitor of the epithelial sodium channel (ENaC), ENaC has been proposed to function as a component of the salt-taste-receptor system. Previously, we showed that four of the five basic taste qualities-sweet, sour, bitter and umami-are mediated by separate taste-receptor cells (TRCs) each tuned to a single taste modality, and wired to elicit stereotypical behavioural responses. Here we show that sodium sensing is also mediated by a dedicated population of TRCs. These taste cells express the epithelial sodium channel ENaC, and mediate behavioural attraction to NaCl. We genetically engineered mice lacking ENaCalpha in TRCs, and produced animals exhibiting a complete loss of salt attraction and sodium taste responses. Together, these studies substantiate independent cellular substrates for all five basic taste qualities, and validate the essential role of ENaC for sodium taste in mice.

Impact of intratumoral microbiome on tumor immunity and prognosis in human pancreatic ductal adenocarcinoma.

BACKGROUND: Recent evidence suggests that the presence of microbiome within human pancreatic ductal adenocarcinoma (PDAC) tissue potentially influences cancer progression and prognosis. However, the significance of tumor-resident microbiome remains unclear. We aimed to elucidate the impact of intratumoral bacteria on the pathophysiology and prognosis of human PDAC. METHODS: The presence of intratumoral bacteria was assessed in 162 surgically resected PDACs using quantitative polymerase chain reaction (qPCR) and in situ hybridization (ISH) targeting 16S rRNA. The intratumoral microbiome was explored by 16S metagenome sequencing using DNA extracted from formalin-fixed paraffin-embedded tissues. The profile of intratumoral bacteria was compared with clinical information, pathological findings including tumor-infiltrating T cells, tumor-associated macrophage, fibrosis, and alterations in four main driver genes (KRAS, TP53, CDKN2A/p16, SMAD4) in tumor genomes. RESULTS: The presence of intratumoral bacteria was confirmed in 52 tumors (32%) using both qPCR and ISH. The 16S metagenome sequencing revealed characteristic bacterial profiles within these tumors, including phyla such as Proteobacteria and Firmicutes. Comparison of bacterial profiles between cases with good and poor prognosis revealed a significant positive correlation between a shorter survival time and the presence of anaerobic bacteria such as Bacteroides, Lactobacillus, and Peptoniphilus. The abundance of these bacteria was correlated with a decrease in the number of tumor-infiltrating T cells positive for CD4, CD8, and CD45RO. CONCLUSIONS: Intratumoral infection of anaerobic bacteria such as Bacteroides, Lactobacillus, and Peptoniphilus is correlated with the suppressed anti-PDAC immunity and poor prognosis.

A sex-specific thermogenic neurocircuit induced by predator smell recruiting cholecystokinin neurons in the dorsomedial hypothalamus.

Olfactory cues are vital for prey animals like rodents to perceive and evade predators. Stress-induced hyperthermia, via brown adipose tissue (BAT) thermogenesis, boosts physical performance and facilitates escape. However, many aspects of this response, including thermogenic control and sex-specific effects, remain enigmatic. Our study unveils that the predator odor trimethylthiazoline (TMT) elicits BAT thermogenesis, suppresses feeding, and drives glucocorticoid release in female mice. Chemogenetic stimulation of olfactory bulb (OB) mitral cells recapitulates the thermogenic output of this response and associated stress hormone corticosterone release in female mice. Neuronal projections from OB to medial amygdala (MeA) and dorsomedial hypothalamus (DMH) exhibit female-specific cFos activity toward odors. Cell sorting and single-cell RNA-sequencing of DMH identify cholecystokinin (CCK)-expressing neurons as recipients of predator odor cues. Chemogenetic manipulation and neuronal silencing of DMHCCK neurons further implicate these neurons in the propagation of predator odor-associated thermogenesis and food intake suppression, highlighting their role in female stress-induced hyperthermia.

New chrodrimanin congeners, chrodrimanins D-H, from YO-2 of Talaromyces sp.

Four new meroterpenoids, named chrodrimanins D-G (4-7), and one known compound, renamed chrodrimanin H (8), were isolated from okara (the insoluble residue of whole soybean) that had been fermented with the YO-2 strain of Talaromyces sp. Their structures were elucidated by spectroscopic methods. Chrodrimanins D (4), E (5), and F (6) showed insecticidal activity against silkworms with respective LD(50) values of 20, 10, and 50 µg/g of diet.

A new meroterpenoid, chrodrimanin C, from YO-2 of Talaromyces sp.

The new meroterpenoid, chrodrimanin C (3), together with chrodrimanins A (2) and B (1) were isolated from okara (the insoluble residue of whole soybean) that had been fermented with strain YO-2 of Talaromyces sp. Their structures were elucidated by spectroscopic methods. The partial structures of 1 essential for exhibiting insecticidal activity were investigated by using a silkworm assay. The absolute configuration of 1 was also determined.

Precision synthesis of linear oligorotaxanes and polyrotaxanes achieving well-defined positions and numbers of cyclic components on the axle.

Interest in macromolecules has increased because of their functional properties, which can be tuned using precise organic synthetic methods. For example, desired functions have been imparted by controlling the nanoscale structures of such macromolecules. In particular, compounds with interlocked structures, including rotaxanes, have attracted attention because of their unique supramolecular structures. In such supramolecular structures, the mobility and freedom of the macrocycles are restricted by an axle and dependent on those of other macrocycles, which imparts unique functions to these threaded structures. Recently, methods for the ultrafine engineering and synthesis, as well as functions, of "defined" rotaxane structures that are not statistically dispersed on the axle (i.e., control over the number and position of cyclic molecules) have been reported. Various synthetic strategies allow access to such well-defined linear oligo- and polyrotaxanes, including [1]rotaxanes and [n]rotaxanes (mostly n > 3). These state-of-the-art synthetic methods have resulted in unique functions of these oligo-and polyrotaxane materials. Herein, we review the effective synthetic protocols and functions of precisely constructed one-dimensional oligomers and polymers bearing defined threaded structures, and discuss the latest reports and trends.