5-HT Neurons Integrate GABA and Dopamine Inputs to Regulate Meal Initiation.

Obesity is a growing global health epidemic with limited effective therapeutics. Serotonin (5-HT) is one major neurotransmitter which remains an excellent target for new weight-loss therapies, but there remains a gap in knowledge on the mechanisms involved in 5-HT produced in the dorsal Raphe nucleus (DRN) and its involvement in meal initiation. Using a closed-loop optogenetic feeding paradigm, we showed that the 5-HTDRN→arcuate nucleus (ARH) circuit plays an important role in regulating meal initiation. Incorporating electrophysiology and ChannelRhodopsin-2-Assisted Circuit Mapping, we demonstrated that 5-HTDRN neurons receive inhibitory input partially from GABAergic neurons in the DRN, and the 5-HT response to GABAergic inputs can be enhanced by hunger. Additionally, deletion of the GABAA receptor subunit in 5-HT neurons inhibits meal initiation with no effect on the satiation process. Finally, we identified the instrumental role of dopaminergic inputs via dopamine receptor D2 in 5-HTDRN neurons in enhancing the response to GABA-induced feeding. Thus, our results indicate that 5-HTDRN neurons are inhibited by synergistic inhibitory actions of GABA and dopamine, which allows for the initiation of a meal.

Modeling of flaxseed protein, oil content, linoleic acid, and lignan content prediction based on hyperspectral imaging.

Protein, oil content, linoleic acid, and lignan are several key indicators for evaluating the quality of flaxseed. In order to optimize the testing methods for flaxseed's nutritional quality and enhance the efficiency of screening high-quality flax germplasm resources, we selected 30 flaxseed species widely cultivated in Northwest China as the subjects of our study. Firstly, we gathered hyperspectral information regarding the seeds, along with data on protein, oil content, linoleic acid, and lignan, and utilized the SPXY algorithm to classify the sample set. Subsequently, the spectral data underwent seven distinct preprocessing methods, revealing that the PLSR model exhibited superior performance after being processed with the SG smoothing method. Feature wavelength extraction was carried out using the Successive Projections Algorithm (SPA) and the Competitive Adaptive Reweighted Sampling (CARS). Finally, four quantitative analysis models, namely Partial Least Squares Regression (PLSR), Support Vector Regression (SVR), Multiple Linear Regression (MLR), and Principal Component Regression (PCR), were individually established. Experimental results demonstrated that among all the models for predicting protein content, the SG-CARS-MLR model predicted the best, with and of 0.9563 and 0.9336, with the corresponding Root Mean Square Error Correction (RMSEC) and Root Mean Square Error Prediction (RMSEP) of 0.4892 and 0.5616, respectively. In the optimal prediction models for oil content, linoleic acid and lignan, the Rp2 was 0.8565, 0.8028, 0.9343, and the RMSEP was 0.8682, 0.5404, 0.5384, respectively. The study results show that hyperspectral imaging technology has excellent potential for application in the detection of quality characteristics of flaxseed and provides a new option for the future non-destructive testing of the nutritional quality of flaxseed.

A study on the recognition of monkeypox infection based on deep convolutional neural networks.

Introduction: The World Health Organization (WHO) has assessed the global public risk of monkeypox as moderate, and 71 WHO member countries have reported more than 14,000 cases of monkeypox infection. At present, the identification of clinical symptoms of monkeypox mainly depends on traditional medical means, which has the problems of low detection efficiency and high detection cost. The deep learning algorithm is excellent in image recognition and can extract and recognize image features quickly and reliably. Methods: Therefore, this paper proposes a residual convolutional neural network based on the λ function and contextual transformer (LaCTResNet) for the image recognition of monkeypox cases. Results: The average recognition accuracy of the neural network model is 91.85%, which is 15.82% higher than that of the baseline model ResNet50 and better than the classical convolutional neural networks models such as AlexNet, VGG16, Inception-V3, and EfficientNet-B5. Discussion: This method realizes high-precision identification of skin symptoms of the monkeypox virus to provide a fast and reliable auxiliary diagnosis method for monkeypox cases for front-line medical staff.

Anoctamin 4 channel currents activate glucose-inhibited neurons in the mouse ventromedial hypothalamus during hypoglycemia.

Glucose is the basic fuel essential for maintenance of viability and functionality of all cells. However, some neurons - namely, glucose-inhibited (GI) neurons - paradoxically increase their firing activity in low-glucose conditions and decrease that activity in high-glucose conditions. The ionic mechanisms mediating electric responses of GI neurons to glucose fluctuations remain unclear. Here, we showed that currents mediated by the anoctamin 4 (Ano4) channel are only detected in GI neurons in the ventromedial hypothalamic nucleus (VMH) and are functionally required for their activation in response to low glucose. Genetic disruption of the Ano4 gene in VMH neurons reduced blood glucose and impaired counterregulatory responses during hypoglycemia in mice. Activation of VMHAno4 neurons increased food intake and blood glucose, while chronic inhibition of VMHAno4 neurons ameliorated hyperglycemia in a type 1 diabetic mouse model. Finally, we showed that VMHAno4 neurons represent a unique orexigenic VMH population and transmit a positive valence, while stimulation of neurons that do not express Ano4 in the VMH (VMHnon-Ano4) suppress feeding and transmit a negative valence. Together, our results indicate that the Ano4 channel and VMHAno4 neurons are potential therapeutic targets for human diseases with abnormal feeding behavior or glucose imbalance.

Hypothalamic Grb10 enhances leptin signalling and promotes weight loss.

Leptin acts on hypothalamic neurons expressing agouti-related protein (AgRP) or pro-opiomelanocortin (POMC) to suppress appetite and increase energy expenditure, but the intracellular mechanisms that modulate central leptin signalling are not fully understood. Here we show that growth factor receptor-bound protein 10 (Grb10), an adaptor protein that binds to the insulin receptor and negatively regulates its signalling pathway, can interact with the leptin receptor and enhance leptin signalling. Ablation of Grb10 in AgRP neurons promotes weight gain, while overexpression of Grb10 in AgRP neurons reduces body weight in male and female mice. In parallel, deletion or overexpression of Grb10 in POMC neurons exacerbates or attenuates diet-induced obesity, respectively. Consistent with its role in leptin signalling, Grb10 in AgRP and POMC neurons enhances the anorexic and weight-reducing actions of leptin. Grb10 also exaggerates the inhibitory effects of leptin on AgRP neurons via ATP-sensitive potassium channel-mediated currents while facilitating the excitatory drive of leptin on POMC neurons through transient receptor potential channels. Our study identifies Grb10 as a potent leptin sensitizer that contributes to the maintenance of energy homeostasis by enhancing the response of AgRP and POMC neurons to leptin.

Identification of wheat seedling varieties based on MssiapNet.

Introduction: In the actual planting of wheat, there are often shortages of seedlings and broken seedlings on long ridges in the field, thus affecting grain yield and indirectly causing economic losses. Variety identification of wheat seedlings using physical methods timeliness and is unsuitable for universal dissemination. Recognition of wheat seedling varieties using deep learning models has high timeliness and accuracy, but fewer researchers exist. Therefore, in this paper, a lightweight wheat seedling variety recognition model, MssiapNet, is proposed. Methods: The model is based on the MobileVit-XS and increases the model's sensitivity to subtle differences between different varieties by introducing the scSE attention mechanism in the MV2 module, so the recognition accuracy is improved. In addition, this paper proposes the IAP module to fuse the identified feature information. Subsequently, training was performed on a self-constructed real dataset, which included 29,020 photographs of wheat seedlings of 29 varieties. Results: The recognition accuracy of this model is 96.85%, which is higher than the other nine mainstream classification models. Although it is only 0.06 higher than the Resnet34 model, the number of parameters is only 1/3 of that. The number of parameters required for MssiapNet is 29.70MB, and the single image Execution time and the single image Delay time are 0.16s and 0.05s. The MssiapNet was visualized, and the heat map showed that the model was superior for wheat seedling variety identification compared with MobileVit-XS. Discussion: The proposed model has a good recognition effect on wheat seedling varieties and uses a few parameters with fast inference speed, which makes it easy to be subsequently deployed on mobile terminals for practical performance testing.

Human loss-of-function variants in the serotonin 2C receptor associated with obesity and maladaptive behavior.

Serotonin reuptake inhibitors and receptor agonists are used to treat obesity, anxiety and depression. Here we studied the role of the serotonin 2C receptor (5-HT2CR) in weight regulation and behavior. Using exome sequencing of 2,548 people with severe obesity and 1,117 control individuals without obesity, we identified 13 rare variants in the gene encoding 5-HT2CR (HTR2C) in 19 unrelated people (3 males and 16 females). Eleven variants caused a loss of function in HEK293 cells. All people who carried variants had hyperphagia and some degree of maladaptive behavior. Knock-in male mice harboring a human loss-of-function HTR2C variant developed obesity and reduced social exploratory behavior; female mice heterozygous for the same variant showed similar deficits with reduced severity. Using the 5-HT2CR agonist lorcaserin, we found that depolarization of appetite-suppressing proopiomelanocortin neurons was impaired in knock-in mice. In conclusion, we demonstrate that 5-HT2CR is involved in the regulation of human appetite, weight and behavior. Our findings suggest that melanocortin receptor agonists might be effective in treating severe obesity in individuals carrying HTR2C variants. We suggest that HTR2C should be included in diagnostic gene panels for severe childhood-onset obesity.

SK3 in POMC neurons plays a sexually dimorphic role in energy and glucose homeostasis.

BACKGROUND: Pro-opiomelanocortin (POMC) neurons play a sexually dimorphic role in body weight and glucose balance. However, the mechanisms for the sex differences in POMC neuron functions are not fully understood. RESULTS: We detected small conductance calcium-activated potassium (SK) current in POMC neurons. Secondary analysis of published single-cell RNA-Seq data showed that POMC neurons abundantly express SK3, one SK channel subunit. To test whether SK3 in POMC neurons regulates POMC neuron functions on energy and glucose homeostasis, we used a Cre-loxP strategy to delete SK3 specifically from mature POMC neurons. POMC-specific deletion of SK3 did not affect body weight in either male or female mice. Interestingly, male mutant mice showed not only decreased food intake but also decreased physical activity, resulting in unchanged body weight. Further, POMC-specific SK3 deficiency impaired glucose balance specifically in female mice but not in male mice. Finally, no sex differences were detected in the expression of SK3 and SK current in total POMC neurons. However, we found higher SK current but lower SK3 positive neuron population in male POMC neurons co-expressing estrogen receptor α (ERα) compared to that in females. CONCLUSION: These results revealed a sexually dimorphic role of SK3 in POMC neurons in both energy and glucose homeostasis independent of body weight control, which was associated with the sex difference of SK current in a subpopulation of POMC + ERα + neurons.

Gabra5 plays a sexually dimorphic role in POMC neuron activity and glucose balance.

Pro-opiomelanocortin (POMC) neurons are important for the regulation of body weight and glucose balance. The inhibitory tone to POMC neurons is mediated primarily by the GABA receptors. However, the detailed mechanisms and functions of GABA receptors are not well understood. The α5 subunit of GABAA receptor, Gabra5, is reported to regulate feeding, and we found that Gabra5 is highly expressed in POMC neurons. To explore the function of Gabra5 in POMC neurons, we knocked down Gabra5 specifically from mature hypothalamic POMC neurons using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 strategy. This POMC-specific knock-down of Gabra5 did not affect body weight or food intake in either male or female mice. Interestingly, the loss of Gabra5 caused significant increases in the firing frequency and resting membrane potential, and a decrease in the amplitude of the miniature inhibitory postsynaptic current (mIPSC) in male POMC neurons. However, the loss of Gabra5 only modestly decreased the frequency of mIPSC in female POMC neurons. Consistently, POMC-specific knock-down of Gabra5 significantly improved glucose tolerance in male mice but not in female mice. These results revealed a sexually dimorphic role of Gabra5 in POMC neuron activity and glucose balance, independent of body weight control.

A D2 to D1 shift in dopaminergic inputs to midbrain 5-HT neurons causes anorexia in mice.

Midbrain dopamine (DA) and serotonin (5-HT) neurons regulate motivated behaviors, including feeding, but less is known about how these circuits may interact. In this study, we found that DA neurons in the mouse ventral tegmental area bidirectionally regulate the activity of 5-HT neurons in the dorsal raphe nucleus (DRN), with weaker stimulation causing DRD2-dependent inhibition and overeating, while stronger stimulation causing DRD1-dependent activation and anorexia. Furthermore, in the activity-based anorexia (ABA) paradigm, which is a mouse model mimicking some clinical features of human anorexia nervosa (AN), we observed a DRD2 to DRD1 shift of DA neurotransmission on 5-HTDRN neurons, which causes constant activation of these neurons and contributes to AN-like behaviors. Finally, we found that systemic administration of a DRD1 antagonist can prevent anorexia and weight loss in ABA. Our results revealed regulation of feeding behavior by stimulation strength-dependent interactions between DA and 5-HT neurons, which may contribute to the pathophysiology of AN.

Hypothalamic Perineuronal Nets Are Regulated by Sex and Dietary Interventions.

Perineuronal nets (PNNs) are widely present in the hypothalamus, and are thought to provide physical protection and ion buffering for neurons and regulate their synaptic plasticity and intracellular signaling. Recent evidence indicates that PNNs in the mediobasal hypothalamus play an important role in the regulation of glucose homeostasis. However, whether and how hypothalamic PNNs are regulated are not fully understood. In the present study, we examined whether PNNs in various hypothalamic regions in mice can be regulated by sex, gonadal hormones, dietary interventions, or their interactions. We demonstrated that gonadal hormones are required to maintain normal PNNs in the arcuate nucleus of hypothalamus in both male and female mice. In addition, PNNs in the terete hypothalamic nucleus display a sexual dimorphism with females higher than males, and high-fat diet feeding increases terete PNNs only in female mice but not in male mice. On the other hand, PNNs in other hypothalamic regions are not influenced by sex, gonadal hormones or dietary interventions. In summary, we demonstrated that hypothalamic PNNs are regulated in a region-specific manner and these results provide a framework to further investigate the potential functions of PNNs in regulating energy/glucose homeostasis at the interplay of sex, gonadal hormones and diets.

A monoclonal antibody with broad specificity for the ligands of insulin B:9-23 reactive T cells prevents spontaneous type 1 diabetes in mice.

Activation of T cells specific for insulin B chain amino acids 9 to 23 (B:9-23) is essential for the initiation of type 1 diabetes (T1D) in non-obese diabetic mice. We previously reported that peptide/MHC complexes containing optimized B:9-23 mimotopes can activate most insulin-reactive pathogenic T cells. A monoclonal antibody (mAb287) targeting these complexes prevented disease in 30-50% of treated animals (compared to 10% of animals given an isotype control). The incomplete protection is likely due to the relatively low affinity of the antibody for its ligand and limited specificity. Here, we report an enhanced reagent, mAb757, with improved specificity, affinity, and efficacy in modulating T1D. Importantly, mAb757 bound with nanomolar affinity to agonists of both "type A" and "type B" cells and suppressed "type B" cells more efficiently than mAb287. When given weekly starting at 4 weeks of age, mAb757 protected ~70% of treated mice from developing T1D for at least 35 weeks, while mAb287 only delayed disease in 25% of animals under the same conditions. Consistent with its higher affinity, mAb757 was also able to stain antigen-presenting cells loaded with B:9-23 mimotopes in vivo. We conclude that monoclonal antibodies that can block the presentation of pathogenic T cell receptor epitopes are viable candidates for antigen-specific immunotherapy for T1D.

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model.

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential. Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

Dual Opposing Roles of Metallothionein Overexpression in C57BL/6J Mouse Pancreatic ß-Cells.

BACKGROUND: Growing evidence indicates that oxidative stress (OS), a persistent state of excess amounts of reactive oxygen species (ROS) along with reactive nitrogen species (RNS), plays an important role in insulin resistance, diabetic complications, and dysfunction of pancreatic ß-cells. Pancreatic ß-cells contain exceptionally low levels of antioxidant enzymes, rendering them susceptible to ROS-induced damage. Induction of antioxidants has been proposed to be a way for protecting ß-cells against oxidative stress. Compared to other antioxidants that act against particular ß-cell damages, metallothionein (MT) is the most effective in protecting ß-cells from several oxidative stressors including nitric oxide, peroxynitrite, hydrogen peroxide, superoxide and streptozotocin (STZ). We hypothesized that MT overexpression in pancreatic ß-cells would preserve ß-cell function in C57BL/6J mice, an animal model susceptible to high fat diet-induced obesity and type 2 diabetes. RESEARCH DESIGN AND METHODS: The pancreatic ß-cell specific MT overexpression was transferred to C57BL/6J background by backcrossing. We studied transgenic MT (MT-tg) mice and wild-type (WT) littermates at 8 weeks and 18 weeks of age. Several tests were performed to evaluate the function of islets, including STZ in vivo treatment, intraperitoneal glucose tolerance tests (IPGTT) and plasma insulin levels during IPGTT, pancreatic and islet insulin content measurement, insulin secretion, and islet morphology assessment. Gene expression in islets was performed by quantitative real-time PCR and PCR array analysis. Protein levels in pancreatic sections were evaluated by using immunohistochemistry. RESULTS: The transgenic MT protein was highly expressed in pancreatic islets. MT-tg overexpression significantly protected mice from acute STZ-induced ROS at 8 weeks of age; unexpectedly, however, MT-tg impaired glucose stimulated insulin secretion (GSIS) and promoted the development of diabetes. Pancreatic ß-cell function was significantly impaired, and islet morphology was also abnormal in MT-tg mice, and more severe damage was detected in males. The unique gene expression pattern and abnormal protein levels were observed in MT-tg islets. CONCLUSIONS: MT overexpression protected ß-cells from acute STZ-induced ROS damages at young age, whereas it impaired GSIS and promoted the development of diabetes in adult C57BL/6J mice, and more severe damage was found in males.

[Research and thinking on needling sensation of acupoint Huantiao (GB 30)].

Taken Huantiao (GB 30) as breakthrough point, acupuncture manipulations of generating various needling sensations by different physicians are sorted. Types of acupoint needling sensations and conducting directions after acupuncture and all kinds of factors that affect needling sensations are analyzed from new perspectives. It is considered that attention should be paid to acupoint location, postures of patients, manipulation methods, types of needling sensations, transmission lines and duration time of needling sensations, etc.

Cloning of a heat shock protein 90 (HSP90) gene and expression analysis in the ridgetail white prawn Exopalaemon carinicauda.

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. In this study, a heat shock protein 90 cDNA named EcHSP90 was cloned from the hepatopancreas of ridgetail white prawn Exopalaemon carinicauda by reverse transcription polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EcHSP90 was of 2695 bp, including an open reading frame (ORF) of 2163 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 82.73 kDa and an estimated isoelectric point of 4.83. BLAST analysis revealed that the EcHSP90 shared high similarity (87.6%-75.24%) with other known HSP90s. The five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in EcHSP90, which indicated that EcHSP90 should be a cytosolic member of the HSP90 family. Quantitative real-time RT-PCR analysis revealed that EcHSP90 transcript could be detected in all the tested tissues, and strongly expressed in ovary of E. carinicauda. The transcript of EcHSP90 in hepatopancreas of E. carinicauda showed different expression profiles after pH and ammonia-N stresses. The results indicated that EcHSP90 was a constitutive and inducible expressed protein and could be induced by various stresses from environment.

Suppressed glucose metabolism in acinar cells might contribute to the development of exocrine pancreatic insufficiency in streptozotocin-induced diabetic mice.

High prevalence of exocrine pancreatic insufficiency has been observed in diabetic patients. However, the underlying mechanisms are not well known. Reduced cytosolic Ca(2+) signals in pancreatic acinar cells may contribute to lower digestive enzyme secretion. It is well known that adenosine triphosphate (ATP) regulates cytosolic Ca(2+) signals in acinar cells; however, little is known as to whether diabetes impairs glucose metabolism that produces ATP in acinar cells. Streptozotocin (STZ)-induced diabetic C57BL/6 mouse model was used. Four weeks after being diabetic, pancreatic acinar cells were isolated; and amylase secretion and contents, glucose utilization and oxidation, the activities of several key enzymes for glucose metabolism, and ATP and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) contents were determined. Compared with controls, diabetic mice had lower body weight. Cholecystokinin-8- and acetylcholine-stimulated amylase secretion was significantly impaired, and total amylase activity in acinar cells of STZ-diabetic mice was markedly reduced. Glucose utilization and oxidation were suppressed; measured enzyme activities for glucose metabolism and the ATP and NADPH contents were significantly reduced. These data indicate that glucose metabolism and ATP and NADPH productions are very important for maintaining acinar cell normal function. Reduction of ATP (reduces cytosolic Ca(2+) signals) and NADPH (reduces cell capability for antioxidative stress) productions may contribute to the development of exocrine pancreatic insufficiency in STZ-diabetic mice.

Voltammetric investigation on interaction of protein with chromotrope 2R and its analytical application.

The electrochemical behaviors of the interaction of chromotrope 2R (CH2R) with human serum albumin (HSA) are investigated on the hanging mercury drop electrode with linear sweep voltammetry. In the acidic buffer solution (pH 2.5) CH2R has a well-defined voltammetric reductive wave at -0.34 V (SCE). On the addition of HSA into the CH2R solution, the reductive peak current of CH2R decreases with little movement of the peak potential. The voltammetric study shows that the electrochemical parameters of interaction solution do not change and a new electrochemically non-active complex is formed via interaction of CH2R with HSA, which cannot be reduced on the Hg electrode and results in the decrease of the free concentration of CH2R. The decrease of reductive peak current is proportional to HSA concentration and further used for protein detection. The binding ratio and the binding constant are further calculated with the experimental voltammetric data.

Rat maternal diabetes impairs pancreatic beta-cell function in the offspring.

It has been shown that maternal diabetes increases the risk for obesity, glucose intolerance, and Type 2 diabetes mellitus in the adult life of the offspring. Mechanisms for these effects on the offspring are not well understood, and little information is available to reveal the mechanisms. We studied the effect of maternal diabetes on beta-cell function in the offspring of streptozotocin (STZ)-induced diabetic rat mothers (STZ-offspring). STZ-offspring did not become glucose intolerant up to 15 wk of age. At this age, however, insulin secretion was significantly impaired, as measured by in vivo and in vitro studies. Consistent with these changes, islet glucose metabolism and some important glucose metabolic enzyme activities were reduced. No significant changes were found in islet morphological analysis. These data indicate that beta-cell function is impaired in adult STZ-offspring; these changes may contribute to the development of type 2 diabetes mellitus in adulthood.