Functional labeling of individualized postsynaptic neurons using optogenetics and trans-Tango in Drosophila (FLIPSOT).

A population of neurons interconnected by synapses constitutes a neural circuit, which performs specific functions upon activation. It is essential to identify both anatomical and functional entities of neural circuits to comprehend the components and processes necessary for healthy brain function and the changes that characterize brain disorders. To date, few methods are available to study these two aspects of a neural circuit simultaneously. In this study, we developed FLIPSOT, or functional labeling of individualized postsynaptic neurons using optogenetics and trans-Tango. FLIPSOT uses (1) trans-Tango to access postsynaptic neurons genetically, (2) optogenetic approaches to activate (FLIPSOTa) or inhibit (FLIPSOTi) postsynaptic neurons in a random and sparse manner, and (3) fluorescence markers tagged with optogenetic genes to visualize these neurons. Therefore, FLIPSOT allows using a presynaptic driver to identify the behavioral function of individual postsynaptic neurons. It is readily applied to identify functions of individual postsynaptic neurons and has the potential to be adapted for use in mammalian circuits.

Induction of MTHFD2 in Macrophages Inhibits Reactive Oxygen Species-mediated NF-κB Activation and Protects against Inflammatory Responses.

The one-carbon metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is critical for cancer cell proliferation and immune cell phenotypes, but whether it can contribute to macrophage inflammatory responses remains unclear. In this study, we show that MTHFD2 was upregulated by LPS in murine macrophages upon activation of the TLR4-MyD88-IKKα/ß-NF-κB signaling pathway. MTHFD2 significantly attenuated LPS-induced macrophage proinflammatory cytokine production through its enzymatic activity. Notably, ablation of myeloid MTHFD2 rendered mice more sensitive to septic shock and CCl4-induced acute hepatitis. Mechanistically, MTHFD2 restrained IKKα/ß-NF-κB activation and macrophage inflammatory phenotype by scavenging reactive oxygen species through the generation of NADPH. Our study reveals MTHFD2 as a "self-control" mechanism in macrophage-mediated inflammatory responses.

A novel homozygous splice-site mutation of JK gene leads to Jk(a-b-) phenotype.

OBJECTIVES: This study aimed to investigate the molecular mechanism of the Jk(a-b-) phenotype in a Chinese transfusion patient. BACKGROUND: Many different mutation types relating to Jk(a-b-) phenotype have been reported. However, the splice-site mutation is relatively rare and the related functional verification is lacking. MATERIALS AND METHODS: In this study, the blood sample was collected from a transfusion patient with the Jk(a-b-) phenotype. Serotyping was performed using routine serological methods. The exons sequences and coding regions of the JK gene were amplified using polymerase chain reaction and directly sequenced. To perform a minigene splicing assay, the intronic mutation sequences were cloned into a pSPL3 splice reporting vector. The splicing reporter minigene assay was performed in HEK 293T cells. RESULTS: The Jk(a-b-) phenotype of the blood sample was identified through serological testing. Sequencing results revealed that the sample had a novel homozygous splice-site mutation JK*02N (NM_015865.7: c.663+3A>C). Further analysis, including cDNA sequencing and minigene splicing assay, confirmed that the novel splice-site mutation resulted in exon skipping. Interestingly, different numbers of exons being skipped were obtained by the two methods. CONCLUSION: This study revealed a novel homozygous splicing-site mutation associated with the Jk(a-b-) phenotype in Chinese population. Our results emphasise the importance of the in vitro functional method minigene splicing assay, while also acknowledging its potential limitations when compared to cDNA sequencing.

The expression and prognostic value of disulfidptosis progress in lung adenocarcinoma.

Disulfidptosis is a new cell death model caused by accumulating intracellular disulfides bonding to actin cytoskeleton proteins. This study aimed to investigate the expression and prognostic value of disulfidptosis-related genes (DRGs) in lung adenocarcinoma (LUAD). The data of expression profiles and scRNA-seq were collected from TCGA and GEO databases. The different expressions of DRGs between normal and LUAD tissues were compared. The LASSO analysis and multivariate Cox regression analysis were utilized to develop a DRGs model for the prognosis evaluation in LUAD. The model's predictive accuracy was evaluated with the area under the receiver operating characteristic curve (AUC) and C-index. Survival analysis, univariate and multivariate Cox regression analysis were used to assessing the predictive value of the DRGs model. ScRNA-seq data were analyzed with "Seurat" and "Monocle 2" packages. There were significant differences in 22 DRGs between normal and tumor tissues. A model with five DRGs (ACTB, FLNB, NCKAP1, SLC3A2, SLC7A11) was constructed. The AUC and C-index of the model were significantly higher than that based on clinical parameters. Survival analysis, univariate and multivariate Cox regression analysis demonstrated risk score was an independent prognostic predictor. In the scRNA-seq study, we identified 14 clusters and 11 cell types. Clusters 2, 8, and 13 were annotated into Epithelial cells. SLC7A11 and SLC3A2, NCKAP1 and FLNB, ACTB expressed most abundantly in Epithelial cells, Endothelial cells, Naive CD4 T, respectively. We explored the expression of DRGs in LUAD and constructed a predictive DRGs model, which was stable and reliable for predicting LUAD prognosis.

Clinical Application of Thromboelastography in Patients With Severe Fever With Thrombocytopenia Syndrome.

AIM: To investigate the clinical application of thromboelastography (TEG) in severe fever with thrombocytopenia syndrome (SFTS). METHODS: One hundred and fifty-seven patients with SFTS were included in the study. The participants were distributed into 3 groups; A, B, and C. And 103 patients in group A met the clinical criteria as they exhibited slight liver and kidney dysfunction. Group B consisted of 54 patients with SFTS who were critically ill while group C was a healthy control group with 58 participants. RESULTS: Patients with SFTS exhibited lower coagulation than the healthy participants. Group B patients exhibited significantly lower coagulation compared to group A. There was no significant difference in platelet count and fibrinogen content between patients in group A and group B, but platelet aggregation function and fibrinogen activity were significantly lower in group B patients. CONCLUSION: Our results suggest that it is risky to solely rely on platelet count and the fibrinogen in SFTS. Monitoring of TEG and other coagulation indexes should be emphasized.

MTHFD2 reprograms macrophage polarization by inhibiting PTEN.

The one-carbon metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is involved in the regulation of tumor oncogenesis and immune cell functions, but whether it can contribute to macrophage polarization remains elusive. Here, we show that MTHFD2 suppresses polarization of interferon-γ-activated macrophages (M(IFN-γ)) but enhances that of interleukin-4-activated macrophages (M(IL-4)) both in vitro and in vivo. Mechanistically, MTHFD2 interacts with phosphatase and tensin homolog (PTEN) to suppress PTEN's phosphatidylinositol 3,4,5-trisphosphate (PIP3) phosphatase activity and enhance downstream Akt activation, independent of the N-terminal mitochondria-targeting signal of MTHFD2. MTHFD2-PTEN interaction is promoted by IL-4 but not IFN-γ. Furthermore, amino acid residues (aa 215-225) of MTHFD2 directly target PTEN catalytic center (aa 118-141). Residue D168 of MTHFD2 is also critical for regulating PTEN's PIP3 phosphatase activity by affecting MTHFD2-PTEN interaction. Our study suggests a non-metabolic function of MTHFD2 by which MTHFD2 inhibits PTEN activity, orchestrates macrophage polarization, and alters macrophage-mediated immune responses.

Using Drosophila Two-Choice Assay to Study Optogenetics in Hands-On Neurobiology Laboratory Activities.

Optogenetics has made a significant impact on neuroscience, allowing activation and inhibition of neural activity with exquisite spatiotemporal precision in response to light. In this lab session, we use fruit flies to help students understand the fundamentals of optogenetics through hands-on activities. The CsChrimson channelrhodopsin, a light-activated cation channel, is expressed in sweet and bitter sensory neurons. Sweet sensory neurons guide animals to identify nutrient-rich food and drive appetitive behaviors, while bitter sensory neurons direct animals to avoid potentially toxic substances and guide aversive behavior. Students use two-choice assays to explore the causality between the stimulation activation of these neurons and the appetitive and avoidance behaviors of the fruit flies. To quantify their observations, students calculate preference indices and use the Student's t-test to analyze their data. After this lab session, students are expected to have a basic understanding of optogenetics, fly genetics, sensory perception, and how these relate to sensory-guided behaviors. They will also learn to conduct, quantify, and analyze two-choice behavioral assays.

Cool and warm ionotropic receptors control multiple thermotaxes in Drosophila larvae.

Animals are continuously confronted with different rates of temperature variation. The mechanism underlying how temperature-sensing systems detect and respond to fast and slow temperature changes is not fully understood in fly larvae. Here, we applied two-choice behavioral assays to mimic fast temperature variations and a gradient assay to model slow temperature changes. Previous research indicates that Rhodopsin 1 (Rh1) and its phospholipase C (PLC) cascade regulate fast and slow temperature responses. We focused on the ionotropic receptors (IRs) expressed in dorsal organ ganglions (DOG), in which dorsal organ cool-activated cells (DOCCs) and warm-activated cells (DOWCs) rely on IR-formed cool and warm receptors to respond to temperature changes. In two-choice assays, both cool and warm IRs are sufficient for selecting 18°C between 18°C and 25°C but neither function in cool preferences between 25°C and 32°C. The Rh1 pathway, on the other hand, contributes to choosing preferred temperatures in both assays. In a gradient assay, cool and warm IR receptors exert opposite effects to guide animals to ∼25°C. Cool IRs drive animals to avoid cool temperatures, whereas warm IRs guide them to leave warm regions. The Rh1 cascade and warm IRs may function in the same pathway to drive warm avoidance in gradient assays. Moreover, IR92a is not expressed in temperature-responsive neurons but regulates the activation of DOWCs and the deactivation of DOCCs. Together with previous studies, we conclude that multiple thermosensory systems, in various collaborative ways, help larvae to make their optimal choices in response to different rates of temperature change.

Serine metabolism orchestrates macrophage polarization by regulating the IGF1-p38 axis.

Serine metabolism is reportedly involved in immune cell functions, but whether and how serine metabolism regulates macrophage polarization remain largely unknown. Here, we show that suppressing serine metabolism, either by inhibiting the activity of the key enzyme phosphoglycerate dehydrogenase in the serine biosynthesis pathway or by exogenous serine and glycine restriction, robustly enhances the polarization of interferon-γ-activated macrophages (M(IFN-γ)) but suppresses that of interleukin-4-activated macrophages (M(IL-4)) both in vitro and in vivo. Mechanistically, serine metabolism deficiency increases the expression of IGF1 by reducing the promoter abundance of S-adenosyl methionine-dependent histone H3 lysine 27 trimethylation. IGF1 then activates the p38-dependent JAK-STAT1 axis to promote M(IFN-γ) polarization and suppress STAT6-mediated M(IL-4) activation. This study reveals a new mechanism by which serine metabolism orchestrates macrophage polarization and suggests the manipulation of serine metabolism as a therapeutic strategy for macrophage-mediated immune diseases.

Responses of different Drosophila species to temperature changes.

Temperature is a critical environmental variable that affects the distribution, survival and reproduction of most animals. Although temperature receptors have been identified in many animals, how these receptors respond to temperature is still unclear. Here, we describe an automated tracking method for studying the thermotactic behaviors of Drosophila larvae and adults. We built optimal experimental setups to capture behavioral recordings and analyzed them using free software, Fiji and TrackMate, which do not require programming knowledge. Then, we applied the adult thermotactic two-choice assay to examine the movement and temperature preferences of nine Drosophila species. The ability or inclination to move varied among these species and at different temperatures. Distinct species preferred various ranges of temperatures. Wild-type D. melanogaster flies avoided the warmer temperature in the warm avoidance assay and the cooler temperature in the cool avoidance assay. Conversely, D. bipectinata and D. yakuba did not avoid warm or cool temperatures in the respective assays, and D. biarmipes and D. mojavensis did not avoid the warm temperature in the warm avoidance assay. These results demonstrate that Drosophila species have different mobilities and temperature preferences, which will benefit further research in exploring molecular mechanisms of temperature responsiveness.

TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis.

Research in neuroscience has evolved to use complex imaging and computational tools to extract comprehensive information from data sets. Calcium imaging is a widely used technique that requires sophisticated software to obtain reliable results, but many laboratories struggle to adopt computational methods when updating protocols to meet modern standards. Difficulties arise due to a lack of programming knowledge and paywalls for software. In addition, cells of interest display movements in all directions during calcium imaging. Many approaches have been developed to correct the motion in the lateral (x/y) direction. This paper describes a workflow using a new ImageJ plugin, TrackMate Analysis of Calcium Imaging (TACI), to examine motion on the z-axis in 3D calcium imaging. This software identifies the maximum fluorescence value from all the z-positions a neuron appears in and uses it to represent the neuron's intensity at the corresponding t-position. Therefore, this tool can separate neurons overlapping in the lateral (x/y) direction but appearing on distinct z-planes. As an ImageJ plugin, TACI is a user-friendly, open-source computational tool for 3D calcium imaging analysis. We validated this workflow using fly larval thermosensitive neurons that displayed movements in all directions during temperature fluctuation and a 3D calcium imaging dataset acquired from the fly brain.

Establishment of Normal Range for Thromboelastography in Healthy Middle-Aged and Elderly People of Weihai in China.

TEG can monitor the dynamic changes of blood clot formation and lysis by activating the coagulation system of a small sample of whole blood in vitro. The parameters can reflect the level of coagulation factors, the function of fibrinogen and platelet, and the presence or absence of hyperfibrinolysis. At present, the normal reference range of the parameters of TEG is mainly based on the reference values established by the Western population. Due to the differences in the distribution of ethnic groups, many countries have established their reference ranges for healthy populations. In China, some scholars have tried to establish the corresponding TEG reference range according to the characteristics of the population in different regions. This study tried to establish the reference range for thromboelastography in healthy middle-aged and elderly people of Weihai in China and compare it with the reference range provided by the manufacturer. The fasting venous blood of 454 healthy middle-aged and elderly people was collected, including 239 males and 215 females. The thromboelastography TEG-5000 was used to measure the reaction time (R), coagulation formation time (K), coagulation angle (Angle), and maximum amplitude (MA). The reference range of TEG parameters of middle-aged and elderly healthy males was R: 4.38-8.27 min, K: 1.44-2.82 min, Angle: 48.53-72.17 deg, and MA: 51.95-72.02 mm; respectively, in the females, the normal value was R: 3.43-7.40 min, K: 1.07-2.53 min, Angle: 48.22-77.22 deg, and MA: 53.10-74.58 mm; The difference of R, K, Angle, and MA between the male group and the female group was statistically significant (P < 0.05); In this study, if we use the reference range established by the manufacturer, the R specificity for males was 91.6%, K specificity was 98.7%, Angle specificity was 85.8%, and MA specificity was 93.7%; the range for females was 68.4%, 99.5%, 75.8%, and 87.4%, respectively. There are statistically significant differences between R, K, Angle, and MA in middle-aged and elderly healthy women and men. It is necessary to establish a TEG reference range for healthy females and males.

Genetic Transsynaptic Techniques for Mapping Neural Circuits in Drosophila.

A neural circuit is composed of a population of neurons that are interconnected by synapses and carry out a specific function when activated. It is the structural framework for all brain functions. Its impairments often cause diseases in the nervous system. To understand computations and functions in a brain circuit, it is of crucial importance to identify how neurons in this circuit are connected. Genetic transsynaptic techniques provide opportunities to efficiently answer this question. These techniques label synapses or across synapses to unbiasedly label synaptic partners. They allow for mapping neural circuits with high reproducibility and throughput, as well as provide genetic access to synaptically connected neurons that enables visualization and manipulation of these neurons simultaneously. This review focuses on three recently developed Drosophila genetic transsynaptic tools for detecting chemical synapses, highlights their advantages and potential pitfalls, and discusses the future development needs of these techniques.

In vitro and in vivo evaluation of virus-induced innate immunity in mouse.

Innate immunity is the first line of host defense against viral infection. As one of the innate immune cell types, antigen-presenting cells play an important role in the process of antiviral immunity. This protocol describes the analysis of innate immunity induced by vesicular stomatitis virus infection of peritoneal macrophages in vitro and in vivo detection of IFN-ß production and lung injury. For complete details on the use and execution of this protocol, please refer to Shen et al. (2021).

GCWOAS2: Multiobjective Task Scheduling Strategy Based on Gaussian Cloud-Whale Optimization in Cloud Computing.

An important challenge facing cloud computing is how to correctly and effectively handle and serve millions of users' requests. Efficient task scheduling in cloud computing can intuitively affect the resource configuration and operating cost of the entire system. However, task and resource scheduling in a cloud computing environment is an NP-hard problem. In this paper, we propose a three-layer scheduling model based on whale-Gaussian cloud. In the second layer of the model, a whale optimization strategy based on the Gaussian cloud model (GCWOAS2) is used for multiobjective task scheduling in a cloud computing which is to minimize the completion time of the task via effectively utilizing the virtual machine resources and to keep the load balancing of each virtual machine, reducing the operating cost of the system. In the GCWOAS2 strategy, an opposition-based learning mechanism is first used to initialize the scheduling strategy to generate the optimal scheduling scheme. Then, an adaptive mobility factor is proposed to dynamically expand the search range. The whale optimization algorithm based on the Gaussian cloud model is proposed to enhance the randomness of search. Finally, a multiobjective task scheduling algorithm based on Gaussian whale-cloud optimization (GCWOA) is presented, so that the entire scheduling strategy can not only expand the search range but also jump out of the local maximum and obtain the global optimal scheduling strategy. Experimental results show that compared with other existing metaheuristic algorithms, our strategy can not only shorten the task completion time but also balance the load of virtual machine resources, and at the same time, it also has a better performance in resource utilization.

Serine metabolism antagonizes antiviral innate immunity by preventing ATP6V0d2-mediated YAP lysosomal degradation.

Serine metabolism promotes tumor oncogenesis and regulates immune cell functions, but whether it also contributes to antiviral innate immunity is unknown. Here, we demonstrate that virus-infected macrophages display decreased expression of serine synthesis pathway (SSP) enzymes. Suppressing the SSP key enzyme phosphoglycerate dehydrogenase (PHGDH) by genetic approaches or by treatment with the pharmaceutical inhibitor CBR-5884 and by exogenous serine restriction enhanced IFN-ß-mediated antiviral innate immunity in vitro and in vivo. Mechanistic experiments showed that virus infection or serine metabolism deficiency increased the expression of the V-ATPase subunit ATP6V0d2 by inhibiting S-adenosyl methionine-dependent H3K27me3 occupancy at the promoter. ATP6V0d2 promoted YAP lysosomal degradation to relieve YAP-mediated blockade of the TBK1-IRF3 axis and, thus, enhance IFN-ß production. These findings implicate critical functions of PHGDH and the key immunometabolite serine in blunting antiviral innate immunity and also suggest manipulation of serine metabolism as a therapeutic strategy against virus infection.

Ionotropic Receptor-dependent cool cells control the transition of temperature preference in Drosophila larvae.

Temperature sensation guides animals to avoid temperature extremes and to seek their optimal temperatures. The larval stage of Drosophila development has a dramatic effect on temperature preference. While early-stage Drosophila larvae pursue a warm temperature, late-stage larvae seek a significantly lower temperature. Previous studies suggest that this transition depends on multiple rhodopsins at the late larval stage. Here, we show that early-stage larvae, in which dorsal organ cool cells (DOCCs) are functionally blocked, exhibit similar cool preference to that of wild type late-stage larvae. The molecular thermoreceptors in DOCCs are formed by three members of the Ionotropic Receptor (IR) family, IR21a, IR93a, and IR25a. Early-stage larvae of each Ir mutant pursue a cool temperature, similar to that of wild type late-stage larvae. At the late larval stage, DOCCs express decreased IR proteins and exhibit reduced cool responses. Importantly, late-stage larvae that overexpress IR21a, IR93a, and IR25a in DOCCs exhibit similar warm preference to that of wild type early-stage larvae. These data suggest that IR21a, IR93a, and IR25a in DOCCs navigate early-stage larvae to avoid cool temperatures and the reduction of these IR proteins in DOCCs results in animals remaining in cool regions during the late larval stage. Together with previous studies, we conclude that multiple temperature-sensing systems are regulated for the transition of temperature preference in fruit fly larvae.

Correlation of APOBEC3G expression with liver function indexes of patients with chronic hepatitis B and comparison in chronic hepatitis B, liver cirrhosis and liver cancer.

Correlation of APOBEC3G expression with liver function indexes of patients with chronic hepatitis B and its expression in chronic hepatitis B, liver cirrhosis and liver cancer were investigated to evaluated the significance of APOBEC3G. Fifty-eight patients with chronic hepatitis B were selected, including 20 cases of chronic hepatitis B, 19 cases of liver cirrhosis and 19 cases of liver cancer. Liver function indexes were detected and analyzed, and messenger ribonucleic acid (mRNA) and protein expression levels of APOBEC3G in liver tissues were detected via reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunohistochemistry, followed by correlation analysis. Certain liver function indexes had significant differences among the three groups of patients (P<0.05). Results of RT-PCR, Western blotting and immunohistochemistry confirmed that the content of APOBEC3G in liver tissues was the highest in patients with chronic hepatitis B, slightly lower in patients with liver cirrhosis and the lowest in patients with liver cancer. The content of APOBEC3G mRNA in liver tissues had a certain correlation with the content of alanine aminotransferase (ALT) (r2 =0.34, P<0.05). Other liver function indexes had no significant correlations with APOBEC3G (P>0.05). APOBEC3G expression has a certain correlation with some liver function indexes of patients with chronic hepatitis B. There are significant differences in the expression level of APOBEC3G in patients with hepatitis, liver cirrhosis and liver cancer.

The Structure and Function of Ionotropic Receptors in Drosophila.

Ionotropic receptors (IRs) are a highly divergent subfamily of ionotropic glutamate receptors (iGluR) and are conserved across Protostomia, a major branch of the animal kingdom that encompasses both Ecdysozoa and Lophothrochozoa. They are broadly expressed in peripheral sensory systems, concentrated in sensory dendrites, and function in chemosensation, thermosensation, and hygrosensation. As iGluRs, four IR subunits form a functional ion channel to detect environmental stimuli. Most IR receptors comprise individual stimulus-specific tuning receptors and one or two broadly expressed coreceptors. This review summarizes the discoveries of the structure of IR complexes and the expression and function of each IR, as well as discusses the future direction for IR studies.

PINK1/Parkin Influences Cell Cycle by Sequestering TBK1 at Damaged Mitochondria, Inhibiting Mitosis.

PINK1 and Parkin are established mediators of mitophagy, the selective removal of damaged mitochondria by autophagy. PINK1 and Parkin have been proposed to act as tumor suppressors, as loss-of-function mutations are correlated with enhanced tumorigenesis. However, it is unclear how PINK1 and Parkin act in coordination during mitophagy to influence the cell cycle. Here we show that PINK1 and Parkin genetically interact with proteins involved in cell cycle regulation, and loss of PINK1 and Parkin accelerates cell growth. PINK1- and Parkin-mediated activation of TBK1 at the mitochondria during mitophagy leads to a block in mitosis due to the sequestration of TBK1 from its physiological role at centrosomes during mitosis. Our study supports a diverse role for the far-reaching, regulatory effects of mitochondrial quality control in cellular homeostasis and demonstrates that the PINK1/Parkin pathway genetically interacts with the cell cycle, providing a framework for understanding the molecular basis linking PINK1 and Parkin to mitosis.