Signal Transduction Components Research Articles

Emerging biosensor probes for glycated hemoglobin (HbA1c) detection.

Glycated hemoglobin (HbA1c), originating from the non-enzymatic glycosylation of βVal1 residues in hemoglobin (Hb), is an essential biomarker indicating average blood glucose levels over a period of 2 to 3 months without external environmental disturbances, thereby serving as the gold standard in the management of diabetes instead of blood glucose testing. The emergence of HbA1c biosensors presents affordable, readily available options for glycemic monitoring, offering significant benefits to small-scale laboratories and clinics. Utilizing nanomaterials coupled with high-specificity probes as integral components for recognition, labeling, and signal transduction, these sensors demonstrate exceptional sensitivity and selectivity in HbA1c detection. This review mainly focuses on the emerging probes and strategies integral to HbA1c sensor development. We discussed the advantages and limitations of various probes in sensor construction as well as recent advances in diverse sensing strategies for HbA1c measurement and their potential clinical applications, highlighting the critical gaps in current technologies and future needs in this evolving field.

Comprehensive genomic features indicative for Notch responsiveness.

Transcription factor RBPJ is the central component in Notch signal transduction and directly forms a coactivator complex together with the Notch intracellular domain (NICD). While RBPJ protein levels remain constant in most tissues, dynamic expression of Notch target genes varies depending on the given cell-type and the Notch activity state. To elucidate dynamic RBPJ binding genome-wide, we investigated RBPJ occupancy by ChIP-Seq. Surprisingly, only a small set of the total RBPJ sites show a dynamic binding behavior in response to Notch signaling. Compared to static RBPJ sites, dynamic sites differ in regard to their chromatin state, binding strength and enhancer positioning. Dynamic RBPJ sites are predominantly located distal to transcriptional start sites (TSSs), while most static sites are found in promoter-proximal regions. Importantly, gene responsiveness is preferentially associated with dynamic RBPJ binding sites and this static and dynamic binding behavior is repeatedly observed across different cell types and species. Based on the above findings we used a machine-learning algorithm to predict Notch responsiveness with high confidence in different cellular contexts. Our results strongly support the notion that the combination of binding strength and enhancer positioning are indicative of Notch responsiveness.

Calcium signaling induces partial EMT and renal fibrosis in a Wnt4mCherry knock-in mouse model

The renal tubular epithelial cells (TEC) have a strong capacity for repair after acute injury, but when this mechanism becomes uncontrollable, it leads to chronic kidney diseases (CKD). Indeed, in progress toward CKDs, the TECs may dedifferentiate, undergo epithelial-to-mesenchyme transition (EMT), and promote inflammation and fibrosis. Given the critical role of Wnt4 signaling in kidney ontogenesis, we addressed whether changes in this signaling are connected to renal inflammation and fibrosis by taking advantage of a knock-in Wnt4mCh/mCh mouse. While the Wnt4mCh/mCh embryos appeared normal, the corresponding mice, within one month, developed CKD-related phenotypes, such as pro-inflammatory responses including T-cell/macrophage influx, expression of fibrotic markers, and epithelial cell damage with a partial EMT. The Wnt signal transduction component β-catenin remained unchanged, while calcium signaling is induced in the injured TECs involving Nfat and Tfeb transcription factors. We propose that the Wnt4 signaling pathway is involved in repairing the renal injury, and when the signal is overdriven, CKD is established.

Oral administration of IPI549 protects mice from neuropathology and an overwhelming inflammatory response during experimental cerebral malaria

Infection with Plasmodium falciparum is often deadly when it results in cerebral malaria, which is associated with neuropathology described as an overwhelming inflammatory response and mechanical obstruction of cerebral microvascular. PI3Kγ is a critical component of intracellular signal transduction and plays a central role in regulating cell chemotaxis, migration, and activation. The purpose of this study was to examine the relationship between inhibiting the PI3Kγ pathway and the outcome of experimental cerebral malaria (ECM) in C57BL/6J mice infected with the mouse malaria parasite, Plasmodium berghei ANKA. We observed that oral administration of the PI3Kγ inhibitor IPI549 after infection completely protected mice from ECM. IPI549 treatment significantly dampened the magnitude of inflammatory responses, with reduced production of pro-inflammatory factors, decreased T cell activation, and altered differentiation of antigen-presenting cells. IPI549 treatment protected the infected mice from neuropathology, as assessed by an observed reduction of pathogenic T cells in the brain. Treating the infected mice with IPI549 three days after parasite inoculation improved the murine blood brain barrier (BBB) integrity and helped the mice pass the onset of ECM. Together, these data indicate that oral administration of the PI3Kγ inhibitor IPI549 has a suppressive role in host inflammation and alleviates cerebral pathology, which supports IPI549 as a new malaria treatment option with potential therapeutic implications for cerebral malaria.

Identification of mitogen-activated protein kinases substrates in Arabidopsis using kinase client assay

ABSTRACT Mitogen-activated protein kinase (MPK) cascades are essential signal transduction components that control a variety of cellular responses in all eukaryotes. MPKs convert extracellular stimuli into cellular responses by the phosphorylation of downstream substrates. Although MPK cascades are predicted to be very complex, only limited numbers of MPK substrates have been identified in plants. Here, we used the kinase client (KiC) assay to identify novel substrates of MPK3 and MPK6. Recombinant MPK3 or MPK6 were tested against a large synthetic peptide library representing in vivo phosphorylation sites, and phosphorylated peptides were identified by high-resolution tandem mass spectrometry. From this screen, we identified 23 and 21 putative client peptides of MPK3 and MPK6, respectively. To verify the phosphorylation of putative client peptides, we performed in vitro kinase assay with recombinant fusion proteins of isolated client peptides. We found that 13 and 9 recombinant proteins were phosphorylated by MPK3 and MPK6. Among them, 11 proteins were proven to be the novel substrates of two MPKs. This study suggests that the KiC assay is a useful method to identify new substrates of MPKs.

Subtilisin-like proteases from Fusarium graminearum induce plant cell death and contribute to virulence.

Fusarium head blight (FHB), caused by Fusarium graminearum, causes huge annual economic losses in cereal production. To successfully colonize host plants, pathogens secrete hundreds of effectors that interfere with plant immunity and facilitate infection. However, the roles of most secreted effectors of F. graminearum in pathogenesis remain unclear. We analyzed the secreted proteins of F. graminearum and identified 255 candidate effector proteins by liquid chromatography-mass spectrometry (LC-MS). Five subtilisin-like family proteases (FgSLPs) were identified that can induce cell death in Nicotiana benthamiana leaves. Further experiments showed that these FgSLPs induced cell death in cotton (Gossypium barbadense) and Arabidopsis (Arabidopsis thaliana). A signal peptide and light were not essential for the cell death-inducing activity of FgSLPs. The I9 inhibitor domain and the entire C-terminus of FgSLPs were indispensable for their self-processing and cell death-inducing activity. FgSLP-induced cell death occurred independent of the plant signal transduction components BRI-ASSOCIATED KINASE 1 (BAK1), SUPPRESSOR OF BIR1 1 (SOBIR1), ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), and PHYTOALEXIN DEFICIENT 4 (PAD4). Reduced virulence was observed when FgSLP1 and FgSLP2 were simultaneously knocked out. This study reveals a class of secreted toxic proteins essential for F. graminearum virulence.

Curation, nomenclature, and topological classification of receptor-like kinases from 528 plant species for novel domain discovery and functional inference

Receptor-like kinases (RLKs) are the most numerous signal transduction components in plants and play important roles in determining how different plants adapt to their ecological environments. Research on RLKs has focused mainly on a small number of typical RLK members in a few model plants. There is an urgent need to study the composition, distribution, and evolution of RLKs at the holistic level to increase our understanding of how RLKs assist in the ecological adaptations of different plant species. In this study, we collected the genome assemblies of 528 plant species and constructed an RLK dataset. Using this dataset, we identified and characterized 524 948 RLK family members. Each member underwent systematic topological classification and was assigned a gene ID based on a unified nomenclature system. Furthermore, we identified two novel extracellular domains in some RLKs, designated Xiao and Xiang. Evolutionary analysis of the RLK family revealed that the RLCK-XVII and RLCK-XII-2 classes were present exclusively in dicots, suggesting that diversification of RLKs between monocots and dicots may have led to differences in downstream cytoplasmic responses. We also used an interaction proteome to help empower data mining for inference of new RLK functions from a global perspective, with the ultimate goal of understanding how RLKs shape the adaptation of different plants to the environments/ecosystems. The assembled RLK dataset, together with annotations and analytical tools, forms an integrated foundation of multiomics data that is publicly accessible via the metaRLK web portal (http://metaRLK.biocloud.top).

Construction of Membraneless and Multicompartmentalized Coacervate Protocells Controlling a Cell Metabolism-like Cascade Reaction.

In recent years, there has been growing attention to designing synthetic protocells, capable of mimicking micrometric and multicompartmental structures and highly complex physicochemical and biological processes with spatiotemporal control. Controlling metabolism-like cascade reactions in coacervate protocells is still challenging since signal transduction has to be involved in sequential and parallelized actions mediated by a pH change. Herein, we report the hierarchical construction of membraneless and multicompartmentalized protocells composed of (i) a cytosol-like scaffold based on complex coacervate droplets stable under flow conditions, (ii) enzyme-active artificial organelles and a substrate nanoreservoir capable of triggering a cascade reaction between them in response to a pH increase, and (iii) a signal transduction component based on the urease enzyme capable of the conversion of an exogenous biological fuel (urea) into an endogenous signal (ammonia and pH increase). Overall, this strategy allows a synergistic communication between their components within the membraneless and multicompartment protocells and, thus, metabolism-like enzymatic cascade reactions. This signal communication is transmitted through a scaffold protocell from an "inactive state" (nonfluorescent protocell) to an "active state" (fluorescent protocell capable of consuming stored metabolites).

Genome-wide identification of the mitogen-activated kinase gene family from Limonium bicolor and functional characterization of LbMAPK2 under salt stress

BackgroundMitogen-activated protein kinases (MAPKs) are ubiquitous signal transduction components in eukaryotes. In plants, MAPKs play an essential role in growth and development, phytohormone regulation, and abiotic stress responses. The typical recretohalophyte Limonium bicolor (Bunge) Kuntze has multicellular salt glands on its stems and leaves; these glands secrete excess salt ions from its cells to mitigate salt damage. The number, type, and biological function of L. bicolor MAPK genes are unknown.ResultsWe identified 20 candidate L. bicolor MAPK genes, which can be divided into four groups. Of these 20 genes, 17 were anchored to 7 chromosomes, while LbMAPK18, LbMAPK19, and LbMAPK20 mapped to distinct scaffolds. Structure analysis showed that the predicted protein LbMAPK19 contains the special structural motif TNY in its activation loop, whereas the other LbMAPK members harbor the conserved TEY or TDY motif. The promoters of most LbMAPK genes carry cis-acting elements related to growth and development, phytohormones, and abiotic stress. LbMAPK1, LbMAPK2, LbMAPK16, and LbMAPK20 are highly expressed in the early stages of salt gland development, whereas LbMAPK4, LbMAPK5, LbMAPK6, LbMAPK7, LbMAPK11, LbMAPK14, and LbMAPK15 are highly expressed during the late stages. These 20 LbMAPK genes all responded to salt, drought and ABA stress. We explored the function of LbMAPK2 via virus-induced gene silencing: knocking down LbMAPK2 transcript levels in L. bicolor resulted in fewer salt glands, lower salt secretion ability from leaves, and decreased salt tolerance. The expression of several genes [LbTTG1 (TRANSPARENT TESTA OF GL1), LbCPC (CAPRICE), and LbGL2 (GLABRA2)] related to salt gland development was significantly upregulated in LbMAPK2 knockdown lines, while the expression of LbEGL3 (ENHANCER OF GL3) was significantly downregulated.ConclusionThese findings increase our understanding of the LbMAPK gene family and will be useful for in-depth studies of the molecular mechanisms behind salt gland development and salt secretion in L. bicolor. In addition, our analysis lays the foundation for exploring the biological functions of MAPKs in an extreme halophyte.

Functionalized carbon nano-enabled plant ROS signal engineering for growth / defense balance

The reactive oxygen species (ROS) wave plays a crucial role as an early systemic signal in nearly all environmental response pathways. Engineering on ROS signaling may be a promising strategy to modulate plant systemic acquired acclimation and systemic acquired resistance. Recently, artificial designed materials mimicking signal transduction components have attracted attention to improve plant immunity and stress acclimation. Here, we report the bioengineering of functional carbon nanoparticles (FCN) through their regulation on ROS signaling. FCN with a C:O atom ratio of 1:2.2 were synthesized by low voltage electrolysis with graphite and carboxyl were the main oxygen-containing groups on the surface of sp2 carbon core. The active redox properties resulting from the carboxyl–rich modification provided FCN superior ROS-triggering effects compared to other carbon nanoparticles with similar core structures but different types and quantities of surface modifications. Moreover, the ROS induced by FCN were quickly scavenged to homeostasis without causing accumulation and then leading to oxidative stress. The findings from transcriptional footprinting in Arabidopsis’s genes revealed a typical and quick ROS signal transduction events and also the regulation of stress responses and adaptions to biotic and abiotic stresses. In addition, the application of FCN to roots have been shown to promote the growth and development of Arabidopsis, rice and wheat, and also the yields particularly when suffering stresses. In summary, our findings confirmed that the ROS burst triggered by FCN modulated the balance between growth and defense through transcriptome reprogramming. It indicates that nano-enabled plant engineering for ROS can provide an economical and efficient way to promote the crops yields, especially under stress conditions.

Characterization of SEC14 family in wheat and the function of TaSEC14-7B in salt stress tolerance

Phospholipids are important components of plant biofilms and signal transduction. They are divided into glycerophospholipids and sphingolipids. Phosphatidylinositol (PI) is an intracellular glycerophospholipid. SEC14s are PI transporter proteins that are widely presented in eukaryotic. They take part in membrane transportation, inositol phosphate metabolism and adversity stress response. To date, systematic analysis of the SEC14 gene family in wheat, especially the function of SEC14 in salt stress tolerance has not been reported. In this study, 106 SEC14 family members have been identified in wheat. Then, a salt inducible Sec14 family member TaSEC14-7B was selected for further functional study in response to salt stress. Expression analysis demonstrated TaSEC14-7B was induced by NaCl, PEG treatment and localized both in the cell membrane and nucleus. TaSEC14-7B over-expressing Arabidopsis increased salt stress tolerance. Under salt stress, the transgenic plants displayed higher germination rate, longer primary root length, more soluble sugar accumulation, higher antioxidant enzyme activity and lower oxidative damage than the wild type plants. Also, at the presence of NaCl stress, the expression level of ABF4, P5CS, PLC4 and AtPLC7 genes was higher in TaSEC14 transgenic Arabidopsis than in the wild type ones. All these results lay a foundation for further study of Sec14 in wheat.

The Hippo-Yki pathway downstream transcription factor Scalloped negatively regulates immune defense against Vibrio parahaemolyticus infection in shrimp.

The Hippo-Yki signaling pathway plays a crucial role in numerous biological processes. Previous studies have demonstrated the significance of signal transduction components of the Hippo pathway in the immune response of shrimp. In this study, the downstream transcription factor of the Hippo signaling pathway, Scalloped, was analyzed in the context of Vibrio parahaemolyticus infection in Pacific white shrimp, Penaeus vannamei. Upon bacterial and fungal infections, the expression of Scalloped was upregulated in hemocytes. Scalloped was found to localize in the nucleus and interact with the Hippo pathway downstream transcriptional co-activator Yki. With the assistance of Yki, Scalloped activated the promoter of Cactus, which is a cytoplasmic inhibitor of the NF-κB pathway, leading to the inhibition of the nuclear translocation of the NF-κB family member Dorsal in shrimp. By inhibiting the Dorsal pathway, Scalloped reduced the expression of immune functional proteins and negatively regulated the immune response against bacterial infection in shrimp. RNAi-mediated silencing of Scalloped significantly enhanced the survival rate of V. parahaemolyticus-infected shrimp and reduced the bacterial load in tissues. These findings demonstrate the potential of Scalloped as a therapeutic target for vibriosis in crustaceans and contribute to our understanding of the shrimp's antibacterial defense and the functional roles of Hippo signaling in animal immunity.

Association between Polymorphism in the Janus Kinase 2 (JAK2) Gene and Selected Performance Traits in Cattle and Sheep.

The Janus Kinase 2 (JAK2) tyrosine kinase is an essential component of signal transduction of the class II cytokine receptors, including the growth hormone receptor. Therefore, it may play a crucial role in the signaling pathway of the somatotropic axis, which influences growth, development, and reproductive traits in ruminants. For this purpose, for three breeds of cattle (Hereford, Angus, and Limousin; a total of 781 individuals), two polymorphic sites located in exon 16 (rs210148032; p.Ile704Val, within pseudokinase (JH2)) and exon 23 (silent mutation rs211067160, within JH1 kinase domain) were analyzed. For two breeds of sheep (Pomeranian and Suffolk; 333 individuals in total), two polymorphic sites in exon 6 (rs160146162 and rs160146160; encoding the FERM domain) and one polymorphic site in exon 24 of the JAK2 gene (rs160146116; JH1 kinase domain) were genotyped. In our study, the associations examined for cattle were inconclusive. However, Hereford and Limousin cattle with genotypes AA (e16/RsaI) and AA (e23/HaeIII) tended to have the highest body weight and better daily gains (p ≤ 0.05). No clear tendency was observed in the selected reproductive traits. In the case of sheep, regardless of breed, individuals with the AA (e6/EarI), GG (e6/seq), and AA (e24/Hpy188III) genotypes had the highest body weights and daily gains in the study periods (p ≤ 0.01). The same individuals in the Pomeranian breed also had better fertility and lamb survival (p ≤ 0.01). To the best of our knowledge, these are the first association studies for all these polymorphic sites. Single-nucleotide polymorphisms in the JAK2 gene can serve as genetic markers for growth and selected reproductive traits in ruminants given that they are further investigated in subsequent populations and analyzed using haplotype and/or combined genotype systems.

The Spliceosome Factor EFTUD2 Promotes IFN Anti-HBV Effect through mRNA Splicing.

Using a CRISPR/Cas9 gene-editing system, EFTUD2 single allele knockout HepG2.2.15 cells were constructed. Subsequently, the HBV biomarkers in EFTUD2+/- HepG2.2.15 cells and wild-type (WT) cells with or without IFN-α treatment were detected. And the EFTUD2-regulated genes were then identified using mRNA sequence. Selected gene mRNA variants and their proteins were examined by qRT-PCR and Western blotting. To confirm the effects of EFTUD2 on HBV replication and IFN-stimulated gene (ISG) expression, a rescue experiment in EFTUD2+/- HepG2.2.15 cells was performed by EFTUD2 overexpression. IFN-induced anti-HBV activity was found to be restricted in EFTUD2+/- HepG2.2.15 cells. The mRNA sequence showed that EFTUD2 could regulate classical IFN and virus response genes. Mechanistically, EFTUD2 single allele knockout decreased the expression of ISG-encoded proteins, comprising Mx1, OAS1, and PKR (EIF2AK2), through mediated gene splicing. However, EFTUD2 did not affect the expression of Jak-STAT pathway genes. Furthermore, EFTUD2 overexpression could restore the attenuation of IFN anti-HBV activity and the reduction of ISG resulting from EFTUD2 single allele knockout. EFTUD2, the spliceosome factor, is not IFN-inducible but is an IFN effector gene. EFTUD2 mediates IFN anti-HBV effect through regulation of gene splicing for certain ISGs, including Mx1, OAS1, and PKR. EFTUD2 does not affect IFN receptors or canonical signal transduction components. Therefore, it can be concluded that EFTUD2 regulates ISGs using a novel, nonclassical mechanism.

The blue light signal transduction module FaCRY1-FaCOP1-FaHY5 regulates anthocyanin accumulation in cultivated strawberry.

Anthocyanins have important physiological functions and are beneficial to the improvement of fruit quality in strawberry. Light is important for anthocyanin biosynthesis, and specific light quality was identified to promote anthocyanin accumulation in many fruits. However, research on the molecular mechanisms of anthocyanin accumulation regulated by light quality in strawberry remains limited. Here we described the effects of red- and blue-light irradiation on anthocyanin accumulation in strawberry. The results showed that blue light, rather than red light, could lead to the rapid accumulation of anthocyanins after exposure to light for 48 hours. The transcriptional levels of anthocyanin structural and regulatory genes displayed similar trend to the anthocyanin content. To investigate the mechanism of blue light-induced anthocyanin accumulation, the homologs of Arabidopsis blue light signal transduction components, including the blue light photoreceptor FaCRY1, an E3 ubiquitin ligase FaCOP1 and light-responsive factor FaHY5, were cloned from the strawberry cultivar 'Benihoppe'. The protein-protein interaction of FaCRY1-FaCOP1-FaHY5 was revealed by yeast two-hybrid and fluorescence signal assays. Functional complementation analysis showed that overexpression of either FaCOP1 or FaHY5 restored the anthocyanin content and hypocotyl length in corresponding Arabidopsis mutants under blue light. Moreover, dual-luciferase assays showed that FaHY5 could increase the activity of FaRAP (anthocyanin transport gene) promoter and that this function relied on other, likely B-box protein FaBBX22, factors. The overexpression of FaHY5-VP16 (chimeric activator form of FaHY5) and FaBBX22 promoted the accumulation of anthocyanins in transgenic strawberry plants. Further, transcriptomic profiling indicated that the genes involved in the phenylpropanoid biosynthesis pathway were enriched in both FaHY5-VP16-OX and FaBBX22-OX strawberry plants. In summary, our findings provide insights into a mechanism involving the regulation of blue light-induced anthocyanin accumulation via a FaCRY1-FaCOP1-FaHY5 signal transduction module in strawberry.

Comparison of gene expression changes in two wheat varieties with different phenotype to strip rust using RNA-Seq analysis

The fungus Puccinia striiformis f. sp. tritici (Pst) is an important threat to wheat production because it can cause wheat stripe rust. The present study aimed to identify new stripe rust resistance genes and to provide a theoretical and practical basis for breeding wheat varieties with broad spectrum, stable, and durable resistance. Wheat leaves inoculated with wheat stripe rust fungus Chinese yellow rust 34 were collected at different time points for transcriptomic analysis based on the wheat stripe rust susceptible varieties AVOCET S (AVS) and AVSYr15NIL [near-isogenic line (NIL) derived from AVS]. The results showed that the number of upregulated genes in the two varieties was 294, 364, 398, and 604, and the number of downregulated genes was 520, 178, 570, and 345 on the 1st, 3rd, 5th, and 7th days post inoculation, respectively. Gene Ontology and Kyoto Encyclopedia of Gene and Genomes enrichment analyses found enrichment of differentially expressed genes in the peroxisome proliferators-activated receptor signaling pathways, plant–pathogen interaction, and styrene acrylic acid biosynthesis that encoded protein kinases, signal transduction, transcription factors, and functional protein components. Differentially expressed genes were randomly selected for quantitative reverse transcription PCR analysis, and the change trend was the same as in the transcriptome data. The results of this study suggest that genes in AVSYr15NIL related to the stripe rust response could be valuable for understanding the mechanisms involved in stripe rust resistance.

Sphingosine kinase-2 silencing modulates sensitivity of HT-29 human colorectal cancer cells to paclitaxel-induced cell death through altering apoptosis-related proteins.

The sphingosine kinase-2 (SphK2), a main component of sphingolipid signal transduction, is reported as an innovative therapeutic candidate for cancer treatment. This study was conducted to investigate the suppression of SphK2 in increasing HT-29 colorectal cancer cells sensitivity to paclitaxel via inducing apoptosis and targeting c-FLIPS, MCL-1, and survivin expressions. Cells were transfected with siRNA against SphK2, PTX, or SphK2 activator. Proliferation and apoptosis were evaluated by MTT assay, and trypan blue staining and ELISA, respectively. SphK2, c-FLIPS, MCL-1, and survivin expression were determined by qRT-PCR and Western blotting. SphK2 siRNA increased cell death induced PTX. SphK2 agonist abolished silencing impact on cell survival. We found down-expression of C-FLIPS, MCL-1, and survivin by SphK2 siRNA transfection either alone or in paclitaxel treated cells, as well as elevation in cell apoptosis. Our results showed that SphK2 suppression may be an effective important modality to enhance cell sensitivity to paclitaxel via the induction of apoptosis in colorectal cancers.

Phosphatidylinositol 4,5-Bisphosphate Sensing Lipid Raft via Inter-Leaflet Coupling Regulated by Acyl Chain Length of Sphingomyelin

Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important molecule located at the inner leaflet of cell membrane, where it serves as anchoring sites for a cohort of membrane-associated molecules and as a broad-reaching signaling intermediate. The lipid raft is thought as the major platform recruiting proteins for signal transduction and also known to mediate PIP2 accumulation across the membrane. While the significance of this cross-membrane coupling is increasingly appreciated, it remains unclear whether and how PIP2 senses the dynamic change of the ordered lipid domains over the packed hydrophobic core of the bilayer. Herein, by means of molecular dynamic simulation, we reveal that inner PIP2 molecules can sense the outer lipid domain via inter-leaflet coupling, and the coupling manner is dictated by the acyl chain length of sphingomyelin (SM) partitioned to the lipid domain. Shorter SM promotes membrane domain registration, whereby PIP2 accumulates beneath the domain across the membrane. In contrast, the anti-registration is thermodynamically preferred if the lipid domain has longer SM due to the hydrophobic mismatch between the corresponding acyl chains in SM and PIP2. In this case, PIP2 is expelled by the domain with a higher diffusivity. These results provide molecular insights into the regulatory mechanism of correlation between the outer lipid domain and inner PIP2, both of which are critical components for cell signal transduction.

SCREENING OF POTENTIAL CORE GENES IN THE PERIPHERAL BLOOD OF ADULT PATIENTS WITH SEPSIS BASED ON IMMUNOREGULATION AND SIGNAL TRANSDUCTION FUNCTIONS.

Objective: Based on the functions of immunoregulation and signal transduction, septic peripheral blood sequencing and bioinformatics technology were used to screen potential core targets. Methods: Peripheral blood of 23 patients with sepsis and 10 normal volunteers underwent RNA-seq processing within 24 hours after admission to the hospital. Data quality control and differential gene screening were performed based on R language ( P < 0.01; log2FC ≥ 2). Gene function enrichment analysis was conducted on differentially expressed genes (DEGs). Then, target genes were submitted to STRING to constitute the PPI network, and GSE65682 were used to explore the prognostic relevance of potential core genes. Meta-analysis was used to verify the expression trends of core genes in the sepsis group. Then, cell line localization analysis of core genes in the 5 peripheral blood mononuclear cell samples (normal control = 2; systemic inflammatory response syndrome = 1; SEPSIS = 2) was performed. Results: A total of 1,128 DEGs were obtained between sepsis and normal group, of which 721 were upregulated and 407 downregulated. These DEGs were mainly enriched in leukocyte-mediated cytotoxicity, cell killing regulation, adaptive immune response regulation, lymphocyte-mediated immune regulation, and negative regulation of adaptive immune response. PPI network analysis results showed that CD160, KLRG1, S1PR5, and RGS16 were located in the core area, which are related to adaptive immune regulation, signal transduction, and intracellular components. The above four genes in the core area were found to be related to the prognosis of patients with sepsis, of which RGS16 was negatively correlated with the survival rate, and CD160, KLRG1, and S1PR5 were positively correlated. However, several public data sets showed that CD160, KLRG1, and S1PR5 were all downregulated in the peripheral blood of patients with sepsis, while RGS16 was upregulated in the sepsis group. Single-cell sequencing analysis showed that they were mainly expressed in NK-T cells. Conclusions: CD160, KLRG1, S1PR5, and RGS16 were mainly located in human peripheral blood NK-T cells. Sepsis participants expressed lower levels of S1PR5, CD160, and KLRG1, while sepsis participants expressed higher levels of RGS16. This suggests that they may be potential research targets for sepsis.

The oscillating mechanism of a circadian clock.

Circadian clocks provide a biochemical representation of local time inside cells and control the timing of gene expression in anticipation of sunrise and sunset. In cyanobacteria, a circadian oscillator comprised of three Kai proteins — KaiA, KaiB, and KaiC — relay temporal information downstream through two kinases, SasA and CikA, to regulate the transcription factor RpaA. Identifying the mechanisms by which circadian clocks exert temporal control over gene expression has been challenging in the complex milieu of cells. Thus, we reassembled an intact clock including oscillator and signal transduction components under defined conditions in vitro. Together with structural studies and biochemical analyses of partial clock reactions, we acquired new insights into mechanisms by which the cyanobacterial circadian clock functions to control gene expression. The in vitro oscillator is known to function under a relatively narrow set of Kai protein concentrations; we show here that a KaiABC-only mixture that fails to oscillate in a sustained manner due to limiting levels of KaiB can be rescued by SasA, which acts to recruit KaiB to the KaiC hexamer through heterotropic cooperativity. Cooperativity is based on structural mimicry between SasA and KaiB, and mutations that eliminate heterocooperativity in vitro profoundly affect circadian rhythms in vivo. CikA also rescues period defects under low levels of KaiA; together, our data help explain how the clock compensates in vivo for changes in concentrations of oscillator components that occur as part of the transcription-translation feedback loop and protein turnover. The coupling between oscillator and input-output components blurs their distinction. We developed the in vitro clock to establish causal links between clock biochemistry and in vivo phenotypes, and it provides a platform to explore how changes in factors such as temperature or ATP levels are propagated to regulate transcription.