Catalytic hairpin self-assembly amplification fluorescence detection of chloramphenicol based on cross-shaped DNA and UiO-66.

A catalytic hairpin self-assembly (CHA) amplification method was developed for CAP detection based on cross-shaped DNA and UiO-66. MOF was used to quench the fluorescent signal of FAM labeled DNA. Cross-shaped DNA with four fluorophore group (FAM) was utilized to enhance the fluorescent intensity. CAP could open hairpin structure of H-apt and induce CHA reaction. The product of CHA hybridized with cross-shaped DNA, resulting its leaving from the surface of UiO-66 and recovery of fluorescent signal. The limit of detection (LOD) was low to 0.87 pM. This method had been successfully applied for the detection of CAP in actual samples. Importantly, the high sensitivity was attributed to the great amplification efficiency of CHA, strong fluorescent intensity of cross-shaped DNA structure and great fluorescent quenched efficiency of UiO-66.

LncRNA xist regulates sepsis associated neuroinflammation in the periventricular white matter of CLP rats by miR-122-5p/PKCη Axis.

Background: Neuroinflammation is a common feature of many neurological diseases, and remains crucial for disease progression and prognosis. Activation of microglia and astrocytes can lead to neuroinflammation. However, little is known about the role of lncRNA xist and miR-122-5p in the pathogenesis of sepsis-associated neuroinflammation (SAN). This study aims to investigate the role of lncRNA xist and miR-122-5p in the pathogenesis of SAN. Methods: Levels of miR-122-5p and proinflammatory mediators were detected in the cerebrospinal fluid (CSF) of patients with intracranial infection (ICI) by ELISA and qRT-PCR. miRNA expression in the periventricular white matter (PWM) in rats was analyzed by high-throughput sequencing. Levels of lncRNA xist, miR-122-5p and proinflammatory mediators in the PWM were measured using qRT-PCR and western blot. Bioinformatics analysis was used to predict the upstream and downstream of miR-122-5p. The interaction between miR-122-5p and its target protein was validated using luciferase reporter assay. BV2 and astrocytes were used to detect the expression of lncRNA xist, miR-122-5p. Results: The level of miR-122-5p was significantly decreased in the CSF of ICI patients, while the expression of IL-1ß and TNF-α were significantly upregulated. Furthermore, it was found that the expression of IL-1ß and TNF-α were negatively correlated with the level of miR-122-5p. A high-throughput sequencing analysis showed that miR-122-5p expression was downregulated with 1.5-fold changes in the PWM of CLP rats compared with sham group. Bioinformatics analysis found that lncRNA xist and PKCη were the upstream and downstream target genes of miR-122-5p, respectively. The identified lncRNA xist and PKCη were significantly increased in the PWM of CLP rats. Overexpression of miR-122-5p or knockdown of lncRNA xist could significantly downregulate the level of PKCη and proinflammatory mediators from activated microglia and astrocytes. Meanwhile, in vitro investigation showed that silencing lncRNA xist or PKCη or enhancing the expression of miR-122-5p could obviously inhibit the release of proinflammatory mediators in activated BV2 cells and astrocytes. Conclusion: LncRNA xist could regulate microglia and astrocytes activation in the PWM of CLP rats via miR-122-5p/PKCη axis, further mediating sepsis associated neuroinflammation.

Ras-mediated homeostatic control of front-back signaling dictates cell polarity.

Studies in the model systems, Dictyostelium amoebae and HL-60 neutrophils, have shown that local Ras activity directly regulates cell motility or polarity. Localized Ras activation on the membrane is spatiotemporally regulated by its activators, RasGEFs, and inhibitors, RasGAPs, which might be expected to create a stable 'front' and 'back', respectively, in migrating cells. Focusing on C2GAPB in amoebae and RASAL3 in neutrophils, we investigated how Ras activity along the cortex controls polarity. Since existing gene knockout and overexpression studies can be circumvented, we chose optogenetic approaches to assess the immediate, local effects of these Ras regulators on the cell cortex. In both cellular systems, optically targeting the respective RasGAPs to the cell front extinguished existing protrusions and changed the direction of migration, as might be expected. However, when the expression of C2GAPB was induced globally, amoebae polarized within hours. Furthermore, within minutes of globally recruiting either C2GAPB in amoebae or RASAL3 in neutrophils, each cell type polarized and moved more rapidly. Targeting the RasGAPs to the cell backs exaggerated these effects on migration and polarity. Overall, in both cell types, RasGAP-mediated polarization was brought about by increased actomyosin contractility at the back and sustained, localized F-actin polymerization at the front. These experimental results were accurately captured by computational simulations in which Ras levels control front and back feedback loops. The discovery that context-dependent Ras activity on the cell cortex has counterintuitive, unanticipated effects on cell polarity can have important implications for future drug-design strategies targeting oncogenic Ras.

Therapeutics of Extracellular Vesicles in Cardiocerebrovascular and Metabolic Diseases.

Extracellular vesicles (EVs) are nanoscale membranous vesicles containing DNA, RNA, lipids, and proteins, which play versatile roles in intercellular communications. EVs are increasingly being recognized as the promising therapeutic agents for many diseases, including cardiocerebrovascular and metabolic diseases, due to their ability to deliver functional and therapeutical molecules. In this chapter, the biological characteristics and functions of EVs are briefly summarized. Importantly, the current state of applying EVs in the prevention and treatment of cardiocerebrovascular and metabolic diseases, including myocardial infarction, atrial fibrillation, myocardial hypertrophy, stroke, diabetes, Alzheimer's disease, fatty liver, obesity, thyroid diseases, and osteoporosis, is discussed. Lastly, the challenges and prospects related to the preclinical and clinical application of EVs receive a particular focus.

MiRNA-338-3p Inhibits Neuroinflammation in the Corpus Callosum of LCV-LPS Rats Via STAT1 Signal Pathway.

Neuroinflammation is a common characteristic of intracranial infection (ICI), which is associated with the activation of astrocytes and microglia. MiRNAs are involved in the process of neuroinflammation. This study aimed to investigate the potential mechanism by which miR-338-3p negatively modulate the occurrence of neuroinflammation. We here reported that the decreased levels of miR-338-3p were detected using qRT-PCR and the upregulated expression of TNF-α and IL-1ß was measured by ELISA in the cerebrospinal fluid (CSF) in patients with ICI. A negative association between miR-338-3p and TNF-α or IL-1ß was revealed by Pearson correlation analysis. Sprague-Dawley (SD) rats were injected with LPS (50 µg) into left cerebral ventricule (LCV), following which the increased expression of TNF-α and IL-1ß and the reduction of miR-338-3p expression were observed in the corpus callosum (CC). Moreover, the expression of TNF-α and IL-1ß in the astrocytes and microglia in the CC of LCV-LPS rats were saliently inhibited by the overexpression of miR-338-3p. In vitro, cultured astrocytes and BV2 cells transfected with mimic-miR-338-3p produced less TNF-α and IL-1ß after LPS administration. Direct interaction between miR-338-3p and STAT1 mRNA was validated by biological information analysis and dual luciferase assay. Furthermore, STAT1 pathway was found to be implicated in inhibition of neuroinflammation induced by mimic miR-338-3p in the astrocytes and BV2 cells. Taken together, our results suggest that miR-338-3p suppress the generation of proinflammatory mediators in astrocyte and BV2 cells induced by LPS exposure through the STAT1 signal pathway. MiR-338-3p could act as a potential therapeutic strategy to reduce the neuroinflammatory response. Diagram describing the cellular and molecular mechanisms associated with LPS-induced neuroinflammation via the miR-338-3p/STAT1 pathway. LPS binds to TLRs on astrocytes or microglia to activate the STAT1 pathway and upregulate the production of pro-inflammatory cytokines. However, miR-338-3p inhibits the expression of STAT1 and reduces the production of inflammatory mediators.

Optogenetic modulation of guanine nucleotide exchange factors of Ras superfamily proteins directly controls cell shape and movement.

In this article, we provide detailed protocols on using optogenetic dimerizers to acutely perturb activities of guanine nucleotide exchange factors (GEFs) specific to Ras, Rac or Rho small GTPases of the migratory networks in various mammalian and amoeba cell lines. These GEFs are crucial components of signal transduction networks which link upstream G-protein coupled receptors to downstream cytoskeletal components and help cells migrate through their dynamic microenvironment. Conventional approaches to perturb and examine these signaling and cytoskeletal networks, such as gene knockout or overexpression, are protracted which allows networks to readjust through gene expression changes. Moreover, these tools lack spatial resolution to probe the effects of local network activations. To overcome these challenges, blue light-inducible cryptochrome- and LOV domain-based dimerization systems have been recently developed to control signaling or cytoskeletal events in a spatiotemporally precise manner. We illustrate that, within minutes of global membrane recruitment of full-length GEFs or their catalytic domains only, widespread increases or decreases in F-actin rich protrusions and cell size occur, depending on the particular node in the networks targeted. Additionally, we demonstrate localized GEF recruitment as a robust assay system to study local network activation-driven changes in polarity and directed migration. Altogether, these optical tools confirmed GEFs of Ras superfamily GTPases as regulators of cell shape, actin dynamics, and polarity. Furthermore, this optogenetic toolbox may be exploited in perturbing complex signaling interactions in varied physiological contexts including mammalian embryogenesis.

Dual-amplification colorimetric detection of bisphenol A based on catalytic hairpin assembly and DNAzyme-caused fragment self-assembly hybridization chain reaction.

An efficient and innovative strategy for colorimetric detection of bisphenol A (BPA) is shown here based on target-induced catalytic hairpin assembly (CHA) and DNAzyme-caused fragment self-assembly hybridization chain reaction (HCR). BPA can bind with its aptamer hairpin to trigger CHA, thus forming Y-shaped DNA nanostructures with an enzyme-strand (E-DNA) tail. Subsequently, the E-DNA can cyclically cleave the substrate hairpin, generating many fragments which can cause self-assembly HCR to form long strand DNA. Finally, the formed long strand DNA can hybridize with short single strand DNA on AuNPs, causing the color change of AuNPs from red to blue. Six important detection conditions of the proposed aptasensor were optimized. Under optimal conditions, the biosensor has high sensitivity for BPA detection at concentrations ranging from 0.8 pM to 500 pM and the detection limit is as low as 0.2 pM, providing a promising prospective ultrasensitive detection of BPA.

Actuation of single downstream nodes in growth factor network steers immune cell migration.

Ras signaling is typically associated with cell growth, but not direct regulation of motility or polarity. By optogenetically targeting different nodes in the Ras/PI3K/Akt network in differentiated human HL-60 neutrophils, we abruptly altered protrusive activity, bypassing the chemoattractant receptor/G-protein network. First, global recruitment of active KRas4B/HRas isoforms or a RasGEF, RasGRP4, immediately increased spreading and random motility. Second, activating Ras at the cell rear generated new protrusions, reversed pre-existing polarity, and steered sustained migration in neutrophils or murine RAW 264.7 macrophages. Third, recruiting a RasGAP, RASAL3, to cell fronts extinguished protrusions and changed migration direction. Remarkably, persistent RASAL3 recruitment at stable fronts abrogated directed migration in three different chemoattractant gradients. Fourth, local recruitment of the Ras-mTORC2 effector, Akt, in neutrophils or Dictyostelium amoebae generated new protrusions and rearranged pre-existing polarity. Overall, these optogenetic effects were mTORC2-dependent but relatively independent of PI3K. Thus, receptor-independent, local activations of classical growth-control pathways directly control actin assembly, cell shape, and migration modes.

Gut Microbiota in Children with Hand Foot and Mouth Disease on 16S rRNA Gene Sequencing.

Hand foot and mouth disease (HFMD) is a contagious and seasonal viral disease in children. The gut microbiota of HFMD children is not clear now. The study aimed to explore the gut microbiota of HFMD children. The 16S rRNA gene of the gut microbiota of ten HFMD patients and ten healthy children were sequenced on the NovaSeq and PacBio platforms respectively. There were significant differences in gut microbiota between the patients and healthy children. The diversity and abundance of gut microbiota in HFMD patients were lower than that in healthy children. The species Roseburia inulinivorans and Romboutsia timonensis were more abundant in healthy children than those in HFMD patients, which suggests that the two species may be used as probiotics for adjusting the gut microbiota of HFMD patients. Meanwhile, the results of 16S rRNA gene sequences from the two platforms were different. The NovaSeq platform identified more microbiota and has the characteristics of high throughput, short time and low price. However, the NovaSeq platform has low resolution at the species level. The PacBio platform has high resolution based on its long reads length, which is more suitable for species-level analysis. But, the shortcomings of the high price and low throughput of PacBio still need to be overcome. With the development of sequencing technology, the reduction in sequencing price and the increase in throughput will promote the third-generation sequencing technology used in the study of gut microbes.

Intrahippocampal injection of IL-1ß upregulates Siah1-mediated degradation of synaptophysin by activation of the ERK signaling in male rat.

Interleukin-1ß (IL-1ß) has been described to exert important effect on synapses in the brain. Here, we explored if the synapses in the hippocampus would be adversely affected following intracerebral IL-1ß injection and, if so, to clarify the underlying molecular mechanisms. Adult male Sprague-Dawley rats were divided into control, IL-1ß, IL-1ß + PD98059, and IL-1ß + MG132 groups and then sacrificed for detection of synaptophysin (syn) protein level, synaptosome glutamate release, and synapse ultrastructure by western blotting, glutamate kit and electron microscopy, respectively. These rats were tested by Morris water maze for learning and memory ability. It was determined by western blotting whether IL-1ß exerted the effect of on syn and siah1 expression in primary neurons via extracellular regulated protein kinases (ERK) signaling pathway. Intrahippocampal injection of IL-1ß in male rats and sacrificed at 8d resulted in a significant decrease in syn protein, damage of synapse structure, and abnormal release of neurotransmitters glutamate. ERK inhibitor and proteosome inhibitor treatment reversed the above changes induced by IL-1ß both in vivo and in vitro. In primary cultured neurons incubated with IL-1ß, the expression level of synaptophysin was significantly downregulated coupled with abnormal glutamate release. Furthermore, use of PD98059 had confirmed that ERK signaling pathway was implicated in synaptic disorders caused by IL-1ß treatment. The present results suggest that exogenous IL-1ß can suppress syn protein level and glutamate release. A possible mechanism for this is that IL-1ß induces syn degradation that is regulated by the E3 ligase siah1 via the ERK signaling pathway.

Target controlled alternative hybridization chain reaction for fluorescent detection of dual mycotoxins.

A target controlled alternative hybridization chain reaction (HCR) was developed for fluorescent detection of multiple mycotoxin. Ochratoxin A (OTA) and aflatoxin B1 (AFB1) can bind with their specific aptamer on the gold nanoparticles and cause the releasing of the short DNA sequences. The short DNA sequences can trigger different reaction route of HCR and thus produce two kinds of side-chain sequences. The side-chain sequences can cause the opening of the DNA tweezers and result the recovery of fluorescent signals. Good linear relationships were obtained in the range of 0.06-2 ng/mL (R2 = 0.994) for OTA and 0.005-1 ng/mL (R2 = 0.992) for AFB1 with limit of detection of 0.02 ng/mL for OTA and 0.002 ng/mL for AFB1. Importantly, it showed great sensitivity and excellent selectivity in practical food sample analysis for simultaneous detection of OTA and AFB1.

Using Live-Cell Imaging and Synthetic Biology to Probe Directed Migration in Dictyostelium.

For decades, the social amoeba Dictyostelium discoideum has been an invaluable tool for dissecting the biology of eukaryotic cells. Its short growth cycle and genetic tractability make it ideal for a variety of biochemical, cell biological, and biophysical assays. Dictyostelium have been widely used as a model of eukaryotic cell motility because the signaling and mechanical networks which they use to steer and produce forward motion are highly conserved. Because these migration networks consist of hundreds of interconnected proteins, perturbing individual molecules can have subtle effects or alter cell morphology and signaling in major unpredictable ways. Therefore, to fully understand this network, we must be able to quantitatively assess the consequences of abrupt modifications. This ability will allow us better control cell migration, which is critical for development and disease, in vivo. Here, we review recent advances in imaging, synthetic biology, and computational analysis which enable researchers to tune the activity of individual molecules in single living cells and precisely measure the effects on cellular motility and signaling. We also provide practical advice and resources to assist in applying these approaches in Dictyostelium.

An extracellular matrix protein promotes anillin-dependent processes in the Caenorhabditis elegans germline.

Cell division requires constriction of an actomyosin ring to segregate the genetic material equally into two daughter cells. The spatial and temporal regulation of the contractile ring at the division plane primarily depends on intracellular signals mediated by the centralspindlin complex and astral microtubules. Although much investigative work has elucidated intracellular factors and mechanisms controlling this process, the extracellular regulation of cytokinesis remains unclear. Thus far, the extracellular matrix protein Hemicentin (HIM-4) has been proposed to be required for cleavage furrow stabilization. The underlying molecular mechanism, however, has remained largely unknown. Here, we show that HIM-4 and anillin (ANI-1) genetically act in the same pathway to maintain the rachis bridge stability in the germline. Our FRAP experiments further reveal that HIM-4 restricts the motility of ANI-1. In addition, we demonstrate that HIM-4 is recruited to the cleavage site in dividing germ cells and promotes the proper ingression of the cleavage membrane. Collectively, we propose that HIM-4 is an extracellular factor that regulates ANI-1 for germ cell membrane stabilization and contractile ring formation in Caenorhabditis elegans germline cells.