<em>In Vivo</em> Circadian Oscillation of dCREB2 and NF-κB Activity in the <em>Drosophila</em> Nervous System

<div><p>cAMP response element-binding protein (CREB) and nuclear factor kappa-B (NF-κB) are two ubiquitous transcription factors involved in a wide number of cellular processes, including the circadian system. Many previous studies on these factors use cellular assays that provide limited information on circadian activity or anatomical specificity. The ability to study transcription factors in defined tissue within intact animals will help to bridge the gap between cellular and <em>in vivo</em> data. We have used the GAL4-UAS and FLP-FRT systems to gain spatial control over reporter gene expression. Using a luciferase-based reporter, we show <em>in vivo</em> that <em>Drosophila</em> dCREB2- and NF-κB-mediated transcription oscillates in neuronal cells, glia, and in the mushroom body, a higher-order brain center in flies. This oscillation is under circadian control, cycling with a 24-hour rhythm, under both light-dark and dark-dark conditions. In light-light conditions, dCREB2 and NF-κB reporter flies exhibit a suppression of rhythmic activity. Furthermore, neuronal cycling of dCREB2 and NF-κB activity are modulated in <em>period</em> mutant flies, indicating these oscillations are controlled through the central clock. This study shows for the first time region-specific circadian oscillation of dCREB2/NF-κB activity in the <em>Drosophila</em> nervous system.</p> </div

Expression of the NF-κB-F-luc reporter is FLP dependent, oscillatory and NF-κB-dependent.

<p>(A) NF-κB-F-luc reporter activity is FLP-dependent. Reporter activity (Y-axis, relative light units) is plotted as a function of genotype (singly [NFκB-F-luc] or doubly transgenic flies [hs-FLP/+;NFκB-F-luc/+] and treatment (heat shock HS+ or not, HS−). The histogram bars show the mean hourly counts over a 3-day window (n = 24). (B) The ubiquitously expressed <i>actin</i>-GAL4 driver produces oscillatory NFκB-F-luc activity over a 24 h period. Luciferase activity is plotted as a function of time for singly (denoted no driver; NF-κB-F-luc) or doubly transgenic (denoted <i>actin</i>-GAL4; NFκB-F-luc/<i>actin</i>-GAL4) flies. n = 24 for each group (C) Bacterial infection affects reporter activity. Top panel: Reporter activity in the fat body is plotted for the first 48 hours after infection (filled circles) or sham treatment (open circles). The <i>cg</i>-GAL4 line (together with UAS-FLP and NFκB-F-luc) is used to activate the reporter in the fat body. n = 48 for each group. Bottom panel: The difference in reporter activity between the infected and sham-treated groups (infected RLU-sham RLU) over the same duration post-injection. (D) Quantification of reporter activity (mean hourly count) during the first and second days following infection. The mean luminescence counts in RLU are plotted as a function of time after infection. Values for each day are averaged and binned together. (E) There is a dose-dependent decrease in reporter activity in flies fed PDTC. The relative luminescence is plotted as a function of the PDTC dose fed to flies. Triply transgenic flies (UAS-FLP/+;NF-κB-F-luc/<i>actin</i>-GAL4) were fed different dosages of PDTC for 24 hours and then measured for luminescence 1 h after the end of feeding. n = 24 for each group, Error bars = S.E.M, **p<.001.</p

The NF-κB reporter activity oscillates under circadian control.

<p>(A) NF-κB reporter activity in multiple tissues oscillates in constant darkness. Reporter activity is plotted as a function of time for different genotypes. Triply transgenic flies, where the reporter is activated in all neurons (using the <i>elav</i>^c155-GAL4 driver, black squares), in a pan-glial pattern (using the <i>repo</i>-GAL4 driver, open circles), or in the mushroom body (using the <i>ok107</i>-GAL4 driver, open triangles), exhibit oscillatory activity when flies are maintained in light∶dark (first 24 h period) or dark∶dark (subsequent time) conditions. These plots are compared to those from doubly transgenic flies (solid line; UAS-FLP/+; NFκB-F-luc/+) that do not contain the tissue-specific driver. (B) The activity of the NF-κB reporter in different tissues is dampened under constant light. Reporter activity is shown as a function of time, with the shift to constant light occurring during the second 24 h period. The reporter is activated in triply transgenic flies in neurons (denoted <i>elav^c155</i>-GAL4, <i>elav^c155</i>-GAL4; UAS-FLP/+; NFκB-F-luc/+), glia (denoted <i>repo</i>-GAL4, UAS-FLP; NFκB-F-luc/<i>repo</i>-GAL4) or the mushroom body (denoted <i>ok107-GAL4</i>; UAS-FLP; NFκB-F-luc/+; <i>ok107</i>-GAL4/+) when compared to doubly transgenic flies without a GAL4 driver (solid line, UAS-FLP/+; NFκB-F-luc/+). (C–F) Neuronal reporter activity is plotted over time as flies are shifted from light∶dark to constant darkness. For all of these panels, the same transgenes (UAS-FLP/+; NFκB-F-luc/<i>elav</i>-GAL4) exist in all flies, but the flies are examined in a wild type (C), <i>per⁰</i> (D), <i>per^S</i> (E) or <i>per^L</i> (F) genetic background.</p

Oscillations in dCREB2 and NF-κB activity persist after luciferin removal.

<p>(A) Reporter activity in UAS-FLP/+;CRE-F-luc/<i>actin</i>-GAL4 flies starting ∼1 h following luciferin removal, plotted as a function of time. The inset shows a scaled view of the same data, with the first 24 h dropped. (B) Reporter activity in UAS-FLP/+;NFκB-F-luc/<i>actin</i>-GAL4 flies starting ∼1 h following luciferin removal. Inset shows a scaled view of the same data, with the first 24 h dropped. n = 24 flies for each group.</p

Developing a spatially restricted reporter system.

<p>(A) A cartoon of the reporter system. Three DNA binding sites are placed upstream of the CaSpeR TATAA sequence, followed by transcription (arrow) and translation (arrowhead) initiation sites, an FRT-flanked open reading frame for mCherry (ORF) including two tandem stop codons, and the luciferase ORF (#1). Targeted GAL4 expression (#3) drives the expression of UAS-FLP (#2), which catalyzes site-specific recombination at the FRT sites, activating the reporter. (B) CRE-F-luc reporter activity is FLP-dependent. Singly (CRE-F-luc) or doubly transgeneic flies (<i>hs</i>-FLP/+;CRE-F-luc/+) are maintained under 12∶12 LD conditions. Flies are exposed to heat-shock (+HS) or not (−HS), and measured for <i>in vivo</i> luminescence. The relative luminescence is plotted as a function of time, with daytime (white bars) and nighttime (black bars) durations indicated below the graph. Each data point represents the average hourly luminescence counts in relative light units (RLU) of 24 flies. (C) FLP protein expressed using the GAL4-UAS system can activate the reporter. Reporter activity (Y-axis, the mean hourly relative light units [RLUs] over a 4-day window) is plotted as a function of genotype (indicated with different colored bars) and treatment (+/− HS, heat shock). Error bars = S.E.M. *** p<0.001, Student' T-test, n = 24; n.s. signifies not significant. Similar statistical comparisons are made for the remaining figures. (D) Anatomical specificity of <i>gmr^long</i>-GAL4 driven reporters. <i>In vitro</i> luciferase activity measured in extracts made from isolated heads and bodies. The relative light units (Y-axis) are plotted as a function of the genotype (shown in gray [UAS-FLP/+; CRE-F-luc/+ or black [UAS-FLP/<i>gmr^long</i>-GAL4; CRE-F-luc/+]) or tissue source (head versus body).</p

Table_2_Molecular cloning, characterization, and expression of two 5-HTRs from the pearl oyster Pinctada fucata martensii.xlsx

The receptors of serotonin, also known as 5-hydroxytryptamine receptor (5-HTR) can mediate regulatory metamorphosis processes in a variety of mollusks. Studying the mechanisms of metamorphosis of the pearl oyster is significant to elucidate breeding, resource recovery and marine pearl production. In this study, two 5-HTR genes from Pinctada fucata martensii (Pm5-HTR2 and Pm5-HTR4) were cloned. A total of 1623 bp open reading frame was identified in Pm5-HTR2, and a 1185 bp open reading frame was detected in Pm5-HTR4; these open reading frames encoded a 540-residue polypeptide and a 394-residue polypeptide, respectively. We also conducted a domain analysis, which indicated that Pm5-HTR2 and Pm5-HTR4 contained a seven-transmembrane domain and revealed that the receptors had high similarity to Crassostrea gigas 5-HTR2 (54.62%) and 5-HTR4 (66.23%). Sequence analysis demonstrated conserved advanced structure and motifs (the DRY/ERY and NPXXY motifs). The expression pattern analysis revealed high expression levels of Pm5-HTR2 and Pm5-HTR4 during the developmental stages. ISH analysis showed that Pm5-HTR2 was primarily expressed in the FE, B, T, EU, and EL stages and Pm5-HTR4 was mainly expressed in the FE, B, T, D, EU, and EL stages. These results suggest that 5-HTRs may play key roles in P. f. martensii larval metamorphosis.</p

Table_1_Molecular cloning, characterization, and expression of two 5-HTRs from the pearl oyster Pinctada fucata martensii.xlsx

The receptors of serotonin, also known as 5-hydroxytryptamine receptor (5-HTR) can mediate regulatory metamorphosis processes in a variety of mollusks. Studying the mechanisms of metamorphosis of the pearl oyster is significant to elucidate breeding, resource recovery and marine pearl production. In this study, two 5-HTR genes from Pinctada fucata martensii (Pm5-HTR2 and Pm5-HTR4) were cloned. A total of 1623 bp open reading frame was identified in Pm5-HTR2, and a 1185 bp open reading frame was detected in Pm5-HTR4; these open reading frames encoded a 540-residue polypeptide and a 394-residue polypeptide, respectively. We also conducted a domain analysis, which indicated that Pm5-HTR2 and Pm5-HTR4 contained a seven-transmembrane domain and revealed that the receptors had high similarity to Crassostrea gigas 5-HTR2 (54.62%) and 5-HTR4 (66.23%). Sequence analysis demonstrated conserved advanced structure and motifs (the DRY/ERY and NPXXY motifs). The expression pattern analysis revealed high expression levels of Pm5-HTR2 and Pm5-HTR4 during the developmental stages. ISH analysis showed that Pm5-HTR2 was primarily expressed in the FE, B, T, EU, and EL stages and Pm5-HTR4 was mainly expressed in the FE, B, T, D, EU, and EL stages. These results suggest that 5-HTRs may play key roles in P. f. martensii larval metamorphosis.</p

Chemoenzymatic Synthesis of Unnatural Nucleotide Sugars for Enzymatic Bioorthogonal Labeling

In recent years, the development of the enzymatic bioorthogonal labeling strategy has offered exciting possibilities in the probing of structure-defined glycan epitopes. This strategy takes advantage of relaxed donor specificity and strict acceptor specificity of glycosyltransferases to label target glycan epitopes with bioorthogonal reactive groups carried by unnatural nucleotide sugars in vitro. The subsequent covalent conjugation by bioorthogonal chemical reactions with either fluorescent or affinity tags allows further visualization, quantification, or enrichment of target glycan epitopes. However, the application and development of the enzymatic labeling strategy have been hindered due to the limited availability of unnatural nucleotide sugars. Herein, a platform that combines chemical synthesis and enzymatic synthesis for the facile preparation of unnatural nucleotide sugars modified with diverse bioorthogonal reactive groups is described. By this platform, a total of 25 UDP-GlcNAc and UDP-GalNAc derivatives, including the most well explored bioorthogonal functional groups, were successfully synthesized. Furthermore, the potential application of these compounds for use in enzymatic bioorthogonal labeling reactions was also evaluated

Silk Fibroin-Coated Nano-MOFs Enhance the Thermal Stability and Immunogenicity of HBsAg

Vaccines are widely regarded as one of the most effective weapons in the fight against infectious diseases. Currently, vaccines must be stored and transported at low temperatures as high temperatures can lead to a loss of vaccine conformation and reduced therapeutic efficacy. Metal–organic frameworks (MOFs), such as zeolitic imidazole framework-8 (ZIF-8), are a new class of hybrid materials with large specific surface areas, high loading rates, and good biocompatibility and are successful systems for vaccine delivery and protection. Silk fibroin (SF) has a good biocompatibility and thermal stability. In this study, the hepatitis B surface antigen (HBsAg) was successfully encapsulated in ZIF-8 to form HBsAg@ZIF-8 (HZ) using a one-step shake and one-pot shake method. Subsequently, the SF coating modifies HZ through hydrophobic interactions to form HBsAg/SF@ZIF-8 (HSZ), which enhanced the thermal stability and immunogenicity of HBsAg. Compared to free HBsAg, HZ and HSZ improved the thermostability of HBsAg, promoted the antigen uptake and lysosomal escape, stimulated dendritic cell maturation and cytokine secretion, formed an antigen reservoir to promote antibody production, and activated CD4+ T and CD8+ T cells to enhance memory T-cell production. Importantly, HSZ induced a strong immune response even after 14 days of storage at 25 °C. Furthermore, the nanoparticles prepared by the one-step shake method exhibited superior properties compared to those prepared by the one-pot shake method. This study highlights the importance of SF-coated ZIF-8, which holds promise for investigating thermostable vaccines and breaking the vaccine cold chain