AANAT1 regulates insect midgut detoxification through the ROS/CncC pathway.

Insecticide resistance has been a problem in both the agricultural pests and vectors. Revealing the detoxification mechanisms may help to better manage insect pests. Here, we showed that arylalkylamine N-acetyltransferase 1 (AANAT1) regulates intestinal detoxification process through modulation of reactive oxygen species (ROS)-activated transcription factors cap"n"collar isoform-C (CncC): muscle aponeurosis fibromatosis (Maf) pathway in both the oriental fruit fly, Bactrocera dorsalis, and the arbovirus vector, Aedes aegypti. Knockout/knockdown of AANAT1 led to accumulation of biogenic amines, which induced a decreased in the gut ROS level. The reduced midgut ROS levels resulted in decreased expression of CncC and Maf, leading to lower expression level of detoxification genes. AANAT1 knockout/knockdown insects were more susceptible to insecticide treatments. Our study reveals that normal functionality of AANAT1 is important for the regulation of gut detoxification pathways, providing insights into the mechanism underlying the gut defense against xenobiotics in metazoans.

Characterization of an agmatine N-acetyltransferase from Bactrocera dorsalis that modulates ovary development.

Agmatine N-acetyltransferase (AgmNAT), which catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine, is a member of the GCN5-related N-acetyltransferase family. So far, knowledge of the physiological roles of AgmNAT in insects is limited. Here, we identified one gene encoding protein homologous to that of Drosophila AgmNAT using sequence information from an activity-verified Drosophila AgmNAT in a BLAST search of the Bactrocera dorsalis genome. We expressed and purified B. dorsalis AgmNAT in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Our application of the screening strategy to BdorAgmNAT led to the identification of agmatine as the best amine substrate for this enzyme, with the highest kcat/Km value. We successfully obtained a BdorAgmNAT knockout strain based on a wild-type strain (WT) using the CRISPR/Cas9 technique. The ovary development of the BdorAgmNAT knockout mutants was delayed for 10 days compared with the WT specimens. Moreover, mutants had a much smaller mature ovary size and laid far fewer eggs than WT. Loss of function of BdorAgmNAT caused by RNAi with mature WT females did not affect their fecundity. These findings indicate that BdorAgmNAT is critical for oogenesis. Our data provide the first evidence for AgmNAT in regulating ovary development.

Elasto-plastic effects on shape-shifting electron-beam-patterned gel-based micro-helices.

Shape-shifting helical gels have been created by various routes, notably by photolithography. We explore electron-beam lithography as an alternative to prescribe microhelix formation in tethered patterns of pure poly(acrylic acid). Simulations indicate the nanoscale spatial distribution of deposited energy that drives the loss of acid groups and crosslinking. Upon exposure to buffer, a patterned line converts to a 3D helix whose cross section comprises a crosslinked and hydrophobic core surrounded by a high-swelling pH-responsive corona. Through-thickness asymmetries generate out-of-plane bending to drive helix formation. The relative core and corona fractions are determined by the electron dose which in turn controls the helical radius and pitch. Increasing pH substantially raises the swelling stress and the rod elongates plastically. The pitch concurrently changes from minimal to non-minimal. The in-plane asymmetry driving this change can be attributed to shear-band formation in the hydrophobic core. Subsequent pH cycling drives elastic cycling of the helical properties. These findings illustrate the effects of elastoplastic deformation on helical properties and elaborate unique attributes of electron lithography as an alternate means to create shape-shifting structures.

Distribution analysis of TRH in Bactrocera dorsalis using a CRISPR/Cas9-mediated reporter knock-in strain.

Although the study of many genes and their protein products is limited by the availability of high-quality antibodies, this problem could be solved by fusing a tag/reporter to an endogenous gene using a gene-editing approach. The type II bacterial CRISPR/Cas system has been demonstrated to be an efficient gene-targeting technology for many insects, including the oriental fruit fly Bactrocera dorsalis. However, knocking in, an important editing method of the CRISPR/Cas9 system, has lagged in its application in insects. Here, we describe a highly efficient homology-directed genome editing system for B. dorsalis that incorporates coinjection of embryos with Cas9 protein, guide RNA and a short single-stranded oligodeoxynucleotide donor. This one-step procedure generates flies carrying V5 tag (42 bp) in the BdorTRH gene. In insects, as in other invertebrates and in vertebrates, the neuronal tryptophan hydroxylase (TRH) gene encodes the rate-limiting enzyme for serotonin biosynthesis in the central nervous system. Using V5 monoclonal antibody, the distribution of TRH in B. dorsalis at different developmental stages was uncovered. Our results will facilitate the generation of insects carrying precise DNA inserts in endogenous genes and will lay foundation for the investigation of the neural mechanisms underlying the serotonin-mediated behaviour of B. dorsalis.

Overexpression of human ACE2 protein in mouse fibroblasts stably transfected with the intact ACE2 gene.

Infection by SARS-CoV-2 is dependent on binding of the viral spike protein to angiotensin converting enzyme 2 (ACE2), a membrane glycoprotein expressed on epithelial cells in the human upper respiratory tract. Recombinant ACE2 protein has potential application for anti-viral therapy. Here we co-transfected mouse fibroblasts (A9 cells) with a cloned fragment of human genomic DNA containing the intact ACE2 gene and an unlinked neomycin phosphotransferase gene, and then selected stable neomycin-resistant transfectants. Transfectant clones expressed ACE2 protein at levels that were generally proportional to the number of ACE2 gene copies integrated in the cell genome, ranging up to approximately 50 times the level of ACE2 present of Vero-E6 cells. Cells overexpressing ACE2 were hypersensitive to infection by spike-pseudotyped vesicular stomatitis virus (VSV-S), and adsorption of VSV-S to these cells occurred at an accelerated rate compared to Vero-E6 cells. The transfectant cell clones described here therefore have favorable attributes as feedstocks for large-scale production of recombinant human ACE2 protein.

Arylalkylamine N-acetyltransferase 1 gene (AANAT1) regulates cuticle pigmentation and ovary development of the adult oriental fruit fly, Bactrocera dorsalis.

The arylalkylamine N-acetyltransferase (AANAT) enzymes catalyze the acetyl-CoA-dependent acetylation of an amine or arylalkylamine, which is involved in important biological processes of insects. Here, we carried out the molecular and biochemical identification of an arylalkylamine N-acetyltransferase (AANAT) from the oriental fruit fly, Bactrocera dorsalis. Using a bacterial expression system, we expressed and purified the encoded recombinant BdorAANAT1-V3 protein. The purified recombinant protein acts on a wide range of substrates, including dopamine, tyramine, octopamine, serotonin, methoxytryptamine, and tryptamine, and shows similar substrate affinity (i.e., Km values: 0.16-0.26 mM) except for serotonin (Km = 0.74 mM) and dopamine (Km = 0.84 mM). Transcriptional profile analysis of BdorAANAT1 revealed that this gene is most prevalent in adults and abundant in the adult brain, gut, and ovary. Using the CRISPR/Cas9 technique, we successfully obtained a BdorAANAT1 knockout strain based on a wild-type strain (WT). Compared with the WT, the cuticle color of larvae and pupae is normal; however, in adult mutants, the yellow region of their thorax is darkly pigmented, and two black spots were evident at the abdomen's end. Moreover, the female BdorAANAT1 knockout mutant had a smaller ovary than the WT, and laid far fewer eggs. Loss of function of BdorAANAT1 caused by RNAi with mature adult females in which the reproductive system is fully developed had no effect on their fecundity. Altogether, these results indicate that BdorAANAT1 regulates ovary development. Our findings provide evidence for the insect AANAT1 modulating adult cuticle pigmentation and female fecundity.

A polypeptide model for toxic aberrant proteins induced by aminoglycoside antibiotics.

Aminoglycoside antibiotics interfere with the selection of cognate tRNAs during translation, resulting in the synthesis of aberrant proteins that are the ultimate cause of cell death. However, the toxic potential of aberrant proteins and how they avoid degradation by the cell's protein quality control (QC) machinery are not understood. Here we report that levels of the heat shock (HS) transcription factor σ32 increased sharply following exposure of Escherichia coli to the aminoglycoside kanamycin (Kan), suggesting that at least some of the aberrant proteins synthesized in these cells were recognized as substrates by DnaK, a molecular chaperone that regulates the HS response, the major protein QC pathway in bacteria. To further investigate aberrant protein toxic potential and interaction with cell QC factors, we studied an acutely toxic 48-residue polypeptide (ARF48) that is encoded by an alternate reading frame in a plant cDNA. As occurred in cells exposed to Kan, σ32 levels were strongly elevated following ARF48 expression, suggesting that ARF48 was recognized as a substrate by DnaK. Paradoxically, an internal 10-residue region that was tightly bound by DnaK in vitro also was required for the ARF48 toxic effect. Despite the increased levels of σ32, levels of several HS proteins were unchanged following ARF48 expression, suggesting that the HS response had been aborted. Nucleoids were condensed and cell permeability increased rapidly following ARF48 expression, together suggesting that ARF48 disrupts DNA-membrane interactions that could be required for efficient gene expression. Our results are consistent with earlier studies showing that aberrant proteins induced by aminoglycoside antibiotics disrupt cell membrane integrity. Insights into the mechanism for this effect could be gained by further study of the ARF48 model system.

Integrating nucleic acid sequence-based amplification and microlensing for high-sensitivity self-reporting detection.

We use electron-beam patterned functional microgels to integrate self-reporting molecular beacons, dielectric microlenses, and solid-phase and/or solution-phase nucleic acid amplification in a viral-detection microarray model. The detection limits for different combinations of these elements range from 10-10 M for direct target-beacon hybridization alone to 10-18 M when all elements are integrated simultaneously.

Chemical Orthogonality in Surface-Patterned Poly(ethylene glycol) Microgels.

Because of its widely known antifouling properties, a variety of lithographic approaches has been used to pattern surfaces with poly(ethylene glycol) (PEG) to control surface interactions with biomolecules and cells over micro- and nanolength scales. Often, however, particular applications need additional functions within PEG-patterned surfaces. Monofunctional films can be generated using PEG modified to include a chemically functional group. We show that patterning with focused electron beams, in addition to cross-linking a monofunctional PEG homopolymer thin-film precursor and grafting the resulting patterned microgels to an underlying substrate, induces additional chemical functionality by radiation chemistry along the polymer main chain and that this second functionality can be orthogonal to the initial one. Specifically, we explore the reactivity of biotin-terminated PEG (PEG-B) as a function of electron dose using 2 keV electrons. At low doses (∼4-10 µC/cm2), the patterned PEG-B microgels are reactive with streptavidin (SA). As dose increases, the SA reactivity decays as biotin is damaged by the incident electrons. Independently, amine reactivity appears at higher doses (∼150-500 µC/cm2). At both extremes, the patterned PEG microgels retain their ability to resist fibronectin adsorption. We confirm that the amine reactivity derives from the PEG main chain by demonstrating similar dose response in hydroxy-terminated PEG (PEG-OH), and we attribute this behavior to the formation of ketones, aldehydes, and/or carboxylic acids during and after electron-beam (e-beam) patterning. Based on relative fluorescent intensities, we estimate that the functional contrast between the differentially patterned areas is about a factor of six or more. This approach provides the ability to easily pattern biospecific functionality while preserving the ability to resist nonspecific adsorption at length scales relevant to controlling protein and cell interactions.

Functional Changes during Electron-Beam Lithography of Biotinylated Poly(ethylene glycol) Thin Films.

In contrast to photolithography where particular wavelengths of light can couple to specific photochemistries, electron-beam lithography can drive competing chemistries. To separate surface-grafting, cross-linking, and chemical functionality, we studied the effects of 2 keV electrons on thin films of poly(ethylene glycol) end-functionalized with hydroxyls (PEG-OH) or biotins (PEG-B). Similarities in the dose-dependent thickness changes of the patterned PEGs indicate that surface grafting and cross-linking primarily involve the ethylene oxide main chain. While higher doses create thicker patterns with more biotin, the concurrent increase in thiol reactivity indicates that cross-linking competes with biotin degradation. The dose window for optimal e-beam patterning of biotinylated PEG is very narrow. Biotin is entirely consumed at higher doses. Its modified functionality is reactive with 5-((2-(and-3)-S-(acetylmercapto) succinoyl) amino) (SAMSA). This effect creates a dose-dependent orthogonal functionality that can be patterned from a single precursor thin film.

Microlens Enhancement of Surface-Tethered Molecular Beacons.

The tethering of molecular beacon oligonucleotide detection probes to surface-patterned poly(ethylene glycol) (PEG) microgels has enabled the integration of molecular beacons into a microarray format. The microgels not only localize the probes to specific surface positions but also maintain them in a waterlike environment. Here we extend the concept of microgel tethering to include dielectric microlenses. We show that streptavidin-functionalized polystyrene microspheres (3 µm diameter) can be colocalized with molecular beacons using biotinylated PEG gels in patterns ranging from pseudocontinuous microgel pads with lateral dimensions on the order of tens of micrometers to individual microgels with lateral dimensions on the order of 400-500 nm. We use a simplex assay based on Influenza A detection to study the lensing behavior. The microspheres increase the effective numerical aperture of the collection optics, and we find that a tethered microsphere increases the peak intensity collected from hybridized beacons between 1.5 and 10 times depending on the specific pattern size and areal density of microgels. The highest signal increase occurs when a single microsphere is tethered to a single isolated microgel. The tethering is highly self-directed and occurs in the individual-microgel case only when the microgel is close to the optic axis of the microsphere. This alignment minimizes spherical aberration and maximizes coupling of emitted fluorescent intensity into the collection optics.

Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification.

Solid-phase oligonucleotide amplification is of interest because of possible applications to next-generation sequencing, multiplexed microarray-based detection, and cell-free synthetic biology. Its efficiency is, however, less than that of traditional liquid-phase amplification involving unconstrained primers and enzymes, and understanding how to optimize the solid-phase amplification process remains challenging. Here, we demonstrate the concept of solid-phase nucleic acid sequence-based amplification (SP-NASBA) and use it to study the effect of tethering density on amplification efficiency. SP-NASBA involves two enzymes, avian myeloblastosis virus reverse transcriptase (AMV-RT) and RNase H, to convert tethered forward and reverse primers into tethered double-stranded DNA (ds-DNA) bridges from which RNA- amplicons can be generated by a third enzyme, T7 RNA polymerase. We create microgels on silicon surfaces using electron-beam patterning of thin-film blends of hydroxyl-terminated and biotin-terminated poly(ethylene glycol) (PEG-OH, PEG-B). The tethering density is linearly related to the PEG-B concentration, and biotinylated primers and molecular beacon detection probes are tethered to streptavidin-activated microgels. While SP-NASBA is very efficient at low tethering densities, the efficiency decreases dramatically with increasing tethering density due to three effects: (a) a reduced hybridization efficiency of tethered molecular beacon detection probes; (b) a decrease in T7 RNA polymerase efficiency;

[Analysis of out-of-hospital emergency treatment for ventricular fibrillation between 2013 and 2016 in Shanghai].

OBJECTIVE: To investigate the epidemiological features of out-of-hospital patients with ventricular fibrillation (VF) in Shanghai and to analysis factors associated with outcomes, and to provide evidence for improving the success rate of VF. METHODS: The data of patients with VF admitted to Shanghai Medical Emergency Center from January 2013 to December 2016 were analyzed retrospectively. All the data were recorded including the clinical data, medical service time, return of spontaneous circulation (ROSC) at scene/en route, survival to hospital discharge. Factors that associated with successful resuscitation were analyzed by Logistic regression. RESULTS: From 2013 to 2016, 21 096 patients with suspected cardiac arrest were admitted to the Shanghai Medical Emergency Center. After excluding ventricular tachycardia (13 cases) and ventricular asystole (20 995 cases), 88 patients with VF were enrolled, with 62 male and 26 female; the average age was (63.22±16.15) years old. While bystander cardiopulmonary resuscitation (CPR) was performed in only 21 cases (23.86%). Fifty-seven cases occurred during the day (08:00-20:00), while 31 cases occurred in the night. And the average emergency response time was (6.47±4.13) minutes; the average on-site time was (14.76±10.88) minutes; the average transport to hospital time was (5.95±4.00) minutes. There were no significant differences in response time, on-site time and transport to hospital time each year, and there were no significant differences in emergency medical service time between day and night either. From 2013 to 2016, prehospital successful resuscitation rate was decreased by years [95.65% (22/23), 87.50% (14/16), 83.33% (20/24) vs. 80.00% (20/25), respectively, χ2 = 1.895, P = 0.595]. Survival to hospital discharge rate was increased by years [21.74% (5/23), 31.25% (5/16), 37.50% (9/24), 40.00% (10/25), respectively, χ 2 = 2.862, P = 0.413]. The success rate of prehospital resuscitation for patients with 1, 2, ≥3 defibrillation was 35.23% (31/88), 23.08% (12/52), 89.19% (33/37), respectively (χ2 = 42.811, P = 0.000). The on-site time in successful final resuscitation group was shorter than that in final resuscitation failure group (minutes: 10.85±8.83 vs. 16.79±11.36, t = 2.367, P = 0.020), the ROSC time in successful final resuscitation group was shorter than that of final resuscitation failure group (minutes: 3.24±3.17 vs. 7.43±6.64, t = 3.175, P = 0.002). It was shown by Logistic regression that long ROSC time was the risk factor for final resuscitation failure [odds ratio (OR) = 0.771, P = 0.024]. Gender, age, availability of witnesses CPR, call time, emergency response time, on-site time and transport to hospital time had no significant impact on the prehospital successful resuscitation and final successful resuscitation. In prehospital successful resuscitation group, there was significant difference in survival to hospital discharge rate among different defibrillation times group [48.39% (15/31), 58.33% (7/12) vs. 21.21% (7/33), χ2 = 7.460, P = 0.024]. CONCLUSIONS: From 2013 to 2016, there were no significant changes in the emergency response time, prehospital successful resuscitation rate and survival to hospital discharge rate of patients with VF in Shanghai. Though, repeated defibrillation could significantly increased prehospital successful resuscitation rate, multiple defibrillation indicated decline of survival to hospital discharge rate in prehospital successful resuscitation group. Additionally, long on-site time and long ROSC time indicated poor prognosis.