Thermal and light-driven soft actuators based on a conductive polypyrrole nanofibers integrated poly(N-isopropylacrylamide) hydrogel with intelligent response.

The development of soft hydrogel actuators with outstanding mechanical properties, fast actuation speed, and available quantification of self-sensing actuation remains a challenging endeavor. In this work, dopamine-decorated polypyrrole nanofibers (DAPPy) were introduced into the polyethylene glycol diacrylate (PEGDA)-crosslinked poly(N-isopropyl acrylamide) network to generate a stretchable, NIR-responsive, and strain sensitive DAPPy/PNIPAM hydrogel layer. Besides, this active layer was combined with the passive ligninsulfonate sodium/polyacrylamide (LS/PAAM) to give DAPPy/PNIPAM//LS/PAAM bilayer hydrogel actuator, which exhibits ultrafast thermo-responsive actuation (19°/s) and underwater grasping and lifting performance. Moreover, the DAPPy/PNIPAM layer has excellent electrical conductivity (0.29 S/m) and thermal conversion ability (10.8 °C/min), which enable such a conductive hydrogel to act as a highly sensitive strain and temperature sensor with real-time resistance change in response to tensile strain (gauge factor up to 3.4), applied pressure, temperature, and remote NIR light irradiation. More importantly, the bilayer hydrogel actuator can integrate both actuation and self-sensing functions through the bending angle-surface temperature-relative resistance change relationship of the photothermal process. With excellent mechanical actuation and self-sensing ability, the resulting bilayer hydrogel showed a promising application potential as soft biomimetic actuating materials and soft intelligent actuators.

Insect cells of Spodoptera frugiperda support WSSV gene replication but not progeny virion assembly.

The lacking of stable and susceptible cell lines has hampered research on pathogenic mechanism of crustacean white spot syndrome virus (WSSV). To look for the suitable cell line which can sustain WSSV infection, we performed the studies on WSSV infection in the Spodoptera frugiperda (Sf9) insect cells. In consistent with our previous study in vitro in crayfish hematopoietic tissue cells, the WSSV envelope was detached from nucleocapsid around 2 hpi in Sf9 cells, which was accompanied with the cytoplasmic transport of nucleocapsid toward the cell nucleus within 3 hpi. Furthermore, the expression profile of both gene and protein of WSSV was determined in Sf9 cells after viral infection, in which a viral immediate early gene IE1 and an envelope protein VP28 exhibited gradually increased presence from 3 to 24 hpi. Similarly, the significant increase of WSSV genome replication was found at 3-48 hpi in Sf9 cells after infection with WSSV, indicating that Sf9 cells supported WSSV genome replication. Unfortunately, no assembled progeny virion was observed at 24 and 48 hpi in Sf9 cell nuclei as determined by transmission electron microscope, suggesting that WSSV progeny could not be assembled in Sf9 cell line as the viral structural proteins could not be transported into cell nuclei. Collectively, these findings provide a cell model for comparative analysis of WSSV infection mechanism with crustacean cells.

Kaposiform hemangioendothelioma presented with raynaud phenomenon: a case report.

BACKGROUND: Kaposiform hemangioendothelioma (KHE) is a rare vascular neoplasm affecting infants or young children. KHE includes a spectrum of lesions, ranging from small and superficial tumors to large and invasive lesions with Kasabach-Merritt phenomenon (KMP). Currently, no published studies have reported a KHE presenting as thrombocytopenia and Raynaud phenomenon. CASE PRESENTATION: A 2-year-old boy with right hand swelling and thrombocytopenia was admitted to our hospital. His right hand turned swelling and red, even occasionally cyanotic. This condition became worse in response to cool environments and improved with warming, and platelet counts were between 50 ~ 80 × 10^9/L. Physical examination on admission revealed the swelling and frostbite-like rash of the right-hand fingers, and the skin temperature of the right hand was lower than the left. On day 3 of admission, chest CT results showed an irregular mass on the right side of the spine. The puncture biopsy demonstrated positive CD31, D2-40, and FLI1 immunohistochemical staining, but negative GLUT1 staining, confirming the diagnosis of KHE. Furthermore, endothelin-1 (ET1) expression levels significantly increased, and eNOS and A20 expression levels significantly decreased comparing with control patients. The patient received methylprednisolone and sirolimus treatments, and his condition gradually improved during the follow-up. CONCLUSIONS: We reported the first case of KHE presenting with thrombocytopenia and Raynaud phenomenon. The development of Raynaud phenomenon could be associated with increased ET-1 and reduced eNOS and A20 expressions. Careful differential diagnosis of hidden KHE should be considered in children with thrombocytopenia and Raynaud phenomenon.

A Deacetylase CqSIRT1 Promotes WSSV Infection by Binding to Viral Envelope Proteins in Cherax quadricarinatus.

Sirtuin 1 (SIRT1), a member of the class III lysine deacetylases, exhibits powerful functional diversity in physiological processes and disease occurrences. However, the potential molecular mechanism underlying the role of SIRT1 during viral infection in crustaceans is poorly understood. Herein, SIRT1 was functionally characterized from the red claw crayfish Cherax quadricarinatus, which possesses typically conserved deacetylase domains and strong evolutionary relationships across various species. Moreover, gene knockdown of CqSIRT1 in crayfish haematopoietic tissue (Hpt) cell culture inhibited white spot syndrome virus (WSSV) late envelope gene vp28 transcription. In contrast, enhancement of deacetylase activity using a pharmacological activator promoted the replication of WSSV. Mechanically, CqSIRT1 was co-localized with viral envelope protein VP28 in the nuclei of Hpt cells and directly bound to VP28 with protein pulldown and co-immunoprecipitation assays. Furthermore, CqSIRT1 also interacted with another two viral envelope proteins, VP24 and VP26. To the best of our knowledge, this is the first report that WSSV structural proteins are linked to lysine deacetylases, providing a better understanding of the role of CqSIRT1 during WSSV infection and novel insights into the basic mechanism underlying the function of lysine deacetylases in crustaceans.

Invasion and Propagation of White Spot Syndrome Virus: Hijacking of the Cytoskeleton, Intracellular Transport Machinery, and Nuclear Import Transporters.

The pathogenesis of white spot syndrome virus (WSSV) is largely unclear. In this study, we found that actin nucleation and clathrin-mediated endocytosis were recruited for internalization of WSSV into crayfish hematopoietic tissue (Hpt) cells. This internalization was followed by intracellular transport of the invading virions via endocytic vesicles and endosomes. After envelope fusion within endosomes, the penetrated nucleocapsids were transported along microtubules toward the periphery of the nuclear pores. Furthermore, the nuclear transporter CqImportin α1/ß1, via binding of ARM repeat domain within CqImportin α1 to the nuclear localization sequences (NLSs) of viral cargoes and binding of CqImportin ß1 to the nucleoporins CqNup35/62 with the action of CqRan for docking to nuclear pores, was hijacked for both targeting of the incoming nucleocapsids toward the nuclear pores and import of the expressed viral structural proteins containing NLS into the cell nucleus. Intriguingly, dysfunction of CqImportin α1/ß1 resulted in significant accumulation of incoming nucleocapsids on the periphery of the Hpt cell nucleus, leading to substantially decreased introduction of the viral genome into the nucleus and remarkably reduced nuclear import of expressed viral structural proteins with NLS; both of these effects were accompanied by significantly inhibited viral propagation. Accordingly, the survival rate of crayfish post-WSSV challenge was significantly increased after dysfunction of CqImportin α1/ß1, also showing significantly reduced viral propagation, and was induced either by gene silencing or by pharmacological blockade via dietary administration of ivermectin per os. Collectively, our findings improve our understanding of WSSV pathogenesis and support future antiviral designing against WSSV. IMPORTANCE As one of the largest animal DNA viruses, white spot syndrome virus (WSSV) has been causing severe economical loss in aquaculture due to the limited knowledge on WSSV pathogenesis for an antiviral strategy. We demonstrate that the actin cytoskeleton, endocytic vesicles, endosomes, and microtubules are hijacked for WSSV invasion; importantly, the nuclear transporter CqImportin α1/ß1 together with CqRan were recruited, via binding of CqImportin ß1 to the nucleoporins CqNup35/62, for both the nuclear pore targeting of the incoming nucleocapsids and the nuclear import of expressed viral structural proteins containing the nuclear localization sequences (NLSs). This is the first report that NLSs from both viral structure proteins and host factor are elaborately recruited together to facilitate WSSV infection. Our findings provide a novel explanation for WSSV pathogenesis involving systemic hijacking of host factors, which can be used for antiviral targeting against WSSV disease, such as the blockade of CqImportin α1/ß1 with ivermectin.

CqPP2A inhibits white spot syndrome virus infection by up-regulating antimicrobial substances expression in red claw crayfish Cherax quadricarinatus.

Protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase, a highly conserved enzyme widely expressed in eukaryotic cells, which accounts for a majority of the serine/threonine phosphatase activity in cells implicated in regulation of immune signaling pathways and antiviral response. However, most of studies about PP2A have been conducted in mammals but few in crustaceans. In this study, two subunits of PP2A (named as CqPP2Ab and CqPP2Ac) were characterized to be involved in white spot syndrome virus (WSSV) infection in the haematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus. The open reading frame (ORF) of CqPP2Ab was 1341 bp encoding 446 amino acids with seven WD40 domains, and the ORF of CqPP2Ac was 930 bp encoding 309 amino acids with a PP2Ac domain. Tissue distribution analysis showed that the mRNA transcript of CqPP2Ab and CqPP2Ac were both widely expressed in all the tested tissues with the highest expression in hemocyte, followed by high expression in Hpt. The gene expressions of CqPP2Ab and CqPP2Ac were both significantly down-regulated at 6 h post WSSV infection (6 hpi) in Hpt cells. Importantly, the expression of viral immediate early gene IE1 and late viral gene envelope protein VP28 were both significantly increased post WSSV infection after gene silencing of CqPP2Ab or CqPP2Ac in Hpt cells, suggesting that CqPP2Ab and CqPP2Ac could inhibit WSSV infection in Hpt cells, probably by increasing the antimicrobial substances expression in consideration to the significantly reduced expression of anti-lipopolysaccharide factor, crustin, and lysozyme after gene silencing of CqPP2Ab or CqPP2Ac, respectively. These findings provide a new light on the mechanism of WSSV infection and the antiviral response in crustaceans.

White Spot Syndrome Virus Benefits from Endosomal Trafficking, Substantially Facilitated by a Valosin-Containing Protein, To Escape Autophagic Elimination and Propagate in the Crustacean Cherax quadricarinatus.

As the most severely lethal viral pathogen for crustaceans in both brackish water and freshwater, white spot syndrome virus (WSSV) has a mechanism of infection that remains largely unknown, which profoundly limits the control of WSSV disease. By using a hematopoietic tissue (Hpt) stem cell culture from the red claw crayfish Cherax quadricarinatus suitable for WSSV propagation in vitro, the intracellular trafficking of live WSSV, in which the acidic-pH-dependent endosomal environment was a prerequisite for WSSV fusion, was determined for the first time via live-cell imaging. When the acidic pH within the endosome was alkalized by chemicals, the intracellular WSSV virions were detained in dysfunctional endosomes, resulting in appreciable blocking of the viral infection. Furthermore, disrupted valosin-containing protein (C. quadricarinatus VCP [CqVCP]) activity resulted in considerable aggregation of endocytic WSSV virions in the disordered endosomes, which subsequently recruited autophagosomes, likely by binding to CqGABARAP via CqVCP, to eliminate the aggregated virions within the dysfunctional endosomes. Importantly, both autophagic sorting and the degradation of intracellular WSSV virions were clearly enhanced in Hpt cells with increased autophagic activity, demonstrating that autophagy played a defensive role against WSSV infection. Intriguingly, most of the endocytic WSSV virions were directed to the endosomal delivery system facilitated by CqVCP activity so that they avoided autophagy degradation and successfully delivered the viral genome into Hpt cell nuclei, which was followed by the propagation of progeny virions. These findings will benefit anti-WSSV target design against the most severe viral disease currently affecting farmed crustaceans.IMPORTANCE White spot disease is currently the most devastating viral disease in farmed crustaceans, such as shrimp and crayfish, and has resulted in a severe ecological problem for both brackish water and freshwater aquaculture areas worldwide. Efficient antiviral control of WSSV disease is still lacking due to our limited knowledge of its pathogenesis. Importantly, research on the WSSV infection mechanism is also quite meaningful for the elucidation of viral pathogenesis and virus-host coevolution, as WSSV is one of the largest animal viruses, in terms of genome size, that infects only crustaceans. Here, we found that most of the endocytic WSSV virions were directed to the endosomal delivery system, strongly facilitated by CqVCP, so that they avoided autophagic degradation and successfully delivered the viral genome into the Hpt cell nucleus for propagation. Our data point to a virus-sorting model that might also explain the escape of other enveloped DNA viruses.

An Ns1abp-like gene promotes white spot syndrome virus infection by interacting with the viral envelope protein VP28 in red claw crayfish Cherax quadricarinatus.

Influenza A virus non-structural-1A binding protein (named as Ns1abp) was originally identified as a host protein from human that bound to the viral NS-1 protein. In our previous study, the expression of an Ns1abp-like gene (denoted as CqNs1abp-like gene) was found to be up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To elucidate the role of CqNs1abp-like gene involved in WSSV infection, we cloned the CqNs1abp-like gene in which the open reading frame was 2232 bp, encoding 743 amino acids with two typical domains of one BTB (Broad-Complex, Tramtrack and Bric a brac) domain at N-terminal and six Kelch domains at C-terminal. The gene expression profile showed that the mRNA transcript of CqNs1abp-like gene was widely expressed in all the tested tissues with highest expression in nerve, relatively high expression in Hpt and lowest expression in eyestalk. Importantly, both the WSSV entry and the viral replication were significantly reduced in Hpt cells after gene silencing of CqNs1abp-like gene. By using protein pull-down assay, we found that the recombinant BTB domain, six Kelch domains and CqNs1abp-like intact protein were all bound to the WSSV envelope protein VP28, respectively, in which the BTB domain showed slightly less binding affinity than that of the six Kelch domains or the recombinant intact protein. Besides, the WSSV entry into Hpt cells was clearly decreased when the virus was pre-incubated with the recombinant BTB domain, six Kelch domains, or the recombinant CqNs1abp-like intact protein, respectively, suggesting that the CqNs1abp-like gene was likely to function as a putative recognition molecular towards WSSV infection in a crustacean C. quadricarinatus. Taken together, these data shed new light on the mechanism of WSSV infection and a putatively novel target on anti-WSSV infection in crustacean farming.

A laminin-receptor-like protein regulates white spot syndrome virus infection by binding to the viral envelope protein VP28 in red claw crayfish Cherax quadricarinatus.

White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, which has been causing huge economic losses in global aquaculture. Laminin receptor (LR) is a cell surface receptor which participates in the interactions between cells as well as cells and extracellular matrix. Previously, we found that a CqLR-like gene was responsive to WSSV infection in the hematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus. To further reveal the role of CqLR-like gene involved in WSSV infection, the full-length cDNA of CqLR-like gene was cloned with 1000 bp, and the open reading frame encoded 308 amino acids with a conserved laminin-binding domain. Importantly, both the WSSV entry and viral replication were strongly reduced in Hpt cells after loss-of-function of CqLR-like gene by gene silencing. Protein interaction assay demonstrated that the recombinant CqLR-like protein could bind to WSSV virion in vitro by enzyme-linked immunosorbent assay and the binding affinity was in a dose-dependent manner. Furthermore, recombinant CqLR-like protein was found to bind to WSSV envelop protein VP28, but not other envelop proteins tested including VP19, VP24, and VP26, by pull down assay in HEK293T cells. In regarding to that LR is mainly localized on many types of cells' membrane, these data together suggested that CqLR-like protein was likely to function as a putative recognition molecule towards WSSV and act in the viral entry into a crustacean host cell, which may benefit the elucidation of the WSSV pathogenesis and further the pharmaceutical target for the possibly effective control of WSSV disease.