Logic-gated tumor-microenvironment nanoamplifier enables targeted delivery of CRISPR/Cas9 for multimodal cancer therapy.

Recent innovations in nanomaterials inspire abundant novel tumor-targeting CRISPR-based gene therapies. However, the therapeutic efficiency of traditional targeted nanotherapeutic strategies is limited by that the biomarkers vary in a spatiotemporal-dependent manner with tumor progression. Here, we propose a self-amplifying logic-gated gene editing strategy for gene/H2O2-mediated/starvation multimodal cancer therapy. In this approach, a hypoxia-degradable covalent-organic framework (COF) is synthesized to coat a-ZIF-8 in which glucose oxidase (GOx) and CRISPR system are packaged. To intensify intracellular redox dyshomeostasis, DNAzymes which can cleave catalase mRNA are loaded as well. When the nanosystem gets into the tumor, the weakly acidic and hypoxic microenvironment degrades the ZIF-8@COF to activate GOx, which amplifies intracellular H+ and hypoxia, accelerating the nanocarrier degradation to guarantee available CRISPR plasmid and GOx release in target cells. These tandem reactions deplete glucose and oxygen, leading to logic-gated-triggered gene editing as well as synergistic gene/H2O2-mediated/starvation therapy. Overall, this approach highlights the biocomputing-based CRISPR delivery and underscores the great potential of precise cancer therapy.

M6PR interacts with the HA2 subunit of influenza A virus to facilitate the fusion of viral and endosomal membranes.

Influenza A virus (IAV) commandeers numerous host cellular factors for successful replication. However, very few host factors have been revealed to be involved in the fusion of viral envelope and late endosomal membranes. In this study, we identified cation-dependent mannose-6-phosphate receptor (M6PR) as a crucial host factor for the replication of IAV. We found that siRNA knockdown of M6PR expression significantly reduced the growth titers of different subtypes of IAV, and that the inhibitory effect of M6PR siRNA treatment on IAV growth was overcome by the complement of exogenously expressed M6PR. When A549 cells were treated with siRNA targeting M6PR, the nuclear accumulation of viral nucleoprotein (NP) was dramatically inhibited at early timepoints post-infection, indicating that M6PR engages in the early stage of the IAV replication cycle. By investigating the role of M6PR in the individual entry and post-entry steps of IAV replication, we found that the downregulation of M6PR expression had no effect on attachment, internalization, early endosome trafficking, or late endosome acidification. However, we found that M6PR expression was critical for the fusion of viral envelope and late endosomal membranes. Of note, M6PR interacted with the hemagglutinin (HA) protein of IAV, and further studies showed that the lumenal domain of M6PR and the ectodomain of HA2 mediated the interaction and directly promoted the fusion of the viral and late endosomal membranes, thereby facilitating IAV replication. Together, our findings highlight the importance of the M6PR-HA interaction in the fusion of viral and late endosomal membranes during IAV replication.

ABTB1 facilitates the replication of influenza A virus by counteracting TRIM4-mediated degradation of viral NP protein.

Influenza A viruses (IAVs) continue to cause tremendous economic losses to the global animal industry and respiratory diseases and deaths among humans. The nuclear import of the vRNP complex, composed of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), nucleoprotein (NP), and viral RNA, is essential for the efficient replication of IAV. Host factors involved in this process can be targeted for the development of countermeasures against IAV infection. Here, we found that Ankyrin Repeat and BTB Domain Containing 1 (ABTB1) promotes the replication of IAV, and positively regulates the nuclear import of the vRNP complex. ABTB1 did not interact directly with NP, indicating that ABTB1 plays an indirect role in facilitating the nuclear import of the vRNP complex. Immunoprecipitation and mass spectrometry revealed that Tripartite Motif Containing 4 (TRIM4) interacts with ABTB1. We found that TRIM4 relies on its E3 ubiquitin ligase activity to inhibit the replication of IAV by targeting and degrading NP within the incoming vRNP complex as well as the newly synthesized NP. ABTB1 interacted with TRIM4, leading to TRIM4 degradation through the proteasome system. Notably, ABTB1-mediated degradation of TRIM4 blocked the effect of TRIM4 on NP stability, and largely counteracted the inhibitory effect of TRIM4 on IAV replication. Our findings define a novel role for ABTB1 in aiding the nuclear import of the vRNP complex of IAV by counteracting the destabilizing effect of TRIM4 on the viral NP protein.

Biodegradable Hollow Nanoscavengers Restore Liver Functions to Reverse Insulin Resistance in Type 2 Diabetes.

Type 2 diabetes (T2D) results from the cells' insulin resistance, and to date, insulin therapy and diabetes medications targeting glycemic management have failed to reverse the increase in T2D prevalence. Restoring liver functions to improve hepatic insulin resistance by reducing oxidative stress is a potential strategy for T2D treatment. Herein, the liver-targeted biodegradable silica nanoshells embedded with platinum nanoparticles (Pt-SiO2) are designed as reactive oxygen species (ROS) nanoscavengers and functional hollow nanocarriers. Then, 2,4-dinitrophenol-methyl ether (DNPME, mitochondrial uncoupler) is loaded inside Pt-SiO2, followed by coating a lipid bilayer (D@Pt-SiO2@L) for long-term effective ROS removal (platinum nanoparticles scavenge overproduced ROS, while DNPME inhibits ROS production) in the liver tissue of T2D models. It is found that D@Pt-SiO2@L reverses elevated oxidative stress, insulin resistance, and impaired glucose consumption in vitro, and significantly improves hepatic steatosis and antioxidant capacity in diabetic mice models induced by a high-fat diet and streptozotocin. Moreover, intravenous administration of D@Pt-SiO2@L indicates therapeutic effects on hyperlipidemia, insulin resistance, hyperglycemia, and diabetic nephropathy, which provides a promising approach for T2D treatment by reversing hepatic insulin resistance through long-term ROS scavenging.

In-Tumor Biosynthetic Construction of Upconversion Nanomachines for Precise Near-Infrared Phototherapy.

Targeted construction of therapeutic nanoplatforms in tumor cells with specific activation remains appealing but challenging. Here, we design a cancer-motivated upconversion nanomachine (UCNM) based on porous upconversion nanoparticles (p-UCNPs) for precise phototherapy. The nanosystem is equipped with a telomerase substrate (TS) primer and simultaneously encapsulates 5-aminolevulinic acid (5-ALA) and d-arginine (d-Arg). After coating with hyaluronic acid (HA), it can readily get into tumor cells, where 5-ALA induces efficient accumulation of protoporphyrin IX (PpIX) via the inherent biosynthetic pathway, and the overexpressed telomerase prolonged the TS to form G-quadruplexes (G4) for binding the resulting PpIX as a nanomachine. This nanomachine can respond to near-infrared (NIR) light and promote the active singlet oxygen (1O2) production due to the efficiency of Förster resonance energy transfer (FRET) between p-UCNPs and PpIX. Intriguingly, such oxidative stress can oxidize d-Arg into nitric oxide (NO), which relieves the tumor hypoxia and in turn improves the phototherapy effect. This in situ assembly approach significantly enhances targeting in cancer therapy and might be of considerable clinical value.

Influenza A virus use of BinCARD1 to facilitate the binding of viral NP to importin α7 is counteracted by TBK1-p62 axis-mediated autophagy.

As a major component of the viral ribonucleoprotein (vRNP) complex in influenza A virus (IAV), nucleoprotein (NP) interacts with isoforms of importin α family members, leading to the import of itself  and vRNP complex into the nucleus, a process pivotal in the replication cycle of IAV. In this study, we found that BinCARD1, an isoform of Bcl10-interacting protein with CARD (BinCARD), was leveraged by IAV for efficient viral replication. BinCARD1 promoted the nuclear import of the vRNP complex and newly synthesized NP and thus enhanced vRNP complex activity. Moreover, we found that BinCARD1 interacted with NP to promote NP binding to importin α7, an adaptor in the host nuclear import pathway. However, we also found that BinCARD1 promoted RIG-I-mediated innate immune signaling by mediating Lys63-linked polyubiquitination of TRAF3, and that TBK1 appeared to degrade BinCARD1. We showed that BinCARD1 was polyubiquitinated at residue K103 through a Lys63 linkage, which was recognized by the TBK1-p62 axis for autophagic degradation. Overall, our data demonstrate that IAV leverages BinCARD1 as an important host factor that promotes viral replication, and two mechanisms in the host defense system are triggered-innate immune signaling and autophagic degradation-to mitigate the promoting effect of BinCARD1 on the life cycle of IAV.

A Single Amino Acid Residue R144 of SNX16 Affects Its Ability to Inhibit the Replication of Influenza A Virus.

Influenza A virus (IAV) is an important zoonotic pathogen, posing a severe burden for the health of both animals and humans. Many host factors are involved in the life cycle of IAV to regulate its replication. Herein, we identified sorting nexin-16 (SNX16) as a new host factor that negatively modulates the replication of IAV. When transiently overexpressed in cells, SNX16 appears to be expressed as two obvious bands. Mutagenesis analysis indicated that the amino acid residue R144 of SNX16 was responsible for its two-band expression phenotype. We found that the R144A mutation of SNX16 changed its cellular distribution in A549 cells and partially weakened the inhibitory effect of SNX16 on IAV replication. Further investigation revealed that SNX16 could negatively regulate the early stage of the replication cycle of IAV. Taken together, our results demonstrated that SNX16 is a novel restriction host factor for the replication of IAV by engaging in the early stage of IAV life cycle, and a single amino acid residue at position 144 plays an important role in the cellular distribution and anti-influenza function of SNX16.

PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence.

Host defense systems employ posttranslational modifications to protect against invading pathogens. Here, we found that protein inhibitor of activated STAT 1 (PIAS1) interacts with the nucleoprotein (NP), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) of influenza A virus (IAV). Lentiviral-mediated stable overexpression of PIAS1 dramatically suppressed the replication of IAV, whereas siRNA knockdown or CRISPR/Cas9 knockout of PIAS1 expression significantly increased virus growth. The expression of PIAS1 was significantly induced upon IAV infection in both cell culture and mice, and PIAS1 was involved in the overall increase in cellular SUMOylation induced by IAV infection. We found that PIAS1 inhibited the activity of the viral RNP complex, whereas the C351S or W372A mutant of PIAS1, which lacks the SUMO E3 ligase activity, lost the ability to suppress the activity of the viral RNP complex. Notably, the SUMO E3 ligase activity of PIAS1 catalyzed robust SUMOylation of PB2, but had no role in PB1 SUMOylation and a minimal role in NP SUMOylation. Moreover, PIAS1-mediated SUMOylation remarkably reduced the stability of IAV PB2. When tested in vivo, we found that the downregulation of Pias1 expression in mice enhanced the growth and virulence of IAV. Together, our findings define PIAS1 as a restriction factor for the replication and pathogenesis of IAV.

Optimization of microbial cell factories for astaxanthin production: Biosynthesis and regulations, engineering strategies and fermentation optimization strategies.

The global market demand for natural astaxanthin is rapidly increasing owing to its safety, the potential health benefits, and the diverse applications in food and pharmaceutical industries. The major native producers of natural astaxanthin on industrial scale are the alga Haematococcus pluvialis and the yeast Xanthopyllomyces dendrorhous. However, the natural production via these native producers is facing challenges of limited yield and high cost of cultivation and extraction. Alternatively, astaxanthin production via metabolically engineered non-native microbial cell factories such as Escherichia coli, Saccharomyces cerevisiae and Yarrowia lipolytica is another promising strategy to overcome these limitations. In this review we summarize the recent scientific and biotechnological progresses on astaxanthin biosynthetic pathways, transcriptional regulations, the interrelation with lipid metabolism, engineering strategies as well as fermentation process control in major native and non-native astaxanthin producers. These progresses illuminate the prospects of producing astaxanthin by microbial cell factories on industrial scale.

Genome-guided investigation of anti-inflammatory sesterterpenoids with 5-15 trans-fused ring system from phytopathogenic fungi.

Fungal terpenoids catalyzed by bifunctional terpene synthases (BFTSs) possess interesting bioactive and chemical properties. In this study, an integrated approach of genome mining, heterologous expression, and in vitro enzymatic activity assay was used, and these identified a unique BFTS sub-clade critical to the formation of a 5-15 trans-fused bicyclic sesterterpene preterpestacin I (1). The 5-15 bicyclic BFTS gene clusters were highly conserved but showed relatively wide phylogenetic distribution across several species of the diverged fungal classes Dothideomycetes and Sordariomycetes. Further genomic organization analysis of these homologous biosynthetic gene clusters from this clade revealed a glycosyltransferase from the graminaceous pathogen Bipolaris sorokiniana isolate BS11134, which was absent in other 5-15 bicyclic BFTS gene clusters. Targeted isolation guided by BFTS gene deletion led to the identification of two new sesterterpenoids (4, and 6) from BS11134. Compounds 2 and 4 showed moderate effects on LPS-induced nitrous oxide production in the murine macrophage-like cell line RAW264.7 with in vitro inhibition rates of 36.6 ± 2.4% and 24.9 ± 2.1% at 10 µM, respectively. The plausible biosynthetic pathway of these identified compounds was proposed as well. This work revealed that phytopathogenic fungi can serve as important sources of active terpenoids via systematic analysis of the genomic organization of BFTS biosynthetic gene clusters, their phylogenetic distribution in fungi, and cyclization properties of their metabolic products. KEY POINTS: • Genome mining of the first BFTS BGC harboring a glycosyltransferase. • Gene-deletion guided isolation revealed three novel 5-15 bicyclic sesterterpenoids. • Biosynthetic pathway of isolated sesterterpenoids was proposed.

The novel caprine parainfluenza virus type 3 showed pathogenicity in Guinea pigs.

Caprine parainfluenza virus type 3 (CPIV3) is one of the important viral respiratory tract agents in goats. The pathogenicity of CPIV3 has been examined in goats but it has not been explored in other laboratory animals. In the present study, an experimental infection of guinea pigs with CPIV3 was performed. The virus-inoculated guinea pigs displayed clinical signs related to the respiratory disease at 2-12 days post inoculation (dpi). Five infected guinea pigs died during 2 and 7 dpi. Apparent gross pneumonic lesions including consolidation and congestion in one or more lung lobes were observed in necropsied and dead animals. Histo-pathological changes in lungs including expansions of the alveolar interstitium, congestion, macrophage infiltration and compensatory emphysema were also observed. Virus was detectable at 2-10 dpi, 2-10 dpi and 2-7 dpi, as detected by virus isolation, real-time RT-PCR and immunohistochemistry staining, respectively. Viremia was also confirmed after CPIV3 infection during 3-7 dpi. The severe pathological lesions and highest viral load were observed before 7 dpi. Viral specific hemagglutination inhibition and neutralizing antibodies were produced from 7 dpi and 10 dpi, respectively, which related to the clearance of virus. The results present here indicated that guinea pig could be an ideal laboratory animal model for CPIV3 studies in the future.

Studies on the operative outcomes and mechanisms of microvascular decompression in treating typical and atypical trigeminal neuralgia.

OBJECTIVE: To evaluate the operative outcomes and mechanisms of microvascular decompression in treating typical and atypical trigeminal neuralgia. METHODS: A group of 45 patients with typical trigeminal neuralgia and 17 patients with atypical trigeminal neuralgia treated by micro-vascular decompression from 2000 to 2002 were reviewed, including their clinical presentations, operative findings, and outcomes. RESULTS: Of 45 patients with typical trigeminal neuralgia, the mean duration was 3.1 years, and the mean age of pain onset was 60.3 years. Single trigeminal division was involved in 20 patients (44.4%), and 2 or 3 divisions were involved in the other 25 patients (55.6%). During the operation, artery compression was found in 39 patients (86.7%), and the combined artery and venous compression was found in 6 patients (13.3%). Postoperatively, complete pain relief was achieved in 44 patients (97.8%), and significant pain relief was achieved in 1 patient (2.2%). As for 17 patients with atypical trigeminal neuralgia, the mean duration and the mean age of pain onset was 8.7 years and 55.5 years, respectively. Two or 3 trigeminal divisions were involved in all of these patients. During operation, artery compression occurred in 10 patients (58.8%), and the combined artery and venous compression was found in 7 patients (41.2%). Postoperatively, complete pain relief was achieved in 5 patients (29.4%), and partial pain relief was achieved in 10 patients (58.8%), and 2 patients showed no response to microvascular decompression. CONCLUSIONS: The operative outcome of microvascular decompression in patients with typical trigeminal neuralgia was better than that of patients with atypical trigeminal neuralgia, which perhaps related to short duration, late onset of pain, limited distribution, artery compression, and complete operative decompression.