Supramolecular Nanofibers Ameliorate Bleomycin-Induced Pulmonary Fibrosis by Restoring Autophagy.

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal interstitial lung disease, with limited therapeutic options available. Impaired autophagy resulting from aberrant TRB3/p62 protein-protein interactions (PPIs) contributes to the progression of IPF. Restoration of autophagy by modulating the TRB3/p62 PPIs has rarely been reported for the treatment of IPF. Herein, peptide nanofibers are developed that specifically bind to TRB3 protein and explored their potential as a therapeutic approach for IPF. By conjugating with the self-assembling fragment (Ac-GFFY), a TRB3-binding peptide motif A2 allows for the formation of nanofibers with a stable α-helix secondary structure. The resulting peptide (Ac-GFFY-A2) nanofibers exhibit specific high-affinity binding to TRB3 protein in saline buffer and better capacity of cellular uptake to A2 peptide. Furthermore, the TRB3-targeting peptide nanofibers efficiently interfere with the aberrant TRB3/p62 PPIs in activated fibroblasts and fibrotic lung tissue of mice, thereby restoring autophagy dysfunction. The TRB3-targeting peptide nanofibers inhibit myofibroblast differentiation, collagen production, and fibroblast migration in vitro is demonstrated, as well as bleomycin-induced pulmonary fibrosis in vivo. This study provides a supramolecular method to modulate PPIs and highlights a promising strategy for treating IPF diseases by restoring autophagy.

Enzyme-Instructed Peptide Assembly Favored by Preorganization for Cancer Cell Membrane Engineering.

Innovative methods for engineering cancer cell membranes promise to manipulate cell-cell interactions and boost cell-based cancer therapeutics. Here, we illustrate an in situ approach to selectively modify cancer cell membranes by employing an enzyme-instructed peptide self-assembly (EISA) strategy. Using three phosphopeptides (pY1, pY2, and pY3) targeting the membrane-bound epidermal growth factor receptor (EGFR) and differing in just one phosphorylated tyrosine, we reveal that site-specific phosphorylation patterns in pY1, pY2, and pY3 can distinctly command their preorganization levels, self-assembling kinetics, and spatial distributions of the resultant peptide assemblies in cellulo. Overall, pY1 is the most capable of producing preorganized assemblies and shows the fastest dephosphorylation reaction in the presence of alkaline phosphatase (ALP), as well as the highest binding affinity for EGFR after dephosphorylation. Consequently, pY1 exhibits the greatest capacity to construct stable peptide assemblies on cancer cell membranes with the assistance of both ALP and EGFR. We further use peptide-protein and peptide-peptide co-assembly strategies to apply two types of antigens, namely ovalbumin (OVA) protein and dinitrophenyl (DNP) hapten respectively, on cancer cell membranes. This study demonstrates a very useful technique for the in situ construction of membrane-bound peptide assemblies around cancer cells and implies a versatile strategy to artificially enrich cancer cell membrane components for potential cancer immunotherapy.

Enzyme-instructed and mitochondria-targeting peptide self-assembly to efficiently induce immunogenic cell death.

Immunogenic cell death (ICD) plays a major role in cancer immunotherapy by stimulating specific T cell responses and restoring the antitumor immune system. However, effective type II ICD inducers without biotoxicity are still very limited. Herein, a tentative drug- or photosensitizer-free strategy was developed by employing enzymatic self-assembly of the peptide F-pY-T to induce mitochondrial oxidative stress in cancer cells. Upon dephosphorylation catalyzed by alkaline phosphatase overexpressed on cancer cells, the peptide F-pY-T self-assembled to form nanoparticles, which were subsequently internalized. These affected the morphology of mitochondria and induced serious reactive oxygen species production, causing the ICD characterized by the release of danger-associated molecular patterns (DAMPs). DAMPs enhanced specific immune responses by promoting the maturation of DCs and the intratumoral infiltration of tumor-specific T cells to eradicate tumor cells. The dramatic immunotherapeutic capacity could be enhanced further by combination therapy of F-pY-T and anti-PD-L1 agents without visible biotoxicity in the main organs. Thus, our results revealed an alternative strategy to induce efficient ICD by physically promoting mitochondrial oxidative stress.

Can respondent uncertainty be ignored when exploring heterogeneity in public preferences for air pollution treatment policies? Comparative results from choice experiment analysis.

Based on choice experiment (CE), evaluating the public's heterogeneous preferences and willingness to pay (WTP) for air pollution treatment policies can provide useful social views for the reasonable formulation of treatment schemes. However, the application of CE contains an implicit assumption that respondents understand their real preferences and can make choices with complete certainty. In reality, for a variety of reasons, not all respondents are absolutely certain about their responses, this assumption distinctly is hard to be consistent with reality. To explore the impact of respondent uncertainty on the public's WTP and heterogeneous preferences for air pollution treatment policies, this study introduces the critical point and exponential weighting methods to deal with this uncertainty in the context of CE and conducts comparative analysis based on the random parameters logit (RPL) and latent classes models (LCM). The results show that, ignoring uncertainty leads to distortions in the public's WTP and preference characteristics. In the RPL models, on average, the WTP for attributes is overstated by 32.10%. Our results also reveal that, whether to consider uncertainty does not affect the ranking of the implicit prices of these attributes. After incorporating uncertainty into the analysis, respondents were divided into two potential groups with different preferences, namely the environment-focused group (79.44%) and the price-focused group (20.56%), which is quite distinct from research results of ignoring the uncertainty. Contribution of this study is not only to provide theoretical insights for exploring the effects of uncertainty on public preferences based on CE, but also to provide valuable guidance for policy makers to formulate more accurate and effective treatment measures.

Tandem molecular self-assembly for selective lung cancer therapy with an increase in efficiency by two orders of magnitude.

In situ self-assembly of prodrug molecules into nanomedicine can elevate the therapeutic efficacy of anticancer medications by enhancing the targeting and enrichment of anticancer drugs at tumor sites. However, the disassembly and biodegradation of nanomedicine after enrichment prevents the further improvement of the efficiency, and avoiding such disassembly and biodegradation remains a challenge. Herein, we rationally designed a tandem molecular self-assembling prodrug that could selectively improve the therapeutic efficacy of HCPT against lung cancer by two orders of magnitude. The tandem molecular self-assembly utilized an elevated level of alkaline phosphatase and reductase in lung cancer cells. The prodrug first self-assembled into nanofibers by alkaline phosphatase catalysis and was internalized more efficiently by lung cancer cells than free HCPT. The resulting nanofiber was next catalyzed by intracellular reductase to form a more hydrophobic nanofiber that prevented the disassembly and biodegradation, which further significantly improved the efficacy of HCPT against lung cancer both in vitro and in vivo.

Genome assembly and annotation at the chromosomal level of first Pleuronectidae: Verasper variegatus provides a basis for phylogenetic study of Pleuronectiformes.

High quality genome is of great significance for the mining of biological information resources of species. Up to now, the genomic information of several important economic flatfishes has been well explained. All these fishes are eyes on left side-type, and no high-quality genome of eyes on right side-type species has been reported. In this study, we applied a combined strategy involving stLFR and Hi-C technologies to generate sequencing data for constructing the chromosomal genome of Verasper variegates, which belongs to Pleuronectidae with characteristic of eyes on right side. The size of genome of V. variegatus is 556 Mb. More than 97.2% of BUSCO genes were detected, and N50 lengths of the contigs and scaffolds reached 79.8 Kb and 23.8 Mb, respectively, demonstrating the outstanding completeness and sequence continuity of the genome. A total of 22,199 protein-coding genes were predicted in the assembled genome, and more than 95% of those genes could be functionally annotated. Meanwhile, the genomic collinearity, gene family and phylogenetic analyses of similar species in Pleuronectiformes were also investigated and portrayed for metamorphosis and benthic adaptation. Sex related genes mapping has also been achieved at the chromosome level. This study is the first chromosomal level genome of a Pleuronectidae fish (V. variegatus). The chromosomal genome assembly constructed in this work will not only be valuable for conservation and aquaculture studies of the V. variegatus but will also be of general interest in the phylogenetic and taxonomic studies of Pleuronectiformes.

Children with Obesity Experience Different Age-Related Changes in Plantar Pressure Distributions: A Follow-Up Study in China.

Age is a key factor in plantar pressure distributions during the development of obese children. However, the existing evidence for age-related plantar pressures of obese children is not sufficient to make clear how the plantar pressures would change with the increasing age. This study aimed to evaluate the plantar pressure redistributions of obese children after a three-year follow-up and to further compare these changes with normal-weighted children. Ten obese children and eleven normal-weighted counterparts were involved in this study. Plantar pressure measurements were undertaken using a Footscan® plantar pressure plate on two test sessions three years apart. Peak pressure, pressure-time integral, standard maximum force, and z-scores of these variables were analyzed. Loading transference analyses were applied to detect the different loading transferring mechanisms between obese and normal-weighted children. Significantly increased plantar pressures were observed at the lateral forefoot and midfoot for obese children, which gradually deviated from those of normal-weighted children over the 3 years. With the increasing age, obese children displayed a lateral loading shift at the forefoot in contrast to the normal-weighted. Early interventions are cautiously recommended for obese children before the plantar loading deviation gets worse as they grow older.

Nuclear delivery of dual anticancer drug-based nanomedicine constructed by cisplatinum-induced peptide self-assembly.

Nuclear delivery of anticancer drugs, particularly dual complementary anticancer drugs, can significantly improve chemotherapy efficacy. However, successful examples are rare. We reported a novel dual anticancer drug-based nanomedicine with nuclear accumulation properties. The nanomedicine was formed by chelation between a drug peptide amphiphile Rh-GFFYERGD (Rh represents Rhein, 1,8-dihydroxy-3-carboxy anthraquinonea) and cisplatinum (Pt). A single molecule of the drug peptide amphiphile could chelate up to 8 equiv. of cisplatinum in the resulting nanofibers. The nanofibers with a 1 : 4 ratio of Rh-GFFYERGD to cisplatinum demonstrated remarkable cellular uptake, and more significantly, superior nuclear accumulation properties. Additionally, the nanofibers could also bind to the DNA molecule more efficiently than those formed by the drug peptide amphiphile. Thus the nanofibers exhibited excellent anticancer properties both in vitro and in vivo. We envision a significant therapeutic potential of the dual anticancer drug-based nanomedicine with cisplatinum in cancer.

Preorganization Increases the Self-Assembling Ability and Antitumor Efficacy of Peptide Nanomedicine.

Inspired by the biological process of phosphorylation for which different sites of the same protein may have different activities and functions, we utilized phosphatase-based enzyme-instructed self-assembly (EISA) to construct self-assembled nanomedicine from the precursors with different phosphorylated sites. We found that, although the obtained self-assembling molecules after EISA were identical, the changes of EISA catalytic sites could determine the outcome of molecular self-assembly. The precursor with the phosphorylated site in the middle preorganized before EISA, while the ones with other phosphorylated sites could not preorganize before EISA. After EISA, the preorganized precursor then resulted in more stable and ordered assemblies than those of the others, which showed increased cellular uptake and up to 1.7-fold higher efficacy in an antitumor therapeutic compared to those assembled from unorganized precursors.

Tandem Molecular Self-Assembly Selectively Inhibits Lung Cancer Cells by Inducing Endoplasmic Reticulum Stress.

The selective formation of nanomaterials in cancer cells and tumors holds great promise for cancer diagnostics and therapy. Until now, most strategies rely on a single trigger to control the formation of nanomaterials in situ. The combination of two or more triggers may provide for more sophisticated means of manipulation. In this study, we rationally designed a molecule (Comp. 1) capable of responding to two enzymes, alkaline phosphatase (ALP), and reductase. Since the A549 lung cancer cell line showed elevated levels of extracellular ALP and intracellular reductase, we demonstrated that Comp. 1 responded in a stepwise fashion to those two enzymes and displayed a tandem molecular self-assembly behavior. The selective formation of nanofibers in the mitochondria of the lung cancer cells led to the disruption of the mitochondrial membrane, resulting in an increased level of reactive oxygen species (ROS) and the release of cytochrome C (Cyt C). ROS can react with proteins, resulting in endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This severe ER stress led to disruption of the ER, formation of vacuoles, and ultimately, apoptosis of the A549 cells. Therefore, Comp. 1 could selectively inhibit lung cancer cells in vitro and A549 xenograft tumors in vivo. Our study provides a novel strategy for the selective formation of nanomaterials in lung cancer cells, which is powerful and promising for the diagnosis and treatment of lung cancer.

Responsive peptide-based supramolecular hydrogels constructed by self-immolative chemistry.

Peptide-based supramolecular hydrogels that are stimuli-responsive under aqueous conditions have many potential biological applications, including drug delivery and sensing. Herein, we reported a series of responsive peptide-based supramolecular hydrogels that respond to glutathione (GSH), nitric oxide (NO) and hydrogen sulfide (H2S), which are biologically important signaling molecules. The responsive hydrogelators were designed by "self-immolative" chemistry and constructed by using self-immolative groups to modify short peptides. The self-immolative capping group could be removed in the presence of a corresponding trigger, thus causing gel-sol phase transitions. The potential of our responsive hydrogels for drug release was also demonstrated in this study. Our study offered several candidates of responsive hydrogels for sensing and drug delivery.

Enzyme-assisted peptide folding, assembly and anti-cancer properties.

The α-helix is the most prevalent conformation in proteins. However, formation of the α-helical conformation remains a challenge for short peptides with less than 5 amino acids. We demonstrated in this study that enzyme-instructed self-assembly (EISA) provides a unique pathway to assist the self-assembly of peptides into the α-helical conformation, while a heating-cooling process leads to a conformation more similar to a ß-sheet. The same peptide with different conformations self-assembled into different nanostructures. The peptide with α-helical conformation self-assembled into stable nanofibers and hydrogels, while the other one assembled into an unstable nanoparticle suspension. The nanofiber solution exhibited better stability against proteinase K digestion and an enhanced cellular uptake compared to the nanoparticle solution. Therefore, the nanomedicine formed by the α-helical peptide showed a better inhibition capacity against cancer cells in vitro and significantly inhibited tumor growth in vivo compared to the one formed by the ß-sheet peptide. Our study demonstrates the unique advantages of EISA to assist peptide folding and self-assembly into biofunctional nanomaterials.

Molecular cloning, characterization and recombinant expression of crustacean hyperglycemic hormone in white shrimp Litopenaeus vannamei.

Crustacean hyperglycemic hormone (CHH) plays an important role in crustacean. In the present study, a full-length cDNA of CHH was cloned from the eyestalk of Litopenaeus vannamei by RACE approach for the first time. The full-length cDNA of LvCHH was 846 bp, containing a 5' untranslated region (UTR) of 65 bp, a 3' UTR of 436 bp with a canonical polyadenylation signal-sequence AATAA and a poly (A) tail, and an open reading frame (ORF) of 345 bp. The ORF encoded a polypeptide of 114 amino acids including a 24 amino acid signal peptide. The calculated molecular mass of the mature protein (74 amino acids) was 8.76 kDa with an estimated pI of 6.78. The sequence of LvCHH was submitted in NCBI GenBank under the accession number HM748790.2. Phylogenetic analysis revealed that LvCHH was clustered with CHH of other crustaceans. Tissue distribution analysis revealed that the expression of LvCHH mRNA was observed in all tissues but gill, and was highest in heart. Specific primers containing Xho I and BamH I restriction sites respectively, were designed based on the obtained ORF sequence of LvCHH gene and the cloning sites of expression vector pET-32a (+). The recombinant plasmid LvCHH-pET32a, was used to transform Escherichia coli BL21 (DE3). LvCHH was successfully expressed by means of SDS-PAGE and western blot analysis. We detected gill Na(+)/K(+)-ATPase activity after rLvCHH protein injection and found that All the experimental group Na(+)/K(+)-ATPase activity presented peak change among 0-6h, and the peaks of all treated groups occurred in 1 h. 20 and 30 µg/shrimp(-1) groups showed significant increase (P<0.05) in 1h post-injection. L. vannamei were exposed for 96h to hypo- and hyper-salinity challenge. Hypo-salinity caused a significant rise (P<0.05) in the mRNA expression of CHH and gill Na(+)/K(+)-ATPase activity at 12h and 24h respectively, then the CHH mRNA expression declining by 24h, and returned to control group level by 48 h, and the Na(+)/K(+)-ATPase activity tended to be stable after 72 h, and higher than that of control. The hyper-salinity challenge had the same trend at mRNA expression with the hypo-salinity group. The Na(+)/K(+)-ATPase activity had no significant change under the low salinity challenge. All these results indicate that LvCHH is an important hormone involved in the osmosis responses of swimming shrimps, and can provide further information of crustacean osmoregulation physiological mechanism.

Immune responses and expression of immune-related genes in swimming crab Portunus trituberculatus exposed to elevated ambient ammonia-N stress.

The effects of ammonia-N (0, 1, 5 and 20mgL(-1)) on immune responses and immune-related gene expression were determined in swimming crab Portunus trituberculatus. The results showed that the THC and phagocytic activity of P. trituberculatus exposed to 1, 5 and 20mgL(-1) ammonia-N decreased significantly during the experimental time. The antibacterial and bacteriolytic activities decreased significantly to the minimum at 6 or 12h respectively, then recovered to the control level except bacteriolytic activity exposed to 5 and 20mgL(-1) ammonia-N. alpha(2)-macroglobulin (alpha(2)-M) activity of all ammonia-N exposure groups decreased significantly, then recovered to the control level rapidly and tended to be stable after 12h. With crabs exposed to 20mgL(-1) ammonia-N, the gene expression levels of crustin and lysozyme decreased significantly, then recovered to the control level after 12h. ALF expression also decreased significantly when exposed to 5 and 20mgL(-1) ammonia-N, then remained stable and significantly lower than the control group after 6h. In contrast, alpha(2)-M gene expression was induced by ammonia-N exposure significantly. The results suggest that high concentration ammonia-N exposure could reduce the crab immunity severely, and induce the short-term response in terms of immune gene regulation.

Effects of injection of biogenic amines on expression of gill related ion transporter mRNA and alpha-subunit protein in Litopenaeus vannamei.

We used semi-quantitative reverse transcription and the polymerase chain reaction (RT-PCR) to identify and characterize the mRNA of Na(+)/K(+)-ATPase alpha-subunit and Vacuolar-type H/-ATPase (V-ATPase) beta-subunit prepared from gills of the white shrimp Litopenaeus vannamei. Western blot and immunocytochemistry analysis were employeed to investigate the effects of biogenic amines such as dopamine (DA) and 5-hydroxytryptamine (5-HT) injection on expression of protein abundance and immunostained sites. Our results show that the gill Na(+)/K(+)-ATPase of 5-HT treatments increased lag to those of DA treatments. However, the DA and 5-HT have no significant effects on the activity of V-ATPase. The biogenic amines could induce the synthesis of Na(+)/K(+)-ATPase alpha-subunit mRNA in the first 12 h. The degree of change in Na(+)/K(+)-ATPase alpha-subunit mRNA expression were different between DA and 5-HT treated groups which depend on the dose. The expression of V-ATPase beta-subunit mRNA was not markedly affected by the biogenic amines. Concomitant elevation of Na(+)/K(+)-ATPase activity, alpha-subunit mRNA as well as protein abundance in response to injected biogenic amines was also found in Na(+)/K(+)-ATPase immunostained sites within the gill epithelium.

Injection of biogenic amines modulates osmoregulation of Litopenaeus vannamei: response of hemolymph osmotic pressure, ion concentration and osmolality effectors.

In this paper, we compared systematically the temporal and dose response relationship and physiological significance among biogenic amines injection, changes of ion concentration, FAA concentrations and composition and protein in context of osmoregulatory ability in marine euryhaline shrimp: Litopenaeus vannamei. The dopamine (DA) and 5-hydroxytryptamine (5-HT) injection all had transient effects on hemolymph osmolality, ion concentrations but which occurred in different time and were dose-dependent. The highest concentrations of FAAs in hemolymph of L. vannamei were alanine, glycine, argnine, proline, lysine which were considered to be specific osmotic effectors. Contrary to the reduction of hemocyanin, injection of DA 10(-6) mol shrimp(-1) and 5-HT 10(-6) mol shrimp(-1) induced notable protein increase respectively, which led to the rapid reduction of hemocyanin/protein ratio in range of 63.2% to 78.3%. The increase of hemolymph FAAs might come from the new amino acid synthesis or degradation of muscle protein to FAAs or denovo synthesis of FAAs. Our study showed that dopamine plays an important role in neurotransmission and causes osmoregulation response modulation and 5-HT has different activation mechanism on osmoregulation.