The quality evaluation of 30 Asparagus officinalis L. varieties.

Asparagus, a vital economic contributor, is a well-liked vegetable grown around the globe, and some secondary metabolites in its spear are beneficial to human health. Asparagus spears possess a significant quantity of nutrients and phytochemicals; however, the difference in these chemical compositions among various varieties has not been sufficiently studied. This work aimed to detect the chemical compositions of 30 varieties of asparagus and to assess them by principal component analysis (PCA). The results showed that the contents of these chemical compositions varied in varieties. Selenium (Se, 1.12-2.9 µg/100 g dry-weight [DW]) was abundant in asparagus, with an average dry matter content of 8.25%. Free amino acids (5.60-9.98 g/100 g DW) and polyphenols (6.34-8.67 mg/g DW) were both present in high amounts, along with flavonoids (4.218-8.22 mg/g DW) and protodioscin (0.44-1.96 mg/g DW). Correlation analysis, PCA, and hierarchical cluster analysis were used to conduct a comprehensive evaluation of asparagus. Atlas, Appolo, Jinggang 111, Jingke 2, and WS-1 were the top five varieties with comprehensive scores. This study provided valuable data for the breeding, quality improvement, processing, and utilization of asparagus varieties in the future.

Silencing gram-negative bacteria binding protein 1 decreases the immunity of Tribolium castaneum against bacteria.

Gram-negative bacteria binding proteins (GNBPs) have the ability to recognize molecular patterns associated with microbial pathogens (PAMPs), leading to the activation of immune responses downstream. In the genome of Tribolium castaneum, three GNBP genes have been identified; however, their immunological roles remain unexplored. In our study, a GNBP1, designated as TcGNBP1, were identified from the cDNA library of T. castaneum. The coding sequence of TcGNBP1 consisted of 1137 bps and resulted in the synthesis of a protein comprising 378 amino acids. This protein encompasses a signal peptide, a low-complexity region, and a glycoside hydrolase 16 domain. TcGNBP1 was strongly expressed in early adult stages, and mainly distributed in hemolymph and gut. Upon being challenged with Escherichia coli or Staphylococcus aureus, the transcript levels of TcGNBP1 were significantly changed at different time points. Through molecular docking and ELISA analysis, it was observed that TcGNBP1 has the ability to interact with lipopolysaccharides, peptidoglycan, and ß-1, 3-glucan. Based on these findings, it was further discovered that recombinant TcGNBP1 can directly bind to five different bacteria in a Ca2+-dependent manner. After knockdown of TcGNBP1 with RNA interference, expression of antimicrobial peptide genes and prophenoloxidase (proPO) activity were suppressed, the susceptibility of T. castaneum to E. coli or S. aureus infection was enhanced, leading to low survival rate. These results suggest a regulatory mechanism of TcGNBP1 in innate immunity of T. castaneum and provide a potential molecular target for dsRNA-based insect pest management.

Development of an analytic method for organosulfur compounds in Welsh onion and its use for nutritional quality analysis of five typical varieties in China.

A reliable, simple, and sensitive method capable of quantifying six organosulfur compounds (OSCs) was established. The samples were extracted by water containing 3 % formic acid with a simple vortex, ultrasound, and centrifugation step, and the solutions were analyzed by ultra-high-performance liquid chromatography separation system coupled with a triple-quadrupole mass spectrometry (UHPLC - MS/MS). Then the method was applied for the analysis of six OSCs in five varieties of two types Welsh onions in China, and the moisture content, reducing sugar, total polyphenols, and 21 free amino acids were also analyzed to study the characters of these Welsh onions intensively. Multivariate statistical analysis was used to investigate the differences in OSCs and free amino acids profiles among the samples. This study showed that enzymatic inhibition method combined with UHPLC - MS/MS is an effective technique to analyze OSCs in Welsh onion, and could be valuable for the routine quantitation of OSCs in other foods.

Harnessing Liquid Crystal Sensors for High-Throughput Real-Time Detection of Structural Changes in Lysozyme during Refolding Processes.

Despite the rapid advances in process analytical technology, the assessment of protein refolding efficiency has largely relied on off-line protein-specific assays and/or chromatographic procedures such as reversed-phase high-performance liquid chromatography and size exclusion chromatography. Due to the inherent time gap pertaining to traditional methods, exploring optimum refolding conditions for many recombinant proteins, often expressed as insoluble inclusion bodies, has proven challenging. The present study describes a novel protein refolding sensor that utilizes liquid crystals (LCs) to discriminate varying protein structures during unfolding and refolding. An LC layer containing 4-cyano-4'-pentylbiphenyl (5CB) intercalated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is used as a sensing platform, and its proof-of-concept performance is demonstrated using lysozyme as a model protein. As proteins unfold or refold, a local charge fluctuation at their surfaces modulates their interaction with zwitterionic phospholipid DOPE. This alters the alignment of DOPE molecules at the aqueous/LC interface, affecting the orientational ordering of bulk LC (i.e., homeotropic to planar for refolding and planar to homeotropic for unfolding). Differential polarized optical microscope images of the LC layer are subsequently generated, whose brightness directly linked to conformational changes of lysozyme molecules is quantified by gray scale analysis. Importantly, our LC-based refolding sensor is compatible with diverse refolding milieus for real-time analysis of lysozyme refolding and thus likely to facilitate the refolding studies of many proteins, especially those lacking a method to determine structure-dependent biological activity.

Study of self-assembling properties of HBc-VLP derivatives aided by molecular dynamic simulations from a thermodynamic perspective.

Self-assembling protein nanoparticles showed promise for vaccine design due to efficient antigen presentations and safety. However, the unpredictable formations of epitopes-fused protein assemblies remain challenging in the upstream design. This study suggests employing molecular dynamic (MD) simulations to investigate the assembly properties of Hepatitis B core protein (HBc) from thermodynamic perspectives. Eight HBc derivatives were expressed in E. coli, with their self-assembly properties characterised by high-performance liquid chromatography and transmission electron microscopy. MD simulations on the dimers, based on AlphaFold-predicted 3D structures, analysed the derivative at the atomic level. Results revealed that HBc derivatives can form dissociative polymers or large multi-subunit structures due to assembly failures. The instability of the dimer in aqueous solvents or inappropriate intradimer distances could cause major assembly failures. Polar solvation energies played a vital role too in forming assemble-incompetent dimers. Importantly, our study demonstrated that MD simulations on dimers can provide preliminary predictions on the assembly properties of HBc derivatives, thus aiding vaccine design by lowering the risk of self-assembling failures in engineered proteins.Communicated by Ramaswamy H. Sarma.

Latrophilin, an adhesion GPCR with galactose-binding lectin domain involved in the innate immune response of Tribolium castaneum.

Latrophilin is a member of adhesion GPCRs involved in various physiological pro1cesses. The extracellular fragment of Tribolium castaneum Latrophilin (TcLph) contains a galactose-binding lectin (GBL) domain. However, the biological function of GBL domain remains mysterious. Here, we initially studied the role of TcLph in recognizing pathogens through its GBL domain and then triggering immune defense in invertebrates. Results showed that GBL domain was highly conserved, and its predicted 3D structure was similar to rhamnose-binding lectin domain of mouse Latrophilin-1 with a unique α/ß fold and two long loops. Molecular docking and ELISA results revealed the GBL domain can bind to D-galactose, L-rhamnose, lipopolysaccharide and peptidoglycan. The recombinant extracellular segment of TcLph and the recombinant GBL exhibited strong agglutinating and binding activities to all tested bacteria in a Ca2+-dependent manner. Moreover, TcLph was markedly induced after infection by Escherichia coli or Staphylococcus aureus, while its silencing exacerbated bacterial loads and larvae mortality. TcLph-deficient larvae significantly decreased the transcription levels of antimicrobial peptides and prophenoloxidase activating system-related genes, leading to a significant reduction in phenoloxidase activity. It indicated that TcLph functioned as a pattern recognition receptor in pathogen recognition and activated immune responses to eliminate invasive microbes, suggesting a potential target for insecticides.

Identification of two galectin-4 proteins (PcGal4-L and PcGal4-L-CRD) and their function in AMP expression in Procambarus clarkii.

Galectins, a family of lectins that bind to ß-galactoside, possess conserved carbohydrate recognition domains (CRDs) and play a crucial role in recognizing and eliminating pathogens in invertebrates. Two galectin-4 genes (PcGal4) isoforms, named PcGal4-L and PcGal4-L-CRD, were cloned from the cDNA library of Procambarus clarkia in our study. PcGal4-L contains an open reading frame (ORF, 1089 bp), which encodes a protein consisting of 362 amino acids including a single CRD and six low complexity regions. The full-length cDNA of PcGal4-L-CRD contains a 483 bp ORF that encodes a protein of 160 amino acids, with a single CRD and a low-complexity region. The difference between the two PcGal4 isoforms is that PcGal4-L has 202 additional amino acids after the CRD compared to the PcGal4-L-CRD. These two isoforms are grouped together with other galectins from crustaceans through phylogenetic analysis. Further study revealed that total PcGal4 (including PcGal4-L and PcGal4-L-CRD) was primarily expressed in the muscle, gills and intestine. The mRNA levels of total PcGal4 in gills and hemocytes were significantly induced after challenge with Aeromonas hydrophila. Both recombinant PcGal4-L and its spliced isoform, PcGal4-L-CRD, could directly bind to lipopolysaccharides, peptidoglycan and five tested microorganisms, inducing a wide spectrum of microbial agglutination. The spliced isoform PcGal4-L-CRD showed a stronger binding ability than PcGal4-L. In addition, when the PcGal4 was knockdown, transcriptions of seven antimicrobial peptides (AMPs) genes (ALF5, ALF6, ALF8, CRU1, CRU2, CRU3 and CRU4) in gills and seven AMPs genes (ALF5, ALF6, ALF8, ALF9, CRU1, CRU3 and CRU4) in hemocytes were significantly decreased. Meanwhile, the survival rate of P. clarkii decreased in the PcGal4-dsRNA group. In summary, these results indicate that PcGal4 can mediate the innate immunity in P. clarkii by bacterial recognition and agglutination, as well as regulating AMP expression, thus recognition and understanding of the functions of galectin in crustaceans in immune resistance.

Functional Analysis of a CTL-X-Type Lectin CTL16 in Development and Innate Immunity of Tribolium castaneum.

C-type lectins (CTLs) are a class of proteins containing carbohydrate recognition domains (CRDs), which are characteristic modules that recognize various glycoconjugates and function primarily in immunity. CTLs have been reported to affect growth and development and positively regulate innate immunity in Tribolium castaneum. However, the regulatory mechanisms of TcCTL16 proteins are still unclear. Here, spatiotemporal analyses displayed that TcCTL16 was highly expressed in late pupae and early adults. TcCTL16 RNA interference in early larvae shortened their body length and narrowed their body width, leading to the death of 98% of the larvae in the pupal stage. Further analysis found that the expression level of muscle-regulation-related genes, including cut, vestigial, erect wing, apterous, and spalt major, and muscle-composition-related genes, including Myosin heavy chain and Myosin light chain, were obviously down-regulated after TcCTL16 silencing in T. castaneum. In addition, the transcription of TcCTL16 was mainly distributed in the hemolymph. TcCTL16 was significantly upregulated after challenges with lipopolysaccharides, peptidoglycans, Escherichia coli, and Staphylococcus aureus. Recombinant CRDs of TcCTL16 bind directly to the tested bacteria (except Bacillus subtilis); they also induce extensive bacterial agglutination in the presence of Ca2+. On the contrary, after TcCTL16 silencing in the late larval stage, T. castaneum were able to develop normally. Moreover, the transcript levels of seven antimicrobial peptide genes (attacin2, defensins1, defensins2, coleoptericin1, coleoptericin2, cecropins2, and cecropins3) and one transcription factor gene (relish) were significantly increased under E. coli challenge and led to an increased survival rate of T. castaneum when infected with S. aureus or E. coli, suggesting that TcCTL16 deficiency could be compensated for by increasing AMP expression via the IMD pathways in T. castaneum. In conclusion, this study found that TcCTL16 could be involved in developmental regulation in early larvae and compensate for the loss of CTL function by regulating the expression of AMPs in late larvae, thus laying a solid foundation for further studies on T. castaneum CTLs.

Partner of neuropeptide bursicon homodimer pburs mediates a novel antimicrobial peptide Ten3LP via Dif/Dorsal2 in Tribolium castaneum.

Bursicon is a cystine knot family neuropeptide, composed of two subunits, bursicon (burs) and partner of burs (pburs). The subunits can form heterodimers to regulate cuticle tanning and wing maturation and homodimers to signal different biological functions in innate immunity, midgut stem cell proliferation and energy homeostasis, and reproductive physiology in the model insects Drosophila melanogaster or Tribolium castaneum. Here, we report on the role of the pburs homodimer in signaling innate immunity in T. castaneum larvae. Through transcriptome analysis we identified a set of immune-related genes that respond to pburs RNAi. Treating larvae with recombinant-pburs protein led to up-regulation of antimicrobial peptide (AMP) genes in vivo and in vitro. The upregulation of most AMP genes was dependent on the NF-κB transcription factor Relish. Most importantly, we identified a novel AMP, Tenecin 3-like peptide (Ten3LP), regulated by pburs via NF-κB transcription factor Dorsal-related immunity factor (Dif)/Dorsal2, but not Relish. We conducted Ten3LP RNAi, synthesized recombinant Ten3LP protein for microbial inhibition assays and functionally characterized Ten3LP as an AMP specific for fungi and Gram-positive bacteria. We demonstrate that expression of Ten3LP is activated by pburs via the Toll pathway. These findings identify new molecular targets for development of potential antibiotics for treating microbial infections and perhaps for RNAi based pest management technology.

Functional Analysis of a Multiple-Domain CTL15 in the Innate Immunity, Eclosion, and Reproduction of Tribolium castaneum.

C-type lectin X (CTL-X) plays critical roles in immune defense, cell adhesion, and developmental regulation. Here, a transmembrane CTL-X of Tribolium castaneum, TcCTL15, with multiple domains was characterized. It was highly expressed in the early and late pupae and early adults and was distributed in all examined tissues. In addition, its expression levels were significantly induced after being challenged with pathogen-associated molecular patterns (PAMPs) and bacteria. In vitro, the recombinant TcCTL15 could recognize bacteria through binding PAMPs and exhibit agglutinating activity against a narrow range of bacteria in the presence of Ca2+. RNAi-mediated TcCTL15-knockdown-larvae infected with Escherichia coli and Staphylococcus aureus showed less survival, had activated immune signaling pathways, and induced the expression of antimicrobial peptide genes. Moreover, silencing TcCTL15 caused eclosion defects by impairing ecdysone and crustacean cardioactive peptide receptors (CCAPRs). Suppression of TcCTL15 in female adults led to defects in ovary development and fecundity, accompanied by concomitant reductions in the mRNA levels of vitellogenin (TcVg) and farnesol dehydrogenase (TcFDH). These findings imply that TcCTL15 has extensive functions in developmental regulation and antibacterial immunity. Uncovering the function of TcCTL15 will enrich the understanding of CTL-X in invertebrates. Its multiple biological functions endow the potential to be an attractive target for pest control.

2D Phthalocyanine-Assembled Porous Nanostructure-Based Electrochemical Platform for High-Efficiency Detection of Ascorbic Acid.

In this work, a novel two-dimensional (2D) porous nanostructure is constructed upon air/water interfacial assembly of 12-crown-ether-4-incorporated double-decker phthalocyanine (Pc2). The combination of the good electroconductivity of phthalocyanine and the great surface area of the porous structure endows the assembled film with excellent chemical sensing property for ascorbic acid (AA). The low limit of detection can be 0.15 µM with a large linear concentration range and strong anti-interfering ability, which can be comparable to the best results of tetrapyrrole-based electrochemical sensors for AA. Furthermore, the obtained 2D porous assembled film sensor can be applied in real-time monitoring of AA in commercial drinks, indicating its application potential in accurate detection of AA in real samples.

Full-length transcriptome sequencing and comparative transcriptome analysis of Eriocheir sinensis in response to infection by the microsporidian Hepatospora eriocheir.

As a new generation of high-throughput sequencing technology, PacBio Iso-Seq technology (Iso-Seq) provides a better alternative sequencing method for the acquisition of full-length unigenes. In this study, a total of 22.27 gigabyte (Gb) subread bases and 128,614 non-redundant unigenes (mean length: 2,324 bp) were obtained from six main tissues of Eriocheir sinensis including the heart, nerve, intestine, muscle, gills and hepatopancreas. In addition, 74,732 unigenes were mapped to at least one of the following databases: Non-Redundant Protein Sequence Database (NR), Gene Ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG), KEGG Orthology (KO) and Protein family (Pfam). In addition, 6696 transcription factors (TFs), 28,458 long non-coding RNAs (lncRNAs) and 94,230 mRNA-miRNA pairs were identified. Hepatospora eriocheir is the primary pathogen of E. sinensis and can cause hepatopancreatic necrosis disease (HPND); the intestine is the main target tissue. Here, we attempted to identify the key genes related to H. eriocheir infection in the intestines of E. sinensis. By combining Iso-Seq and Illumina RNA-seq analysis, we identified a total of 12,708 differentially expressed unigenes (DEUs; 6,696 upregulated and 6,012 downregulated) in the crab intestine following infection with H. eriocheir. Based on the biological analysis of these DEUs, several key processes were identified, including energy metabolism-related pathways, cell apoptosis and innate immune-related pathways. Twelve selected genes from these DEUs were subsequently verified by quantitative real-time PCR (qRT-PCR) analysis. Our findings enhance our understanding of the E. sinensis transcriptome and the specific association between E. sinensis and H. eriocheir infection.

Distinct Quality Changes of Asparagus during Growth by Widely Targeted Metabolomics Analysis.

Asparagus is a popular vegetable and traditional medicine consumed worldwide due to its health benefits. The quality of asparagus, mainly attributed to small components like flavonoids and steroid, is quite differential as a result of different environments and maturities. However, the accumulation pattern and regulatory mechanism of metabolites in asparagus remain unclear so far. Herein, widely targeted metabolomics analysis was employed to study the quality and chemical composition variances of four asparagus, including three green asparagus of different maturities and one white asparagus. A total of 1045 metabolites were annotated in asparagus in which flavonoids and phenolic acids accounted for 37.51% of the total. Green asparagus was found to be rich in flavonoids, while white asparagus contained more steroids. Additionally, 461 biomarkers were screened between matured green and white asparagus, which is much more than that filtered among three green asparagus at different growth stages. These results indicated that sunlight has a stronger effect on the metabolism of asparagus compared to the general development of asparagus. Linoleic acid metabolism and alpha-linolenic acid metabolism were active during green asparagus growth, while flavone and flavonol biosynthesis and flavonoid biosynthesis resulted as two of the most important pathways when asparagus was exposed to sunlight.

The TGF-ß Receptor Gene Saxophone Influences Larval-Pupal-Adult Development in Tribolium castaneum.

The transforming growth factor-ß (TGF-ß) superfamily encodes a large group of proteins, including TGF-ß isoforms, bone morphogenetic proteins and activins that act through conserved cell-surface receptors and signaling co-receptors. TGF-ß signaling in insects controls physiological events, including growth, development, diapause, caste determination and metamorphosis. In this study, we used the red flour beetle, Tribolium castaneum, as a model species to investigate the role of the type I TGF-ß receptor, saxophone (Sax), in mediating development. Developmental and tissue-specific expression profiles indicated Sax is constitutively expressed during development with lower expression in 19- and 20-day (6th instar) larvae. RNAi knockdown of Sax in 19-day larvae prolonged developmental duration from larvae to pupae and significantly decreased pupation and adult eclosion in a dose-dependent manner. At 50 ng dsSax/larva, Sax knockdown led to an 84.4% pupation rate and 46.3% adult emergence rate. At 100 ng and 200 ng dsSax/larva, pupation was down to 75.6% and 50%, respectively, with 0% adult emergence following treatments with both doses. These phenotypes were similar to those following knockdowns of 20-hydroxyecdysone (20E) receptor genes, ecdysone receptor (EcR) or ultraspiracle protein (USP). Expression of 20E biosynthesis genes disembodied and spookier, 20E receptor genes EcR and USP, and 20E downstream genes BrC and E75, were suppressed after the Sax knockdown. Topical application of 20E on larvae treated with dsSax partially rescued the dsSax-driven defects. We can infer that the TGF-ß receptor gene Sax influences larval-pupal-adult development via 20E signaling in T. castaneum.

Eriocheir sinensis vesicle-associated membrane protein can enhance host cell phagocytosis to resist Spiroplasma eriocheiris infection.

Vesicle-associated membrane protein (VAMP) belongs to the receptor protein on the membrane of the secretory transport vesicle and involves in host immune function. The intracellular pathogen Spiroplasma eriocheiris could cause Eriocheir sinensis tremor disease. In a previous study, it was found E. sinensis VAMP (EsVAMP) was differently expressed in S. eriocheiris infection by proteomics analysis. This study mainly aims at the function of EsVAMP in the process of the S. eriocheiris infection. The length of EsVAMP gene was 1681 bp, which contained a 395 bp open reading frame, 90 bp 5'-non-coding region (UTR) and 1277 bp 3'-UTR. The results of qPCR showed that EsVAMP was expressed highly in hemocytes and nerves, followed by gills, intestines and hepatopancreas, and lowly expressed in heart and muscles. EsVAMP in hemocytes was up-regulated after S. eriocheiris infection. After EsVAMP over-expression and S. eriocheiris infection, the RAW264.7 cell morphology and cell viability of the experiment group were significantly better than the control group. Meanwhile, the copy number of S. eriocheiris in the experiment group was significantly lower than that in the control group. After EsVAMP and pCMV-Cre-mCherry were ligated and transfected into RAW264.7 cells, it was found that EsVAMP and lysosome co-localized. Meanwhile, the phagocytosed inactivated S. eriocheiris number and phagocytosed efficiency in RAW264.7 cells were increased significantly. The interference experiment was carried out by synthesizing EsVAMP dsRNA to verify that the EsVAMP transcriptions were successfully suppressed. The S. eriocheiris copy number and the mortality of crab increased significantly after EsVAMP RNAi and S. eriocheiris infection. Meanwhile, the phagocytosed inactivated S. eriocheiris number and phagocytosed efficiency in hemocytes decreased significantly after EsVAMP RNAi and S. eriocheiris infection. These results showed that VAMP was involved in the cell phagocytosis to resist pathogen infection.

Molybdenum-iron-cobalt oxyhydroxide with rich oxygen vacancies for the oxygen evolution reaction.

The sluggish kinetics of the oxygen evolution reaction (OER) restrains the development of water splitting technologies and the efficiency of producing sustainable resources. To this end, the introduction of iron and molybdenum in catalytic systems has been employed as a crucial strategy for the enhancement of catalytic activity toward the oxygen evolution reaction (OER), but the relationship between catalyst components and catalytic performance is still evasive. In this study, by doping iron and molybdenum into cobalt hydroxide via a cation-exchange method, rich oxygen vacancies and active metal centers are introduced to the trimetallic oxyhydroxide, endowing the catalyst with a low overpotential of 223 mV at 10 mA cm-2, a low Tafel slope of 43.6 mV dec-1, and a long stable operation time (>50 h) in alkaline media, comparable to the current best OER catalyst. Moreover, it is demonstrated that the doping of iron favors the generation of oxygen vacancies. It is also found in this work that using a certain amount (5 mg) of iron dopant can alter the electronic structure of the catalyst by tuning the electronic density around the metal ions, thus optimizing the binding energy of intermediates. The present work unveils the doping effect of iron and molybdenum on the construction of trimetallic oxyhydroxide catalysts, and sheds light on the relationship between the catalyst components and catalytic performance of the OER.

Engineered Design of the E-Helix Structure on Ferritin Nanoparticles.

Insertion of an immunogenic epitope at the C-terminus of ferritin has shown the potential to produce a stable and efficacious vaccine. There is however limited understanding of how C-terminus insertion affects ferritin protein stability. The E-helix at the C-terminus has attracted interest because there are contradictory reports as to whether it has a role in protein stabilization. Here, we report, for the first time, combining molecular dynamics simulation (MDS) with experiment to engineer the design of the E-helix at the C-terminus of engineered human ferritin heavy chain (F1) inserted with Epstein-Barr nuclear antigen 1 (EBNA1, E1) and flexible linker (L3) residues (to afford F1L3E1). Hot spots on the E-helix of the C-terminus were predicted by MDS at aa 167 (Glu) and aa 171 (Asp). Five (5) variants of F1L3E1 were constructed by considering hot spots and alteration of electrostatic or hydrophobic interfaces, namely, (1) C1, hot spots substituted with noncharged residue Gln; (2) C2, hot spots substituted with positively charged residue Arg; (3) C3, hydrophobic residues substituted with the most hydrophobic residues Val and Ile; (4) C4, hydrophobic residues substituted with the most hydrophilic residues Gln and Asn; and (5) C5, a heptad repeat structure in the E-helix disrupted by substituting "a" and "d" heptad residues with noncharged polar residue Gln. It was found that the E-helix is essential to maintain integrated protein stability and that changing the hydrophobic interface (C3 and C4) had more significant effects on protein folding and stability than changing the electrostatic interface (C1 and C2). It was confirmed by both MDS and experiment that variants C1, C2, and C5 were able to fold to form stable conformational structures with protein surface hydrophobicity similar to that of F1L3E1. However, they are less thermally stable than F1L3E1. Significant changes in hydrophobicity drove significant protein aggregation for variants C3 and C4. It is concluded that the molecular design of the C-terminus in engineered ferritin, especially the E-helix, is important to ensure the epitope-based chimeric vaccine is safe (aggregate free) and efficacious.

Green synthesis of DOX-loaded hollow MIL-100 (Fe) nanoparticles for anticancer treatment by targeting mitochondria.

Fe-based metal-organic frameworks (MOFs) are promising drug delivery materials due to their large surface area, high stability, and biocompatibility. However, their drug loading capacity is constrained by their small pore size, and a further improvement in their drug capacity is needed. In this work, we report an effective and green structural modification strategy to improve drug loading capacity for Fe-based MOFs. Our strategy is to grow MIL-100 (Fe) on carboxylate-terminated polystyrene (PS-COOH) via a sustainable route, which creates a large inner cavity as well as exposure to more functional groups that benefit drug loading capacity. We employ the scanning electron microscope and transmission electron microscope to confirm the hollow structure of MIL-100 (Fe). Up to 30% of drug loading capacity has been demonstrated in our study. We also conduct cell viability tests to investigate its therapeutic effects on breast cancer cells (MDA-MB-231). Confocal laser scanning microscopy imaging confirms cellular uptake and mitochondrial targeting function of doxorubicin-loaded H-M (DOX@H-M) nanoparticles. JC-1 staining of cancer cells reveals a significant change in the mitochondrial membrane potential, indicating the mitochondrial dysfunction and apoptosis of tumor cells. Our study paves the way for the facile synthesis of hollow structural MOFs and demonstrates the potential of applying Fe-based MOFs in breast cancer treatment.

An integrated and continuous downstream process for microbial virus-like particle vaccine biomanufacture.

In this study, we present the first integrated and continuous downstream process for the production of microbial virus-like particle vaccines. Modular murine polyomavirus major capsid VP1 with integrated J8 antigen was used as a model virus-like particle vaccine. The integrated continuous downstream process starts with crude cell lysate and consists of a flow-through chromatography step followed by periodic counter-current chromatography (PCC) (bind-elute) using salt-tolerant mixed-mode resin and subsequent in-line assembly. The automated process showed a robust behavior over different inlet feed concentrations ranging from 1.0 to 3.2 mg ml-1 with only minimal adjustments needed, and produced continuously high-quality virus-like particles, free of nucleic acids, with constant purity over extended periods of time. The average size remained constant between 44.8 ± 2.3 and 47.2 ± 2.9 nm comparable to literature. The process had an overall product recovery of 88.6% and a process productivity up to 2.56 mg h-1 mlresin-1 in the PCC step, depending on the inlet concentration. Integrating a flow through step with a subsequent PCC step allowed streamlined processing, showing a possible continuous pathway for a wide range of products of interest.

RR-1 cuticular protein TcCPR69 is required for growth and metamorphosis in Tribolium castaneum.

Cuticle is not only critical for protecting insects from noxious stimuli but is also involved in a variety of metabolic activities. Cuticular proteins (CPs) affect cuticle structure and mechanical properties during insect growth, reproduction, and environmental adaptation. Here, we describe the identification and characterization of a member of the RR-1 subfamily of CPs with an R&R consensus (CPR) in Tribolium castaneum (TcCPR69). Although it was previously reported to be highly expressed in the wings, we found that knocking down TcCPR69 by RNA interference (RNAi) did not cause obvious wing abnormalities but markedly disrupted the growth and metamorphosis of beetles with 100% cumulative mortality; additionally, the chitin content of the pharate adult was decreased and the new abdominal cuticle was significantly thinner before molting. TcCPR69 showed chitin-binding ability and the expression levels of key genes involved in chitin metabolism (trehalase [TcTRE], chitin synthase [TcCHSA and TcCHSB], and chitinase [TcCHT5 and TcCHT10]) were also decreased by TcCPR69 knockdown. TcCPR69 gene expression peaked shortly after molting and was increased 2.61 fold at 12 h after 20-hydroxyecdysone (20E) injection. This was reversed by RNAi of the ecdysone-related genes ecdysone receptor (TcECR) and fushi tarazu transcription factor 1 (TcFTZ-F1). These results indicate that TcCPR69 is positively regulated by 20E signaling to contribute to cuticle formation and maintain chitin accumulation during the growth and metamorphosis of beetles.