Effects of Individual Amino Acids on the Blood Circulation of Biosynthetic Protein Nanocages: Toward Guidance on Surface Engineering.

Protein nanocages (PNCs) hold great promise for developing multifunctional nanomedicines. Long blood circulation is a key requirement of PNCs for most in vivo application scenarios. In addition to the classical PEGylation strategy, short peptides with a specific sequence screened via phage display are also very effective in prolonging the blood half-life (t1/2 ) of PNCs. However, there is a lack of knowledge on how individual amino acids affect the circulation of PNCs. Here the effects of the 20 proteinogenic amino acids in the form of an X3 or X5 tag (X represents an amino acid) are explored on the pharmacokinetics of PNCs, which lead to the formation of a heatmap illustrating the extent of t1/2 prolongation by each proteinogenic amino acid. Significantly, oligo-lysine and oligo-arginine can effectively prolong the t1/2 of strongly negatively charged PNCs through charge neutralization, while oligo-cysteine can also do so, but via a different mechanism, mediating the covalent binding of PNCs with plasma albumin as a stealth material. These findings are extendible and offer guidance for surface-engineering biosynthetic PNCs and other nanoparticles.

Probing cell membrane integrity using a histone-targeting protein nanocage displaying precisely positioned fluorophores.

Cell membrane integrity is fundamental to the normal activities of cells and is involved in both acute and chronic pathologies. Here, we report a probe for analyzing cell membrane integrity developed from a 9 nm-sized protein nanocage named Dps via fluorophore conjugation with high spatial precision to avoid self-quenching. The probe cannot enter normal live cells but can accumulate in dead or live cells with damaged membranes, which, interestingly, leads to weak cytoplasmic and strong nuclear staining. This differential staining is found attributed to the high affinity of Dps for histones rather than DNA, providing a staining mechanism different from those of known membrane exclusion probes (MEPs). Moreover, the Dps nanoprobe is larger in size and thus applies a more stringent criterion for identifying severe membrane damage than currently available MEPs. This study shows the potential of Dps as a new bioimaging platform for biological and medical analyses. Electronic Supplementary Material: Supplementary material (Figs. S1-S12 including distance information between neighboring fluorophores on Dps, TEM images, MALDI-TOF analysis, fluorescence spectra, confocal images, gel retardation analysis, tissue staining, and additional data) is available in the online version of this article at 10.1007/s12274-022-4785-5.

Passive cancer targeting with a viral nanoparticle depends on the stage of tumorigenesis.

Tumor targeting with nanoparticles is a promising strategy for cancer diagnosis and treatment, especially for drug delivery to solid tumors. Previous studies mainly focused on nanoparticle design to improve their targeting efficiency, but few have investigated the impact of tumor progression stages on the targeting efficiency. Here, we used PEGylated viral nanoparticles (VNPs) of bacteriophage P22 to explore the relationship between targeting efficiency and tumor progression stages using a colorectal cancer model. We found an 8.1-fold increase in the accumulation of P22 VNPs systematically injected 7 days after tumor inoculation compared with those injected 21 days after tumor inoculation. Most tumor-targeted P22 VNPs were concentrated in tumor-associated macrophages in the tumor blood vessels, the density of which decreased with the progression of tumors. These results reveal that the tumor targeting efficiency of P22 VNPs decreased with tumor progression. These findings provide valuable information for not only the understanding of controversial observations regarding targeted cancer therapy in experimental and clinical studies but also the design of nanoparticle-based tumor targeting probes or therapeutics.

A ROS scavenging protein nanocage for in vitro and in vivo antioxidant treatment.

Elevated levels of reactive oxygen species (ROS) are implicated in the onset and progression of many diseases, e.g., virus infection, ischemic stroke and neurodegenerative diseases. ROS-scavenging nanomaterials have attracted particular interest. Here, we report the development of a natural protein nanocage named Dps for in vitro and in vivo antioxidant treatment by inhibiting the Fenton reaction, a critical step in ROS generation and interconversion. Systematic surface engineering enabled cell penetration, good colloidal stability, and facile purification of Dps. With its intrinsic ferroxidase activity consuming both H2O2 and Fe2+, Dps not only protects human cells from oxidative stress but also effectively alleviates ROS-induced inflammation in a mouse dermatitis model. The protection is triggered by elevated H2O2 and thereby, in principle, avoids ROS imbalances. Thus, Dps has potential as a new bionano platform for different purposes, such as antiaging, anti-inflammation and cosmetics.

AIE nanodots scaffolded by mini-ferritin protein for cellular imaging and photodynamic therapy.

Photodynamic therapy (PDT) is one of the most elegant cancer treatment strategies that can be controlled by a beam of light with non-invasion, precise control, and high spatiotemporal accuracy. An ideal photosensitizer (PS) is the key to ensure the efficacy of PDT. Due to their hydrophobic and rigid planar structures, most traditional PSs are prone to aggregate under physiological conditions, which causes fluorescence quenching and significantly reduces reactive oxygen species (ROS) generation. Fortunately, the emergence of aggregation-induced emission (AIE) dyes offers a potential opportunity to overcome these limitations. When AIE PS molecules are in the aggregation state, the fluorescence intensity and ROS production can be increased. We herein use red AIE PS molecules to prepare stable AIE nanodots for cell imaging and PDT via a simple method with a highly negatively charged mini-ferritin protein as the scaffold. The as-prepared protein-AIE nanodots show strong fluorescence emission and efficient singlet oxygen generation, with good stability, relatively long wavelengths of absorption and emission, and negligible dark toxicity. The mini-ferritin-AIE system may be useful in developing novel functional probes for tumour nanotheranostics.

Role of Surface RGD Patterns on Protein Nanocages in Tumor Targeting Revealed Using Precise Discrete Models.

The effectiveness of active targeting in cancer nanomedicine is becoming increasingly more debatable. Here, the role of the ligand functionalization patterns (number and distribution) on nanoparticle surfaces in tumor targeting is investigated using a 9 nm sized miniferritin protein nanocage, Dps modified with Arg-Gly-Asp (RGD) ligands whose functionalization patterns are precisely controlled. In vitro and in vivo experiments show that RGD modification endows Dps with tumor targeting capacity no matter what the surface pattern is. The tumor targeting of 2-ligand Dps, which is better than that of 1-ligand Dps, rivals or surpasses that of the 12- or 24-ligand Dps. The 12-ligand Dps with clustered RGD distribution shows 2.3 times the in vivo targeting efficiency of that with even distribution. The surface ligand pattern effects are correlated at least to receptor clustering and opsonization. This study provides insights into the understanding of the controversial findings on active tumor targeting in the literature and highlights the necessity of precise functionalization to achieve optimal active targeting in developing cancer nanomedicine.

Characterization of a New Cyclohexylamine Oxidase From Acinetobacter sp. YT-02.

Cyclohexylamine (CHAM) is widely used in various industries, but it is harmful to human beings and the environment. Acinetobacter sp. YT-02 can degrade CHAM via cyclohexanone as an intermediate. In this study, the cyclohexylamine oxidase (CHAO) gene from Acinetobacter sp. YT-02 was cloned. Amino acid sequence alignment indicated that the cyclohexylamine oxidase (CHAOYT-02) was 48% identical to its homolog from Brevibacterium oxydans IH-35A (CHAOIH-35). The enzyme was expressed in Escherichia coli BL21 (DE3), and purified to apparent homogeneity by Ni-affinity chromatography. The purified enzyme was proposed to be a dimer of molecular mass of approximately 91 kDa. The enzyme exhibited its maximum activity at 50°C and at pH 7.0. The enzyme was thermolabile as demonstrated by loss of important percentage of its maximal activity after 30 min incubation at 50°C. Metal ions Mg2+, Co2+, and K+ had certain inhibitory effect on the enzyme activity. The kinetic parameters K m and V max were 0.25 ± 0.02 mM and 4.3 ± 0.083 µM min-1, respectively. The biochemical properties, substrate specificities, and three-dimensional structures of CHAOYT-02 and CHAOIH-35 were compared. Our results are helpful to elucidate the mechanism of microbial degradation of CHAM in the strain YT-02. In addition, CHAOYT-02, as a potential biocatalyst, is promising in controlling CHAM pollution and deracemization of chiral amines.

CTLA-4 methylation regulates the pathogenesis of myasthenia gravis and the expression of related cytokines.

BACKGROUND: Myasthenia gravis (MG) is a progressive autoimmune disease that occurs as a result of the failure of neuromuscular transmission and is characterized by muscle weakness. There has been evidence on the correlations between the genetic predisposition of cytotoxic T lymphocyte and the antigen-4 (CTLA-4) and MG. Thus, the present study was conducted to study is designed to examine the effects of CTLA-4 methylation on the pathogenesis of MG and the expressions of related cytokines. METHODS: The CTLA-4 methylation levels in peripheral blood were quantified in 103 samples collected from MG patients and 86 samples from healthy individiuals. The expression of serum-related cytokines as well as the Treg cell ratio were examined so as to define the contributory role of CTLA-4 methylation in MG and to identify the interaction between CTLA-4 methylation and related factors, the expressions of DNA methyltransferase (DNMT)l, DNMT3A and DNMT3B, CTLA-4, AchR-Ab, Titin-Ab, RyR-Ab, IL-2, IL-10, IFN-γ, and TGF-ß, activity of P- acetylcholinesterase (AchE) and E-AchE. RESULTS: The results indicated that the incidence of CTLA-4 methylation was significantly higher in the control group when compared with the MG group, and CTLA-4 methylation was also found to be associated with the thymus status of MG patients. It was also observed from the experiment data that the expressions of DNMTl, DNMT3A, and DNMT3B, along with the expressions of AchR-Ab, Titin-Ab, RyR-Ab, IL-2, IL-10, IFN-γ and TGF-ß, and the activity of P-AchE and E-AchE were all higher in the MG group than in the control group, with a reduction of CTLA-4 expression. Another key finding from this study revealed that methylation interference can lead to the suppression in the expression of AchR-Ab, the activity of E-AchE, the expression of IL-2, IL-10, IFN-γ, and TGF-ß and the Treg cell ratio in lymphocytes. CONCLUSION: In conclusion, the results obtained from the present study highly indicated that CTLA-4 methylation might play a role in facilitating the occurrence of MG and increasing the expressions of related cytokines through the upregulation of AchR-Ab and E-Ach.

Intracellular delivery of peptide drugs using viral nanoparticles of bacteriophage P22: covalent loading and cleavable release.

Peptides have great potential in the treatment of various diseases because of their high specificity and safety. However, their application has been limited by the lack of appropriate delivery techniques. In this study, we have demonstrated the intracellular delivery of peptides using viral nanoparticles (VNPs) of bacteriophage P22 through covalent loading of two different peptides with synergistic cytotoxic effects and controllable release triggered by Cathepsin B, a highly over-expressed protease in many tumors. Fluorescence imaging of the intracellular localization of the P22 VNPs and the peptides evidenced that the delivery system could be transported as designed. Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis revealed the cytotoxic effects of the peptides on MDA-MB-231 tumor cells. These results have confirmed the feasibility of genetically fusing cargo peptides to the capsid protein of P22 and cleavable release thereafter. This study may be instructive for the design of drug delivery systems and multifunctional theranostic devices based on P22 VNPs.

EXOC3L2 rs597668 variant contributes to Alzheimer's disease susceptibility in Asian population.

Recent genome-wide association studies have established the association between EXOC3L2 rs597668 variant and Alzheimer's disease (AD) in European population. However, recent studies reported inconsistent results in Asian population. Here, we performed a systematic review and meta-analysis to evaluate the impact of rs597668 on AD risk in Asian population using a total of 8686 samples including 2855 cases and 5831 controls. Meanwhile, we selected 17,008 AD cases and 37,154 controls in European population to evaluate the potential heterogeneity between East Asian and European populations. In East Asian population, we identified no potential heterogeneity with P=0.31 and I2 = 15.8%. By meta-analysis, we identified positive association between rs597668 and AD risk with P=0.023, OR=0.93, 95% CI 0.87-0.99. We further found significant heterogeneity in pooled Asian and European populations with P<0.0001 and I2 = 87.7%. The meta-analysis indicated negative association with P=0.66, OR=0.97, 95% CI 0.85-1.11. In summary, all these findings indicate that rs597668 C allele is a risk factor for AD in European population with OR=1.18 and P=2.49E-13. However the rs597668 C allele played a protective role in AD with OR=0.93 and P=0.023 in East Asian population.

HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866.

In addition to growing on p-cresol, Pseudomonas putida NCIMB 9866 is the only reported strain capable of aerobically growing on 2,4-xylenol, which is listed as a priority pollutant by the U.S. Environmental Protection Agency. Several enzymes involved in the oxidation of the para-methyl group, as well as the corresponding genes, have previously been reported. The enzyme catalyzing oxidation of the catabolic intermediate 4-hydroxyisophthalate to the ring cleavage substrate protocatechuate was also purified from strain NCIMB 9866, but its genetic determinant is still unavailable. In this study, the gene hipH, encoding 4-hydroxyisophthalate hydroxylase, from strain NCIMB 9866 was cloned by transposon mutagenesis. Purified recombinant HipH-His6 was found to be a dimer protein with a molecular mass of approximately 110 kDa. HipH-His6 catalyzed the hydroxylation of 4-hydroxyisophthalate to protocatechuate with a specific activity of 1.54 U mg(-1) and showed apparent Km values of 11.40 ± 3.05 µM for 4-hydroxyisophthalate with NADPH and 11.23 ± 2.43 µM with NADH and similar Km values for NADPH and NADH (64.31 ± 13.16 and 72.76 ± 12.06 µM, respectively). The identity of protocatechuate generated from 4-hydroxyisophthalate hydroxylation by HipH-His6 has also been confirmed by high-performance liquid chromatography and mass spectrometry. Gene transcriptional analysis, gene knockout, and complementation indicated that hipH is essential for 2,4-xylenol catabolism but not for p-cresol catabolism in this strain. This fills a gap in our understanding of the gene that encodes a critical step in 2,4-xylenol catabolism and also provides another example of biochemical and genetic diversity of microbial catabolism of structurally similar compounds.

Controlled Self-Assembly of Proteins into Discrete Nanoarchitectures Templated by Gold Nanoparticles via Monovalent Interfacial Engineering.

Designed rational assembly of proteins promises novel properties and functionalities as well as new insights into the nature of life. De novo design of artificial protein nanostructures has been achieved using protein subunits or peptides as building blocks. However, controlled assembly of protein nanostructures into higher-order discrete nanoarchitectures, rather than infinite arrays or aggregates, remains a challenge due to the complex or symmetric surface chemistry of protein nanostructures. Here we develop a facile strategy to control the hierarchical assembly of protein nanocages into discrete nanoarchitectures with gold nanoparticles (AuNPs) as scaffolds via rationally designing their interfacial interaction. The protein nanocage is monofunctionalized with a polyhistidine tag (Histag) on the external surface through a mixed assembly strategy, while AuNPs are modified with Ni(2+)-NTA chelates, so that the protein nanocage can controllably assemble onto the AuNPs via the Histag-Ni(2+) affinity. Discrete protein nanoarchitectures with tunable composition can be generated by stoichiometric control over the ratio of protein nanocage to AuNP or change of AuNP size. The methodology described here is extendable to other protein nanostructures and chemically synthesized nanomaterials, and can be borrowed by synthetic biology for biomacromolecule manipulation.

Purification and characterization of salicylate 5-hydroxylase, a three-component monooxygenase from Ralstonia sp. strain U2.

Salicylate is an important intermediate in the bacterial degradation of polycyclic aromatic hydrocarbons and salicylate hydroxylases play essential roles in linking the peripheral and ring-cleavage catabolic pathways. Unlike the well-characterized salicylate 1-hydroxylases, the rarely occurred salicylate 5-hydroxylase (S5H) has not been characterized in detail. In this study, the three-component Fe-S protein complex (NagAaGHAb) of S5H from Ralstonia sp. strain U2 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component NagGH exhibited an α3ß3 heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. NagAa is the ferredoxin-NADP⁺ reductase component containing flavin and plant type [2Fe-2S] cluster. The ferredoxin component NagAb was characterized as a [2Fe-2S] dimer which remains remarkably stable in denaturing gel electrophoresis after being heated at 100 °C for 1 h. Purified NagAa and NagAb, NagGH catalyzed the hydroxylation of salicylate to gentisate with a specific activity of 107.12 ± 14.38 U/g and showed an apparent K(m) for salicylate of 102.79 ± 27.20 µM and a similar K(m) value for both NADH and NADPH (59.76 ± 7.81 µM versus 56.41 ± 12.76 µM). The hydroxylase exhibited different affinities for two hydroxysalicylates (2,4-dihydroxybenzoate K(m) of 93.54 ± 18.50 µM versus 2,6-dihydroxybenzoate K(m) of 939.80 ± 199.46 µM). Interestingly, this S5H also showed catalytic activity to the pollutant 2-nitrophenol and exhibited steady-state kinetic data of the same order of magnitude as those for salicylate. This study will allow further comparative studies of structure-function relationships of the ring hydroxylating mono- and di-oxygenase systems.

Identification and clarification of the role of key active site residues in bacterial glutathione S-transferase zeta/maleylpyruvate isomerase.

The maleylpyruvate isomerase NagL from Ralstonia sp. strain U2, which has been structurally characterized previously, catalyzes the isomerization of maleylpyruvate to fumarylpyruvate. It belongs to the class zeta glutathione S-transferases (GSTZs), part of the cytosolic GST family (cGSTs). In this study, site-directed mutagenesis was conducted to probe the functions of 13 putative active site residues. Steady-state kinetic information for mutants in the reduced glutathione (GSH) binding site, suggested that (a) Gln64 and Asp102 interact directly with the glutamyl moiety of glutathione, (b) Gln49 and Gln64 are involved in a potential electron-sharing network that influences the ionization of the GSH thiol. The information also suggests that (c) His38, Asn108 and Arg109 interact with the GSH glycine moiety, (d) His104 has a role in the ionization of the GSH sulfur and the stabilization of the maleyl terminal carboxyl group in the reaction intermediate and (e) Arg110 influences the electron distribution in the active site and therefore the ionization of the GSH thiolate. Kinetic data for mutants altered in the substrate-binding site imply that (a) Arg8 and Arg176 are critical for maleylpyruvate orientation and enolization, and (b) Arg109 (exclusive to NagL) participates in k(cat) regulation. Surprisingly, the T11A mutant had a decreased GSH K(m) value, whereas little impact on maleylpyruvate kinetics was observed, suggesting that this residue plays an important role in GSH binding. An evolutionary trend in this residue appears to have developed not only in prokaryotic and eukaryotic GSTZs, but also among the wider class of cGSTs.

Cut-off points of fasting fingertip capillary blood glucose for detecting both undiagnosed diabetes and pre-diabetes / 中华流行病学杂志

Objective To determine the efficient cut-off points of fasting fingertip blood glucose test for undiagnosed diabetes mellitus(DM), impaired glucose tolerance(IGT), and impaired fasting glucose(IFG)in community-based residents aged above 45 years old. Methods A cluster-randomized study was conducted from May 2008 to January 2009. A total of 3250 subjects aged above 45 years in two communities of Baoding city received questionnaire investigation and tested for fingertip blood glucose. Those subjects whose capillary blood glucose level ≥5.1 mmol/L were subjected to 75 g oral glucose tolerance test. Undiagnosed diabetes mellitus and pre- diabetes were identified by fasting plasma glucose and OGTT. In this study, the cut-off points of fasting capillary blood glucose for detecting undiagnosed diabetes and pre-diabetes were evaluated, using receiver operator characteristic curve(ROC). Results Of 1351 subjects that having had oral glucose tolerance test, 230 cases were diagnosed as diabetes mellitus(7.3%), 166 cases(5.2%)as IFG, and 204(6.7%)as IGT under fasting capillary blood glucose as test variable and state variables according to the following criteria.(1)FPG≥7.0 mmol/L or/and 2hPG≥11.1 mmol/L(2)FPG<5.6 mmol/L (3)FPG<7.0 mmol/L and 7.8 mmol/L≤2hPG≤ 11.1 mmol/L, areas under three ROC curves were 0.905, 0.633 and 0.719, respectively. The cut-offvalues of screening for undiagnosed DM, IGT and IFG were 6.0 mmol/L, 5.7 mmol/L, and 5.7 mmol/L, respectively. When cut-off value of screening for undiagnosed DM was 6.0 mmol/L, the maximal sensitivity was 78.0% and specificity was 89.3%.But there were both lower sensitivity and specificity in screening for IFG and IGT according to the best predicting value(5.7 mmol/L)from the ROC curves(50.3% and 28.0% vs. 60.8% and 28.0%). Conclusion Fasting capillary blood glucose with the lower cut-point of 6.0 mmol/L in screening for undiagnosed diabetes mellitus alone, was relatively reliable, whereas for both IFG and IGT the fasting fingertip blood glucose tests were fallible. It was convenient and could be used in screening the DM at the community level.