Engineering diaryl alcohol dehydrogenase KpADH reveals importance of retaining hydration shell in organic solvent tolerance.

Alcohol dehydrogenases (ADHs) are synthetically important biocatalysts for the asymmetric synthesis of chiral alcohols. The catalytic performance of ADHs in the presence of organic solvents is often important since most prochiral ketones are highly hydrophobic. Here, the organic solvent tolerance of KpADH from Kluyveromyces polyspora was semi-rationally evolved. Using tolerant variants obtained, meticulous experiments and computational studies were conducted to explore properties including stability, activity and kinetics in the presence of various organic solvents. Compared with WT, variant V231D exhibited 1.9-fold improvement in ethanol tolerance, while S237G showed a 6-fold increase in catalytic efficiency, a higher T 50 15 $$ {\mathrm{T}}_{50}^{15} $$ , as well as 15% higher tolerance in 7.5% (v/v) ethanol. Based on 3 × 100 ns MD simulations, the increased tolerance of V231D and S237G against ethanol may be ascribed to their enhanced ability in retaining water molecules and repelling ethanol molecules. Moreover, 6.3-fold decreased KM value of V231D toward hydrophilic ketone substrate confirmed its capability of retaining hydration shell. Our results suggest that retaining hydration shell surrounding KpADH is critical for its tolerance to organic solvents, as well as catalytic performance. This study provides useful guidance for engineering organic solvent tolerance of KpADH and other ADHs.

CD44-targeted melanin-based nanoplatform for alleviation of ischemia/reperfusion-induced acute kidney injury.

Ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) is a serious kidney disease with high morbidity and mortality. However, there is no effective clinical treatment strategy. Herein, we developed a CD44 targeting nanoplatform based on HA-assembled melanin NPs covalently coupled with dexamethasone for I/R-induced AKI therapy by alleviating oxidative/inflammatory- induced damage. The constructed HA-MNP-DXM NPs had good dispersion, stability, and broad-spectrum scavenging capabilities against multiple reactive free radicals. Moreover, the NPs could be efficiently internalized and exhibited antioxidative, anti-inflammatory, and antiapoptotic effects in CoCl2-stimulated renal tubular epithelial NRK-52E cells. Furthermore, the I/R-induced AKI murine model was established to evaluate the in vivo performance of NPs. The results suggested the NPs could specifically target impaired kidneys upon intravenous administration according to NIR-II fluorescence imaging and showed high biosafety. Importantly, the NPs could improve renal function, alleviate oxidative stress and inflammatory reactions, inhibit apoptosis of tubular cells, and restore mitochondrial structure and function, exhibiting excellent therapeutic effects. Further therapeutic mechanism indicated the NPs maintained the cellular/mitochondrial redox balance by modulating the Nrf2 and HO-1 expression. Therefore, the NPs can be a promising therapeutic candidate for the treatment of I/R-induced AKI.

Biodegradable Silica-Based Nanotheranostics for Precise MRI/NIR-II Fluorescence Imaging and Self-Reinforcing Antitumor Therapy.

Multi-modality cancer diagnosis techniques based on the second near-infrared window fluorescence (NIR-II FL, 1000-1700 nm) imaging have become the focus of research attention. For such multimodality probes, how to take advantage of the tumor microenvironments (TME) characteristics to better image diseases and combine efficient therapeutics to achieve theranostics is still a big challenge. Herein, a novel TME-activated nanosystem (FMSN-MnO2 -BCQ) employing degradable silica-based nanoplatform is designed, adjusting the ratio of intratumoral hydrogen peroxide (H2 O2 )/glutathione (GSH) for magnetic resonance imaging (MRI)/NIR-II FL imaging and self-reinforcing chemodynamic therapy (CDT). Innovative bovine serum albumin (BSA)-modified fusiform-like mesoporous silica nanoparticles (FMSN) is fabricated as a carrier for NIR-II small molecule (CQ4T) and MRI reporter MnO2 . Remarkably, the BSA modification helped to achieve the dual-functions of high biocompatibility and enhance NIR-II fluorescence. The FMSN-MnO2 -BCQ with FMSN framework featuring a stepwise degradability in tumor interior released MnO2 and BCQ nanoparticles. Through the specific degradation of MnO2 by the TME, the produced Mn2+ ions are effectively exerted Fenton-like activity to generate hydroxyl radical (•OH) from endogenous H2 O2 to eradicate tumor cells. More importantly, the GSH depletion due to the synergistic effect of tetrasulfide bond and MnO2 in turn induced the oxidative cytotoxicity for self-reinforcing CDT.

Biodegradable Multifunctional Nanotheranostic Based on Ag2S-Doped Hollow BSA-SiO2 for Enhancing ROS-Feedback Synergistic Antitumor Therapy.

Stimuli-responsive silica nanoparticles are an attractive therapeutic agent for effective tumor ablation, but the responsiveness of silica nanoagents is limited by intrastimulation level and silica framework structure. Herein, a biodegradable hollow SiO2-based nanosystem (Ag2S-GOx@BHS NYs) is developed by a novel one-step dual-template (bovine serum albumin (BSA) and cetyltrimethylammonium bromide (CTAB)) synthetic strategy for image-guided therapy. The Ag2S-GOx@BHS NYs can be specifically activated in the tumor microenvironment via a self-feedback mechanism to achieve reactive oxygen species (ROS)-induced multistep therapy. In response to the inherent acidity and H2O2 at the tumor sites, Ag2S-GOx@BHS would accelerate the structural degradation while releasing glucose oxidase (GOx), which could efficiently deplete intratumoral glucose to copious amounts of gluconic acid and H2O2. More importantly, the sufficient H2O2 not only acts as a reactant to generate Ag+ from Ag2S for metal-ion therapy and improves the oxidative stress but also combines with gluconic acid results in the self-accelerating degradation process. Moreover, the released Ag2S nanoparticles can help the Ag2S-GOx@BHS NYs realize the second near-infrared window fluorescence (NIR-II FL) and photoacoustic (PA) imaging-guided precise photothermal therapy (PTT). Taken together, the development of a self-feedback nanosystem may open up a new dimension for a highly effective multistep tumor therapy.

A tumor-targeted theranostic nanomedicine with strong absorption in the NIR-II biowindow for image-guided multi-gradient therapy.

Developing new strategies to enhance drug accumulation in the tumor and therapeutic efficacy is of great importance in the field of tumor therapy. Herein, a peanut-like multifunctional nanomedicine (CuS-PGH NMs) made of CuS nanoparticles encapsulated in poly(l-lysine)(PLL)/glucose oxidase (GOx)-hyaluronic acid (HA) shells has been constructed via layer-by-layer (LbL) assembly, and shows good biocompatibility and effective multi-gradient therapy. Because of the enhanced permeability and retention (EPR) effect, the CuS-PGH NMs could significantly enhance the cellular uptake by tumors overexpressing CD44 receptors, which respond to hyaluronidase (HAase)-triggered surface charge conversion. Once internalized by the tumor, GOx was the first to be exposed and could effectively deplete endogenous glucose for starvation therapy, and the excess H2O2 was then converted into highly toxic hydroxyl radicals (˙OH) via a Cu+-mediated Fenton-like reaction for chemodynamic therapy (CDT). Meanwhile, the as-obtained Cu+ ions accompanied the regenerated less-active Cu2+ ions. Interestingly, the high content of H2O2 could, in turn, accelerate Cu2+/Cu+ conversion to promote the Cu+-H2O2 reaction for enhanced chemodynamic therapy (CDT), thereby achieving efficient tumor growth suppression via synergistic starvation/CDT therapy. Subsequently, owing to the strong NIR-II absorption capability of CuS-PGH NMs, effective photothermal tumor ablation of the weakened tumor cells could be realized with the precise guidance of NIR-II PAI. This multi-gradient therapeutic strategy has been demonstrated to have excellent antitumor activity with minimal nonspecific damages, and offers a new avenue to precise tumor therapy.

Two-stage activated nano-truck enhanced specific aggregation and deep delivery for synergistic tumor ablation.

Although nanomedicines have shown high performance in tumor theranostics, their anticancer activity is still limited by the drug delivery capacity, especially lack of targeting capability, poor tumor accumulation, and insufficient tumor deep-penetration. To address this challenge, a high biocompatibility nano-truck (BMP NT) with a two-stage delivery mechanism is designed and developed to achieve the precision therapeutic efficacy of cancer. In view of the enhanced permeability retention (EPR) effect, the surface cleavable layer of BMP NTs can be selectively removed by the overexpressed MMP-2 in a tumor-microenvironment to expose the hydrophobic segments for an induced "braking effect" strategy, resulting in a significant increase in tumor accumulation. Once internalized into cancer cells with the overproduced glutathione (GSH) and H2O2, the BMP NTs undergo the second-stage "unloading process" to release Mn2+ ions and ultrasmall Bi2S3@BSA nanoparticles, and the obtained Mn2+ ions can act as a Fenton-like catalyst for continuously catalyzing the endogenous H2O2 into highly toxic hydroxyl radicals (˙OH) for CDT. The GSH depletion will in turn improve the Mn2+-H2O2 reaction, further enhancing CDT efficiency. Meanwhile, the ultrasmall Bi2S3@BSA endows BMP NTs with excellent photothermal conversion ability to generate local hyperthermia and accelerate the intratumoral Fenton process, thus leading to an effective tumor therapeutic outcome in the synergistic function of CDT/photothermal therapy (PTT). Moreover, the BMP NTs can be used for in situ self-generation magnetic resonance imaging (MRI) and photoacoustic (PA) imaging to guide precision cancer therapy.

Iontophoresis-assisted accelerated riboflavin/ultraviolet A scleral cross-linking: A potential treatment for pathologic myopia.

Scleral collagen cross-linking is one of the most promising treatments to control the pathologic process of myopia. However, the exact procedure and its impact on animal models of myopia are still to be explored. We modified the scleral riboflavin/ultraviolet A (UVA) cross-linking procedure with an iontophoresis-assisted drug delivery system and an accelerated UVA irradiation (10 mW/cm2, 9 min) and applied this treatment to an animal model of myopia. Ninety-six New Zealand White rabbits developed relatively stable myopia by visual deprivation and then underwent the modified scleral cross-linking surgery. All the statistics and sample collection were obtained from 4 postoperative time points (1-day, 10-day, 1-month and 3-month groups). We found that the ultimate stress, Young's modulus and physiological Young's modulus of treated myopia sclera were significantly increased and maintained in 4 groups. The abnormal elongation of the myopic eye was effectively controlled 1 month after the treatment and even almost halted 3 months after the treatment. The histochemical assay revealed no notable post-surgery damage or apoptosis in the retina and choroid. Vigorous collagen synthesis was observed in scleral fibroblasts of the treated samples but were rarely observed in the untreated ones under electron microscopy. Furthermore, the remarkable difference in collagen gene expression and protein content between treated and untreated samples also indicated that an alteration in collagen metabolism may be triggered by the treatment. The effectiveness and safety exploration suggested that the modified scleral cross-linking procedure may be a potential method to control the pathologic process of myopia.

[Gene expression of down-regulation of lysyl oxidases family in keratoconus corneal fibroblasts induced by combination of mechanical stretching and prostaglandin E 2].

In order to investigate the effects of mechanical stretching combined with prostaglandin E 2 (PGE 2) on the gene expression of lysyl oxidases (LOXs) in keratoconus, we treated cultured corneal fibroblasts from healthy human cornea and keratoconus patient cornea with PGE 2 and/or cyclic stretch (12% elongation, 0.1 Hz, 12 h). Real-time fluorescent quantitative polymerase chain reaction was used to detect the gene expression of LOXs. The results showed that the gene expression of LOXs in keratoconus group was significantly lower than that in the healthy one. Compared to the static control group, 12% stretching alone up-regulated gene expression of LOXL-2, LOXL-4 in the healthy group, whereas it down-regulated LOXL-3, LOXL-4 in the keratoconus group. Combination of 12% stretching and PEG 2 induced LOXL-4 down-regulation in in healthy group, and all LOXs except LOXL-1 in keratoconus group. The results suggested that combination of mechanical stretching and PGE 2 down-regulate the gene expression of LOXs in keratoconus. Lower LOXs expression may lead to impaired cross-linking, and thus to a loss of cohesion between collagen fibrils, affecting corneal structural stability by collagen lamellae slippage. This may facilitate the development of keratoconus. Exploring the effects of mechanical stretching and inflammatory factor on the expression LOXs in this paper will help us to understand the possible mechanism of how the keratoconus occurs and develops well, and provide the reference for the prevention and treatment of keratoconus.

Combined effects of interleukin-1ß and cyclic stretching on metalloproteinase expression in corneal fibroblasts in vitro.

BACKGROUND: Corneal tensile strain increases if the cornea becomes thin or if intraocular pressure increases. However, the effects of mechanical stress on extracellular matrix (ECM) remodelling in the corneal repair process and the corneal anomalies are unknown. METHODS: In this study, the combined effects of interleukin-1ß (IL-1ß) on matrix metalloproteinases (MMPs) in corneal fibroblasts under cyclic stretching were investigated in vitro. Cultured rabbit corneal fibroblasts were subjected to 5, 10 or 15 % cyclic equibiaxial stretching at 0.1 Hz for 36 h in the presence of IL-1ß. Conditioned medium was harvested for the analysis of MMP2 and MMP9 protein production using the gelatin zymography and western blot techniques. RESULTS AND CONCLUSIONS: Cyclic equibiaxial stretching changed the cell morphology by increasing the contractility of F-actin fibres. IL-1ß alone induced the expression of MMP9 and increased the production of MMP2, and 5 % stretching alone decreased the production of MMP2, which indicates that a low stretching magnitude can reduce ECM degradation. In the presence of IL-1ß, 5 and 10 % stretching increased the production of MMP2, whereas 15 % stretching increased the production of MMP9. These results indicate that MMP expression is enhanced by cyclic mechanical stimulation in the presence of IL-1ß, which is expected to contribute to corneal ECM degradation, leading to the development of post-refractive surgery keratectasia.

RNA interference to reveal roles of ß-N-acetylglucosaminidase gene during molting process in Locusta migratoria.

ß-N-acetylglucosaminidases are crucial enzymes involved in chitin degradation in insects. We identified a ß-N-acetylglucosaminidase gene (LmNAG1) from Locusta migratoria. The full-length complementary DNA (cDNA) of LmNAG1 consists of 2 667 nucleotides, including an open reading frame (ORF) of 1 845 nucleotides encoding 614 amino acid residues, and 233- and 589-nucleotide non-coding regions at the 5'- and 3'-ends, respectively. Phylogenetic analysis grouped the cDNA-deduced LmNAG1 protein with the enzymatically characterized ß-N-acetylglucosaminidases in group I. Analyses of stage- and tissue-dependent expression patterns of LmNAG1 were carried out by real-time quantitative polymerase chain reaction. Our results showed that LmNAG1 transcript level in the integument was significantly high in the last 2 days of the fourth and fifth instar nymphs. LmNAG1 was highly expressed in foregut and hindgut. RNA interference of LmNAG1 resulted in an effective silence of the gene and a significantly reduced total LmNAG enzyme activity at 48 and 72 h after the injection of LmNAG1 double-stranded RNA (dsRNA). As compared with the control nymphs injected with GFP dsRNA, 50% of the dsLmNAG1-injected nymphs were not able to molt successfully and eventually died. Our results suggest that LmNAG1 plays an essential role in molting process of L. migratoria.