Expression and significance of Fractalkine/CX3CL1 in MPO-AAV-associated glomerulonephritis rats.

OBJECTIVE: To investigate the expression and significance of Fractalkine (CX3CL1, FKN) in serum and renal tissue of myeloperoxidase and anti-neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV) rats. METHODS: Thirty Wistar-Kyoto (WKY) rats were randomly divided into: Control group, MPO-AAV group (400 µg/kg MPO mixed with Freund's complete adjuvant i.p), MPO-AAV + Anti-FKN group (400 µg/kg MPO mixed with Freund's complete adjuvant i.p), anti-FKN group (1 µg/ rat /day, i.p) after 6 weeks. MPO-AAV associated glomerulonephritis model was established by intraperitoneal injection of MPO + Freund's complete adjuvant with 10 mice in each group. The concentration of MPO-ANCA and FKN in serum was detected by Enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was used to detect pathological changes of kidney tissue. Western blot and immunofluorescence staining were used to detect the expression and localization of FKN protein in kidney tissue. Renal function test indicators: 24-hour urinary protein (UAER), blood urea nitrogen (BUN), serum creatinine (Scr). The expression levels of p65NF-κB and IL-6 was detected by Immunohistochemical assays. RESULTS: Compared with the control group, the serum MPO-ANCA antibody expression level in the MPO-AAV group was significantly increased (P < 0.01), and the contents of UAER, BUN and Scr were significantly up-regulated at 24 h (P < 0.01). Compared with the control group, the glomeruli in the MPO-AAV group had different degrees of damage, infiltration of inflammatory cell, and membrane cell hyperplasia and renal tubule edema. Compared with the control group, rats in the MPO-AAV group had significantly higher levels of FKN in serum and renal tissues (P < 0.01), and high expression of p65NF-κB and IL-6 in renal tissues (P < 0.01) (P < 0.05), whereas anti-FKN reversed the expression of the above factors. In MPO-AAV renal tissue, FKN was mainly expressed in the cytoplasm of renal tubular epithelial cells and glomerular podocytes. In addition, the contents of 24 h UAER, BUN and Scr of renal function in MPO-AAV rats were significantly decreased (P < 0.01) and the damage of renal tissue was significantly ameliorated after the administration of antagonistic FKN. CONCLUSION: FKN may play a key role in the pathogenesis of MPO-AAV associated glomerulonephritis.

Probing non-equilibrium inner structure of polymeric nanoparticle via aggregation-induced emission of luminogen.

A molecular segregation inside a nanoparticle was crucial for its properties but usually hard to be determined, especially for organic particles. Herein, non-equilibrium polymeric nanoparticles loading a luminogen via an aggregation-induced emission (AIE) were prepared via an instant formation process, flash nanoprecipitation (FNP). Small organic molecules, polymeric excipients, and oily compounds were used as coprecipitants to reveal effects of conjugate moiety, glass transition temperature (Tg), and a condensed state of a coprecipitant on the fluorescence (FL) intensity of the suspension. The results indicated that the addition of a small molecule in a solid state without any conjugate moiety or a polymeric excipient with high Tg would facilitate enhancing the FL intensity, while a coprecipitant with a conjugate moiety or low Tg or in liquid would decrease the intensity. Moreover, this study revealed that the nanoparticle formed via FNP had a randomly packed inner structure where different compositions tended to evenly distribute inside rather than a micellar structure with a phase-separated core-shell one. These findings provided a guide to selecting a suitable coprecipitant for AIE-luminogen nanoparticles in applications. The developed probing method would also benefit for better understanding the particle formation kinetics in FNP.

Cyr61 Mediates Angiotensin II-Induced Podocyte Apoptosis via the Upregulation of TXNIP.

Purpose: It is well documented that angiotensin II (Ang II) elevation promotes apoptosis of podocytes in vivo and vitro, but the potential mechanism is still oscular. The current study is aimed at probing into the assignment of cysteine-rich protein 61 (Cyr61) in Ang II-induced podocyte apoptosis. Methods: Podocytes were treated with Ang II (10-6 mol/L) for 48 hours to establish an injury model in vitro. Western blot assays were detected the expression of Cyr61, Cyt-c, Bax, and Bcl-2. Gene microarray was used to analyze the expression of mRNAs after treatment with Ang II. CRISPR/Cas9 technology was used to knock down Cyr61 and overexpress TXNIP gene, respectively. Results: The expression of Cyr61, TXNIP, Cyt-c, and Bax in podocytes treated with Ang II were upregulated, but the expression and apoptotic rates of Bcl-2 in podocytes were inhibited. The level of the above factors was not significantly different after the knockdown of Cyr61 with Ang II in podocytes. In Ang II group, when knocked down Cyr61, the expressed level of TXNIP, Cyt-c, and Bax was diminished after Ang II treatment; interestingly Bcl-2 expression and podocyte apoptotic rate were reduced. Under the stimulation of Ang II, the expression of Cyt-c and Bax were growing, whereas Bcl-2 was reduced, and the apoptotic rates were higher in the TXNIP overexpression group. Cyt-c and Bax were put on, whereas that of Bcl-2 was to be cut down when the Cyr61 was knockdown, and the apoptotic rates were gained in the TXNIP overexpression+Cyr61 knockdown group. Conclusions: The results of the study extrapolate that Cyr61 plays a dominant role in Ang II-induced podocyte apoptosis. Additionally, Cyr61 may mediate the Ang II-induced podocyte apoptosis by promoting the expression of TNXIP.

Bioinformatics analysis identifies diagnostic biomarkers and their correlation with immune infiltration in diabetic nephropathy.

Background: Diabetic nephropathy (DN) is a major cause of end-stage renal disease (ESRD). Currently, microalbuminuria is mainly used as a diagnostic indicator of DN, but there are still limitations and lack of immune-related diagnostic markers. In this study, we aimed to explore diagnostic biomarkers associated with immune infiltration of DN. Methods: Immune-related differentially expressed genes (DEGs) were derived from those at the intersection of the ImmPort database and DEGs identified from 3 datasets, which were based on the Gene Expression Omnibus (GEO). Functional enrichment analyses were performed; a protein-protein interaction (PPI) network was constructed; and hub genes were identified by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). After screening the key genes using least absolute shrinkage and selection operator (LASSO) and support vector machine recursive feature elimination (SVM-RFE), a prediction model for DN was constructed. The predictive performance of the model was quantified by receiver-operating characteristic curve, decision curve analysis, and nomogram. Next, infiltration of 22 types of immune cells in DN kidney tissue was evaluated using cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT). Expression of diagnostic markers was analyzed in DN and control patient groups to determine the genes with the maximum diagnostic potential. Finally, we explored the correlation between diagnostic markers and immune cells. Results: Overall, 191 immune-related DEGs were identified, that primarily positively regulated with cell adhesion, T cell activation, leukocyte proliferation and migration, urogenital system development, lymphocyte differentiation and proliferation, and mononuclear cell proliferation. Gene sets were related to the PI3K-Akt, MAPK, Rap1, and WNT signaling pathways. Finally, CCL19, CD1C, and IL33 were identified as diagnostic markers of DN and recognized in the 3 datasets [area under the curve (AUC) =0.921]. Immune cell infiltration analysis demonstrated that CCL19 was positively correlated with macrophages M1 (R=0.47, P<0.001) and macrophages M2 (R=0.75, P<0.001). CD1C was positively correlated with macrophages M1 (R=0.47, P<0.05), macrophages M2 (R=0.75, P<0.01), and monocytes (R=0.42, P<0.01). IL33 was positively correlated with macrophages M1 (R=0.45, P<0.05), macrophages M2 (R=0.74, P<0.01), and monocytes (R=0.41, P<0.01). Conclusions: Our results provide evidence that CCL19, CD1C, and IL33, which are associated with immune infiltration, are the potential diagnostic biomarkers for DN candidates.

Evolutionary analyses of emerging GII.2[P16] and GII.4 Sydney [P16] noroviruses.

GII.2[P16] and GII.4 Sydney [P16] are currently the two predominant norovirus genotypes. This study sought to clarify their evolutionary patterns by analyzing the major capsid VP1 and RNA-dependent RNA polymerase (RdRp) genes. Sequence diversities were analyzed at both nucleotide and amino acid levels. Selective pressures were evaluated with the Hyphy package in different models. Phylogenetic trees were constructed by the maximum likelihood method from full VP1 sequences, and evolutionary rates were estimated by the Bayesian Markov Chain Monte Carlo approach. The results showed that (1) several groups of tightly linked mutations between the RdRp and VP1 genes were detected in the GII.2[P16] and GII.4[P16] noroviruses, and most of these mutations were synonymous, which may lead to a better viral fitness to the host; (2) although the pattern of having new GII.4 variants every 2-4 years has been broken, both the pre- and the post-2015 Sydney VP1 had comparable evolutionary rates to previously epidemic GII.4 variants, and half of the major antigenic sites on GII.4 Sydney had residue substitutions and several caused obvious changes in the carbohydrate-binding surface that may potentially alter the property of the virus; and (3) GII.4 Sydney variants during 2018-21 showed geographical specificity in East Asia, South Asia, and North America; the antigenic sites of GII.2 are strictly conserved, but the GII.2 VP1 chronologically evolved into nine different sublineages over time, with sublineage IX being the most prevalent one since 2018. This study suggested that both VP1 and RdRp of the GII.2[P16] and GII.4 Sydney [P16] noroviruses exhibited different evolutionary directions. GII.4[P16] is likely to generate potential novel epidemic variants by accumulating mutations in the P2 domain, similar to previously epidemic GII.4 variants, while GII.2[P16] has conserved predicted antigenicity and may evolve by changing the properties of nonstructural proteins, such as polymerase replicational fidelity and efficiency. This study expands the understanding of the evolutionary dynamics of GII.2[P16] and GII.4[P16] noroviruses and may predict the emergence of new variants.

Preparation of bioactive glass nanoparticles with highly and evenly doped calcium ions by reactive flash nanoprecipitation.

Nanoscale bioactive glass particles have greater bioactivity than microscale bioactive glass particles, due to their high-specific surface area and fast ion release rate in body fluid. However, preparation of bioactive glass nanoparticles (BGNPs) is difficult since calcium is not easy to be highly doped into the forming silica atom network, leading to an uneven distribution and a low content of calcium. In addition, BGNPs are usually prepared in a dilute solution to avoid agglomeration of the nanoparticles, which decreases the production efficiency and increases the cost. In this work, BGNPs are prepared by a method of the reactive flash nanoprecipitation (RFNP) as well as a traditional sol-gel method. The results indicate that the BGNPs by the RFNP present a smaller size, narrower size distribution, more uniform composition, and better bioactivity than those by the traditional sol-gel method. The obtained BGNPs have uniform compositions close to the feed values. The high and even doping of calcium in the BGNPs is achieved. This successful doping of calcium into nanoparticles by the RFNP demonstrates a promising way to effectively generate high-quality BGNPs for bone repairs.

Facile Fabrication of Highly Porous Bioelectrode Membrane by Combining Breath Figure Process and Self-Assembly Process with Amphiphilic Diblock Copolymer and Hydrophilic Biocatalyst.

This study demonstrated a facile method to fabricate a bioelectrode immobilizing a hydrophilic biocatalyst via self-assembling of the biocatalyst with an amphiphilic diblock copolymer (di-BCP) in the breath figure process. Poly(methyl methacrylate)-block-poly(acrylic acid) (PMMA-b-PAA) as an amphiphilic di-BCP and glucose oxidase (GOD) as a hydrophilic biocatalyst were used in this model study. The electrode membrane presented a highly porous morphology, bringing a large surface area and high permeability to reactants. The sensing performances of the bioelectrode were tested to rapidly and conveniently detect glucose. The results showed that PMMA-b-PAA and GOD had good synergism on the electrocatalytic activity of GOD, and its current response to glucose was remarkably enhanced. The fabricated electrode presented a good reproducibility and durability. The blood glucose test provided a value well consistent with the one measured by an automatic biochemical analyzer with an enzymatic method from a hospital. This technique would be anticipated to build a platform to rapidly and conveniently construct a highly sensitive biosensor immobilizing a hydrophilic biocatalyst.

Porous polystyrene-block-poly(acrylic acid)/hemoglobin membrane formed by dually driven self-assembly and electrochemical application.

This study demonstrated a facile method to form a porous polymeric membrane, immobilizing a biocatalyst. A polyelectrolyte-based amphiphilic diblock copolymer, i.e., polystyrene-block-poly(acrylic acid) (PS-b-PAA), self-assembled with hemoglobin (Hb) dually driven by charge and amphiphilicity during solution-casting and evaporation. XPS and contact angle measurements suggested that the PS block enriched on the membrane surface. The PAA block pointed toward the internal membrane as well as ordered the Hb arrangement at the interface of the polymer and electrode. The obtained PS-b-PAA/Hb electrode showed a remarkably enhanced direct electron transfer (ET), which was revealed to be a surface-controlled process accompanied by single-proton transfer. The membrane was tested to catalyze the reduction of hydrogen peroxide, and exhibited an excellent reproducibility and stability. This method with a charge and amphiphilicity dually driven (CADD) self-assembly opened up a new way to construct a third-generation electrochemical biosensor.

Flash nanoprecipitation: prediction and enhancement of particle stability via drug structure.

Flash nanoprecipitation (FNP) can generate hydrophobic drug nanoparticles in ∼ 100 nm with a much higher drug loading (e.g., > 40 wt %) than traditional nanocarriers (e.g., < 20 wt %). This paper studies the effects of drug molecules on nanoparticle stability made via FNP and demonstrates that chemically bonding a drug compound (e.g., paclitaxel) with a cleavable hydrophobic moiety of organosilicate (e.g., triethoxysilicate) is able to enhance the particle size stability. A nonionic amphiphilic diblock copolymer, poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG), is used as a model surfactant to provide steric stabilization. The experiments here show that the lower the drug solubility in the aqueous medium, the more stable the particles in terms of Ostwald ripening, which are consistent with the prediction by the LSW theory. The initial particle size distribution is sufficiently narrow and of insignificance to Ostwald ripening. To correlate the particle stability with hydrophobicity, this study introduces the n-octanol/water partition coefficient (LogP), a hydrophobicity indication, into the FNP technique. A comparison of various drugs and their analogues shows that LogP of a drug is a better hydrophobicity indication than the solubility parameter (δ) and correlates well with the particle stability. Empirically, with ACDLogP > ∼ 12, nanoparticles have good stability; with ∼ 2 < ACDLogP < ∼ 9, nanoparticles show fast Ostwald ripening and interparticle recrystallization; with ACDLogP < ∼ 2, the drug is very likely difficult to form nanoparticles. This rule creates a quick way to predict particle stability for a randomly selected drug structure and helps to enable a fast preclinical drug screen.

Effects of amphiphilic diblock copolymer on drug nanoparticle formation and stability.

This study systematically compares the effects of amphiphilic diblock copolymer (di-BCP) on stabilizing hydrophobic drug nanoparticles formed by flash nanoprecipitation (FNP), and provides a guideline on choosing suitable di-BCPs. Four widely used di-BCPs, i.e., polystyrene-block-poly(ethylene glycol) (PS-b-PEG), polycaprolactone-block-poly(ethylene glycol) (PCL-b-PEG), polylactide-block-poly(ethylene glycol) (PLA-b-PEG), and poly(lactic-co-glycolic acid) (PLGA-b-PEG), and ß-carotene as a model drug were used. The study showed that PLGA-b-PEG was the most suitable one, whose hydrophobic block was biodegradable and noncrystallizable as well as had relatively high glass transition temperature (Tg) and a right solubility parameter (δ). The molecular weight of PLGA block over the range from 5k to 15k showed an insignificant effect on controlling the particle size. Amorphous drug particles with a high drug loading of over 83 wt% can be achieved. Much remarkable evidence supported the nanoparticles with kinetically frozen and non-equilibrium packing structures of polymer chains rather than either the micelles or micellar nanoparticles with two well segregated polymer blocks. The thermodynamic effects of the drug and BCP on the particle stability, size and structures were discussed by using solubility parameters.

A simple confined impingement jets mixer for flash nanoprecipitation.

Johnson and Prud'homme (2003. AICHE J 49:2264-2282) introduced the confined impingement jets (CIJ) mixer to prepare nanoparticles loaded with hydrophobic compounds (e.g., drugs, inks, fragrances, or pheromones) via flash nanoprecipitation (FNP). We have modified the original CIJ design to allow hand operation, eliminating the need for a syringe pump, and we added a second antisolvent dilution stage. Impingement mixing requires equal flow momentum from two opposing jets, one containing the drug in organic solvent and the other containing an antisolvent, typically water. The subsequent dilution step in the new design allows rapid quenching with high antisolvent concentration that enhances nanoparticle stability. This new CIJ with dilution (CIJ-D) mixer is a simple, cheap, and efficient device to produce nanoparticles. We have made 55 nm diameter ß-carotene nanoparticles using the CIJ-D mixer. They are stable and reproducible in terms of particle size and distribution. We have also compared the performance of our CIJ-D mixer with the vortex mixer, which can operate at unequal flow rates (Liu et al., 2008. Chem Eng Sci 63:2829-2842), to make ß-carotene-containing particles over a series of turbulent conditions. On the basis of dynamic light scattering measurements, the new CIJ-D mixer produces stable particles of a size similar to the vortex mixer. Our CIJ-D design requires less volume and provides an easily operated and inexpensive tool to produce nanoparticles via FNP and to evaluate new nanoparticle formulation.

Polyelectrolyte stabilized drug nanoparticles via flash nanoprecipitation: a model study with beta-carotene.

Polyelectrolyte protected beta-carotene nanoparticles (nanosuspensions) with average diameter of <100 nm were achieved by turbulent mixing and flash nanoprecipitation (FNP). Three types of multi-amine functional polyelectrolytes, epsilon-polylysine (epsilon-PL), poly(ethylene imine) (PEI), and chitosan, were investigated to electrosterically protect the nanoparticles. Particle size and distribution were measured by dynamic light scattering (DLS); particles were imaged via scanning electron microscopy (SEM) and cryogenic transmission electron microscopy (cryo-TEM). Low pH and high polyelectrolyte molecular weight gave the smallest and most stable particles. High drug loading capacity, >80 wt%, was achieved by using either PEI or chitosan. X-ray diffraction (XRD) patterns showed that beta-carotene nanoparticles were amorphous. These findings open the way for utilization of FNP for preparation of nanoparticles with enhanced bioavailability for highly water insoluble drugs.

Maleimide Functionalized Poly(ε-caprolactone)-b-poly(ethylene glycol) (PCL-PEG-MAL): Synthesis, Nanoparticle Formation, and Thiol Conjugation.

Carboxylic acid terminated poly(ε-caprolactone)s (PCL-COOHs) with narrow polydispersity were synthesized and coupled with poly(ethylene glycol) (HO-PEG-OH) to afford PCL-PEG-OH copolymers. The hydroxyl groups in the PCL-PEG-OHs were then converted to maleimide groups to afford maleimide terminated PCL-PEG-MALs that contained 70-90% maleimide functionality. Nanoparticles with maleimide functionality on their surfaces were prepared by impingement mixing. Particle sizes and size distributions were determined by dynamic light scattering. Conjugation of reduced glutathione with model maleimides and two MAL-functional nanoparticles was also demonstrated. The amount of accessible maleimide on the particle surface was measured using Ellman's reagent to range between ~51-67%.

Formation of block copolymer-protected nanoparticles via reactive impingement mixing.

Reactive impingement mixing was employed to produce polymer-protected nanoparticles. Amphiphilic block copolymer was formed in situ by reactive coupling of hydrophobic and hydrophilic blocks. Simultaneously, a hydrophobic compound and the copolymer coprecipitated to form nanoparticles in the range of 100 nm. Specifically, beta-carotene was stabilized by the amphiphilic diblock copolymer, formed from the reaction of an amino-terminated hydrophilic block, poly(ethylene glycol) (PEG-NH2), with an acid chloride-terminated hydrophobic block, either poly(epsilon-caprolactone) (PCL-COCl) or polystyrene (PS-COCl). Spherical particles were observed by scanning and cryogenic transmission electron microscopy. Process conditions, including feed concentration of beta-carotene and feed concentrations of polymeric stabilizers, had little or no effect on average particle sizes over the range studied. Further, for Reynolds numbers greater than 500 the feed flow rates also had no effect. The effect of glass transition temperature (Tg) of the hydrophobic polymer on morphology and particle formation mechanism is discussed.