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Background: The micro-inflammatory state is important for the occurrence of diabetic kidney disease (DKD). Here, we aimed to explore the expression of pyroptosis related indicators and ultrastructural characteristics in DKD, and investigate pyroptosis in renal tubular epithelial cells induced by high glucose., Methods: Immunohistochemistry was used to detect expression of the inflammation-related protein NOD-like receptor protein 3 (NLRP3) and pyroptosis key protein gasdermin D (GSDMD) in kidney tissues of DKD patients. HK-2 cells were cultured in vitro and stimulated with different concentrations of glucose. The changes in HK-2 cell ultrastructure were observed using electronmicroscopy, and western blot was used to detect NLRP3, caspase-1 p20, GSDMD-N, interleukin (IL)-1β, and IL-18 expression., Results: NLRP3 and GSDMD expression in kidney tissues of DKD patients was higher than that in control subjects. Further, GSDMD expression was positively correlated with that of NLRP3 ( r = 0.847, P = 0.02). After stimulating HK-2 cells for 24 h with different glucose concentrations, compared with the control group, the 15 and 30 mmol/L glucose groups showed typical ultrastructural changes of pyroptosis. The protein expression of NLRP3, caspase-1 p20, GSDMD-N, IL-1β, and IL-18 expression in high glucose group increased significantly compared with the control group, and was glucose-concentration-dependent., Conclusion: High glucose can activate inflammasome, cause inflammatory cytokines release, and induce pyroptosis in HK-2 cells. NLRP3-caspase-1 may be involved in GSDMD-mediated pyroptosis. This study shows a novel relationship between glucose concentration and pyroptosis, which can be studied further to design better therapies for patients with DKD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, He, Xiong and Yuan.)

For phase change materials (PCMs) confined in porous structure, the interface interaction between the PCM and the porous skeleton is the key factor to determine the thermal storage performance of PCM. In this study, the modified expanded vermiculite (EVM) was prepared by dopamine modification and DTA + intercalation method and a series of EVM-based composite phase change materials with form shape (fs-CPCMs) were prepared by physical impregnation using n -octadecane as PCMs. The heat storage performance of the composite phase change materials were improved obviously, and the encapsulation ratio is increased to 84.7% and 83.1%. The nonisothermal crystallization process of prepared composite phase change material were further studied. The effects of dopamine modification and organic intercalation on the encapsulation performance of EVM-based fs-CPCMs were analyzed. The results of XRD, FT-IR, thermal cycle, and TGA tests showed that EVM-based composite phase change materials had good chemical compatibility, thermal reliability, and thermal stability. This study provides a theoretical basis for the preparation of EVM-based fs-CPCMs with high thermal stability and good heat storage performance., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Zinc oxide nanoparticles (Nano-ZnO) have been widely used in the food, cosmetics, and biomedical fields due to their excellent antibacterial and antioxidant properties. However, with the widespread application of Nano-ZnO, Nano-ZnO inevitably enters the environment and living organisms, causing harm to human health and ecosystem safety. Therefore, the biosafety and toxicological issues of Nano-ZnO are gradually being emphasized. Our study found that Nano-ZnO has superior antibacterial properties compared to ofloxacin in the fight against Staphylococcus aureus ( S. aureus ). Given that ofloxacin can inhibit bacterial-induced inflammation, we constructed a model of bacterial inflammation using S. aureus in zebrafish. We found that Nano-ZnO inhibited the NF-κB-mediated inflammatory signaling pathway. However, in the process, we found that Nano-ZnO caused hepatic steatosis in zebrafish. This suggested that Nano-ZnO had a certain hepatotoxicity, but did not affect liver development. Subsequently, we investigated the mechanism of hepatotoxicity produced by Nano-ZnO. Nano-ZnO triggered oxidative stress in the liver by generating ROS, which then induced endoplasmic reticulum stress to occur. It further activated srebp and its downstream genes fasn and acc1 , which promoted the accumulation of fatty acid synthesis and the development of steatosis, leading to the development of nonalcoholic fatty liver disease (NAFLD). To address the hepatotoxicity of Nano-ZnO, we added carbon dots for the treatment of NAFLD. The carbon dots were found to normalize the steatotic liver. This provided a new strategy to address the hepatotoxicity caused by Nano-ZnO. In this work, we systematically analyzed the antibacterial advantages of Nano-ZnO in vivo and in vitro, explored the mechanism of Nano-ZnO hepatotoxicity, and proposed a new method to treat Nano-ZnO hepatotoxicity.

Iron overload is the direct cause of many ferroptosis diseases, and it is essential to maintain iron homeostasis. In this paper, we report the Fe 3+ chelation and therapy of the iron overload nonalcoholic fatty liver disease (NAFLD) by the fluorescent egg white-based carbon dots (EWCDs) obtained through the microwave-assisted pyrolysis method. As a high-sensitivity sensor, EWCDs show a high correlation between fluorescence emission and the concentration of Fe 3+ ( R 2 = 0.993) in low concentration ranges of 0-25 μM. In vivo and in vitro, the EWCDs show characteristics of high biocompatibility and specific binding of Fe 3+ . As a novel type of the nano-iron-chelator, EWCDs can successfully attenuate the production of lethal reactive oxygen species. EWCDs not only alleviate the endoplasmic reticulum stress response but also regulate the NF-κB signaling pathway downstream of the Nrf2 signaling pathway. EWCDs prevent hepatocyte apoptosis, regulate fatty acid metabolism, and alleviate inflammation. Ultimately, they alleviate NAFLD induced by iron overload in zebrafish. This work may provide a new idea and method for the application of carbon dots in the field of disease detection and treatment.

Broomcorn millet ( Panicum miliaceum L.) is one of the earliest domesticated crops in the world. Weedy broomcorn millet [ Panicum ruderale (Kitag.) Chang or Panicum miliaceum subsp. ruderale (Kitag.) Tzvel] is thought to be the descendant of the wild ancestor or the feral type of this cereal. The genealogical relationships and genetic divergence among these taxa have not been clarified. In this study, the genetic diversity and population structure of weedy and cultivated broomcorn millets were investigated by using the high-throughput sequencing technology, i.e., the specific-locus amplified fragment sequencing (SLAF-seq). Our analyses consistently revealed both the wild and the feral genotypes in the weedy broomcorn millets. The single nucleotide polymorphisms (SNPs) at the genomic level provided useful evidence to distinguish the wild and the endoferal/exoferal types of weedy broomcorn millets. The genetic divergence revealed between the cultivated broomcorn millet from eastern Eurasia and those from central-western Eurasia was probably derived from either the genetic introgression from weedy broomcorn millets along the spread routes or the founder effect, while the limited gene flow of broomcorn millets from eastern and central-western Eurasia was probably due to the different uses of broomcorn millets and eating habits of the local people., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Liu, Sun, Zhao, He, Li, Lu and Xu.)

The prevalence of erectile dysfunction (ED) has increased in recent decades. Although many treatments offer some benefits for patients with ED, unmet therapeutic needs remain, and promising new approaches are under investigation. One of these approaches is the use of stem-cell (SC) therapy for ED. We comprehensively reviewed the published literature and ongoing phase 1 and phase 2 trials and identified 27 trials by using SC therapy to treat ED. Of the 27 trials, three have been withdrawn, nine have published results, six are complete but without published results, and nine trials are ongoing or have an "unknown" status. Our analysis revealed that SC therapy represents a promising option to treat ED, although published data exist for less than 100 patients. Large placebo-controlled trials with longer follow-up are needed to confirm the long-term safety and efficacy of SC therapy for ED.

In this study, we investigated the structure, luminescence properties and morphology of La 6 Ba 4 (SiO 4 ) 6 F 2 :Dy 3+ in detail using X-ray diffraction (XRD), photoluminescence spectroscopy, scanning electron microscopy (SEM) and decay kinetics measurements. The results indicate that La 6 Ba 4 (SiO 4 ) 6 F 2 :Dy 3+ was well crystallized, and its structure is of apatite-type and belongs to the hexagonal system. The prepared samples exhibit two intense characteristic bands in the blue (484 nm) and yellow (579 nm) spectral ranges corresponding to the Dy 3+ transitions 4 F 9/2 → 6 H 15/2 and 4 F 9/2 → 6 H 13/2 , respectively. These dominant photoluminescence bands are accompanied by a weak red band (670 nm) due to the 4 F 9/2 → 6 H 11/2 transition. The emission color of La 6 Ba 4 (SiO 4 ) 6 F 2 :Dy 3+ phosphors are found to fall in the white light region. The Dy 3+ optimal dopant concentration in the La 6 Ba 4 (SiO 4 ) 6 F 2 host was found to be 0.12 (mol). In addition, the phosphors have high thermal stability. Hence, La 6 Ba 4 (SiO 4 ) 6 F 2 :Dy 3+ may have an application in white light-emitting diodes., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Protein microarrays are a miniaturized format for assaying functional protein interactions and proteomics using a high-throughput, multiplexed approach. The primary limitations of this technology include the time and cost involved in the production of highly purified individual proteins for arraying and also the limited stability of functional proteins once arrayed. In light of these difficulties, cell-free protein expression systems are being increasingly used to generate protein arrays in situ from coding DNA sequences. This chapter describes the method DNA array to protein array (DAPA) allowing the repeated "printing" of protein arrays directly from a DNA array template using cell-free protein expression. Once the DNA templates have been spotted, the generation of the protein array involves only simple handling procedures and is relatively time and cost-efficient, and protein arrays can easily be produced "on demand" as and when required. The resultant protein array may be used for any downstream applications as for conventionally spotted protein arrays.

Ribosome display is a cell-free display technology which enables in vitro selection of antibodies from large recombinant DNA libraries. It also allows continuous introduction of mutations into the selected DNA pool by PCR-based mutagenesis in each cycle, enabling selection of antibody variants with improved affinity, specificity, and stability, thus providing a powerful "protein evolution" tool for optimizing antibody therapeutics. Ribosome display selects required molecules by linking individual proteins (phenotype) with their corresponding mRNAs (genotype) through the formation of stable Protein-Ribosome-mRNA (PRM) complexes. By affinity interaction with an immobilized ligand, the captured PRM complexes are recovered as cDNA using RT-PCR from the ribosome-attached mRNA. The DNA is then subjected to subsequent ribosome display cycles for further enrichment of rare species or cloning, expression, and sequencing to identify wanted candidates. Both prokaryotic and eukaryotic cell-free systems have been developed for ribosome display of different proteins. In this chapter, we describe ribosome display of antibodies using the eukaryotic rabbit reticulocyte system with an in situ single-primer DNA recovery method. A high-throughput Escherichia coli expression format is also described for screening of individual antibody binders from the ribosome-selected population.

Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

The development of protein microarrays makes possible interaction-based protein assays in miniaturised, multiplexed formats. A major requirement determining their uptake and use is the availability and stability of purified, functional proteins for immobilisation. With conventional methods, involving individual expression and purification of recombinant proteins, the cost of commercial high-content protein arrays is often found to be prohibitively high. Moreover, due to the need for specialised microarray production equipment, custom-made protein arrays containing more focussed sets of proteins of interest are also in little use. In the DNA array to protein array technology described herein, repeated economical printing of protein microarrays from a reusable template DNA microarray is performed on demand by cell-free -protein synthesis. Once the template DNA microarray has been obtained, protein microarrays are made using purely macro-handling procedures, making protein arraying accessible without sophisticated microarraying apparatus.

Protein arrays are miniaturised and highly parallelised formats of interaction-based functional protein assays. Major bottlenecks in protein microarraying are the limited availability and high cost of purified, functional proteins for immobilisation and the limited stability of immobilised proteins in their functional state. In contrast, protein-coding DNA is readily available by PCR, and DNA arrays can be stored over prolonged times without deterioration. This chapter presents a method for the rapid and economical "printing" of replicate protein microarrays directly from a single DNA array template using cell-free protein synthesis, termed "DNA array to protein array," DAPA. The procedure is a truly enabling technology, making customised protein microarrays affordable for laboratories with no access to routine microarray spotting. The experimental effort involved for the printing of a protein array from the template DNA array is comparable to the assembly of a Western blot.

Polypeptide and protein arrays enable high-throughput screening capabilities for studying molecular interactions and profiling of biomarkers, and provide a powerful functional screening tool for peptidomics. To overcome the limitations of conventional arraying methods, we have exploited cell-free systems for generating arrays of polypeptides by direct on-chip biosynthesis from DNA templates. Here we describe two protocols: (i) Protein In Situ Array (PISA), which allows the generation of polypeptide arrays in a single reaction by spotting cell-free lysate together with PCR DNA on a glass surface pre-coated with a capturing reagent, and (ii) DNA Array to Protein Array (DAPA), which is capable of producing multiple copies of a polypeptide array from a single DNA array template. The main advantage of these methods is in using an in vitro coupled transcription and translation system which circumvents the need to synthesise and purify individual polypeptides. Our methods allow making polypeptide arrays using amplified linear DNA fragments.

We describe a method for identification of protein-protein interactions by combining two cell-free protein technologies, namely ribosome display and protein in situ immobilisation. The method requires only PCR fragments as the starting material, the target proteins being made through cell-free protein synthesis, either associated with their encoding mRNA as ribosome complexes or immobilised on a solid surface. The use of ribosome complexes allows identification of interacting protein partners from their attached coding mRNA. To demonstrate the procedures, we have employed the lymphocyte signalling proteins Vav1 and Grb2 and confirmed the interaction between Grb2 and the N-terminal SH3 domain of Vav1. The method has promise for library screening of pairwise protein interactions, down to the analytical level of individual domain or motif mapping.

Recent advances in life science have led to great motivation for the development of protein arrays to study functions of genome-encoded proteins. While traditional cell-based methods have been commonly used for generating protein arrays, they are usually a time-consuming process with a number of technical challenges. Cell-free protein synthesis offers an attractive system for making protein arrays, not only does it rapidly converts the genetic information into functional proteins without the need for DNA cloning, but also presents a flexible environment amenable to production of folded proteins or proteins with defined modifications. Recent advancements have made it possible to rapidly generate protein arrays from PCR DNA templates through parallel on-chip protein synthesis. This article reviews current cell-free protein array technologies and their proteomic applications.

Antibodies, in particular human antibodies, are a growing class of new agents with potential diagnostic and therapeutic applications. Advanced DNA and protein technologies allow for the generation of recombinant antibodies in vitro from large DNA libraries. Ribosome display is a cell-free protein display technology that selects desirable antibodies through an efficient search for novel diversity from large populations. The ribosome-selected pools can then be screened rapidly to identify individual antibodies possessing the required activity, without involving the need for cloning by Escherichia coli. The combination of ribosome display, selection and cell-free screening provides a rapid discovery tool for the generation, optimization and modification of antibody-combining sites.

The crystal structures of the Fab' fragment of the anti-progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D-JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three-domain single-chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone-BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild-type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 10(4)-fold for progesterone-3-carboxymethyloxime and 10-fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid-binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position.

A cell-free DiscernArray technology is described for making protein arrays directly from polymerase chain reaction deoxyribonucleic acid (PCR DNA) fragments. In this method, individual tagged proteins are synthesized in a cell-free system on a tag-binding surface, such that the tagged proteins are immobilized on the surface as they are produced. This technology is particularly useful for arraying proteins/domains that cannot be functionally produced by heterologous expression or for which the cloned DNA is not available. Protein arrays of single-chain antibodies, ligand-binding domains, and enzymes have been successfully generated by this method.