Chemical and Structural Engineering of Gelatin-Based Delivery Systems for Therapeutic Applications: A Review.

As a biodegradable and biocompatible protein derived from collagen, gelatin has been extensively exploited as a fundamental component of biological scaffolds and drug delivery systems for precise medicine. The easily engineered gelatin holds great promise in formulating various delivery systems to protect and enhance the efficacy of drugs for improving the safety and effectiveness of numerous pharmaceuticals. The remarkable biocompatibility and adjustable mechanical properties of gelatin permit the construction of active 3D scaffolds to accelerate the regeneration of injured tissues and organs. In this Review, we delve into diverse strategies for fabricating and functionalizing gelatin-based structures, which are applicable to gene and drug delivery as well as tissue engineering. We emphasized the advantages of various gelatin derivatives, including methacryloyl gelatin, polyethylene glycol-modified gelatin, thiolated gelatin, and alendronate-modified gelatin. These derivatives exhibit excellent physicochemical and biological properties, allowing the fabrication of tailor-made structures for biomedical applications. Additionally, we explored the latest developments in the modulation of their physicochemical properties by combining additive materials and manufacturing platforms, outlining the design of multifunctional gelatin-based micro-, nano-, and macrostructures. While discussing the current limitations, we also addressed the challenges that need to be overcome for clinical translation, including high manufacturing costs, limited application scenarios, and potential immunogenicity. This Review provides insight into how the structural and chemical engineering of gelatin can be leveraged to pave the way for significant advancements in biomedical applications and the improvement of patient outcomes.

Comparative Transcriptome Analysis of the Molecular Mechanism of the Hairy Roots of Brassica campestris L. in Response to Cadmium Stress.

Brassica campestris L., a hyperaccumulator of cadmium (Cd), is considered a candidate plant for efficient phytoremediation. The hairy roots of Brassica campestris L are chosen here as a model plant system to investigate the response mechanism of Brassica campestris L. to Cd stress. High-throughput sequencing technology is used to identify genes related to Cd tolerance. A total of 2394 differentially expressed genes (DEGs) are identified by RNA-Seq analysis, among which 1564 genes are up-regulated, and 830 genes are down-regulated. Data from the gene ontology (GO) analysis indicate that DEGs are mainly involved in metabolic processes. Glutathione metabolism, in which glutathione synthetase and glutathione S-transferase are closely related to Cd stress, is identified in the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. A Western blot shows that glutathione synthetase and glutathione S-transferase are involved in Cd tolerance. These results provide a preliminary understanding of the Cd tolerance mechanism of Brassica campestris L. and are, hence, of particular importance to the future development of an efficient phytoremediation process based on hairy root cultures, genetic modification, and the subsequent regeneration of the whole plant.

Antimicrobial research of carbohydrate polymer- and protein-based hydrogels as reservoirs for the generation of reactive oxygen species: A review.

Reactive oxygen species (ROS) play an important role in biological milieu. Recently, the rapid growth in our understanding of ROS and their promise in antibacterial applications has generated tremendous interest in the combination of ROS generators with bulk hydrogels. Hydrogels represent promising supporters for ROS generators and can locally confine the nanoscale distribution of ROS generators whilst also promoting cellular integration via biomaterial-cell interactions. This review highlights recent efforts and progress in developing hydrogels derived from biological macromolecules with embedded ROS generators with a focus on antimicrobial applications. Initially, an overview of passive and active antibacterial hydrogels is provided to show the significance of proper hydrogel selection and design. These are followed by an in-depth discussion of the various approaches for ROS generation in hydrogels. The structural engineering and fabrication of ROS-laden hydrogels are given with a focus on their biomedical applications in therapeutics and diagnosis. Additionally, we discuss how a compromise needs to be sought between ROS generation and removal for maximizing the efficacy of therapeutic treatment. Finally, the current challenges and potential routes toward commercialization in this rapidly evolving field are discussed, focusing on the potential translation of laboratory research outcomes to real-world clinical outcomes.

Wintersweet-like Nanohybrids of Titanium-doped Cerium Vanadate Loaded with Polypyrrole for Tumor Theranostic.

Compromises between enhanced on-targeting reactivity and precise real-time monitoring in the tumor microenvironment (TME) are the main roadblocks for catalytic cancer therapy. The hallmark of a high level of hydrogen peroxide (H2O2) and acidic extracellular environment of the hypoxia solid tumor can underpin therapeutic and tracking performance. Herein, this work provides an activatable wintersweet-like nanohybrid consisting of titanium (Ti) doped cerium vanadate nanorods with the modification of polypyrrole (PPy) nanoparticles (CeVO4-Ti@PPy) for combinatorial therapies of breast carcinoma. The Ti dopants in the size-controllable CeVO4 nanorods lower the energy barrier (0.5 eV) of the rate-determining steps and elaborate peroxidase-like (POD-like) activities to improve the generation of toxic hydroxyl radical (·OH) according to the density functional theory (DFT) calculation. The multiple enzyme-like activities, including the intrinsic glutathione peroxidase (GPx) and catalase (CAT), achieve a record-high therapeutic efficiency. Coupling this oxidative stress with the photothermal effects of PPy enables enhanced catalytic tumor necrosis. The exterior PPy heterogeneous structure can be further doped with protons in the local acidic environment to intensify photoacoustic signals, allowing the non-invasive accurate tracking of tumors. The theranostic performance displayed negligible attenuated signals in near-infrared (NIR) windows. This organic-inorganic nanohybrid with a heterogeneous structure provides the potential to improve the overall outcomes of catalytic therapy.

SETD4 Confers Cancer Stem Cell Chemoresistance in Nonsmall Cell Lung Cancer Patients via the Epigenetic Regulation of Cellular Quiescence.

Increasing evidence indicates that quiescent cancer stem cells (CSCs) are a root cause of chemoresistance. SET domain-containing protein 4 (SETD4) epigenetically regulates cell quiescence in breast cancer stem cells (BCSCs), and SETD4-positive BCSCs are chemoradioresistant. However, the role of SETD4 in chemoresistance, tumor progression, and prognosis in nonsmall cell lung cancer (NSCLC) patients is unclear. Here, SETD4-positive cells were identified as quiescent lung cancer stem cells (qLCSCs) since they expressed high levels of ALDH1 and CD133 and low levels of Ki67. SETD4 expression was significantly higher in advanced-stage NSCLC tissues than in early-stage NSCLC tissues and significantly higher in samples from the chemoresistant group than in those from the chemosensitive group. Patients with high SETD4 expression had shorter progression-free survival (PFS) times than those with low SETD4 expression. SETD4 facilitated heterochromatin formation via H4K20me3, thereby leading to cell quiescence. RNA-seq analysis showed upregulation of genes involved in cell proliferation, glucose metabolism, and PI3K-AKT signaling in activated qLCSCs (A-qLCSCs) compared with qLCSCs. In addition, SETD4 overexpression facilitated PTEN-mediated inhibition of the PI3K-mTOR pathway. In summary, SETD4 confers chemoresistance, tumor progression, and a poor prognosis by regulating CSCs in NSCLC patients.

Rapid diagnosis of fatal Nocardia kroppenstedtii bacteremic pneumonia and empyema thoracis by next-generation sequencing: a case report.

Nocardia species do not replicate as rapidly as other pyogenic bacteria and nocardial infections can be highly fatal, particularly in immunocompromised patients. Here, we present the first report of fatal Nocardia kroppenstedtii bacteremic pneumonia and empyema thoracis diagnosed by next-generation sequencing (NGS) using the Oxford Nanopore Technologies' MinION device. The bacterium was not identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Due to its low equipment cost, short turn-around-time, and portable size, the Oxford Nanopore Technologies' MinION device is a useful platform for NGS in routine clinical microbiology laboratories.

A hierarchical porous aerohydrogel for enhanced water evaporation.

Natural solar-powered steam generation provides a promising strategy to deal with deteriorating water resources. However, the practical applications of this strategy are limited by the tedious manufacturing of structures at micro-nano levels to concentrate heat and transport water to heat-localized regions. Herein, this work reports the fabrication of hierarchically porous aerohydrogel with enhanced light absorption and thermal localization at the air-solid interface. This aerohydrogel steam generator is fabricated by a simple yet controllable micropore generation approach to assemble air and hydrogel into hierarchically porous gas-solid hybrids. The tunable micropore size in a wide range from 99±49µm to 316±58µm not only enables contrasting sunlight absorptance (0.2 - 2.5µm) by reducing the reflection of solar light but also harnesses water transportation to the heating region via a capillary force-driven liquid flow. Therefore, a solar-vapor conversion efficiency of 91.3% under one sun irradiation was achieved using this aerohydrogel evaporator, reaching a ready evaporation rate of 2.76kg m-2 h-1 and 3.71kg m-2 h-1 under one and two sun irradiations, respectively. Our work provides a versatile and scalable approach to engineering porous hydrogels for highly efficient steam generation and opens an avenue for other potential practical applications based on this aerohydrogel.

Diverse and atypical manifestations of Q fever in a metropolitan city hospital: Emerging role of next-generation sequencing for laboratory diagnosis of Coxiella burnetii.

Although Q fever has been widely reported in the rural areas of China, there is a paucity of data on the epidemiology and clinical characteristics of this disease in large metropolitan cities. In this study, we profile the epidemiology and clinical manifestations of Q fever from a tertiary hospital in Shenzhen, a Southern Chinese metropolitan city with a large immigrant population from other parts of China. A total of 14 patients were confirmed to have Q fever during a nine-year-and-six-month period, five of whom were retrospectively diagnosed during case review or incidentally picked up because of another research project on unexplained fever without localizing features. Some patients had the typical exposure histories and clinical features, while a few other patients had rare manifestations of Q fever, including one with heart failure and diffuse intracapillary proliferative glomerulonephritis, a patient presenting with a spontaneous bacterial peritonitis-like syndrome, and another one with concomitant Q fever and brucellosis. Using a combination of clinical manifestation, inflammatory marker levels, echocardiographic findings and serological or molecular test results, nine, three and two patients were diagnosed to have acute, chronic and convalescent Q fever, respectively. Seven, five and two patients were diagnosed to have Q fever by serological test, nested real-time PCR and next-generation sequencing respectively. Diverse and atypical manifestations are associated with Q fever. The incidence of Q fever is likely to be underestimated. Next-generation sequencing is becoming an important diagnostic modality for culture-negative infections, particularly those that the physicians fail to recognize clinically, such as Q fever.

Exosomal DEK removes chemoradiotherapy resistance by triggering quiescence exit of breast cancer stem cells.

Tumor therapeutics often target the primary tumor bulk but fail to eradicate therapy-resistant cancer stem cells (CSCs) in quiescent state. These can then become activated to initiate recurrence and/or metastasis beyond therapy. Here, we identified and isolated chemoradiotherapy-resistant CSCs in quiescent state with high capacity of tumor-initiation and tumorsphere formation from three types of breast tumors in mice. Experiments of knockdown and rescue revealed DEK, a nuclear protein, as essential for CSC activation. Exogenous DEK was then used to trigger quiescence exit of CSCs. ChIP-seq and ATAC-seq showed that DEK directly binds to chromatin, facilitating its genome-wide accessibility. The resulting epigenetic events upregulate the expression of cellular activation-related genes including MYC targets, whereas cellular quiescence-related genes including the p53 signaling pathway are silenced. However, twinned with DEK-induced activation, formerly resistant CSCs were then destroyed by chemotherapy in vitro. In mice, traditional chemoradiotherapy concurrent with the injection of DEK-containing exosomes resulted in eradication of primary tumors together with formerly resistant CSCs without recurrence or metastasis. Our findings advance knowledge of the mechanism of quiescent CSC activation and may provide novel clinical opportunities for removal of quiescence-linked therapy resistance.

Setd4 controlled quiescent c-Kit+ cells contribute to cardiac neovascularization of capillaries beyond activation.

Blood vessels in the adult mammal exist in a highly organized and stable state. In the ischemic heart, limited expansion capacity of the myocardial vascular bed cannot satisfy demands for oxygen supply and the myocardium eventually undergoes irreversible damage. The predominant contribution of endogenous c-Kit+ cells is understood to be in the development and homeostasis of cardiac endothelial cells, which suggests potential for their targeting in treatments for cardiac ischemic injury. Quiescent cells in other tissues are known to contribute to the long-term maintenance of a cell pool, preserve proliferation capacity and, upon activation, facilitate tissue homeostasis and regeneration in response to tissue injury. Here, we present evidence of a Setd4-expressing quiescent c-Kit+ cell population in the adult mouse heart originating from embryonic stages. Conditional knock-out of Setd4 in c-Kit-CreERT2;Setd4f/f;Rosa26TdTomato mice induced an increase in vascular endothelial cells of capillaries in both neonatal and adult mice. We show that Setd4 regulates quiescence of c-Kit+ cells by the PI3K-Akt-mTOR signaling pathway via H4K20me3 catalysis. In myocardial infarction injured mice, Setd4 knock-out resulted in attenuated cardiomyocyte apoptosis, decreased infarction size and improved cardiac function. Lineage tracing in Setd4-Cre;Rosa26mT/mG mice showed that Setd4+ cells contribute to each cardiac lineage. Overall, Setd4 epigenetically controls c-Kit+ cell quiescence in the adult heart by facilitating heterochromatin formation via H4K20me3. Beyond activation, endogenous quiescent c-Kit+ cells were able to improve cardiac function in myocardial infarction injured mice via the neovascularization of capillaries.

5'-UTR SNP of FGF13 causes translational defect and intellectual disability.

The congenital intellectual disability (ID)-causing gene mutations remain largely unclear, although many genetic variations might relate to ID. We screened gene mutations in Chinese Han children suffering from severe ID and found a single-nucleotide polymorphism (SNP) in the 5'-untranslated region (5'-UTR) of fibroblast growth factor 13 (FGF13) mRNA (NM_001139500.1:c.-32c>G) shared by three male children. In both HEK293 cells and patient-derived induced pluripotent stem cells, this SNP reduced the translation of FGF13, which stabilizes microtubules in developing neurons. Mice carrying the homologous point mutation in 5'-UTR of Fgf13 showed delayed neuronal migration during cortical development, and weakened learning and memory. Furthermore, this SNP reduced the interaction between FGF13 5'-UTR and polypyrimidine-tract-binding protein 2 (PTBP2), which was required for FGF13 translation in cortical neurons. Thus, this 5'-UTR SNP of FGF13 interferes with the translational process of FGF13 and causes deficits in brain development and cognitive functions.

SET Domain-Containing Protein 4 Epigenetically Controls Breast Cancer Stem Cell Quiescence.

Quiescent cancer stem cells (CSC) play important roles in tumorigenesis, relapse, and resistance to chemoradiotherapy. However, the determinants of CSC quiescence and how they sustain themselves to generate tumors and relapse beyond resistance to chemoradiotherapy remains unclear. Here, we found that SET domain-containing protein 4 (SETD4) epigenetically controls breast CSC (BCSC) quiescence by facilitating heterochromatin formation via H4K20me3 catalysis. H4K20me3 localized to the promoter regions and regulated the expression of a set of genes in quiescent BCSCs (qBCSC). SETD4-defined qBCSCs were resistant to chemoradiotherapy and promoted tumor relapse in a mouse model. Upon activation, a SETD4-defined qBCSC sustained itself in a quiescent state by asymmetric division and concurrently produced an active daughter cell that proliferated to produce a cancer cell population. Single-cell sequence analysis indicated that SETD4+ qBCSCs clustered together as a distinct cell type within the heterogeneous BCSC population. SETD4-defined quiescent CSCs were present in multiple cancer types including gastric, cervical, ovarian, liver, and lung cancers and were resistant to chemotherapy. SETD4-defined qBCSCs had a high tumorigenesis potential and correlated with malignancy and chemotherapy resistance in clinical breast cancer patients. Taken together, the results from our previous study and current study on six cancer types reveal an evolutionarily conserved mechanism of cellular quiescence epigenetically controlled by SETD4. Our findings provide insights into the mechanism of tumorigenesis and relapse promoted by SETD4-defined quiescent CSCs and have broad implications for clinical therapies. SIGNIFICANCE: These findings advance our knowledge on the epigenetic determinants of quiescence in cancer stem cell populations and pave the way for future pharmacologic developments aimed at targeting drug-resistant quiescent stem cells.

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.