Psychometric property and measurement invariance of perceived benefits and barriers of physical activity in Chinese adolescents.

Background: The perceived benefits and barriers to physical activity play crucial roles in determining daily physical activity levels. However, previous studies have employed tools lacking adequate validation, leading to inconsistent conclusions about the impact of these two factors. Therefore, this national, population-based study was conducted to assess the psychometric properties, measurement invariance, and predictive validity of the Chinese versions of the perceived benefits (C-PBEPA) and barriers to physical activity (C-PBAPA) scales. Methods: The final sample (N = 2942, 49.3 % for boys) was randomly split into two subsamples. The first subsample was used for exploratory factor analysis and the second subsample was used for confirmatory factor analysis. Measurement invariance across gender and age groups were examined. Structural equation models were developed to examine the predictive validity of the revised C-PBEPA and C-PBAPA on moderate to vigorous PA. Results: The results showed that both scales were unidimensional, had excellent model fit (e.g., X 2/df < 3, CFI >0.9, RMSEA <0.06) and demonstrated convergent validity. Findings also revealed lack of scalar invariance for C-PBAPA between preadolescents and adolescents' groups (ΔCFI >0.01) and supported the predictive validity of both scales (p < 0.001). Conclusion: The study demonstrated that the revised C-PBEPA and C-PBAPA are valid scales for measuring Chinese adolescents' perceived benefits and barriers to PA.

Spatiotemporally Controllable Covalent Bonding of RNA for Multi-Protein Interference.

Many diseases are associated with genetic mutation and expression of mutated proteins, such as cancers. Therapeutic approaches that selectively target the synthesis process of multiple proteins show greater potential compared to single-protein approaches in oncological diseases. However, conventional agents to regulate the synthesis of multiple protein still suffer from poor spatiotemporal selectivity and stability. Here, a new method using a dye-peptide conjugate, PRFK, for multi-protein interference with spatiotemporal selectivity and reliable stability, is reported. By using the peptide sequence that targets tumor cells, PRFK can be efficiently taken up, followed by specific binding to the KDELR (KDEL receptor) protein located in the endoplasmic reticulum (ER). The dye generates 1O2 under light irradiation, enabling photodynamic therapy. This process converts the furan group into a cytidine-reactive intermediate, which covalently binds to mRNA, thereby blocking protein synthesis. Upon treating 4T1 cells, the proteomics data show alterations in apoptosis, ferroptosis, proliferation, migration, invasion, and immune infiltration, suggesting that multi-protein interference leads to the disruption of cellular physiological activities, ultimately achieving tumor treatment. This study presents a multi-protein interference probe with the potential for protein interference within various subcellular organelles in the future.

Identification of neutralizing antibodies against monkeypox virus using high-throughput sequencing of A35+H3L+B cells from patients with convalescent monkeypox.

The global monkeypox virus (MPXV) outbreak in 2022 emphasizes the urgent need for effective and accessible new-generation vaccines and neutralizing antibodies. Herein, we identified MPXV-neutralizing antibodies using high-throughput single-cell RNA and V(D)J sequencing of antigen-sorted B cells from patients with convalescent monkeypox. IgG1-expressing B cells were obtained from 34 paired heavy- and light-chain B cell receptor sequences. Subsequently, three potent neutralizing antibodies, MV127, MV128, and MV129, were identified and reacted with the MPXV A35 protein. Among these, MV129, which has a half-maximal inhibitory concentration of 2.68µg/mL against authentic MPXV, was considered to be the putative candidates for MPXV neutralization in response to monkeypox disease.

A novel nanobody broadly neutralizes SARS-CoV-2 via induction of spike trimer dimers conformation.

The ongoing mutations of the SARS-CoV-2 pose serious challenges to the efficacy of the available antiviral drugs, and new drugs with fantastic efficacy are always deserved investigation. Here, a nanobody called IBT-CoV144 is reported, which exhibits broad neutralizing activity against SARS-CoV-2 by inducing the conformation of spike trimer dimers. IBT-CoV144 was isolated from an immunized alpaca using the RBD of wild-type SARS-CoV-2, and it showed strong cross-reactive binding and neutralizing potency against diverse SARS-CoV-2 variants, including Omicron subvariants. Moreover, the prophylactically and therapeutically intranasal administration of IBT-CoV144 confers fantastic protective efficacy against the challenge of Omicron BA.1 variant in BALB/c mice model. The structure analysis of the complex between spike (S) protein, conducted using Cryo-EM, revealed a special conformation known as the trimer dimers. This conformation is formed by two trimers, with six RBDs in the "up" state and bound by six VHHs. IBT-CoV144 binds to the lateral region of the RBD on the S protein, facilitating the aggregation of S proteins. This aggregation results in steric hindrance, which disrupts the recognition of the virus by ACE2 on host cells. The discovery of IBT-CoV144 will provide valuable insights for the development of advanced therapeutics and the design of next-generation vaccines.

Ovarian microenvironment: challenges and opportunities in protecting against chemotherapy-associated ovarian damage.

BACKGROUND: Chemotherapy-associated ovarian damage (CAOD) is one of the most feared short- and long-term side effects of anticancer treatment in premenopausal women. Accumulating detailed data show that different chemotherapy regimens can lead to disturbance of ovarian hormone levels, reduced or lost fertility, and an increased risk of early menopause. Previous studies have often focused on the direct effects of chemotherapeutic drugs on ovarian follicles, such as direct DNA damage-mediated apoptotic death and primordial follicle burnout. Emerging evidence has revealed an imbalance in the ovarian microenvironment during chemotherapy. The ovarian microenvironment provides nutritional support and transportation of signals that stimulate the growth and development of follicles, ovulation, and corpus luteum formation. The close interaction between the ovarian microenvironment and follicles can determine ovarian function. Therefore, designing novel and precise strategies to manipulate the ovarian microenvironment may be a new strategy to protect ovarian function during chemotherapy. OBJECTIVE AND RATIONALE: This review details the changes that occur in the ovarian microenvironment during chemotherapy and emphasizes the importance of developing new therapeutics that protect ovarian function by targeting the ovarian microenvironment during chemotherapy. SEARCH METHODS: A comprehensive review of the literature was performed by searching PubMed up to April 2024. Search terms included 'ovarian microenvironment' (ovarian extracellular matrix, ovarian stromal cells, ovarian interstitial, ovarian blood vessels, ovarian lymphatic vessels, ovarian macrophages, ovarian lymphocytes, ovarian immune cytokines, ovarian oxidative stress, ovarian reactive oxygen species, ovarian senescence cells, ovarian senescence-associated secretory phenotypes, ovarian oogonial stem cells, ovarian stem cells), terms related to ovarian function (reproductive health, fertility, infertility, fecundity, ovarian reserve, ovarian function, menopause, decreased ovarian reserve, premature ovarian insufficiency/failure), and terms related to chemotherapy (cyclophosphamide, lfosfamide, chlormethine, chlorambucil, busulfan, melphalan, procarbazine, cisplatin, doxorubicin, carboplatin, taxane, paclitaxel, docetaxel, 5-fluorouraci, vincristine, methotrexate, dactinomycin, bleomycin, mercaptopurine). OUTCOMES: The ovarian microenvironment shows great changes during chemotherapy, inducing extracellular matrix deposition and stromal fibrosis, angiogenesis disorders, immune microenvironment disturbance, oxidative stress imbalances, ovarian stem cell exhaustion, and cell senescence, thereby lowering the quantity and quality of ovarian follicles. Several methods targeting the ovarian microenvironment have been adopted to prevent and treat CAOD, such as stem cell therapy and the use of free radical scavengers, senolytherapies, immunomodulators, and proangiogenic factors. WIDER IMPLICATIONS: Ovarian function is determined by its 'seeds' (follicles) and 'soil' (ovarian microenvironment). The ovarian microenvironment has been reported to play a vital role in CAOD and targeting the ovarian microenvironment may present potential therapeutic approaches for CAOD. However, the relation between the ovarian microenvironment, its regulatory networks, and CAOD needs to be further studied. A better understanding of these issues could be helpful in explaining the pathogenesis of CAOD and creating innovative strategies for counteracting the effects exerted on ovarian function. Our aim is that this narrative review of CAOD will stimulate more research in this important field. REGISTRATION NUMBER: Not applicable.

Towards broad-spectrum protection: the development and challenges of combined respiratory virus vaccines.

In the post-COVID-19 era, the co-circulation of respiratory viruses, including influenza, SARS-CoV-2, and respiratory syncytial virus (RSV), continues to have significant health impacts and presents ongoing public health challenges. Vaccination remains the most effective measure for preventing viral infections. To address the concurrent circulation of these respiratory viruses, extensive efforts have been dedicated to the development of combined vaccines. These vaccines utilize a range of platforms, including mRNA-based vaccines, viral vector vaccines, and subunit vaccines, providing opportunities in addressing multiple pathogens at once. This review delves into the major advancements in the field of combined vaccine research, underscoring the strategic use of various platforms to tackle the simultaneous circulation of respiratory viruses effectively.

Surface-engineered erythrocyte membrane-camouflage fluorescent bioprobe for precision ovarian cancer surgery.

PURPOSE: Fluorescence imaging-guided surgery has been used in oncology. However, for tiny tumors, the current imaging probes are still difficult to achieve high-contrast imaging, leading to incomplete resection. In this study, we achieved precise surgical resection of tiny metastatic cancers by constructing an engineering erythrocyte membrane-camouflaged bioprobe (AR-M@HMSN@P). METHODS: AR-M@HMSN@P combined the properties of aggregation-induced emission luminogens (AIEgens) named PF3-PPh3 (P), with functional erythrocyte membrane modified by a modular peptide (AR). Interestingly, AR was composed of an asymmetric tripodal pentapeptide scaffold (GGKGG) with three appended modulars: KPSSPPEE (A6) peptide, RRRR (R4) peptide and cholesterol. To verify the specificity of the probe in vitro, SKOV3 cells with overexpression of CD44 were used as the positive group, and HLF cells with low expression of CD44 were devoted as the control group. The AR-M@HMSN@P fluorescence imaging was utilized to provide surgical guidance for the removal of micro-metastatic lesions. RESULTS: In vivo, the clearance of AR-M@HMSN@P by the immune system was reduced due to the natural properties inherited from erythrocytes. Meanwhile, the A6 peptide on AR-M@HMSN@P was able to specifically target CD44 on ovarian cancer cells, and the electrostatic attraction between the R4 peptide and the cell membrane enhanced the firmness of this targeting. Benefiting from these multiple effects, AR-M@HMSN@P achieved ultra-precise tumor imaging with a signal-to-noise ratio (SNR) of 15.2, making it possible to surgical resection of tumors < 1 mm by imaging guidance. CONCLUSION: We have successfully designed an engineered fluorescent imaging bioprobe (AR-M@HMSN@P), which can target CD44-overexpressing ovarian cancers for precise imaging and guide the resection of minor tumors. Notably, this work holds significant promise for developing biomimetic probes for clinical imaging-guided precision cancer surgery by exploiting their externally specified functional modifications.

Multi-MXene assisted large-scale manufacturing of electrochemical biosensors based on enzyme-nanoflower enhanced electrodes for the detection of H2O2 secreted from live cancer cells.

In situ monitoring of H2O2 in cellular microenvironments plays a critical role in the early diagnosis and pretreatment of cancer, but is limited by the lack of efficient and low-cost strategies for the large-scale preparation of real-time biosensors. Herein, a universal strategy for MXene-based composite inks combined with a scalable screen-printing process is validated in large-scale manufacturing of electrochemical biosensors for in situ detection of H2O2 secreted from live cells. Compositing biocompatible carboxymethyl cellulose (CMCS) with excellent conductive MXene, a water-based ink electrode (MXene/CMCS) with tunable viscosity is efficiently printed with desirable printing accuracy. Subsequently, the MXene/CMCS@HRP electrochemical biosensor exhibits stable electrochemical performance through HRP nanoflower modification, showing rapid electron transport and high electrocatalytic capacity, and demonstrating a low limit of detection (0.29 µM) with a wide linear detection range (0.5 µM-3 mM), superior sensitivity (56.45 µA mM-1 cm-2), long-term stability and high anti-interference ability. Moreover, this electrochemical biosensor is effectively employed for in situ detection of H2O2 secreted from HeLa cells, revealing good biocompatibility and outstanding biosensing capability. This proposed strategy not only extends the possibility of low-cost biomedical devices, but also provides a promising approach for early diagnosis and treatment of cancer.

Exploring the oncogenic potential of circSOD2 in clear cell renal cell carcinoma: a novel positive feedback loop.

BACKGROUND: Existing studies have found that circular RNAs (circRNAs) act as sponges for micro RNAs (miRNAs) to control downstream genes. However, the specific functionalities and mechanisms of circRNAs in human clear cell renal cell carcinoma (ccRCC) have yet to be thoroughly investigated. METHODS: Patient cohorts from online databases were used to screen candidate circRNAs, while another cohort from our hospital was obtained for validation. CircSOD2 was identified as a potential oncogenic target, and its relevant characteristics were investigated during ccRCC progression through various assays. A positive feedback loop containing downstream miRNA and its target gene were identified using bioinformatics and validated by luciferase reporter assays, RNA pull-down, and high-throughput sequencing. RESULTS: CircSOD2 expression was elevated in tumor samples and significantly correlated with overall survival (OS) and the tumor stage of ccRCC patients, which appeared in the enhanced proliferation, invasion, and migration of tumor cells. Through competitive binding to circSOD2, miR-532-3p can promote the expression of PAX5 and the progression of ccRCC, and such regulation can be salvaged by miR-532-3p inhibitor. CONCLUSION: A novel positive feedback loop, PAX5/circSOD2/miR-532-3p/PAX5 was identified in the study, indicating that the loop may play an important role in the diagnosis and prognostic prediction in ccRCC patients.

Virus infection and sphingolipid metabolism.

Cellular sphingolipids have vital roles in human virus replication and spread as they are exploited by viruses for cell entry, membrane fusion, genome replication, assembly, budding, and propagation. Intracellular sphingolipid biosynthesis triggers conformational changes in viral receptors and facilitates endosomal escape. However, our current understanding of how sphingolipids precisely regulate viral replication is limited, and further research is required to comprehensively understand the relationships between viral replication and endogenous sphingolipid species. Emerging evidence now suggests that targeting and manipulating sphingolipid metabolism enzymes in host cells is a promising strategy to effectively combat viral infections. Additionally, serum sphingolipid species and concentrations could function as potential serum biomarkers to help monitor viral infection status in different patients. In this work, we comprehensively review the literature to clarify how viruses exploit host sphingolipid metabolism to accommodate viral replication and disrupt host innate immune responses. We also provide valuable insights on the development and use of antiviral drugs in this area.

A Dual-Polarization Programmable Metasurface for Green and Secure Wireless Communication.

Dual-polarization programmable metasurfaces can flexibly manipulate electromagnetic (EM) waves while providing approximately twice the information capacity. Therefore, they hold significant applications in next-generation communication systems. However, there are three challenges associated with the existing dual-polarization programmable metasurfaces. This article aims to propose a novel design to address them. First, the design overcomes the challenge of element- and polarization-independent controls, enabling more powerful manipulations of EM waves. Second, by using more energy-efficient tunable components and reducing their number, the design can be nearly passive (maximum power consumption of 27.7 mW), leading to a significant decrease in the cost and power consumption of the system (at least two orders of magnitude lower than the power consumption of conventional programmable metasurfaces). Third, the design can operate in a broad bandwidth, which is attractive for practical engineering applications. Both the element and array of the metasurface are meticulously designed, and their performance has been carefully studied. The experiments demonstrate that 2D wide-angle beam scanning can be realized. Moreover, secure communication based on directional information modulation can be implemented by exploiting the metasurface and an efficient discrete optimization algorithm, showing its programmable, multiplexing, broadband, green, and secure features.

TMVP1448, a novel peptide improves detection of primary tumors and metastases by specifically targeting VEGFR-3.

Lymphangiogenesis at primary tumor and draining lymph nodes plays a pivotal role in tumor metastasis, which has been demonstrated to be regulated by the vascular endothelial growth factor receptor 3 (VEGFR-3) pathway. However, the effect of molecular imaging peptides, which specifically bind VEGFR-3, in tracing tumors remains unclear. We prepared a novel peptide, TMVP1448, with high-affinity to VEGFR-3. The dissociation constant (KD) of TMVP1448 with VEGFR-3 was 7.07 ×10-7 M. In vitro cellular assay showed that TMVP1448 could bind specifically with VEGFR-3. Near infrared imaging results showed that Cy7-TMVP1448 was able to accurately trace primary and metastatic cancers, and PET/CT results showed that [68Ga]Ga-DOTA-TMVP1448 was superior to commonly used radiotracers 18F-FDG. Additionally, no significant negative effect of TMVP1448 was found in mice. Our results suggested that TMVP1448 had great potential for future clinical applications in fluorescence imaging and nuclear imaging of tumors.

68Ga-labeled TMTP1 radiotracer for PET imaging of cervical cancer.

Molecular imaging enables visualization and characterization of biological processes that influence tumor behavior and response to therapy. The TMTP1 (NVVRQ) peptide has shown remarkable affinity to highly metastatic tumors and and its potential receptor is aminopeptidase P2. In this study, we have designed and synthesized a 68Ga-labeled cyclic TMTP1 radiotracer (68Ga-DOTA-TMTP1), for PET imaging of cervical cancer. The goal of this study was to investigate the properties of this radiotracer and its tumor diagnostic potential. The radiochemical yield of 68Ga-DOTA-TMTP1 was high and the radiochemical purity was greater than 95%. The octanol-water partition coefficient for 68Ga-DOTA-TMTP1 was -2.76 ± 0.08 and 68Ga-DOTA-TMTP1 has showed excellent stability in in vitro studies. The cellular uptake and efflux of 68Ga-DOTA-TMTP1 in paired highly metastatic and lowly metastatic cervical cancer cell line HeLa and C-33A as well as normal cervical epithelial cell line End1 were measured in a γ counter. 68Ga-DOTA-TMTP1 exhibited higher uptake in HeLa cells than in C-33A cells. The binding to HeLa and C-33A cells could be blocked by excess TMTP1. On microPET images, HeLa tumors were clearly visualized within 60 min and the uptake of the radiotracer in HeLa tumors was higher than that of C-33A tumors. After blocking with TMTP1, HeLa tumors uptake was significantly reduced and the specificity 68Ga-DOTA-TMTP1 was thus validated. Overall, we have successfully synthesized 68Ga-DOTA-TMTP1 with high yield and high specific activity and have demonstrated its potential role for highly metastatic tumor-targeted diagnosis.

Management of PICC rupture in a multidisciplinary collaborative model: A case report.

A peripherally inserted central catheter (PICC) is commonly used for fluid infusion in patients. However, rupture is one of the most serious complications associated with PICC placement. We investigated an 81-year-old patient who experienced a catheter break nearly 11 months after the placement of PICC, followed by a catheter break during catheter capture that was removed after accessing the superior vena cava with a catcher. This case suggests that silicone-based PICCs are fragile and have a high risk of spontaneous dislocation. Therefore, they should be replaced with polyurethane-based PICCs.

Reactive oxygen species-scavenging nanomaterials for the prevention and treatment of age-related diseases.

With increasing proportion of the elderly in the population, age-related diseases (ARD) lead to a considerable healthcare burden to society. Prevention and treatment of ARD can decrease the negative impact of aging and the burden of disease. The aging rate is closely associated with the production of high levels of reactive oxygen species (ROS). ROS-mediated oxidative stress in aging triggers aging-related changes through lipid peroxidation, protein oxidation, and DNA oxidation. Antioxidants can control autoxidation by scavenging free radicals or inhibiting their formation, thereby reducing oxidative stress. Benefiting from significant advances in nanotechnology, a large number of nanomaterials with ROS-scavenging capabilities have been developed. ROS-scavenging nanomaterials can be divided into two categories: nanomaterials as carriers for delivering ROS-scavenging drugs, and nanomaterials themselves with ROS-scavenging activity. This study summarizes the current advances in ROS-scavenging nanomaterials for prevention and treatment of ARD, highlights the potential mechanisms of the nanomaterials used and discusses the challenges and prospects for their applications.

Effects of foliar selenium, biochar, and pig manure on cadmium accumulation in rice grains and assessment of health risk.

Addressing cadmium (Cd) contamination in agricultural lands is crucial, given its health implications and accumulation in crops. This study used pot experiments to evaluate the impact of foliar selenium spray (Se) (0.40 mM), corn straw biochar (1%), and pig manure (1%) on the growth of rice plants, the accumulation of Cd in rice grain, and to examine their influence on health risk indices associated with Cd exposure. The treatments were designated as follows: a control group without any amendment (CK), biochar (T1), pig manure (T2), Se (T3), Se and biochar (T4), Se and pig manure (T5), and Se along with biochar and pig manure (T6). Our results indicated that the treatments affected soil pH and redox potential and improved growth and the nitrogen and phosphorus content in rice plants. The soil-plant analysis development (SPAD) meter readings of leaves during the tillering stage indicated a 5.27%-15.86% increase in treatments T2 to T6 compared to CK. The flag leaves of T2 exhibited increases of 12.06%-38.94% for electrolyte leakage and an 82.61%-91.60% decline in SOD compared to treatments T3 to T6. Treatments T1 to T6 increased protein content; however, amylose content was significantly reduced in T6. Treatment T6 recorded the lowest Cd concentration in rice grains (0.018 mg/kg), while T2 recorded the highest (0.051 mg/kg). The CK treatment group showed a grain Cd content reduction of 29.30% compared to T2. The assessment of acceptable daily intake, hazard quotient, and carcinogenic risk revealed an ascending order as follows: T6 < T3 < T5 < T4 < T1 < CK < T2. In conclusion, the application of treatment T6 demonstrates the potential to lower oxidative stress, enhance production, reduce cancer risk, and ensure the safe cultivation of rice in environments affected by Cd contamination.

Gradient gyroid Ti6Al4V scaffolds with TiO2 surface modification: Promising approach for large bone defect repair.

Large bone defects, particularly those exceeding the critical size, present a clinical challenge due to the limited regenerative capacity of bone tissue. Traditional treatments like autografts and allografts are constrained by donor availability, immune rejection, and mechanical performance. This study aimed to develop an effective solution by designing gradient gyroid scaffolds with titania (TiO2) surface modification for the repair of large segmental bone defects. The scaffolds were engineered to balance mechanical strength with the necessary internal space to promote new bone formation and nutrient exchange. A gradient design of the scaffold was optimized through Finite Element Analysis (FEA) and Computational Fluid Dynamics (CFD) simulations to enhance fluid flow and cell adhesion. In vivo studies in rabbits demonstrated that the G@TiO2 scaffold, featuring a gradient structure and TiO2 surface modification, exhibited superior healing capabilities compared to the homogeneous structure and TiO2 surface modification (H@TiO2) and gradient structure (G) scaffolds. At 12 weeks post-operation, in a bone defect representing nearly 30 % of the total length of the radius, the implantation of the G@TiO2 scaffold achieved a 27 % bone volume to tissue volume (BV/TV) ratio, demonstrating excellent osseointegration. The TiO2 surface modification provided photothermal antibacterial effects, enhancing the scaffold's biocompatibility and potential for infection prevention. These findings suggest that the gradient gyroid scaffold with TiO2 surface modification is a promising candidate for treating large segmental bone defects, offering a combination of mechanical strength, bioactivity, and infection resistance.

Connexin 43 Prevents Radiation-Induced Intestinal Damage via the Ca2+-Dependent PI3K/Akt Signaling Pathway.

Radiation-induced intestinal damage (RIID) is a common side effect of radiotherapy in patients with abdominopelvic malignancies. Gap junctions are special structures consisting of connexins (Cxs). This study aimed to investigate the expression and role of connexins in RIID and underlying mechanism. In this study, a calcein-AM fluorescence probe was used to detect changes in gap junctional intercellular communication in intestinal epithelial IEC-6 cells. Our results show that gap junctional intercellular communication of IEC-6 cells was reduced at 6, 12, 24, and 48 h after irradiation, with the most pronounced effect at 24 h. Western blotting and immunofluorescence results showed that the expression of Cx43, but not other connexins, was reduced in irradiated intestinal epithelial cells. Silencing of Cx43 reduced gap junctional intercellular communication between irradiated intestinal epithelial cells with increased ROS and intracellular Ca2+ levels. Furthermore, knockdown of Cx43 reduced the number of clonal clusters, decreased cell proliferation with increased cytotoxicity and apoptosis. Western blotting results showed that silencing of Cx43 resulted in changed γ-H2AX and PI3K/AKT pathway proteins in irradiated intestinal epithelial cells. Administration of the PI3K/AKT pathway inhibitor LY294002 inhibited the radioprotective effects in Cx43-overexpressing intestinal epithelial cells. Our study demonstrated that Cx43 expression is decreased by ionizing radiation, which facilitates the radioprotection of intestinal epithelial cells.

Expanding the CRISPR Toolbox for Engineering Lycopene Biosynthesis in Corynebacterium glutamicum.

Lycopene represents one of the central compounds in the carotenoid pathway and it exhibits a potent antioxidant ability with wide potential applications in medicine, food, and cosmetics. The microbial production of lycopene has received increasing concern in recent years. Corynebacterium glutamicum (C. glutamicum) is considered to be a safe and beneficial industrial production platform, naturally endowed with the ability to produce lycopene. However, the scarcity of efficient genetic tools and the challenge of identifying crucial metabolic genes impede further research on C. glutamicum for achieving high-yield lycopene production. To address these challenges, a novel genetic editing toolkit, CRISPR/MAD7 system, was established and developed. By optimizing the promoter, ORI and PAM sequences, the CRISPR/MAD7 system facilitated highly efficient gene deletion and exhibited a broad spectrum of PAM sites. Notably, 25 kb of DNA from the genome was successfully deleted. In addition, the CRISPR/MAD7 system was effectively utilized in the metabolic engineering of C. glutamicum, allowing for the simultaneous knockout of crtEb and crtR genes in one step to enhance the accumulation of lycopene by blocking the branching pathway. Through screening crucial genes such as crtE, crtB, crtI, idsA, idi, and cg0722, an optimal carotenogenic gene combination was obtained. Particularly, cg0722, a membrane protein gene, was found to play a vital role in lycopene production. Therefore, the CBIEbR strain was obtained by overexpressing cg0722, crtB, and crtI while strategically blocking the by-products of the lycopene pathway. As a result, the final engineered strain produced lycopene at 405.02 mg/L (9.52 mg/g dry cell weight, DCW) in fed-batch fermentation, representing the highest reported lycopene yield in C. glutamicum to date. In this study, a powerful and precise genetic tool was used to engineer C. glutamicum for lycopene production. Through the modifications between the host cell and the carotenogenic pathway, the lycopene yield was stepwise improved by 102-fold as compared to the starting strain. This study highlights the usefulness of the CRISPR/MAD7 toolbox, demonstrating its practical applications in the metabolic engineering of industrially robust C. glutamicum.

Cellulose Nanofibrils of High Immunoaffinity for Efficient Enrichment of Small Extracellular Vesicles.

Extracellular vesicles (EVs), crucial in facilitating the transport of diverse molecular cargoes for intercellular communication, have shown great potential in diagnostics, therapeutics, and drug delivery. The challenge of developing effective preparation methods for EVs is heightened by their intrinsic heterogeneity and complexity. Here, a novel strategy for high EV enrichment is developed by utilizing EV-affinitive-modified cellulose nanofibrils. Specifically, modified cellulose with rich carboxyl groups has outstanding dispersing properties, able to be dispersed into cellulose nanofibrils in solution. These cellulose nanofibrils are utilized as scaffolds for the immobilization of EV-affinitive antibody of CD63 by chemical conjugation. The CD63-modified nanofibrils demonstrate a superior EV capture efficiency of 86.4% compared with other reported methods. The high performance of this system is further validated by the efficient capture of EVs from biological blood plasma, allowing the detection of bioactive markers from EV-derived miRNAs and proteins. The authors envision that these modified cellulose nanofibrils of enhanced capability on EV enrichment will open new avenues in various biomedical applications.