Lysosome Targeting Chimaeras for Glut1-Facilitated Targeted Protein Degradation.

Targeted protein degradation technology holds great potential in biomedicine, particularly in treating tumors and other protein-related diseases. Research on intracellular protein degradation using molecular glues and PROTAC technology is leading, while research on the degradation of membrane proteins and extracellular proteins through the lysosomal pathway is still in the preclinical stage. The scarcity of useful targets is an immense limitation to technological advancement, making it essential to explore novel, potentially effective approaches for targeted lysosomal degradation. Here, we employed the glucose transporter Glut1 as an innovative lysosome-targeting receptor and devised the Glut1-Facilitated Lysosomal Degradation (GFLD) strategy. We synthesized potential Glut1 ligands via reversible addition-fragmentation chain transfer (RAFT) polymerization and acquired antibody-glycooligomer conjugates through bioorthogonal reactions as lysosome-targeting protein degradation molecules, utilized in the management of PD-L1 high-expressing triple-negative breast cancer. The glucose transporter Glut1 as a lysosome-targeting receptor exhibits potential for the advancement of a broader array of medications in the future.

CRISPR/Cas9-mediated efficient white genome editing in the black soldier fly Hermetia illucens.

The CRISPR/Cas9 system is the most straightforward genome-editing technology to date, enabling genetic engineering in many insects, including the black soldier fly, Hermetia illucens. The white gene plays a significant role in the multifarious life activities of insects, especially the pigmentation of the eyes. In this study, the white gene of H. illucens (Hiwhite) was cloned, identified, and bioinformatically analysed for the first time. Using quantitative real-time polymerase chain reaction (qPCR), we found that the white gene was expressed in the whole body of the adult flies, particularly in Malpighian tubules and compound eyes. Furthermore, we utilised CRISPR/Cas9-mediated genome-editing technology to successfully generate heritable Hiwhite mutants using two single guide RNAs. During Hiwhite genome editing, we determined the timing, method, and needle-pulling parameters for embryo microinjection by observing early embryonic developmental features. We used the CasOT program to obtain highly specific guide RNAs (gRNAs) at the genome-wide level. According to the phenotypes of Hiwhite knockout strains, the pigmentation of larval stemmata, imaginal compound eyes, and ocelli differed from those of the wild type. These phenotypes were similar to those observed in other insects harbouring white gene mutations. In conclusion, our results described a detailed white genome editing process in black soldier flies, which lays a solid foundation for intensive research on the pigmentation pathway of the eyes and provides a methodological basis for further genome engineering applications in black soldier flies.

Amphiphilic Block Copolymers Containing Benzenesulfonyl Azide Groups as Visible Light-Responsive Drug Carriers for Image-Guided Delivery.

Nanoparticles (NPs) containing light-responsive polymers and imaging agents show great promise for controlled drug delivery. However, most light-responsive NPs rely on short-wavelength excitation, resulting in poor tissue penetration and potential cytotoxicity. Moreover, excessively sensitive NPs may prematurely release drugs during storage and circulation, diminishing their efficacy and causing off-target toxicity. Herein, we report visible-light-responsive NPs composed of an amphiphilic block copolymer containing responsive 4-acrylamide benzenesulfonyl azide (ABSA) and hydrophilic N,N'-dimethylacrylamide (DMA) units. The polymer pDMA-ABSA was loaded with the chemotherapy drug dasatinib and zinc tetraphenylporphyrin (ZnTPP). ZnTPP acted as an imaging reagent and a photosensitizer to reduce ABSA upon visible light irradiation, converting hydrophobic units to hydrophilic units and disrupting NPs to trigger drug release. These NPs enabled real-time fluorescence imaging in cells and exhibited synergistic chemophotodynamic therapy against multiple cancer cell lines. Our light-responsive NP platform holds great promise for controlled drug delivery and cancer theranostics, circumventing the limitations of traditional photosensitive nanosystems.

RNF34 functions in immunity and selective mitophagy by targeting MAVS for autophagic degradation.

Viral infection triggers the formation of mitochondrial antiviral signaling protein (MAVS) aggregates, which potently promote immune signaling. Autophagy plays an important role in controlling MAVS-mediated antiviral signaling; however, the exact molecular mechanism underlying the targeted autophagic degradation of MAVS remains unclear. Here, we investigated the mechanism by which RNF34 regulates immunity and mitophagy by targeting MAVS. RNF34 binds to MAVS in the mitochondrial compartment after viral infection and negatively regulates RIG-I-like receptor (RLR)-mediated antiviral immunity. Moreover, RNF34 catalyzes the K27-/K29-linked ubiquitination of MAVS at Lys 297, 311, 348, and 362 Arg, which serves as a recognition signal for NDP52-dependent autophagic degradation. Specifically, RNF34 initiates the K63- to K27-linked ubiquitination transition on MAVS primarily at Lys 311, which facilitates the autophagic degradation of MAVS upon RIG-I stimulation. Notably, RNF34 is required for the clearance of damaged mitochondria upon viral infection. Thus, we elucidated the mechanism by which RNF34-mediated autophagic degradation of MAVS regulates the innate immune response, mitochondrial homeostasis, and infection.

Supramolecular Self-Assembly in Living Cells.

Supramolecular interactions rely on non-covalent forces, such as hydrophobic effects, hydrogen-bonding, and electrostatic interactions, which govern many intracellular biological pathways. In cellulo supramolecular self-assembly is mainly based on host-guest interactions, changes in pH, enzymes, and polymerization-induced self-assembly to accurately induce various unnatural reactions without disturbing natural biological processes. This process can produce synthetic biocompatible macromolecules to control cell properties and regulate biological functions, such as cell proliferation and differentiation. This Minireview focuses on the latest reports in the field of in cellulo supramolecular self-assembly and anticipates future advances regarding its activation in response to internal and external stimuli, such as pH changes, reactive oxygen species, and enzymes, as well as external light illumination.

C=C Bond Oxidative Cleavage of BODIPY Photocages by Visible Light.

Photocages for protection and the controlled release of bioactive compounds have been widely investigated. However, the vast majority of these photocages employ the cleavage of single bonds and high-energy ultraviolet light. The construction of a photoactivation system that uses visible light to cleave unsaturated bonds still remains a challenge. Herein, we report a regioselective oxidative cleavage of C=C bonds from a boron-dipyrrolemethene (BODIPY)-based photocage by illumination at 630 nm, resulting in a free aldehyde and a thiol fluorescent probe. This strategy was demonstrated in live HeLa cells, and the generated α-formyl-BODIPY allowed real-time monitoring of aldehyde release in the cells. In particular, it is shown that a mannose-functionalized photocage can target HepG2 cells.

PM2.5 induces endothelial dysfunction via activating NLRP3 inflammasome.

PM2.5 (particulate matter <2.5 µm in diameter) is proven to contribute to the development of atherosclerosis. Endothelial cell dysfunction is the initial step of atherosclerosis. The underlying mechanisms of endothelial cell damage exposed to PM2.5 are still obscure. In our study, PM2.5 was administrated to C57BL/6 male mice by intranasal instillation for 2 weeks. Human umbilical vein endothelial cells (HUVECs) were also treated with PM2.5 to evaluate the adverse effect in vitro. The immunohistochemical staining of aortas showed that the expressions of proinflammatory cytokines and endothelial adhesion markers were significantly increased in PM2.5-exposed mice than that in saline-exposed mice. In vitro, PM2.5 could inhibit HUVECs viability and impair cell migration in a concentration-dependent manner. Besides, PM2.5 exposure downregulated eNOS expression while upregulated reactive oxygen species (ROS) levels. Mechanistically, PM2.5 activated the NLRP3 inflammasome in HUVECs while knockdown of NLRP3 could effectively reverse the downregulation of eNOS expression and production of ROS after PM2.5 exposure. In summary, our data showed that PM2.5 could cause endothelial dysfunction, and probably via NLRP3 inflammasome activation.

Bioorthogonal Swarming: In Situ Generation of Dendrimers.

With the aid of bioorthogonal chemistry, we demonstrate the fabrication of synthetic dendrimers in situ around living cells. Using tetrazine dienophile and aminooxyl/hydrazide aldehyde chemistries, the density of functional groups on the dendrimers exponentially amplified intensities of fluorescent markers in antibody-targeted live cell imaging. This novel "swarming" approach highlights the power of bioorthogonal chemistry and provides a route to non-natural chemical structures on cells, paving the way for the generation of various artificial cellular nanostructures and scaffolds.

Long-term coexistence of SARS-CoV-2 with antibody response in COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing coronavirus disease 2019 (COVID-19) has spread worldwide. Whether antibodies are important for the adaptive immune responses against SARS-CoV-2 infection needs to be determined. Here, 26 cases of COVID-19 in Jinan, China, were examined and shown to be mild or with common clinical symptoms, and no case of severe symptoms was found among these patients. Strikingly, a subset of these patients had SARS-CoV-2 and virus-specific IgG coexist for an unexpectedly long time, with two cases for up to 50 days. One COVID-19 patient who did not produce any SARS-CoV-2-bound IgG successfully cleared SARS-CoV-2 after 46 days of illness, revealing that without antibody-mediated adaptive immunity, innate immunity alone may still be powerful enough to eliminate SARS-CoV-2. This report may provide a basis for further analysis of both innate and adaptive immunity in SARS-CoV-2 clearance, especially in nonsevere cases.

Tumor Microenvironment Regulation by the Endoplasmic Reticulum Stress Transmission Mediator Golgi Protein 73 in Mice.

The unfolded protein response (UPR) signal in tumor cells activates UPR signaling in neighboring macrophages, which leads to tumor-promoting inflammation by up-regulating UPR target genes and proinflammatory cytokines. However, the molecular basis of this endoplasmic reticulum (ER) stress transmission remains largely unclear. Here, we identified the secreted form of Golgi protein 73 (GP73), a Golgi-associated protein functional critical for hepatocellular carcinoma (HCC) growth and metastasis, is indispensable for ER stress transmission. Notably, ER stressors increased the cellular secretion of GP73. Through GRP78, the secreted GP73 stimulated ER stress activation in neighboring macrophages, which then released cytokines and chemokines involved in the tumor-associated macrophage (TAM) phenotype. Analysis of HCC patients revealed a positive correlation of GP73 with glucose-regulated protein 78 (GRP78) expression and TAM density. High GP73 and CD206 expression was associated with poor prognosis. Blockade of GP73 decreased the density of TAMs, inhibited tumor growth, and prolonged survival in two mouse HCC models. Conclusion: Our findings provide insight into the molecular mechanisms of extracellular GP73 in the amplification and transmission of ER stress signals.

Long non-coding RNA MEG3 knockdown attenuates endoplasmic reticulum stress-mediated apoptosis by targeting p53 following myocardial infarction.

Mounting evidence has indicated that long non-coding RNA maternally expressed gene 3 (lncRNA MEG3) regulates cell apoptosis, and is involved in a variety of diseases. However, its exact role in myocardial infarction (MI) has not been fully elucidated. In the present study, we firstly observed that the expression levels of the lncRNA MEG3 in infarct hearts and hypoxic neonatal mice ventricular myocytes (NMVMs) were up-regulated by quantitative real-time PCR (qRT-PCR). Then, we knocked down lncRNA MEG3 by lentiviral delivery in the myocardial border region following multipoint injection. Following 28 days of MI, the lncRNA MEG3 knockdown mice indicated better cardiac function, and less cardiac remodelling by ultrasonic cardiogram and histological analysis. In addition, we indicated that lncRNA MEG3 knockdown reduced myocyte apoptosis and reactive oxygen species production in MI mice model and hypoxic NMVMs. Furthermore, we revealed that knockdown of lncRNA MEG3 protected against endoplasmic reticulum stress (ERS)-mediated myocardial apoptosis including the induction of PERK-eIF2α and caspase 12 pathways. At last, we provided evidence that p53 was identified as a protein target of lncRNA MEG3 to regulate NF-κB- and ERS-associated apoptosis. Taken collectively, our findings demonstrated that lncRNA MEG3 knockdown exerted cardioprotection by reducing ERS-mediated apoptosis through targeting p53 post-MI.

Neutrophil Gelatinase-Associated Lipocalin2 Exaggerates Cardiomyocyte Hypoxia Injury by Inhibiting Integrin ß3 Signaling.

BACKGROUND The neutrophil inflammatory protein, lipocalin-2 (NGAL), is elevated in certain forms of cardiac hypertrophy and acute heart failure. However, the specific role of NGAL in cardiac hypoxia injury is unclear. This study aimed to elucidate the functional role of NGAL in cardiomyocyte hypoxia injury. MATERIAL AND METHODS Neonatal rat cardiomyocytes were transfected with adenovirus [(Ad-NGAL] to overexpress human-NGAL and then were exposed to hypoxia for 24 h to establish a hypoxia model. Cell inflammation was detected by RT-PCT and ELISA assay. Cell apoptosis was detected by TUNEL assay. Oxidative stress was also detected by commercial kits. RESULTS An increased inflammatory response, apoptosis, and augmented oxidative stress were observed after exposure to hypoxia, while NGAL overexpression in cells increased the expression and release of inflammatory cytokines. NGAL overexpression also increased the number of apoptotic cells and the imbalance of Bax/Bcl-2 protein expression. Moreover, NGAL overexpression increased the levels of reactive oxygen species and oxidase activity, but reduced anti-oxidase activity. Mechanistically, we found that NGAL decreased the expression of integrin ß3, but not the expression of integrin avß3 and avß5, thus inhibiting the downstream protein AKT. When we used the constitutively activated AKT overexpression adenovirus to activate AKT, the deteriorated phenotype by NGAL was counteracted. CONCLUSIONS NGAL can directly affect cardiomyocytes and cause cardiomyocyte deteriorated hypoxia injury through inhibiting integrin ß3 signaling.

PM2.5 promotes plaque vulnerability at different stages of atherosclerosis and the formation of foam cells via TLR4/MyD88/NFκB pathway.

Clinical evidence has shown an elevated myocardial infarction (MI) risk after PM2.5 (particulate matter < 2.5 µm) exposure. Incident MI may result from rupture of vulnerable plaques. To test whether PM2.5 could promote plaque vulnerability, we exposed PM2.5 to apoe-/- mice by intranasal instillation. We detected the lipid, collagen, macrophage and smooth muscle cells (SMCs) content, and fibrous cap thickness to evaluate the plaque vulnerability. Plaques in HFD-fed mice with PM2.5 treatment for 24 weeks had increased lipid content and macrophage recruitment, and reduced collagen content, fibrous cap thickness and SMCs infiltration. Besides, 4-week exposure to PM2.5 could reduce the fibrous cap thickness, collagen content, but increase the macrophage infiltration and SMCs loss in a rapid atherosclerosis model. In existing plaques, PM2.5 could also decrease the fibrous cap thickness, collagen content. In RAW264.7, PM2.5 could promote the transformation of macrophage into foam cells. The expression of TLR4/MyD88/NFκB and CD36 were upregulated by PM2.5 treatment. Besides, the expression of CD36 promoted by PM2.5 was downregulated by the TLR4 inhibitor or MyD88/NFκB SiRNA. In conclusion, our data indicated that short- and long-term PM2.5 exposure increased plaque vulnerability. The underlying mechanism might be the PM2.5-enhanced formation of foam cells via TLR4/MyD88/NFκB pathway.

Rapid Polymer Conjugation Strategies for the Generation of pH-Responsive, Cancer Targeting, Polymeric Nanoparticles.

The combination of controlled living polymerization in association with rapid and highly efficient macromolecule conjugation strategies provides a powerful tool for the synthesis of novel polymeric materials. Here functional block copolymers were rapidly and quantitatively conjugated using an efficient reaction between polymers containing a phenolic group and the 4-phenyl-3 H-1,2,4-triazole-3,5(4 H)-dione (PTAD) moiety and used to generate nanoparticles that encapsulated drugs. pH responsive amphiphilic block copolymers, which self-assemble into nanoparticles, were fabricated using our novel polymer conjugation strategy with the resulting system designed to promote drug release within the acidic milieu of the cancer microenvironment. The conjugation strategy also enabled the direct tagging of the nanoparticles with a range of fluorophores, targeting assets, or both with cargo release demonstrated in cancer cells.

Exposure to particulate matter induces cardiomyocytes apoptosis after myocardial infarction through NFκB activation.

Clinical evidence has indicated an increased myocardial infarction (MI) morbidity and mortality after exposure to air pollution (particulate matter<2.5 µm, PM2.5). However, the mechanisms by which PM2.5 aggravates MI remain unknown. Present study was to explore the adverse effect of PM2.5 on myocardium after MI and the potential mechanisms. Male mice with MI surgery were treated with PM2.5 by intranasal instillation. Neonatal mice ventricular myocytes (NMVMs) subjected to hypoxia were also incubated with PM2.5 to determine the role of PM2.5 in vitro. Exposure to PM2.5 significantly impaired the cardiac function and increased the infarct size in MI mice. TUNEL assay, flow cytometry and western blotting of Caspase 3, Bax and BCl-2 indicated that PM2.5 exposure could cause cellular apoptosis in vivo and in vitro. Besides, PM2.5 activated NFκB pathway and increased gene expression of IL-1ß and IL-6 in NMVMs with hypoxia, which could be effectively reversed by SN-50-induced blockade of NFκB translocation to the nucleus. In summary, air pollution induces myocardium apoptosis and then impairs cardiac function and aggravates MI via NFκB activation.

Two ghrelin receptor agonists for adults with malnutrition: a systematic review and meta-analysis.

BACKGROUND: Ghrelin receptor agonists have been established to be important in ameliorating the nutritional conditions in patients with malnutrition. However, some studies have reported inconsistent results. We aimed to coalesce the available evidence on the efficacy of ghrelin receptor agonists for the treatment of malnutrition. METHODS: We searched PubMed, the Cochrane Central Register of Controlled Trials, and EMBASE for relevant articles published through March 2016. Studies comparing the efficacy of ghrelin receptor agonists versus placebo in malnourished patients were eligible for inclusion. RESULTS: A total of 12 studies involving 1377 patients were included. Compared with placebo, ghrelin receptor agonists could increase the energy intake (standard mean difference [SMD] 2.67, 95% confidence interval [CI] 1.48 to 3.85, P < 0.001), lean body mass (weighted mean difference [WMD] 0.25 kg, 95% CI 0.07 to 0.42, P = 0.006), fat mass (WMD 0.92 kg, 95% CI 0.05 to 1.8, P = 0.038), and grip strength (WMD 0.31 kg, 95% CI 0.207 to 0.414, P < 0.001) of patients with malnutrition. CONCLUSION: Our analysis indicated that ghrelin receptor agonists could improve the poor nutritional state of malnourished patients by increasing their energy intake, ameliorating their irregular body composition and improving their grip strength. However, these results might be less conclusive due to the limited sample sizes and one potential publication that has not been released.

Statistical properties and error threshold of quasispecies on single-peak Gaussian-distributed fitness landscapes.

The stochastic Eigen model proposed by Feng et al. (2007) (Journal of Theoretical Biology, 246, 28) showed that error threshold is no longer a phase transition point but a crossover region whose width depends on the strength of the random fluctuation in an environment. The underlying cause of this phenomenon has not yet been well examined. In this article, we adopt a single peak Gaussian distributed fitness landscape instead of a constant one to investigate and analyze the change of the error threshold and the statistical property of the quasi-species population. We find a roughly linear relation between the width of the error threshold and the fitness fluctuation strength. For a given quasi-species, the fluctuation of the relative concentration has a minimum with a normal distribution of the relative concentration at the maximum of the averaged relative concentration, it has however a largest value with a bimodal distribution of the relative concentration near the error threshold. The above results deepen our understanding of the quasispecies and error threshold and are heuristic for exploring practicable antiviral strategies.

The role of Toll-like receptor 4 on inflammation and Aß formation in cortex astrocytes.

To investigate the role and possible molecular mechanism of astrocytes in inflammation and amyloid ß-protein (Aß) formation, in this research, by using LPS to stimulate cultured rat astrocytes in vitro with or without anti-Toll-like receptor 4 (TLR4) antibody pretreatment, we first detected the TLR4, TNF-α, IL-1ß, ß-amyloid precursor protein (ß-APP) and ß-site APP clearing enzyme 1 (BACE1) mRNA with real-time PCR, and TLR4, NF-κB/P65 protein in cultured astrocytes by Western blot, and then further probed the translocation of NF-κB/P65 using immunofluorescence and the contents of TNF-α, IL-1ß and Aß in culture supernatant through ELISA. We found that all of these indexes increased at different degrees after LPS-stimulation. However, if pretreatment with anti- TLR4 antibody, such stimulating effects of LPS on the nuclear translocation of NF-κB/P65 and TNF-α, IL-1ß, Aß contents in astrocytic culture supernatant were reduced significantly or disappeared in comparison with the group with only LPS-administration. Our results suggest that TLR4 in astrocytes might play an important role in the inflammation and Aß formation through the TLR4/NF-κB signaling pathway, thus providing new knowledge and understanding of the inflammatory hypothesis of AD pathogenesis.

Protocol to synthesize sequence-controlled glycooligomers for tumor targeting in mice.

Due to the higher and more rapid consumption of carbohydrates by cancer cells compared to normal cells, carbohydrates can be effectively employed as a targeted therapeutic strategy for tumor treatment. Here, we present a protocol for synthesizing sequence-controlled glycooligomers using both solution-phase and solid-phase systems. We outline detailed procedures for evaluating the safety and tumor-targeting properties of the sequence-controlled glycooligomers in vivo. For complete details on the use and execution of this protocol, please refer to Chen et al.1.

Light-mediated intracellular polymerization.

The synthesis of synthetic intracellular polymers offers groundbreaking possibilities in cellular biology and medical research, allowing for novel experiments in drug delivery, bioimaging and targeted cancer therapies. These macromolecules, composed of biocompatible monomers, are pivotal in manipulating cellular functions and pathways due to their bioavailability, cytocompatibility and distinct chemical properties. This protocol details two innovative methods for intracellular polymerization. The first one uses 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) as a photoinitiator for free radical polymerization under UV light (365 nm, 5 mW/cm2). The second method employs photoinduced electron transfer-reversible addition-fragmentation chain-transfer polymerization with visible light (470 nm, 100 mW/cm2). We further elaborate on isolating these intracellular polymers by streptavidin/biotin interaction or immobilized metal ion affinity chromatography for polymers tagged with biotin or histidine. The entire process, from polymerization to isolation, takes ~48 h. Moreover, the intracellular polymers thus generated demonstrate significant potential in enhancing actin polymerization, in bioimaging applications and as a novel avenue in cancer treatment strategies. The protocol extends to animal models, providing a comprehensive approach from cellular to systemic applications. Users are advised to have a basic understanding of organic synthesis and cell biology techniques.