SOCS5-RBMX stimulates SREBP1-mediated lipogenesis to promote metastasis in steatotic HCC with HBV-related cirrhosis.

Abnormal lipid metabolism promotes hepatocellular carcinoma (HCC) progression, which engenders therapeutic difficulties owing to unclear mechanisms of the phenomenon. We precisely described a special steatotic HCC subtype with HBV-related cirrhosis and probed its drivers. Hematoxylin-eosin (HE) staining of 245 HCC samples revealed a special HCC subtype (41 cases) characterized by HBV-related cirrhosis and intratumoral steatosis without fatty liver background, defined as steatotic HCC with HBV-related cirrhosis (SBC-HCC). SBC-HCC exhibits a larger tumor volume and worse prognosis than non-SBC-HCC. Screening for driver genes promoting fatty acid (FA) biosynthesis in the Gao's HBV-related cirrhosis HCC cases and GSE121248' HBV-related HCC cases revealed that high expression of SOCS5 predicts increased FA synthesis and that SOCS5 is upregulated in SBC-HCC. Through proteomics, metabolomics, and both in vivo and in vitro experiments, we demonstrated that SOCS5 induces lipid accumulation to promote HCC metastasis. Mechanistically, through co-IP and GST-pulldown experiments, we found that the SOCS5-SH2 domain, especially the amino acids Y413 and D443, act as critical binding sites for the RBMX-RRM domain. SOCS5-RBMX costimulates the promoter of SREBP1, inducing de novo lipogenesis, while mutations in the SH2 domain, Y413, and D443 reverse this effect. These findings precisely identified SBC-HCC as a special steatotic HCC subtype and highlighted a new mechanism by which SOCS5 promotes SBC-HCC metastasis.

nSEA: n-Node Subnetwork Enumeration Algorithm Identifies Lower Grade Glioma Subtypes with Altered Subnetworks and Distinct Prognostics.

Advances in molecular characterization have reshaped our understanding of low-grade glioma (LGG) subtypes, emphasizing the need for comprehensive classification beyond histology. Lever-aging this, we present a novel approach, network-based Subnetwork Enumeration, and Analysis (nSEA), to identify distinct LGG patient groups based on dysregulated molecular pathways. Using gene expression profiles from 516 patients and a protein-protein interaction network we generated 25 million sub-networks. Through our unsupervised bottom-up approach, we selected 92 subnetworks that categorized LGG patients into five groups. Notably, a new LGG patient group with a lack of mutations in EGFR, NF1, and PTEN emerged as a previously unidentified patient subgroup with unique clinical features and subnetwork states. Validation of the patient groups on an independent dataset demonstrated the robustness of our approach and revealed consistent survival traits across different patient populations. This study offers a comprehensive molecular classification of LGG, providing insights beyond traditional genetic markers. By integrating network analysis with patient clustering, we unveil a previously overlooked patient subgroup with potential implications for prognosis and treatment strategies. Our approach sheds light on the synergistic nature of driver genes and highlights the biological relevance of the identified subnetworks. With broad implications for glioma research, our findings pave the way for further investigations into the mechanistic underpinnings of LGG subtypes and their clinical relevance.Availability: Source code and supplementary data are available at https://github.com/bebeklab/nSEA.

Engineering Nucleotidoproteins for Base-Pairing-Assisted Cytosolic Delivery and Genome Editing.

Protein therapeutics targeting intracellular machineries hold profound potential for disease treatment, and hence robust cytosolic protein delivery technologies are imperatively demanded. Inspired by the super-negatively charged, nucleotide-enriched structure of nucleic acids, adenylated pro-proteins (A-proteins) with dramatically enhanced negative surface charges have been engineered for the first time via facile green synthesis. Then, thymidine-modified polyethyleneimine is developed, which exhibits strong electrostatic attraction, complementary base pairing, and hydrophobic interaction with A-proteins to form salt-resistant nanocomplexes with robust cytosolic delivery efficiencies. The acidic endolysosomal environment enables traceless restoration of the A-proteins and consequently promotes the intracellular release of the native proteins. This strategy shows high efficiency and universality for a variety of proteins with different molecular weights and isoelectric points in mammalian cells. Moreover, it enables highly efficient delivery of CRISPR-Cas9 ribonucleoproteins targeting fusion oncogene EWSR1-FLI1, leading to pronounced anti-tumor efficacy against Ewing sarcoma. This study provides a potent and versatile platform for cytosolic protein delivery and gene editing, and may benefit the development of protein pharmaceuticals.

One-pot synthesis of dynamically cross-linked polymers for serum-resistant nucleic acid delivery.

Cationic polymers used for nucleic acid delivery often suffer from complicated syntheses, undesired intracellular cargo release and low serum stability. Herein, a series of ternary polymers were synthesized via facile green chemistry to achieve efficient plasmid DNA and mRNA delivery in serum. During the one-pot synthesis of the ternary polymer, acetylphenylboric acid (APBA), polyphenol and low-molecular weight polyethyleneimine (PEI 1.8k) were dynamically cross-linked with each other due to formation of an imine between PEI 1.8k and APBA and formation of a boronate ester between APBA and polyphenol. Series of polyphenols, including ellagic acid (EA), epigallocatechin gallate (EGCG), nordihydroguaiaretic acid (NDGA), rutin (RT) and rosmarinic acid (RA), and APBA molecules, including 2-acetylphenylboric acid (2-APBA), 3-acetylphenylboric acid (3-APBA) and 4-acetylphenylboric acid (4-APBA), were screened and the best-performing ternary polymer, 2-PEI-RT, constructed from RT and 2-APBA, was identified. The ternary polymer featured efficient DNA condensation to favor cellular internalization, and the acidic environment in endolysosomes triggered effective degradation of the polymer to promote cargo release. Thus, 2-PEI-RT showed robust plasmid DNA transfection efficiencies in various tumor cells in serum, outperforming the commercial reagent PEI 25k by 1-3 orders of magnitude. Moreover, 2-PEI-RT mediated efficient cytosolic delivery of Cas9-mRNA/sgRNA to enable pronounced CRISPR-Cas9 genome editing in vitro. Such a facile and robust platform holds great potential for non-viral nucleic acid delivery and gene therapy.

STX5 Inhibits Hepatocellular Carcinoma Adhesion and Promotes Metastasis by Regulating the PI3K/mTOR Pathway.

Background and Aims: Syntaxin 5 (STX5) is a member of the syntaxin or target-soluble SNAP receptor (t-SNARE) family and plays a critical role in autophagy. However, its function and molecular mechanism in tumor cell migration are still unknown. The role of STX5 in influencing hepatocellular carcinoma (HCC) is an important topic in our research. Methods: By using quantitative reverse transcription polymerase chain reaction (qPCR), western blotting, and immunohistochemical analysis of RNA and protein in tissues, we comprehensively evaluated data sets from public databases and clinical patient cohorts for STX5. The correlation of STX5 expression with the clinicopathological characteristics of HCC patients were assessed. In addition, we predicted signal pathways from differentially expressed genes (DEGs) and the Cancer Genome Atlas (TCGA) databases, and confirmed the prediction using integrated transcriptome and RNA-seq. We further investigated the underlying mechanisms of STX5 in the migration and adhesion of HCC cells both in vitro and in vivo. Results: In the TCGA dataset and our patient cohort, STX5 levels were significantly higher in HCC tissues than in adjacent normal liver tissues. At the same time, high expression of STX5 predicted worse prognosis in patients with liver cancer. High expression of STX5 indicates the decrease of adhesion and the increase of migration of HCC cells, and the conversion of epithelial-mesenchymal transition (EMT) in vitro via PI3K/mTOR pathway activation. Conversely, when Sirolimus, a phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) inhibitor acts on cells simultaneously, STX5 overexpression-mediated enhancement of HCC metastasis is reversed. Double-negative regulation of STX5 and mTOR further enhanced the inhibitory effect of STX5 on HCC metastasis. In vivo, STX5 knockdown inhibited the metastasis of HCC cells. Conclusions: Our study demonstrates a novel research result that STX5 promotes HCC metastasis through PI3K/mTOR pathway. We believe that combined inhibition of STX5 and mTOR is a potential treatment for effectively prolonging patient survival and inhibiting HCC metastasis.

Efficient 38.8 W/m2 solar pumped laser with a Ce:Nd:YAG crystal and a Fresnel lens.

Herein, we report a significant improvement in solar-pumped laser collection efficiency based on end-side pumping a 6-mm-diameter 95-mm-length Ce:Nd:YAG/YAG grooved bonded crystal rod. A Fresnel lens, quartz cooling-water tube, and gold-plated conical cavity constituted the solar-energy collection and concentration system, which was designed to maximum pump light absorption and minimize thermal effects in the Ce:Nd:YAG laser medium. To the best of our knowledge, this is the first time that a Ce:Nd:YAG crystal has been pumped by a Fresnel-lens solar-energy collection and concentration system. The 0.69-m2 effective solar-collection area produced 26.93 W of continuous-wave laser power, corresponding to 6.33% slope efficiency. The collection efficiency (38.8 W/m2) was 1.21 times higher than the highest previously reported value for Fresnel-lens solar collection, and is a record for single-beam solar-pumped lasers.

Reactive oxygen species-responsive branched poly (ß-amino ester) with robust efficiency for cytosolic protein delivery.

Protein therapy targeting the intracellular machinery holds great potentials for disease treatment, and therefore, effective cytosolic protein delivery technologies are highly demanded. Herein, we developed reactive oxygen species (ROS)-degradable, branched poly(ß-amino ester) (PBAE) with built-in phenylboronic acid (PBA) in the backbone and terminal-pendent arginine for the efficient cytosolic protein delivery. The PBAE could form stable and cell-ingestible nanocomplexes (NCs) with proteins via electrostatic interaction, nitrogen-boronate (N-B) coordination, and hydrogen bonding, while it can be degraded into small segments by the over-produced H2O2 in tumor cells to enable cytoplasmic protein release. As thus, PBAE exhibited high efficiency in delivering varieties of proteins with distinct molecular weights (12.4-430 kDa) and isoelectric points (4.7-10.5) into tumor cells, including enzymes, toxins, and antibodies. Moreover, PBAE mediated efficient delivery of saporin into tumor cells in vivo, provoking pronounced anti-tumor outcomes. This study provides a robust and versatile platform for cytosolic protein delivery, and the elaborately tailored PBAE may find promising applications for protein-based biological research and disease management. STATEMENT OF SIGNIFICANCE: Cytosolic delivery of native proteins holds great therapeutic potentials, which however, is limited by the lack of robust delivery carriers that can simultaneously feature strong protein encapsulation yet effective intracellular protein release. Herein, ROS-degradable, branched poly(ß-amino ester) (PBAE) with backbone-embedded phenylboronic acid (PBA) and terminal-pendent arginine was developed to synchronize these two processes. PBA and arginine moieties allowed PBAE to encapsulate proteins via N-B coordination, electrostatic interaction, hydrogen bonding, and salt bridging, while PBA could be oxidized by over-produced H2O2 inside cancer cells to trigger PBAE degradation and intracellular protein release. As thus, the top-performing PBAE mediated efficient cytosolic delivery of various proteins including enzymes, toxins, and antibodies. This study provides a powerful platform for cytosolic protein delivery, and may find promising utilities toward intracellular protein therapy against cancer and other diseases such as inflammation.

Cardiomyocyte-targeted anti-inflammatory nanotherapeutics against myocardial ischemia reperfusion (IR) injury.

Myocardial ischemia reperfusion (IR) injury is closely related to the overwhelming inflammation in the myocardium. Herein, cardiomyocyte-targeted nanotherapeutics were developed for the reactive oxygen species (ROS)-ultrasensitive co-delivery of dexamethasone (Dex) and RAGE small interfering RNA (siRAGE) to attenuate myocardial inflammation. PPTP, a ROS-degradable polycation based on PGE2-modified, PEGylated, ditellurium-crosslinked polyethylenimine (PEI) was developed to surface-decorate the Dex-encapsulated mesoporous silica nanoparticles (MSNs), which simultaneously condensed siRAGE and gated the MSNs to prevent the Dex pre-leakage. Upon intravenous injection to IR-injured rats, the nanotherapeutics could be efficiently transported into the inflamed cardiomyocytes via PGE2-assisted recognition of over-expressed E-series of prostaglandin (EP) receptors on the cell membranes. Intracellularly, the over-produced ROS degraded PPTP into small segments, promoting the release of siRAGE and Dex to mediate effective RAGE silencing (72%) and cooperative antiinflammatory effect. As a consequence, the nanotherapeutics notably suppressed the myocardial fibrosis and apoptosis, ultimately recovering the systolic function. Therefore, the current nanotherapeutics represent an effective example for the co-delivery and on-demand release of nucleic acid and chemodrug payloads, and might find promising utilities toward the synergistic management of myocardial inflammation. Electronic Supplementary Material: Supplementary material (experimental methods, RNA and primer sequences, 1H NMR spectra, FTIR spectrum, TEM images, zeta potential, drug loading content, RNA and drug release, cytotoxicity, etc.) is available in the online version of this article at 10.1007/s12274-022-4553-6.

Endocytosis-Independent and Cancer-Selective Cytosolic Protein Delivery via Reversible Tagging with LAT1 substrate.

Protein drugs targeting intracellular machineries have shown profound therapeutic potentials, but their clinical utilities are greatly hampered by the lack of efficient cytosolic delivery techniques. Existing strategies mainly rely on nanocarriers or conjugated cell-penetrating peptides (CPPs), which often have drawbacks such as materials complexity/toxicity, lack of cell specificity, and endolysosomal entrapment. Herein, a unique carrier-free approach is reported for mediating cancer-selective and endocytosis-free cytosolic protein delivery. Proteins are sequentially modified with 4-nitrophenyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzyl carbonate as the H2 O2 -responsive domain and 3,4-dihydroxy-l-phenylalanine as the substrate of l-type amino acid transporter 1 (LAT1). Thus, the pro-protein can be directly transported into tumor cells by overexpressed LAT1 on cell membranes, bypassing endocytosis and endolysosomal entrapment. In the cytosol, overproduced H2 O2 restores the protein structure and activity. Using this technique, versatile proteins are delivered into tumor cells with robust efficiency, including toxins, enzymes, CRISPR-Cas9 ribonucleoprotein, and antibodies. Furthermore, intravenously injected pro-protein of saporin shows potent anticancer efficacy in 4T1-tumor-bearing mice, without provoking systemic toxicity. Such a facile and versatile pro-protein platform may benefit the development of protein pharmaceuticals.

A new nomogram model for prognosis of hepatocellular carcinoma based on novel gene signature that regulates cross-talk between immune and tumor cells.

BACKGROUND: The combined application of immune cells and specific biomarkers related to the tumor immune microenvironment has a better predictive value for the prognosis of HCC. The purpose of this study is to construct a new prognostic model based on immune-related genes that regulate cross-talk between immune and tumor cells to assess the prognosis and explore possible mechanisms. METHOD: The immune cell abundance ratio of 424 cases in the TCGA-LIHC database is obtained through the CIBERSORT algorithm. The differential gene analysis and cox regression analysis is used to screen IRGs. In addition, the function of IRGs was preliminarily explored through the co-culture of M2 macrophages and HCC cell lines. The clinical validation, nomogram establishment and performing tumor microenvironment score were validated. RESULTS: We identified 4 immune cells and 9 hub genes related to the prognosis. Further, we identified S100A9, CD79B, TNFRSF11B as an IRGs signature, which is verified in the ICGC and GSE76427 database. Importantly, IRGs signature is closely related to the prognosis, tumor microenvironment score, clinical characteristics and immunotherapy, and nomogram combined with clinical characteristics is more conducive to clinical promotion. In addition, after co-culture with M2 macrophages, the migration capacity and cell pseudopod of MHCC97H increased significantly. And CD79B and TNFRSF11B were significantly down-regulated in MHCC97H, Huh7 and LM3, while S100A9 was up-regulated. CONCLUSION: We constructed an IRGs signature and discussed possible mechanisms. The nomogram established based on IRGs can accurately predict the prognosis of HCC patients. These findings may provide a suitable therapeutic target for HCC.

Macrophage-targeting gene silencing orchestrates myocardial microenvironment remodeling toward the anti-inflammatory treatment of ischemia-reperfusion (IR) injury.

Ischemia-reperfusion (IR) injury represents a major cause of myocardial dysfunction after infarction and thrombolytic therapy, and it is closely related to the free radical explosion and overwhelming inflammatory responses. Herein, macrophage-targeting nanocomplexes (NCs) are developed to mediate efficient co-delivery of siRNA against MOF (siMOF) and microRNA-21 (miR21) into myocardial macrophages, cooperatively orchestrating the myocardial microenvironment against IR injury. Bioreducible, branched poly(ß-amino ester) (BPAE-SS) is designed to co-condense siMOF and miR21 into NCs in a multivalency-reinforced approach, and they are surface-decorated with carboxylated mannan (Man-COOH) to shield the positive surface charges and enhance the serum stability. The final MBSsm NCs are efficiently internalized by myocardial macrophages after systemic administration, wherein BPAE-SS is degraded into small segments by intracellular glutathione to promote the siMOF/miR21 release, finally provoking efficient gene silencing. Thus, cardiomyocyte protection and macrophage modulation are realized via the combined effects of ROS scavenging, inflammation inhibition, and autophagy attenuation, which ameliorates the myocardial microenvironment and restores the cardiac function via positive cellular crosstalk. This study renders promising solutions to address the multiple systemic barriers against in vivo nucleic acid delivery, and it also offers new options for IR injury by manipulating multiple reciprocal bio-reactions.

Endothelial cell-targeting, ROS-ultrasensitive drug/siRNA co-delivery nanocomplexes mitigate early-stage neutrophil recruitment for the anti-inflammatory treatment of myocardial ischemia reperfusion injury.

Neutrophils serve as a key contributor to the pathophysiology of myocardial ischemia reperfusion injury (MIRI), because the unregulated activation and infiltration of neutrophils lead to overwhelming inflammation in the myocardium to cause tissue damage. Herein, endothelial cell-targeting and  reactive oxygen species (ROS)-ultrasensitive nanocomplexes (NCs) were developed to mediate efficient co-delivery of VCAM-1 siRNA (siVCAM-1) and dexamethasone (DXM), which cooperatively inhibited neutrophil recruitment by impeding neutrophil migration and adhesion. RPPT was first synthesized via crosslinking of PEI 600 with ditellurium followed by modification with PEG and the endothelial cell-targeting peptide cRGD. RPPT was allowed to envelope the DXM-loaded PLGA nanoparticles and condense the siVCAM-1. After systemic administration in rats experiencing MIRI, the cRGD-modified NCs efficiently targeted and entered the inflamed endothelial cells, wherein RPPT was sensitively degraded by over-produced ROS to trigger intracellular siVCAM-1 release and potentiate the VCAM-1 silencing efficiency. As a consequence of the complementary function of DXM and siVCAM-1, the NCs notably mitigated neutrophil infiltration into ischemic myocardium, provoking potent anti-inflammatory efficacy to reduce MIRI and recover cardiac function. The present study offers an effective approach for the controlled co-delivery of siRNA and drug cargoes, and it also highlights the importance of multi-dimensional manipulation of neutrophils in anti-inflammatory treatment. STATEMENT OF SIGNIFICANCE: The unregulated activation and infiltration of neutrophils lead to overwhelming inflammation in the myocardium after myocardial ischemia reperfusion injury (MIRI). Here, endothelial cell-targeting and ROS-ultrasensitive nanocomplexes (NCs), comprised of PLGA NPs decorated with cRGD-poly(ethylene glycol) (PEG)-modified, ditellurium-crosslinked PEI (RPPT), were developed to mediate efficient co-delivery of VCAM-1 siRNA (siVCAM-1) and dexamethasone (DXM). DXM and siVCAM-1 with complementary functions inhibited both the migration and adhesion of neutrophils, efficiently interventing the neutrophil recruitment and interrupting the self-amplified inflammation cascade in the injured myocardium. The molecular design of RPPT renders an effective example for constructing polymeric materials with high ROS sensitivity, and it resolves the critical dilemma related to polycation-mediated siRNA delivery, such as siRNA encapsulation versus release, and transfection efficiency versus toxicity.

Immuno-Engineered Nanodecoys for the Multi-Target Anti-Inflammatory Treatment of Autoimmune Diseases.

Overactivated T cells and overproduced pro-inflammatory cytokines form a self-amplified signaling loop to continuously exacerbate the dysregulated inflammatory response and propel the progression of autoimmune diseases (AIDs). Herein, immuno-engineered nanodecoys (NDs) based on poly(lactic-co-glycolic acid) nanoparticles coated with programmed death-ligand 1 (PD-L1)-expressing macrophage membrane (PRM) are developed to mediate multi-target interruption of the self-promoted inflammatory cascade in AIDs. The PRM collected from IFN-γ-treated RAW 264.7 cells possesses elevated surface levels of adhesion molecule receptors and pro-inflammatory cytokine receptors, and, thus, systemically administered PRM NDs afford higher accumulation level in inflamed tissues and stronger scavenging efficiency toward multiple pro-inflammatory cytokines. More importantly, IFN-γ treatment induces remarkable PD-L1 expression on PRM, thereby allowing PRM NDs to bind membrane-bound programmed death-1 (PD-1) on CD4+ T cell surfaces or neutralize free soluble PD-1, which reconstructs the PD-1/PD-L1 inhibitory axis to suppress CD4+ T cell activation and restore immune tolerance. As such, PRM NDs provoke potent and cooperative anti-inflammatory and immune-suppressive efficacies to alleviate autoimmune damages in Zymosan A-induced arthritis mice and dextran sulfate sodium-induced ulcerative colitis mice. This study provides an enlightened example for the immuno-engineering of cell-membrane-based NDs, rendering promising implications into the treatment of AIDs via multi-target immune-modulation.

Tailoring Hyperbranched Poly(ß-amino ester) as a Robust and Universal Platform for Cytosolic Protein Delivery.

Cytosolic protein delivery is a prerequisite for protein-based biotechnologies and therapeutics on intracellular targets. Polymers that can complex with proteins to form nano-assemblies represent one of the most important categories of materials, because of the ease of nano-fabrication, high protein loading efficiency, no need for purification, and maintenance of protein bioactivity. Stable protein encapsulation and efficient intracellular liberation are two critical yet opposite processes toward cytosolic delivery, and polymers that can resolve these two conflicting challenges are still lacking. Herein, hyperbranched poly(ß-amino ester) (HPAE) with backbone-embedded phenylboronic acid (PBA) is developed to synchronize these two processes, wherein PBA enhanced protein encapsulation via nitrogen-boronate (N-B) coordination while triggered polymer degradation and protein release upon oxidation by H2 O2 in cancer cells. Upon optimization of the branching degree, charge density, and PBA distribution, the best-performing A2-B3-C2-S2 -P2 is identified, which mediates robust delivery of various native proteins/peptides with distinct molecular weights (1.6-430 kDa) and isoelectric points (4.1-10.3) into cancer cells, including enzymes, toxins, antibodies, and CRISPR-Cas9 ribonucleoproteins (RNPs). Moreover, A2-B3-C2-S2 -P2 mediates effective cytosolic delivery of saporin both in vitro and in vivo to provoke remarkable anti-tumor efficacy. Such a potent and universal platform holds transformative potentials for protein pharmaceuticals.

Cytokine-scavenging nanodecoys reconstruct osteoclast/osteoblast balance toward the treatment of postmenopausal osteoporosis.

Imbalance between osteoblasts and osteoclasts accounts for the incidence and deterioration of postmenopausal osteoporosis. Abnormally elevated RANKL and TNF-α levels after menopause promote osteoclast formation and inhibit osteoblast differentiation, respectively. Here, nanodecoys capable of scavenging RANKL and TNF-α were developed from preosteoclast (RAW 264.7 cell) membrane­coated poly(lactic-co-glycolic acid) (PLGA) nanoparticles, which inhibited osteoporosis and maintained bone integrity. The nanodecoys effectively escaped from macrophage capture and enabled prolonged blood circulation after systemic administration. The abundant RANK and TNF-α receptor (TNF-αR) on the cell membranes effectively neutralized RANKL and TNF-α to prevent osteoclastogenesis and promote osteoblastogenesis, respectively, thus reversing the progression of osteoporosis in the ovariectomized (OVX) mouse model. These biomimetic nanodecoys provide an effective strategy for reconstructing the osteoclast/osteoblast balance and hold great potentials for the clinical management of postmenopausal osteoporosis.

A sulfonate-based polypeptide toward infection-resistant coatings.

Multifunctional coatings have gained significant attention for their promising potential to address the issue of medical device-related infections. However, they usually have multiple components in one layer which decreases the density of functional groups on surfaces and hence reduces the biological properties. Herein, we report a mono-component and sulfonate-based anionic polypeptide coating with on-demand antibacterial activity, antifouling property, and biocompatibility. The anionic polypeptide was prepared by ring-opening polymerization of L-cysteine-based N-carboxyanhydride (NCA) with allyl groups and a subsequent thiol-ene reaction to incorporate the sulfonate pendants. It adopted a 17.1-19.5% ß-sheet conformation and self-assembled into a spherical nanoparticle. The polypeptide coating showed excellent in vitro antibacterial activity against both Gram-positive (i.e., S. aureus) and Gram-negative bacteria (i.e., E. coli) with >99% killing efficacy after acidic solution treatment and prominent antifouling property and biocompatibility after weak base treatment. An in vivo study revealed that the sulfonate-based polypeptide-coated polydimethylsiloxane (PDMS) exhibited good anti-infection property and histocompatibility.

Murine Inducible Nitric Oxide Synthase Expression Is Essential for Antifungal Defenses in Kidneys during Disseminated Cryptococcus deneoformans Infection.

Disseminated cryptococcosis has a nearly 70% mortality, mostly attributed to CNS infection, with lesser-known effects on other organs. Immune protection against Cryptococcus relies on Th1 immunity with M1 polarization, rendering macrophages fungicidal. The importance of M1-upregulated inducible NO synthase (iNOS) has been documented in pulmonary anticryptococcal defenses, whereas its role in disseminated cryptococcosis remains controversial. Here we examined the effect of iNOS deletion in disseminated (i.v.) C. deneoformans 52D infection, comparing wild-type (C57BL/6J) and iNOS-/- mice. iNOS-/- mice had significantly reduced survival and nearly 100-fold increase of the kidney fungal burden, without increases in the lungs, spleen, or brain. Histology revealed extensive lesions and almost complete destruction of the kidney cortical area with a loss of kidney function. The lack of fungal control was not due to a failure to recruit immune cells because iNOS-/- mice had increased kidney leukocytes. iNOS-/- mice also showed no defect in T cell polarization. We conclude that iNOS is critically required for local anticryptococcal defenses in the kidneys, whereas it appears to be dispensable in other organs during disseminated infection. This study exemplifies a unique phenotype of local immune defenses in the kidneys and the organ-specific importance of a single fungicidal pathway.

Cytosolic protein delivery via metabolic glycoengineering and bioorthogonal click reactions.

Cytosolic protein delivery holds great potential for the development of protein-based biotechnologies and therapeutics. Currently, cytosolic protein delivery is mainly achieved with the assistance of various carriers. Herein, we present a universal and effective strategy for carrier-free cytosolic protein delivery via metabolic glycoengineering and bioorthogonal click reactions. Ac4ManNAz (AAM), an azido-modified N-acetylmannosamine analogue, was first employed to label tumor cell surfaces with abundant azido groups via glycometabolism. Then, proteins including RNase A, cytochrome C (Cyt C), and bovine serum albumin (BSA) were covalently modified with dibenzocyclooctyne (DBCO). Based on the highly efficient bioorthogonal click reactions between DBCO and azido, DBCO-modified proteins could be efficiently internalized by azido-labeled cancer cells. RNase A-DBCO could largely maintain its enzymatic activity and, thus, led to notable anti-tumor efficacy in HeLa and B16F10 cells in vitro and in B16F10 xenograft tumors in vivo. This study therefore provides a simple and powerful approach for carrier-free protein delivery and would have broad applicability in anti-tumor protein therapy.

Temperature Plays an Essential Regulatory Role in the Tumor Immune Microenvironment.

In recent years, emerging immunotherapy has been included in various malignant tumor treatment standards. Temperature has been considered to affect different pathophysiological reactions such as inflammation and cancer for a long time. However, in tumor immunology research, temperature is still rarely considered a significant variable. In this review, we discuss the effects of room temperature, body temperature, and the local tumor temperature on the tumor immune microenvironment from multiple levels and perspectives, and we discuss changes in the body's local and whole-body temperature under tumor conditions. We analyze the current use of ablation treatment-the reason for the opposite immune effect. We should pay more attention to the therapeutic potential of temperature and create a better antitumor microenvironment that can be combined with immunotherapy.

Guanidine-rich helical polypeptides bearing hydrophobic amino acid pendants for efficient gene delivery.

Non-viral gene delivery vectors with high transfection efficiency both in vitro and in vivo and low cytotoxicity are highly desirable for clinical applications. Herein, a series of guanidine-rich polypeptides bearing hydrophobic amino acid pendants was efficiently prepared via the 1,3-dipolar cycloaddition between azido decorated polypeptide and propargyl functionalized guanidinium and N-acetylamino acids. CD analysis indicated α-helical conformations of all resulting polypeptides in aqueous solution. The guanidine-rich polypeptide/DNA complexes showed significantly enhanced cellular internalization and high cell viability (>90%) in different mammalian cell lines (i.e., HeLa and RAW 264.7) at concentrations of the best performance. The top-performing guanidine-rich polypeptide containing 10% N-acetyl-l-valine pendants outperformed the commercial transfection reagent PEI by 400 times in vitro and 6 times in vivo. This study provides a new guidance for future molecular design of non-viral gene vectors with high delivery efficiency and low cytotoxicity.