Arginine-assembly as NO nano-donor prevents the negative feedback of macrophage repolarization by mitochondrial dysfunction for cancer immunotherapy.

Repolarizing the tumor-associated macrophages (TAMs) towards the antitumoral M1-like phenotype has been a promising approach for cancer immunotherapy. However, the anti-cancer immune response is severely limited mainly by the repolarized M1-like macrophages belatedly returning to the M2-like phenotype (i.e., negative feedback). Inspired by nitric oxide (NO) effectively preventing repolarization of inflammatory macrophages in inflammatory diseases, herein, we develop an arginine assembly, as NO nano-donor for NO generation to prevent the negative feedback of the macrophage repolarization. The strategy is to first apply reversible tagging of hydrophobic terephthalaldehyde to create an arginine nano-assembly, and then load a toll-like receptor 7/8 agonist resiquimod (R848) (R848@Arg). Through this strategy, a high loading efficiency of 40 % for the arginine and repolarization characteristics for TAMs can be achieved. Upon the macrophage repolarization by R848, NO can be intracellularly generated from the released arginine by the upregulated inducible nitric oxide synthase. Mechanistically, NO effectively prevented the negative feedback of the repolarized macrophage by mitochondrial dysfunction via blocking oxidative phosphorylation. Notably, R848@Arg significantly increased the tumor inhibition ratio by 3.13-fold as compared to the free R848 by maintaining the M1-like phenotype infiltrating into tumor. The Arg-assembly as NO nano-donor provides a promising method for effective repolarization of macrophages.

Application of bacteriophage φPaP11-13 attenuates rat Cutibacterium acnes infection lesions by promoting keratinocytes apoptosis via inhibiting PI3K/Akt pathway.

Acne vulgaris caused by antibiotic-resistant Cutibacterium acnes (C. acnes) infection is difficult to treat conventionally. Phages have been suggested as a potential solution, but research on the mechanism of phage treatment is inadequate. This research investigates the underlying molecular mechanisms of phage φPaP11-13 attenuating C. acnes-induced inflammation in rat models. We found that rats infected with C. acnes had higher average ear thickness, greater enrichment of inflammatory cells as shown by hematoxylin-eosin (HE) staining, and fewer TUNEL (TdT-mediated dUTP Nick-End Labeling)-positive keratinocytes visualized by IF staining. Moreover, an increase of IGF-1 and IGF-1 receptor (IGF-1r) was detected using the immunohistochemical (IHC) staining method, Western blot (WB), and quantitative real-time PCR (qRT-PCR) when infected with C. acnes, which was decreased after the application of phage φPaP11-13. By applying the IGF-1 antibody, it was demonstrated that the severity of C. acnes-induced inflammation was relevant to the expression of IGF-1. Through WB and qRT-PCR, activation of the PI3K/Akt pathway and a down-regulation of the BAD-mediated apoptosis pathway were discovered after C. acnes infection. Subsequently, it was shown that the activation of the PI3K/Akt pathway against BAD-mediated apoptosis pathway was alleviated after applying phage φPaP11-13. Furthermore, applying the IGF-1r inhibitor, Pan-PI3K inhibitor, and Akt inhibitor reversed the changing trends of BAD induced by C. acnes and phage φPaP11-13. This study demonstrates that one of the critical mechanisms underlying the attenuation of acne vulgaris by phage φPaP11-13 is lysing C. acnes and regulating keratinocyte apoptosis via the PI3K/Akt signaling pathway.IMPORTANCECutibacterium acnes infection-induced acne vulgaris may cause severe physical and psychological prognosis. However, the overuse of antibiotics develops drug resistance, bringing challenges in treating Cutibacterium acnes. Bacteriophages are currently proven effective in MDR (multiple drug-resistant) Cutibacterium acnes, but there is a significant lack of understanding of phage therapy. This study demonstrated a novel way of curing acne vulgaris by using phages through promoting cell death of excessive keratinocytes in acne lesions by lysing Cutibacterium acnes. However, the regulation of this cell cycle has not been proven to be directly mediated by phages. The hint of ternary relation among "phage-bacteria-host" inspires huge interest in future phage therapy studies.

Nanotechnology-enabled M2 macrophage polarization and ferroptosis inhibition for targeted inflammatory bowel disease treatment.

Transforming macrophages into the anti-inflammatory M2 phenotype could markedly strengthen inflammatory bowel disease (IBD) treatment, which is considered as a promising strategy. However, the high ferroptosis sensitivity of M2 macrophages, which decreases their activity, is a major stumbling block to this strategy. Therefore, promoting M2 polarization while simultaneously inhibiting ferroptosis to tackle this challenge is indispensable. Herein, a calcium­carbonate (CaCO3) mineralized liposome encapsulating a ferroptosis inhibitor (Fer-1) was developed (CaCO3@Lipo@Fer-1, CLF). The CaCO3 mineralized coating shields the liposomes to prevent the release of Fer-1 in circulation, while releasing Ca2+ in the acidic-inflammatory environment. This released Ca2+ promotes M2 polarization through the CaSR/AKT/ß-catenin pathway. The subsequently released Fer-1 effectively upregulates GSH and GPX4, scavenges reactive oxygen species, and inhibits ferroptosis in M2 macrophages. In vivo, CLF improved the targeting efficiency of IBD lesions (about 4.17-fold) through the epithelial enhanced permeability and retention (eEPR) effect and enhanced IBD therapy by increasing the M2/M1 macrophage ratio and inhibiting ferroptosis. We demonstrate that the synergistic regulation of macrophage polarization and ferroptosis sensitivity by this mineralized nanoinhibitor is a viable strategy for IBD therapy.

Source and health risk of urinary neonicotinoids in Tibetan pregnant women.

High altitude could influence the level of exposure to neonicotinoids, but relevant data remain limited for people living in Tibet. We investigated 476 Tibetan pregnant women from Lhasa of Tibet, China in 2021 and measured eight neonicotinoids and four metabolites in urine. Food consumption was investigated by a food frequency questionnaire. Health risk was assessed by using hazard quotient (HQ) and hazard index (HI) based on acceptable daily dose or chronic reference dose. Neonicotinoids and metabolites were overall detected in 56.5% of urine samples with a median concentration being 0.73 µg g-1 creatinine. Four neonicotinoids or metabolites were detected in more than 10% of urine samples, including N-desmethyl-acetamiprid (47.5%), clothianidin (15.5%), thiamethoxam (16.0%), and imidacloprid (10.5%). Annual household income, family smoking, and pre-pregnancy body mass index were associated with the detection frequencies of neonicotinoids. Pregnant women with a higher consumption frequency of wheat, rice, fresh vegetable, fresh fruit, beef and mutton, fresh milk, yoghourt, candy and chocolate, or carbonated drinks had a higher detection frequency of neonicotinoids. Both HQ and HI were less than one. There was an evident exposure to neonicotinoids in Tibetan pregnant women with both plant- and animal-derived food items as exposure sources, but a low health risk was found based on current safety thresholds.

Exposure of Tibetan pregnant women to antibiotics in China: A biomonitoring-based study.

Tibetan people are one Chinese ethnic minority living in Qinghai-Tibet Plateau with an average altitude of more than 4500 m. High altitude could cause a different antibiotic exposure, but relevant information is limited in Tibetan people. We investigated 476 Tibetan pregnant women in Lhasa, Tibet in 2021 and measured 30 antibiotics from five categories in urine, including 13 veterinary antibiotics (VAs), five human antibiotics (HAs), and 12 human/veterinary antibiotics (H/VAs). Food consumption was investigated by a brief food frequency questionnaire. Health risk was assessed by hazard quotient (HQ) and hazard index (HI) based on acceptable daily intakes (ADIs). All antibiotics were overall detected in 34.7% of urine samples with the 75th percentile concentration of 0.19 ng/mL (0.35 µg/g creatinine). HAs, VAs, and H/VAs were respectively detected in 5.3%, 13.0%, and 25.0% of urine samples, with the 95th percentiles of 0.01 ng/mL (0.01 µg/g creatinine), 0.50 ng/mL (0.99 µg/g creatinine), and 3.58 ng/mL (5.02 µg/g creatinine), respectively. Maternal age, smoking of family members, and housework time were associated with detection frequencies of HAs, VAs, or sum of all antibiotics. Pregnant women with a more frequent consumption of fresh milk, egg, yoghourt, poultry meat, and fish had a higher detection frequency of VAs or H/VAs. Only ciprofloxacin and tetracycline had a HQ of larger than one based on microbiological effect in 1.26% and 0.21% of pregnant women, respectively and a HI of larger than one was found in 1.47% of pregnant women. The findings suggested that there was an evident antibiotic exposure from various sources in Tibetan pregnant women with some basic characteristics of pregnant women as potential predictors and several animal-derived food items were important sources of exposure to antibiotic with a fraction of pregnant women in the health risk related to microbiological disruption of gut microbiota.

Pancreatic lipase inhibitory effects of peptides derived from sesame proteins: In silico and in vitro analyses.

Pancreatic lipase (PL) is the main digestive enzyme that is responsible for breaking triglycerides into smaller components for absorption. Inhibition of PL can effectively reduce triglyceride absorption, helping to prevent the development of obesity. The objective of this study was to investigate the PL inhibitory activity of peptides derived from sesame (Sesamum indicum L.) in silico and in vitro. In silico proteolysis of sesame proteins with pepsin, trypsin and chymotrypsin was performed with ExPASy PeptideCutter. Six peptides (TF, EW, QWM, NIF, AGY and PIF) were screened out by PeptideRanker, SwissADME and AutoDock. Molecular docking analysis showed that these six peptides could interact directly with Phe77, His151, Ser152, Phe215 and His263 at the key sites of PL. The six peptides were further synthesized to verify their PL-inhibitory effects in vitro, and TF, EW, QWM, NIF and AGY exhibited inhibitory activity on PL with IC50 values of 751 ± 75, 907 ± 91, 986 ± 170, 1044 ± 179 and 1183 ± 179 µM, respectively. Inhibitory kinetics indicated that TF, QWM and NIF were mixed-type inhibitors of PL, while EW and AGY were uncompetitive inhibitors. Our results suggest that peptides from sesame could potentially inhibit the activity of PL.

Construction of QSAR model based on cysteine-containing dipeptides and screening of natural tyrosinase inhibitors.

Considering that natural products as tyrosinase inhibitors are considered to be safe, with little or no toxic side effects and friendly to the environment, it is urgent to develop a new recognition strategy for natural tyrosinase inhibitors. In current study, an integrated computational analysis was conducted on Cys-containing dipeptides with high tyrosinase inhibitory activity. Firstly, molecular fingerprint similarity (FS) clustering analysis was performed on the target molecule using machine learning. Secondly, genetic algorithm was used to construct two kinds of highly accurate QSAR models (R2  = .978 and .984, respectively) with Cys at C-terminal and N-terminal. Finally, three novel natural candidate inhibitors (NP1, NP2, NP3) were discovered using Molnatsim natural product cluster library, automated screening process and QSAR based on the maximum common substructure (MCS) algorithm, their IC50pre were 260.96, 3.37 and 0.05 µm/mol. Pharmacokinetic predictions showed that NP2 and NP3 had high Bioavailability Score (BS) and Gastrointestinal (GI) absorption, and molecular dynamics simulations further validated the stability of these novel natural candidate inhibitors in binding to tyrosinase. In conclusion, our results provide new ideas for discovering new activities of natural products, and provide an accurate QSAR model for developing novel tyrosinase inhibitors based on MCS Cys-containing dipeptides. PRACTICAL APPLICATIONS: Tyrosinase is related to the occurrence of diseases such as excessive melanin deposition such as freckles and chloasma, and studies have shown that neurodegeneration associated with Parkinson's disease and Huntington's disease is also related. In addition, enzymatic browning on the surface of fresh fruit and vegetable slices will shorten the shelf life and affect their quality. Therefore, screening, designing and developing efficient tyrosinase inhibitors is very important in the fields of medicine, cosmetics, food and so on.

Late-Onset Acute Kidney Injury is a Poor Prognostic Sign for Severe Burn Patients.

Background: Acute kidney injury (AKI) is a morbid complication and the main cause of multiple organ failure and death in severely burned patients. The objective of this study was to explore epidemiology, risk factors, and outcomes of AKI for severely burned patients. Methods: This retrospective study was performed with prospectively collected data of severely burned patients from the Institute of Burn Research in Southwest Hospital during 2011-2017. AKI was diagnosed according to Kidney Disease Improving Global Outcomes (KDIGO) criteria (2012), and it was divided into early and late AKIs depending on its onset time (within the first 3 days or >3 days post burn). The baseline characteristics, clinical data, and outcomes of the three groups (early AKI, late AKI and non-AKI) were compared using logistic regression analysis. Mortality predictors of patients with AKI were assessed. Results: A total of 637 adult patients were included in analysis. The incidence of AKI was 36.9% (early AKI 29.4%, late AKI 10.0%). Multiple logistic regression analysis revealed that age, gender, total burn surface area (TBSA), full-thickness burns of TBSA, chronic comorbidities (hypertension or/and diabetes), hypovolemic shock of early burn, and tracheotomy were independent risk factors for both early and late AKIs. However, sepsis was only an independent risk factor for late AKI. Decompression escharotomy was a protective factor for both AKIs. The mortality of patients with AKI was 32.3% (early AKI 25.7%, late AKI 56.3%), and that of patients without AKI was 2.5%. AKI was independently associated with obviously increased mortality of severely burned patients [early AKI, OR = 12.98 (6.08-27.72); late AKI, OR = 34.02 (15.69-73.75)]. Compared with patients with early AKI, patients with late AKI had higher 28-day mortality (34.9% vs. 19.4%, p = 0.007), 90-day mortality (57.1% vs. 27.4%, p < 0.0001). Conclusions: AKI remains prevalent and is associated with high mortality in severely burned patients. Late-onset acute kidney injury had greater severity and worse prognosis.

Identification of novel saltiness-enhancing peptides from yeast extract and their mechanism of action for transmembrane channel-like 4 (TMC4) protein through experimental and integrated computational modeling.

Excessive consumption of sodium salt is one of the important inducers of cardiovascular and cerebrovascular diseases. The reduction of physical labor and attention to health make research on low-sodium salt imminent. Ultrafiltration, gel filtration, preparative high-performance liquid chromatography, and liquid chromatography with tandem mass spectrometry were employed for further purification and identification of the salty enhancing peptides in yeast extracts. Moreover, human transmembrane channel-like 4 (TMC4) was constructed and evaluated by computer-based methods, and salt-enhancing peptides were identified based on its allosteric sites. PN, NSE, NE and SPE were further determined to be salty enhancing peptides through sensory evaluation, and their taste mechanism was investigated. The results presented here suggest that silicon screening focused on TMC4 allosteric sites and sensory evaluation experiments can greatly increase the discoverability and identifiability of salty enhancer peptides, and this strategy is the first to be applied to the development of salty enhancer peptides.

Molecular mechanism mediating enteric bacterial translocation after severe burn: the role of cystic fibrosis transmembrane conductance regulator.

BACKGROUND: Gut ischemia and hypoxia post severe burn leads to breakdown of intestinal epithelial barrier and enteric bacterial translocation (EBT), resulting in serious complications, such as systemic inflammatory response syndrome, sepsis and multiple organ failure. Cystic fibrosis transmembrane conductance regulator (CFTR) is known to be downregulated by hypoxia and modulate junctional complexes, which are crucial structures maintaining the intestinal barrier. This study aimed to investigate whether CFTR plays a role in both regulating the intestinal barrier and mediating EBT post severe burn, as well as the signaling pathways involved in these processes. METHODS: An in vitro Caco-2 cell model subjected to hypoxic injury and an in vivo mouse model with a 30% total body surface area full-thickness dermal burn were established. DF 508 mice (mice with F508del CFTR gene mutation) were used as an in vivo model to further demonstrate the role of CFTR in maintaining normal intestinal barrier function. QRT-PCR, western blot, ELISA, TER assay and immunofluorescence staining were used to detect the expression and localization of CFTR and tight junction proteins, as well as the function of tight junctions. RESULTS: Our data indicated that, in Caco-2 cells, the hypoxia condition significantly reduced CFTR expression; activated extracellular signal-regulated kinase and nuclear factor-κB signaling; elevated secretion of inflammatory factors (tumor necrosis factor-α, interleukin-1ß and interleukin-8); downregulated zonula occludens-1, occludin and E-cadherin expression; decreased transepithelial electrical resistance values; and led to a cellular mislocation of ZO-1. More importantly, knockdown of CFTR caused similar alterations. The upregulation of inflammatory factors and downregulation of tight junction proteins (ZO-1 and occludin) induced by knockdown of CFTR could be reversed by specific extracellular signal-regulated kinase or nuclear factor-κB inhibition. In support of the in vitro data, exuberant secretion of pro-inflammatory mediators and EBT was observed in the intestine of severely burnt mice in vivo. EBT occurred in DF508 mice (mice with the F508del CFTR gene mutation), accompanied by augmented tumor necrosis factor-α, interleukin-1ß and interleukin-8 levels in the ileum compared to wildtype mice. In addition, vitamin D3 was shown to protect the intestinal epithelial barrier from hypoxic injury. CONCLUSIONS: Collectively, the present study illustrated that CFTR and downstream signaling were critical in modulating the intestinal epithelial junction and EBT post severe burn.

Lytic Bacteriophage Screening Strategies for Multidrug-Resistant Bloodstream Infections in a Burn Intensive Care Unit.

BACKGROUND Increasing antibiotic resistance and multidrug resistance (MDR) in patients with bloodstream infection (BSI) has resulted in treatment using bacteriophage. This study aimed to identify Gram-negative bacilli and Gram-positive cocci and antibiotic resistance in patients with BSI in a burn intensive care unit (BICU). The environment, including sewage systems, were investigated for the presence of lytic bacteriophage. MATERIAL AND METHODS Between January 2011 to December 2017, 486 patients with BSI were admitted to the BICU. Blood culture identified the main infectious organisms. Bacterial screening tests for antibiotic resistance included the D test and the modified Hodge test (MHT). Lytic bacteriophage was isolated from the environment. RESULTS In 486 patients with BSI, the main causative organisms were Gram-negative bacilli (64.6%), Gram-positive cocci (27.7%), and fungi (7.7%). The main pathogenic organisms that showed multidrug resistance (MDR) were Acinetobacter baumannii (26.0%), Staphylococcus aureus (16.8%), and Pseudomonas aeruginosa (14.2%). Bacteriophage was mainly isolated from Gram-negative bacilli. Screening of hospital and residential sewage systems identified increased levels of bacteriophage in hospital sewage. CONCLUSIONS The causative organisms of BSI and the presence of MDR in a hospital BICU were not typical, which supports the need for routine bacterial monitoring. Hospital sewage provides a potential source of bacteriophage for the treatment of MDR pathogenic bacteria.

The Interaction of N-Acetylcysteine and Serum Transferrin Promotes Bacterial Biofilm Formation.

BACKGROUND/AIMS: N-acetylcysteine (NAC) is a novel and promising agent with activity against bacterial biofilms. Human serum also inhibits biofilm formation by some bacteria. We tested whether the combination of NAC and human serum offers greater anti-biofilm activity than either agent alone. METHODS: Microtiter plate assays and confocal laser scanning microscopy were used to evaluate bacterial biofilm formation in the presence of NAC and human serum. qPCR was used to examine expression of selected biofilm-associated genes. Extracellular matrix (ECM) was observed by transmission electron microscopy. The antioxidants GSH or ascorbic acid were used to replace NAC, and human transferrin, lactoferrin, or bovine serum albumin were used to replace serum proteins in biofilm formation assays. A rat central venous catheter model was developed to evaluate the effect of NAC on biofilm formation in vivo. RESULTS: NAC and serum together increased biofilm formation by seven different bacterial strains. In Staphylococcus aureus, expression of genes for some global regulators and for genes in the ica-dependent pathway increased markedly. In Pseudomonas aeruginosa, transcription of las, the PQS quorum sensing (QS) systems, and the two-component system GacS/GacA increased significantly. ECM production by S. aureus and P. aeruginosa was also enhanced. The potentiation of biofilm formation is due mainly to interaction between NAC and transferrin. Intravenous administration of NAC increased colonization by S. aureus and P. aeruginosa on implanted catheters. CONCLUSIONS: NAC used intravenously or in the presence of blood increases bacterial biofilm formation rather than inhibits it.

Concurrent CCR7 Overexpression and RelB Knockdown in Immature Dendritic Cells Induces Immune Tolerance and Improves Skin-Graft Survival in a Murine Model.

BACKGROUND/AIMS: Skin transplantation aims to cover skin defects but often fails due to immune rejection of the transplantated tissue. Immature dendritic cells (imDCs) induce immune tolerance but have a low migration rate. After stimulation, imDCs transform into mature DCs, which activate immune rejection. Thus, inducing imDC to obtain a high migration counteracts development of immune tolerance. METHODS & RESULTS: We transfected imDCs with a recombinant adenovirus carrying the CCR7 gene (Ad-CCR7) and a small interfering RNA targeting RelB (RelB-siRNA) to concurrently overexpress CCR7 and downregulate RelB expression. Functionally, such cells showed a significantly enhanced migration rate in the chemotactic assay and decreased T-cell proliferation after lipopolysaccharide stimulation in mixed lymphocyte reactions. Cotransfected cells showed an increased ability to induce immune tolerance by upregulating T regulatory (Treg) cells and shifting the Th1/Th2 ratio. Cotransfection of Ad-CCR7 and RelB-siRNA endowed imDCs with resistance to apoptosis and cell death. CCR7 overexpression and RelB knockdown (KD) in imDCs improve skin-graft survival in a murine skin-transplantation model. CONCLUSION: Transfection with Ad-CCR7 and RelB KD in imDCs may be an effective approach inducing immune tolerance, thus being potentially valuable for inhibiting allograft rejection.

P68 RNA helicase as a molecular target for cancer therapy.

The DEAD-box family of RNA helicase is known to be required in virtually all cellular processes involving RNA, and p68 is a prototypic one of the family. Reports have indicated that in addition to ATPase and RNA helicase ability, p68 can also function as a co-activator for transcription factors such as estrogen receptor alpha, tumor suppressor p53 and beta-catenin. More than that, post-translational modification of p68 including phosphorylation, acetylation, sumoylation, and ubiquitylation can regulate the coactivation effect. Furthermore, aberrant expression of p68 in cancers highlights that p68 plays an important role for tumorgenesis and development. In this review, we briefly introduce the function and modulation of p68 in cancer cells, and put forward envisagement about future study about p68.