The phytopathogen Xanthomonas campestris senses and effluxes salicylic acid via a sensor HepR and an RND family efflux pump to promote virulence in host plants.

Salicylic acid (SA) plays an essential role in plant defense against biotrophic and semi-biotrophic pathogens. Following pathogen recognition, SA biosynthesis dramatically increases at the infection site of the host plant. The manner in which pathogens sense and tolerate the onslaught of SA stress to survive in the plant following infection remains to be understood. The objective of this work was to determine how the model phytopathogen Xanthomonas campestris pv. campestris (Xcc) senses and effluxes SA during infection inside host plants. First, RNA-Seq analysis identified an SA-responsive operon Xcc4167-Xcc4171, encoding a MarR family transcription factor HepR and an RND (resistance-nodulation-cell division) family efflux pump HepABCD in Xcc. Electrophoretic mobility shift assays and DNase I footprint analysis revealed that HepR negatively regulated hepABCD expression by specifically binding to an AT-rich region of the promoter of the hepRABCD operon, Phep. Second, isothermal titration calorimetry and further genetic analysis suggest that HepR is a novel SA sensor. SA binding released HepR from its cognate promoter Phep and then induced the expression of hepABCD. Third, the RND family efflux pump HepABCD was responsible for SA efflux. The hepRABCD cluster was also involved in the regulation of culture pH and quorum sensing signal diffusible signaling factor turnover. Finally, the hepRABCD cluster was transcribed during the XC1 infection of Chinese radish and was required for the full virulence of Xcc in Chinese radish and cabbage. These findings suggest that the ability of Xcc to co-opt the plant defense signal SA to activate the multidrug efflux pump may have evolved to ensure Xcc survival and virulence in susceptible host plants.

Antifungal Potential and Mechanism of Bacillus velezensis HeN-7 Isolated from Tobacco Leaves on Bipolaris sorokiniana.

Wheat leaf blight caused by Bipolaris sorokiniana is a widespread fungal disease that poses a serious risk to wheat. Biological control without causing environmental pollution is one of the safest and most effective method to control plant diseases. The antagonistic bacterial strain HeN-7 (identified as Bacillus velezensis) was isolated from tobacco leaves cultivated in Henan province, China. The results of different concentrations of cell-free supernatant (CFS) from HeN-7 culture against B. sorokiniana mycelia showed that 20% HeN-7 CFS (v/v) reached the maximum inhibition rate of 96%. In the potted plants control assay, B. velezensis HeN-7 CFS exhibited remarkable biocontrol activity on the wheat infected with B. sorokiniana, the best pot control efficacy was 65% at 20% CFS. The research on the mechanism of action demonstrated that HeN-7 CFS induced the membrane lipid peroxidation in B. sorokiniana, leading to the disruption of cell membrane integrity and resulting in the leakage of cell contents; in addition, the intracellular mitochondrial membrane potential in mycelium dissipated and reactive oxygen species accumulated, thereby inhibiting the growth of B. sorokiniana. These results indicate that B. velezensis HeN-7 is a promising candidate as a biological control agent against Bipolaris sorokiniana infection.

Efficacy of Different Steroid Therapy in Preventing Esophageal Stricture after Endoscopic Submucosal Dissection: A Comparative Meta-analysis.

BACKGROUND AND AIMS: Endoscopic submucosal dissection (ESD) is the standard therapy for superficial esophageal cancer (SEC) presently. However, postoperative mucosal defects often lead to esophageal stricture. Although steroid application is effective prophylaxis, the efficacy and safety of various steroid administration modes remain unclear. Thus, this study aimed to evaluate the efficacy and safety of different steroid administrations for SEC patients post-ESD. METHODS: A search for relevant studies was conducted on China National Knowledge Infrastructure, Wanfang Database, PubMed, Embase, and Web of Science up to March 25, 2024. Treatment strategies were categorized into four groups: no prevention as control (CON), steroid injection (SI), oral steroids (OS), and SI combined with OS (SI+OS). Comparative meta-analysis was conducted to assess outcomes, including postoperative esophageal stricture rate and the number of endoscopic balloon dilatation (EBD) sessions required after stricture. RESULTS: A total of 25 studies, involving 1555 patients, were included. The SUCRA rankings were as follows: SI+OS (98.9%) > OS (59.9%) > SI (41.2%) > CON (0.0%) in preventing postoperative esophageal stricture rate, and OS (76.9%) > SI+OS (62.1%) > SI (61.0%) > CON (0.0%) in the number of EBD sessions required. Forest plot results indicated that compared with the non-steroid group, steroid interventions were associated with lower rates of postoperative stricture and fewer EBD sessions. Additionally, SI+OS was superior to SI or OS alone in preventing stricture, with no significant differences observed between different steroid administrations in terms of EBD sessions. The incidence of adverse reactions was less than 10% for all interventions, mostly mild and resolvable upon discontinuation. CONCLUSION: This study suggests that combined administration appears preferable for preventing esophageal stricture in patients post-ESD, and steroids could enhance stricture prognosis. However, due to the lack of large-sample RCT studies comparing different steroid administrations, more high-quality research is necessary to confirm these findings in the future.

Seasonal variations and the prevalence of phenolic profiles in ambient fine particulate matter and their impact on oxidative potential.

Exposure to fine particulate matter (PM2.5) poses numerous health risks, with oxidative potential (OP) serving as a critical marker of its toxicity. Synthetic phenolic antioxidants (SPAs) and bisphenols (BPs) influence reactive oxygen species (ROS) levels in PM2.5, and exposure to these compounds induces oxidative stress in organisms, thereby potentially affecting the OP of PM2.5. We detected 26 phenols (including 12 SPAs, 5 transformation products (TPs), and 9 BPs) in PM2.5 sample collected from October 2018 to September 2021 in Wuhan, China. Among them, 19 substances were detected at a detection frequency greater than 50 % in PM2.5 sample. AO 2246 and BHT were the main components of SPAs, and BHT-Q and BPA had the highest concentrations in TPs and BPs, respectively. PM2.5 mass concentrations and phenolic levels were higher in winter and autumn. Substances within groups were strongly correlated, suggesting the same or similar source of exposure. This finding aid in more precise pollution source identification and is crucial for comprehensively evaluating their combined health effects. Furthermore, we determined the OP of PM2.5 and found that BPs were related to increased OP and ROS. This suggests that the toxicity of PM2.5 is influenced not only by its concentration but also by its chemical composition, with BPs potentially enhancing its toxic effects. These factors should be fully considered when assessing the health impacts of PM2.5.

Ultrasound-Activatable In Situ Vaccine for Enhanced Antigen Self- and Cross-Presentation to Overcome Cancer Immunotherapy Resistance.

Insufficient antigen self-presentation of tumor cells and ineffective antigen cross-presentation by dendritic cells (DCs) contribute to diminished immune recognition and activation, which cause resistance to immunotherapies. Herein, we present an ultrasound-activatable in situ vaccine by utilizing a hybrid nanovesicle composed of a thylakoid (TK)/platelet (PLT) membrane and a liposome encapsulating DNA methyltransferase inhibitor zebularine (Zeb) and sonosensitizer hematoporphyrin monomethyl ether (HMME). Upon local exposure to ultrasound, reactive oxygen species (ROS) are generated and induce the sequential release of the payloads. Zeb can efficiently inhibit tumor DNA hypermethylation, promoting major histocompatibility complex class I (MHC-I) molecules-mediated antigen self-presentation to improve immune recognition. Meanwhile, the catalase on the TK membrane can decompose the tumoral overexpressed H2O2 into O2, which boosts the generation of ROS and the destruction of tumor cells, resulting in the in situ antigen release and cross-presentation of tumor antigens by DCs. This in situ vaccine simultaneously promotes antigen self-presentation and cross-presentation, resulting in heightened antitumor immunity to overcome resistance.

Fibroblast activation protein (FAP) as a prognostic biomarker in multiple tumors and its therapeutic potential in head and neck squamous cell carcinoma.

Background: Fibroblast activation protein (FAP), a cell surface serine protease, plays roles in tumor invasion and immune regulation. However, there is currently no pan-cancer analysis of FAP. Objective: We aimed to assess the pan-cancer expression profile of FAP, its molecular function, and its potential role in head and neck squamous cell carcinoma (HNSC). Methods: We analyzed gene expression, survival status, immune infiltration, and molecular functional pathways of FAP in The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx) tumors. Furthermore, to elucidate the role of FAP in HNSC, we performed proliferation, migration, and invasion assays post-FAP overexpression or knock-down. Results: FAP expression was elevated in nine tumor types and was associated with poor survival in eight of them. In the context of immune infiltration, FAP expression negatively correlated with CD8+ T-cell infiltration in five tumor types and positively with regulatory T-cell infiltration in four tumor types. Our enrichment analysis highlighted FAP's involvement in the PI3K-Akt signaling pathway. In HNSC cells, FAP overexpression activated the PI3K-Akt pathway, promoting tumor proliferation, migration, and invasion. Conversely, FAP knockdown showed inhibitory effects. Conclusion: Our study unveils the association of FAP with poor tumor prognosis across multiple cancers and highlights its potential as a therapeutic target in HNSC.

Activity and mechanism of action of antimicrobial peptide ACPs against Candida albicans.

AIMS: Candida albicans is the most prevalent pathogenic fungus, exhibiting escalating multidrug resistance (MDR). Antimicrobial peptides (AMPs) represent promising candidates for addressing this issue. In this research, five antimicrobial peptides, ACP1 to ACP5 which named ACPs were studied as alternative fungicidal molecules. MAIN METHODS: CD assay was used to analyze the 2D structures, Absorbance method was used to test the antimicrobial activity, haemolytic activity, time-kill kinetics, biofilm inhibition and reduction activity, resistance induction activity and assessment against fluconazole-resistant C. albicans. SEM, TEM, CLSM, flow cytometer and FM were carried out to provide insight into the mechanisms of anti-Candida action. KEY FINDINGS: ACPs possessed an α-helical structure and strong anti-Candida activities, with minimum inhibitory concentrations (MICs) from 3.9 to 15.6 µg/mL. In addition, ACPs did not produce hemolysis at concentrations lower than 10 or 62 × MIC, indicating their low cytotoxicity. Fungicidal kinetics showed that they completely killed C. albicans within 8 h at 2 to 4 × MIC. Notably, ACPs were highly fungicidal against fluconazole-resistant C. albicans and showed low resistance. In addition, they were effective in inhibiting mycelium and biofilm formation. Fluorescence microscopy revealed that while fluconazole had minimal to no inhibitory effect on biofilm-forming cells, ACPs induced apoptosis in all of them. The research on mechanism of action revealed that ACPs disrupted the cell membranes, with ROS increasing and cellular mitochondrial membrane potential decreasing. SIGNIFICANCE: ACPs could be promising candidates for combating fluconazole-resistant C. albicans infections.

The Transcriptional Regulator TfmR Directly Regulates Two Pathogenic Pathways in Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola (Xoc) is a notorious plant pathogen. Like most bacterial pathogens, Xoc has evolved a complex regulatory network to modulate the expression of various genes related to pathogenicity. Here, we have identified TfmR, a transcriptional regulator belonging to the TetR family, as a key player in the virulence mechanisms of this phytopathogenic bacterium. We have demonstrated genetically that tfmR is involved in the hypersensitive response (HR), pathogenicity, motility and extracellular polysaccharide production of this phytopathogenic bacterium. Our investigations extended to exploring TfmR's interaction with RpfG and HrpX, two prominent virulence regulators in Xanthomonas species. We found that TfmR directly binds to the promoter region of RpfG, thereby positively regulating its expression. Notably, constitutive expression of RpfG partly reinstates the pathogenicity compromised by TfmR-deletion mutants. Furthermore, our studies revealed that TfmR also exerts direct positive regulation on the expression of the T3SS regulator HrpX. Similar to RpfG, sustained expression of HrpX partially restores the pathogenicity of TfmR-deletion mutants. These findings underscore TfmR's multifaceted role as a central regulator governing key virulence pathways in Xoc. Importantly, our research sheds light on the intricate molecular mechanisms underlying the regulation of pathogenicity in this plant pathogen.

Prevalence of synthetic phenolic antioxidants in food contact materials from China and their implications for human dietary exposure through take-away food.

The application of disposable tableware has increased substantially in recent times due to the rapidly growing food delivery business in China. Synthetic phenolic antioxidants (SPAs) are widely used in food contact materials (FCMs) to delay the process of oxidation; however, their compositions, concentrations, and potential health hazards remain unclear. Therefore, FCMs comprised of five materials obtained from 19 categories (n = 118) in China were analyzed for SPAs concentrations. FCMs have been found to contain a variety of SPAs, with ∑SPAs concentrations ranging from 44.18 to 69,485.12 µg/kg (median: 2615.63 µg/kg). The predominant congeners identified in the sample include 2,4-di-tert-butylphenol (2,4-DTBP), 2,6-di-tert-butylphenol (2,6-DTBP), and 2,6-di-tert-butyl-p-benzoquinone (BHT-Q) with a median concentration of 885.75, 555.45 and of 217.44 µg/kg, respectively. Milky tea paper cups, instant noodle buckets, milky teacups, and disposable cups showed high levels of SPAs. 2,2'-methylenebis(4-methyl-6-tert-butylphenol) (AO 2246) was predominantly detected in polyethylene and polyethylene terephthalate-based products. The migration test identified disposable plastic cups and bowls as the predominant FCMs and 2,4-DTBP as the dominant SPA. The exposure risk of SPAs decreased with age. In children, the estimated daily intake of ∑SPAs from FCMs was determined to be 17.56 ng/kg body weight/day, which was 8.3 times higher than that of phthalic acid esters. The current findings indicate the potential ingestion risk of SPAs during the daily life application of multiple FCM categories.

Human Biomonitoring of Environmental Chemicals among Elderly in Wuhan, China: Prioritizing Risks Using EPA's ToxCast Database.

In line with the "healthy aging" principle, we aim to assess the exposure map and health risks of environmental chemicals in the elderly. Blood samples from 918 elderly individuals in Wuhan, China, were analyzed using the combined gas/liquid-mass spectrometry technology to detect levels of 118 environmental chemicals. Cluster analysis identified exposure profiles, while risk indexes and bioanalytical equivalence percentages were calculated using EPA's ToxCast database. The detection rates for 87 compounds exceeded 70%. DEHP, DiBP, naphthalene, phenanthrene, DnBP, pyrene, anthracene, permethrin, fluoranthene, and PFOS showed the highest concentrations. Fat-soluble pollutants varied across lifestyles. In cluster 2, which was characterized by higher concentrations of fat-soluble substances, the proportion of smokers or drinkers was higher than that of nonsmokers or nondrinkers. Pesticides emerged as the most active environmental chemicals in peroxisome proliferator-activated receptor gamma antagonist, thyroid hormone receptor (TR) antagonist, TR agonist, and androgen receptor (AR) agonist activity assays. Additionally, PAEs and polycyclic aromatic hydrocarbons played significant roles as active contaminants for the corresponding targets of AR antagonists and estrogen receptor alpha. We proposed a list of priority pollutants linked to endocrine-disrupting toxic effects in the elderly, which may provide the groundwork for further research into environmental etiology.

Density Functional Theory Study of the Mechanism of Ni-Catalyzed Carboxylation of Aryl C(sp2)-S Bonds with CO2: Computational Evidence for the Multifaceted Role of Additive Zn.

The mechanism of Ni-catalyzed carboxylation of aryl C(sp2)-S bonds with CO2 was studied for the first time by density functional theory calculations. We first proposed another possible reaction pathway in which CO2 insertion occurs prior to reduction. Then, we performed calculations on all proposed reaction pathways, and our calculation results show that the pathway in which reduction occurs prior to CO2 insertion is the favored pathway for this reaction. Additionally, our calculations disclose that additive Zn0 acts in multifaceted roles. (1) Zn0 acts as a reductant to reduce the NiI and NiII intermediates. (2) The simultaneously formed ZnIIBr2 can undergo transmetalation with NiI or NiII intermediates to produce an aryl reservoir by forming arylzinc species. (3) ZnIIBr2 can also coordinate to the CO2 to lower the energy barrier of the CO2 insertion step. Moreover, the calculation results suggest that CO2 insertion is the rate-determining step of the reaction, and CO2 is easier to insert into the NiI-Ph bond rather than into the NiII-Ph bond. These calculation results can improve our understanding of the mechanism of the carboxylation process and the multifaceted roles of metal additive Zn0 and provide theoretical guidance for improving the carboxylation reaction.

Density Functional Theory Study on the Mechanism of Nickel-Catalyzed 3,3-Dialkynylation of 2-Aryl Acrylamides Via Double Vinylic C-H Bond Activation.

The mechanisms of Ni-catalyzed 3,3-dialkynylation of 2-aryl acrylamide have been investigated by using density functional theory calculations. The result shows that this reaction includes double alkynylation, which involves sequential key steps of vinylic C-H bond activation, successive oxidative addition, and reductive elimination, with the second C-H bond activation being the rate-determining step. C-H and N-H bond activation occurs via the concerted metalation-deprotonation mechanism. The calculations show that no transition state exists in the first reductive elimination process, and a negative free energy barrier in the second reductive elimination process though a transition state is identified, indicating that the nickel-catalyzed vinylic C(sp2)-C(sp) bond formation does not require activation energy. Z-E isomerization is the prerequisite for the second alkynylation. In addition, our spin-flip TDDFT (SF-TDDFT) computational result discloses that the actual process of Z-E isomerization occurs on the potential energy surface of the first excited singlet state S1.

Differential Characteristics of the Metabolic Profiles and Microbial Community between Superior and Normal Grades of Nongxiangxing-daqu.

Nongxiangxing-daqu (NXDQ), as a saccharification and fermentation agent, directly affects the flavor and yield of fresh Nongxiangxing Baijiu (NXBJ). The difference in fermentation temperature owing to the artificial turning operation leads to the formation of superior (S) and normal (N) grades of NXDQ. Here, aiming to explore the discriminant characteristics of two grades of NXDQ, we studied the physicochemical properties, volatile compounds and microbial communities using HS-SPME-GC/MS and high-throughput sequencing technology. The NXDQ grades presented different physicochemical properties. Staphylococcus, Weissella, Lactobacillus and Thermoascus were dominant in the S grade (S-NXDQ), while Bacillus, Thermoactinomyces and Aspergillus were predominant in the N grade (N-NXDQ). Higher alcohols, aldehydes and ketones positively correlated with the bacterial biomarkers could be used as metabolic biomarkers for N-NXDQ; the S-NXDQ had a higher abundance of key enzymes involved in lactic acid and ethanol fermentation, while N-NXDQ had a higher abundance of key enzymes involved in amino acid synthesis and long-chain fatty acid and lipid metabolism. N-NXDQ and S-NXDQ had different microbial and metabolic biomarkers. These findings provide insight into the discriminant characteristics of different grades of NXDQ, a theoretical basis for rational evaluation of NXDQ, and effective information for quality improvement of daqu.

Efficacy and safety of endoscopic cardia peripheral tissue scar formation (ECSF) for the treatment of refractory gastroesophageal reflux disease: A systematic review with meta-analysis.

BACKGROUND: Endoscopic treatment is increasingly used for refractory gastroesophageal reflux disease (rGERD). Unlike the mechanism of conventional surgical fundoplication, gastroesophageal junction ligation, anti-reflux mucosal intervention, and radiofrequency ablation have extremely similar anti-reflux mechanisms; hence, we collectively refer to them as endoscopic cardia peripheral tissue scar formation (ECSF). We conducted a systematic review and meta-analysis to assess the safety and efficacy of ECSF in treating rGERD. METHODS: We performed a comprehensive search of several databases, including PubMed, Embase, Medline, China Knowledge Network, and Wanfang, to ensure a systematic approach for data collection between January 2011 and July 2023. Forest plots were used to summarize and combine the GERD-health-related quality of life (HRQL), gastroesophageal reflux questionnaire score, and DeMeester scores, acid exposure time, lower esophageal sphincter pressure, esophagitis, proton pump inhibitors use, and patient satisfaction. RESULTS: This study comprised 37 studies, including 1732 patients. After ECSF, significant improvement in gastroesophageal reflux disease health-related quality of life score (mean difference [MD] = 18.27 95% CI: 14.81-21.74), gastroesophageal reflux questionnaire score (MD = 4.85 95% CI: 3.96-5.75), DeMeester score (MD = 42.34, 95% CI: 31.37-53.30), acid exposure time (MD = 7.98, 95% CI: 6.03-9.92), and lower esophageal sphincter pressure was observed (MD = -5.01, 95% CI: -8.39 to 1.62). The incidence of serious adverse effects after ECSF was 1.1% (95% CI: 0.9%-1.2%), and postoperatively, 67.4% (95% CI: 66.4%-68.2%) of patients could discontinue proton pump inhibitor-like drugs, and the treatment outcome was observed to be satisfactory in over 80% of the patients. Subgroup analyses of the various procedures showed that all 3 types improved several objective or subjective patient indicators. CONCLUSIONS: Based on the current meta-analysis, we conclude that rGERD can be safely and effectively treated with ECSF as an endoscopic procedure.

Exploring the potential prompting role of cervical human papilloma virus detection in vulvar lesions: a cross-sectional study in China.

Introduction: The etiology and clinical presentation of vulvar carcinomas, especially vulvar lesions, are not fully understood. Because the vulva and cervix are anatomically connected, human papillomavirus (HPV) is the main cause of cervical lesions. Thus, this study explored the potential characteristics and effects of specific HPV infection types across vulvar lesions and concurrent cervical lesions. Methods: This retrospective, cross-sectional study analyzed patients with cervical HPV or cytological results and concurrent vulvar biopsy who were seen in our hospital colposcopy clinic in Shanxi Province, China, between 2013 and 2023. Data on age, menopause status, vulvar manifestations, and cytology and HPV infection testing results were collected. Attributable fractions and multinominal logistic models were used to evaluate HPV genotyping and clinical characteristics across vulvar lesions. Results: Among the 1,027 participants, 83 (8.1%) had vulvar intraepithelial neoplasia (VIN) of high grade or worse (VIN2+), and 127 (12.4%) had non-neoplastic epithelial disorders of the vulva (NNEDV). A total of 175 patients had either VIN2+ or cervical intraepithelial neoplasia (CIN) lesions of grade 2 or worse (CIN2+). The most common HPV genotypes for VIN2+ or concurrent VIN2+/CIN2+ were HPV16, HPV52, and HPV58, although attributable fractions differed among lesions. Patients with normal cytological or histopathological result were more likely to have NNEDV detected, while abnormal cervical diagnosis was associated with higher detection of VIN2+. Multinominal logistic modeling showed that age and HPV16 infection were risk factors for VIN2+ or concurrent VIN2+/CIN2+; however, only vulvar presentation with depigmentation was a risk factor for NNEDV. Among patients with low-grade CIN1/VIN1, compared with those who were HPV16 negative, those who were HPV16 positive were at 6.63-fold higher risk of VIN2+/CIN2+ [95% confidence interval (CI): 3.32, 13.21]. Vulvar depigmentation was also associated with increased risk of NNEDV (odds ratio: 9.98; 95% CI: 3.02, 33.04). Conclusions: Chinese women may be at specific, high risk for HPV infection types associated with VIN or CIN. The use of cervical cell HPV detection along with vulvar presentation during cervical cancer screening may also contribute to vulvar lesion detection.

Biocompatible formulation of a hydrophobic antimicrobial peptide L30 through nanotechnology principles and its potential role in mouse pneumonia model infected with Staphylococcus aureus.

Hydrophobic antimicrobial peptide L30, a potential antibiotic candidate, has poor water solubility and hemolytic activity. Herein, a biocompatible nano-formulation composed of liposomes and dendritic mesoporous silica encapsulation (LDMSNs@L30) was constructed for L30 to solve the limits for its clinical development. The characterization, antimicrobial activity and therapeutic effect of LDMSNs@L30 on Staphylococcus aureus 9 (cfr+) infected mice models were investigated. LDMSNs@L30 displayed a smooth, spherical, and monodisperse nanoparticle with a hydrodynamic diameter of 177.40 nm, an encapsulation rate of 56.13%, a loading efficiency of 32.26%, a release rate of 66.5%, and effective slow-release of L30. Compared with free L30, the formulation could significantly increase the solubility of L30 in PBS with the maximum concentration from 8 µg/mL to 2.25 mg/mL and decrease the hemolytic activity of hydrophobic peptide L30 with the HC5 from 65.36 µg/mL to more than 500 µg/mL. The nano delivery system LDMSNs@L30 also exhibited higher therapeutic effects on mice models infected with S. aureus 9 (cfr+) than those of free L30 after 7 days of treatment by reducing the lung inflammation and the inflammatory cytokines levels in plasma, showing better health score and pulmonary pathological improvement. Our research suggests that nano-formulation can be expected to be a promising strategy for peptide drugs in therapeutic applications.