Cathelicidin boosts the antifungal activity of neutrophils and improves prognosis during Aspergillus fumigatus keratitis.

Aspergillus fumigatus (A. fumigatus) is one of the common pathogens of fungal keratitis. Fungal growth and invasion cause excessive inflammation and corneal damage, leading to severe vision loss. Neutrophils are the primary infiltrating cells critical for fungal clearance. Cathelicidin [LL-37 in humans and cathelicidin-related antimicrobial peptide (CRAMP) in mice], a natural antimicrobial peptide, can directly inhibit the growth of many pathogens and regulate immune responses. However, the role of cathelicidin and its effect on neutrophils in A. fumigatus keratitis remain unclear. By establishing A. fumigatus keratitis mouse models, we found that cathelicidin was increased in A. fumigatus keratitis. It could reduce fungal loads, lower clinical scores, and improve corneal transparency. Restriction of CRAMP on fungal proliferation was largely counteracted in CD18-/- mice, in which neutrophils cannot migrate into infected sites. When WT neutrophils were transferred into CD18-/- mice, corneal fungal loads were distinctly reduced, indicating that neutrophils are vital for CRAMP-mediated resistance. Furthermore, cathelicidin promoted neutrophils to phagocytose and degrade conidia both in vitro and in vivo. CXC chemokine receptor 2 (CXCR2) was reported to be a functional receptor of LL-37 on neutrophils. CXCR2 antagonist SB225002 or phospholipase C (PLC) inhibitor U73122 weakened LL-37-induced phagocytosis. Meanwhile, LL-37 induced PLC γ phosphorylation, which was attenuated by SB225002. SB225002 or the autophagy inhibitors Bafilomycin-A1 and 3-Methyladenine weakened LL-37-induced degradation of conidia. Transmission electron microscopy (TEM) observed that LL-37 increased autophagosomes in Aspergillus-infected neutrophils. Consistently, LL-37 elevated autophagy-associated protein expressions (Beclin-1 and LC3-II), but this effect was weakened by SB225002. Collectively, cathelicidin reduces fungal loads and improves the prognosis of A. fumigatus keratitis. Both in vitro and in vivo, cathelicidin promotes neutrophils to phagocytose and degrade conidia. LL-37/CXCR2 activates PLC γ to amplify neutrophils' phagocytosis and induces autophagy to eliminate intracellular conidia.

Extracellular vesicles from Candida albicans modulate immune cells function and play a protective role in fungal keratitis.

Fungal keratitis (FK) is a highly blinding infectious corneal disease caused by pathogenic fungi. Candida albicans (C. albicans) is one of the main pathogens of fungal keratitis. Extracellular vesicles (EVs), lipid bilayer compartments released by almost all living cells, including fungi, have garnered attention for their role in pathogenic microbial infection and host immune responses in recent years. Studies have reported that pretreating the host with fungal EVs can reduce the inflammatory response of the host when attacked by fungi and reduce the lethality of fungal infection. However, there are no studies that have evaluated whether C. albicans EVs can modulate the inflammatory response associated with C. albicans keratitis. Our study revealed that C. albicans EVs could activate the polymorphonuclear cells (PMNs) and promote their secretion of proinflammatory cytokines and nitric oxide (NO), enhance their phagocytic and fungicidal abilities against C. albicans. C. albicans EVs also induced a proinflammatory response in RAW264.7 cells, which was characterized by increased production of inflammatory cytokines and elevated expression of the chemokine CCL2. Similarly, stimulation of C. albicans EVs to RAW264.7 cells also enhanced the phagocytosis and killing ability of cells against C. albicans. Besides, in our in vivo experiments, after receiving subconjunctival injection of C. albicans EVs, C57BL/6 mice were infected with C. albicans. The results demonstrated that pre-exposure to C. albicans EVs could effectively diminish the severity of keratitis, reduce fungal load and improve prognosis. Overall, we conclude that C. albicans EVs can modulate the function of immune cells and play a protective role in C. albicans keratitis.

The Therapeutic Role and Mechanism of Glabridin Under Aspergillus fumigatus Infection.

Purpose: To characterize the efficiency of glabridin alone and in combination with clinical antifungals in Aspergillus fumigatus keratitis. Methods: The broth microdilution method was performed to investigate whether glabridin exerted an antifungal role on planktonic cells and immature and mature biofilm. Antifungal mechanism was evaluated by Sorbitol and Ergosterol Assays. The synergistic effect of glabridin and antifungals was assessed through the checkerboard microdilution method and time-killing test. Regarding anti-inflammatory role, inflammatory substances induced by A. fumigatus were assessed by real-time quantitative polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Drug toxicity was assessed by Draize test in vivo. Macrophage phenotypes were examined by flow cytometry. Results: Regarding antifungal activity, glabridin destroyed fungal cell wall and membrane on planktonic cells and suppressed immature and mature biofilm formation. After combining with natamycin or amphotericin B, glabridin possessed a potent synergistic effect against A. fumigatus. Regarding anti-inflammatory aspects, Dectin-1, toll­like receptor (TLR)-2 and TLR-4 expression of human corneal epithelial cells were significantly elevated after A. fumigatus challenge and reduced by glabridin. The elevated expression of interleukin-1ß and tumor necrosis factor-alpha induced by A. fumigatus or corresponding agonists were reversed by glabridin, equivalent to the effect of corresponding inhibitors. Glabridin could also contribute to anti-inflammation by downregulating inflammatory mediator expression to suppress macrophage infiltration. Conclusions: Glabridin contributed to fungal clearance by destroying fungal cell wall and membrane, and disrupting biofilm. Combining glabridin with clinical antifungals was superior in reducing A. fumigatus growth. Glabridin exerted an anti-inflammatory effect by downregulating proinflammatory substance expression and inhibiting macrophage infiltration, which provide a potential agent and treatment strategies for fungal keratitis.

Aspergillus fumigatus-Derived Extracellular Vesicles Mitigate Fungal Keratitis by Modulating the Immune Cell Function.

Fungal keratitis (FK) is a refractory global disease characterized by a high incidence of blindness and a lack of effective therapeutic options, and Aspergillus fumigatus (A. fumigatus, AF) is one of the most common causative fungi. This study aimed to investigate the role of extracellular vesicles (EVs) from A. fumigatus in the immune cell function and their protective role in A. fumigatus keratitis in order to explore their therapeutic potential. First, we isolated and characterized the EVs (AF-derived EVs). In vitro, we stimulated RAW264.7 cells and polymorphonuclear cells with AF-derived EVs. The expression levels of inflammatory factors increased in both immune cells along with an M1 polarization variation of RAW264.7 cells. After being incubated with AF-derived EVs, both immune cells exhibited an increased conidia-phagocytic index and a decreased conidia survival rate. In vivo, we injected EVs subconjunctivally on mice resulting in a heightened production of secretory immunoglobulin A (sIgA) in tear fluid. By the injection of EVs on mice in advance, a significant reduction in severity of A. fumigatus FK was witnessed by lower clinical scores, inflammatory appearances, and mitigated fungal load. Collectively, these results positioned AF-derived EVs as a promising and innovative immune therapy for combating FK, while also providing a platform for further investigation into developing an optimal formulation for modulating inflammation in the context of FK.

Role of Inhibiting Inflammation of LC3-Associated Phagocytosis in Dry Eye Disease.

PURPOSE: To confirm the expression and investigate the role of LC3-associated phagocytosis (LAP) in dry eye disease (DED). METHODS: The DED model of mice was established by scopolamine subcutaneous injection in a low-humidity environment chamber. Tear secretion test and corneal fluorescein sodium staining were used to evaluate the severity of DED. Expression levels of Rubicon, microtubule-associated protein light chain 3-II (LC3-II), Beclin-1 and autophagy-related gene-7 (Atg-7) in corneas of mice with DED were tested by western blot. Cell Counting Kit-8 (CCK-8) assay was used to detect the effects of different concentrations of hypertonic solutions on the proliferation activity of human corneal epithelial cells (HCECs). The expression levels of Dectin-1, IL-6 and IL-1ß in HCECs after stimulation with different concentrations of hypertonic solutions were tested. The expressions of Rubicon, LC3-II, Beclin-1 and ATG-7 in HCECs were detected by reverse transcription polymerase chain reaction (RT-PCR). After being pretreated with 10 µM si-Rubicon, the severity of the disease was documented by corneal fluorescein sodium staining. And the expression levels of IL-6 and IL-1ß were also tested by RT-PCR. RESULTS: Compared with the normal control group, the corneal fluorescein sodium staining scores and tear secretion were significantly reduced. Rubicon, LC3-II, Beclin-1 and ATG-7 were significantly elevated. CCK-8 showed that the 400 and 450 mOsM hypertonic solutions did not affect the proliferation activity of HCECs. The expression of Dectin-1, IL-1ß and IL-6 were elevated after stimulation with 450 mOsM solution. LC3-II, Rubicon, ATG-7 and Beclin-1 increased after stimulation with 450 mOsM hyperosmolar solution in HCECs. Corneal fluorescein staining showed that si-Rubicon increased the severity of DED in mice. Moreover, the mRNA expressions of inflammatory factors IL-1ß and IL-6 in the cornea of mice were significantly increased. CONCLUSION: DED increased the expression of proteins associated with LAP. LAP could play an anti-inflammatory effect in DED.

Dopamine and cyclic adenosine monophosphate-regulated phosphoprotein with an apparent Mr of 32000 promotes colorectal cancer growth.

BACKGROUND: Dopamine and cyclic adenosine monophosphate (cAMP)-regulated phosphoprotein with an apparent Mr of 32000 (DARPP-32) is a protein that is involved in regulating dopamine and cAMP signaling pathways in the brain. However, recent studies have shown that DARPP-32 is also expressed in other tissues, including colorectal cancer (CRC), where its function is not well understood. AIM: To explore the effect of DARPP-32 on CRC progression. METHODS: The expression levels of DARPP-32 were assessed in CRC tissues using both quantitative polymerase chain reaction and immunohistochemistry assays. The proliferative capacity of CRC cell lines was evaluated with Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays, while apoptosis was measured by flow cytometry. The migratory and invasive potential of CRC cell lines were determined using wound healing and transwell chamber assays. In vivo studies involved monitoring the growth rate of xenograft tumors. Finally, the underlying molecular mechanism of DARPP-32 was investigated through RNA-sequencing and western blot analyses. RESULTS: DARPP-32 was frequently upregulated in CRC and associated with abnormal clinicopathological features in CRC. Overexpression of DARPP-32 was shown to promote cancer cell proliferation, migration, and invasion and reduce apoptosis. DARPP-32 knockdown resulted in the opposite functional effects. Mechanistically, DARPP-32 may regulate the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway in order to carry out its biological function. CONCLUSION: DARPP-32 promotes CRC progression via the PI3K/AKT signaling pathway.

Application of the 'online assessment + FOBT at home' to improve participation and the efficacy of opportunistic screening for colorectal cancer: a retrospective cohort study.

BACKGROUND: Colorectal cancer (CRC) screening faces two major challenges: insufficient screening coverage and poor adherence. A smartphone applet named "Early Screening Assistant (ESA)" was developed to create an online risk-assessment and fecal occult blood test (FOBT) at home. This retrospective study was designed to evaluate whether the new CRC screening strategy can improve the colonoscopy participation rate (PR) and lesion detection rate (DR). METHODS: In total, 6194 individuals who accepted normal health examinations and CRC screening based on the ESA from June 2020 to May 2022 were assigned to the ESA group. Accordingly, 7923 inhabitants who only accepted normal health examinations were assigned to the control group. The colonoscopy PR and neoplastic lesion DR were then compared between the two groups. RESULTS: Overall, a higher proportion of subjects in the ESA group (285 of 6194 [4.6%]) completed colonoscopy than in the control group (126 of 7923, [1.6%]), p < 0.01). The neoplastic lesion DR also significantly increased in the ESA group (76 of 6194 [1.22%]) compared with the control group (15 of 7923 [0.19%]) (p < 0.01). The adjusted diagnostic sensitivity and specificity of the "Online assessment + FOBT at home" were 41.5% and 62.6% for neoplastic lesions, respectively. CONCLUSIONS: This retrospective cohort study confirmed that the new CRC screening strategy based on the "Online assessment + FOBT at home" can improve colonoscopy participation and the neoplastic lesion detection rate and may represent a promising screening strategy for CRC. TRIAL REGISTRATION: This study was registered in China Clinical Trial Registry ( https://www.chictr.org.cn ) on 29/09/2022. REGISTRATION NUMBER: ChiCTR2200064186.

Quercetin protects against Aspergillus fumigatus keratitis by reducing fungal load and inhibiting TLR-4 induced inflammatory response.

PURPOSE: To investigate the antifungal and anti-inflammatory effects of quercetin in Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: Draize eye test was performed in mice to evaluate the toxicity of quercetin, and the antifungal effects on A. fumigatus were assessed via scanning electron microscopy (SEM), propidium iodide uptake, and adherence assay. In fungal keratitis (FK) mouse models, immunostaining was performed for investigating toll-like receptor 4 (TLR-4) expression and macrophage infiltration. Real-time PCR, ELISA, and Western blot were used to evaluate the expression of pro-inflammatory factors IL-1ß, TNF-α, and IL-6 in infected RAW264.7 cells. Cells were also treated with TLR-4 siRNA or agonist CRX-527 to investigate mechanisms underlying the anti-inflammatory activity of quercetin. RESULTS: Quercetin at 32 µM was non-toxic to corneal epithelial and significantly inhibited A. fumigatus growth and adhesion, and also altered the structure and reduced the number of mycelia. Quercetin significantly reduced macrophage infiltration in the mouse cornea, and attenuated the expression of TLR-4 in the corneal epithelium and stroma of mice with keratitis caused by A. fumigatus. In RAW264.7 cells infected by A. fumigatus, quercetin downregulated TLR-4 along with pro-inflammatory factors IL-1ß, TNF-α, and IL-6. RAW cells with TLR-4 knockdown had reduced expression of factors after A. fumigatus infection, which was decreased even further with quercetin treatment. In contrast, cells with CRX-527 had elevated inflammatory factors compared to control, which was significantly attenuated in the presence of quercetin. CONCLUSION: Quercetin plays a protective role in mouse A. fumigatus keratitis by inhibiting fungal load, disrupting hyphae structure, macrophage infiltration, and suppressing inflammation response in macrophages via TLR-4 mediated signaling pathway.

Effects of chronic liver disease on the outcomes of simultaneous resection of colorectal cancer with synchronous liver metastases: a propensity score matching study.

Introduction: Given the rising prevalence of chronic liver disease (CLD), it is increasingly important to understand its impact on surgical outcomes. Our aim was to evaluate the impact of CLD on short-term outcomes in patients with colorectal cancer and synchronous liver metastases undergoing simultaneous surgery. Methods: We retrospectively reviewed patients with colorectal cancer and liver metastases who underwent simultaneous resection between January 2013 and June 2022. Patients were divided into the CLD and non-CLD groups. Data regarding short-term surgical outcomes were compared between the two groups. Results: A total of 187 patients were included. After propensity score matching, there were 42 patients in each group, and the basic characteristics of the two groups were similar. Patients with CLD had a significantly greater incidence of postoperative complications (47.6% vs. 26.2%; P = 0.042). The operation times of the CLD and non-CLD groups were similar (297 vs. 307.5 min, P = 0.537), and the blood loss was comparable between the two groups (250 vs. 155 ml, P = 0.066). No significant differences were observed between the two groups in pneumonia (P > 0.999), urinary infection rate (P > 0.999), ileus rate (P = 0.474), wound infection rates (P > 0.999), abdominal infection rate (P = 0.533), anastomotic leakage rate (P > 0.999), digestive hemorrhage rate (P > 0.999), bile leakage rate (P > 0.999), hepatic hemorrhage rate (P > 0.999), reoperation rate (P > 0.999), intensive care rate (P > 0.999), or severe liver failure (P > 0.999). There were no deaths in the two groups. CLD significantly prolonged the length of hospital stay (P = 0.011). Discussion: CLD is an important factor affecting postoperative complications in patients with colorectal cancer liver metastases undergoing simultaneous surgery. Considering the large number of patients with CLD in China, more attention and medical care should be provided to patients with CLD who require simultaneous resection of colorectal cancer with synchronous liver metastases.

The influence of smoking on retinal ganglion cell-inner plexiform layer complex in male diabetes.

PURPOSE: This study aims to evaluate the influence of smoking on ganglion cell-inner plexiform layer complex (GC-IPL) thickness and central macular thickness (CMT) measured by spectral domain optical coherence tomography (OCT) in male diabetes. METHODS: 90 smoking and 90 never-smoking male subjects were included in this study. They were divided into six groups based on the diagnostic criteria for diabetes and the Early Treatment Diabetic Retinopathy Study (ETDRS) classification: smoking healthy subjects (SH, n = 20), non-smoking healthy subjects (NSH, n = 20), smoking diabetic patients without diabetic retinopathy (SNDR, n = 40), non-smoking diabetic patients without diabetic retinopathy (NSNDR, n = 40), smoking diabetic patients with diabetic retinopathy (SDR, n = 30), and non-smoking diabetic patients with diabetic retinopathy (NSDR, n = 30). After a full ophthalmologic examination, GC-IPL thickness and central macular thickness (CMT) were measured by OCT. Statistical analysis was performed to compare GC-IPL thickness and CMT between groups. Multiple linear regression equations were constructed to explore the potential risk factors of mean GC-IPL thickness. RESULTS: There were no significant differences in GC-IPL thickness and CMT between SH and NSH (all p > 0.05). Mean, superonasal, superior, superotemporal, inferonasal, inferior GC-IPL (p＜0.001, p＜0.001, p＜0.001, p = 0.003, p = 0.001, and p = 0.005, respectively) were thinner in the SNDR than NSNDR except for inferotemporal GC-IPL thickness and CMT (p = 0.066, p = 0.605, respectively). Mean, superonasal, superior, and inferonasal GC-IPL were thinner in the SDR than NSDR (p = 0.019, p = 0.045, p = 0.037, and p = 0.049, respectively). Multiple regression analysis demonstrated that age (ß [SE], -0.141 [0.060]; p = 0.020) and smoking (ß [SE], -4.470 [1.015]; p＜0.001) were the most important determinants for mean GC-IPL thickness. CONCLUSION: Smoking is associated with reduced retinal GC-IPL thickness in male diabetes. Smoking behavior and age are important determinants of mean GC-IPL thickness.

The preparation and therapeutic effects of ß-glucan-specific nanobodies and nanobody-natamycin conjugates in fungal keratitis.

Fungal keratitis (FK) is a severe infectious corneal disease. Since traditional eye drops exhibit poor dissolution and high corneal toxicity, the efficacy of current treatments for FK remains limited. It is needed to develop new approaches to control the cornea damage from FK. In this study, a nanobody (Nb) specific to ß-glucan in the fungal cell wall was prepared. The conjugate of the Nb with natamycin (NAT), a traditional antifungal drug, was synthesized. Firstly, we found the Nb specific to ß-glucan inhibited fungal growth by disrupting cell wall and biofilm formation.. In addition, the content of ß-glucan in the fungal cell wall decreased after Nb treatment. The Nb also reduced the adhesion ability of fungal conidia to human corneal epithelial cells (HCECs). Further, we examined the difference between NAT and Nb-NAT in antifungal growth. Nb-NAT showed better antifungal effects than NAT which was caused by the interaction between Nb and ß-glucan. Moreover, Nb concentration below 0.5 mg/mL did not affect the viability of HCECs. Nb-NAT had less cytotoxicity and ocular surface irritation than NAT. Nb specific to ß-glucan attenuated Aspergillus fumigatus (A. fumigatus) virulence and relieved inflammatory responses in FK. Nb-NAT treatment of the cornea improved therapeutic effects compared with NAT. It decreased clinical scores and expression level of inflammatory factors. To our knowledge, this study is the first to report a Nb specific to ß-glucan and Nb-NAT for the treatment of FK. These unique functions of the Nb specific to ß-glucan and Nb-NAT would render it as an alternative molecule to control fungal infections including FK. STATEMENT OF SIGNIFICANCE: Fungal keratitis is a corneal disease with a high rate of blindness. Due to the poor dissolution and high corneal toxicity exhibited by traditional eye drops, the efficacy of current therapeutic treatments for fungal keratitis (FK) remains limited. To enhance the therapeutic effect of natamycin in treating fungal keratitis, this study developed an innovative approach by preparing a ß-glucan-specific nanobody and loading it with the antifungal drug natamycin. The ß-glucan-specific nanobody has the ability to control both fungal pathogen invasion and inflammation, which can cause damage to the cornea in FK. The conjugation with the ß-glucan-specific nanobody significantly increased the antifungal capacity of natamycin and reduced its toxicity. The further application of natamycin conjugated with the ß-glucan-specific nanobody could be expanded to other diseases caused by fungal pathogen infections.

An artificial interfacial layer with biomimetic ionic channels towards highly stable Li metal anodes.

Lithium (Li) metal with low electrochemical potential and high theoretical capacity is a promising anode material for next-generation batteries. However, the low reversibility and safety problems caused by the notorious dendrite growth significantly impede the development of high-energy-density lithium metal batteries (LMBs). Here, to enable a dendrite-free and highly reversible Li metal anode (LMA), we develop a cytomembrane-inspired artificial layer (CAL) with biomimetic ionic channels using a scalable spread coating method. The negatively charged CAL with uniform intraparticle and interparticle ionic channels facilitates the Li-ion transport and redistributes the Li-ion flux, contributing to stable and homogeneous Li stripping and plating. Furthermore, a robust underneath transition layer with abundant lithiophilic inorganic components is in-situ formed through the transformation of CAL during cycling, which promotes Li-ion diffusion and suppresses the continuous side reactions with the electrolyte. Additionally, the resulting cytomembrane-inspired artificial Janus layer (CAJL) displays an ultrahigh Young's modulus (≥10.7 GPa) to inhibit the dendrite growth. Consequently, the CAJL-protected LMA (Li@CAJL) is stably cycled with a high areal capacity of 10 mAh cm-2 at a high current density of 10 mA cm-2. More importantly, the effective CAJL modification realizes the stable operation of a practical 429.2 Wh kg-1 lithium-sulfur (Li-S) pouch cell using a low electrolyte/sulfur (E/S) ratio of 3 µL mg-1. The facile yet effective protection strategy of LMAs can promote the practical application of LMBs.

Hinokitiol inhibits Aspergillus fumigatus by interfering with the cell membrane and cell wall.

Hinokitiol (ß-thujaplicin) is an important component of the essential oil extracted from Chamaecyparis obtuse, which prevents the decay and decomposition of temple and shrine buildings in Japan. Hinokiol has been shown to have a detrimental effect on various fungi such as Candida albicans and saprophytic fungi. However how hinokitiol works against Aspergillus fumigatus (A. fumigatus) has not been claimed. This study aims to investigate the adverse effects of hinokitiol on the disruption of the cell wall and cell membrane of A. fumigatus and to explore possible potential mechanisms or pathways. According to our results, hinokitiol negatively altered mycelium morphology, growth density, and cell plasma composition content. When incubated with human corneal epithelial cells (HCECs), hinokitiol saw a safe effect with concentrations below 12 µg/ml. Hinokitiol was shown to increase the cell membrane's permeability by decreasing the cell membrane's ergosterol content. The integrity of the cell wall was disrupted, as well as a significant increase in chitin degradation and chitinase activity. As determined by RNA-seq results, subsequent analysis, and qRT-PCR, altered transcript levels of cell walls and cell membranes-related genes (such as eglC) illustrated how hinokitiol affected the genetic profile of A. fumigatus. With this study, we recommend hinokitiol as an effective anti-A. fumigatus agent by reducing the amounts of key components in the cell wall and membrane by preventing production and accelerating breakdown.

Honokiol reduces fungal burden and ameliorate inflammation lesions of Aspergillus fumigatus keratitis via Dectin-2 down-regulation.

PURPOSE: To screen and identify the mechanism of honokiol on anti-fungi and anti-inflammation in fungal keratitis (FK) through bioinformatic analysis and biological experiments. METHODS: Transcriptome profile demonstrated differential expression genes (DEGs) of Aspergillus fumigatus keratitis between PBS-treated and honokiol-treated groups via bioinformatics analyses. Inflammatory substances were quantified by qRT-PCR, Western blot and ELISA, and macrophage polarization was examined by flow cytometry. Periodic acid Schiff staining and morphological interference assay were used to detect hyphal distribution in vivo and fungal germination in vitro, respectively. Electron microscopy was to illustrate hyphal microstructure. RESULTS: Illumina sequencing demonstrated that compared with the honokiol group, 1175 up-regulated and 383 down-regulated genes were induced in C57BL/6 mice Aspergillus fumigatus keratitis with PBS treatment. Through GO analysis, some differential expression proteins (DEPs) played major roles in biological processes, especially fungal defense and immune activation. KEGG analysis provided fungus-related signaling pathways. PPI analysis demonstrated that DEPs from multiple pathways form a close-knit network, providing a broader context for FK treatment. In biological experiments, Dectin-2, NLRP3 and IL-1ß were upregulated by Aspergillus fumigatus to evaluate immune response. Honokiol could reverse the trend, comparable to Dectin-2 siRNA interference. Meanwhile, honokiol could also play an anti-inflammatory role via promoting M2 phenotype polarization. Moreover, honokiol reduced hyphal distribution in the stroma, delayed germination, and destroyed the hyphal cell membrane in-vitro. CONCLUSIONS: Honokiol possesses anti-fungal and anti-inflammatory effects in Aspergillus fumigatus keratitis and may develop a potential and safe therapeutic modality for FK.

Glabridin Inhibits Aspergillus fumigatus Growth and Alleviate Inflammation Mediated by Dectin-2 and NLRP3 Inflammasome.

PURPOSE: The research was used to uncover the mechanism of glabridin in Aspergillus fumigatus keratitis in anti-fungus and anti-inflammation. METHODS: In vitro, RAW 264.7 cells were infected with A. fumigatus with incubation of glabridin in different concentrations. Real-time quantitative polymerase chain reaction (RT­qPCR), Western blot, and enzyme-linked immunosorbent assay (ELISA) were used to assess the inflammatory severe and alternation with the intervention of Dectin-2 siRNA and glabridin. In vivo, A. fumigatus keratitis mouse models were established by spore intra-stromal injection and treated with glabridin or PBS. And disease scores, inflammatory mediators, and periodic acid-schiff (PAS) staining were exhibited to demonstrate the therapeutic efficiency of glabridin in vivo. Morphological interference assay monitored fungal germination. Scanning and transmission electron microscopy were used to observe the growth of fungi. RESULTS: In RAW 264.7 cells and mouse keratitis models, noncytotoxic 16 µg/mL glabridin showed significant inhibition in the expression of Dectin-2, NLRP3, Caspase-1, IL-1ß, and TNF-α after A. fumigatus infection, almost similar to the intervention of Dectin-2 siRNA. PAS staining illustrated the reduced hyphal distribution in cornea stroma with glabridin treatment. Glabridin remarkably inhibited A. fumigatus growth through delaying germination and disrupting the integrity of the hyphae membrane. CONCLUSION: Glabridin plays an anti-inflammatory role in A. fumigatus challenge via suppression of the Dectin-2 and NLRP3 inflammasome, and plays an anti-fungal role through delaying germination and changing the hyphal integrity.KEY MESSAGESGlabridin plays an anti-inflammatory role in A. fumigatus infection of RAW264.7 cells in a concentration-dependent manner and through Dectin-2 mediation.Glabridin decreases fungal distribution and inflammation in mouse A. fumigatus keratitis.Glabridin inhibits A. fumigatus growth by delaying germination and disrupting cellular structure in vitro.

Exploring the generality of ligands for Silica-Encapsulated nanoclusters as SERS labels.

The surface enhanced Raman scattering (SERS) reporters are rather limited, and the Raman peaks still overlap in varying degrees, making SERS multiplex coding a critical bottleneck in the exploration of SERS nanotechnology. Herein, we design a general strategy to expand the SERS probe scope to 26 probes of six types, which can be further expanded within a limited range, with stable performance and structure. By constructing (Au-aggregate)@Ag@silica and (Au-aggregate)@silica nanocomposites, we develop optimal enhancement strategies for each Raman molecules. Mixed signal-ligand SERS probes improve the complexity of Raman spectra and expand the coding capacity. By integrating the strategies, SERS inks are produced and applied in anti-counterfeiting. With these improvements, this work breaks the constrains of probe selection, bringing SERS one step closer to the sensor or anti-counterfeiting application.

Gallic Acid Ameliorates Aspergillus Fumigatus Keratitis Through Reducing Fungal Load and Suppressing the Inflammatory Response.

Purpose: The purpose of this study was to explore the antifungal and anti-inflammatory effects of gallic acid (GA) on Aspergillus fumigatus (A. fumigatus) keratitis. Methods: CCK-8 assay and Draize eye test were used to determine the non-cytotoxic concentration of GA in RAW264.7 cells and an A. fumigatus keratitis mouse model. The antifungal effects of GA were analyzed using minimal inhibitory concentration (MIC), biofilm formation test, fungal adherence assay, calcofluor white staining, and propidium iodide staining. The therapeutic effects of GA were estimated by slit lamp photographs, clinical score, hematoxylin and eosin (H&E) staining, and Periodic acid-Schiff staining in vivo. Immunofluorescence staining and myeloperoxidase assay were conducted to identify neutrophil infiltration and activity. RT-PCR, ELISA, and Western blot were performed to detect the expression of pro-inflammatory cytokines and Nrf2/HO-1. Results: In HCECs and A. fumigatus keratitis mouse model, GA at 100 µg/mL did not affect cell viability, thus this concentration was applied to subsequent experiments. In vitro, GA significantly inhibited A. fumigatus growth, biofilm formation, and adhesion. In vivo, 100 µg/mL GA alleviated the severity of fungal keratitis (FK) by repressing fungal load, reducing neutrophil infiltration, and lowering MPO activity. Besides, the expression of IL-1ß, TNF-α, LOX-1, and COX-2 was inhibited, whereas Nrf2 and HO-1 expression was enhanced at both mRNA and protein levels in the 100 µg/mL GA treated group in comparison to PBS control. Conclusions: GA ameliorates FK severity through inhibiting A. fumigatus load, reducing neutrophils infiltration, downregulating the expression of pro-inflammatory cytokines, and enhancing the Nrf2/HO-1 pathway, which provides new insight into A. fumigatus keratitis treatment.

Transanal total mesorectal excision port-assisted perineal hernia repair: A case report.

Perineal hernia after abdominoperineal resection (APR) is a troublesome problem, and severe cases require surgical treatment. However, perineal hernia repair is challenging, especially when combined with intestinal adhesions. The difficulty of the operation lies in performing adhesiolysis and mesh placement under poor visibility. While there are traditional, laparoscopic and even robotic methods of performing this procedure, no easy and minimally-invasive approach has been reported. Here, we report the case of a patient with perineal hernia, who underwent transanal total mesorectal excision (TaTME) port-assisted laparoscopic perineal hernia repair. The operation was successful, the postoperative recovery was uneventful, the patient's symptoms improved significantly, and no recurrence was found during the 4-month follow-up. The availability and safety of TaTME port-assisted perineal hernia repair provide a promising approach for hernia repair. Compared with traditional perineal or laparoscopic abdominal approaches, this procedure is less invasive and results in a better field of vision.

Flavopiridol Protects against Fungal Keratitis due to Aspergillus fumigatus by Alleviating Inflammation through the Promotion of Autophagy.

Fungal keratitis is a serious infectious keratopathy related to fungal virulence and excessive inflammatory responses. Autophagy exhibits a potent ability to resolve inflammation during fungal infection. This study aimed to investigate the protective function of flavopiridol in Aspergillus fumigatus keratitis and explore its effects on autophagy. In our study, the corneas of the fungal keratitis mouse model were treated with 5 µM flavopiridol. In vitro, RAW 264.7 cells were pretreated with 200 nM flavopiridol before fungal stimulation. A. fumigatus was incubated with flavopiridol, and the antifungal activity of flavopiridol was detected. Our results indicated that flavopiridol treatment notably reduced clinical scores as well as cytokines expression of infected corneas. In infected RAW 264.7 cells, flavopiridol treatment inhibited IL-1ß, IL-6, and TNF-α expression but promoted IL-10 expression. Transmission electron microscopy (TEM) images showed that more autolysosomes were present in infected corneas and RAW 264.7 cells after flavopiridol treatment. Flavopiridol treatment notably upregulated the protein expression of LC3, Beclin-1, and Atg-7. 3-Methyladenine (3-MA, an inhibitor of autophagy) pretreatment counteracted the cytokine regulation induced by flavopiridol. Moreover, flavopiridol promoted the phagocytosis of RAW 264.7 cells. Flavopiridol also exhibited antifungal activity by restricting fungal growth and limiting fungal biofilm formation and conidial adhesion. In conclusion, flavopiridol significantly alleviated the inflammation of fungal keratitis by activating autophagy. In addition, flavopiridol promoted the phagocytosis of RAW 264.7 cells and exhibited antifungal function, indicating the potential therapeutic role of flavopiridol in fungal keratitis.

CRISPR-based knockout screening identifies the loss of MIEF2 to enhance oxaliplatin resistance in colorectal cancer through inhibiting the mitochondrial apoptosis pathway.

The first-line anticancer agent oxaliplatin (OXL) is the preferred drug for treating colorectal cancer (CRC); however, the development of drug resistance is common in patients treated with OXL, which considerably reduces the efficacy of OXL-based regimens. By performing genome-wide CRISPR/Cas9 library knockdown screening, we found that mitochondrial elongation factor 2 (MIEF2) was among the top candidate genes. The OXL-resistant cell lines and organoids developed in the present study showed stable but low expression of MIEF2. Reduced MIEF2 expression may enhance CRC resistance to OXL by reducing mitochondrial stability and inhibiting apoptosis by decreasing cytochrome C release. In conclusion, among the different biomarkers of OXL resistance in CRC, MIEF2 may serve as a specific biomarker of OXL responsiveness and a potential target for the development of therapies to improve chemotherapeutic effectiveness.