CCPG1 involved in corneal Aspergillus fumigatus infection

INTRODUCTION

Corneal diseases, especially infectious corneal diseases,are one of the leading causes of blindness worldwide

.Its importance in the prevention and treatment of eye diseases is second only to cataracts

. Fungal keratitis (FK), as a kind of blinding keratopathy, has been paid more attention globally

.In developing countries and tropical and subtropical regions,FK usually occurs after vegetative trauma

. In developed countries, improper wearing of contact lenses is the main reason for FK

. Due to the lack of effective drugs and surgical treatments, the cure of FK is still challenging.

after 4h infection, the protein expression of CCPG1 increased significantly with time (Figure 1B). The above results show that CCPG1 participated in the antifungal immune response of HCECs.

Bernales

and Schuck

found that in yeast, the endoplasmic reticulum provided a membrane for autophagosomes and was also a target of autophagy. As a key intracellular organelle, the endoplasmic reticulum is responsible for overseeing the synthesis of transmembrane proteins and secreted proteins. The unfolded protein response (UPR) occurs when the endoplasmic reticulum is under stress. UPR is a response that resolves the defects of endoplasmic reticulum cavity protein inhibition through transcriptional activation.When a protein misfolds and accumulates in the endoplasmic reticulum, some signaling pathways are activated, and then a cascade reaction is triggered to inhibit translation

.

Use RNAiso plus reagent (Takara) to extract total RNA. PrimeScript RT kit (Takara) was used for reverse transcription of total RNA (2 μg). SYBR Green was used for real-time PCR.The sequences of all oligonucleotide primers used can be as follows: hβ-actinF-GCTCCTCCTGAGCGCAAG and R-CATCTGCTGGAAGGTGGACA, hCCPG1 F-GTCACACTTTTTCCCCTCCA and R-CTCAGTGGCC ATAAAGCACA.

The role of CCPG1 in UPR is still under further study. Smith and Wilkinson

, by inhibiting the expression of CCPG1,found that the expansion of endoplasmic reticulum (ER) and the destruction of cell distribution in mouse retinal acinar cells were aggravated; at the same time, molecular markers of ER stress also increased. That is to say, it is related to the loss of UPR-regulated ER phagocytosis above the maintenance of the ER.

This type of selective autophagy promotes the targeted elimination of specific organelles or cargo through the action of specific autophagy receptors. These substances are recruited and isolated into autophagosomes

.

This experiment predicts whether CCPG1 can participate in the current harmful FK, which can prevent excessive inflammation, protect the host from collateral damage and increase the treatment of FK.

MATERIALS AND METHODS

(

) strain 3.0772 was purchased from the China General Microbial Culture Collection Center (Beijing, China)and was cultured for 3-4d. Remove impurities, filter with sterile cotton gauze to obtain a pure conidia suspension, and adjust the concentration to 5×10

cfu/mL for retention.

对照组124例患者中，单独用药91例，占73.39%；二联用药8例，占6.45%；三联用药1例，占0.81%。干预组139例患者中，单独用药18例，占12.95%；无联合用药情况。预防性应用抗菌药物的联合用药情况，见表3。

Human corneal epithelial cells(HCECs) and THP-1 macrophages were seeded into 6-well plates and 12-well plates, respectively, and then grown to 80%confluence in an incubator (37℃, 5% CO

). After stimulation with

conidia for 4, 8, and 16h later, the cells were collected and lysed, and then qRT-PCR and Western blot tests were performed.

disease, a common inflammatory bowel disease. This noncanonical autophagy pathway can prevent inflammation during the process of removing dead cells and protect autoimmunity and inflammatory bowel disease

.

Cell cycle progression 1 (CCPG1) is an endoplasmic reticulum protein induced by endoplasmic reticulum stress. Its expression is up-regulated under the induction of UPR. It can interact with GABARAP and LC3 family proteins

.

夹层加热系统的投入减少机组启动时间，降低上下缸温差，改善机组启动条件，有效避免因加热膨胀不均可能发生的碰磨引起振动。锅炉点火起压后，炉侧压力为0.2～0.5 MPa，凝汽器建立真空后稍开联箱进汽门，维持联箱压力0.1～0.3 MPa，对汽轮机汽缸夹层加热供汽及联箱暖管疏水；汽轮机冲转到500 r/min投入汽缸夹层加热，控制汽缸温升率小于1.5 ℃/min，使汽缸内外加热均匀；高压外缸下半外壁金属温度达到320 ℃时停用夹层加热系统。

The HCECs and THP-1 were collected and placed in RIPA buffer (Solarbio, China): PMSF (Solarbio,China): phosphatase inhibitor cocktail I (MedChemExpress,USA) at a ratio of 98:1:1 and lysed on ice. A BCA kit (Solarbio,China) was used to determine the protein concentration.

Then used 8%-16% SDS-PAGE gel (GenScript, China)for total protein electrophoresis, and transfferd to PVDF membrane. The PVDF membrane was blocked in blocking buffer (Beyotime, China) at 37℃ for 2h, and with antiinterleukin-1β (IL-1β) primary antibody incubated (R&D,USA) or anti-β-actin primary antibody (CST, USA) or anti-CCPG1 primary antibody (Santa Cruz Biotechnology, USA)overnight at 4℃. The membrane was then incubated with HRP-labeled secondary antibody. Western ECL blotting substrate (Bio-Rad, USA) was added to the PVDF membrane to develop blots. Digital images were analyzed using a Vilber Solo 4S chemiluminescence imaging system.

The slides with THP-1 cells were soaked in phosphate buffer saline (PBS) for 3 times,and fixed with 4% paraformaldehyde. Then the slides were permeated with 0.5% Triton X-100 (prepared in PBS) for 20min at room temperature, and immersed in PBS 3 times.Next, normal goat serum was added to the slides, blocking for 30min at 37℃; then the slides were incubated overnight with the following antibodies: anti-CCPG1 (Santa Cruz Biotechnology, USA), anti-CLEC1 (Novus, USA).

Honest()(Ŝ)⊃∃X∃x.Computes(X,{RB}K)∧Send(X,x)∧Contains(x,{RB}K)∧After(Send(X,x),Receive(B,{,

According to STRING Interaction Network Preview(Figure 4A), we predicted that C-type lectin-like receptor-1(CLEC-1) and CCPG1 might be interacting proteins.Immunofluorescence results showed that CCPG1 and CLEC1 proteins were distributed in macrophages, and there was obvious intracytoplasmic co-localization (Figure 4B).

The statistical significance of each score and qRT-PCR was determined by

-test. The data are expressed as mean±standard deviation (SD) and analyzed by GraphPad 5.0 software. When

＜0.05, the data is considered significant.

RESULTS

After stimulation by

, the mRNA expression of CCPG1 increased indistinctively in THP-1 pretreated with dectin-1 neutralizing antibody (Figure 3A). But compared with the control group, there was no statistical difference.

Recent studies have shown that autophagy plays an immunomodulatory role in innate and adaptive immune responses by selectively targeting signal transduction molecules. Autophagy is a highly conserved cellular process in eukaryotes. Its function of maintaining cell homeostasis is achieved by supporting cell survival and regulating inflammation. Autophagy can degrade unnecessary or functionally abnormal intracellular components, such as abnormal proteins, old organelles

and pathogens

, and has been extensively studied in various immune cells including T cells

, B cells

, and macrophages

. More and more evidences show that autophagy plays an important role in the host's defense against microbial infections and inflammation.For example, it can inhibit the activation of inflammasomes in macrophages

, and may eliminate active inflammasomes through p62 ubiquitination

.

Similarly,

stimulated THP-1 to establish cell models. The mRNA expression of CCPG1 did not change significantly after 4h, and increased significantly after 8 and 16h (Figure 2A). Through the Western blot experiment, differently, we observed that the protein level of CCPG1 began to increase after 4h stimulation, and increased significantly after 8 and 16h (Figure 2B). Accordingly, CCPG1 also participated in the THP-1 antifungal immune response.

stimulated HCECs to establish fungal infection cell models. qRT-PCR results showed that the mRNA expression of CCPG1 increased after 4h, and the CCPG1 mRNA levels increased significantly with time (Figure 1A). Western blot experiments showed that

The protein expression level of IL-1β was significantly decreased compared with the control group. However,treatment with dectin-1 neutralizing antibody did not affect the protein expression of CCPG1 (Figure 3B). Although all regulate the immune response by participating in noncanonical autophages, there was no necessary connection between dectin-1 and CCPG1.

Fluorescent secondary antibody (ProteinTech) was added in the dark for 1h, and then the slides were soaked with PBST 3 times. Next, DAPI solution (Solarbio, China) was added dropwise and incubated for 5min in the dark, and then the specimens were stained with nuclei and immersed in PBST 3 times. Finally, a fluorescence micrograph was taken by Zeiss Axiovert microscope.

DISCUSSION

Autophagy was originally described as a process of selfdecomposition, but now it is known that it plays a key role in the face of many aspects such as bacteria, viruses and parasitic pathogens

. Recent mechanism studies have shown that autophagy played an immunomodulatory role in innate and adaptive immune responses by selectively supplementing signal molecules

. In the past decade, another form of autophagy had emerged, called LC3- (microtubule-associated protein 1A/1b light chain 3-)-associated phagocytosis (LAP)or non-canonical autophagy. LAP is a unique pathway that participates in cell surface receptor signaling by recruiting the LC3-phosphatidylethanolamine (PE) binding system during phagocytosis

.

精神分裂症属于精神疾病的一种,其发病率在6.55%左右,阴性症状、阳性症状以及认知功能障碍为患者主要临床症状,早期患者主要有发呆、不理睬人及反应不灵敏等临床表现,随着病情逐渐发展,患者可出现妄想、幻觉等严重表现。本文主要选取了74例急性期精神分裂症患者为研究对象,分别给予氨磺必利与奥氮平治疗,对比两种方法的临床疗效,现报告如下。

LAP has recently become a major anti-inflammatory pathway,playing an important role in intracellular and physiological

.Jostins

found that LAP was associated with Crohn's

THP-1 macrophages were pretreated with dectin-1 neutralizing antibody (R&D) and IgG neutralizing antibody (R&D) for 2h.After 16h of stimulation with

conidia, the cells were collected for qRT-PCR and Western blot.

权筝今天在小花园跟何东分手后就打电话找发小儿丁香。丁香一听出了这么大的事儿，马上请假来见她。丁香是精神病医生，俩人分析结果，何东是恐婚，给他点时间让他不恐。

拮抗真菌种类较多，主要为木霉属，其次是酵母菌，另外也发现担子菌、炭角菌、拟茎点霉属和拟盘多毛孢属等对果蔬病原菌有拮抗作用[5]。颜霞等[7]从菊科植物假橐吾中分离得到86株内生真菌，分为10个属，交链孢霉属和丝核菌属占优势 (分别为41.9%和16.3%)，其中31株内生真菌分别对细菌、植物病原菌有显著的抑制作用。

The ER is the largest membrane-bound organelle in eukaryotic cells. Its complex morphology, including flakes, tubules and impurities

, reflects its different roles in various physiological processes including autophagosomes

.Studies have confirmed that the four receptors RETREG1,SEC62, CCPG1, and RTN3 helped to isolate the ER separation products into autophagosomes. Among them, CCGP1 and RTN3 were mainly responsible for the conversion of ER tubules

.

When the non-canonical autophagy pathway is activated,UPR induction leads to an increase in the ER CCPG1 protein.When CCPG1 has FIR and LIR motifs, it can interact with autophagy proteins of the RB1CC1/FIP200 and ATG8 families, respectively. It helps to isolate part of the ER to the phagosome, thereby restoring the ER replacement

.

Both corneal epithelial cells and macrophages were important cells involved in the antifungal infection of the cornea. When HCECs and THP-1 stimulated by

, the expression of CCPG1 increased significantly. Our experiment proved that CCPG1, as an alternative autophagy protein for non-canonical autophagy pathways, participated in corneal antifungal immunity. Since THP-1 cell line is the most common in FK,follow-up experiments use THP-1 cells as the cell model.

Previous experiments

have found that dectin-1 was one of the most important host anti-fungal pattern recognition receptors. The members of the dectin-1 cluster include CLEC-1 receptors

. We found that using dectin-1 neutralizing antibody to inhibit the expression of dectin-1 down-regulated the expression of IL-1β, indicating that the neutralization was effective. Surprisingly, there was no significant change in the expression of CCPG1. This experiment proved that there was no necessary connection between dectin-1 and the generation of CCPG1.

在动态MCS场景下，将AdaCode与RainbowRate[3]进行了比较.RainbowRate是针对长距离无线链路设计的速率选择算法，优于其它适用于短距离链路的速率选择算法.

Alice将要给Bob发送一条消息，要求消息包含数字签名来进行身份识别，那么可以定义一组参数(CURVE,G,n)，其中CURVE表示椭圆曲线的点域以及它所使用的几何方程，G表示椭圆曲线基点，大素数n是椭圆曲线的阶数[7]。接下来我们介绍数字签名的具体过程和验证数字签名的具体过程。

According to the STRING Interaction Network Preview,we predicted that CLEC-1 and CCPG1 might interact with proteins. We verified the expression positions of the two were basically coincident by immunofluorescence experiments:CCPG1 and CLEC-1 were co-expressed in macrophages. At present, there are few studies on CLEC-1 and CCPG1, so it is difficult to buy commercial antibodies, and there is no way to do further research such as co-IP.

This study emphasizes that CCPG1 is involved in corneal antifungal immunity, but more in-depth research is needed.Future research directions will continue to explore the mechanism of CCPG1 in the corneal research response. In summary, CCPG1 as a non-canonical autophagy cargo receptor is involved in corneal antifungal immunity.

Supported by the National Natural Science Foundation of China (No.82171019); the Natural Science Foundation of Shandong Province (No.ZR2021MH368);Traditional Chinese Medicine Research Project of Qingdao(No.2020-zyy055); Shandong Qingdao Outstanding Health Professional Development Fund.

分析框架是以“螺旋式”组织形式为指导思想，在对研究对象与相关文献反复对照分析的基础上，主要将孔凡哲提出的“教材运用‘螺旋式上升’可以从深度、广度和应用等维度予以实现”[6]与李卓、于波研究小学数学教材螺旋式结构编排的“螺旋的时间间隔”等维度[9]，综合化和具体化而成．具体包括3个一级维度：螺旋间隔、内容广度、内容深度．

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