Investigating Retinal Explant Models Cultured in Static and Perfused Systems to Test the Performance of Exosomes Secreted from Retinal Organoids.

BACKGROUND: Ex vivo cultures of retinal explants are appropriate models for translational research. However, one of the difficult problems of retinal explants ex vivo culture is that their nutrient supply needs cannot be constantly met. NEW METHOD: This study evaluated the effect of perfused culture on the survival of retinal explants, addressing the challenge of insufficient nutrition in static culture. Furthermore, exosomes secreted from retinal organoids (RO-Exos) were stained with PKH26 to track their uptake in retinal explants to mimic the efficacy of exosomal drugs in vivo. RESULTS: We found that the retinal explants cultured with perfusion exhibited significantly higher viability, increased NeuN+ cells, and reduced apoptosis compared to the static culture group at Days Ex Vivo (DEV) 4, 7, and 14. The perfusion-cultured retinal explants exhibited reduced mRNA markers for gliosis and microglial activation, along with lower expression of GFAP and Iba1, as revealed by immunostaining. Additionally, RNA-sequencing analysis showed that perfusion culture mainly upregulated genes associated with visual perception and photoreceptor cell maintenance while downregulating the immune system process and immune response. RO-Exos promoted the uptake of PKH26-labelled exosomes and the growth of retinal explants in perfusion culture. COMPARISON WITH EXISTING METHODS: Our perfusion culture system can provide a continuous supply of culture medium to achieve steady-state equilibrium in retinal explant culture. Compared to traditional static culture, it better preserves the vitality, provides better neuroprotection, and reduces glial activation. CONCLUSIONS: This study provides a promising ex vivo model for further studies on degenerative retinal diseases and drug screening.

Unveiling the role of interleukin-6 in pancreatic cancer occurrence and progression.

Pancreatic cancer is difficult to diagnose early and progresses rapidly. Researchers have found that a cytokine called Interleukin-6 (IL-6) is involved in the entire course of pancreatic cancer, promoting its occurrence and development. From the earliest stages of pancreatic intraepithelial neoplasia to the invasion and metastasis of pancreatic cancer cells and the appearance of tumor cachexia, IL-6 drives oncogenic signal transduction pathways and immune escape that accelerate disease progression. IL-6 is considered a biomarker for pancreatic cancer diagnosis and prognosis, as well as a potential target for treatment. IL-6 antibodies are currently being explored as a hot topic in oncology. This article aims to systematically explain how IL-6 induces the deterioration of normal pancreatic cells, with the goal of finding a breakthrough in pancreatic cancer diagnosis and treatment.

Two new phenylpropanoids from the whole plant of Ainsliaea bonatii.

Two new phenylpropanoids, ainsbons A and B (1 and 2), along with a known analogue coniferyl diisovalerate (3) were isolated from the whole plant of Ainsliaea bonatii. Their structures were elucidated by analysis of NMR spectroscopic data and HRESIMS, and the absolute configuration of 2 was established by the optical rotation calculations. Compounds 1-3 were evaluated for their effects on LPS-induced nitric oxide production, and 1 and 3 showed inhibitory activities with IC50 values of 43.43 and 7.57 µM, respectively.

Deciphering the role of transcription factors in glioblastoma cancer stem cells.

Glioblastoma (GBM), the most aggressive and fatal brain malignancy, is largely driven by a subset of tumor cells known as cancer stem cells (CSCs). CSCs possess stem cell-like properties, including self-renewal, proliferation, and differentiation, making them pivotal for tumor initiation, invasion, metastasis, and overall tumor progression. The regulation of CSCs is primarily controlled by transcription factors (TFs) which regulate the expressions of genes involved in maintaining stemness and directing differentiation. This review aims to provide a comprehensive overview of the role of TFs in regulating CSCs in GBM. The discussion encompasses the definitions of CSCs and TFs, the significance of glioma stem cells (GSCs) in GBM, and how TFs regulate GSC self-renewal, proliferation, differentiation, and transformation. The potential for developing TF-targeted GSC therapies is also explored, along with future research directions. By understanding the regulation of GSCs by TFs, we may uncover novel diagnostic and therapeutic strategies against this devastating disease of GBM.

Differences in insomnia-related self-reported outcomes among elderly hospitalized patients.

BACKGROUND: Insomnia is among the most common sleep disorders worldwide. Insomnia in older adults is a social and public health problem. Insomnia affects the physical and mental health of elderly hospitalized patients and can aggravate or induce physical illnesses. Understanding subjective feelings and providing reasonable and standardized care for elderly hospitalized patients with insomnia are urgent issues. AIM: To explore the differences in self-reported outcomes associated with insomnia among elderly hospitalized patients. METHODS: One hundred patients admitted to the geriatric unit of our hospital between June 2021 and December 2021 were included in this study. Self-reported symptoms were assessed using the Athens Insomnia Scale (AIS), Generalized Anxiety Disorder Scale-7 (GAD-7), Geriatric Depression Scale-15 (GDS-15), Memorial University of Newfoundland Scale of Happiness (MUNSH), Barthel Index Evaluation (BI), Morse Fall Scale (MFS), Mini-Mental State Examination, and the Short Form 36 Health Survey Questionnaire (SF-36). Correlation coefficients were used to analyze the correlation between sleep quality and self-reported symptoms. Effects of insomnia was analyzed using Logistic regression analysis. RESULTS: Nineteen patients with AIS ≥ 6 were included in the insomnia group, and the incidence of insomnia was 19% (19/100). The remaining 81 patients were assigned to the non-insomnia group. There were significant differences between the two groups in the GDA-7, GDS-15, MUNSH, BI, MFS, and SF-36 items (P < 0.05). Patients in the insomnia group were more likely to experience anxiety, depression, and other mental illnesses, as well as difficulties with everyday tasks and a greater risk of falling (P < 0.05). Subjective well-being and quality of life were poorer in the insomnia group than in the control group. The AIS scores positively correlated with the GAD-7, GDS-15, and MFS scores in elderly hospitalized patients with insomnia (P < 0.05). Logistic regression analysis showed that GDS-15 ≥ 5 was an independent risk factor for insomnia in elderly hospitalized patients (P < 0.05). CONCLUSION: The number of self-reported symptoms was higher among elderly hospitalized patients with insomnia. Therefore, we should focus on the main complaints of patients to meet their care needs.

A novel exosome-based multifunctional nanocomposite platform driven by photothermal-controlled release system for repair of skin injury.

Currently, exosomes showed appropriate potential in the repair of skin injury. However, the functions of the exosomes could be compromised rapidly due to their short half-life and high clearance rate in vivo. In addition, the controlled release of effective concentrations of exosomes could increase the utilization efficiency of exosomes in wound healing. Accordingly, the design of an effective system for the controlled delivery of exosomes during the wound treatment period was necessary. In this contribution, we designed a novel exosome-based multifunctional nanocomposite platform with photothermal-controlled release performance for the repair of skin injury. Based on the agarose hydrogel, two-dimensional Ti3C2 (Ti3C2 MXene) and human umbilical cord mesenchymal stem cell (hucMSC)-derived exosomes, the as-prepared platform (i.e., hucMSC-derived exosome/Ti3C2 MXene hydrogel) was synthesized for the first time. Apart from possessing injectability, the hucMSC-derived exosome/Ti3C2 MXene hydrogel utilized the excellent photothermal effect of Ti3C2 MXene and proper phase transition performance of agarose hydrogel to provide a photothermal-controlled release system for the hucMSC-derived exosomes, which was beneficial for the personalized on-demand drug delivery. Importantly, the hucMSC-derived exosomes maintained their inherent structure and activity after being released from the Ti3C2 MXene hydrogel. Additionally, the as-prepared hydrogel with multifunctional performance also presented remarkable biocompatibility and photothermal-antibacterial property, and could efficiently accelerate wound healing by promoting cell proliferation, angiogenesis, collagen deposition, and reducing the level of inflammation at the wound site. The results suggested that the exosome-based multifunctional nanocomposite platform with great potential for wound healing would make significant advances in the revolution of traditional treatment methods in skin injury.

Silver Nanoparticles Encapped by Dihydromyricetin: Optimization of Green Synthesis, Characterization, Toxicity, and Anti-MRSA Infection Activities for Zebrafish (Danio rerio).

To achieve the environmentally friendly and rapid green synthesis of efficient and stable AgNPs for drug-resistant bacterial infection, this study optimized the green synthesis process of silver nanoparticles (AgNPs) using Dihydromyricetin (DMY). Then, we assessed the impact of AgNPs on zebrafish embryo development, as well as their therapeutic efficacy on zebrafish infected with Methicillin-resistant Staphylococcus aureus (MRSA). Transmission electron microscopy (TEM) and dynamic light-scattering (DLS) analyses revealed that AgNPs possessed an average size of 23.6 nm, a polymer dispersity index (PDI) of 0.197 ± 0.0196, and a zeta potential of -18.1 ± 1.18 mV. Compared to other published green synthesis products, the optimized DMY-AgNPs exhibited smaller sizes, narrower size distributions, and enhanced stability. Furthermore, the minimum concentration of DMY-AgNPs required to affect zebrafish hatching and survival was determined to be 25.0 µg/mL, indicating the low toxicity of DMY-AgNPs. Following a 5-day feeding regimen with DMY-AgNP-containing food, significant improvements were observed in the recovery of the gills, intestines, and livers in MRSA-infected zebrafish. These results suggested that optimized DMY-AgNPs hold promise for application in aquacultures and offer potential for further clinical use against drug-resistant bacteria.

Single-cell transcriptomics dissects the transcriptome alterations of hematopoietic stem cells in myelodysplastic neoplasms.

BACKGROUND: Myelodysplastic neoplasms (MDS) are myeloid neoplasms characterized by disordered differentiation of hematopoietic stem cells and a predisposition to acute myeloid leukemia (AML). The underline pathogenesis remains unclear. METHODS: In this study, the trajectory of differentiation and mechanisms of leukemic transformation were explored through bioinformatics analysis of single-cell RNA-Seq data from hematopoietic stem and progenitor cells (HSPCs) in MDS patients. RESULTS: Among the HSPC clusters, the proportion of common myeloid progenitor (CMP) was the main cell cluster in the patients with excess blasts (EB)/ secondary AML. Cell cycle analysis indicated the CMP of MDS patients were in an active proliferative state. The genes involved in the cell proliferation, such as MAML3 and PLCB1, were up-regulated in MDS CMP. Further validation analysis indicated that the expression levels of MAML3 and PLCB1 in patients with MDS-EB were significantly higher than those without EB. Patients with high expression of PLCB1 had a higher risk of transformation to AML. PLCB1 inhibitor can suppress proliferation, induce cell cycle arrest, and activate apoptosis of leukemic cells in vitro. CONCLUSION: This study revealed the transcriptomic change of HSPCs in MDS patients along the pseudotime and indicated that PLCB1 plays a key role in the transformation of MDS into leukemia.

DMC-siERCC2 hybrid nanoparticle enhances TRAIL sensitivity by inducing cell cycle arrest for glioblastoma treatment.

ERCC2 plays a pivotal role in DNA damage repair, however, its specific function in cancer remains elusive. In this study, we made a significant breakthrough by discovering a substantial upregulation of ERCC2 expression in glioblastoma (GBM) tumor tissue. Moreover, elevated levels of ERCC2 expression were closely associated with poor prognosis. Further investigation into the effects of ERCC2 on GBM revealed that suppressing its expression significantly inhibited malignant growth and migration of GBM cells, while overexpression of ERCC2 promoted tumor cell growth. Through mechanistic studies, we elucidated that inhibiting ERCC2 led to cell cycle arrest in the G0/G1 phase by blocking the CDK2/CDK4/CDK6/Cyclin D1/Cyclin D3 pathway. Notably, we also discovered a direct link between ERCC2 and CDK4, a critical protein in cell cycle regulation. Additionally, we explored the potential of TRAIL, a low-toxicity death ligand cytokine with anticancer properties. Despite the typical resistance of GBM cells to TRAIL, tumor cells undergoing cell cycle arrest exhibited significantly enhanced sensitivity to TRAIL. Therefore, we devised a combination strategy, employing TRAIL with the nanoparticle DMC-siERCC2, which effectively suppressed the GBM cell proliferation and induced apoptosis. In summary, our study suggests that targeting ERCC2 holds promise as a therapeutic approach to GBM treatment.

More potential uses of specific perforator flaps in the calf - A cadaveric study on the subdermal vascular structure of the lower leg.

BACKGROUND: The perforator flap has garnered significant interest since its inception due to its advantage of not needing a vascular network at the deep fascial level. Perforator flaps are commonly utilized in different flap transplant surgeries, and the thigh flap is presently the most widely used perforator flap. Is it possible for the calf to replace the thigh as a more suitable site for harvesting materials? Currently, there is a lack of relevant anatomical research. This study aims to address this question from an anatomical and imaging perspective. METHODS: This study used cadavers to observe the branches and courses of perforators on the calf and the distribution of skin branches using microdissection techniques, digital X-ray photography, and micro-computed tomography techniques. RESULTS: The perforators had three main branches: the vertical cutaneous branch, the oblique cutaneous branch, and the superficial fascial branch. The superficial fascial branch traveled in the superficial fascia and connected with the nearby perforators. The vertical and oblique cutaneous branches entered the subdermal layer and connected with each other to create the subdermal vascular network. CONCLUSIONS: We observed an intact calf cutaneous branch chain between the cutaneous nerve and the perforator of the infrapopliteal main artery at the superficial vein site. Utilizing this anatomical structure, the calfskin branch has the potential to serve as a substitute for thigh skin flap transplantation and may be applied to perforator flap transplantation in more locations.

Imaging and Liquid Biopsy for Distinguishing True Progression From Pseudoprogression in Gliomas, Current Advances and Challenges.

RATIONALE AND OBJECTIVES: Gliomas are aggressive brain tumors with a poor prognosis. Assessing treatment response is challenging because magnetic resonance imaging (MRI) may not distinguish true progression (TP) from pseudoprogression (PsP). This review aims to discuss imaging techniques and liquid biopsies used to distinguish TP from PsP. MATERIALS AND METHODS: This review synthesizes existing literature to examine advances in imaging techniques, such as magnetic resonance diffusion imaging (MRDI), perfusion-weighted imaging (PWI) MRI, and liquid biopsies, for identifying TP or PsP through tumor markers and tissue characteristics. RESULTS: Advanced imaging techniques, including MRDI and PWI MRI, have proven effective in delineating tumor tissue properties, offering valuable insights into glioma behavior. Similarly, liquid biopsy has emerged as a potent tool for identifying tumor-derived markers in biofluids, offering a non-invasive glimpse into tumor evolution. Despite their promise, these methodologies grapple with significant challenges. Their sensitivity remains inconsistent, complicating the accurate differentiation between TP and PSP. Furthermore, the absence of standardized protocols across platforms impedes the reliability of comparisons, while inherent biological variability adds complexity to data interpretation. CONCLUSION: Their potential applications have been highlighted, but gaps remain before routine clinical use. Further research is needed to develop and validate these promising methods for distinguishing TP from PsP in gliomas.

Efficacy of the posterior nasal nerve resection combined with hormone transnasal nebulization on difficult-to-treat rhinosinusitis: a retrospective analysis.

OBJECTIVE: A retrospective analysis was performed to explore the clinical effect of the Posterior Nasal Nerve (PNN) resection combined with hormone transnasal nebulization on Difficult-to-Treat Rhinosinusitis (DTRS). METHODS: A total of 120 DTRS patients were selected and divided into a control group (n = 60) and a study group (n = 60) according to different treatments. The control group patients were treated via PNN resection, followed by normal saline transnasal nebulization; the study group patients were given PNN resection and then treated with budesonide suspension transnasal nebulization. Subsequently, the comparison was performed between the two groups in terms of (1) Clinical baseline characteristics; (2) Sino-nasal Outcome Test (SNOT)-22 scores before treatment and after 3-months, 6-months and 12-months of treatment; (3) Lund-MacKay scores before treatment and after 10, 30, 90, and 180 days of treatment; (4) Incidence of adverse reactions during treatment. RESULTS: There was no significant difference in SNOT-22 or Lund-Kennedy scores between the two groups before treatment (p > 0.05). After treatment, the SNOT-22 and Lund-Kennedy scores of the control and the study groups were decreased, and compared with the control group, the SNOT-22 and Lund-Kennedy scores in the study group improved more significantly (p < 0.05). In addition, the study group and the control group presented with 1 and 4 cases of nasal adhesion, 2 and 3 cases of epistaxis, 1 and 4 cases of sinus orifice obstruction, 1 and 3 cases of lacrimal duct injuries, respectively. The incidence of adverse reactions in the study group was significantly lower than that in the control group (8.3% vs. 23.3%) (p < 0.05). CONCLUSION: PNN resection combined with hormone transnasal nebulization treatment can improve the symptoms and quality of life of DTRS patients, with good clinical efficacy but few adverse reactions. Therefore, such combination treatment deserves a promotion and application clinically. LEVEL OF EVIDENCE: Level 3.

Comparison of the Efficacy of Different Radiofrequency Techniques for the Treatment of Lumbar Facet Joint Pain: Combined with Anatomy.

PURPOSE OF REVIEW: Lumbar facet pain is generally considered to be one of the major causes of chronic low back pain. Each lumbar facet joint is innervated by the medial branch of the posterior spinal nerve from its own level and above. Radiofrequency (RF) of the medial branch of the posterior branch of the spinal nerve is an effective method for the treatment of lumbar facet pain. RF technology is diverse, including traditional radiofrequency (TRF), pulsed radiofrequency (PRF), cooled radiofrequency (CRF), low-temperature plasma radiofrequency ablation (CA), and other treatment methods. The purpose of this paper is to compare the efficacy of different radiofrequency techniques and to analyze the reasons for this in the context of anatomy. RECENT FINDINGS: There have been studies confirming the differences in efficacy of different RF techniques. However, most of the studies only compared two RF techniques, not four techniques, TRF, CRF, PRF, and CA, and did not analyze the reasons for the differences in efficacy. This article reviews the differences in the efficacy of the above four RF techniques, clarifies that the differences are mainly due to the inability to precisely localize the medial branch of the posterior branch of the spinal nerve, analyzes the reasons for the inability to precisely localize the posterior branch of the spinal nerve in conjunction with anatomy, and proposes that the development of RF technology for lumbar facet pain requires more in-depth anatomical, imaging, and clinical studies.

Hepatic danger signaling triggers TREM2+ macrophage induction and drives steatohepatitis via MS4A7-dependent inflammasome activation.

Metabolic dysfunction-associated steatohepatitis (MASH), formerly known as nonalcoholic steatohepatitis (NASH), is an advanced stage of metabolic fatty liver disease. The pathogenic mechanisms of MASH center on hepatocyte injury and the ensuing immune response within the liver microenvironment. Recent work has implicated TREM2+ macrophages in various disease conditions, and substantial induction of TREM2+ NASH-associated macrophages (NAMs) serves as a hallmark of metabolic liver disease. Despite this, the mechanisms through which NAMs contribute to MASH pathogenesis remain poorly understood. Here, we identify membrane-spanning 4-domains a7 (MS4A7) as a NAM-specific pathogenic factor that exacerbates MASH progression in mice. Hepatic MS4A7 expression was strongly induced in mouse and human MASH and associated with the severity of liver injury. Whole-body and myeloid-specific ablation of Ms4a7 alleviated diet-induced MASH pathologies in male mice. We demonstrate that exposure to lipid droplets (LDs), released upon injury of steatotic hepatocytes, triggered NAM induction and exacerbated MASH-associated liver injury in an MS4A7-dependent manner. Mechanistically, MS4A7 drove NLRP3 inflammasome activation via direct physical interaction and shaped disease-associated cell states within the liver microenvironment. This work reveals the LD-MS4A7-NLRP3 inflammasome axis as a pathogenic driver of MASH progression and provides insights into the role of TREM2+ macrophages in disease pathogenesis.

SEL1L-HRD1 interaction is required to form a functional HRD1 ERAD complex.

The SEL1L-HRD1 protein complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD). Despite recent advances in both mouse models and humans, in vivo evidence for the importance of SEL1L in the ERAD complex formation and its (patho-)physiological relevance in mammals remains limited. Here we report that SEL1L variant p.Ser658Pro (SEL1LS658P) is a pathogenic hypomorphic mutation, causing partial embryonic lethality, developmental delay, and early-onset cerebellar ataxia in homozygous mice carrying the bi-allelic variant. Biochemical analyses reveal that SEL1LS658P variant not only reduces the protein stability of SEL1L, but attenuates the SEL1L-HRD1 interaction, likely via electrostatic repulsion between SEL1L F668 and HRD1 Y30 residues. Proteomic screens of SEL1L and HRD1 interactomes reveal that SEL1L-HRD1 interaction is a prerequisite for the formation of a functional HRD1 ERAD complex, as SEL1L is required for the recruitment of E2 enzyme UBE2J1 as well as DERLIN to HRD1. These data not only establish the disease relevance of SEL1L-HRD1 ERAD, but also provide additional insight into the formation of a functional HRD1 ERAD complex.

Highly Efficient Loading of Procaine on Water-Soluble Carbon Dots toward Long-Acting Anesthesia.

Safe and efficient local anesthetic delivery carriers are crucial for long-term anesthesia and analgesics in clinical treatment. But currently, most of the local anesthetic carriers still have some disadvantages such as low drug-loading capacity, drug leakage, and potential side effects. Here, we report red-emissive carbon dots (Cys-CDs) synthesized by choosing cysteine and citric acid as precursors, which contain a large and intact sp2-domain with rich hydrophilic groups around the edge. The special structure of Cys-CDs is conducive to the efficient loading of procaine (PrC) via strong π-π stacking interactions. Based on the strong noncovalent interactions between them, the PrC loaded on Cys-CDs achieved slow release in vitro and had a long-lasting nerve blocking effect in vivo, which is 4-fold more than that of free PrC. More importantly, PrC/Cys-CDs do not cause any toxicity and inflammation during treatment owing to slow release of PrC and good water solubility of Cys-CDs, thus demonstrating the potential clinical application of CDs in long-lasting analgesia.

Z-Scheme Heterojunction Excited by DNA-Programmed Upconversion Nanotransducers for a Near-Infrared Light-Actuated Lab-on-Paper Device.

Herein, a flexible near-infrared (NIR) light-actuated photoelectrochemical (PEC) lab-on-paper device was constructed toward miRNA-122 detection, utilizing the combination of DNA-programmed NaYF4/Yb,Tm upconversion nanoparticles (UCNPs) and the Z-scheme AgI/WO3 heterojunction grown in situ on gold nanoparticle-decorated 3D cellulose fibers. The UCNPs were employed as light transducers for converting NIR light into ultraviolet/visible (UV/vis) light to excite the nanojunction. The multiple diffraction of NaYF4/Yb,Tm matched the absorption band of the Z-scheme AgI/WO3 heterojunction, resulting in enhanced PEC photocurrent output. This prepared Z-scheme heterojunction effectively directed charge migration and highly facilitated the electron-hole pair separation. Target miRNA-122 activated the nonenzyme catalytic hairpin assembly signal amplification strategy, generating duplexes which caused the exfoliation of NaYF4/Yb,Tm UCNPs from the biosensor electrode and lowered the photocurrent under 980 nm irradiation. Under optimized circumstances, the proposed NIR-actuated PEC lab-on-paper device presented accurate miRNA-122 detection within a wide linear range of 10 fM-100 nM with a low limit of detection of 2.32 fM, providing a reliable strategy in the exploration of NIR-actuated PEC biosensors for low-cost, high-performance bioassay in clinical applications.

Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover.

Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors of ECM clearance, relatively little is understood regarding the upstream regulation of this process. Collagen is the most abundant constituent of normal and fibrotic ECM in mammalian tissues. Its catabolism occurs through extracellular proteolysis and cell-mediated uptake of collagen fragments for intracellular degradation. Given the paucity of information regarding the regulation of this latter process, here we execute unbiased genome-wide screens to understand the molecular underpinnings of cell-mediated collagen clearance. Using this approach, we discover a mechanism through which collagen biosynthesis is sensed by cells internally and directly regulates clearance of extracellular collagen. The sensing mechanism appears to be dependent on endoplasmic reticulum-resident protein SEL1L and occurs via a noncanonical function of this protein. This pathway functions as a homeostatic negative feedback loop that limits collagen accumulation in tissues. In human fibrotic lung disease, the induction of this collagen clearance pathway by collagen synthesis is impaired, thereby contributing to the pathological accumulation of collagen in lung tissue. Thus, we describe cell-autonomous, rheostatic collagen clearance as an important pathway of tissue homeostasis.

Proteomic screens of SEL1L-HRD1 ER-associated degradation substrates reveal its role in glycosylphosphatidylinositol-anchored protein biogenesis.

Endoplasmic reticulum-associated degradation (ERAD) plays indispensable roles in many physiological processes; however, the nature of endogenous substrates remains largely elusive. Here we report a proteomics strategy based on the intrinsic property of the SEL1L-HRD1 ERAD complex to identify endogenous ERAD substrates both in vitro and in vivo. Following stringent filtering using a machine learning algorithm, over 100 high-confidence potential substrates are identified in human HEK293T and mouse brown adipose tissue, among which ~88% are cell type-specific. One of the top shared hits is the catalytic subunit of the glycosylphosphatidylinositol (GPI)-transamidase complex, PIGK. Indeed, SEL1L-HRD1 ERAD attenuates the biogenesis of GPI-anchored proteins by specifically targeting PIGK for proteasomal degradation. Lastly, several PIGK disease variants in inherited GPI deficiency disorders are also SEL1L-HRD1 ERAD substrates. This study provides a platform and resources for future effort to identify proteome-wide endogenous substrates in vivo, and implicates SEL1L-HRD1 ERAD in many cellular processes including the biogenesis of GPI-anchored proteins.