Paradoxical Mitophagy Regulation by PINK1 and TUFm.

Aberrant mitophagy has been implicated in a broad spectrum of disorders. PINK1, Parkin, and ubiquitin have pivotal roles in priming mitophagy. However, the entire regulatory landscape and the precise control mechanisms of mitophagy remain to be elucidated. Here, we uncover fundamental mitophagy regulation involving PINK1 and a non-canonical role of the mitochondrial Tu translation elongation factor (TUFm). The mitochondrion-cytosol dual-localized TUFm interacts with PINK1 biochemically and genetically, which is an evolutionarily conserved Parkin-independent route toward mitophagy. A PINK1-dependent TUFm phosphoswitch at Ser222 determines conversion from activating to suppressing mitophagy. PINK1 modulates differential translocation of TUFm because p-S222-TUFm is restricted predominantly to the cytosol, where it inhibits mitophagy by impeding Atg5-Atg12 formation. The self-antagonizing feature of PINK1/TUFm is critical for the robustness of mitophagy regulation, achieved by the unique kinetic parameters of p-S222-TUFm, p-S65-ubiquitin, and their common kinase PINK1. Our findings provide new mechanistic insights into mitophagy and mitophagy-associated disorders.

A universal and scalable transformation of bulk metals into single-atom catalysts in ionic liquids.

Single-atom catalysts (SACs) with maximized metal atom utilization and intriguing properties are of utmost importance for energy conversion and catalysis science. However, the lack of a straightforward and scalable synthesis strategy of SACs on diverse support materials remains the bottleneck for their large-scale industrial applications. Herein, we report a general approach to directly transform bulk metals into single atoms through the precise control of the electrodissolution-electrodeposition kinetics in ionic liquids and demonstrate the successful applicability of up to twenty different monometallic SACs and one multimetallic SAC with five distinct elements. As a case study, the atomically dispersed Pt was electrodeposited onto Ni3N/Ni-Co-graphene oxide heterostructures in varied scales (up to 5 cm × 5 cm) as bifunctional catalysts with the electronic metal-support interaction, which exhibits low overpotentials at 10 mA cm-2 for hydrogen evolution reaction (HER, 30 mV) and oxygen evolution reaction (OER, 263 mV) with a relatively low Pt loading (0.98 wt%). This work provides a simple and practical route for large-scale synthesis of various SACs with favorable catalytic properties on diversified supports using alternative ionic liquids and inspires the methodology on precise synthesis of multimetallic single-atom materials with tunable compositions.

Test-Time Training for Deep MS/MS Spectrum Prediction Improves Peptide Identification.

In bottom-up proteomics, peptide-spectrum matching is critical for peptide and protein identification. Recently, deep learning models have been used to predict tandem mass spectra of peptides, enabling the calculation of similarity scores between the predicted and experimental spectra for peptide-spectrum matching. These models follow the supervised learning paradigm, which trains a general model using paired peptides and spectra from standard data sets and directly employs the model on experimental data. However, this approach can lead to inaccurate predictions due to differences between the training data and the experimental data, such as sample types, enzyme specificity, and instrument calibration. To tackle this problem, we developed a test-time training paradigm that adapts the pretrained model to generate experimental data-specific models, namely, PepT3. PepT3 yields a 10-40% increase in peptide identification depending on the variability in training and experimental data. Intriguingly, when applied to a patient-derived immunopeptidomic sample, PepT3 increases the identification of tumor-specific immunopeptide candidates by 60%. Two-thirds of the newly identified candidates are predicted to bind to the patient's human leukocyte antigen isoforms. To facilitate access of the model and all the results, we have archived all the intermediate files in Zenodo.org with identifier 8231084.

Electronic Modulation in Cu Doped NiCo LDH/NiCo Heterostructure for Highly Efficient Overall Water Splitting.

Layered double hydroxides (LDHs), promising bifunctional electrocatalysts for overall water splitting, are hindered by their poor conductivity and sluggish electrochemical reaction kinetics. Herein, a hierarchical Cu-doped NiCo LDH/NiCo alloy heterostructure with rich oxygen vacancies by electronic modulation is tactfully designed. It extraordinarily effectively drives both the oxygen evolution reaction (151 mV@10 mA cm-2 ) and the hydrogen evolution reaction (73 mV@10 mA cm-2 ) in an alkaline medium. As bifunctional electrodes for overall water splitting, a low cell voltage of 1.51 V at 10 mA cm-2 and remarkable long-term stability for 100 h are achieved. The experimental and theoretical results reveal that Cu doping and NiCo alloy recombination can improve the conductivity and reaction kinetics of NiCo LDH with surface charge redistribution and reduced Gibbs free energy barriers. This work provides a new inspiration for further design and construction of nonprecious metal-based bifunctional electrocatalysts based on electronic structure modulation strategies.

Relationship between dietary knowledge, food preference, and long-short term health status among Chinese adults.

BACKGROUND AND OBJECTIVES: In recent years, with the improvement of people's living standards and changes in dietary patterns, dietary knowledge and food preference have been playing an increasingly crucial role in health. The aim of our study was to examine the relationship between dietary knowledge, food preference, and long-short term health status among Chinese adults aged 18-70. METHODS AND STUDY DESIGN: This study employed cross-sectional data from the 2015 China Health and Nutrition Survey obtained from 4822 adults. We utilized self-assessed health status as an indicator of long-term health status and utilized sickness in the last four weeks as a measure of short-term health status. Taking advantage of ordered probit regression, long-term health status was regressed on all predictors, while the binary logistic regression was used to analyze the factors influencing short-term health status. The propensity score matching is employed to account for potential selection bias in analysis, thereby increasing the robustness and credibility of results. RESULTS: The analysis revealed that dietary knowledge and food preference can improve an individual's long-term health status significantly. However, there is no evidence to show that short-term health status is affected by food preference. Furthermore, dietary knowledge is negatively associated with short-term health status. CONCLUSIONS: These findings highlight the importance of dietary education and healthy eating habits in improving the long-term health status of Chinese adults. The study suggests implications for public health strategies aimed at enhancing the health and well-being of Chinese adults.

One-step Synthesis of Organic Terminal 2D Ti3C2Tx MXene Nanosheets by Etching of Ti3AlC2 in an Organic Lewis Acid Solvent.

The surface and interface chemistry are critical for controlling the properties of two-dimensional transition metal carbides and nitrides (MXenes). Numerous efforts have been devoted to the functionalization of MXenes with small inorganic ligands; however, few etching methods have been reported on the direct bonding of organic groups to MXene surfaces. In this work, we demonstrated an efficient and rapid strategy for the direct synthesis of 2D Ti3C2Tx MXene nanosheets with organic terminal groups in an organic Lewis acid (trifluoromethanesulfonic acid) solvent, without introducing additional intercalations. The dissolution of aluminum and the subsequent in situ introduction of trifluoromethanesulfonic acid resulted in the extraction of Ti3C2Tx MXene (T=CF3SO3 -) (denoted as CF3SO3H-Ti3C2Tx) flakes with sizes reaching 15 µm and high productivity (over 70 %) of monolayers or few layers. More importantly, the large CF3SO3H-Ti3C2Tx MXene nanosheets had high colloidal stability, making them promising as efficient electrocatalysts for the hydrogen evolution reaction.

HA15 inhibits binding immunoglobulin protein and enhances the efficacy of radiation therapy in esophageal squamous cell carcinoma.

Proteomic profiling is a promising approach to identify novel predictors of radiation response. The present study aimed to identify potential biomarkers of radiation response by serum proteomics in esophageal squamous cell carcinoma (ESCC) patients and find efficacious therapeutic drugs to enhance the efficacy of radiation therapy (RT). Serum binding immunoglobulin protein (BIP) was identified and validated as a treatment response predictor in ESCC patients treated with RT. Novel BIP inhibitor HA15 showed antitumor activity in ESCC cells by viability assay. Tumor cell colony formation and apoptosis assay revealed targeting BIP was associated with significant improvements of radiation sensitivity. Further analyses revealed that HA15 enhanced radiation-induced endoplasmic reticulum (ER) stress and immunogenic cell death (ICD) in ESCC. Clinical data indicated that high expression of BIP was associated with poor survival in patients of ESCC. In conclusion, proteomics analysis suggested BIP was a promising predictor of radiation response in locally advanced ESCC. The BIP inhibitor HA15 acted as an ER stress inducer and ICD stimulator; RT combined with HA15 was effective in suppressing the growth of ESCC in vitro and in vivo. Pretreatment BIP was an essential prognostic biomarker in locally advanced ESCC patients treated with RT.

A New Design to Rayleigh Wave EMAT Based on Spatial Pulse Compression.

The main disadvantage of the electromagnetic acoustic transducer (EMAT) is low energy-conversion efficiency and low signal-to-noise ratio (SNR). This problem can be improved by pulse compression technology in the time domain. In this paper, a new coil structure with unequal spacing was proposed for a Rayleigh wave EMAT (RW-EMAT) to replace the conventional meander line coil with equal spacing, which allows the signal to be compressed in the spatial domain. Linear and nonlinear wavelength modulations were analyzed to design the unequal spacing coil. Based on this, the performance of the new coil structure was analyzed by the autocorrelation function. Finite element simulation and experiments proved the feasibility of the spatial pulse compression coil. The experimental results show that the received signal amplitude is increased by 2.3~2.6 times, the signal with a width of 20 µs could be compressed into a δ-like pulse of less than 0.25 µs and the SNR is increased by 7.1-10.1 dB. These indicate that the proposed new RW-EMAT can effectively enhance the strength, time resolution and SNR of the received signal.

Staphylococcus aureus virulence attenuation and immune clearance mediated by a phage lysin-derived protein.

New anti-infective approaches are much needed to control multi-drug-resistant (MDR) pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). Here, we found for the first time that a recombinant protein derived from the cell wall binding domain (CBD) of the bacteriophage lysin PlyV12, designated as V12CBD, could attenuate S. aureus virulence and enhance host immune defenses via multiple manners. After binding with V12CBD, S. aureus became less invasive to epithelial cells and more susceptible to macrophage killing. The expressions of multiple important virulence genes of S. aureus were reduced 2.4- to 23.4-fold as response to V12CBD More significantly, V12CBD could activate macrophages through NF-κB pathway and enhance phagocytosis against S. aureus As a result, good protections of the mice from MRSA infections were achieved in therapeutic and prophylactic models. These unique functions of V12CBD would render it a novel alternative molecule to control MDRS. aureus infections.

TNN is first linked to auditory neuropathy.

Autosomal recessive nonsyndromic auditory neuropathy is attributed to a genetic etiology. We identified a compound heterozygous missense variant, c.G736A (p.G246S) and c.C2954T (p.T985 M) in TNN of affected patients in a pedigree via candidate gene screening and exome sequencing. To determine the genetic etiology of deafness in the pedigree with a heterozygous missense variant in the gene TNN encoding tenascin-W associated with autosomal recessive nonsyndromic auditory neuropathy, the cochlear expression of tenascin-W was evaluated at mRNA and protein levels in mice, and Tnn knock out mice were generated and utilized to study the function of Tnn in the auditory system. Immunofluorescence stainings showed that tenascin-W was mainly expressed in the somatic cytoplasm of spiral ganglion neurons of mice. Homozygous Tnn knockout was lethal in mice, whereas Tnn heterozygous mice showed decreases in spiral ganglion neuron density and progressive hearing loss. We demonstrate that tenascin-W is expressed in the murine cochleae and is essential for the development of spiral ganglion neurons. An abnormal expression of tenascin-W can influence the development and function of SGNs and affect the function of the auditory system.

Tumor-suppressive MEG3 induces microRNA-493-5p expression to reduce arabinocytosine chemoresistance of acute myeloid leukemia cells by downregulating the METTL3/MYC axis.

BACKGROUND: Chemoresistance serves as a huge obstacle for acute myeloid leukemia (AML) patients. To counteract the chemoresistance in AML cells, we discussed the role of maternally expressed gene 3 (MEG3) in arabinocytosine (AraC) chemoresistance in AML cells. METHODS: MEG3, microRNA (miR)-493-5p, methyltransferase-like 3 (METTL3) and MYC expression in AML cells was determined and then their interactions were also analyzed. Then, the viability and apoptosis of AML cells were determined through loss- and gain- function assay. The level of m6A modification in AML cells was examined. AML mouse models were also established to validate the potential roles of MEG3. RESULTS: MEG3 and miR-493-5p were downregulated in AML cells, and they were lower in resistant cells than in parental cells. MEG3 led to elevated expression of miR-493-5p which targeted METTL3. METTL3 increased expression of MYC by promoting its m6A levels. Overexpression of MEG3 and miR-493-5p or knockdown of METTL3 inhibited HL-60 and Molm13 cell proliferation and promoted their apoptosis. Overexpressed MEG3 induced heightened sensitivity of AML cells to AraC. However, the suppression of miR-493-5p reversed the effects of overexpressed MEG3 on AML cells. CONCLUSIONS: Collectively, MEG3 could upregulate miR-493-5p expression and suppress the METTL3/MYC axis through MYC m6A methylation, by which MEG3 promoted the chemosensitivity of AML cells.

Downregulation of SMIM3 inhibits growth of leukemia via PI3K-AKT signaling pathway and correlates with prognosis of adult acute myeloid leukemia with normal karyotype.

BACKGROUND: Acute myeloid leukemia (AML) patients with normal karyotype (NK-AML) have significant variabilities in outcomes. The European Leukemia Net stratification system and some prognostic models have been used to evaluate risk stratification. However, these common standards still have some limitations. The biological functions and mechanisms of Small Integral Membrane Protein 3 (SMIM3) have seldomly been investigated. To this date, the prognostic value of SMIM3 in AML has not been reported. This study aimed to explore the clinical significance, biological effects and molecular mechanisms of SMIM3 in AML. METHODS: RT-qPCR was applied to detect the expression level of SMIM3 in bone marrow specimens from 236 newly diagnosed adult AML patients and 23 healthy volunteers. AML cell lines, Kasumi-1 and THP-1, were used for lentiviral transfection. CCK8 and colony formation assays were used to detect cell proliferation. Cell cycle and apoptosis were analyzed by flow cytometry. Western blot was performed to explore relevant signaling pathways. The biological functions of SMIM3 in vivo were validated by xenograft tumor mouse model. Survival rate was evaluated by Log-Rank test and Kaplan-Meier. Cox regression model was used to analyze multivariate analysis. The correlations between SMIM3 and drug resistance were also explored. RESULTS: Through multiple datasets and our clinical group, SMIM3 was shown to be significantly upregulated in adult AML compared to healthy subjects. SMIM3 overexpression conferred a worse prognosis and was identified as an independent prognostic factor in 95 adult NK-AML patients. Knockdown of SMIM3 inhibited cell proliferation and cell cycle progression, and induced cell apoptosis in AML cells. The reduced SMIM3 expression significantly suppressed tumor growth in the xenograft mouse model. Western blot analysis showed downregulation of p-PI3K and p-AKT in SMIM3-knockdown AML cell lines. SMIM3 may also be associated with some PI3K-AKT and first-line targeted drugs. CONCLUSIONS: SMIM3 was highly expressed in adult AML, and such high-level expression of SMIM3 was associated with a poor prognosis in adult AML. Knockdown of SMIM3 inhibited the proliferation of AML through regulation of the PI3K-AKT signaling pathway. SMIM3 may serve as a potential prognostic marker and a therapeutic target for AML in the future.

Comprehensive Effect of Oxidant Addition in an FGD Slurry on the Removal and Distribution of Selenium: A Field Study.

Flue gas desulfurization (FGD) scrubbers capture selenium in coal-fired power plants, leading to a high concentration of selenium in the slurry. This research proves that SO32- is preferentially oxidized compared to SeO32- by S2O82-. With the increase in the oxidation-reduction potential (ORP) caused by S2O82- addition, the conversion rate of SO32- increased and the size of gypsum grains grew from 31.2 to 34.6 µm. SeO32- migrates into gypsum grains during the growth of CaSO4·2H2O, leading to selenium fixation in gypsum. In a field study of a 350 MW unit, the ORP increased from 142 to 450 mV when Na2S2O8 was fed into the FGD slurry. With the addition of the oxidant, 65.1% of selenium in the liquid phase migrated into gypsum. The concentration of selenium in the leachate of gypsum after oxidant addition decreased by 68.0%. A 2.34% increase in the selenium removal rate was observed in the scrubber. This study focuses on the migration and conversion of selenium in an actual FGD slurry via a field test. The results found in the 350 MW unit are consistent with laboratory results. The change in ORP has been proven to be effective in adjusting the selenium distribution in the FGD slurry.

Inactivated STAT5 pathway underlies a novel inhibitory role of EBF1 in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) is a disease caused by gradual accumulation of functionally incompetent lymphocytes. The majority of CLL cases are accompanied by chemoresistance. Early B cell factor 1 (EBF1) is a crucial contributor to B-cell lymphopoiesis. This study is to explore the effect of EBF1 on CLL cell progression and its involvement in regulating the signal transducers and activators of transcription 5 (STAT5) pathway. We conducted a correlation analysis between EBF1 and the clinical characteristics of CLL patients. Subsequently, EBF1 was overexpressed by transfection with EBF1 overexpression plasmid and the STAT5 pathway was also blocked by treatment with SH-4-54 in isolated CD20+ B lymphocytes to investigate their roles in the regulation of cellular functions. STAT5, Janus kinase 2 (JAK2) expression and their phosphorylation levels were determined by quantitative PCR and Western blot analyses. The in vivo effects of EBF1 on tumor growth were evaluated using a xenotransplant model. Downregulation of EBF1 was observed in CD20+ B lymphocytes of CLL patients. EBF1 overexpression disrupted the activation of STAT5 pathway, as evidenced by decreased expression and phosphorylation levels of STAT5 and JAK2. Furthermore, overexpression of EBF1 repressed viability and cell cycle entry, and increased apoptosis of CD20+ B lymphocytes by inhibiting the STAT5 pathway. Finally, EBF1 exerted antitumor effects in nude mice. Overall, our study elucidates the inhibitory role of EBF1 in CLL through inactivation of the STAT5 pathway, which may provide new targets for CLL treatment.

Highly sensitive and selective detection of cytochrome P450 46A1 activity by a ultra-high-performance liquid chromatography-tandem mass spectrometry method.

Cytochrome P450 46A1 (CYP46A1) is a key enzyme responsible for metabolizing cholesterol to 24-hydroxycholesterol in the brain, and thus might serve as a therapeutic target for several neurodegenerative disorders including Parkinson's disease, Alzheimer's disease and Huntington's disease. However, an applicable, sensitive and reliable method for the precise measurement of CYP46A1 activities in complex biological samples remains limited. In this study, a novel ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for highly sensitive and selective determination of 24-hydroxycholesterol was developed to characterize CYP46A1 activity. The mass spectrometric detection was performed using multiple reaction monitoring for 24-hydroxcholesterol at m/z 385.2 → 367.2. The limit of quantification for 24-hydroxycholesterol using this UPLC-MS/MS method was as low as 10 nM, which is lower than those reported previously. The method also showed favorable accuracy and precision. Meanwhile, the short- and long-term stability of this method was fully validated. In addition, the method was successfully applied to investigate the kinetic properties of 24-hydroxycholesterol formation by CYP46A1.

De novo Splice Site Mutation of the CHD7 Gene in a Chinese Patient with Typical CHARGE Syndrome.

INTRODUCTION: CHARGE syndrome (CS, OMIM 214800) is a rare genetic disease characterized by multiple congenital abnormalities, including coloboma, heart defect, atresia of the choanae, retardation of development, genital anomalies, and ear anomalies/deafness. The syndrome is mainly caused by a heterozygous variant in the chromodomain helicase DNA-binding protein 7 (CHD7) gene that encodes the CHD7 protein, involved in the ATP-dependent remodeling of chromatin. METHODS: In this study, the next-generation sequencing targeted panel was used to detect a de novo variant c.3523-2A>G in the CHD7 gene in a patient with severe CS, congenital heart disease, left coloboma of the choroid, cryptorchidism, and congenital deafness. The Sanger sequencing confirmed the variant and clarified it as de novo variant by short tandem repeat analysis in the patient family. We analyzed the effect of a variant by Minigene assay to evaluate the pathogenicity of the variant. RESULTS: In summary, cDNA analysis confirmed that c.3523-2A>G variant activates a cryptic splice site, resulting in 172 base pair missing in exon 15, leading to the premature truncation of the CHD7 protein (p.V1175Afs*11). CONCLUSION: The present study functionally characterized the novel c.3523-2A>G variant in CHD7, providing further confirmatory evidence that it is associated with CS.

Extending the Incidence Angle of Shear Vertical Wave Electromagnetic Acoustic Transducer with Horizontal Magnetization.

Angled shear vertical (SV) waves have been successfully employed in the non-destructive testing of welds, pipes, and railways. Non-contact meander-line coil electromagnetic acoustic transducers (EMAT) have many benefits in generating angled SV waves. The most important benefit is that the incidence angle of an SV wave can be controlled by the excitation frequency. However, the incidence angle of a traditional SV-wave EMAT is reported to be under 45 degrees in many cases. In this work, such cases are tested, and the problems of the received signal at large incidence angles are found to be due to wave interference and small signal amplitudes. An equivalent finite element (FE) model is established to analyze the problem, and the main reason is found to be the head wave. An alternative configuration of angled SV-wave EMAT with horizontal magnetization is proposed to reduce the influence of the head wave. Finally, the results from simulations and experiments show that the proposed EMAT has a larger signal amplitude and significantly reduced interference in large-incidence angle scenarios. Moreover, an incidence angle of an SV wave of up to 60 degrees can be achieved, which will help improve the performance and capability of nondestructive testing.

Analysis of the risk of traumatic brain injury and evaluation neurogranin and myelin basic protein as potential biomarkers of traumatic brain injury in postmortem examination.

In forensic pathology, traumatic brain injury (TBI) is a frequently encountered cause of death. Unfortunately, the statistic autopsy data, risk investigation about injury patterns, and circumstances of TBI are still sparse. Estimates of survival time post-TBI and postmortem diagnosis of TBI are especially important implications in forensic medicine. Neurogranin (Ng) and myelin basic protein (MBP) represent potential biomarkers of TBI. The present study analyzed retrospectively the forensic autopsy records of TBI cases at a university center of medico-legal investigation from 2008 to 2020. Immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) were used to investigate the expression changes of Ng and MBP in the cortical brain injury adjacent tissues and serum, respectively, from cases of TBI at autopsy with different survival times post-TBI. The results show that the major mechanism of death of TBI is assault, and accident was the major manner of death. Ng and MBP are mainly expressed in the cortical nerve cells and the myelin sheath, respectively. The serum levels of Ng and MBP in each TBI group were higher compared with those in the controls. The brain cortical levels of Ng and MBP decreased at first and then steadily increased with extended survival time post-TBI. The immunopositive ratios and serum concentration of Ng and MBP have shown significant differences among control group and all TBI group (p < 0.001). Collectively, the immunohistochemical analyses of Ng and MBP in human brain tissues may be useful to determine the survival time after TBI, and Ng and MBP level in the human blood specimens could be considered as a postmortem diagnostic tools of TBI in forensic practice.

Strongylocentrotus nudos Egg Polysaccharide induces autophagy and apoptosis in leukaemia cells by regulating mitochondrial function.

In this study, we investigated the ability of the Polysaccharide from the Eggs of Strongylocentrotus nudus (SEP) to regulate cellular autophagy and apoptosis in leukaemia cells. Human acute myeloid leukaemia (AML) cells (HL60) and murine AML cells (L1210) treated with SEP were used to assess viability using Cell Counting Kit-8, cytotoxicity by measuring lactate dehydrogenase release, the generation of reactive oxygen species (ROS) by DCFH-DA staining. In addition, we utilized a mouse model of leukaemia in which L1210 cells were injected into DBA/2 mice by sub-axillary injection. Treatment with SEP decreased cell viability, increased in cytotoxicity and increased the release of ROS in a dose-dependent manner. SEP treatment was also associated with the activation of pro-apoptotic proteins cleaved caspase-3, cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase (PARP). Activation of the apoptotic pathway led to the release of cytochrome C (CytoC) into the cytosol of the cell resulting in decreased membrane potential. The effect of SEP treatment was depended on the activation of the nuclear factor kappa-B (NF-κB) signalling pathway as SEP treatment led to an increase in NF-κB phosphorylation, and inhibition of NF-κB signalling using PDTC blocked SEP-mediated activation of apoptosis. Treatment with SEP also prolonged survival time in our leukaemia mouse model and was associated with diminished tumour volume, increased leucocyte and lymphocyte proliferation, promoted pro-inflammatory factor release in serum and enhanced immune function. Taken together, these data suggest that SEP inhibits the progression of leukaemia by initiating mitochondrial dysfunction, autophagy, and apoptosis via the NF-κB signalling pathway.

A System-Wide Spatiotemporal Characterization of ErbB Receptor Complexes by Subcellular Fractionation Integrated Quantitative Mass Spectrometry.

Precise spatiotemporal regulation of protein complex assembly is essential for cells to achieve a meaningful rely of information flow via intracellular signaling networks in response to extracellular cues, whose disruption would lead to disease. Although various attempts have been made for spatial and/or temporal analysis of protein complexes, it is still a challenge to track cell-wide dynamics of a particular protein complex under physiological conditions. Here we describe a workflow that combines endogenous expression of tagged proteins, organelle marker distribution-directed subcellular fractionation, scaffold protein-mediated receptor complex purification, and targeted proteomics for spatiotemporal quantification of protein complexes in whole cell scale. We applied our method to investigate the assembly kinetics of EGF-dependent ErbB receptor complexes. After fractionation using the density gradient centrifugation and organelle assignment based on organelle markers, endogenous ErbB complex in different subcellular fractionation was efficiently enriched. By using targeted mass spectrometry, ErbB complex components that expressed medium to low level was precisely quantified with in-depth coverage, simultaneously in time and subcellular spaces. Our results revealed a sophisticated scheme of complex behaviors characterized by multiple subcomplexes with distinct molecular composition formed across subcellular fractions enriched with cytosol, plasma membrane, endosome, or mitochondria, implying organelle-specific ErbB functions. Remarkably, our results demonstrated for the first time that activated ErbB receptors might increase their signaling range through promoting a cytosolic, receptor-free subcomplex, consisting of Shc1, Grb2, Arhgef5, Garem1, and Lrrk1. These findings emphasize the potential of our strategy as a powerful tool to study spatiotemporal dynamics of protein complexes.