Endothelial mesenchymal transition promotes pannus formation in ankylosing spondylitis by activation of TGF-ß/SMAD signalling pathway.

OBJECTIVES: To explore the role of endothelial-mesenchymal transition (EndMT) mediated by the TGF-ß/SMAD signalling pathway in the pathogenesis of ankylosing spondylitis (AS). METHODS: Serum levels of TGF-ß1 were measured by enzyme-linked immunosorbent assay (ELISA) in 48 patients with AS and 15 healthy subjects. The expression levels of TGF-ß1, SMAD7, CTGF, CD34 and EndMT-related markers (α-SMA, vimentin, FSP-1, VE-cadherin) in the sacroiliac joint (SIJ) of three AS patients were detected by immunohistochemistry, and three non-spondyloarthritis (SpA) autopsy samples were used as controls. RESULTS: Serum TGF-ß1 level of AS patients was significantly higher than that of healthy controls (22971 ± 7667 pg/ml vs. 14837±4653 pg/ml, p<0.01). Compared with the non-SpA control group, the microvascular density (MVD) at the pannus formation site of SIJ in AS patients was significantly increased, accompanied by respectively increased expressions of TGF-ß1, CTGF, α-SMA, vimentin, and FSP-1 (all p<0.05), whereas respectively decreased expressions of VE-cadherin and SMAD7 (p<0.01). The expression level of FSP-1 was positively correlated with levels of TGF-ß1 and MVD, and negatively correlated with SMAD7. CONCLUSIONS: Our findings show that EndMT is involved in the promotion of pannus formation by TGF-ß/SMAD signalling pathway activation in AS.

Lysosome-targeted ruthenium(II) complex encapsulated with pluronic® F-127 induces oncosis in A549 cells.

Transition metal complexes with characteristics of unique packaging in nanoparticles and remarkable cancer cell cytotoxicity have emerged as potential alternatives to platinum-based antitumor drugs. Here we report the synthesis, characterization, and antitumor activities of three new Ruthenium complexes that introduce 5-fluorouracil-derived ligands. Notably, encapsulation of one such metal complex, Ru3, within pluronic® F-127 micelles (Ru3-M) significantly enhanced Ru3 cytotoxicity toward A549 cells by a factor of four. To determine the mechanisms underlying Ru3-M cytotoxicity, additional in vitro experiments were conducted that revealed A549 cell treatment with lysosome-targeting Ru3-M triggered oxidative stress, induced mitochondrial membrane potential depolarization, and drastically reduced intracellular ATP levels. Taken together, these results demonstrated that Ru3-M killed cells mainly via a non-apoptotic pathway known as oncosis, as evidenced by observed Ru3-M-induced cellular morphological changes including cytosolic flushing, cell swelling, and cytoplasmic vacuolation. In turn, these changes together caused cytoskeletal collapse and activation of porimin and calpain1 proteins with known oncotic functions that distinguished this oncotic process from other cell death processes. In summary, Ru3-M is a potential anticancer agent that kills A549 cells via a novel mechanism involving Ru(II) complex triggering of cell death via oncosis.

Cell state dependent effects of Bmal1 on melanoma immunity and tumorigenicity.

The circadian clock regulator Bmal1 modulates tumorigenesis, but its reported effects are inconsistent. Here, we show that Bmal1 has a context-dependent role in mouse melanoma tumor growth. Loss of Bmal1 in YUMM2.1 or B16-F10 melanoma cells eliminates clock function and diminishes hypoxic gene expression and tumorigenesis, which could be rescued by ectopic expression of HIF1α in YUMM2.1 cells. By contrast, over-expressed wild-type or a transcriptionally inactive mutant Bmal1 non-canonically sequester myosin heavy chain 9 (Myh9) to increase MRTF-SRF activity and AP-1 transcriptional signature, and shift YUMM2.1 cells from a Sox10high to a Sox9high immune resistant, mesenchymal cell state that is found in human melanomas. Our work describes a link between Bmal1, Myh9, mouse melanoma cell plasticity, and tumor immunity. This connection may underlie cancer therapeutic resistance and underpin the link between the circadian clock, MRTF-SRF and the cytoskeleton.

Exploring sustainable food packaging: Nanocellulose composite films with enhanced mechanical strength, antibacterial performance, and biodegradability.

Food packaging films play a vital role in preserving and protecting food. However, due to their non-biodegradability, conventional packaging materials have led to significant environmental pollution. To overcome this hurdle, we have developed safe, innovative, sustainable and biodegradable packaging materials that can effectively extend the shelf life of food. In this study, two types of cellulose materials cellulose nanofibers (CNF) and carboxymethyl cellulose (CMC) with complementary roles were combined to prepare nanocellulose composite films with high transparency (90.3 %) of a certain thickness (30 ± 0.019 µm) by solution casting method, and their mechanical properties were further optimized by the addition of plasticizer-glycerol (Gly) and cross-linking agent-glutaraldehyde (GA), so as to maintain the strong tensile strength (≈112.60 MPa) and better malleability (4.12 %). In addition, we loaded the natural active agent tea polyphenols (TPs) with different concentrations to study the inhibition effect on E.coli and S.aureus and to simulate food packaging. Finally, we also found that the synthesized nanocellulose composite films can also achieve rapid degradation in a short time through soil burial, water flushing and immersion. The excellent performance demonstrated in this study provides reference value for further replacing petroleum-based materials with biomass materials in the field of food packaging.

The first Chinese with Hb Chile leading to chronic anemia and methemoglobinemia: a case report.

BACKGROUND: Hemoglobin (Hb) Chile [ß28(B10) Leu > Met; HBB: c.85 C > A] is a rare hemoglobin variant caused by a missense mutation in the HBB gene. Only one case of Hb Chile has been reported worldwide so far. It is an unstable hemoglobin, characterized by cyanosis associated with chronic methemoglobinemia and hemolytic anemia induced by sulfonamides or methylene blue. CASE PRESENTATION: A 9-year-3-month-old girl had mild anemia of unknown etiology for more than 6 years. She had a slight pallor without other symptoms or signs. The complete blood count revealed normocytic normochromic anemia with a sometimes-elevated reticulocyte count, and the bone marrow cytology showed marked erythroid hyperplasia, but the tests related to hemolysis were normal. Therefore, the whole exome sequencing was performed and showed a heterozygous mutation for HBB: c.85 C > A. With asymptomatic methemoglobinemia confirmed later, she was eventually diagnosed with Hb Chile. CONCLUSIONS: This is the first report of Hb Chile in China and the second worldwide. This case shows that Hb Chile is clinically heterogeneous and difficult to diagnose and expands our understanding on the clinical and hematological traits of the disease.

Transvaginal natural orifice transluminal endoscopic surgery-assisted versus transumbilical laparoendoscopic single-site ovarian cystectomy for ovarian mature cystic teratoma. A randomized controlled trial.

OBJECTIVES: Transvaginal natural orifice transluminal endoscopic surgery (vNOTES) and transumbilical laparoendoscopic single-site surgery (LESS) have shown the prospection as minimally invasive procedures. Here we aimed to compare ovarian cystectomy assisted by vNOTES and by LESS for ovarian mature cystic teratoma (OMCT). MATERIAL AND METHODS: A total of 81 premenopausal women with OMCT were randomized to undergo ovarian cystectomy assisted by either vNOTES (n = 41) or LESS (n = 40). The main outcome was the operative time. Secondary outcomes included the length of hospital stay, visual analog scale (VAS) pain scores, abdominal contamination by teratoma contents, and intraoperative and postoperative complications. RESULTS: There were no intergroup differences in age, body mass index, tumor size, or bilaterality of tumor. The operative time for the vNOTES group was significantly shorter than that for the LESS group (68.41 ± 20.92 min vs 85.05 ± 32.94 min, p = 0.008). The highest VAS pain score 24 hours postoperatively was 1.21 ± 0.48 in the vNOTES group and 2.43 ± 0.57 in the LESS group (p < 0.001). Twenty-four of the 40 patients in the LESS group experienced teratoma rupture intraoperatively, leading to abdominal contamination by the teratoma content, while 5 abdominal contamination was observed in the vNOTES group （p = 0.005. No significant differences between the two groups were observed in the other outcomes. CONCLUSIONS: vNOTES assisted ovarian cystectomy has short operative time, fast recovery, no scarring, less pain, and low rate of abdominal contamination. Consequently, vNOTES might be superior to LESS for treating OMCTs.

Rapid in situ RNA imaging based on Cas12a thrusting strand displacement reaction.

RNA In situ imaging through DNA self-assembly is advantaged in illustrating its structures and functions with high-resolution, while the limited reaction efficiency and time-consuming operation hinder its clinical application. Here, we first proposed a new strand displacement reaction (SDR) model (Cas12a thrusting SDR, CtSDR), in which Cas12a could overcome the inherent reaction limitation and dramatically enhance efficiency through energy replenishment and by-product consumption. The target-initiated CtSDR amplification was established for RNA analysis, with order of magnitude lower limit of detection (LOD) than the Cas13a system. The CtSDR-based RNA in situ imaging strategy was developed to monitor intra-cellular microRNA expression change and delineate the landscape of oncogenic RNA in 66 clinic tissue samples, possessing a clear advantage over classic in situ hybridization (ISH) in terms of operation time (1 h versus 14 h) while showing comparable sensitivity and specificity. This work presents a promising approach to developing advanced molecular diagnostic tools.

8-Hydroxyquinoline ruthenium(II) complexes induce ferroptosis in HeLa cells by down-regulating GPX4 and ferritin.

Ruthenium complexes are one of the most promising anticancer drugs triggered extensive research. Here, the synthesis and characterization of two ruthenium(II) polypyridine complexes containing 8-hydroxylquinoline as ligand, [Ru(dip)2(8HQ)]PF6 (Ru1), [Ru(dpq)2(8HQ)]PF6 (Ru2) (8HQ = 8-hydroxylquinoline; dip = 4,7-diphenyl-1,10-phenanthroline; dpq = pyrazino[2,3-f][1,10]phenanthroline) were reported. On the basis of cytotoxicity tests, Ru1 (IC50 = 1.98 ± 0.02 µM) and Ru2 (IC50 = 10.02 ± 0.19 µM) both showed good anticancer activity in a panel of cell lines, especially in HeLa cells. Researches on mechanism indicated that Ru1 and Ru2 acted on mitochondria and nuclei and induced reactive oxygen species (ROS) accumulation, while the morphology of nuclei and cell cycle had no significant change. Western blot assay further proved that GPX4 and Ferritin were down-regulated, which eventually triggered ferroptosis in HeLa cells. In addition, the toxicity test of zebrafish embryos showed that the concentrations of Ru1 and Ru2 below 120 µM and 60 µM were safe and did not have obvious effect on the normal development of zebrafish embryos.

Cyclometalated iridium(III) complexes induce immunogenic cell death in HepG2 cells via paraptosis.

Immunotherapy has been shown to provide superior antitumor efficacy by activating the innate immune system to recognize, attack and eliminate tumor cells without seriously harming normal cells. Herein, we designed and synthesized three new cyclometalated iridium(III) complexes (Ir1, Ir2, Ir3) then evaluated their antitumor activity. When co-incubated with HepG2 cells, the complex Ir1 localized in the lysosome, where it induced paraptosis and endoplasmic reticulum stress (ER stress). Notably, Ir1 also induced immunogenic cell death (ICD), promoted dendritic cell maturation that enhanced effector T cell chemotaxis to tumor tissues, down-regulated proportions of immunosuppressive regulatory T cells within tumor tissues and triggered activation of antitumor immunity throughout the body. To date, Ir1 is the first reported iridium(III) complex-based paraptosis inducer to successfully induce tumor cell ICD. Furthermore, Ir1 induced ICD of HepG2 cells without affecting cell cycle or reactive oxygen species levels.

Feasibility of a Brief Intervention to Decrease Harmful Alcohol Use Among Methadone Maintenance Treatment Clients in Shanghai: A Randomized Controlled Trial.

OBJECTIVES: In this study, we aimed to examine the prevalence of alcohol consumption among methadone maintenance treatment (MMT) clients in Shanghai and to determine whether a brief intervention (BI) affects drinking among them. METHODS: A total of 837 clients from 14 local MMT clinics were invited to complete the Alcohol Use Disorders Identification Test (AUDIT). One hundred one were included in the study and randomly assigned to the BI group or the control group. Clients in the BI group received a BI and general health education, whereas clients in the control group received the general health education only. Baseline and postintervention assessments were conducted by using the AUDIT, the Drinking Attitude Questionnaire, the Depression Module of the Patient Health Questionnaire, the Generalized Anxiety Disorder Scale, and the General Well-Being Schedule. RESULTS: Two hundred fifty-nine (30.9%) reported drinking during the last year, and 103 (12.3%) met the criteria for harmful use. At the 3-month follow-up, the AUDIT scores of the 2 groups were significantly decreased, and the time effect was statistically significant ( F = 6.224, P = 0.018), but there was no group difference in AUDIT scores ( F = 1.953, P = 0.172). Both groups had a main time effect of time on the improvement of depression ( F = 8.044, P = 0.008), anxiety ( F = 9.650, P = 0.004), and general well-being ( F = 5.056, P = 0.033). However, there was no statistical difference between the 2 groups ( P > 0.05), and no statistical difference in the time ( F = 1.738, P = 0.198) and group ( F = 0.658, P = 0.424) effect of drinking attitude. CONCLUSIONS: Alcohol consumption is common among MMT clients in China. Brief intervention, in its current form, could not effectively help them reduce their alcohol consumption.

A one-pot CRISPR-Cas12a-based toolbox enables determination of terminal deoxynucleotidyl transferase activity for acute leukemia screening.

An isothermal, one-pot toolbox (called OPT-Cas) based on CRISPR-Cas12a collateral cleavage capability is proposed for highly sensitive and selective determination of terminal deoxynucleotidyl transferase (TdT) activity. Oligonucleotide primers with 3'-hydroxyl (OH) terminal were randomly introduced for TdT-induced elongation. In the presence of TdT, dTTP nucleotides polymerized at the 3' terminals of the primers to generate abundant polyT-tails, which function as triggers for the synchronous activation of Cas12a proteins. Finally, the activated Cas12a trans-cleaved FAM and BHQ1 dual-labeled single-stranded DNA (ssDNA-FQ) reporters, producing significantly amplified fluorescence signals. This one-pot assay, that is primer, crRNA, Cas12a protein and ssDNA-FQ reporter are all in one tube, allows simple but high-sensitive quantification of TdT activity with a low detection limit of 6.16 × 10-5 U µL-1 in the concentration scope from 1 × 10-4 U µL-1 to 1 × 10-1 U µL-1, and achieves extraordinary selectivity with other interfering proteins. Furthermore, the OPT-Cas was successfully used to detect TdT in complex matrices and accurate determination of TdT activity in acute lymphoblastic leukemia cells, which might be a reliable technique platform for the diagnosis of TdT-related diseases and biomedical research applications.

Levels of vitamin A and selenium of children with cystic echinococcosis in Aba Tibetan and Qiang and Garze Tibetan Autonomous Prefecture, Sichuan, China.

OBJECTIVE: The aim of this study was to review the levels of vitamin A and selenium in children with echinococcosis in Ganzi and Aba. METHODS: Twenty-six children with cystic echinococcosis and 104 apparently healthy controls from the Aba and Garze Tibetan Autonomous Prefecture of Sichuan province in China were recruited. The serum levels of selenium and vitamin A of the cases and controls were detected and stratified by sex and age. RESULTS: The results showed that the ratio of boys to girls was 1:1.36. Compared with the healthy controls, the vitamin A serum levels of the cases significantly declined. The rate of selenium deficiency was high in the cases, but there was no significant difference compared with the control group. Additionally, the lower the vitamin A serum level, the higher the risk for developing echinococcosis. CONCLUSIONS: To improve management strategies in children with cystic echinococcosis, or possibly to prevent the disease, children living in high-risk areas for cystic echinococcosis should be given supplemental vitamin A and encouraged to consume a vitamin A-rich diet containing paw-paw, carrot, palm oil, or fish. Children with vitamin A deficiency in high-risk areas should be screened for cystic echinococcosis.

[Accuracy evaluation of static guided implant placement using an intraoral scan method].

PURPOSE: To evaluate the accuracy of static guided implant placement with intraoral scanning technology and to analyze the influencing factors of guided surgery. METHODS: Totally 27 cases were included in this retrospective study. The implant designs were made in 3Shape Implant Studio and then guided implant surgeries were performed with CAD-CAM templates. Postoperative implant positions were detected with an intraoral scanner (3Shape TRIOS) and deviation of implantation was evaluated using established CAD/CAM based evaluation method. SAS 9.4 software package was used for data analysis. RESULTS: The mean deviation of entrance point and apical point was (1.182±0.609) mm and (1.658±0.741) mm, respectively. Angular deviation was (5.712±3.347)°. Implant quadrant, location of the implant site, guidance degree, supporting type and implant size influenced direction deviation, while angular deviation was mainly affected by guidance degree and number of missing teeth. CONCLUSIONS: Accuracy of static guided implant placement can be influenced by many factors. More research is needed to improve the accuracy of static guided implantation.

TGF-ß1 promotes human breast cancer angiogenesis and malignant behavior by regulating endothelial-mesenchymal transition.

Background: Endothelial-mesenchymal transition (EndMT) is an important process of angiogenesis, which plays a significant role in in tumor invasion and metastasis, while its regulatory mechanisms in breast cancer remain to be fully elucidated. We previously demonstrated that tumor-associated macrophages (TAMs) can induce EndMT in endothelial cells by secreting CCL18 through the activation of the TGF-ß and Notch signaling pathways in breast cancer. This study was designed to study the role of EndMT in breast cancer angiogenesis and progression in order to explore the underlying mechanism. Methods: Immunohistochemistry (IHC) was used to evaluate the expression of microvascular density (MVD) and EndMT markers in breast cancer. TGF-ß1 was used to induce EndMT models of differentiated-endothelial breast cancer stem-like cells (BCSLCs). In vitro cell migration, proliferation and matrigel tube-formation assays, as well as in vivo nude mouse tumor-bearing model and nude mouse dorsal skinfold window chamber (DSWC) model, were utilized to investigate the effects in order to explore the mechanism of EndMT induced by TGF-ß1 on breast cancer progression. Results: In this study, we demonstrated that the EndMT markers were positively associated with MVD indicating unfavorable prognosis of invasive ductal carcinoma (IDC) patients. Functionally, TGF-ß1 promoted migration, proliferation and angiogenesis of differentiated-endothelial BCSLCs by inducing EndMT in vitro and promoted tumor growth and angiogenesis in vivo. Mechanically, we revealed TGF-ß1 induced EndMT by activation of TGF-ß and Notch signaling pathways with increase of p-Smad2/3 and Notch1 expression. Moreover, we found Snail and Slug were key factors of TGF-ß and Notch signaling pathways. Conclusion: Our findings elucidated the mechanism of TGF-ß1 in the promotion of angiogenesis and progression by EndMT in breast cancer.

Na+/H+-exchanger 1 enhances antitumor activity of engineered NK-92 natural killer cells.

Adoptive cell transfer (ACT) immunotherapy has remarkable efficacy against some hematological malignancies. However, its efficacy in solid tumors is limited by the adverse tumor microenvironment (TME) conditions, most notably that acidity inhibits T and natural killer (NK) cell mTOR complex 1 (mTORC1) activity and impairs cytotoxicity. In several reported studies, systemic buffering of tumor acidity enhanced the efficacy of immune checkpoint inhibitors. Paradoxically, we found in a c-Myc-driven hepatocellular carcinoma model that systemic buffering increased tumor mTORC1 activity, negating inhibition of tumor growth by anti-PD1 treatment. Therefore, in this proof-of-concept study, we tested the metabolic engineering of immune effector cells to mitigate the inhibitory effect of tumor acidity while avoiding side effects associated with systemic buffering. We first overexpressed an activated RHEB in the human NK cell line NK-92, thereby rescuing acid-blunted mTORC1 activity and enhancing cytolytic activity. Then, to directly mitigate the effect of acidity, we ectopically expressed acid extruder proteins. Whereas ectopic expression of carbonic anhydrase IX (CA9) moderately increased mTORC1 activity, it did not enhance effector function. In contrast, overexpressing a constitutively active Na+/H+-exchanger 1 (NHE1; SLC9A1) in NK-92 did not elevate mTORC1 but enhanced degranulation, target engagement, in vitro cytotoxicity, and in vivo antitumor activity. Our findings suggest the feasibility of overcoming the inhibitory effect of the TME by metabolically engineering immune effector cells, which can enhance ACT for better efficacy against solid tumors.

Effects of Umbilical Preparation Before Trans-umbilical Laparo-endoscopic Single-site Surgery on Umbilical Wounds Healing: a Randomized Controlled Trial.

OBJECTIVE: The umbilicus is the only anatomic entrance and incision site for trans-umbilical laparoendoscopic single-site surgery (TU-LESS). Data on incisional surgical site infections (ISSI) and incision healing in TU-LESS are lacking. Therefore, we aimed to observe umbilical incision healing and possible hernia after TU-LESS and explore the efficacy of preoperative umbilicus preparation on ISSI. SUBJECTS AND METHODS: Consecutive patients aged 18 to 65 years, who were scheduled to undergo TU-LESS at a teaching hospital between March 2020 and November 2021, were enrolled in this prospective study. All patients were randomized to the study group with preoperative umbilicus preparation 30 minutes before patients were sent to the operating room and to the control group without preparation. The umbilical dimple was disinfected twice using povidone-iodine in both groups before the skin incision. The primary outcome was ISSI within 30 days of surgery. Umbilical hernia at 3 months after surgery and perioperative data such as operation time, complications, and incision healing were recorded and compared. RESULTS: A total of 400 patients were recruited for this study. TU-LESS was performed in all patients without major complications. ISSI occurred in 5 patients in the study group (2.5%) and 3 patients in the control group (1.5%), with no significant differences between both groups ( P =0.479). No umbilical hernia occurred in any patient during the 3 months follow-up. Six patients in the study group (3.1%) and 1 in the control group (0.5%) experienced excessive scarring, a relatively high incidence in the study group, though the difference was not statistically significant ( P =0.067). CONCLUSIONS: TU-LESS-related umbilical hernias are rare with existing suturing methods. Umbilicus preparation before TU-LESS could not decrease ISSI; however, it increased the nursing workload, which should be avoided.

HypDB: A functionally annotated web-based database of the proline hydroxylation proteome.

Proline hydroxylation (Hyp) regulates protein structure, stability, and protein-protein interaction. It is widely involved in diverse metabolic and physiological pathways in cells and diseases. To reveal functional features of the Hyp proteome, we integrated various data sources for deep proteome profiling of the Hyp proteome in humans and developed HypDB (https://www.HypDB.site), an annotated database and web server for Hyp proteome. HypDB provides site-specific evidence of modification based on extensive LC-MS analysis and literature mining with 14,413 nonredundant Hyp sites on 5,165 human proteins including 3,383 Class I and 4,335 Class II sites. Annotation analysis revealed significant enrichment of Hyp on key functional domains and tissue-specific distribution of Hyp abundance across 26 types of human organs and fluids and 6 cell lines. The network connectivity analysis further revealed a critical role of Hyp in mediating protein-protein interactions. Moreover, the spectral library generated by HypDB enabled data-independent analysis (DIA) of clinical tissues and the identification of novel Hyp biomarkers in lung cancer and kidney cancer. Taken together, our integrated analysis of human proteome with publicly accessible HypDB revealed functional diversity of Hyp substrates and provides a quantitative data source to characterize Hyp in pathways and diseases.

A dual identification strategy based on padlock ligation and CRISPR/Cas14a for highly specific detection of BRAF V600E mutation in clinical samples.

BRAF V600E mutation is a single-nucleotide variation (SNV) that is widely found in various cancers and has been demonstrated to have a strong association with the prognosis and development of some diseases. Thus, we developed a strategy based on rolling circle amplification (RCA) and CRISPR/Cas14a to meet the great need for detecting highly specific BRAF V600E mutation in fine-needle biopsy samples. In this study, a padlock probe was designed to recognize and trigger subsequent ligase chain reactions (LCR). And due to the Taq DNA ligase, a great number of ligated annular padlock probes were generated in the presence of BRAF V600E mutation, subsequently generating long repeated single-strand DNA by RCA. The obtained amplicons were activators triggering the trans-cleavage of CRISPR/Cas14a. CRISPR/Cas14a shows outstanding performance in identifying ssDNA with single base mutation, which significantly increases the specificity of mutation discrimination. Under the optimal conditions, our strategy can identify BRAF V600E mutation down to 0.307 fM with a wide linear range from 1 fM to 10 pM. On the other hand, the dual identification strategy endows the method with terrific specificity for the detection of SNV. Furthermore, our method has been successfully employed to identify BRAF V600E mutation in clinical fine-needle aspiration samples, proving great potential for ultra-specific identification of low abundance BRAF V600E mutation and providing a novel method for diagnosis and treatment of cancer.

UbE3-APA: a bioinformatic strategy to elucidate ubiquitin E3 ligase activities in quantitative proteomics study.

MOTIVATION: Ubiquitination is widely involved in protein homeostasis and cell signaling. Ubiquitin E3 ligases are critical regulators of ubiquitination that recognize and recruit specific ubiquitination targets for the final rate-limiting step of ubiquitin transfer reactions. Understanding the ubiquitin E3 ligase activities will provide knowledge in the upstream regulator of the ubiquitination pathway and reveal potential mechanisms in biological processes and disease progression. Recent advances in mass spectrometry-based proteomics have enabled deep profiling of ubiquitylome in a quantitative manner. Yet, functional analysis of ubiquitylome dynamics and pathway activity remains challenging. RESULTS: Here, we developed a UbE3-APA, a computational algorithm and stand-alone python-based software for Ub E3 ligase Activity Profiling Analysis. Combining an integrated annotation database with statistical analysis, UbE3-APA identifies significantly activated or suppressed E3 ligases based on quantitative ubiquitylome proteomics datasets. Benchmarking the software with published quantitative ubiquitylome analysis confirms the genetic manipulation of SPOP enzyme activity through overexpression and mutation. Application of the algorithm in the re-analysis of a large cohort of ubiquitination proteomics study revealed the activation of PARKIN and the co-activation of other E3 ligases in mitochondria depolarization-induced mitophagy process. We further demonstrated the application of the algorithm in the DIA (data-independent acquisition)-based quantitative ubiquitylome analysis. AVAILABILITY AND IMPLEMENTATION: Source code and binaries are freely available for download at URL: https://github.com/Chenlab-UMN/Ub-E3-ligase-Activity-Profiling-Analysis, implemented in python and supported on Linux and MS Windows. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Quantitative Proteome and Transcriptome Dynamics Analysis Reveals Iron Deficiency Response Networks and Signature in Neuronal Cells.

Iron and oxygen deficiencies are common features in pathophysiological conditions, such as ischemia, neurological diseases, and cancer. Cellular adaptive responses to such deficiencies include repression of mitochondrial respiration, promotion of angiogenesis, and cell cycle control. We applied a systematic proteomics analysis to determine the global proteomic changes caused by acute hypoxia and chronic and acute iron deficiency (ID) in hippocampal neuronal cells. Our analysis identified over 8600 proteins, revealing similar and differential effects of each treatment on activation and inhibition of pathways regulating neuronal development. In addition, comparative analysis of ID-induced proteomics changes in cultured cells and transcriptomic changes in the rat hippocampus identified common altered pathways, indicating specific neuronal effects. Transcription factor enrichment and correlation analysis identified key transcription factors that were activated in both cultured cells and tissue by iron deficiency, including those implicated in iron regulation, such as HIF1, NFY, and NRF1. We further identified MEF2 as a novel transcription factor whose activity was induced by ID in both HT22 proteome and rat hippocampal transcriptome, thus linking iron deficiency to MEF2-dependent cellular signaling pathways in neuronal development. Taken together, our study results identified diverse signaling networks that were differentially regulated by hypoxia and ID in neuronal cells.