Collagen synthase P4HA3 as a novel biomarker for colorectal cancer correlates with prognosis and immune infiltration.

Objective: In this study, we aimed to determine whether proly4-hydroxylase-III (P4HA3) could be used as a biomarker for the diagnosis of colorectal cancer (CRC) as well as for determining prognosis. Methods: We used The Cancer Genome Atlas (TCGA) database to analyze P4HA3 expression in CRC and further investigated the association between P4HA3 and clinicopathological parameters, immune infiltration, and prognosis of patients with CRC. Enrichment analysis was conducted to investigate the potential biological role of P4HA3 in CRC. To verify the results of TCGA analysis, we performed immunohistochemical staining of 180 clinical CRC tissue samples to probe into the relationship of P4HA3 expression with lymphocyte infiltration and immune checkpoints expression. Results: The expression of P4HA3 was significantly higher in CRC tissues and associated with a higher degree of malignancy and poorer prognosis in CRC. The results of enrichment analysis indicated that P4HA3 may be associated with the epithelial-mesenchymal transition process and the immune response. Immunohistochemical staining results showed that high P4HA3 expression was associated with high infiltration levels of CD8+ and Foxp3+ TILs and high PD-1/PD- L1 expression. Lastly, patients with CRC co-expressing P4HA3 and PD-1 had a significantly worse prognosis. Conclusion: High expression of P4HA3 is associated with adverse clinical features and immune cell infiltration in CRC, and has the potential to serve as a biomarker for predicting CRC prognosis.

A cardiomyocyte-based biosensing platform for dynamic and quantitative investigation of excessive autophagy.

Autophagy is an important physiological phenomenon in eukaryotes that helps maintain the cellular homeostasis. Autophagy is involved in the development of various cardiovascular diseases, affecting the maintenance of cardiac function and disease prognosis. Physiological levels of autophagy serve as a defense mechanism for cardiomyocytes against environmental stimuli, but an overabundance of autophagy may contribute to the development of cardiovascular diseases. However, conventional biological methods are difficult to monitor the autophagy process in a dynamic and chronic manner. Here, we developed a cardiomyocyte-based biosensing platform that records electrophysiological evolutions in action potentials to reflect the degree of autophagy. Different concentrations of rapamycin-mediated autophagy were administrated in the culture environment to simulate the autophagy model. Moreover, the 3-methyladenine (3-MA)-mediated autophagy inhibition was also investigated the protection on the autophagy. The recorded action potentials can precisely reflect different degrees of autophagy. Our study confirms the possibility of visualizing and characterizing the process of cardiomyocyte autophagy using cardiomyocyte-based biosensing platform, allowing to monitor the whole autophagy process in a non-invasive, real-time, and continuous way. We believe it will pave a promising avenue to precisely study the autophagy-related cardiovascular diseases.

A Validated Set of Ascorbate Peroxidase-Based Organelle Markers for Electron Microscopy of Saccharomyces cerevisiae.

Genetically encoded tags, such as engineered ascorbate peroxidase APEX2, offer unique advantages for the specific labeling of subcellular structures in electron microscopy (EM). However, the use of APEX2 in EM investigation of yeast has been limited. Here we describe the development of APEX2-based organelle markers for Saccharomyces cerevisiae. We found that with regard to APEX2 -catalyzed formation of diaminobenzidine precipitation, cell wall removal was not essential during sample preparation, yet the presence of fluorescent proteins in APEX2 chimeras had a negative impact. We showed that major organelles including endoplasmic reticulum, early Golgi, late Golgi/early endosomes, late endosomes, mitochondria, peroxisomes, and lipid droplets could be labeled by appropriate APEX2 chimeras. The subcellular localization of our APEX2 chimeras was verified by EM visualization and supplemented with immunofluorescence colocalization analysis when necessary, validating their feasibility as organelle markers. IMPORTANCE Yeast is an excellent single cellular model system for studying basic cellular processes. However, yeast cells are much smaller than most animal and plant cells, making the observation and recognition of yeast subcellular structures challenging. Here we developed a set of yeast organelle markers for use in electron microscopy and documented our technical approach for using this method.

Anti-HMGB1 antibody is a potential characteristic autoantibody for Sjögren's syndrome.

Sjögren's syndrome (SS) is a common chronic inflammatory autoimmune disease that affects about 0.33-0.77% population in China. The positive for antinuclear antibodies (ANA) is one of the key features of SS, which shows a nuclear fine speckled (AC-4) pattern in an indirect immunofluorescent antibody test (IIFT). About 70% of ANA-positive SS patients have detectable anti-SS-A and/or SS-B antibodies, which indicates that other autoantibodies may present in SS patients. The anti-HMGB1 antibodies in 93 SS patients and 96 healthy controls were investigated with in-house developed ELISA and immunoblotting, and the locations of HMGB1 and fluorescent pattern of anti-HMGB1 antibody were investigated with IIFT. The contribution of anti-HMGB1 antibody in ANA-IF was evaluated with Cas9-induce HMGB1 knockout B16 cells. The anti-HMGB1 antibody level is higher in SS patients (9.96 ± 5.55 RU/ml) than in healthy controls (4.9 ± 1.4 RU/ml). With ROC curve analysis, when taking 8 RU/ml as the cutoff value, the sensitivity, specificity, and the area under the curve were 64.5%, 96.9%, and 0.83, respectively. A total of 18 patients (20.7%) with nuclear fine speckled (AC-4) pattern in ANA-IF test were anti-HMGB1 antibody positive only. With commercial antibody, anti-HMGB1 antibody showed the same nuclear fine speckled (AC-4) pattern. The serum from ANA-IF (+), SS-A (-), and SS-B (-) SS patients showed nuclear fine speckled (AC-4) pattern in wildtype B16 cells, but no fluorescence in HMGB1 knockout B16 cells. Anti-HMGB1 antibody may be one of the characteristic autoantibodies of SS in addition to anti-SS-A and SS-B. The detection of anti-HMGB1 antibody can provide more laboratory evidence for clinical diagnosis of SS.

Yeast Lipid Extraction and Analysis by HPTLC.

The diversity of lipid structures, properties, and combinations in biological tissues makes their extraction and analysis an experimental challenge. Accordingly, even for one of the simplest single-cellular fungi, the budding yeast (Saccharomyces cerevisiae), numerous extraction and analysis protocols have been developed to separate and quantitate the different molecular lipid species. Among them, most are quite sophisticated and tricky to follow. Herein, we describe a yeast total lipids extraction procedure with a relatively good yield, which is appropriate for subsequent thin-layer chromatography (TLC) or liquid chromatography-mass (LC-MS) analysis. We then discuss the most widely used solvent systems to separate yeast phospholipids and neutral lipids by TLC. Finally, we describe an easy and rapid method for silica gel staining by a Coomassie Brilliant Blue-methanol mixture. The stained lipid species can then be quantitated using imaging software such as ImageJ. Overall, the methods described in this protocol are time-saving and novice-friendly.

Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase.

BACKGROUND: The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways. RESULTS: Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation. CONCLUSIONS: Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.

Atg9-centered multi-omics integration reveals new autophagy regulators in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Atg9 is an important autophagy-related (Atg) protein, and interacts with hundreds of other proteins. How many Atg9-interacting proteins are involved in macroautophagy/autophagy is unclear. Here, we conducted a multi-omic profiling of Atg9-dependent molecular landscapes during nitrogen starvation-induced autophagy, and identified 290 and 256 genes to be markedly regulated by ATG9 in transcriptional and translational levels, respectively. Unexpectedly, we found most of known Atg proteins and autophagy regulators that interact with Atg9 were not significantly changed in the mRNA or protein level during autophagy. Based on a hypothesis that proteins with similar molecular characteristics might have similar functions, we developed a new method named inference of functional interacting partners (iFIP) to integrate the transcriptomic, proteomic and interactomic data, and predicted 42 Atg9-interacting proteins to be potentially involved in autophagy, including 15 known Atg proteins or autophagy regulators. We validated 2 Atg9-interacting partners, Glo3 and Scs7, to be functional in both bulk and selective autophagy. The mRNA and protein expressions but not subcellular localizations of Glo3 and Scs7 were affected with or without ATG9 during autophagy, whereas the colocalizations of the 2 proteins and Atg9 were markedly enhanced at early stages of the autophagic process. Further analyses demonstrated that Glo3 but not Scs7 regulates the retrograde transport of Atg9 during autophagy. A working model was illustrated to highlight the importance of the Atg9 interactome. Taken together, our study not only provided a powerful method for analyzing the multi-omics data, but also revealed 2 new players that regulate autophagy.Abbreviations: ALP: alkaline phosphatase; Arf1: ADP-ribosylation factor 1; Atg: autophagy-related; Co-IP: co-immunoprecipitation; Cvt: cytoplasm-to-vacuole targeting; DEM: differentially expressed mRNA; DEP: differentially expressed protein; DIC: differential interference contrast; E-ratio: enrichment ratio; ER: endoplasmic reticulum; ES: enrichment score; FC: fold change; FPKM: fragments per kilobase of exon per million fragments mapped; GAP: GTPase-activating protein; GFP: green fluorescent protein; GO: gene ontology; GSEA: gene set enrichment analysis; GST: glutathione S-transferase; HA: hemagglutinin; iFIP: inference of functional interacting partners; KO: knockout; LR: logistic regression; OE: over-expression; PAS: phagophore assembly site; PPI: protein-protein interaction; RFP: red fluorescence protein; RNA-seq: RNA sequencing; RT-PCR: real-time polymerase chain reaction; SCC: Spearman's correlation coefficient; SD-N: synthetic minimal medium lacking nitrogen; THANATOS: The Autophagy, Necrosis, ApopTosis OrchestratorS; Vsn: variance stabilization normalization; WT: wild-type.

Constrained L1-Norm Minimization Method for Range-Based Source Localization under Mixed Sparse LOS/NLOS Environments.

Under mixed sparse line-of-sight/non-line-of-sight (LOS/NLOS) conditions, how to quickly achieve high positioning accuracy is still a challenging task and a critical problem in the last dozen years. To settle this problem, we propose a constrained L1 norm minimization method which can reduce the effects of NLOS bias for improve positioning accuracy and speed up calculation via an iterative method. We can transform the TOA-based positioning problem into a sparse optimization one under mixed sparse LOS/NLOS conditions if we consider NLOS bias as outliers. Thus, a relatively good method to deal with sparse localization problem is L1 norm. Compared with some existing methods, the proposed method not only has the advantages of simple and intuitive principle, but also can neglect NLOS status and corresponding NLOS errors. Experimental results show that our algorithm performs well in terms of computational time and positioning accuracy.

Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy.

BACKGROUND: When stressed, eukaryotic cells produce triacylglycerol (TAG) to store nutrients and mobilize autophagy to combat internal damage. We and others previously reported that in yeast, elimination of TAG synthesizing enzymes inhibits autophagy under nitrogen starvation, yet the underlying mechanism has remained elusive. RESULTS: Here, we show that disruption of TAG synthesis led to diacylglycerol (DAG) accumulation and its relocation from the vacuolar membrane to the endoplasmic reticulum (ER). We further show that, beyond autophagy, ER-accumulated DAG caused severe defects in the endomembrane system, including disturbing the balance of ER-Golgi protein trafficking, manifesting in bulging of ER and loss of the Golgi apparatus. Genetic or chemical manipulations that increase consumption or decrease supply of DAG reversed these defects. In contrast, increased amounts of precursors of glycerolipid synthesis, including phosphatidic acid and free fatty acids, did not replicate the effects of excess DAG. We also provide evidence that the observed endomembrane defects do not rely on Golgi-produced DAG, Pkc1 signaling, or the unfolded protein response. CONCLUSIONS: This work identifies DAG as the critical lipid molecule responsible for autophagy inhibition under condition of defective TAG synthesis and demonstrates the disruption of ER and Golgi function by excess DAG as the potential cause of the autophagy defect.

The influence of different blood samples treatment methods on pro-gastrin-releasing peptide.

Pro-gastrin-releasing peptide (ProGRP) is the promising molecular tumor marker of small cell lung cancer (SCLC). Here we study the influence of different blood samples treatment methods on ProGRP.Serum with and without separation gel and heparin plasma from 10 SCLC patients and 5 healthy individuals were assayed for ProGRP immediately and 2, 4, 6, 8, 24, and 48 hours after collection.ProGRP of serum with and without separation gel and heparin plasma detected immediately was basically consistent, whereas there was a significant difference in the level of them assayed after 2 hours. No significant variation with time was observed in heparin plasma, but in serum with and without separation gel, ProGRP concentrations gradually declined with time, with statistical significance. When assayed within 2 hours, each time point of ProGRP in heparin plasma had no significant difference and the difference of PrpGRP in serum separating gel existed at 1.5 hours.Heparin plasma is the best option for clinical test of ProGRP. If serum with separation gel is used, optimization methods of turn-around-time which guarantee samples detected within 1 hour after collection can make results more instructive for clinical treatment.

[Retracted] Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts.

The authors wish to retract their article entitled "Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts" published in Molecular Medicine Reports 15, 3690­3698, 2017. Following the publication of this article, the authors noted that the description of the reprogramming method and cell cultured conditions in the article was inconsistent with the experimental facts, due to their oversight and personnel issues in terms of who performed the experimental work and who wrote up the paper. Some of the experimental approaches were wrongly described in the Materials and methods section. Namely, the feeder cells which were used in this reprogramming were mouse embryonic fibroblasts, not guinea fibroblast cells, as was reported; the reprogramming medium contained 15% knockout serum replacement; the culture medium contained 10% ESC qualified fetal bovine serum and 10% knockout serum replacement; and 293T cells were transfected using the Quick Shuttle­293 system. Due to the authors' oversight, Figs 2A and C, and Fig. 3 on p. 3,694 do not match with the textual description. In view of these serious errors, which the authors are not able to rectify, they have requested that this article be retracted from the publication. All the named authors agree to this retraction. The authors regret any inconvenience to the readers that this retraction will cause.

Genetic Analysis of Floral Symmetry Transition in African Violet Suggests the Involvement of Trans-acting Factor for CYCLOIDEA Expression Shifts.

With the growing demand for its ornamental uses, the African violet (Saintpaulia ionantha) has been popular owing to its variations in color, shape and its rapid responses to artificial selection. Wild type African violet (WT) is characterized by flowers with bilateral symmetry yet reversals showing radially symmetrical flowers such as dorsalized actinomorphic (DA) and ventralized actinomorphic (VA) peloria are common. Genetic crosses among WT, DA, and VA revealed that these floral symmetry transitions are likely to be controlled by three alleles at a single locus in which the levels of dominance are in a hierarchical fashion. To investigate whether the floral symmetry gene was responsible for these reversals, orthologs of CYCLOIDEA (CYC) were isolated and their expressions correlated to floral symmetry transitions. Quantitative RT-PCR and in situ results indicated that dorsal-specific CYCs expression in WT S. ionantha (SiCYC and SiCYC1B) shifted in DA with a heterotopically extended expression to all petals, but in VA, SiCYC1s' dorsally specific expressions were greatly reduced. Selection signature analysis revealed that the major high-expressed copy of SiCYC had been constrained under purifying selection, whereas the low-expressed helper SiCYC1B appeared to be relaxed under purifying selection after the duplication into SiCYC and SiCYC1B. Heterologous expression of SiCYC in Arabdiopsis showed petal growth retardation which was attributed to limited cell proliferation. While expression shifts of SiCYC and SiCYC1B correlate perfectly to the resulting symmetry phenotype transitions in F1s of WT and DA, there is no certain allelic combination of inherited SiCYC1s associated with specific symmetry phenotypes. This floral transition indicates that although the expression shifts of SiCYC/1B are responsible for the two contrasting actinomorphic reversals in African violet, they are likely to be controlled by upstream trans-acting factors or epigenetic regulations.

Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts.

Induced pluripotent stem cells (iPS) represent an important tool to develop disease­modeling assays, drug testing assays and cell­based replacement therapies. The application of iPS in these fields requires the development of suitable animal models. Of the suitable species, guinea pigs are particularly important and offer significant advantages. Successful iPS generation has been accomplished in a number of species; however, it has not been reported in the guinea pig. The present study successfully generated iPS from guinea pigs (giPS) using single polycistronic virus transduction with mouse octamer­binding transcription factor 4 (Oct4), sex determining region Y­box 2 (Sox2), Kruppel­like factor 4 and c­Myc. The giPS cell lines were cultured in media containing leukemia inhibitory factor and guinea pig fibroblast cells were used as feeder cells. These cultures were expanded under feeder­free culture conditions using ESGRO Complete Plus Clonal Grade medium containing 15% fetal bovine serum on gelatin­coated dishes. The resultant cells had a normal karyotype, exhibited alkaline phosphatase activity and expressed the pluripotency markers Oct4, Sox2 and Nanog. The cells differentiated in vivo to form teratomas that contained all three germ layers of the tissue cells. The generation of giPS may facilitate future studies investigating the mechanisms underlying innate immunity, particularly for tuberculosis. These experiments provide proof of principle that iPS technology may be adapted to use the guinea pig as a model of human diseases.

ROCK inhibitor Y27632 promotes proliferation and diminishes apoptosis of marmoset induced pluripotent stem cells by suppressing expression and activity of caspase 3.

Previous studies reported that Rho-associated kinase inhibitor Y27632 markedly diminishes human embryonic stem cell and induced pluripotent stem cell (iPSC) dissociation-induced apoptosis and increases cloning efficiency in a feeder-free culture system. However, the mechanisms by which Y27632 protects pluripotent stem cells from apoptosis remain unknown. In the present study, we tested the effects of Y27632 on single dissociated marmoset iPSCs in a feeder-free culture. The results showed that Y27632 promoted the number of cells proliferating after passage by single-cell dissociation in a dose-dependent manner. The Rho-associated kinase inhibitor Y27632 markedly increased the cloning efficiency of marmoset iPSCs without affecting their karyotype and the expression of pluripotency markers. Meanwhile, Y27632 markedly diminished apoptosis of the marmoset iPSCs under even more severe conditions by suppressing the expression and activity of caspase 3. Taken together, the present results suggest that this reagent is effective in improving the cultural system of primate iPSCs.