irGSEA: the integration of single-cell rank-based gene set enrichment analysis.

irGSEA is an R package designed to assess the outcomes of various gene set scoring methods when applied to single-cell RNA sequencing data. This package incorporates six distinct scoring methods that rely on the expression ranks of genes, emphasizing relative expression levels over absolute values. The implemented methods include AUCell, UCell, singscore, ssGSEA, JASMINE and Viper. Previous studies have demonstrated the robustness of these methods to variations in dataset size and composition, generating enrichment scores based solely on the relative gene expression of individual cells. By employing the robust rank aggregation algorithm, irGSEA amalgamates results from all six methods to ascertain the statistical significance of target gene sets across diverse scoring methods. The package prioritizes user-friendliness, allowing direct input of expression matrices or seamless interaction with Seurat objects. Furthermore, it facilitates a comprehensive visualization of results. The irGSEA package and its accompanying documentation are accessible on GitHub (https://github.com/chuiqin/irGSEA).

Comparison of Biological Characteristics of Human Umbilical Cord Wharton's Jelly-Derived Mesenchymal Stem Cells from Extremely Preterm and Term Infants.

BACKGROUND: Despite the progress in perinatal-neonatal medicine, complications of extremely preterm infants continue to constitute the major adverse outcomes in neonatal intensive care unit. Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells (HUMSCs) may offer new hope for the treatment of intractable neonatal disorders. This study will explore the functional differences of HUMSCs between extremely preterm and term infants. METHODS: UMSCs from 5 extremely preterm infants(weeks of gestation: 22+5 w,24+4 w,25+3 w,26 w,28 w) and 2 term infants(39 w,39+2 w) were isolated, and mesenchymal markers, pluripotent genes, proliferation rate were analyzed. HUVECs were injured by treated with LPS and repaired by co-cultured with HUMSCs of different gestational ages. RESULTS: All HUMSCs showed fibroblast-like adherence to plastic and positively expressed surface marker of CD105,CD73 and CD90, but did not expressed CD45,CD34,CD14,CD79a and HLA-DR; HUMSCs in extremely preterm exhibited significant increase in proliferation as evidenced by CCK8, pluripotency markers OCT-4 tested by RT-PCR also showed increase. Above all, in LPS induced co-cultured inflame systerm, HUMSCs in extremely preterm were more capable to promote wound healing and tube formation in HUVEC cultures, they promoted TGFß1 expression and inhibited IL6 expression. CONCLUSIONS: Our results suggest that HUMSCs from extremely preterm infants may be more suitable as candidates in cell therapy for the preterm infants.

Tone and vowel disruptions in Mandarin aphasia and apraxia of speech.

In this study, we investigated the lexical tones and vowels produced by ten speakers diagnosed with aphasia and coexisting apraxia of speech (A-AOS) and ten healthy participants, to compare their tone and vowel disruptions. We first judged the productions of both A-AOS and healthy participants and classified them into three categories, i.e. those by healthy speakers and rated as correct, those by A-AOS participants and rated as correct, and those by A-AOS participants and rated as incorrect. We then compared the perceptual results for the three groups based on their respective acoustic correlates to reveal the relations among different accuracy groups. Results showed that the numbers of tone and vowel disruptions by A-AOS speakers occurred on a comparable scale. In perception, approximately equal numbers of tones and vowels produced by A-AOS participants were identified as correct; however, acoustic parameters showed that, unlike vowels, the patients' tones categorised as correct by native Mandarin listeners differed considerably from those of the healthy speakers, suggesting that for Mandarin A-AOS patients, tones were in fact more disrupted than vowels in acoustic terms. Native Mandarin listeners seemed to be more tolerant of less well-targeted tones than less-well targeted vowels. The clinical implication is that tonal and segmental practice should be incorporated for Mandarin A-AOS patients to enhance their overall motor speech control.

Adaptation of Melodic Intonation Therapy to a Tone Language: A Pilot Study of Tone-Rhythmic Therapy in Mandarin Chinese.

INTRODUCTION: Although melodic intonation therapy (MIT) has proven effective in individuals with non-fluent aphasia in a variety of western languages, its application to Mandarin-speaking aphasic patients has not been thoroughly studied. The adaptation is complicated because Mandarin Chinese is a tone language with specific prosodic elements that differ from Indo-European languages. This study developed a Chinese-specific variant of MIT, i.e., tone-rhythmic therapy (TRT), and tested its efficacy in individuals with non-fluent aphasia. METHODS: Six non-fluent aphasic patients were recruited; all of them were admitted to the study over 6 months after stroke and had received a standard program of language therapy. In the current research, tone and rhythmic practice were incorporated into the training procedures, and the adaptation was then examined in patients. The TRT treatment lasted 6 weeks, with five 50-min sessions per week. The Boston Diagnostic Aphasia Examination (BDAE) and the Functional Assessment of Communication Skills for Adults (FACS) tests were used to measure the change in the speech and language skills of patients. RESULTS: The results showed that the patients had increased BDAE and FACS scores after intervention, and the treatment effect lasted for 6 months. DISCUSSION: The modified MIT proved effective for Mandarin-speaking patients with non-fluent aphasia with lasting effects. Further studies evaluating its efficacy are needed for other types of aphasia and other tone languages.

Single-cell RNA sequencing reveals the potential mechanism of heterogeneity of immunomodulatory properties of foreskin and umbilical cord mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) are heterogeneous populations. Heterogeneity exists within the same tissue and between different tissues. Some studies have found enormous heterogeneity in immunomodulatory function among MSCs derived from different tissues. Moreover, the underlying mechanism of heterogeneity in immunomodulatory abilities is still unclear. METHODS: Foreskin mesenchymal stromal cells (FSMSCs) and human umbilical cord mesenchymal stromal cells (HuMSCs) were isolated and cultured until the third passage. According to the International Association for Cell Therapy standard, we confirmed the cell type. Then, FSMSCs and HuMSCs were cocultured with human peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide (LPS) in vitro. Furthermore, the supernatant was sampled for an enzyme-linked immunosorbent assay to investigate the secretion of IL-1ß, IL-6, IL-10, TNF-α, and TGF-ß1. Finally, we performed single-cell RNA sequencing (scRNA-seq) of FSMSCs and HuMSCs. RESULTS: We successfully identified FSMSCs and HuMSCs as MSCs. When cocultured with LPS pretreated PBMCs, FSMSCs and HuMSCs could effectively reduced the secretion of IL-1ß and TNF-α. However, FSMSCs stimulated the PBMCs to secrete more IL-10, TGF-ß1, and IL-6. Furthermore, 4 cell subsets were identified from integrated scRNA-seq data, including proliferative MSCs (MKI67+, CD146low+, NG2+, PDGFRB-), pericytes (CD146high+, PDGFRB+, MKI67-, CD31-, CD45-, CD34-), immune MSCs (CXCL12high+, PTGIShigh+, PDGFRB+, CD146-, MKI67-) and progenitor proliferative MSCs (CXCL12low+, PTGISlow+, PDGFRB+, CD146-, MKI67-). Among them, we found that immune MSCs with strengthened transcriptional activity were similar to pericytes with regard to the degree of differentiated. Various of immune-related genes, gene sets, and regulons were also enriched in immune MSCs. Moreover, immune MSCs were determined to be close to other cell subsets in cell-cell communication analysis. Finally, we found that the proportion of immune MSCs in foreskin tissue was highest when comparing the subset compositions of MSCs derived from different tissues. CONCLUSIONS: FSMSCs show better immunomodulatory capacity than HuMSCs in vitro. Moreover, immune MSCs may play a vital role in the heterogeneity of immunoregulatory properties. This study provides new insights suggesting that immune MSCs can be isolated to exert stable immunoregulatory functions without being limited by the heterogeneity of MSCs derived from different tissues.

Single-Cell Transcriptome Integration Analysis Reveals the Correlation Between Mesenchymal Stromal Cells and Fibroblasts.

Background: Mesenchymal stromal cells (MSCs) and fibroblasts show similar morphology, surface marker expression, and proliferation, differentiation, and immunomodulatory capacities. These similarities not only blur their cell identities but also limit their application. Methods: We performed single-cell transcriptome sequencing of the human umbilical cord and foreskin MSCs (HuMSCs and FSMSCs) and extracted the single-cell transcriptome data of the bone marrow and adipose MSCs (BMSCs and ADMSCs) from the Gene Expression Omnibus (GEO) database. Then, we performed quality control, batch effect correction, integration, and clustering analysis of the integrated single-cell transcriptome data from the HuMSCs, FMSCs, BMSCs, and ADMSCs. The cell subsets were annotated based on the surface marker phenotypes for the MSCs (CD105 + , CD90 +, CD73 +, CD45 -, CD34 -, CD19 -, HLA-DRA -, and CD11b -), fibroblasts (VIM +, PECAM1 -, CD34 -, CD45 -, EPCAM -, and MYH11 -), and pericytes (CD146 +, PDGFRB +, PECAM1 -, CD34 -, and CD45 -). The expression levels of common fibroblast markers (ACTA2, FAP, PDGFRA, PDGFRB, S100A4, FN1, COL1A1, POSTN, DCN, COL1A2, FBLN2, COL1A2, DES, and CDH11) were also analyzed in all cell subsets. Finally, the gene expression profiles, differentiation status, and the enrichment status of various gene sets and regulons were compared between the cell subsets. Results: We demonstrated 15 distinct cell subsets in the integrated single-cell transcriptome sequencing data. Surface marker annotation demonstrated the MSC phenotype in 12 of the 15 cell subsets. C10 and C14 subsets demonstrated both the MSC and pericyte phenotypes. All 15 cell subsets demonstrated the fibroblast phenotype. C8, C12, and C13 subsets exclusively demonstrated the fibroblast phenotype. We identified 3,275 differentially expressed genes, 305 enriched gene sets, and 34 enriched regulons between the 15 cell subsets. The cell subsets that exclusively demonstrated the fibroblast phenotype represented less primitive and more differentiated cell types. Conclusion: Cell subsets with the MSC phenotype also demonstrated the fibroblast phenotype, but cell subsets with the fibroblast phenotype did not necessarily demonstrate the MSC phenotype, suggesting that MSCs represented a subclass of fibroblasts. We also demonstrated that the MSCs and fibroblasts represented highly heterogeneous populations with distinct cell subsets, which could be identified based on the differentially enriched gene sets and regulons that specify proliferating, differentiating, metabolic, and/or immunomodulatory functions.

Regrowth of Escherichia coli in environmental waters after chlorine disinfection: shifts in viability and culturability.

Bacterial regrowth after water/wastewater disinfection poses severe risks to public health. However, regrowth studies under realistic water conditions that might critically affect bacterial regrowth are scarce. This study aimed to assess for the first time the regrowth of Escherichia coli (E. coli) in terms of its viability and culturability in environmental waters after chlorine disinfection, which is the most widely used disinfection method. Post-chlorination regrowth tests were conducted in 1) standard 0.85% NaCl solution, 2) river water receiving domestic wastewater effluents, and 3) river water that is fully recharged by domestic wastewater effluents. The multiplex detection of plate count and fluorescence-based viability test was adopted to quantify the culturable and viable E. coli to monitor the regrowth process. The results confirmed that chlorine treatment (0.2, 0.5 and 1.0 mg L-1 initial free chlorine) induced more than 99.95% of E. coli to enter a viable but non-culturable (VBNC) state and the reactivation of VBNC E. coli is presumably the major process of the regrowth. A second-order regrowth model well described the temporal shift of the survival ratio of culturable E. coli after the chlorination (R2: 0.73-1.00). The model application also revealed that the increase in initial chlorine concentration and chlorine dose limited the maximum regrowth rate and the maximum survival ratio, and the regrowth rate and percentage also changed with the water type. This study gives a better understanding of the potential regrowth after chlorine disinfection and highlights the need for investigating the detailed relation of the regrowth to environmental conditions such as major components of water matrices.

Novel fluorescence-based method for rapid quantification of live bacteria in river water and treated wastewater.

Monitoring bacteria is essential for ensuring microbial safety of water sources, including river water and treated wastewater. The plate count method is common for monitoring bacterial abundance, although it cannot detect all live bacteria such as viable but non-culturable bacteria, causing underestimation of microbial risks. Live/Dead BacLight kit, involving fluorochromes SYTO 9 and propidium iodide (PI), provides an alternative to assess bacterial viability using flow cytometry or microscopy. However, its application is limited due to the high cost of flow cytometry and the inapplicability of microscopy to most environmental waters. Thus, this study introduces the combination of BacLight kit and fluorescence spectroscopy for quantifying live bacteria in river water and treated wastewater. Mixtures of live and dead Escherichia coli (E. coli) with various ratios and total cell concentrations were stained with SYTO 9 and PI and measured by fluorescence spectroscopy. The fluorescence emission peak area of SYTO 9 in the range of 500-510 nm at the excitation wavelength of 470 nm correlates linearly with the viable cell counts (R 2 > 0.99, p < 0.0001) with only slight variations in the complex water matrix. The tested method can quantify the live E. coli from 3.67 × 104 to 2.70 × 107 cells per mL. This method is simple, sensitive and reliable for quantifying live bacteria in environmental water, which can be later integrated into real-time monitoring systems.

Regrowth of bacteria after light-based disinfection - What we know and where we go from here.

Regrowth of bacteria after water/wastewater disinfection is a serious risk to public health, particularly when such pathogens carry antibiotic resistance genes. Despite increasing interest in light-based disinfection using ultraviolet or solar radiation, the mechanism of bacterial regrowth and their concentration upon light exposure (i.e., during storage, or after discharge into rivers or lakes) remain poorly understood. Therefore, we present a focused critical review to 1) elucidate regrowth mechanisms, 2) summarize the pros and cons of available experimental designs and detection techniques for regrowth evaluation, and 3) provide an outlook of key research directions for further investigations of post-disinfection bacterial regrowth. Bacterial regrowth can occur through reactivation from a viable but non-culturable state, repair of photo-induced DNA damage, and reproduction of bacteria surviving disinfection. Many studies have underestimated the degree of actual regrowth because of the use of simple experimental designs and plate count methods, which cannot quantify actual abundance of viable bacteria. Further research should investigate the effects of various factors on bacterial regrowth in realistic conditions in regrowth tests and adopt multiplex detection methods that combine culture-based and culture-independent approaches. An accurate understanding of the mechanisms involved in bacterial regrowth following disinfection is critical for safeguarding public health and aquatic environments.

p53-dependent elimination of aneuploid mitotic offspring by entosis.

Entosis was proposed to promote aneuploidy and genome instability by cell-in-cell mediated engulfment in tumor cells. We reported here, in epithelial cells, that entosis coupled with mitotic arrest functions to counteract genome instability by targeting aneuploid mitotic progenies for engulfment and elimination. We found that the formation of cell-in-cell structures associated with prolonged mitosis, which was sufficient to induce entosis. This process was controlled by the tumor suppressor p53 (wild-type) that upregulates Rnd3 expression in response to DNA damages associated with prolonged metaphase. Rnd3-compartmentalized RhoA activities accumulated during prolonged metaphase to drive cell-in-cell formation. Remarkably, this prolonged mitosis-induced entosis selectively targets non-diploid progenies for internalization, blockade of which increased aneuploidy. Thus, our work uncovered a heretofore unrecognized mechanism of mitotic surveillance for entosis, which eliminates newly born abnormal daughter cells in a p53-dependent way, implicating in the maintenance of genome integrity.

Mechanical Ring Interfaces between Adherens Junction and Contractile Actomyosin to Coordinate Entotic Cell-in-Cell Formation.

Entosis is a cell-in-cell (CIC)-mediated death program. Contractile actomyosin (CA) and the adherens junction (AJ) are two core elements essential for entotic CIC formation, but the molecular structures interfacing them remain poorly understood. Here, we report the characterization of a ring-like structure interfacing between the peripheries of invading and engulfing cells. The ring-like structure is a multi-molecular complex consisting of adhesive and cytoskeletal proteins, in which the mechanical sensor vinculin is highly enriched. The vinculin-enriched structure senses mechanical force imposed on cells, as indicated by fluorescence resonance energy transfer (FRET) analysis, and is thus termed the mechanical ring (MR). The MR actively interacts with CA and the AJ to help establish and maintain polarized actomyosin that drives cell internalization. Vinculin depletion leads to compromised MR formation, CA depolarization, and subsequent CIC failure. In summary, we suggest that the vinculin-enriched MR, in addition to CA and AJ, is another core element essential for entosis.

Subtype-Based Prognostic Analysis of Cell-in-Cell Structures in Early Breast Cancer.

Though current pathological methods are greatly improved, they provide rather limited functional information. Cell-in-cell structures (CICs), arising from active cell-cell interaction, are functional surrogates of complicated cell behaviors within heterogeneous cancers. In light of this, we performed the subtype-based CIC profiling in human breast cancers by the "EML" multiplex staining method, and accessed their values as prognostic factors by Cox univariate, multivariate, and nomogram analysis. CICs were detected in cancer specimens but not in normal breast tissues. A total of five types of CICs were identified with one homotypic subtype (91%) and four heterotypic subtypes (9%). Overall CICs (oCICs) significantly associated with patient overall survival (OS) (P = 0.011) as an independent protective factor (HR = 0.423, 95% CI, 0.227-0.785; P = 0.006). Remarkably, three CICs subtypes (TiT, TiM, and MiT) were also independent prognostic factors. Among them, higher TiT, from homotypic cannibalism between tumor cells, predicted longer patient survival (HR = 0.529, 95% CI, 0.288-0.973; P = 0.04) in a way similar to that of oCICs and that (HR = 0.524, 95% CI, 0.286-0.962; P = 0.037) of heterotypic TiM (tumor cell inside macrophage); conversely, the presence of MiT (macrophage inside tumor cell) predicted a death hazard of 2.608 (95% CI, 1.344-5.063; P = 0.05). Moreover, each CIC subtype tended to preferentially affect different categories of breast cancer, with TiT (P < 0.0001) and oCICs (P = 0.008) targeting luminal B (Her2+), TiM (P = 0.011) targeting HR- (Her2+/HR- and TNBC), and MiT targeting luminal A (P = 0.017) and luminal B (Her-) (P = 0.006). Furthermore, nomogram analysis suggested that CICs impacted patient outcomes in contributions comparable (for oCICs, TiT, and TiM), or even superior (for MiT), to TNM stage and breast cancer subtype, and incorporating CICs improved nomogram performance. Together, we propose CICs profiling as a valuable way for prognostic analysis of breast cancer and that CICs and their subtypes, such as MiT, may serve as a type of novel functional markers assisting clinical practices.

Disinfection mechanism of E. coli by CNT-TiO2 composites: Photocatalytic inactivation vs. physical separation.

Magnetic carbon nanotube (MCNT) composites with titanium dioxide (TiO2) have an enhanced photocatalytic disinfection efficiency (i.e. higher disinfection rate) and better applicability (i.e. solar light applicability and catalyst separation using its magnetic property) than bare TiO2 and/or MCNT. However, the role and mechanism of MCNT in the disinfection process are still unclear. Therefore, this study aimed at investigating the disinfection mechanism of Escherichia coli using MCNT-TiO2 nanocomposites under various conditions (i.e. the presence and absence of light and reactive oxygen species scavengers, and different MCNT-TiO2 ratio) and photocatalytic disinfection models. The results showed that (i) MCNT and its nanocomposites with TiO2 had much higher disinfection efficiencies than bare TiO2, (ii) the physical bacterial capture was the dominant disinfection mechanism, (iii) the higher disinfection rate was found at an optimum MCNT:TiO2 ratio of 5:1 under the tested experimental conditions, (iv) hydroxyl radical (OH) was the influencing reactive oxygen species on the photocatalytic disinfection using MCNT-TiO2, and (v) good correlation between experimental parameters (i.e. carbon contents, surface area and concentration of MCNT-TiO2) and the contribution rate of physical and photocatalysis reactions. The finding from this study and the methods proposed herein are essential for understanding the photocatalytic disinfection processes using TiO2 and its carbonaceous nanocomposites, which can promote the application of photocatalytic disinfection process.

Causality analysis and prediction of 2-methylisoborneol production in a reservoir using empirical dynamic modeling.

2-Methylisobornel (MIB) is one of the most widespread and problematic biogenic compounds causing taste-and-odor problems in freshwater. To investigate the causes of MIB production and develop models to predict the MIB concentration, we have applied empirical dynamic modeling (EDM), a nonlinear approach based on Chaos theory, to the long-term water quality dataset of Kamafusa Reservoir in Japan. The study revealed the dynamic nature of MIB production in the reservoir, and determined causal variables for MIB production, including water temperature, pH, transparency, light intensity, and Green Phormidium. Moreover, EDM established that the system is three-dimensional, and the approach found elevated nonlinearity (from 1.5 to 3) across the whole study period (1996-2015). By taking only one or two candidate predictors with varying time lags, multivariate models for predicting MIB production (best model: r = 0.83, p < 0.001, root mean squared error = 3.1 ng/L) were successfully established. The modeling approach used in this study is a powerful tool for causality identification and odor prediction, thus making important contributions to reservoir management.

CDKN2A inhibits formation of homotypic cell-in-cell structures.

Cell-in-cell (CIC) structures, characterized by enclosure of one or more cells within another cell, were extensively documented in human cancers. Although elevated CIC formation was found in cancers with CDKN2A inactivation, a causal link between them remains to be established. We reported here that inhibiting CDKN2A expression effectively promoted homotypic CIC formation, whereas ectopic overexpression of p16INK4a or p14ARF, two proteins encoded by CDKN2A gene, significantly suppressed CIC formation in MCF7 cells. The regulation of CIC formation by CDKN2A was tightly correlated with subcellular redistribution of E-cadherin, F-actin rearrangement and reduced phosphorylation of myosin light chain 2 (p-MLC2), consistent with which, CDKN2A expression imparted cells winner/outer identity in competition assay. Moreover, CIC formation negatively correlates with p16INK4a expression in human breast cancers. Thus, our work identifies CDKN2A as the first tumor suppressor whose inactivation promotes homotypic CIC formation in human cancer cells.

Cholesterol inhibits entotic cell-in-cell formation and actomyosin contraction.

Cell-in-cell structure is prevalent in human cancer, and associated with several specific pathophysiological phenomena. Although cell membrane adhesion molecules were found critical for cell-in-cell formation, the roles of other membrane components, such as lipids, remain to be explored. In this study, we attempted to investigate the effects of cholesterol and phospholipids on the formation of cell-in-cell structures by utilizing liposome as a vector. We found that Lipofectamine-2000, the reagent commonly used for routine transfection, could significantly reduce entotic cell-in-cell formation in a cell-specific manner, which is correlated with suppressed actomyosin contraction as indicated by reduced ß-actin expression and myosin light chain phosphorylation. The influence on cell-in-cell formation was likely dictated by specific liposome components as some liposomes affected cell-in-cell formation while some others didn't. Screening on a limited number of lipids, the major components of liposome, identified phosphatidylethanolamine (PE), stearamide (SA), lysophosphatidic acid (LPA) and cholesterol (CHOL) as the inhibitors of cell-in-cell formation. Importantly, cholesterol treatment significantly inhibited myosin light chain phosphorylation, which resembles the effect of Lipofectamine-2000, suggesting cholesterol might be partially responsible for liposomes' effects on cell-in-cell formation. Together, our findings supporting a role of membrane lipids and cholesterol in cell-in-cell formation probably via regulating actomyosin contraction.

[Subcellular re-distribution of F-actin in MCF7 cells upon inducible expression of CDKN2A/p16INK4a].

Objective To investigate the effect of CDKN2A/p16INK4a, a cyclin-dependent kinase inhibitor, on cell morphology and cytoskeleton in MCF7 breast cancer cells by tetracycline operon (Tet-on) inducible gene expression system. Methods MCF7 cells, genetically null for p16INK4a expression, were sequentially infected with two viruses of Tet-on systems to make MCF7-pTet-on-p16INK4a cells, which were induced to express p16NK4a by doxycycline (Dox) treatment. Cell growth was evaluated by cell counting. To observe the effect of p16INK4a on cell adhesion and cytoskeleton, intercellular adhesion and intracellular F-actin were examined by fluorescent staining with anti-E-cadherin antibody and phalloidin, respectively. Changes in cell morphology were analyzed by ImageJ software after microscopic imaging. Results Cell line expressing p16INK4a upon Dox induction was successfully made in MCF7 breast cancer cells. The inducible expression of p16INK4a inhibited cell proliferation while significantly increasing cell size as marked by prolonged contour lines. Furthermore, intracellular F-actin were enriched at the cortical peripheries of p16INK4a -expressing cells, which was different from those in control MCF7 cells where F-actin were enriched in cytoplasmic regions. Conclusion p16INK4a expression in MCF7 breast cancer cells leads to increased cell size, weakened intercellular adhesion and cortical re-distribution of cytoskeletal F-actin.

Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression.

Although cell-in-cell structures (CICs) could be detected in a wide range of human tumors, homotypic CICs formed between tumor cells occur at low rate for most of them. We recently reported that tumor cells lacking expression of E- and P-cadherin were incapable of forming homotypic CICs by entosis, and re-expression of E- or P-cadherin was sufficient to induce CICs formation in these tumor cells. In this work, we found that homotypic CICs formation was impaired in some tumor cells expressing high level of E-cadherin due to loss expression of alpha-catenin (α-catenin), a molecular linker between cadherin-mediated adherens junctions and F-actin. Expression of α-catenin in these tumor cells restored cell-cell adhesion and promoted CICs formation in a ROCK kinase-dependent way. Thus, our work identified α-catenin as another molecule in addition to E- and P-cadherin that were targeted to inactivate homotypic CICs formation in human tumor cells.

Detecting cell-in-cell structures in human tumor samples by E-cadherin/CD68/CD45 triple staining.

Although Cell-in-cell structures (CICs) had been documented in human tumors for decades, it is unclear what types of CICs were formed largely due to low resolution of traditional way such as H&E staining. In this work, we employed immunofluorescent method to stain a panel of human tumor samples simultaneously with antibodies against E-cadherin for Epithelium, CD68 for Macrophage and CD45 for Leukocytes, which we termed as "EML method" based on the cells detected. Detail analysis revealed four types of CICs, with tumor cells or macrophage engulfing tumor cells or leukocytes respectively. Interestingly, tumor cells seem to be dominant over macrophage (93% vs 7%) as the engulfer cells in all CICs detected, whereas the overall amount of internalized tumor cells is comparable to that of internalized CD45+ leukocytes (57% vs 43%). The CICs profiles vary from tumor to tumor, which may indicate different malignant stages and/or inflammatory conditions. Given the potential impacts different types of CICs might have on tumor growth, we therefore recommend EML analysis of tumor samples to clarify the correlation of CICs subtypes with clinical prognosis in future researches.

[Nuclear protein 1 knockdown inhibits proliferation and migration of HepG2 cells].

OBJECTIVE: To observe the expression of nuclear protein 1 (Nupr1), a stress-related nuclear protein, in a panel of human hepatocellular carcinoma cell lines, and its effects on the proliferation and migration of hepatocellular carcinoma cells by RNA interference-mediated knockdown. METHODS: Real-time quantitative PCR was employed to detect Nupr1 mRNA levels in hepatocellular carcinoma cell lines, including BEL-7402, QSG-7703, SMMC-7721 and HepG2. After Nupr1 expression was knocked down by RNA interference, cell proliferation was monitored by MTT assay and colony formation in plate. Cell cycle was analyzed by flow cytometry and cell migration was assayed by TranswellTM assay. RESULTS: Higher expression of Nupr1 was detected in HepG2 as compared with the other cell lines. Nupr1 expression in HepG2 cells were efficiently knocked down by two short hairpin RNAs (shRNAs), with inhibitory rates being 72.25% and 84.25%, respectively. HepG2 cells with Nupr1 knockdown displayed lower rate of proliferation, G1 arrest, and significantly decreased abilities of cell migration and colony formation. Western blotting showed that Nupr1 knockdown increased the expressions of p21 and p27, two negative regulators of cell cycle. CONCLUSION: Knockdown of Nupr1 inhibited the proliferation and migration of HepG2 hepatocellular carcinoma cells.