Physical and in silico immunopeptidomic profiling of a cancer antigen prostatic acid phosphatase reveals targets enabling TCR isolation.

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

Single-Layer Hexagonal Boron Nitride Nanopores as High-Performance Ionic Gradient Power Generators.

Atomically thin two-dimensional (2D) materials have emerged as promising candidates for efficient energy harvesting from ionic gradients. However, the exploration of robust 2D atomically thin nanopore membranes, which hold sufficient ionic selectivity and high ion permeability, remains challenging. Here, the single-layer hexagonal boron nitride (hBN) nanopores are demonstrated as various high-performance ion-gradient nanopower harvesters. Benefiting from the ultrathin atomic thickness and large surface charge (also a large Dukhin number), the hBN nanopore can realize fast proton transport while maintaining excellent cation selectivity even in highly acidic environments. Therefore, a single hBN nanopore achieves the pure osmosis-driven proton-gradient power up to ≈3 nW under 1000-fold ionic gradient. In addition, the robustness of hBN membranes in extreme pH conditions allows the ionic gradient power generation from acid-base neutralization. Utilizing 1 m HCl/KOH, the generated power can be promoted to an extraordinarily high level of ≈4.5 nW, over one magnitude higher than all existing ionic gradient power generators. The synergistic effects of ultrathin thickness, large surface charge, and excellent chemical inertness of 2D single-layer hBN render it a promising membrane candidate for harvesting ionic gradient powers, even under extreme pH conditions.

DLG5 connects cell polarity and Hippo signaling protein networks by linking PAR-1 with MST1/2.

Disruption of apical-basal polarity is implicated in developmental disorders and cancer; however, the mechanisms connecting cell polarity proteins with intracellular signaling pathways are largely unknown. We determined previously that membrane-associated guanylate kinase (MAGUK) protein discs large homolog 5 (DLG5) functions in cell polarity and regulates cellular proliferation and differentiation via undefined mechanisms. We report here that DLG5 functions as an evolutionarily conserved scaffold and negative regulator of Hippo signaling, which controls organ size through the modulation of cell proliferation and differentiation. Affinity purification/mass spectrometry revealed a critical role of DLG5 in the formation of protein assemblies containing core Hippo kinases mammalian ste20 homologs 1/2 (MST1/2) and Par-1 polarity proteins microtubule affinity-regulating kinases 1/2/3 (MARK1/2/3). Consistent with this finding, Hippo signaling is markedly hyperactive in mammalian Dlg5-/- tissues and cells in vivo and ex vivo and in Drosophila upon dlg5 knockdown. Conditional deletion of Mst1/2 fully rescued the phenotypes of brain-specific Dlg5 knockout mice. Dlg5 also interacts genetically with Hippo effectors Yap1/Taz Mechanistically, we show that DLG5 inhibits the association between MST1/2 and large tumor suppressor homologs 1/2 (LATS1/2), uses its scaffolding function to link MST1/2 with MARK3, and inhibits MST1/2 kinase activity. These data reveal a direct connection between cell polarity proteins and Hippo, which is essential for proper development of multicellular organisms.

Urinary microRNA in Diabetic Kidney Disease: A Literature Review.

Diabetic kidney disease is the most common primary disease of end-stage kidney disease globally; however, a sensitive and accurate biomarker to predict this disease remains awaited. microRNAs are endogenous single-stranded noncoding RNAs that have intervened in different post-transcriptional regulations of various cellular biological functions. Previous literatures have reported its potential role in the pathophysiology of diabetic kidney disease, including regulation of Transforming Growth Factor-ß1-mediated fibrosis, extracellular matrix and cell adhesion proteins, cellular hypertrophy, growth factor, cytokine production, and redox system activation. Urinary microRNAs have emerged as a novel, non-invasive liquid biopsy for disease diagnosis. In this review, we describe the available experimental and clinical evidence of urinary microRNA in the context of diabetic kidney disease and discuss the future application of microRNA in routine practice.

Spin-Polarized Photocatalytic CO2 Reduction of Mn-Doped Perovskite Nanoplates.

"Spin" has been recently reported as an important degree of electronic freedom to improve the performance of electrocatalysts and photocatalysts. This work demonstrates the manipulations of spin-polarized electrons in CsPbBr3 halide perovskite nanoplates (NPLs) to boost the photocatalytic CO2 reduction reaction (CO2RR) efficiencies by doping manganese cations (Mn2+) and applying an external magnetic field. Mn-doped CsPbBr3 (Mn-CsPbBr3) NPLs exhibit an outstanding photocatalytic CO2RR compared to pristine CsPbBr3 NPLs due to creating spin-polarized electrons after Mn doping. Notably, the photocatalytic CO2RR of Mn-CsPbBr3 NPLs is significantly enhanced by applying an external magnetic field. Mn-CsPbBr3 NPLs exhibit 5.7 times improved performance of photocatalytic CO2RR under a magnetic field of 300 mT with a permanent magnet compared to pristine CsPbBr3 NPLs. The corresponding mechanism is systematically investigated by magnetic circular dichroism spectroscopy, ultrafast transient absorption spectroscopy, and density functional theory simulation. The origin of enhanced photocatalytic CO2RR efficiencies of Mn-CsPbBr3 NPLs is due to the increased number of spin-polarized photoexcited carriers by synergistic doping of the magnetic elements and applying a magnetic field, resulting in prolonged carrier lifetime and suppressed charge recombination. Our result shows that manipulating spin-polarized electrons in photocatalytic semiconductors provides an effective strategy to boost photocatalytic CO2RR efficiencies.

Stabilized High-Membered and Phase-Pure 2D All Inorganic Ruddlesden-Popper Halide Perovskites Nanocrystals as Photocatalysts for the CO2 Reduction Reaction.

In contrast to the 2D organic-inorganic hybrid Ruddlesden-Popper halide perovskites (RPP), a new class of 2D all inorganic RPP (IRPP) has been recently proposed by substituting the organic spacers with an optimal inorganic alternative of cesium cations (Cs+ ). Nevertheless, the synthesis of high-membered 2D IRPPs (n > 1) has been a very challenging task because the Cs+ need to act as both spacers and A-site cations simultaneously. This work presents the successful synthesis of stable phase-pure high-membered 2D IRPPs of Csn+1 Pbn Br3n+1 nanosheets (NSs) with n = 3 and 4 by employing the strategy of using additional strong binding bidentate ligands. The structures of the 2D IRPPs (n = 3 and 4) NSs are confirmed by powder X-ray diffraction and high-resolution aberration-corrected scanning transmission electron microscope measurements. These 2D IRPPs NSs exhibit a strong quantum confinement effect with tunable absorption and emission in the visible light range by varying their n values, attributed to their inherent 2D quantum-well structure. The superior structural and optical stability of the phase-pure high-membered 2D IRPPs make them a promising candidate as photocatalysts in CO2 reduction reactions with outstanding photocatalytic performance and long-term stability.

Atomically Resolved Quantum-Confined Electronic Structures at Organic-Inorganic Interfaces of Two-Dimensional Ruddlesden-Popper Halide Perovskites.

This work demonstrates the direct visualization of atomically resolved quantum-confined electronic structures at organic-inorganic heterointerfaces of two-dimensional (2D) organic-inorganic hybrid Ruddlesden-Popper perovskites (RPPs); this is accomplished with scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS) by using solvent engineering to prepare perpendicularly oriented 2D RPPs. Atomically resolved band mapping images across the organic-inorganic interfaces of 2D RPPs yield typical quantum-well-like type-I heterojunction band alignment with band gaps depending on the thicknesses or n values of the inorganic perovskite slabs. The presence of edge states within the band gap due to organic cation vacancies is also observed. In addition, real-space visualization of atomic-scale structural phase transition behavior and changes in local electronic band structures are obtained simultaneously. Our results provide an unequivocal observation and explanation of the quantum-confined electronic structures formed at organic-inorganic interfaces of 2D RPPs.

Induction Chemotherapy Improved Long Term Outcomes in Stage IV Locoregional Advanced Nasopharyngeal Carcinoma.

Purpose: We aimed to determine whether adding induction chemotherapy (IC) to concurrent chemoradiation (CCRT) improved outcomes in each stage of locally advanced nasopharyngeal carcinoma (LANPC). Methods: From 2007 to 2013, we retrospectively collected 259 histopathologically identified adult LANPC patients from two campuses in south Taiwan. Among the 238 eligibly treated cases, 156 patients received CCRT (CCRT group) upfront and 82 received IC followed by CCRT (IC group). Of these patients, 130 were stage III (92 patients that received CCRT and 38 that received IC adding CCRT) and 108 were stage IV (76 CCRT and 32 IC adding CCRT). Most chemotherapy regimens for IC are composed of cisplatin (P), 5-fluorouracil (F), and ifosfamide (I), while concurrent chemotherapy (CC) was essentially cisplatin-based. For CCRT as the upfront treatment, a P or PF regimen was usually used in CC. Survival outcomes were accessed with a Kaplan-Meier estimate and a p-value by log-rank test to compare the survival distributions of IC added to CCRT or CCRT as the upfront treatment in all LANPC stage III and LANPC IV patients. The failure free survival (FFS), overall survival (OS), local relapse free survival (LRFS), regional recurrence-free survival (RRFS), distant metastasis-free survival (DMFS), first failure site, and other prognostic factors were analyzed. Results: The median follow-up time of all treated LANPC patients was 59 months. For all LANPC patients, there was a significant difference only in the DMFS favoring IC group (91.5% vs 79.4%, p=0.013). In the subgroup study, for the stage III group, there was no significant difference between the groups for overall OS (IC group 71.3% vs CCRT group 78.7%), FFS (71.5% vs 62.4%) and RRFS (91.9% vs 90.9%). However, inferior LRLS (71.7% vs 91.5%; p = 0.03) was noted for the IC group. In contrast, for stage IV, there were significantly longer OS (75.8% vs 52.6%), FFS (66.8% vs 46.8%), and DMFS (86.0% vs 69.6%; p = 0.02, p = 0.04, and p = 0.03, respectively) rates in the IC group. Conclusion: Adding PIF-based IC to CCRT for the LANPC patients resulted in better outcomes for stage IV patients, but not for stage III patients. A future properly designed study should stratify enough LANPC cases under the structure of the AJCC stage grouping system to determine which subgroups truly benefit from adding IC to CCRT.

A circulating miRNA signature for early diagnosis of acute kidney injury following acute myocardial infarction.

BACKGROUND: Acute kidney injury (AKI) is a common complication of acute myocardial infarction (AMI), and is associated with adverse outcomes. The study aimed to identify a miRNA signature for the early diagnosis of post-AMI AKI. METHODS: A total of 108 patients admitted to a coronary care unit (CCU) were divided into four subgroups: AMI-AKI-, AMI+AKI-, AMI+AKI+, and AMI-AKI+. Thirty-six miRNA candidates were selected based on an extensive literature review. Real-time quantitative RT-PCR analysis was used to determine the expression levels of these miRNAs in the serum collected on the day of CCU admittance. TargetScan 7.1 and miRDB databases were used for target prediction and Metacore 6.13 was used for pathway analysis. RESULTS: Through a stepwise selection based on abundance, hemolytic effect and differential expression between four groups, 9 miRNAs were found to have significantly differential expression levels as potential biomarkers for post-AMI AKI specifically. Noticeably, the expression levels of miR-24, miR-23a and miR-145 were significantly down-regulated in AMI+AKI+ patients compared to those in AMI+AKI- patients. Combination of the three miRNAs as a panel showed the best performance in the early detection of AKI following AMI (AUC = 0.853, sensitivity 95.65%), compared to the analysis of serum neutrophil gelatinase-associated lipocalin (AUC = 0.735, sensitivity 63.16%). Furthermore, bioinformatic analysis indicated that these three miRNAs regulate the transforming growth factor beta signaling pathway and involve in apoptosis and fibrosis in AKI. CONCLUSIONS: For the first time, this study identify a unique circulating miRNA signature (miR-24-3p, miR-23a-3p, miR-145-5p) that can potentially early detect AKI following AMI and may be involved in renal injury and fibrosis in post-AMI AKI pathogenesis.

Saliva protein biomarkers to detect oral squamous cell carcinoma in a high-risk population in Taiwan.

Most cases of oral squamous cell carcinoma (OSCC) develop from visible oral potentially malignant disorders (OPMDs). The latter exhibit heterogeneous subtypes with different transformation potentials, complicating the early detection of OSCC during routine visual oral cancer screenings. To develop clinically applicable biomarkers, we collected saliva samples from 96 healthy controls, 103 low-risk OPMDs, 130 high-risk OPMDs, and 131 OSCC subjects. These individuals were enrolled in Taiwan's Oral Cancer Screening Program. We identified 302 protein biomarkers reported in the literature and/or through in-house studies and prioritized 49 proteins for quantification in the saliva samples using multiple reaction monitoring-MS. Twenty-eight proteins were successfully quantified with high confidence. The quantification data from non-OSCC subjects (healthy controls + low-risk OPMDs) and OSCC subjects in the training set were subjected to classification and regression tree analyses, through which we generated a four-protein panel consisting of MMP1, KNG1, ANXA2, and HSPA5. A risk-score scheme was established, and the panel showed high sensitivity (87.5%) and specificity (80.5%) in the test set to distinguish OSCC samples from non-OSCC samples. The risk score >0.4 detected 84% (42/50) of the stage I OSCCs and a significant portion (42%) of the high-risk OPMDs. Moreover, among 88 high-risk OPMD patients with available follow-up results, 18 developed OSCC within 5 y; of them, 77.8% (14/18) had risk scores >0.4. Our four-protein panel may therefore offer a clinically effective tool for detecting OSCC and monitoring high-risk OPMDs through a readily available biofluid.

Low-Threshold Lasing from 2D Homologous Organic-Inorganic Hybrid Ruddlesden-Popper Perovskite Single Crystals.

Organic-inorganic hybrid two-dimensional (2D) perovskites have recently attracted great attention in optical and optoelectronic applications due to their inherent natural quantum-well structure. We report the growth of high-quality millimeter-sized single crystals belonging to homologous two-dimensional (2D) hybrid organic-inorganic Ruddelsden-Popper perovskites (RPPs) of (BA)2(MA) n-1Pb nI3 n+1 ( n = 1, 2, and 3) by a slow evaporation at a constant-temperature (SECT) solution-growth strategy. The as-grown 2D hybrid perovskite single crystals exhibit excellent crystallinity, phase purity, and spectral uniformity. Low-threshold lasing behaviors with different emission wavelengths at room temperature have been observed from the homologous 2D hybrid RPP single crystals. Our result demonstrates that solution-growth homologous organic-inorganic hybrid 2D perovskite single crystals open up a new window as a promising candidate for optical gain media.

Quantitative Analysis of Glucose Metabolic Cleavage in Glucose Transporters Overexpressed Cancer Cells by Target-Specific Fluorescent Gold Nanoclusters.

The glucose metabolism rate in cancer cells is a crucial piece of information for the cancer aggressiveness. A feasible method to monitor processes of oncogenic mutations has been demonstrated in this work. The fluorescent gold nanoclusters conjugated with glucose (glucose-AuNCs) were successfully synthesized as a cancer-targeting probe for glucose transporters (Gluts) overexpressed by U-87 MG cancer cells, which can be observed under confocal microscopy. The structural and optical characterizations of fluorescent glucose-AuNCs were confirmed by transmission electron microscope (TEM) and Fourier transform infrared spectroscopy (FTIR). The MTT assay exhibited the high biocompatibility of water-soluble glucose-AuNCs for further biomedical applications. The glucose metabolic cleavage of glucose-AuNCs by glycolytic enzymes from U-87 MG cancer cell was measured by fluorescence change of glucose-AuNCs. The fluorescence change based on the integrated area under fluorescence spectra ( A t) of glucose-AuNCs was plotted as a function of different reaction time ( t) with glycolytic enzymes. The fitted curve of A t versus t showed the first-order kinetics to explain the mechanism of glucose metabolic cleavage rate of glucose-AuNCs by glycolytic enzymes. The rate constant k could be utilized to determine the glucose metabolism rate of glucose-AuNCs for the quantitative analysis of cancer aggressiveness. Our work provides a practical application of target-specific glucose-AuNCs as a fluorescence probe to analyze the glucose metabolism in Gluts overexpressed cancer cells.

Improving Hydrogen Evolution Activity of Earth-Abundant Cobalt-Doped Iron Pyrite Catalysts by Surface Modification with Phosphide.

Hydrogen is considered as sustainable and environmentally friendly energy for global energy demands in the future. Here a Co-FeS2 catalyst with surface phosphide doping (P/Co-FeS2 ) for hydrogen evolution reaction (HER) in acidic solutions is developed. The P/Co-FeS2 exhibits superior HER electrochemical performance with overpotential of -90 mV at 100 mA cm-2 and Tafel slope of 41 mV/decade and excellent durability.

MicroRNAs in acute kidney injury.

Acute kidney injury (AKI) is an important clinical issue that is associated with significant morbidity and mortality. Despite research advances over the past decades, the complex pathophysiology of AKI is not fully understood. The regulatory mechanisms underlying post-AKI repair and fibrosis have not been clarified either. Furthermore, there is no definitively effective treatment for AKI. MicroRNAs (miRNAs) are endogenous single-stranded noncoding RNAs of 19~23 nucleotides that have been shown to be crucial to the post-transcriptional regulation of various cellular biological functions, including proliferation, differentiation, metabolism, and apoptosis. In addition to being fundamental to normal development and physiology, miRNAs also play important roles in various human diseases. In AKI, some miRNAs appear to act pathogenically by promoting inflammation, apoptosis, and fibrosis, while others may act protectively by exerting anti-inflammatory, anti-apoptotic, anti-fibrotic, and pro-angiogenic effects. Thus, miRNAs have not only emerged as novel biomarkers for AKI; they also hold promise to be potential therapeutic targets.

Highly active and stable hybrid catalyst of cobalt-doped FeS2 nanosheets-carbon nanotubes for hydrogen evolution reaction.

Hydrogen evolution reaction (HER) from water through electrocatalysis using cost-effective materials to replace precious Pt catalysts holds great promise for clean energy technologies. In this work we developed a highly active and stable catalyst containing Co doped earth abundant iron pyrite FeS(2) nanosheets hybridized with carbon nanotubes (Fe(1-x)CoxS(2)/CNT hybrid catalysts) for HER in acidic solutions. The pyrite phase of Fe(1-x)CoxS(2)/CNT was characterized by powder X-ray diffraction and absorption spectroscopy. Electrochemical measurements showed a low overpotential of ∼0.12 V at 20 mA/cm(2), small Tafel slope of ∼46 mV/decade, and long-term durability over 40 h of HER operation using bulk quantities of Fe(0.9)Co(0.1)S(2)/CNT hybrid catalysts at high loadings (∼7 mg/cm(2)). Density functional theory calculation revealed that the origin of high catalytic activity stemmed from a large reduction of the kinetic energy barrier of H atom adsorption on FeS(2) surface upon Co doping in the iron pyrite structure. It is also found that the high HER catalytic activity of Fe(0.9)Co(0.1)S(2) hinges on the hybridization with CNTs to impart strong heteroatomic interactions between CNT and Fe(0.9)Co(0.1)S(2). This work produces the most active HER catalyst based on iron pyrite, suggesting a scalable, low cost, and highly efficient catalyst for hydrogen generation.

Nanodiamond-Mediated Intercellular Transport of Proteins through Membrane Tunneling Nanotubes.

Recently discovered tunneling nanotubes (TNTs) are capable of creating intercellular communication pathways through which transport of proteins and other cytoplasmic components occurs. Intercellular transport is related to many diseases and nanotubes are potentially useful as drug-delivery channels for cancer therapy. Here, we apply fluorescent nanodiamond (FND) as a photostable tracker, as well as a protein carrier, to illustrate the transport events in TNTs of human cells. Proteins, including bovine serum albumin and green fluorescent protein, are first coated on 100-nm FNDs by physical adsorption and then single-particle tracking of the bioconjugates in the transient membrane connections is carried out by fluorescence microscopy. Stop-and-go and to-and-fro motions mediated by molecular motors are found for the active transport of protein-loaded FNDs trapped in the endosomal vehicles of human embryonic kidney cells (HEK293T). Quantitative analysis of the heterotypical transport between HEK293T and SH-SY5Y neuroblastoma cells by flow cytometry confirm the formation of open-ended nanotubes between them, despite that their TNTs differ in structural components. Our results demonstrate the promising applications of this novel carbon-based nanomaterial for intercellular delivery of biomolecular cargo down to the single-particle level.

Graphene-Based Integrated Photovoltaic Energy Harvesting/Storage Device.

Energy scavenging has become a fundamental part of ubiquitous sensor networks. Of all the scavenging technologies, solar has the highest power density available. However, the energy source is erratic. Integrating energy conversion and storage devices is a viable route to obtain self-powered electronic systems which have long-term maintenance-free operation. In this work, we demonstrate an integrated-power-sheet, consisting of a string of series connected organic photovoltaic cells (OPCs) and graphene supercapacitors on a single substrate, using graphene as a common platform. This results in lighter and more flexible power packs. Graphene is used in different forms and qualities for different functions. Chemical vapor deposition grown high quality graphene is used as a transparent conductor, while solution exfoliated graphene pastes are used as supercapacitor electrodes. Solution-based coating techniques are used to deposit the separate components onto a single substrate, making the process compatible with roll-to-roll manufacture. Eight series connected OPCs based on poly(3-hexylthiophene)(P3HT):phenyl-C61-butyric acid methyl ester (PC60 BM) bulk-heterojunction cells with aluminum electrodes, resulting in a ≈5 V open-circuit voltage, provide the energy harvesting capability. Supercapacitors based on graphene ink with ≈2.5 mF cm(-2) capacitance provide the energy storage capability. The integrated-power-sheet with photovoltaic (PV) energy harvesting and storage functions had a mass of 0.35 g plus the substrate.

Continuously tunable and fast-response terahertz metamaterials using in-plane-switching dual-frequency liquid crystal cells.

A metamaterial that is embedded in an in-plane-switching dual-frequency liquid crystal cell is used to develop an electrically controllable terahertz (THz) metamaterial. The resonance peak of the metamaterial can be redshifted and blueshifted as the frequency of an external voltage is switched, and the response times for the redshift and blueshift are 1.044 and 1.376 ms, respectively. A simulation confirms the spectral redshift and blueshift. The variation in peak frequency as a function of applied frequency at the external voltage is presented. Experimental results show that the resonance peak of the metamaterial can be continuously tuned within a frequency range of 15 GHz as the applied frequency is switched between 19 and 22 kHz. Therefore, this metamaterial is a continuously tunable and fast-response THz filter and could be used for THz imaging and THz telecommunications.

Epstein-Barr virus-encoded LMP1 interacts with FGD4 to activate Cdc42 and thereby promote migration of nasopharyngeal carcinoma cells.

Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC), a human malignancy notorious for its highly metastatic nature. Among EBV-encoded genes, latent membrane protein 1 (LMP1) is expressed in most NPC tissues and exerts oncogenicity by engaging multiple signaling pathways in a ligand-independent manner. LMP1 expression also results in actin cytoskeleton reorganization, which modulates cell morphology and cell motility- cellular process regulated by RhoGTPases, such as Cdc42. Despite the prominent association of Cdc42 activation with tumorigenesis, the molecular basis of Cdc42 activation by LMP1 in NPC cells remains to be elucidated. Here using GST-CBD (active Cdc42-binding domain) as bait in GST pull-down assays to precipitate active Cdc42 from cell lysates, we demonstrated that LMP1 acts through its transmembrane domains to preferentially induce Cdc42 activation in various types of epithelial cells, including NPC cells. Using RNA interference combined with re-introduction experiments, we identified FGD4 (FYVE, RhoGEF and PH domain containing 4) as the GEF (guanine nucleotide exchange factor) responsible for the activation of Cdc42 by LMP1. Serial deletion experiments and co-immunoprecipitation assays further revealed that ectopically expressed FGD4 modulated LMP1-mediated Cdc42 activation by interacting with LMP1. Moreover, LMP1, through its transmembrane domains, directly bound FGD4 and enhanced FGD4 activity toward Cdc42, leading to actin cytoskeleton rearrangement and increased motility of NPC cells. Depletion of FGD4 or Cdc42 significantly reduced (∼50%) the LMP1-stimulated cell motility, an effect that was partially reversed by expression of a constitutively active mutant of Cdc42. Finally, quantitative RT-PCR and immunohistochemistry analyses showed that FGD4 and LMP1 were expressed in NPC tissues, supporting the potential physiologically relevance of this mechanism in NPC. Collectively, our results not only uncover a novel mechanism underlying LMP1-mediated Cdc42 activation, namely LMP1 interaction with FGD4, but also functionally link FGD4 to NPC tumorigenesis.

Live and heat-killed Lactobacillus rhamnosus GG upregulate gene expression of pro-inflammatory cytokines in 5-fluorouracil-pretreated Caco-2 cells.

PURPOSE: This study investigates whether post-chemotherapeutic use of live and heat-killed Lactobacillus rhamnosus GG can modulate the expression of three pro-inflammatory cytokines in 5-fluorouracil (5-FU)-induced intestinal mucositis in vitro. METHODS: Live L. rhamnosus GG and heat-killed L. rhamnosus GG were observed using scanning electron microscopy. To establish the duration required for optimal expression of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interleukin-12 (IL-12), 5 µM of 5-FU was selected to treat 10-day-old Caco-2 cells for 4, 6, 8, and 24 h. Caco-2 cells were treated with 5-FU (5 µM) for 4 h, followed by the administration of live L. rhamnosus GG (multiplicity of infection = 25), and heat-killed L. rhamnosus GG for 2 and 4 h. Finally, total cellular RNA was isolated to quantify mRNA expression of TNF-α, MCP-1, and IL-12 using real-time PCR. RESULTS: The results demonstrated that heat-killed L. rhamnosus GG remained structurally intact with elongation. A biphasic upregulated expression of TNF-α, MCP-1, and IL-12 was observed in 5-FU-treated Caco-2 cells at 4 and 24 h. Compared to non-L. rhamnosus GG controls in 5-FU-pretreated Caco-2 cells, a 2-h treatment of heat-killed L. rhamnosus GG significantly upregulated the MCP-1 expression (p < 0.05), and both live and heat-killed L. rhamnosus GG treatments lasting 4 h upregulated the TNF-α and MCP-1 expression (p < 0.05). Only live L. rhamnosus GG upregulated the IL-12 expression (p < 0.05). CONCLUSIONS: Post-chemotherapeutic use of live or heat-killed L. rhamnosus GG can upregulate the gene expression of 5-FU-induced pro-inflammatory cytokines in Caco-2 cells. Human intestinal epithelium may be vulnerable to the post-chemotherapeutic use of L. rhamnosus GG in 5-FU-induced mucositis that requires further in vivo studies for clarification.