An automatic and accurate deep learning-based neuroimaging pipeline for the neonatal brain.

BACKGROUND: Accurate segmentation of neonatal brain tissues and structures is crucial for studying normal development and diagnosing early neurodevelopmental disorders. However, there is a lack of an end-to-end pipeline for automated segmentation and imaging analysis of the normal and abnormal neonatal brain. OBJECTIVE: To develop and validate a deep learning-based pipeline for neonatal brain segmentation and analysis of structural magnetic resonance images (MRI). MATERIALS AND METHODS: Two cohorts were enrolled in the study, including cohort 1 (582 neonates from the developing Human Connectome Project) and cohort 2 (37 neonates imaged using a 3.0-tesla MRI scanner in our hospital).We developed a deep leaning-based architecture capable of brain segmentation into 9 tissues and 87 structures. Then, extensive validations were performed for accuracy, effectiveness, robustness and generality of the pipeline. Furthermore, regional volume and cortical surface estimation were measured through in-house bash script implemented in FSL (Oxford Centre for Functional MRI of the Brain Software Library) to ensure reliability of the pipeline. Dice similarity score (DSC), the 95th percentile Hausdorff distance (H95) and intraclass correlation coefficient (ICC) were calculated to assess the quality of our pipeline. Finally, we finetuned and validated our pipeline on 2-dimensional thick-slice MRI in cohorts 1 and 2. RESULTS: The deep learning-based model showed excellent performance for neonatal brain tissue and structural segmentation, with the best DSC and the 95th percentile Hausdorff distance (H95) of 0.96 and 0.99 mm, respectively. In terms of regional volume and cortical surface analysis, our model showed good agreement with ground truth. The ICC values for the regional volume were all above 0.80. Considering the thick-slice image pipeline, the same trend was observed for brain segmentation and analysis. The best DSC and H95 were 0.92 and 3.00 mm, respectively. The regional volumes and surface curvature had ICC values just below 0.80. CONCLUSIONS: We propose an automatic, accurate, stable and reliable pipeline for neonatal brain segmentation and analysis from thin and thick structural MRI. The external validation showed very good reproducibility of the pipeline.

Signaling protein signature predicts clinical outcome of non-small-cell lung cancer.

BACKGROUND: Non-small-cell lung cancer (NSCLC) is characterized by abnormalities of numerous signaling proteins that play pivotal roles in cancer development and progression. Many of these proteins have been reported to be correlated with clinical outcomes of NSCLC. However, none of them could provide adequate accuracy of prognosis prediction in clinical application. METHODS: A total of 384 resected NSCLC specimens from two hospitals in Beijing (BJ) and Chongqing (CQ) were collected. Using immunohistochemistry (IHC) staining on stored formalin-fixed paraffin-embedded (FFPE) surgical samples, we examined the expression levels of 75 critical proteins on BJ samples. Random forest algorithm (RFA) and support vector machines (SVM) computation were applied to identify protein signatures on 2/3 randomly assigned BJ samples. The identified signatures were tested on the remaining BJ samples, and were further validated with CQ independent cohort. RESULTS: A 6-protein signature for adenocarcinoma (ADC) and a 5-protein signature for squamous cell carcinoma (SCC) were identified from training sets and tested in testing sets. In independent validation with CQ cohort, patients can also be divided into high- and low-risk groups with significantly different median overall survivals by Kaplan-Meier analysis, both in ADC (31 months vs. 87 months, HR 2.81; P <  0.001) and SCC patients (27 months vs. not reached, HR 9.97; P <  0.001). Cox regression analysis showed that both signatures are independent prognostic indicators and outperformed TNM staging (ADC: adjusted HR 3.07 vs. 2.43, SCC: adjusted HR 7.84 vs. 2.24). Particularly, we found that only the ADC patients in high-risk group significantly benefited from adjuvant chemotherapy (P = 0.018). CONCLUSIONS: Both ADC and SCC protein signatures could effectively stratify the prognosis of NSCLC patients, and may support patient selection for adjuvant chemotherapy.

Ubiquitin-associated domain-containing ubiquitin regulatory X (UBX) protein UBXN1 is a negative regulator of nuclear factor κB (NF-κB) signaling.

Excessive nuclear factor κB (NF-κB) activation should be precisely controlled as it contributes to multiple immune and inflammatory diseases. However, the negative regulatory mechanisms of NF-κB activation still need to be elucidated. Various types of polyubiquitin chains have proved to be involved in the process of NF-κB activation. Many negative regulators linked to ubiquitination, such as A20 and CYLD, inhibit IκB kinase activation in the NF-κB signaling pathway. To find new NF-κB signaling regulators linked to ubiquitination, we used a small scale siRNA library against 51 ubiquitin-associated domain-containing proteins and screened out UBXN1, which contained both ubiquitin-associated and ubiquitin regulatory X (UBX) domains as a negative regulator of TNFα-triggered NF-κB activation. Overexpression of UBXN1 inhibited TNFα-triggered NF-κB activation, although knockdown of UBXN1 had the opposite effect. UBX domain-containing proteins usually act as valosin-containing protein (VCP)/p97 cofactors. However, knockdown of VCP/p97 barely affected UBXN1-mediated NF-κB inhibition. At the same time, we found that UBXN1 interacted with cellular inhibitors of apoptosis proteins (cIAPs), E3 ubiquitin ligases of RIP1 in the TNFα receptor complex. UBXN1 competitively bound to cIAP1, blocked cIAP1 recruitment to TNFR1, and sequentially inhibited RIP1 polyubiquitination in response to TNFα. Therefore, our findings demonstrate that UBXN1 is an important negative regulator of the TNFα-triggered NF-κB signaling pathway by mediating cIAP recruitment independent of VCP/p97.

N-(2-Chloro-acet-yl)glycine.

The title compound, C4H6ClNO3, crystallizes with two independent mol-ecules (A and B) in the asymmetric unit. In each mol-ecule, there are N-H⋯O and N-H⋯Cl hydrogen bonds. Both mol-ecules are relatively planar, with the mean plane of the acetamide [N-C(=O)C] group being inclined to the mean plane of the acetate group [C-C(=O)O] by 9.23 (13)° in mol-ecule A and 6.23 (12)° in mol-ecule B. In the crystal, adjacent mol-ecules are linked by O-H⋯O hydrogen bonds and weak C-H⋯O contacts forming -A-A-A- and -B-B-B- parallel chains propagating along the a-axis direction.

[The effect of Bcl-2 gene silencing on the sensitivity of cell line A549 to chemotherapeutic drugs].

OBJECTIVE: To investigate the effects of miRNA-mediated down-regulation of the Bcl-2 gene on the chemotherapeutic sensitivities and mRNA transcriptions of sensitivity associated genes in human lung adenocarcinoma cell line A549 cells, and therefore to provide experimental data for improving the chemotherapeutic effects on non-small cell lung cancer (NSCLC). METHODS: The miRNA recombinant plasmid targeting to human Bcl-2 gene was designed, synthesized and stably transferred into A549 cells by lipofectin technique as the experiment group. The transcription of Bcl-2 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) by agarose gel electrophoresis, real-time PCR, and the protein level of Bcl-2 was measured by Western blot to confirm the function of miRNA plasmid. The cell proliferation was examined by methyl thiazolyl tetrazolium (MTT) assay. Cell cycle was measured by flow cytometry. Drug sensitivities of A549 cells to etoposide, 5-fluorouracil, cisplatin, adriamycin, vincristine, paclitaxel and navelbine were analyzed by MTT assay. The mRNA expressions of excision repair cross-complementing gene 1 (ERCC1), thymidylate synthase (TYMS), Class III ß-tubulin, topoisomerase 2 alpha (TOP2α) genes were detected by RT-PCR and real-time PCR. RESULTS: The recombinant miRNA plasmid was successfully synthesized and stably transferred into A549 cells. The transcription of Bcl-2 mRNA dramatically decreased by 98.1% in the experiment group (RQ = 0.002 ± 0.001) compared to that in the negative control group (RQ = 0.104 ± 0.003) by real-time PCR (t = 98.70, P < 0.05); and the protein level of Bcl-2 in the experiment group decreased by 57.6% by Western blot (t = 7.66, P < 0.05). The cell cycle profile showed that the low expression of Bcl-2 gene led to A549 cell cycle arrest at G1-phase. The results of MTT showed that the growth of A549 cells in the experiment group was markedly inhibited. The sensitivities of A549 cells to etoposide, cisplatin, paclitaxel, and navelbine were significantly enhanced [IC50 values in the experiment group were (107.3 ± 0.1) mg/L, (7.7 ± 0.6) mg/L, (11.5 ± 1.9) mg/L and (10.8 ± 1.6) mg/L; IC50 values in the negative control group were (145.8 ± 0.1) mg/L, (60.7 ± 1.4) mg/L, (80.6 ± 1.7) mg/L and (20.6 ± 1.7) mg/L], the respective t values being 655.33, 108.04, 82.16 and 12.48, all P < 0.05. The mRNA level of ERCC1, TYMS, and TOP2α genes in the experiment group decreased by 99.6%, 92.9% and 96.1% respectively, but Class III ß-tubulin mRNA increased by 122% compared to the negative control group (1.154 ± 0.008, 0.520 ± 0.009), the respective t values being 689.79, 689.37, 768.04 and 160.07, all P < 0.05. CONCLUSION: Targeting to inhibit antiapoptotic mitochondrial gene Bcl-2 expression in A549 cells specifically decreased the mRNA of ERCC1, TYMS, and TOP2α genes, and significantly increased the sensitivities of A549 cells to chemotherapeutic agents such as etoposide, cisplatin, paclitaxel and navelbine.

Molecular Hydrogen Promotes Adipose-derived Stem Cell Myogenic Differentiation via Regulation of Mitochondria.

BACKGROUND: Acute skeletal muscle injuries are common physical or sports traumas. Cellular therapy has excellent potential for regeneration after skeletal muscle injury. Adipose-derived stem cells (ADSCs) are a more accessible type of stem cell. However, it has a low survival rate and differentiation efficiency in the oxidative stress-rich microenvironment after transplantation. Although molecular hydrogen (H2) possesses anti-inflammatory and antioxidant biological properties, its utility in mitochondrial and stem cell research has not been adequately explored. OBJECTIVE: This study aimed to reveal the role of H2 on adipose-derived stem cells' myogenic differentiation. METHODS: The protective effects of H2 in ADSCs were evaluated by MTT assay, live-dead cell staining, western blot analysis, immunofluorescence staining, confocal imaging, and transmission electron microscopy. RESULTS: An appropriate volume fraction of H2 significantly decreased mitochondrial reactive oxygen species (ROS) levels, increased the number of mitochondria, and promoted mitophagy, thus enhancing the survival and myogenic differentiation of ADSCs. CONCLUSION: This study reveals the application potential of H2 in skeletal muscle diseases or other pathologies related to mitochondrial dysfunction.

Combining Porous Se@SiO2 Nanocomposites and dECM Enhances the Myogenic Differentiation of Adipose-Derived Stem Cells.

Background: Volumetric Muscle Loss (VML) denotes the traumatic loss of skeletal muscle, a condition that can result in chronic functional impairment and even disability. While the body can naturally repair injured skeletal muscle within a limited scope, patients experiencing local and severe muscle loss due to VML surpass the compensatory capacity of the muscle itself. Currently, clinical treatments for VML are constrained and demonstrate minimal efficacy. Selenium, a recognized antioxidant, plays a crucial role in regulating cell differentiation, anti-inflammatory responses, and various other physiological functions. Methods: We engineered a porous Se@SiO2 nanocomposite (SeNPs) with the purpose of releasing selenium continuously and gradually. This nanocomposite was subsequently combined with a decellularized extracellular matrix (dECM) to explore their collaborative protective and stimulatory effects on the myogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs). The influence of dECM and NPs on the myogenic level, reactive oxygen species (ROS) production, and mitochondrial respiratory chain (MRC) activity of ADSCs was evaluated using Western Blot, ELISA, and Immunofluorescence assay. Results: Our findings demonstrate that the concurrent application of SeNPs and dECM effectively mitigates the apoptosis and intracellular ROS levels in ADSCs. Furthermore, the combination of dECM with SeNPs significantly upregulated the expression of key myogenic markers, including MYOD, MYOG, Desmin, and myosin heavy chain in ADSCs. Notably, this combination also led to an increase in both the number of mitochondria and the respiratory chain activity in ADSCs. Conclusion: The concurrent application of SeNPs and dECM effectively diminishes ROS production, boosts mitochondrial function, and stimulates the myogenic differentiation of ADSCs. This study lays the groundwork for future treatments of VML utilizing the combination of SeNPs and dECM.

Rhythmic cilia changes support SCN neuron coherence in circadian clock.

The suprachiasmatic nucleus (SCN) drives circadian clock coherence through intercellular coupling, which is resistant to environmental perturbations. We report that primary cilia are required for intercellular coupling among SCN neurons to maintain the robustness of the internal clock in mice. Cilia in neuromedin S-producing (NMS) neurons exhibit pronounced circadian rhythmicity in abundance and length. Genetic ablation of ciliogenesis in NMS neurons enabled a rapid phase shift of the internal clock under jet-lag conditions. The circadian rhythms of individual neurons in cilia-deficient SCN slices lost their coherence after external perturbations. Rhythmic cilia changes drive oscillations of Sonic Hedgehog (Shh) signaling and clock gene expression. Inactivation of Shh signaling in NMS neurons phenocopied the effects of cilia ablation. Thus, cilia-Shh signaling in the SCN aids intercellular coupling.

Porous Se@SiO2 nanoparticles improve oxidative injury to promote muscle regeneration via modulating mitochondria.

Background: Acute skeletal muscle injuries are common among physical or sports traumas. The excessive oxidative stress at the site of injury impairs muscle regeneration. The authors have recently developed porous Se@SiO2 nanoparticles (NPs) with antioxidant properties. Methods: The protective effects were evaluated by cell proliferation, myogenic differentiation and mitochondrial activity. Then, the therapeutic effect was investigated in a cardiotoxin-induced muscle injury rat model. Results: Porous Se@SiO2 NPs significantly protected the morphological and functional stability of mitochondria, thus protecting satellite cells from H2O2-induced damage to cell proliferation and myogenic differentiation. In the rat model, intervention with porous Se@SiO2 NPs promoted muscle regeneration. Conclusion: This study reveals the application potential of porous Se@SiO2 NPs in skeletal muscle diseases related to mitochondrial dysfunction.

Muscle injuries are very common in daily life and in sports. When a muscle is injured, the local response inhibits the regeneration and differentiation of stem cells inside the muscle, thus hindering muscle regeneration. The authors have recently developed a nanoparticle with the ability to protect muscle stem cell function, promote stem cell proliferation and differentiation and facilitate muscle regeneration after skeletal muscle injury in rats. Thus, this study reveals the potential of porous Se@SiO₂ nanoparticles in skeletal muscle diseases associated with mitochondrial dysfunction.

LUBAC regulates ciliogenesis by promoting CP110 removal from the mother centriole.

Primary cilia transduce diverse signals in embryonic development and adult tissues. Defective ciliogenesis results in a series of human disorders collectively known as ciliopathies. The CP110-CEP97 complex removal from the mother centriole is an early critical step for ciliogenesis, but the underlying mechanism for this step remains largely obscure. Here, we reveal that the linear ubiquitin chain assembly complex (LUBAC) plays an essential role in ciliogenesis by targeting the CP110-CEP97 complex. LUBAC specifically generates linear ubiquitin chains on CP110, which is required for CP110 removal from the mother centriole in ciliogenesis. We further identify that a pre-mRNA splicing factor, PRPF8, at the distal end of the mother centriole acts as the receptor of the linear ubiquitin chains to facilitate CP110 removal at the initial stage of ciliogenesis. Thus, our study reveals a direct mechanism of regulating CP110 removal in ciliogenesis and implicates the E3 ligase LUBAC as a potential therapy target of cilia-associated diseases, including ciliopathies and cancers.

CEP55 promotes cilia disassembly through stabilizing Aurora A kinase.

Primary cilia protrude from the cell surface and have diverse roles during development and disease, which depends on the precise timing and control of cilia assembly and disassembly. Inactivation of assembly often causes cilia defects and underlies ciliopathy, while diseases caused by dysfunction in disassembly remain largely unknown. Here, we demonstrate that CEP55 functions as a cilia disassembly regulator to participate in ciliopathy. Cep55-/- mice display clinical manifestations of Meckel-Gruber syndrome, including perinatal death, polycystic kidneys, and abnormalities in the CNS. Interestingly, Cep55-/- mice exhibit an abnormal elongation of cilia on these tissues. Mechanistically, CEP55 promotes cilia disassembly by interacting with and stabilizing Aurora A kinase, which is achieved through facilitating the chaperonin CCT complex to Aurora A. In addition, CEP55 mutation in Meckel-Gruber syndrome causes the failure of cilia disassembly. Thus, our study establishes a cilia disassembly role for CEP55 in vivo, coupling defects in cilia disassembly to ciliopathy and further suggesting that proper cilia dynamics are critical for mammalian development.

LPA signaling acts as a cell-extrinsic mechanism to initiate cilia disassembly and promote neurogenesis.

Dynamic assembly and disassembly of primary cilia controls embryonic development and tissue homeostasis. Dysregulation of ciliogenesis causes human developmental diseases termed ciliopathies. Cell-intrinsic regulatory mechanisms of cilia disassembly have been well-studied. The extracellular cues controlling cilia disassembly remain elusive, however. Here, we show that lysophosphatidic acid (LPA), a multifunctional bioactive phospholipid, acts as a physiological extracellular factor to initiate cilia disassembly and promote neurogenesis. Through systematic analysis of serum components, we identify a small molecular-LPA as the major driver of cilia disassembly. Genetic inactivation and pharmacological inhibition of LPA receptor 1 (LPAR1) abrogate cilia disassembly triggered by serum. The LPA-LPAR-G-protein pathway promotes the transcription and phosphorylation of cilia disassembly factors-Aurora A, through activating the transcription coactivators YAP/TAZ and calcium/CaM pathway, respectively. Deletion of Lpar1 in mice causes abnormally elongated cilia and decreased proliferation in neural progenitor cells, thereby resulting in defective neurogenesis. Collectively, our findings establish LPA as a physiological initiator of cilia disassembly and suggest targeting the metabolism of LPA and the LPA pathway as potential therapies for diseases with dysfunctional ciliogenesis.

Phylogeny and biogeography of Allium (Amaryllidaceae: Allieae) based on nuclear ribosomal internal transcribed spacer and chloroplast rps16 sequences, focusing on the inclusion of species endemic to China.

BACKGROUND AND AIMS: The genus Allium comprises more than 800 species, placing it among the largest monocotyledonous genera. It is a variable group that is spread widely across the Holarctic region. Previous studies of Allium have been useful in identifying and assessing its evolutionary lineages. However, there are still many gaps in our knowledge of infrageneric taxonomy and evolution of Allium. Further understanding of its phylogeny and biogeography will be achieved only through continued phylogenetic studies, especially of those species endemic to China that have often been excluded from previous analyses. Earlier molecular studies have shown that Chinese Allium is not monophyletic, so the goal of the present study was to infer the phylogeny and biogeography of Allium and to provide a classification of Chinese Allium by placement of Chinese species in the context of the entire phylogeny. METHODS: Phylogenetic studies were based on sequence data of the nuclear ribosomal internal transcribed spacer (ITS) and chloroplast rps16 intron, analysed using parsimony and Bayesian approaches. Biogeographical patterns were conducted using statistical dispersal-vicariance analysis (S-DIVA). KEY RESULTS: Phylogenetic analyses indicate that Allium is monophyletic and consists of three major clades. Optimal reconstructions have favoured the ancestors of Amerallium, Anguinum, Vvedenskya, Porphyroprason and Melanocrommyum as originating in eastern Asia. CONCLUSIONS: Phylogenetic analyses reveal that Allium is monophyletic but that some subgenera are not. The large genetic distances imply that Allium is of ancient origin. Molecular data suggest that its evolution proceeded along three separate evolutionary lines. S-DIVA indicates that the ancestor of Amerallium, Anguinum, Vvedenskya, Porphyroprason and Melanocrommyum originated from eastern Asia and underwent different biogeographical pathways. A taxonomic synopsis of Chinese Allium at sectional level is given, which divides Chinese Allium into 13 subgenera and 34 sections.

Plaid Detectors in Macaque V1 Revealed by Two-Photon Calcium Imaging.

Neuronal responses to one-dimensional orientations are combined to represent two-dimensional composite patterns; this plays a key role in intermediate-level vision such as texture segmentation. However, where and how the visual cortex starts to represent composite patterns, such as a plaid consisting of two superimposing gratings of different orientations, remains neurophysiologically elusive. Psychophysical and modeling evidence has suggested the existence of early neural mechanisms specialized in plaid detection [1-6], but the responses of V1 neurons to an optimally orientated grating are actually suppressed by a superimposing grating of different orientation (i.e., cross-orientation inhibition) [7, 8]. Would some other V1 neurons be plaid detectors? Here, we used two-photon calcium imaging [9] to compare the responses of V1 superficial-layer neurons to gratings and plaids in awake macaques. We found that many non-orientation-tuned neurons responded weakly to gratings but strongly to plaids, often with plaid orientation selectivity and cross-angle selectivity. In comparison, most (∼94%) orientation-tuned neurons showed more or less cross-orientation inhibition, regardless of the relative stimulus contrasts. Only a small portion (∼8%) of them showed plaid facilitation at off-peak orientations. These results suggest separate subpopulations of plaid and grating responding neurons. Because most of these plaid neurons (∼95%) were insensitive to motion direction, they were plaid pattern detectors, not plaid motion detectors.

Rab7 regulates primary cilia disassembly through cilia excision.

The primary cilium is a sensory organelle that protrudes from the cell surface. Primary cilia undergo dynamic transitions between assembly and disassembly to exert their function in cell signaling. In this study, we identify the small GTPase Rab7 as a novel regulator of cilia disassembly. Depletion of Rab7 potently induced spontaneous ciliogenesis in proliferating cells and promoted cilia elongation during quiescence. Moreover, Rab7 performs an essential role in cilia disassembly; knockdown of Rab7 blocked serum-induced ciliary resorption, and active Rab7 was required for this process. Further, we demonstrate that Rab7 depletion significantly suppresses cilia tip excision, referred to as cilia ectocytosis, which has been identified as required for cilia disassembly. Mechanically, the failure of F-actin polymerization at the site of excision of cilia tips caused suppression of cilia ectocytosis on Rab7 depletion. Overall, our results suggest a novel function for Rab7 in regulating cilia ectocytosis and cilia disassembly via control of intraciliary F-actin polymerization.

Microtubule asters anchored by FSD1 control axoneme assembly and ciliogenesis.

Defective ciliogenesis causes human developmental diseases termed ciliopathies. Microtubule (MT) asters originating from centrosomes in mitosis ensure the fidelity of cell division by positioning the spindle apparatus. However, the function of microtubule asters in interphase remains largely unknown. Here, we reveal an essential role of MT asters in transition zone (TZ) assembly during ciliogenesis. We demonstrate that the centrosome protein FSD1, whose biological function is largely unknown, anchors MT asters to interphase centrosomes by binding to microtubules. FSD1 knockdown causes defective ciliogenesis and affects embryonic development in vertebrates. We further show that disruption of MT aster anchorage by depleting FSD1 or other known anchoring proteins delocalizes the TZ assembly factor Cep290 from centriolar satellites, and causes TZ assembly defects. Thus, our study establishes FSD1 as a MT aster anchorage protein and reveals an important function of MT asters anchored by FSD1 in TZ assembly during ciliogenesis.

Circulating microRNAs as diagnostic and prognostic tools for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive malignancy and the second leading cause of cancer-related deaths worldwide. Conventional biomarkers exhibit poor performance in the surveillance, diagnosis, and prognosis of HCC. MicroRNAs (miRNAs) are a class of evolutionarily conserved small non-coding RNAs that are involved in the regulation of gene expression and protein translation, and they play critical roles in cell growth, differentiation, and the development of various types of cancers, including HCC. Recent evidence revealed the role of miRNAs as potential novel and ideal biomarkers for HCC. miRNAs are released to extracellular spaces, and they are extremely stable in bodily fluids, including serum or plasma, where they are packaged into various microparticles or associated with RNA-binding proteins. Numerous studies have demonstrated that circulating miRNAs have potential applications as minimally invasive biomarkers for HCC diagnosis and prognosis. The present review highlights current understanding of miRNA biogenesis and the origins and types of circulating miRNAs. We summarize recent progress in the use of circulating miRNAs as diagnostic and prognostic biomarkers for HCC. We also discuss the challenges and perspectives of the clinical utility of circulating miRNAs in HCC.

Identification of suitable reference genes for gene expression studies using quantitative polymerase chain reaction in lung cancer in vitro.

The present study aimed to examine 10 housekeeping genes (HKGs), including 18s ribosomal RNA (18S), glyceraldehyde­3­phosphate dehydrogenase (GAPDH), ribosomal protein large P0 (RPLP0), ß­actin (ACTB), peptidylprolyl isomerase A (PPIA), phosphoglycerate kinase­1 (PGK1), ß­2­microglobulin (B2M), ribosomal protein LI3a (RPL13A), hypoxanthine phosphoribosyl transferase­1 (HPRT1) and TATA box binding protein (TBP) in order to identify the most stable and suitable reference genes for use in expression studies in non­small cell lung cancer. The mRNA expression encoding the panel of the 10 HKGs was determined using reverse transcription­quantitative PCR (RT­qPCR) in human lung cancer cell lines. Three software programs, BestKeeper, NormFinder and geNorm, were used to ascertain the most suitable reference genes to normalize the RNA input. The present study examined three lung cancer cell lines (A549, NCI­H446 and NCI­H460). The analysis of the experimental data using BestKeeper software revealed that all 10 HKGs were stable, with GADPH, followed by 18S being the most stable genes and PPIA and HPRT1 being the least stable genes. The NormFinder software results demonstrated that PPIA followed by ACTB were the most stable and B2M and RPLP0 were the least stable. The geNorm software results revealed that ACTB and PGK1, followed by PPIA were the most stable genes and B2M and RPLP0 were identified as the least stable genes. Due to discrepancies in the ranking orders of the reference genes obtained by different analyzing software programs, it was not possible to determine a single universal reference gene. The suitability of selected reference genes requires unconditional validation prior to each study. Based on the three analyzing programs, ACTB, PPIA and PGK1 were the most stable reference genes in lung cancer cell lines.

RNF4 negatively regulates NF-κB signaling by down-regulating TAB2.

Most of NF-κB (nuclear factor kappa B) signaling molecules have various types of post-translational modifications. In this study, we focused on ubiquitination and designed a siRNA library including most ubiquitin-binding domains. With this library, we identified several candidate regulators of canonical NF-κB pathway, including RNF4. Overexpression of RNF4 impaired NF-κB activation in a dose-dependent manner, whereas RNF4 knockdown potentiated NF-κB activation. We showed that RNF4 interacts with the TAK1-TAB2-TAB3 complex, but not TAB1. Further, we found that RNF4 specifically down-regulated TAB2 through a lysosomal pathway, and knockdown of RNF4 impaired endogenous TAB2 degradation. Therefore, our findings will provide new insights into the negative regulation of NF-κB signaling.

Antitumor activity of lentivirus-mediated interleukin -12 gene modified dendritic cells in human lung cancer in vitro.

OBJECTIVES: Dendritic cell (DC)-based tumor immunotherapy needs an immunogenic tumor associated antigen (TAA) and an effective approach for its presentation to lymphocytes. In this study we explored whether transduction of DCs with lentiviruses (LVs) expressing the human interleukin-12 gene could stimulate antigen- specific cytotoxic T cells (CTLs) against human lung cancer cells in vitro. METHODS: Peripheral blood monocyte- derived DCs were transduced with a lentiviral vector encoding human IL-12 gene (LV-12). The anticipated target of the human IL-12 gene was detected by RT-PCR. The concentration of IL-12 in the culture supernatant of DCs was measured by ELISA.Transduction efficiencies and CD83 phenotypes of DCs were assessed by flow cytometry. DCs were pulsed with tumor antigen of lung cancer cells (DC+Ag) and transduced with LV-12 (DC-LV-12+Ag). Stimulation of T lymphocyte proliferation by DCs and activation of cytotoxic T-lymphocytes (CTL) stimulated by LV-12 transduced DCs pulsed with tumor antigen against A549 lung cancer cells were assessed with methyl thiazolyltetrazolium (MTT). RESULTS: A recombinant lentivirus expressing the IL-12 gene was successfully constructed. DC transduced with LV-12 produced higher levels of IL-12 and expressed higher levels of CD83 than non-transduced. The DC modified by interleukin -12 gene and pulsed with tumor antigen demonstrated good stimulation of lymphocyte proliferation, induction of antigen-specific cytotoxic T lymphocytes and anti- tumor effects. CONCLUSIONS: Dendritic cells transduced with a lentivirus-mediated interleukin-12 gene have an enhanced ability to kill lung cancer cells through promoting T lymphocyte proliferation and cytotoxicity.