The significance of CD4+ and CD8+ T lymphocyte infiltration in esophageal squamous cell carcinoma.

PURPOSE: To investigate the relationship between the abundance of CD4+ and CD8+ T cells in the tumor microenvironment and the prognosis of patients with esophageal squamous cell carcinoma, and to analyze their correlation and explore its clinical value. MATERIALS AND METHODS: In total, we enrolled 120 cases of esophageal squamous cell carcinoma diagnosed. The abundance of CD4+ and CD8+ T lymphocytes in the tissue specimens of esophageal cancer was examined by immunohistochemistry. We measured the correlation between the abundance of CD4+ and CD8+ T lymphocytes and the clinical and pathological characteristics and prognosis of esophageal squamous cell carcinoma. RESULTS: The tissue abundance of CD4+ T lymphocytes was closely related to tumor prognosis (P < 0.05). Similarly, there was a statistically significant relationship between the tissue abundance of CD8+ T lymphocytes and patients' prognosis (P < 0.05), indicating that a high abundance of CD8+ T lymphocytes predicts better prognosis in esophageal squamous cell carcinoma. Surprisingly, we found that a higher CD4+/CD8+ ratio predicted a better prognosis of esophageal squamous cell carcinoma. CONCLUSIONS: The tissue abundance of CD4+ and CD8+ T lymphocytes can serve as an important indicator for predicting the long-term survival of patients with esophageal squamous cell carcinoma. A high CD4+/CD8+ ratio may improve patients' prognosis through several pathways. The association of this ratio with clinical and pathological characteristics may explain the poor efficacy of immunotherapy in patients with esophageal cancer. These findings may help us find new targets for immunotherapy by exploring the immune microenvironment of esophageal squamous cell carcinoma.

Single-cell RNA sequencing reveals aberrant sphingolipid metabolism in non-small cell lung cancer impacts tumor-associated macrophages and stimulates angiogenesis via macrophage inhibitory factor signaling.

BACKGROUND: Sphingolipids not only serve as structural components for maintaining cell membrane fluidity but also function as bioactive molecules involved in cell signaling and the regulation of various biological processes. Their pivotal role in cancer cell development, encompassing cancer cell proliferation, migration, angiogenesis, and metastasis, has been a focal point for decades. However, the contribution of sphingolipids to the complexity of tumor microenvironment promoting cancer progression has been rarely investigated. METHODS: Through the integration of publicly available bulk RNA-seq and single-cell RNA-seq data, we conducted a comprehensive analysis to compare the transcriptomic features between tumors and adjacent normal tissues, thus elucidating the intricacies of the tumor microenvironment (TME). RESULTS: Disparities in sphingolipid metabolism (SLM)-associated genes were observed between normal and cancerous tissues, with the TME characterized by the enrichment of sphingolipid signaling in macrophages. Cellular interaction analysis revealed robust communication between macrophages and cancer cells exhibiting low SLM, identifying the crucial ligand-receptor pair, macrophage inhibitory factor (MIF)-CD74. Pseudo-time analysis unveiled the involvement of SLM in modulating macrophage polarization towards either M1 or M2 phenotypes. Categorizing macrophages into six subclusters based on gene expression patterns and function, the SPP1+ cluster, RGS1+ cluster, and CXCL10+ cluster were likely implicated in sphingolipid-induced M2 macrophage polarization. Additionally, the CXCL10+, AGER+, and FABP4+ clusters were likely to be involved in angiogenesis through their interaction with endothelial cells. CONCLUSION: Based on multiple scRNA-seq datasets, we propose that a MIF-targeted strategy could potentially impede the polarization from M1 to M2 and impair tumor angiogenesis in low-SLM non-small cell lung cancer (NSCLC), demonstrating its potent antitumor efficacy.

Protein expression of nucleolar protein 12 in the retina and its implication in protection of retina from UV irradiation damage.

Nucleolar protein 12 (NOL12), one of the nucleolar proteins which are primarily expressed in the nucleolus and play key roles in RNA metabolism, cell proliferation, cell cycle, and cell survival, is widely expressed in various species and multiple organs. Although it has been reported that the mRNA of Drosophila NOL12 homolog viriato is expressed in the eyes of Drosophila, the protein expression of NOL12 in mammalian eyes remains to be elucidated. In this study, we showed through immunohistochemistry that NOL12 was present in the rat retina, with predominant distribution in the cytoplasm of the retinal neuronal cells. In the human retinoblastoma cell line WERI-Rb1, we found that altering NOL12 expression led to a change in WERI-Rb1 cell viability. Knocking down NOL12 expression decreased cell viability. In contrast, overexpressing NOL12 increased cell viability. Furthermore, increasing NOL12 expression inhibited ultraviolet (UV)-induced apoptosis. These findings demonstrated that NOL12 may play an important protective role in retinal cells. In the WERI-Rb1 cells exposed to UV irradiation, we detected that NOL12 was degraded, but this degradation could be attenuated by a pan-Caspase inhibitor. Notably, the inhibitory effect of NOL12 against UV-induced apoptosis could be restrained by increasing the expression of ATR serine/threonine kinase (ATR), a kinase that, when activated by severe DNA damage, can result in apoptosis. We also found that upregulating NOL12 inhibited the activation of ATR caused by UV irradiation. Additionally, inhibiting ATR activity reduced apoptosis resulting from both silencing NOL12 expression and UV exposure. Thus, NOL12 may protect against UV irradiation-induced retinal damage by inhibiting ATR activity.

Multifunctional composite films based on polyvinyl alcohol, quaternary ammonium salt modified cellulose nanofibers and tannic acid-iron ion coordination complexes for food packaging.

The development of sustainable and well-performing food packaging materials takes on critical significance, whereas it is still challenging. To overcome the shortcomings of polyvinyl alcohol (PVA) as a degradable packaging material, in this work, hydrophobic quaternary ammonium salt (QAS) modified cellulose nanofibers (CNF) and tannic acid­iron ion coordination complexes (TA-Fe) were adopted for the preparation of functional PVA films. The modified CNF (CNF-QAS) not only improved the mechanical properties and water resistance of PVA, but also endowed it with antibacterial ability. In addition, the synergistic antibacterial capability with CNF-QAS was achieved using TA-Fe with photothermal therapy. As a result, the modulus, elongation at break, tensile strength, and water contact angle of the prepared PVA films were examined as 88 MPa, 200 %, 11.7 MPa, and 94.8°, respectively. Furthermore, with the assistance of CNF-QAS and TA-Fe, the films inhibited the growth of E. coli and S. aureus by 99.8 % and 99.7 %, respectively, and they exhibited high cell viability of 90.5 % for L929 fibroblasts. Based on the above encouraging properties, the functional PVA films could significantly extend the shelf life of oranges for over two weeks, proving the excellent application prospects in the food packaging field.

MicroRNA-29a-3p Prevents Drug-Induced Acute Liver Failure through Inflammation-Related Pyroptosis Inhibition.

OBJECTIVE: Little is known about the role of microRNA-29a-3p (miR-29a-3p) in inflammation-related pyroptosis, especially in drug-induced acute liver failure (DIALF). This study aimed to identify the relationship between miR-29a-3p and inflammation-related pyroptosis in DIALF and confirm its underlying mechanisms. METHODS: Thioacetamide (TAA)- and acetaminophen (APAP)-induced ALF mouse models were established, and human samples were collected. The expression levels of miR-29a-3p and inflammation and pyroptosis markers were measured by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, or immunochemical staining in miR-29a-3p knock-in transgenic mouse (MIR29A(KI/KI)) DIALF models. In addition, RNA sequencing was conducted to explore the mechanisms. RESULTS: MiR-29a-3p levels were decreased in TAA- and APAP-induced DIALF models. MiR-29a-3p prevented DIALF caused by TAA and APAP. RNA sequencing and further experiments showed that the protective effect of miR-29a-3p on DIALF was mainly achieved through inhibition of inflammation-related pyroptosis, and the inhibition was dependent on activation of the PI3K/AKT pathway. In addition, miR-29a-3p levels were reduced, and pyroptosis was activated in both peripheral blood mononuclear cells and liver tissues of DIALF patients. CONCLUSION: The study supports the idea that miR-29a-3p inhibits pyroptosis by activating the PI3K/AKT pathway to prevent DIALF. MiR-29a-3p may be a promising therapeutic target for DIALF.

Primary Alveolar Soft-Part Sarcoma of the Lung: A Case Report.

Alveolar soft-part sarcoma is a rare type of soft tissue malignant tumor. Although the tumor can occur in many parts of the body, primary alveolar soft-part sarcoma of the lung is extremely rare. According to previous literature, only 3 cases of primary alveolar soft-part sarcoma of the lung were reported, and no comprehensive analysis was conducted on these cases. Here, we describe another case of alveolar soft-part sarcoma in the lung, where the negative results of immunohistochemical staining cause extreme difficulty in distinguishing this lesion from other tumors. A 30-year-old Chinese male presented with chest pain and dyspnea. Computed tomography revealed a pulmonary mass, and biopsy results showed vacuolar tumor cells with abundant eosinophilic cytoplasm. A number of immunohistochemical markers were negative, but the tumor cells were positive for TFE3 and ASPSCR1::TFE3 fusion gene. No other tumor masses were found in the patient after whole-body scanning. The final diagnosis was primary alveolar soft-part sarcoma of the lung. Pathologists should consider the possibility of alveolar soft-part sarcoma in lung tumors with typical "organ like" or "acinar like" cell nests, where the tumor cells are large, vacuolated, and the nucleolus is obvious. After excluding metastasis from other sites, TFE3 immunohistochemical staining and ASPSCR1::TFE3 fusion gene detection are recommended for the diagnosis of primary alveolar soft-part sarcoma.

Calcium Channel α2δ1 is Essential for Pancreatic Tumor-Initiating Cells through Sequential Phosphorylation of PKM2.

BACKGROUND & AIMS: Tumor-initiating cells (TICs) drive pancreatic cancer tumorigenesis, therapeutic resistance, and metastasis. However, TICs are highly plastic and heterogenous, which impede the robust identification and targeted therapy of such a population. The aim of this study is to identify the surface marker and therapeutic target for pancreatic TICs. METHODS: We isolated voltage-gated calcium channel α2δ1 subunit (isoform 5)-positive subpopulation from pancreatic cancer cell lines and freshly resected primary tissues by fluorescence-activated cell sorting and evaluated their TIC properties by spheroid formation and tumorigenic assays. Coimmunoprecipitation was used to identify the direct substrate of CaMKⅡδ. RESULTS: We demonstrate that the voltage-gated calcium channel α2δ1 subunit (isoform 5) marks a subpopulation of pancreatic TICs with the highest TIC frequency among the known pancreatic TIC markers tested. Furthermore, α2δ1 is functionally sufficient and indispensable to promote TIC properties by mediating Ca2+ influx, which activates CaMKⅡδ to directly phosphorylate PKM2 at T454 that results in subsequent phosphorylation at Y105 to translocate into nucleus, enhancing the stem-like properties. Interestingly, blocking α2δ1 with its specific antibody has remarkably therapeutic effects on pancreatic cancer xenografts by reducing TICs. CONCLUSIONS: α2δ1 promotes pancreatic TIC properties through sequential phosphorylation of PKM2 mediated by CaMKⅡδ, and targeting α2δ1 provides a therapeutic strategy against TICs for pancreatic cancer.

Characteristics and mechanisms of resorption in lumbar disc herniation.

Lumbar disc herniation (LDH) can be spontaneously absorbed without surgical treatment. However, the pathogenesis and physiological indications for predicting protrusion reabsorption are still unclear, which prevents clinicians from preferentially choosing conservative treatment options for LDH patients with reabsorption effects. The purpose of this review was to summarize previous reports on LDH reabsorption and to discuss the clinical and imaging features that favor natural absorption. We highlighted the biological mechanisms involved in the phenomenon of LDH reabsorption, including macrophage infiltration, inflammatory responses, matrix remodeling, and neovascularization. In addition, we summarized and discussed potential clinical treatments for promoting reabsorption. Current evidence suggests that macrophage regulation of inflammatory mediators, matrix metalloproteinases, and specific cytokines in intervertebral disc is essential for the spontaneous reabsorption of LDH.

The tumour neovasculature-homing dimeric peptide GX1 demonstrates antiangiogenic activity in the retinal neovasculature.

Identification of molecules specific to the retinal neovasculature will promote antiangiogenic therapy with enhanced targeting ability. The specificity of phage-displayed peptide GX1 (a cyclic 7-mer peptide motif CGNSNPKSC) to gastric cancer neovasculature has been extensively confirmed both in vitro and in vivo. To investigate the potential application of GX1 in antiangiogenic therapy targeting retinal angiogenesis-related diseases, we performed immunohistochemistry and immunofluorescence analyses. GX1 demonstrated positive staining in the retinal neovasculature in an oxygen-induced mouse model of retinopathy (OIR) as well as in rat retinal microvasculature endothelial cells (RMECs), confirming the major role of the GX1 receptor during retinal angiogenesis. Dimeric GX1 was synthesized to increase the binding affinity to the GX1 receptor, and the antiangiogenic effects were examined in RMECs in vitro and the retinal neovasculature in the OIR in vivo. Cell proliferation was evaluated using a Cell Counting Kit-8 (CCK-8) assay, revealing that compared with the GX1 monomer, dimeric GX1 significantly inhibited RMEC proliferation (P < 0.05). This finding may be attributed to the enhanced (P < 0.05) apoptosis induced by dimeric GX1 in RMECs based on results obtained from TUNEL, flow cytometric and cell cycle analyses. In RMECs, in vitro cell migration and tube formation were significantly inhibited following exposure to dimeric GX1. Intravitreal administration of dimeric GX1 resulted in a greater reduction in the retinal neovascularization in vivo than administration of the GX1 monomer (P < 0.05). In conclusion, dimeric GX1 showed greater inhibition of angiogenesis than monomeric GX1 and could be a promising agent for antiangiogenic therapy in retinal angiogenesis-related diseases.

All-trans retinoic acid (ATRA) inhibits insufficient radiofrequency ablation (IRFA)-induced enrichment of tumor-initiating cells in hepatocellular carcinoma.

OBJECTIVE: Local recurrence of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) treatment remains a serious problem. Tumor-initiating cells (TICs) are thought to be responsible for tumor relapse. Here, we investigated the effect of the TIC differentiation inducer, all-trans retinoic acid (ATRA), on RFA and explored the potential molecular mechanisms. METHODS: The proportions of CD133+ and epithelial cell adhesion molecule (EpCAM)+ TICs in recurrent HCC after RFA and primary HCC were first determined in clinic. Then, the effect of heat intervention or insufficient RFA (IRFA) on the malignant potential of HCC cells, including cell migration, sphere formation ability, tumor growth, the proportion of CD133+ and EpCAM+ TICs and expression of stem cell-related genes, was evaluated in vitro andin vivo. Finally, the effect of ATRA on the tumor growth and the proportion of TICs was evaluated. RESULTS: In clinical data, a higher proportion of CD133+ and EpCAM+ TICs was found in recurrent tumors than in primary tumors. In vitro heat intervention promoted the cell migration and sphere formation ability. Additionally, it increased the proportion of CD133+ and EpCAM+ TICs and the expression of stem cell-related genes. In addition, after IRFA the residual tumors in xenografts grew faster and had more TICs than untreated tumors. ATRA remarkably inhibited residual tumor growth after IRFA by elimination of TICs though the PI3K/AKT pathway. Combination treatment with ATRA resulted in longer survival outcomes in mouse xenografts than RFA alone. CONCLUSIONS: ATRA, as a TIC differentiation inducer, could help to improve the effect of RFA treatment, which was partially attributed to its effect against TICs. The data indicated its potential as an alternative drug in the development of better therapeutic strategies for use in combination with RFA.

Specific Inhibitor of Matrix Metalloproteinase Decreases Tumor Invasiveness After Radiofrequency Ablation in Liver Tumor Animal Model.

OBJECTIVE: To determine whether the specific inhibitor of matrix metalloproteinase (MMP)-batimastat (BB-94)-could decrease the progression of liver tumor after radiofrequency ablation (RFA) and achieve better therapeutic efficacy in an animal model. METHODS: In vitro experiments, the proliferation of H22 liver tumor cells was detected by CCK 8 assay and cell migration was detected by Transwell method. In vivo experiments, H22 murine liver tumors were used. First, 32 mice with one tumor were randomized into four groups (n = 8 each group): control (PBS only), RFA alone (65°C, 5 min), BB-94 (30 mg/kg), RFA+BB-94. The growth rate of the residual tumor and the end point survival were calculated and the pathologic changes were evaluated. Secondly, a total of 48 tumors in 24 animals (paired tumors) were randomized into three groups (n = 8 each group): control, RFA alone, RFA+BB-94. Each mouse was implanted with two tumors subcutaneously, one tumor was treated by RFA and the other was evaluated for distant metastasis after applying BB-94. RESULTS: In vitro, the proliferation assay demonstrated higher proliferation ability after heat treatment (0.82 ± 0.07 vs 1.27 ± 0.08, P = 0.008), and it could be inhibited by BB-94 (1.27 ± 0.08 vs 0.67 ± 0.06, P = 0.001). In the cell migration assay, the H22 cells demonstrated enhanced tumor invasiveness in the heat group than the control group (33.7 ± 2.1 vs 19.7 ± 4.9, P = 0.011). And it could be significantly suppressed after BB-94 incubation (33.7 ± 2.1 vs 23.0 ± 4.6, P = 0.009). With one tumor animal, the growth rate of the residual tumor in the BB-94+RFA group was slower than that in the RFA alone group (P = 0.003). And combination of BB-94 could significantly prolong the survival of the mice (40.3 ± 1.4d vs 47.1 ± 1.3d, P = 0.002). The expression of CD31 and VEGF at the coagulation margin were decreased after combined with BB-94. With two tumors animal, the growth of metastasis tumor in the BB-94+RFA group was slower than that in the RFA group (P < 0.001). CONCLUSION: BB-94 combined with RFA reduced the invasiveness of the liver tumor and improved the end-point survival. Our data suggested that targeting the MMP process with the specific inhibition could help to increase overall ablation efficacy.

The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma.

The diagnosis of lymph node metastasis (LNM) by liquid biopsy is a novel concept prompted by the necessity to develop a more convenient and accurate method to guide the clinical management of early LNM in papillary thyroid carcinoma (PTC). However, the sensitivity and specificity of many biomarkers are not high enough. We aimed to detect circRNAs from peripheral circulation that may be better associated with the prognosis of LNM in PTC. First, Galectin-3 (Gal3) in blood was determined to be highly expressed in LNM patients. Second, based on a bioinformatics analysis and miRNA sequencing analysis from 2 paired primary and LNM tumors, miR-873 was identified to directly target Gal3, which was significantly associated with clinical parameters including LNM. Third, from additional circRNA sequencing, circRNA-UMAD1 was selected as a specific sponge for miR-873 and was correlated with Gal3 levels in peripheral circulation. Fourth, circRNA-UMAD1 and Gal3 were identified to have stronger co-biomarker potential with relatively high expression in the serum of LNM patients compared with primary tumor patients, as demonstrated by the RNA expression levels in the serum of 50 PTC patients with or without LNM by quantitative real-time PCR. Overall, the combination of circRNA-UMAD1 and Gal3 is a useful and effective co-biomarker for the prognosis of LNM in PTC patients. This new molecular typing method for LNM in PTC is more precise.

Palindromic probe-mediated strand displacement amplification for highly sensitive and selective microRNA imaging.

MicroRNAs (miRNAs) are involved in a variety of biological processes, and the accurate detection of miRNAs is of great importance for early diagnosis of various cancers. Herein, we have developed a highly sensitive method for the intracellular imaging of miRNAs based on a palindromic probe-induced strand displacement amplification (pSDA). The sensing element is a partly complementary hybrid consisting of two DNA components: one fluorescent dye-labeled signaling probe containing a palindromic sequence and loop-based target recognition site and one quencher moiety-attached locking probe. In the presence of target miRNA, the target species can hybridize with the loop site and release the terminal palindromic fragment, initiating the pSDA reaction. Thus, a considerable amount of fluorescent moieties are spatially separated from the quenchers, generating a dramatically enhanced fluorescence signal. As a result, the target miRNAs can be quantified down to 25 pM with the linear response range over four orders of magnitude. The detection specificity is high enough to eliminate the interference from nontarget miRNAs and other biospecies co-existing in samples, and thus the diseased cells are easily distinguished from healthy cells. Strikingly, the pSDA-based system possesses the desirable capability to discriminate tumor cells from healthy cells, indicating a promising diagnostic tool for the detection of cancers and other diseases in early stage.

Pharmacogenetic impact of UGT1A1 polymorphisms on pulmonary neuroendocrine tumours treated with metronomic irinotecan-based chemotherapy in Chinese populations.

OBJECTIVES: To evaluate the effects of UGT1A1*6 and UGT1A1*28 polymorphisms on the safety and efficacy of metronomic irinotecan-based chemotherapy (IBC) in Chinese patients with pulmonary neuroendocrine tumours (PNTs). METHODS: Sixty-eight PNT patients who received metronomic IBC were observed. The quantitative fluorescent polymerase chain reaction was used to detect UGT1A1*6 and UGT1A1*28 polymorphisms. The follow-up data were collected to investigate the relationship between different genotypes and adverse drug reactions. The clinical outcomes of metronomic IBC were also evaluated. KEY FINDINGS: In the genotype-toxicity association analysis, patients with homozygous UGT1A1*6 had the highest incidence of grade 3-4 diarrhoea (P = 0.010). Compared to other groups, patients with the haplotype of UGT1A1*28 showed a trend towards an increased incidence of grade 4 neutropaenia (P = 0.047). A higher incidence of grade 3-4 leucopaenia was found in groups with UGT1A1*1/*28 (P = 0.023) and UGT1A1*28/*28 (P = 0.022). Grade 1 total bilirubin elevation was associated with the homozygous UGT1A1*6 mutation (P = 0.027) or any UGT1A1*6 variants (P = 0.047). However, neither UGTA1A*28 nor UGT1A1*6 showed any significant association with tumour response or clinical outcomes. CONCLUSIONS: The impact of UGT1A1 polymorphisms varies in different irinotecan-based chemotherapies. UGT1A1*6 and UGTA1A*28 were useful for the prediction of irinotecan-related severe toxicity in Chinese PNT patients treated with metronomic IBC.

MicroRNA-200b is downregulated and suppresses metastasis by targeting LAMA4 in renal cell carcinoma.

BACKGROUND: Metastasis is the primary cause of tumor death in renal cell carcinoma (RCC). Improved diagnostic markers of metastasis are critically needed for RCC. MicoRNAs are demonstrated to be stable and significant biomarkers for several malignancies. In this study, we aimed to explore the metastasis related microRNAs and its mechanism in RCC. METHODS: The relationship between microRNAs expression and prognosis and metastasis of RCC patients were explored by data mining through expression profiles from The Cancer Genome Atlas (TCGA). A total of 80 RCC tissues and adjacent normal kidney tissues were obtained from Department of Urology, Peking University First Hospital. Expression of microRNA-200b (miR-200b) in RCC tissues and cell lines were determined by bioinformatic data mining and quantitative real-time PCR (qRT-PCR). The effects of miR-200b on cell proliferation, migration and invasion were determined by cell counting kit-8 and colony formation assay, wound healing assay and Boyden chamber assay. Mouse cell-derived xenograft and patient-derived xenograft model were also performed to evaluate the effects of miR-200b on tumor growth and metastasis in vivo. The molecular mechanism of miR-200b function was investigated using bioinformatic target predication and high-throughput cDNA sequencing (RNA-seq) and validated by luciferase reporter assay, qRT-PCR, Western blot and immunostaining in vitro and in vivo. FINDINGS: Our findings indicates that miR-200b is frequently downregulated and have potential utility as a biomarker of metastasis and prognosis in RCC. Interestingly, ectopic expression of miR-200b in the Caki-1 and OSRC-2 cell lines suppresses cell migration and invasion in vitro as well as tumor metastases in vivo. However, miR-200b has no effect on cell proliferation in vitro and tumor growth in vivo. In addition, bioinformatics target predication and RNA-seq results reveals that Laminin subunit alpha 4 (LAMA4) is one target of miR-200b and significantly inhibited by miR-200b in vitro and in vivo. INTERPRETATION: These results demonstrate a previously undescribed role of miR-200b as a suppressor of tumor metastasis in RCC by directly destabilizing LAMA4 mRNA.

Lysyl oxidase assists tumor­initiating cells to enhance angiogenesis in hepatocellular carcinoma.

A highly tumorigenic and malignant sub­population of HCC containing tumor­initiating cells (TICs) are defined by high self­renewal and sphere formation ability. Lysyl oxidase (LOX) regulates various factors involved in extracellular matrix (ECM) maintenance, migration and angiogenesis. Certain reports have demonstrated the role of LOX in ECM crosslinking, however, the cancer­promoting effects of LOX in HCC remain unclear, and whether LOX has a role in the regulation of angiogenesis in HCC TICs has not been elucidated. In the current study, RNA sequencing using next­generation sequencing technology and bioinformatics analyses revealed that LOX gene expression was significantly upregulated in cell spheres. Sphere cells may form tumors with more vascular enrichment compared with tumors produced from adherent cells, as observed in a mouse xenograft model. LOX expression is correlated with increased vascular endothelial growth factor (VEGF) and platelet­derived growth factor, as demonstrated by analyses of The Cancer Genome Atlas and Gene Expression Omnibus databases. Conditioned media obtained from LOX­overexpressing tumor cells stimulated angiogenesis via secreted VEGF and enhanced the tube formation capacity of endothelial cells. Furthermore, these functional behaviors were blocked by the LOX inhibitor ß­aminopropionitrile. These findings provide novel mechanistic insight into the pivotal role of LOX in the regulation of TICs in HCC. Combination of LOX inhibitor with sorafenib is a potentially advantageous strategy for HCC therapy.

Angiogenesis inhibition and cell cycle arrest induced by treatment with Pseudolarix acid B alone or combined with 5-fluorouracil.

Angiogenesis inhibitors combined with chemotherapeutic drugs have significant efficacy in the treatment of a variety of cancers. Pseudolarix acid B (PAB) is a traditional pregnancy-terminating agent, which has previously been shown to reduce tumor growth and angiogenesis. In this study, we used the high content screening assay to examine the effects of PAB on human umbilical vein endothelial cells (HUVECs). Two hepatocarcinoma 22-transplanted mouse models were used to determine PAB efficacy in combination with 5-fluorouracil (5-Fu). Our results suggested that PAB (0.156-1.250 µM) inhibited HUVECs motility in a concentration-dependent manner without obvious cytotoxicity in vitro. In vivo, PAB (25 mg/kg/day) promoted the anti-tumor efficacy of 5-Fu (5 mg/kg/2 days) in combination therapy, resulting in significantly higher tumor inhibition rates, lower microvessel density values, and prolonged survival times. It was also demonstrated that PAB acted by blocking the cell cycle at both the G(1)/S boundary and M phase, down-regulation of vascular endothelial growth factor, hypoxia-inducible factor 1α and cyclin E expression, and up-regulation of cdc2 expression. These observations provide the first evidence that PAB in combination with 5-Fu may be useful in cancer treatment.

Pseudolaric acid B inhibits inducible cyclooxygenase-2 expression via downregulation of the NF-κB pathway in HT-29 cells.

PURPOSE: Pseudolaric acid B (PAB) is a diterpene acid isolated from the root and trunk bark of Pseudolaric kaempferi Gordon. Previous work has found that PAB has anti-inflammatory and anti-tumor effects in xenograft models of human hepatocellular carcinoma. The aim of this study is to evaluate the correlation between anti-cancer and anti-inflammatory effects of PAB and its molecular mechanisms on HT-29 cells. METHODS: Production of prostaglandin E2 (PGE2) in HT-29 cells was evaluated by ELISA. mRNA of cyclooxygenase-2 (COX-2) was analyzed by RT-PCR assay. High-content screening (HCS) method was adopted to detect the cytokine mixture (CM)-induced transcription activity of NF-κB and STAT3. Western blotting was used to evaluate the protein expression levels of inflammatory mediators induced by CM. After treatment with PAB in various concentrations, the inhibition rate of cell proliferation was measured with sulforhodamine B assays. For the in vivo studies, tumor-bearing models xenografted with HT-29 cells were developed in nude mice, and following oral administration with PAB, tumor inhibition rate was calculated. RESULTS: PAB inhibited the PGE2 production in HT-29 cells significantly (P < 0.05) with similar results detected at the COX-2 mRNA level. Furthermore, PAB suppressed the COX-2 protein expression and significant nuclear translocation of NF-κB and STAT3 induced by CM, which correlated with a concomitant degradation of I-κB and a decrease in constitutive STAT3 phosphorylation (P < 0.05). Moreover, various concentrations of PAB inhibited the proliferation of HT-29 cells in a dose- and time-dependent manner. In vivo, after treatment with PAB for 17 days, the tumor weight of the 50 and 100 mg/kg treated groups was 0.62 ± 0.15 and 0.54 ± 0.06 g, respectively. When compared to the control group (0.82 ± 0.16 g), the inhibition rate of tumor weight was 24.2% at 50 mg/kg (P < 0.05) and 34.7% at 100 mg/kg (P < 0.001). CONCLUSIONS: PAB shows potential anti-cancer activity in HT-29 cells, and its molecular mechanisms are related to the anti-inflammatory action.

Transcriptome analysis of female and male Xiphophorus maculatus Jp 163 A.

BACKGROUND: Xiphophorus models are important for melanoma, sex determination and differentiation, ovoviviparity and evolution. To gain a global view of the molecular mechanism(s) whereby gene expression may influence sexual dimorphism in Xiphophorus and to develop a database for future studies, we performed a large-scale transcriptome study. METHODOLOGY/PRINCIPAL FINDINGS: The 454-FLX massively parallel DNA sequencing platform was employed to obtain 742,771 and 721,543 reads from 2 normalized cDNA libraries generated from whole adult female and male X. maculatus Jp 163 A, respectively. The reads assembled into 45,538 contigs (here, a "contig" is a set of contiguous sequences), of which, 11,918 shared homology to existing protein sequences. These numbers estimate that the contigs may cover 53% of the total number of Xiphophorus transcriptome. Putative translations were obtained for 11,918 cDNA contigs, of which, 3,049 amino acid sequences contain Pfam domains and 11,064 contigs encode secretory proteins. A total of 3,898 contigs were associated with 2,781 InterPro (IPR) entries and 5,411 contigs with 132 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. There were 10,446 contigs annotated with 69,778 gene ontology (GO) terms and the three corresponding organizing principles. Fifty-four potential sex differentially expressed genes have been identified from these contigs. Eight and nine of these contigs were confirmed by real-time PCR as female and male predominantly expressed genes respectively. Based on annotation results, 34 contigs were predicted to be differentially expressed in male and female and 17 of them were also confirmed by real-time PCR. CONCLUSIONS/SIGNIFICANCE: This is the first report of an annotated overview of the transcriptome of X. maculatus and identification of sex differentially expressed genes. These data will be of interest to researchers using the Xiphophorus model. This work also provides an archive for future studies in molecular mechanisms of sexual dimorphism and evolution, and can be used in comparative studies of other fish.

Multipathogen infections in hospitalized children with acute respiratory infections.

BACKGROUND: To explore the epidemiologic and clinical features of, and interactions among, multipathogen infections in hospitalized children with acute respiratory tract infection (ARTI). A prospective study of children admitted with ARTI was conducted. Peripheral blood samples were analyzed by indirect immunofluorescence to detect respiratory agents including respiratory syncytial virus; adenovirus; influenza virus (Flu) types A and B; parainfluenza virus (PIV) types 1, 2, and 3; chlamydia pneumonia; and mycoplasma pneumonia. A medical history of each child was taken. RESULTS: Respiratory agents were detected in 164 (51.9%) of 316 children with ARTI. A single agent was identified in 50 (15.8%) children, and multiple agents in 114 (36.1%). Flu A was the most frequently detected agent, followed by Flu B. Coinfection occurred predominantly in August and was more frequent in children between 3 and 6 years of age. A significantly higher proportion of Flu A, Flu B, and PIV 1 was detected in samples with two or more pathogens per sample than in samples with a single pathogen. CONCLUSION: Our study suggests that there is a high occurrence of multipathogen infections in children admitted with ARTI and that coinfection is associated with certain pathogens.