LncRNA HAGLROS contribute to papillary thyroid cancer progression by modulating miR-206/HMGA2 expression.

OBJECTIVE: Papillary thyroid cancer (PTC) is one of the most serious diseases of the endocrine system. In view of the limited therapeutic effects of current medical methods, this study starts from the molecular level and looks for potential treatments. The interaction between HAGLROS/miR-206/HMGA2 was studied using multi-omics methods, which provided new ideas and methods for future treatments. METHOD: Microarray analysis and R language were used for differential analysis to screening experimental targets of lncRNA, miRNA, and mRNA. qRT-PCR was used to detect RNA expression in tissues and cells. Double luciferase reporter assays analyzed and validated binding relationships between different RNAs. Colony formation, flow cytometry, and transwell assays were used to measure the effect of them on cell proliferation, apoptosis, and migration. RESULT: Microarray analysis identified lncRNAs, miRNAs, and mRNAs differentially expressed in PTC and normal cells, and selected lncRNA HAGLROS, miR-206, and mRNA HMGA2 as study subjects. LncRNA HAGLROS and mRNA HMGA2 were highly expressed in PTC cells while miR-206 was lowly expressed in PTC cells. LncRNA HAGLROS/HMGA2 can inhibit apoptosis of PTC cells, promote proliferation and migration, and miR-206 promotes the above process. HAGLROS and HMGA2 were negatively correlated with miR-206. shHAGLROS promoted miR-206 expression, inhibited HMGA2 expression and repressed PTC tumor growth in mice. CONCLUSIONS: HAGLROS promotes the growth of PTC by competitively binding to miR-206 to promote HMGA2 expression.

The STING-IRF3 Signaling Pathway, Mediated by Endoplasmic Reticulum Stress, Contributes to Impaired Myocardial Autophagic Flux After Ischemia/Reperfusion.

ABSTRACT: This study aimed to determine whether endoplasmic reticulum (ER) stress is involved in impaired autophagy after myocardial ischemia/reperfusion (M-I/R) and elucidate the underlying mechanisms. The expression levels of stimulator of interferon gene (STING) and interferon regulatory transcription factor 3 (IRF3) phosphorylation increased in M-I/R heart tissues and hypoxia-treated/reoxygenation-treated H9c2 cells. The ER stress inhibitor 4-phenylbutyric acid (4-PBA) significantly suppressed the stimulation of STING-IRF3 transcription and alleviated cardiac dysfunction caused by M-I/R injury. In addition, 4-PBA reversed ischemia-induced/reperfusion-induced autophagic flux dysfunction, as demonstrated by a decrease in p 62 and LC3 levels. Similarly, the protective effect of STING deficiency on myocardial cell damage was achieved by the recovery of autophagic flux. Conversely, the protective effect of 4-PBA against hypoxia/reoxygenation injury in cardiomyocytes was offset by STING overexpression, wherein the activated STING-IRF3 pathway promoted the expression of Rubicon (a negatively-regulated autophagic molecule) by binding to the Rubicon promoter. Rubicon ablation effectively counteracts the adverse effects of STING overexpression in cardiomyocytes. The data showed that STING-IRF3 signaling of ER stress receptors is particularly important in the progression of physiological M-I/R caused by the inhibition of autophagic flow in vivo and in vitro.

Circular RNA eukaryotic translation initiation factor 6 facilitates TPC-1 cell proliferation and invasion through the microRNA-138-5p/lipase H axis.

Both circular RNA eukaryotic translation initiation factor 6 (circEIF6) and microRNA (miR)-138-5p participate in thyroid cancer (TC) progression. Nevertheless, the relationship between them remains under-explored. Hence, this research ascertained the mechanism of circEIF6 in TC via miR-138-5p. After TC tissues and cells were harvested, circEIF6, miR-138-5p, and lipase H (LIPH) levels were assessed. The binding relationships among circEIF6, miR-138-5p, and LIPH were analyzed. The impacts of circEIF6, miR-138-5p, and LIPH on the invasive and proliferative abilities of TPC-1 cells were examined by Transwell and EdU assays. Tumor xenograft in nude mice was established for in vivo validation of the impact of circEIF6. CircEIF6 expression was high in TC cells and tissues. Additionally, miR-138-5p was poor and LIPH level was high in TC tissues. Mechanistically, circEIF6 competitively bound to miR-138-5p to elevate LIPH via a competitive endogenous RNA mechanism. Silencing of circEIF6 reduced TPC-1 cell proliferative and invasive properties, which was annulled by further inhibiting miR-138-5p or overexpressing LIPH. Likewise, circEIF6 silencing repressed the growth of transplanted tumors, augmented miR-138-5p expression, and diminished LIPH expression in nude mice. Conclusively, circEIF6 silencing reduced LIPH level by competitive binding to miR-138-5p, thus subduing the proliferation and invasion of TPC-1 cells.

Different Prevalence of Neuropathic Pain and Risk Factors in Patients with Knee Osteoarthritis at Stages of Outpatient, Awaiting and after Total Knee Arthroplasty.

OBJECTIVE: Neuropathic pain (NP) plays an important role in patients with knee osteoarthritis (KOA). However, the prevalence of NP at different treatment stages including outpatient, awaiting and after total knee arthroplasty (TKA) have not been compared. The understanding of this issue and identify risk factors can help physicians develop individualized strategies to manage the pain of KOA. Therefore, the aim of the study is to investigate the prevalence and risk factors of NP at different treatment stages of KOA. METHODS: Patients diagnosed as KOA between August 2016 and August 2020 were enrolled in this cross-sectional study and divided into three groups according to treatment stage, including outpatient stage, awaiting TKA stage (pre-TKA) and after TKA stage (post-TKA). A numeric rating scale (NRS) and PainDETECT questionnaire were used to evaluate nociceptive pain and NP. Patient demographics, radiological assessments using Kellgren-Lawrence (K-L) grade, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores were analyzed. Data analysis and statistics were processed using SPSS 20.0 and examined by ANOVA with/without Bonferroni correction or Kruskal-Wallis test. A chi-square test was used to determine cross-table data and calculate the odds ratio (OR) value. RESULTS: Of the 921 patients, the prevalence of possible and likely NP was 17.5% (56/320) and 2.5% (8/320) in the pre-TKA group compared with 3.4% (8/233) and 0.4% (1/233) in the outpatient group and 1.4% (5/368) and 0.5% (2/368) in the post-TKA group, respectively. In the pre-TKA group, higher NRS (NRS >3; OR = 10.65, 95% CI: 3.25-34.92, P < 0.001) and WOMAC pain score (score > 10; OR = 4.88, 95% CI: 2.38-10.01, P < 0.001) conferred an increased risk of unclear pain. Age, gender, BMI and K-L grade showed no significant differences among the unlikely, possible and likely NP groups. CONCLUSION: Prevalence of NP is different at stages of out-patient, awaiting and after TKA in patients with KOA. Patients awaiting TKA have the highest prevalence of NP compared with patients in outpatient and post-TKA groups. In the patients waiting for TKA, higher NRS (NRS >3) and WOMAC pain scores (score > 10) are risk factors of NP.

Ginsenoside Rb3 attenuates skin flap ischemia-reperfusion damage by inhibiting STING-IRF3 signaling.

We investigate the protective effect of ginsenoside Rb3 on skin flap microvasculature following ischemia-reperfusion (I/R) injury and its regulatory mechanism. We used a rat model of I/R injury with the right iliolumbar artery and oxidative stress model of human dermal microvascular endothelial cells. The effects of Rb3 on skin flap tissue and endothelial cell survival, STING-IRF3 pathway activation, and endothelial cell adhesion were measured. Following reperfusion, the survival rate of rat perforator flaps in the Rb3-treated group gradually increased with increasing Rb3 concentration. The treatment also reduced the amount of STING protein, phosphorylated IRF3, and P-selectin in skin flap tissue, with this change being most obvious in microvascular endothelial cells. In vitro, activated IRF3 binds to the P-selectin promoter and induces P-selectin expression. Our results suggest that Rb3 plays a role in reducing I/R flap damage through negatively regulating STING-IRF3 activation to limit leukocyte-endothelial cell adhesion.

MicroRNA-144-3p Represses the Growth and EMT of Thyroid Cancer via the E2F2/TNIK Axis in Cells and Male BALB/c Nude Mice.

CONTEXT: microRNA (miR/miRNA)-144-3p has been implicated in thyroid cancer (TC) progression with poorly identified mechanisms. Furthermore, E2F2 has been documented to assume a role in the development of various cancers. OBJECTIVE: This research sought to ascertain the role of miR-144-3p in growth and epithelial-mesenchymal transition (EMT) in TC in cells and male BALB/c nude mice. METHODS: In the obtained TC cells, miR-144-3p expression was detected by quantitative reverse transcription polymerase chain reaction, and E2F2 and TNIK expression by Western blot analysis. After gain- and loss-of-function assays, cell viability, clone formation, migration, and invasion were assessed by cell counting kit-8, clone formation, scratch, and Transwell assays. The expression of EMT-related proteins (Snail, Vimentin, N-cadherin, and E-cadherin) was tested by Western blot analysis. The targeting relationship between miR-144-3p and E2F2 was evaluated by dual-luciferase reporter and radioimmunoprecipitation assays, and the binding relationship between E2F2 and TNIK by dual-luciferase reporter and chromatin immunoprecipitation assays. TC cell growth in vivo was determined by subcutaneous tumorigenesis assays in nude mice. RESULTS: miR-144-3p was downregulated, whereas E2F2 and TNIK were upregulated in TC cells. Mechanistically, miR-144-3p inversely targeted E2F2, which increased TNIK expression by binding to TNIK promoter in TC cells. Overexpression of miR-144-3p reduced proliferation, migration, invasion, and EMT of FRO and KTC3 cells, which was nullified by overexpressing E2F2 or TNIK expression. Upregulation of miR-144-3p diminished FRO cell growth and EMT in nude mice, which was abrogated by overexpressing TNIK. CONCLUSION: miR-144-3p inhibits cell growth and EMT in TC through E2F2/TNIK axis inactivation in cells and male BALB/c nude mice.

Specneuzhenide Ameliorate Complete Freund Adjuvant Induced Arthritis in Rats: Involvement of NF-κB and HO-1/Nrf-2 Pathway.

Rheumatoid arthritis (RA) is growing autoimmune and inflammatory disease that occur due to self-destruction of immune response inducing joint deformity and bone erosion. During the arthritis inducing the swelling, pain and inflammation in around the joint and other body organs due to injury of tendons and ligaments. Specneuzhenide (SZ) already proved antioxidant and anti-inflammatory effect against the various diseases. In this experimental study, we scrutinized the anti-arthritic effect of SZ against the complete freund adjuvant (CFA) induced arthritic in rats. Subcutaneously injection of CFA was injected into the subplantar region of the left hind paw for induction the arthritis and rats were divided into different groups and rats received the oral administration of SZ (5, 10 and 15 mg/kg) for 28 days. The body weight, paw swelling arthritic score mRNA expression and biochemical parameters were determined at regular time interval. CFA induced arthritic rats treated with SZ significantly (p < 0.001) enhanced the body weight and decreased the paw swelling, arthritic index and organ (spleen and thymus) index, respectively. SZ treated rats significantly (p < 0.001) decreased the hepatic parameter such as SGPT, SGOT and ALP, anti-CII IgG levels (IgG1 and IgG2A) and inflammatory parameters (COX-2 and PGE2). SZ treated rats significantly (p < 0.001) suppressed the level of MDA and increased the level of GSH, SOD and CAT. SZ treated rats suppressed the inflammatory cytokines such as TNF-α, Il-1ß, IL-2, IL-6, IL-16, IL-17 and increased the level of IL-10, TGF-ß. SZ treated rats significantly (p < 0.001) suppressed the mRNA expression of Nrf2, HO-1 and NF-κB. On the basis of result, we can say that specneuzhenide protective effect against CFA induced arthritis in rats via alteration of HO-1/Nrf-2 pathway.

Long noncoding RNA ILF3-AS1 aggravates papillary thyroid carcinoma progression via regulating the miR-4306/PLAGL2 axis.

BACKGROUND: It have been proven that long non-coding RNAs (lncRNAs) serve as regulators in carcinogenesis. Interleukin enhancer binding factor 3 antisense RNA 1 (ILF3-AS1) has been illuminated as a prognostic factor in some cancers. Nevertheless, its expression pattern and possible functions in papillary thyroid carcinoma (PTC) have not been studied. METHODS: The expression of ILF3-AS1 was measured by RT-qPCR and ISH. Colony formation assay and EdU assay were used to probe cell proliferation. TUNEL assay was used for analysis of cell apoptosis. Immunofluorescence and western blot were conducted to evaluate the expression change of E-cadherin and N-cadherin. The RNA interaction was demonstrated by mechanism experiments, including pull down assay and dual luciferase reporter assay. RESULTS: ILF3-AS1 expression was evidently upregulated in PTC cell lines. ILF3-AS1 knockdown restrained the proliferation, migration and invasion of PTC cells. Mechanical investigation revealed that miR-4306 could interact with ILF3-AS1. PLAGL2 was a downstream target of miR-4306. The effects of ILF3-AS1 knockdown on the cellular processes were abrogated by miR-4306 downregulation or pleiomorphic adenoma gene-like 2 (PLAGL2) overexpression. CONCLUSION: ILF3-AS1 plays tumor-promoting role in PTC via targeting miR-4306/PLAGL2 axis.

Characterization of the inhomogeneity of Pt/CeO x /Pt resistive switching devices prepared by magnetron sputtering.

There are unrevealed factors that bring about the performance variations of resistive switching devices. In this work, Pt/CeO x /Pt devices prepared by magnetron sputtering showed rectification in their asymmetrical current-voltage (I-V) curves during voltage sweeps. X-ray photoelectron spectroscopy showed that the deposited CeO x film had an inhomogeneous composition, and more oxygen vacancies existed in CeO x near the top electrode. The asymmetrical resistance change of the Pt/CeO x /Pt devices can be explained by the presence of more charged oxygen vacancies in CeO x near the top electrode, along with the Schottky conduction mechanism. This work reveals that the compositional inhomogeneity is inevitable in the magnetron sputtering of oxide targets like CeO2 and can be an important source of device-to-device and cycle-to-cycle variations of memristors.

Induced membrane technique combined with antibiotic-loaded calcium sulfate-calcium phosphate composite as bone graft expander for the treatment of large infected bone defects: preliminary results of 12 cases.

BACKGROUND: Management of large infected bone defects is a major clinical and socioeconomic problem. The induced membrane technique has been widely used as a solution. However, it has apparent disadvantages such as limited autologous bone graft supply and lack of continuous infection control. Meanwhile, calcium sulfate/calcium phosphate composites have efficacious osteogenesis and antibiotic delivery capacity. For the first time, we analyzed the efficiency of calcium sulfate/calcium phosphate composites as a bone graft expander in the induced membrane technique to treat large infected bone defects. METHODS: We retrospectively analyzed the clinical data of 12 patients with large infected bone defects of 6.1-17.2 cm treated with the induced membrane technique from November 2016 to July 2019. In the second reconstruction stage, the bone defect was filled with a mixture of the autogenous iliac bone and vancomycin-impregnated calcium sulfate/calcium phosphate composites at a ratio of 3:1. We assessed the bony union by Samantha X-ray score and recorded infection recurrence and complications. Paley scale and SF-36 score were used to evaluate the function of adjacent joint and quality of life pre and postoperatively. Pearson's correlation coefficients were calculated for union time and other clinical scores. RESULTS: The mean follow-up was 69 weeks (ranging from 30 to 142) after the second stage of the operation. The mean Samantha X-ray score was 5.1 [3-6], preoperative and postoperative SF-36 scores showed that there were statistical differences in all the nine aspects, and the excellent rate of adjacent joint function was 75% (Paley). All cases were radiologically healed, and none of the 12 patients had infection recurrence or failure of fixation at the last follow-up. Two cases had delayed wound healing and were cured after dress changing. There was a significant correlation between union time and Samantha X-ray score (r =‒0.887; P=0.000), while there was no correlation between filling dose, size of the defect, and other outcomes. CONCLUSIONS: This study provided evidence supporting calcium sulfate/calcium phosphate composites as an effective and safe bone graft expander in the induced membrane technique to treat large infected bone defect. This technique may help decrease the use of autologous bone graft and enhance the anti-infection effect of the induced membrane technique.

Importance of Bi-O Bonds at the Cs2AgBiBr6 Double-Perovskite/Substrate Interface for Crystal Quality and Photoelectric Performance.

Interface interactions between perovskite materials and substrates are of great significance for the development of high-quality perovskite materials. Herein, we have successfully prepared Cs2AgBiBr6 double-perovskite films via a one-step spin-coating process and demonstrated a novel approach that modifies the surface of substrates with an ultrathin metal oxide (MOx) layer to promote the film quality and photoelectric performance. Characterization results strongly suggest that the improvement is attributed to the Bi-O interfacial interaction at substrate/perovskite interface. Benefiting from this interface interaction, the average grain size of Cs2AgBiBr6 films has remarkably risen up to ∼500 nm, which is nearly four times larger than the one directly deposited on a commercial fluorine-doped tin oxide substrate. Meanwhile, the pin hole surface area ratio has reduced from 2.61 to 0.60%. Furthermore, the corresponding photodetectors (PDs) have been fabricated and the performance has significantly improved owing to the enhanced Cs2AgBiBr6 film quality. The on-off ratio of the optimized PD has a boost of almost 10 times. In addition, the minimum detected irradiation has decreased from 9.7 × 10-8 to 1.9 × 10-9 W cm-2, as well as the maximum detectivity has increased from 3.3 × 1011 to 1.2 × 1013 Jones. These results suggest a feasible method for crystallization improvement of double-perovskite films and indicate promising promotion of photoelectric performance.

Comparative genomics revealed the gene evolution and functional divergence of magnesium transporter families in Saccharum.

BACKGROUND: Sugarcane served as the model plant for discovery of the C4 photosynthetic pathway. Magnesium is the central atom of chlorophyll, and thus is considered as a critical nutrient for plant development and photosynthesis. In plants, the magnesium transporter (MGT) family is composed of a number of membrane proteins, which play crucial roles in maintaining Mg homeostasis. However, to date there is no information available on the genomics of MGTs in sugarcane due to the complexity of the Saccharum genome. RESULTS: Here, we identified 10 MGTs from the Saccharum spontaneum genome. Phylogenetic analysis of MGTs suggested that the MGTs contained at least 5 last common ancestors before the origin of angiosperms. Gene structure analysis suggested that MGTs family of dicotyledon may be accompanied by intron loss and pseudoexon phenomena during evolution. The pairwise synonymous substitution rates corresponding to a divergence time ranged from 142.3 to 236.6 Mya, demonstrating that the MGTs are an ancient gene family in plants. Both the phylogeny and Ks analyses indicated that SsMGT1/SsMGT2 originated from the recent ρWGD, and SsMGT7/SsMGT8 originated from the recent σ WGD. These 4 recently duplicated genes were shown low expression levels and assumed to be functionally redundant. MGT6, MGT9 and MGT10 weredominant genes in the MGT family and werepredicted to be located inthe chloroplast. Of the 3 dominant MGTs, SsMGT6 expression levels were found to be induced in the light period, while SsMGT9 and SsMTG10 displayed high expression levels in the dark period. These results suggested that SsMGT6 may have a function complementary to SsMGT9 and SsMTG10 that follows thecircadian clock for MGT in the leaf tissues of S. spontaneum. MGT3, MGT7 and MGT10 had higher expression levels Insaccharum officinarum than in S. spontaneum, suggesting their functional divergence after the split of S. spontaneum and S. officinarum. CONCLUSIONS: This study of gene evolution and expression of MGTs in S. spontaneum provided basis for the comprehensive genomic study of the entire MGT genes family in Saccharum. The results are valuable for further functional analyses of MGT genes and utilization of the MGTs for Saccharum genetic improvement.

Lycopene Improves Insulin Sensitivity through Inhibition of STAT3/Srebp-1c-Mediated Lipid Accumulation and Inflammation in Mice fed a High-Fat Diet.

In the past few years, metabolic disorders, such as type 2 diabetes and metabolic syndrome, have reached global prevalence. Lycopene is one of the major carotenoids in tomatoes, watermelons, red grapefruits, and guava. In the current study, using high fat diet (HFD)-fed mice, we investigated the effect of Lycopene on insulin resistance. We showed that diet containing Lycopene significantly prevented HFD-induced increase of fasting blood glucose and insulin level, glucose and insulin intolerance, and decrease of hepatic glycogen content. We found that Lycopene notably prevented the increase of IL-1ß, TNFα and CRP levels in mice fed HFD. We showed that Lycopene improved the lipid profiles in HFD-fed mice, as evidenced by decrease of systemic and hepatic TC, TG and LDL, and increase of HDL. Lycopene suppressed the increase of the expression of Srebp-1c, FAS and ACC-1 in mice fed HFD. The administration of Lycopene notably prevented the expression and phosphorylation of STAT3 in livers of mice induced by HFD. The treatment of adenovirus carrying STAT3 significantly suppressed the decrease of Srebp-1c expression induced by Lycopene. Furthermore, enhancement of STAT3 signaling by adenovirus markedly blocked the reduction of fasting blood glucose and insulin level. In conclusion, in the current study, we found that Lycopene prevented STAT3 signaling and inhibited Srebp-1c and downstream gene expression, resulting in inhibition of lipid accumulation, inflammation, insulin resistance and metabolic dysfunction. Overall, the data in the study provide better understanding of the beneficial effects of Lycopene against insulin resistance and metabolic disorder.

Antidiabetic Activity of a Lotus Leaf Selenium (Se)-Polysaccharide in Rats with Gestational Diabetes Mellitus.

A selenium (Se)-containing polysaccharide, lotus leaf selenium (Se)-polysaccharide (LLP), was isolated from a lotus leaf. The effects of LLP on antioxidant enzyme activities and insulin resistance in pregnant rats with gestational diabetes mellitus (GDM) were investigated. LLP administered orally at two doses (50 and 100 mg/kg) could significantly reverse the weight loss of pregnant rats before the delivery, fetal rats, and placentas in GDM rats (P < 0.05). Furthermore, LLP treatment induced a decrease of fasting blood glucose (FBG) and fasting blood insulin (FINS) levels in GDM rats, but an increase of hepatic glycogen content, when compared with those in GDM rats (P < 0.05). Also, oral administrations of LLP markedly improved the lipid profile of GDM rats, as evidenced by a reduction of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL) cholesterol levels except for the high-density lipoprotein (HDL) cholesterol level. Additionally, antioxidant enzyme levels, such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione (GSH), in liver tissues of the GDM group were lower than those of the other groups, and following treatment of LLP, these indexes in liver tissues were equivalent to those of the control group (P > 0.05). All the data indicated that LLP may be a promising drug candidate or a healthcare food for GDM therapy or protection.

Effect of hyperthermic CO2-treated dendritic cell-derived exosomes on the human gastric cancer AGS cell line.

The aim of the present study was to determine the antitumor effects of hyperthermic CO2 (HT-CO2)-treated dendritic cell (DC)-derived exosomes (Dex) on human gastric cancer AGS cells. Mouse-derived DCs were incubated in HT-CO2 at 43°C for 4 h. The exosomes in the cell culture supernatant were then isolated. Cell proliferation was analyzed using the cell counting kit-8 (CCK-8) assay. Cell apoptosis was observed using flow cytometry, Hoechst 33258 staining and the analysis of caspase-3 activity. In addition, the proliferation of tumor cells was evaluated in xenotransplant nude mice. HT-CO2 markedly inhibited cell proliferation, as assessed by the CCK-8 assay, and also induced apoptosis in a time-dependent manner, as demonstrated by Annexin V/propidium iodide flow cytometry, caspase-3 activity and morphological analysis using Hoechst fluorescent dye. It was also revealed that HT-CO2-treated Dex decreased the expression of heat shock protein 70 and inhibited tumor growth in nude mice. In conclusion, HT-CO2 exerted an efficacious immune-enhancing effect on DCs. These findings may provide a novel strategy for the elimination of free cancer cells during laparoscopic resection. However, the potential cellular mechanisms underlying this process require further investigation.

Mitochondrial genomes reveal the pattern and timing of marten (Martes), wolverine (Gulo), and fisher (Pekania) diversification.

Despite recent advances in understanding the pattern and timescale of evolutionary diversification in the marten, wolverine, fisher, and tayra subfamily Guloninae (Mustelidae, Carnivora), several important issues still remain contentious. Among these are the phylogenetic position of Gulo relative to the subgenera of Martes (Martes and Charronia), the phylogenetic relationships within the subgenus Martes, and the timing of gulonine divergences. To elucidate these issues we explored nucleotide variation in 11 whole mitochondrial genomes (mitogenomes) from eight gulonine species and two outgroup meline species. Parsimony, maximum likelihood, and Bayesian phylogenetic analyses yielded fully resolved and identical patterns of relationships with high support for all divergences. The generic status of Pekania (P. pennanti), the monophyly of the genus Martes containing M. flavigula (subgenus Charronia) to the exclusion of the genus Gulo (G. gulo), and the M. foina (M. americana (M. melampus (M. zibellina, M. martes))) phylogeny of the subgenus Martes were strongly supported. Dating analyses (BEAST) using a set of five newly applied fossil calibrations provided divergence times considerably younger than previous multigene mitochondrial estimates, but similar to multigene nuclear and nuclear-mitochondrial estimates. The 95% confidence (highest posterior density) intervals of our divergence times fell within those inferred from nuclear and nuclear-mitochondrial sequence data, and were markedly narrower than in earlier studies (whether nuclear, mitochondrial, or combined). Notably, and contrary to long-held beliefs, our findings indicate that fossils older than the Tortonian-Messinian transition (late Late Miocene) do not represent Martes, excluding from this genus its putative members from the Early, Middle, and early Late Miocene. This study demonstrates the high informativeness of the mitogenome for phylogenetic inference and divergence time estimation within Guloninae, and suggests that mitogenomes can be highly informative also for other clades at similar levels of evolutionary divergence.

Chemokine CXC receptor 4--mediated glioma tumor tracking by bone marrow--derived neural progenitor/stem cells.

Malignant gliomas manifest frequent tumor recurrence after surgical resection and/or other treatment because of their nature of invasiveness and dissemination. The recognized brain tumor-tracking property of neural progenitor/stem cells opened the possibility of targeting malignant brain tumors using neural progenitor/stem cells. We and others have previously shown that fetal neural progenitor/stem cells can be used to deliver therapeutic molecules to brain tumors. Our recent work has further shown that gene delivery by bone marrow-derived neural progenitor/stem cells achieves therapeutic effects in a glioma model. In this study, we isolate and characterize bone marrow-derived neural progenitor/stem cells, which also express the chemokine receptor chemokine CXC receptor 4 (CXCR4). We show that CXCR4 is required for their chemotaxis and extracellular matrix invasion against a gradient of glioma soluble factors. Furthermore, beta-galactosidase-labeled bone marrow-derived neural progenitor/stem cells implanted in the contralateral side of the brain were shown to track gliomas as early as day 1 and increased through days 3 and 7. Intracranial glioma tracking by bone marrow-derived neural progenitor/stem cells is significantly inhibited by preincubation of bone marrow-derived neural progenitor/stem cells with a blocking anti-CXCR4 antibody, suggesting a CXCR4-dependent tracking mechanism. Glioma tracking bone marrow-derived neural progenitor/stem cells were found to express progenitor/stem cell markers, as well as CXCR4. Although bromodeoxyuridine incorporation assays and proliferating antigen staining indicated that tumor tracking bone marrow-derived neural progenitor/stem cells were mostly nonproliferating, these cells survive in the local tumor environment with little apoptosis. Elucidating the molecular mechanism of brain tumor tracking by adult source stem cells may provide basis for the development of future targeted therapy for malignant brain tumors.

Pleiotrophin produced by multiple myeloma induces transdifferentiation of monocytes into vascular endothelial cells: a novel mechanism of tumor-induced vasculogenesis.

Enhanced angiogenesis is a hallmark of cancer. Pleiotrophin (PTN) is an angiogenic factor that is produced by many different human cancers and stimulates tumor blood vessel formation when it is expressed in malignant cancer cells. Recent studies show that monocytes may give rise to vascular endothelium. In these studies, we show that PTN combined with macrophage colony-stimulating factor (M-CSF) induces expression of vascular endothelial cell (VEC) genes and proteins in human monocyte cell lines and monocytes from human peripheral blood (PB). Monocytes induce VEC gene expression and develop tube-like structures when they are exposed to serum or cultured with bone marrow (BM) from patients with multiple myeloma (MM) that express PTN, effects specifically blocked with antiPTN antibodies. When coinjected with human MM cells into severe combined immunodeficient (SCID) mice, green fluorescent protein (GFP)-marked human monocytes were found incorporated into tumor blood vessels and expressed human VEC protein markers and genes that were blocked by anti-PTN antibody. Our results suggest that vasculogenesis in human MM may develop from tumoral production of PTN, which orchestrates the transdifferentiation of monocytes into VECs.

Manipulation of proliferation and differentiation of human bone marrow-derived neural stem cells in vitro and in vivo.

Recent evidence has demonstrated that neural stem cells (NSC) can be expanded from a variety of sources, including embryos, fetuses, and adult bone marrow and brain tissue. We have previously reported the generation of adult rat bone marrow-derived cellular spheres that are morphologically and phenotypically similar to neurospheres derived from brain NSC. Here we show that adult human bone marrow-derived neural stem cells (HBM-NSC) are capable of generating spheres that are similar to brain neural-derived neurospheres. Additionally, we sought to promote proliferation and differentiation of HBM-NSC through transduction with nonreplicative recombinant adenovirus encoding the cDNA sequence for Gli, rADV-Gli-1; sonic hedgehog, rADV-Shh; or Nurr1, rADV-Nurr1. Immunocytochemistry and RT-PCR analysis showed that HBM-NSC could be efficiently expanded and differentiated in vitro and that HBM-NSC transduced with rADV-Gli-1 or rADV-Shh dramatically increased NSC time-related proliferation; however, Nurr1 had no effect on proliferation. We also transplanted HBM-NSC into chicken embryos to examine their potential function in vivo. We found that transduction of HBM-NSC with rADV-Gli-1 or rADV-Shh and subsequent transplantation into chicken embryos increased HBM-NSC proliferation, whereas rADV-Nurr1 promoted migration and differentiation in vivo. Our findings suggest that HBM-NSC can be efficiently expanded and differentiated in vitro and in vivo by overexpressing Gli-1, Shh or Nurr1.

Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma.

BACKGROUND: Recently, a small population of cancer stem cells in adult and pediatric brain tumors has been identified. Some evidence has suggested that CD133 is a marker for a subset of leukemia and glioblastoma cancer stem cells. Especially, CD133 positive cells isolated from human glioblastoma may initiate tumors and represent novel targets for therapeutics. The gene expression and the drug resistance property of CD133 positive cancer stem cells, however, are still unknown. RESULTS: In this study, by FACS analysis we determined the percentage of CD133 positive cells in three primary cultured cell lines established from glioblastoma patients 10.2%, 69.7% and 27.5%, respectively. We also determined the average mRNA levels of markers associated with neural precursors. For example, CD90, CD44, CXCR4, Nestin, Msi1 and MELK mRNA on CD133 positive cells increased to 15.6, 5.7, 337.8, 21.4, 84 and 1351 times, respectively, compared to autologous CD133 negative cells derived from cell line No. 66. Additionally, CD133 positive cells express higher levels of BCRP1 and MGMT mRNA, as well as higher mRNA levels of genes that inhibit apoptosis. Furthermore, CD133 positive cells were significantly resistant to chemotherapeutic agents including temozolomide, carboplatin, paclitaxel (Taxol) and etoposide (VP16) compared to autologous CD133 negative cells. Finally, CD133 expression was significantly higher in recurrent GBM tissue obtained from five patients as compared to their respective newly diagnosed tumors. CONCLUSION: Our study for the first time provided evidence that CD133 positive cancer stem cells display strong capability on tumor's resistance to chemotherapy. This resistance is probably contributed by the CD133 positive cell with higher expression of on BCRP1 and MGMT, as well as the anti-apoptosis protein and inhibitors of apoptosis protein families. Future treatment should target this small population of CD133 positive cancer stem cells in tumors to improve the survival of brain tumor patients.