Effect of Cu(II) and Conserved Copper Binding Sites on the Multicopper Oxidase CopA and Characterization of BioMnOx.

The microbial manganese removal process is believed to consist of the catalytic oxidation of Mn(II) by manganese oxidase. In this study, the multicopper oxidase CopA was purified and exhibited high manganese oxidation activity in vitro, and it was found that Cu(II) can significantly enhance its manganese oxidation activity. Gene site-directed mutagenesis was used to mutate four conserved copper binding sites of CopA to obtain four mutant strains. The manganese removal efficiencies of the four strains were determined, and it was found that H120 is the catalytically active site of CopA. The loss of Cu(II) and the mutation of the conserved copper binding site H120 resulted in the loss of ethoxyformyl and quinone modifications, a reduction in the number of modifications, and a change in the position of modifications, eventually causing a decrease in protein activity from 85.87% to 70.1%. These results reveal that Cu(II) and H120 play an indispensable role in manganese oxidation by the multicopper oxidase CopA. X-ray photoelectron spectroscopy (XPS) analysis indicates that biogenic manganese oxides produced by strains and by CopA were both composed of MnO2 and Mn3O4 and that the average valence of Mn was 3.2.

Characterization of the interactions between Fulvic acid and Trypsin with Spectroscopic and Molecular Docking technology.

The interaction mechanism between trypsin and fulvic acid was analyzed by multispectral method and molecular docking simulation. The fluorescence spectra showed that fulvic acid induced static quenching of trypsin. The validity of this conclusion was further substantiated through the computation of the binding constants. The thermodynamic parameters show that the reaction is mainly controlled by van der Waals force and hydrogen bond force, and the reaction is spontaneous. In addition, based on the obtained binding distance, there may be a non-radiative energy transfer between the two. The ultraviolet spectrum showed that fulvic acid could shift the absorption peak of trypsin, indicating that fulvic acid had an effect on the secondary structure of trypsin. According to the synchronous fluorescence spectrum results, fulvic acid primarily interacts with tryptophan residues in trypsin and induces alterations in their microenvironment. Three-dimensional fluorescence spectrum and circular dichroism further proves this conclusion. The molecular docking simulation reveals that the interaction between the two groups primarily arises from hydrogen bonding and van der Waals forces. The findings suggest that FA has the ability to induce conformational changes in trypsin's secondary structure.

Effect of Chloramphenicol as Antibiotic on the Structure and Function of Pepsin and Its Mechanism of Action.

The interaction between chloramphenicol (CHL) and pepsin (PEP), as well as the impact of CHL on PEP conformation, were investigated using spectroscopic techniques and molecular docking simulations in this study. The experimental results demonstrate that CHL exhibits a static quenching effect on PEP. The thermodynamic parameters indicate that the reaction between CHL and PEP is spontaneous, primarily driven by hydrogen bonding and van der Waals forces. Moreover, the binding distance of r<7 nm suggests the occurrence of Förster's non-radiative energy transfer between these two molecules. In the synchronous fluorescence spectrum, the maximum fluorescence intensity of PEP produced a redshift phenomenon, indicating that CHL was bound to tryptophan residues of PEP. The addition of CHL induces changes in the secondary structure of PEP, as confirmed by the observed alterations in peak values in three-dimensional fluorescence spectra. The UV spectra reveal a redshift of 3 nm in the maximum absorption peak, indicating a conformational change in the secondary structure of PEP upon addition of CHL. Circular dichroism analysis demonstrates significant alterations in the α-helix, ß-sheet, ß-turn, and random coil contents of PEP before and after CHL incorporation, further confirming its ability to modulate the secondary structure of PEP.

Isolation of native microorganisms from Shengli lignite and study on their ability to dissolve lignite.

To more greenly and efficiently utilize the abundant lignite resources and explore the microbial degradation and transformation potential of lignite for its environmentally friendly and resourceful utilization, Shengli lignite from the Hulunbuir region of Inner Mongolia, China, was selected as the research subject. Through the dilution plating method and streaking method, 31 native microorganisms were successfully isolated from the Shengli lignite, including 16 bacteria and 15 fungi. After microbial coal dissolution experiments, it was found that certain microorganisms have a significant dissolving effect on lignite, with some bacterial and fungal strains showing strong dissolution capabilities. In particular, the bacterium SH10 Lysinibacillus fusiformis and the fungus L1W Paecilomyces lilacinus demonstrated the best coal-dissolving abilities, with dissolution rates both reaching 60%. The products of microbial dissolution of lignite were analyzed using gas chromatography-mass spectrometry (GC-MS) technology, identifying a variety of small molecular organic compounds, including alkanes, alcohols, esters, and phenols. The results of this study provide a new perspective on the biodegradation of lignite and lay the foundation for the development of new lignite treatment and utilization technologies.

Clinical validation of a 90-gene expression test for tumor tissue of origin diagnosis: a large-scale multicenter study of 1417 patients.

BACKGROUND: Once malignancy tumors were diagnosed, the determination of tissue origin and tumor type is critical for clinical management. Although the significant advance in imaging techniques and histopathological approaches, the diagnosis remains challenging in patients with metastatic and poorly differentiated or undifferentiated tumors. Gene expression profiling has been demonstrated the ability to classify multiple tumor types. The present study aims to assess the performance of a 90-gene expression test for tumor classification (i.e. the determination of tumor tissue of origin) in real clinical settings. METHODS: Formalin-fixed paraffin-embedded samples and associated clinicopathologic information were collected from three cancer centers between January 2016 and January 2021. A total of 1417 specimens that met quality control criteria (RNA quality, tumor cell content ≥ 60% and so on) were analyzed by the 90-gene expression test to identify the tumor tissue of origin. The performance was evaluated by comparing the test results with histopathological diagnosis. RESULTS: The 1417 samples represent 21 main tumor types classified by common tissue origins and anatomic sites. Overall, the 90-gene expression test reached an accuracy of 94.4% (1338/1417, 95% CI: 0.93 to 0.96). Among different tumor types, sensitivities were ranged from 74.2% (head&neck tumor) to 100% (adrenal carcinoma, mesothelioma, and prostate cancer). Sensitivities for the most prevalent cancers of lung, breast, colorectum, and gastroesophagus are 95.0%, 98.4%, 93.9%, and 90.6%, respectively. Moreover, specificities for all 21 tumor types are greater than 99%. CONCLUSIONS: These findings showed robust performance of the 90-gene expression test for identifying the tumor tissue of origin and support the use of molecular testing as an adjunct to tumor classification, especially to those poorly differentiated or undifferentiated tumors in clinical practice.

Novel highly sensitive IL-10-beta-lactamase reporter mouse reveals cells of the innate immune system as a substantial source of IL-10 in vivo.

In this study, we report on a novel, highly sensitive IL-10 reporter mouse based on the reporter enzyme ß-lactamase and the fluorescence resonance energy transfer substrate coumarin-cephalosporin-fluorescein (4). In contrast to an IL-10 reporter mouse model that we generated by using enhanced GFP as reporter and allowed tracking IL-10 expression only in T cells, the IL-10-ß-lactamase reporter (ITIB) mouse enables us to easily analyze and quantify IL-10 production at the single-cell level in all myeloid and lymphoid cell types. Furthermore, the ITIB mouse allows studying of the kinetics of IL-10 expression on a single-cell basis and provides a valuable tool for in vivo screening of cell type-specific IL-10-modulating drugs. Remarkably, the ITIB mouse revealed that, although a significant portion of each myeloid and lymphoid cell type produces IL-10, macrophages represent the major IL-10 producer population in several organs of naive mice. Moreover, using the examples of bacterial infection and transplantable skin melanoma models, we demonstrate the exceptional applicability of the ITIB mouse for the identification of IL-10-producing cells during immune responses in vivo. In this study, we identified tumor-infiltrating F4/80(+) macrophages as the major source for IL-10 in B16-F10 melanoma in vivo. During systemic infection with Yersinia enterocolitica, although the proportion of IL-10(+) cells increased in each myeloid and lymphoid cell type population, infiltrating CD11b(+)Ly6G(+) neutrophils represent a majority among IL-10-producing cells at the site of infection. We conclude that cells of the innate immune system that are involved in immune homeostasis or immune responses are substantial sources of IL-10.

Combined proteomic and transcriptomic analysis of the antimicrobial mechanism of tannic acid against Staphylococcus aureus.

Staphylococcus aureus is a zoonotic opportunistic pathogen that represents a significant threat to public health. Previous studies have shown that tannic acid (TA) has an inhibitory effect on a variety of bacteria. In this study, the proteome and transcriptome of S. aureus were analyzed to comprehensively assess changes in genes and proteins induced by TA. Initial observations of morphological changes revealed that TA damaged the integrity of the cell membrane. Next, proteomic and genetic analyses showed that exposure to TA altered the expression levels of 651 differentially expressed proteins (DEPs, 283 upregulated and 368 downregulated) and 503 differentially expressed genes (DEGs, 191 upregulated and 312 downregulated). Analysis of the identified DEPs and DEGs suggested that TA damages the integrity of the cell envelope by decreasing the expression and protein abundance of enzymes involved in the synthesis of peptidoglycans, teichoic acids and fatty acids, such as murB, murQ, murG, fmhX and tagA. After treatment with TA, the assembly of ribosomes in S. aureus was severely impaired by significant reductions in available ribosome components, and thus protein synthesis was hindered. The levels of genes and proteins associated with amino acids and purine synthesis were remarkably decreased, which further reduced bacterial viability. In addition, ABC transporters, which are involved in amino acid and ion transport, were also badly affected. Our results reveal the molecular mechanisms underlying the effects of TA on S. aureus and provide a theoretical basis for the application of TA as an antibacterial chemotherapeutic agent.

Retrospective Analysis of the Predictive Value of 18F-FDG PET/CT Metabolic Parameters for PD-L1 Expression in Cervical Cancer.

BACKGROUND: Immunotherapy targeting PD-1/PD-L1 has been proven to be effective for cervical cancer treatment. To explore non-invasive examinations for assessing the PD-L1 status in cervical cancer, we performed a retrospective study to investigate the predictive value of 18F-FDG PET/CT. METHODS: The correlations between PD-L1 expression, clinicopathological characteristics and 18F-FDG PET/CT metabolic parameters were evaluated in 74 cervical cancer patients. The clinicopathological characteristics included age, histologic type, tumor differentiation, FIGO stage and tumor size. The metabolic parameters included maximum standard uptake (SUVmax), mean standard uptake (SUVmean), total lesion glycolysis (TLG) and tumor metabolic volume (MTV). RESULTS: In univariate analysis, SUVmax, SUVmean, TLG, tumor size and tumor differentiation were obviously associated with PD-L1 status. SUVmax (rs = 0.42) and SUVmean (rs = 0.40) were moderately positively correlated with the combined positive score (CPS) for PD-L1 in Spearman correlation analysis. The results of multivariable analysis showed that the higher SUVmax (odds ratio = 2.849) and the lower degree of differentiation (Odds Ratio = 0.168), the greater probability of being PD-L1 positive. The ROC curve analysis demonstrated that when the cut-off values of SUVmax, SUVmean and TLG were 10.45, 6.75 and 143.4, respectively, the highest accuracy for predicting PD-L1 expression was 77.0%, 71.6% and 62.2%, respectively. The comprehensive predictive ability of PD-L1 expression, assessed by combining SUVmax with tumor differentiation, showed that the PD-L1-negative rate was 100% in the low probability group, whereas the PD-L1-positive rate was 84.6% in the high probability group. In addition, we also found that the H-score of HIF-1α was moderately positively correlated with PD-L1 CPS (rs = 0.51). CONCLUSIONS: The SUVmax and differentiation of the primary lesion were the optimum predictors for PD-L1 expression in cervical cancer. There was a great potential for 18F-FDG PET/CT in predicting PD-L1 status and selecting cervical cancer candidates for PD1/PD-L1 immune checkpoint therapy.

Epigenetic Profiling of PTPN11 Mutant JMML Hematopoietic Stem and Progenitor Cells Reveals an Aberrant Histone Landscape.

Juvenile myelomonocytic leukemia (JMML) is a deadly pediatric leukemia driven by RAS pathway mutations, of which >35% are gain-of-function in PTPN11. Although DNA hypermethylation portends severe clinical phenotypes, the landscape of histone modifications and chromatin profiles in JMML patient cells have not been explored. Using global mass cytometry, Epigenetic Time of Flight (EpiTOF), we analyzed hematopoietic stem and progenitor cells (HSPCs) from five JMML patients with PTPN11 mutations. These data revealed statistically significant changes in histone methylation, phosphorylation, and acetylation marks that were unique to JMML HSPCs when compared with healthy controls. Consistent with these data, assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis revealed significant alterations in chromatin profiles at loci encoding post-translational modification enzymes, strongly suggesting their mis-regulated expression. Collectively, this study reveals histone modification pathways as an additional epigenetic abnormality in JMML patient HSPCs, thereby uncovering a new family of potential druggable targets for the treatment of JMML.

Pancreatic Serous Cystic Neoplasms and Mucinous Cystic Neoplasms: Differential Diagnosis by Combining Imaging Features and Enhanced CT Texture Analysis.

OBJECTIVE: To establish a diagnostic model by combining imaging features with enhanced CT texture analysis to differentiate pancreatic serous cystadenomas (SCNs) from pancreatic mucinous cystadenomas (MCNs). MATERIALS AND METHODS: Fifty-seven and 43 patients with pathology-confirmed SCNs and MCNs, respectively, from one center were analyzed and divided into a training cohort (n = 72) and an internal validation cohort (n = 28). An external validation cohort (n = 28) from another center was allocated. Demographic and radiological information were collected. The least absolute shrinkage and selection operator (LASSO) and recursive feature elimination linear support vector machine (RFE_LinearSVC) were implemented to select significant features. Multivariable logistic regression algorithms were conducted for model construction. Receiver operating characteristic (ROC) curves for the models were evaluated, and their prediction efficiency was quantified by the area under the curve (AUC), 95% confidence interval (95% CI), sensitivity and specificity. RESULTS: Following multivariable logistic regression analysis, the AUC was 0.932 and 0.887, the sensitivity was 87.5% and 90%, and the specificity was 82.4% and 84.6% with the training and validation cohorts, respectively, for the model combining radiological features and CT texture features. For the model based on radiological features alone, the AUC was 0.84 and 0.91, the sensitivity was 75% and 66.7%, and the specificity was 82.4% and 77% with the training and validation cohorts, respectively. CONCLUSION: This study showed that a logistic model combining radiological features and CT texture features is more effective in distinguishing SCNs from MCNs of the pancreas than a model based on radiological features alone.

PTEN transcript variants caused by illegitimate splicing in "aged" blood samples and EBV-transformed cell lines.

PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. Mutations occur in either heritable or sporadic fashion. Sequencing of cDNA from patients and normal individuals often reveals splicing variants (SVs) of PTEN, some of which are non-mutation related. To investigate whether these SVs were the result of illegitimate splicing (a general decrease of fidelity in splicing site selection in "aged" samples), we tested "aged" blood from individuals who had normal PTEN transcripts in their "fresh" mononuclear cells. Blood from 20 normal individuals was collected and split into two aliquots. Total RNA and DNA were extracted immediately ("fresh") and 48 h later ("aged"), respectively. Using RT-PCR, subcloning and sequencing, we found seven types of SVs. No mutation was detected in the related intron-exon flanking region in genomic DNA in either "fresh" or "aged" samples. Some of the SVs were also consistently present in both the "fresh" and "aged" EBV-transformed lymphoblastoid cells from six normal individuals. Western blot data indicated that the PTEN protein level (in full length) was not altered in the "fresh" EBV-transformed lymphoblastoid cells with SVs. In conclusion, our data demonstrate that PTEN illegitimate splicing often occurs in "aged" blood and EBV-transformed lymphoblastoid cells. Therefore, it is critical to note the time point of RNA extraction when investigating for PTEN aberrant transcripts. We hope that our data will increase awareness about the sample status, because gene expression data may be potentially flawed from "aged" samples, particularly when dealing with clinical samples.

Significantly enhanced dielectric constant and energy storage properties in polyimide/reduced BaTiO3 composite films with excellent thermal stability.

In this work, reduced BaTiO3 (rBT) particles with a large number of defects sintered in a reducing atmosphere (95N2/5H2) were introduced into polyimide (PI) matrix without using any modifier or surfactant components. The rBT/PI composite films fabricated by an in situ polymerization method showed significantly enhanced dielectric constant and energy storage density. The dielectric constant of the rBT/PI composite with 30 wt% rBT reached up to 31.6, while maintaining lower loss (tg δ = 0.031@1000 kHz) compared to pure PI (ε r = 4.1). Its energy storage density (9.7 J cm-3 at 2628 kV cm-1) was enhanced by more than 400% over that of pure PI (1.9 J cm-3 at 3251 kV cm-1), and was greater than the energy density of the best commercial biaxially-oriented-polypropylenes (BOPP) (1.2 J cm-3 at 6400 kV cm-1). The energy storage efficiency was around 90% due to the linear dielectric performance of rBT/PI composite films. The improved dielectric constant and energy storage density could be attributed to the combined effect of the interface interaction between two phases and the surface defects of rBT induced by the reducing atmosphere. Therefore, rBT/PI composite films with high dielectric constant, energy storage density and storage efficiency may have potential applications in the preparation of embedded capacitors.

[Inhibitory effects of emodin on proliferation of rat vascular smooth muscle cell induced by angiotensin II].

OBJECTIVE: To investigate the effects of emodin on the proliferation of cultured rat vascular smooth muscle cell (VSMC) induced by angiotensin II. METHOD: VSMCs were cultured by explant method. Cell proliferation model was established by stimulation with Ang II. Cell proliferation was measured by MTT assay to observe the effects of emodin (10, 20, 40 and 80 micromol x L(-1)) and N(G)-nitro-L-arginine methyl ester (L-NAME, 100 micromol x L(-1)) on VSMC proliferation induced by Ang II. The expression of PCNA was measured by immunohistochemical staining. Nitric oxide (NO) level was measured by Griess reagent. Nitric oxide synthase (NOS) and inducible nitric oxide synthase (iNOS) levels were detected by chemical colorimetric method. mRNA expression of iNOS was measured by reverse transcription polymerase chain reaction (RT-PCR). RESULT: Emodin at the doses range from 10 to 80 mol x L(-1) inhibited cell proliferation in a dose and time-dependent manner. The inhibitory effects were partly blocked by 100 mol x L(-1) of L-NAME. Emodin markedly decreased the expression of PCNA in VSMC, increased NO, NOS and iNOS levels, and increased iNOS mRNA expression in VSMC. CONCLUSION: Emodin could inhibite VSMCs proliferation induced by Ang II. Inhibiting the expression of PCNA, increasing the NO secretion and upregulating the iNOS gene expression might be associated with the inhibitory effects.

Manganese(II) oxidation by the multi-copper oxidase CopA from Brevibacillus panacihumi MK-8.

Manganese contamination of groundwater exists worldwide. Manganese removal is primarily performed through catalytic oxidation by manganese-oxidizing bacteria. In this study, we identified a new manganese(II) oxidase (CopA) from Brevibacillus panacihumi MK-8. The copA gene was cloned and expressed in Escherichia coli strain BL21(DE3), and the recombinant strain BL21-pET-copA was able to remove 85.87% of Mn(II) from LB medium containing 1 mM Mn(II) after seven days. The optimum Mn(II) oxidase CopA activity was obtained at 37 °C in 10 mM HEPES buffer (pH 8.0) containing 0.4 mM CuCl2. Purified CopA removed 51.98% of manganese(II) under the optimal conditions. The copA gene-deleted strain (MK-8-ΔcopA) barely oxidized manganese, further demonstrating that the copA gene is the manganese oxidase gene. Biogenic Mn oxides were analyzed by scanning electron microscopy and X-ray diffraction. Thus, we suggest that the recombinant BL21-pET-copA strain and oxidase CopA have the potential to be used in biological manganese removal technology.

[Apoptosis and ultrastructural lesions in Vero and J774A.1 cells induced by Leptospira interrogans].

OBJECTIVE: To determine the ability of adhering and invading to host cells and the related pathologic changes of Leptospira spp. with different virulence. METHODS: A special Fontana silver staining method was developed to observe the ability of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 and serogroup Pomona serovar pomona strain 56608, and L.biflexa serogroup Samaranga serovar patoc strain Patoc I to adhere Vero and J774A.1 cells. Ultrastructural lesions of the infected cells were examined by electron microscopy. By using flow cytometry with fluorescein labeling of FITC-Annexin V/PI,apoptosis and necrosis of the Vero and J774A.1 cells induced by the leptospiral strains before and after inactivation with ultraviolet treatment were detected, respectively. RESULTS: The adhering rates of L.interrogans strains 56601 and 56608 to the Vero cells were 24.2% and 22.9% (P>0.05), while to the J774A.1 cells were 49.0% and 46.9% (P>0.05), respectively. L.biflexa strain Patoc I did not adhere to these two host cells. After the two strains of L.interrogans invaded two different cell lines, the special phagosomes containing the Leptospira were formed and similar cell ultrastructural lesions, such as chromatilysis and chromatin condensation, vacuolar degeneration, mitochondrion swelling and mitochondrial crista disappearance, and endoplasmic reticulum swelling and paramembranous ribosome disappearance, were observed. The apoptosis rates in the Vero cells caused by the L.interrogans strains 56601 and 56608 before and after ultraviolet inactivation were 84.4%, 82.8% and 77.9%, 86.1%, respectively. The L.interrogans strain 56601 mainly induced the terminal apoptosis in Vero cells with the rates of 68.0% and 52.9% before or after inactivation, while the L.interrogans strain 56608 mainly induced the early apoptosis in Vero cells with apoptosis rates of 64.1% and 50.1% before or after inactivation. In the J774A.1 cells, the L.interrogans strain 56608 caused cell necrosis (before and after inactivation) and apoptosis that was dominated by terminal apoptosis. CONCLUSION: The established Fontana silver staining method can be used to observe adhesion of L.interrogans.L.interrogans can invade host cells through endocytosis which causes untrastructural lesions. The necrosis or apoptosis induced by L.interrogans are affected by different host cell lines but not the virulence of L.interrogans strains.

Regulation of ferrochelatase gene expression by hypoxia.

Ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, catalyzes the insertion of iron into protoporphyrin to form heme. This pathway provides heme for hemoglobin and other essential hemoproteins. The regulatory role of oxygen in the pathway has not been clearly established. In this study, we examined whether FECH gene expression is upregulated during hypoxia by a mechanism which involves the hypoxia-inducible factor 1 (HIF-1). Two HIF-1 binding motifs were identified within the -150 bp FECH minimal promoter sequence. Exposure of HEL, K562, and Hep-G2 cells to hypoxia for 18 hours resulted in a significant increase in FECH mRNA expression (p < 0.05). Hypoxia also transactivated the minimal promoter for the FECH gene in the cells. Transient co-expression of wild-type HIF-1alpha or a dominant negative HIF-1alpha with the FECH minimal promoter luciferase construct stimulated or blocked FECH promoter activity, respectively. Expression of the von Hippel-Lindau (VHL) tumor suppressor factor blocked the expression of both FECH mRNA and HIF-1alpha protein during normoxic culture of renal carcinoma cell line (RCC4). The results suggest that the FECH gene is a target for HIF-1 during hypoxia.

Symmetrical and asymmetrical division analysis provides evidence for a hierarchy of prostate epithelial cell lineages.

Although symmetrical and asymmetrical divisions of stem cells have been extensively studied in invertebrate and mammalian neural epithelia, their role remains largely unknown in mammalian non-neural epithelial development, regeneration and tumorigenesis. Here, using basal and luminal cell-specific markers and cell lineage tracing transgenic mice, we report that in developing prostatic epithelia, basal and luminal cells exhibit distinct division modes. While basal cells display both symmetric and asymmetric divisions leading to different cell fates, luminal cells only exhibit symmetrical divisions. Examination of cell division modes in prostate-specific Pten-null mice indicates that both luminal and basal cells can be cellular origins for prostate cancer. Furthermore, analysis of Sox2-expressing cells in p63 and Pten-null mice suggests that basal cells contribute to the luminal population and tumorigenesis. These findings provide direct evidence for the existence of a hierarchy of epithelial cell lineages during prostate development, regeneration and tumorigenesis.

TOP2Ahigh is the phenotype of recurrence and metastasis whereas TOP2Aneg cells represent cancer stem cells in prostate cancer.

Recurrence and metastasis are the main causes of death for prostate cancer patients and cancer stem cells (CSCs) are proposed to play important roles in cancer recurrence and metastasis. It is generally thought that genes upregulated in recurrent/metastatic disease are likely biomarkers of CSCs. Hence we analyzed multiple microarray datasets on prostate tumor tissues to identify upregulated genes associated with cancer recurrence/metastasis, and tried to explore whether those genes were true biomarkers of prostate CSCs. Our results indicated that TOP2A was the most highly upregulated gene in recurrent/metastatic prostate cancer, and its high expression was positively correlated with poor prognosis in patients. Using a promoter reporter system, we unexpectedly discovered enrichment of CSCs in TOP2Aneg cells. Compared to TOP2Ahigh cells, TOP2Aneg cells formed spheres and tumors more efficiently, and became enriched in the presence of stresses. Analysis of cell divisions by time lapse imaging indicated that more slow-cycling cells were observed in TOP2Aneg cells while the proportion of abnormal divisions was higher in TOP2Ahigh cells. Our studies demonstrate that TOP2Ahigh is the phenotype of recurrence/metastasis but TOP2Aneg cells show slow cycling and have CSCs properties in prostate cancer, which has significant implications for prostate cancer therapy.

Generation of functional organs from stem cells.

We are now well entering the exciting era of stem cells. Potential stem cell therapy holds great promise for the treatment of many diseases such as stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral-sclerosis, myocardial infarction, muscular dystrophy, diabetes, and etc. It is generally believed that transplantation of specific stem cells into the injured tissue to replace the lost cells is an effective way to repair the tissue. In fact, organ transplantation has been successfully practiced in clinics for liver or kidney failure. However, the severe shortage of donor organs has been a major obstacle for the expansion of organ transplantation programs. Toward that direction, generation of transplantable organs using stem cells is a desirable approach for organ replacement and would be of great interest for both basic and clinical scientists. Here we review recent progress in the field of organ generation using various methods including single adult tissue stem cells, a blastocyst complementation system, tissue decellularization/recellularization and a combination of stem cells and tissue engineering.

PTEN deficiency is a common defect in juvenile myelomonocytic leukemia.

The biological hallmark of juvenile myelomonocytic leukemia (JMML) is selective GM-CSF hypersensitivity. We hypothesized that PTEN protein deficiency might lead to insufficient negative growth signals to counter the hyperactive Ras signaling and therefore aid in the acceleration of the malignant transformation of JMML. In screening 34 JMML patients we found: (1) decreased PTEN protein in 67% of patients; (2) significantly lower PTEN mRNA levels in patients compared to controls (p<0.01); (3) a hypermethylated PTEN promoter in 77% of patients; and (4) constitutive-hyperactive Akt and MAPK in 55% and 73% of patients, respectively. These findings suggest that PTEN deficiency is very common in JMML and is in part due to hypermethylation of the PTEN gene promoter.