Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the acquisition of cancer stem cell signatures that are attenuated by BR-DIM

The emergence of castrate-resistant prostate cancer (CRPC) contributes to the high mortality of patients diagnosed with prostate cancer (PCa), which in part could be attributed to the existence and the emergence of cancer stem cells (CSCs). Recent studies have shown that deregulated expression of microRNAs (miRNAs) contributes to the initiation and progression of PCa. Among several known miRNAs, let-7 family appears to play a key role in the recurrence and progression of PCa by regulating CSCs; however, the mechanism by which let-7 family contributes to PCa aggressiveness is unclear. Enhancer of Zeste homolog 2 (EZH2), a putative target of let-7 family, was demonstrated to control stem cell function. In this study, we found loss of let-7 family with corresponding over-expression of EZH2 in human PCa tissue specimens, especially in higher Gleason grade tumors. Overexpression of let-7 by transfection of let-7 precursors decreased EZH2 expression and repressed clonogenic ability and sphere-forming capacity of PCa cells, which was consistent with inhibition of EZH2 3'UTR luciferase activity. We also found that the treatment of PCa cells with BR-DIM (formulated DIM: 3,3'-diindolylmethane by Bio Response, Boulder, CO, abbreviated as BR-DIM) up-regulated let-7 and down-regulated EZH2 expression, consistent with inhibition of self-renewal and clonogenic capacity. Moreover, BR-DIM intervention in our on-going phase II clinical trial in patients prior to radical prostatectomy showed upregulation of let-7 consistent with down-regulation of EZH2 expression in PCa tissue specimens after BR-DIM intervention. These results suggest that the loss of let-7 mediated increased expression of EZH2 contributes to PCa aggressiveness, which could be attenuated by BR-DIM treatment, and thus BR-DIM is likely to have clinical impact.     

Augmenter of liver regeneration (alr) promotes liver outgrowth during zebrafish hepatogenesis

Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of alr in liver organogenesis in zebrafish model. We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive Alr(C131S) mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alr(C131S) is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO-mediated knockdown allows the identification of its late developmental role in vertebrate liver organogenesis.     

Analysis of the N-terminal region of human MLKL,as well as two distinct MLKL isoforms,reveals new insights into necroptotic cell death

The pseudokinase mixed lineage kinase domain-like (MLKL) is an essential effector of necroptotic cell death. Two distinct human MLKL isoforms have previously been reported, but their capacities to trigger cell death have not been compared directly. Herein, we examine these two MLKL isoforms, and further probe the features of the human MLKL N-terminal domain that are required for cell death. Expression in HEK293T cells of the N-terminal 201 amino acids (aa) of human MLKL is sufficient to cause cell death, whereas expression of the first 154 aa is not. Given that aa 1–125 are able to initiate necroptosis, our findings indicate that the helix that follows this region restrains necroptotic activity, which is again restored in longer constructs. Furthermore, MLKL isoform 2 (MLKL2), which lacks much of the regulatory pseudokinase domain, is a much more potent inducer of cell death than MLKL isoform 1 (MLKL1) in ectopic expression studies in HEK293T cells. Modelling predicts that a C-terminal helix constrains the activity of MLKL1, but not MLKL2. Although both isoforms are expressed by human monocyte-derived macrophages at the mRNA level, MLKL2 is expressed at much lower levels. We propose that it may have a regulatory role in controlling macrophage survival, either in the steady state or in response to specific stimuli.     

Solutions for ecosystem‐level protection of ocean systems under climate change

The Paris Conference of Parties (COP21) agreement renewed momentum for action against climate change, creating the space for solutions for conservation of the ocean addressing two of its largest threats: climate change and ocean acidification (CCOA). Recent arguments that ocean policies disregard a mature conservation research field and that protected areas cannot address climate change may be oversimplistic at this time when dynamic solutions for the management of changing oceans are needed. We propose a novel approach, based on spatial meta‐analysis of climate impact models, to improve the positioning of marine protected areas to limit CCOA impacts. We do this by estimating the vulnerability of ocean ecosystems to CCOA in a spatially explicit manner and then co‐mapping human activities such as the placement of renewable energy developments and the distribution of marine protected areas. We test this approach in the NE Atlantic considering also how CCOA impacts the base of the food web which supports protected species, an aspect often neglected in conservation studies. We found that, in this case, current regional conservation plans protect areas with low ecosystem‐level vulnerability to CCOA, but disregard how species may redistribute to new, suitable and productive habitats. Under current plans, these areas remain open to commercial extraction and other uses. Here, and worldwide, ocean conservation strategies under CCOA must recognize the long‐term importance of these habitat refuges, and studies such as this one are needed to identify them. Protecting these areas creates adaptive, climate‐ready and ecosystem‐level policy options for conservation, suitable for changing oceans.     

Differential expression of novel tyrosine kinase substrates during breast cancer development

To identify novel tyrosine kinase substrates that have never been implicated in cancer, we studied the phosphoproteomic changes in the MCF10AT model of breast cancer progression using a combination of phosphotyrosyl affinity enrichment, iTRAQ technology, and LC-MS/MS. Using complementary MALDI- and ESI-based mass spectrometry, 57 unique proteins comprising tyrosine kinases, phosphatases, and other signaling proteins were detected to undergo differential phosphorylation during disease progression. Seven of these proteins (SPAG9, Toll-interacting protein (TOLLIP), WBP2, NSFL1C, SLC4A7, CYFIP1, and RPS2) were validated to be novel tyrosine kinase substrates. SPAG9, TOLLIP, WBP2, and NSFL1C were further proven to be authentic targets of epidermal growth factor signaling and Iressa (gefitinib). A closer examination revealed that the expression of SLC4A7, a bicarbonate transporter, was down-regulated in 64% of the 25 matched normal and tumor clinical samples. The expression of TOLLIP in clinical breast cancers was heterogeneous with 25% showing higher expression in tumor compared with normal tissues and 35% showing the reverse trend. Preliminary studies on SPAG9, on the other hand, did not show differential expression between normal and diseased states. This is the first time SLC4A7 and TOLLIP have been discovered as novel tyrosine kinase substrates that are also associated with human cancer development. Future molecular and functional studies will provide novel insights into the roles of TOLLIP and SLC4A7 in the molecular etiology of breast cancer.     

Influence of noninvasive peripheral arterial blood pressure measurements on assessment of dynamic cerebral autoregulation.

Assessment of dynamic cerebral autoregulation (CA) requires continuous recording of arterial blood pressure (ABP). In humans, noninvasive ABP recordings with the Finapres device have often been used for this purpose. We compared estimates of dynamic CA derived from Finapres with those from invasive recordings in the aorta. Measurements of finger noninvasive ABP (Finapres), intra-aortic ABP (Millar catheter), surface ECG, transcutaneous CO2, and bilateral cerebral blood flow velocity (CBFV) in the middle cerebral arteries were simultaneously and continuously recorded in 27 patients scheduled for percutaneous coronary interventions. Phase, gain, coherence, and CBFV step response from both the Finapres and intra-arterial catheter were estimated by transfer function analysis. A dynamic autoregulation index (ARI) was also calculated. For both hemispheres, the ARI index and the CBFV step response recovery at 4 s were significantly greater for the Finapres-derived estimates than for the values obtained from aortic pressure. The transfer function gain for frequencies <0.1 Hz was significantly smaller for the Finapres estimates. The phase frequency response was significantly greater for the Finapres estimates at frequencies >0.1 Hz, but not at lower frequencies. The Finapres gives higher values for the efficiency of dynamic CA compared with values derived from aortic pressure measurements, as indicated by biases in the ARI index, CBFV step response, gain, and phase. Despite the significance of these biases, their relatively small amplitude indicates a good level of agreement between indexes of CA derived from the Finapres compared with corresponding estimates obtained from invasive measurements of aortic ABP.     

Biosynthesis of beta-substituted furan skeleton in the lower furanoterpenoids: a model study.

Furanoterpenes are widely distributed in the plant kingdom. In this study we have carried out enzymatic synthesis of simple furan compounds from the molecules containing an alpha-isopropylidene ketone unit and the role of cytochrome P450 in this biotransformation has been conclusively established. Eight model compounds (acyclic, monocyclic, and bicyclic, 1-8), having an alpha-isopropylidene ketone unit, were synthesized and incubated with PB-induced rat liver microsomes in the presence of NADPH and O(2). GC-MS and NMR analyses of the product(s) indicated the formation of corresponding furano derivatives (11-18). Cytochrome P450 inhibitors, metyrapone, SKF-525, and carbon monoxide, inhibited the formation of furan (8) to 76, 62, and 97%, respectively. Incubation of dehydrofukinone (7), a naturally occurring terpene, with purified cytochrome P450, NADPH-cytochrome P450 reductase, and dilaurylphosphatidylcholine in the presence of NADPH and O(2) resulted in the formation of 10 and furanodehydrofukinone (19). Based on these observations, we propose one of the probable biosynthetic routes for lower furanoterpenoids in higher plants.     

Synthesis and therapeutic evaluation of pyridyl based novel mTOR inhibitors

A series of novel cyanopyridyl based molecules (1–14) were designed, synthesized and probed for inhibition of mammalian target of rapamycin (mTOR) activity. Compound 14 was found to be a potent inhibitor of mTOR activity as assessed by enzyme-linked immunoassays and Western blot analysis. Most importantly, systemic application (intraperitoneal; ip) of compound 14 significantly suppressed macroscopic and histological abnormalities associated with chemically-induced murine colitis.     

<Emphasis Type="Italic">Chlorophytum gothanense</Emphasis>, a new species of Anthericaceae from the Western Ghats of India

A new species of Chlorophytum is described and illustrated. It is adapted to grow on the open, exposed lateritic plateaus in the Northern Western Ghats of India. The chromosome number of the species is 2n = 28.