Significant changes in subseries means and variances in an 8000-year precipitation reconstruction from tree rings in the southwestern USA

Both algorithms were applied to an 8000-year long time series of annual precipitation that was reconstructed from tree rings in the southwestern USA. One of the algorithms is the scanning <I>t</I>-test, which detects significant changes in subseries means (the first center moments) on various time scales. Another is the scanning <I>F</I>-test, which detects significant changes in subseries variances (the second center moments) on multi-time scales. Firstly, the scanning <I>t</I>-test identified 22 change points in subseries means and partitioned the series into 23 relatively wet, normal or dry episodes. Secondly, the scanning <I>F</I>-test detected 15 change points in subseries variances and divided 16 phases in comparatively steady (with smaller variance) or unsteady (with larger variance) features. Thirdly, the 23 wetness-episodes were characterized as the steady or unsteady situations by jointing the results from the scanning <I>F</I>-test into those from the <I>t</I>-test. Fourthly, the 23 episodes were compared to those in the TIC and δ<sup>18</sup>O records from cored sediments in the deep basin of the Pyramid Lake in Nevada by using a coherency analysis of the <I>t</I>-test between the precipitation reconstruction and the TIC or δ<sup>18</sup>O series. Fifthly, the 23 episodes were collaborated with some published papers in related studies. In addition, the 23 episodes were also compared with studies of the global climate change and with documents of climate changes in China during the same periods. As the TIC and δ<sup>18</sup>O record series are high resolution with unequal sampling intervals between 3 and 14 years, an algorithm in the scanning <I>t</I>-test for dealing with the unequal time intervals was developed in this study

Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC.

Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion of ftsX or envC produces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the ΔftsX and ΔenvC mutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of ΔftsX and ΔenvC mutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions in Fusobacterium cell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated.IMPORTANCE Little is known about the virulence mechanisms and associated factors in F. nucleatum, due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identify F. nucleatum biofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the ΔftsX and ΔenvC mutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen

PtdIns (3,4,5) P3 Recruitment of Myo10 Is Essential for Axon Development

Myosin X (Myo10) with pleckstrin homology (PH) domains is a motor protein acting in filopodium initiation and extension. However, its potential role has not been fully understood, especially in neuronal development. In the present study the preferential accumulation of Myo10 in axon tips has been revealed in primary culture of hippocampal neurons with the aid of immunofluorescence from anti-Myo10 antibody in combination with anti-Tuj1 antibody as specific marker. Knocking down Myo10 gene transcription impaired outgrowth of axon with loss of Tau-1-positive phenotype. Interestingly, inhibition of actin polymerization by cytochalasin D rescued the defect of axon outgrowth. Furthermore, ectopic expression of Myo10 with enhanced green fluorescence protein (EGFP) labeled Myo10 mutants induced multiple axon-like neurites in a motor-independent way. Mechanism studies demonstrated that the recruitment of Myo10 through its PH domain to phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5) P3) was essential for axon formation. In addition, in vivo studies confirmed that Myo10 was required for neuronal morphological transition during radial neuronal migration in the developmental neocortex

Mutant Kras- and p16-regulated NOX4 activation overcomes metabolic checkpoints in development of pancreatic ductal adenocarcinoma

Kras activation and p16 inactivation are required to develop pancreatic ductal adenocarcinoma (PDAC). However, the biochemical mechanisms underlying these double alterations remain unclear. Here we discover that NAD(P)H oxidase 4 (NOX4), an enzyme known to catalyse the oxidation of NAD(P)H, is upregulated when p16 is inactivated by looking at gene expression profiling studies. Activation of NOX4 requires catalytic subunit p22phox, which is upregulated following Kras activation. Both alterations are also detectable in PDAC cell lines and patient specimens. Furthermore, we show that elevated NOX4 activity accelerates oxidation of NADH and supports increased glycolysis by generating NAD+, a substrate for GAPDH-mediated glycolytic reaction, promoting PDAC cell growth. Mechanistically, NOX4 was induced through p16-Rb-regulated E2F and p22phox was induced by KrasG12V-activated NF-κB. In conclusion, we provide a biochemical explanation for the cooperation between p16 inactivation and Kras activation in PDAC development and suggest that NOX4 is a potential therapeutic target for PDAC

Extracellular Vesicles Secreted by Neospora caninum Are Recognized by Toll-Like Receptor 2 and Modulate Host Cell Innate Immunity Through the MAPK Signaling Pathway

Neospora caninum is an obligate intracellular parasite, which causes significant economic losses in the cattle industry. However, the immune mechanism of the parasite–host interaction is not yet fully understood. Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism by which almost all cells, especially immune and tumor cells, participate in intercellular communications. Although studies have indicated that EVs secreted by Toxoplasma gondii or Trypanosoma brucei promote exchanges of biological molecules important for the host–parasite interplay, however, EVs and their biological activities in N. caninum is not clear. Here, we used multiple methods, including electron microscopy, nanoparticle tracking analysis, RT-PCR, immunofluorescence, western blot, proteomics, and cytokine analyses, to examine the properties of N. caninum EVs. We found that N. caninum produced EVs that are similar to mammalian exosomes, which generally range from 30 to 150 nm in diameter. It was shown that N. caninum EVs could remarkably increase the production of pro-inflammatory cytokines IL-12p40, TNF-α, IL-1β, IL-6, and IFN-γ by wild-type (WT) mouse bone marrow-derived macrophages (BMDMs) whereas the secretion of IL-12p40, TNF-α, and IFN-γ was very strongly downregulated in TLR2−/− mouse BMDMs. The levels of IL-6 were not affected, but the secretion of IL-10 was upregulated. We found that the phosphorylation levels of P38, ERK, and JNK were significantly reduced in the TLR2−/− cells compared with those in WT mouse BMDMs and that treatment with chemical inhibiters of P38, ERK, and JNK resulted in upregulation of IL-6, IL-12p40, and IL-10 production. Together, these results demonstrated that N. caninum EVs could be rapidly internalized to deliver proteins to the host cells and modulate the host cell immune responses through MAPK signaling pathway in a TLR2-dependent manner. Our study is the first to reveal potential roles for N. caninum EVs in host communication and immune response in parasite–host interactions

14-3-3 Protein of Neospora caninum Modulates Host Cell Innate Immunity Through the Activation of MAPK and NF-κB Pathways

Neospora caninum is an obligate intracellular apicomplexan parasite, the etiologic agent of neosporosis, and a major cause of reproductive loss in cattle. There is still a lack of effective prevention and treatment measures. The 14-3-3 protein is a widely expressed acidic protein that spontaneously forms dimers within apicomplexan parasites. This protein has been isolated and sequenced in many parasites; however, there are few reports about the N. caninum 14-3-3 protein. Here, we successfully expressed and purified a recombinant fusion protein of Nc14-3-3 (rNc14-3-3) and prepared a polyclonal antibody. Immunofluorescence and immunogold electron microscopy studies of tachyzoites or N. caninum-infected cells suggested that 14-3-3 was localized in the cytosol and the membrane. Western blotting analysis indicated that rNc14-3-3 could be recognized by N. caninum-infected mouse sera, suggesting that 14-3-3 may be an infection-associated antigen that is involved in the host immune response. We demonstrated that rNc14-3-3 induced cytokine expression by activating the MAPK and AKT signaling pathways, and inhibitors of p38, ERK, JNK, and AKT could significantly decrease the production of IL-6, IL-12p40, and TNF-α. In addition, phosphorylated nuclear factor-κB (NF-κB/p65) was observed in wild-type peritoneal macrophages (PMs) treated with rNc14-3-3, and the protein level of NF-κB/p65 was reduced in the cytoplasm but increased correspondingly in the nucleus after 2 h of treatment. These results were also observed in deficient in TLR2-/- PMs. Taken together, our results indicated that the N. caninum 14-3-3 protein can induce effective immune responses and stimulate cytokine expression by activating the MAPK, AKT, and NF-κB signaling pathways but did not dependent TLR2, suggesting that Nc14-3-3 is a novel vaccine candidate against neosporosis

Pseudonocardians A–C, New Diazaanthraquinone Derivatives from a Deap-Sea Actinomycete Pseudonocardia sp. SCSIO 01299

Pseudonocardians A–C (2–4), three new diazaanthraquinone derivatives, along with a previously synthesized compound deoxynyboquinone (1), were produced by the strain SCSIO 01299, a marine actinomycete member of the genus Pseudonocardia, isolated from deep-sea sediment of the South China Sea. The structures of compounds 1–4 were determined by mass spectrometry and NMR experiments (1H, 13C, HSQC, and HMBC). The structure of compound 1, which was obtained for the first time from a natural source, was confirmed by X-ray analysis. Compounds 1–3 exhibited potent cytotoxic activities against three tumor cell lines of SF-268, MCF-7 and NCI-H460 with IC50 values between 0.01 and 0.21 μm, and also showed antibacterial activities on Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and Bacillus thuringensis SCSIO BT01, with MIC values of 1–4 μg mL−1

MD-2 is required for disulfide HMGB1-dependent TLR4 signaling

Innate immune receptors for pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) orchestrate inflammatory responses to infection and injury. Secreted by activated immune cells or passively released by damaged cells, HMGB1 is subjected to redox modification that distinctly influences its extracellular functions. Previously, it was unknown how the TLR4 signalosome distinguished between HMGB1 isoforms. Here we demonstrate that the extracellular TLR4 adaptor, myeloid differentiation factor 2 (MD-2), binds specifically to the cytokine-inducing disulfide isoform of HMGB1, to the exclusion of other isoforms. Using MD-2–deficient mice, as well as MD-2 silencing in macrophages, we show a requirement for HMGB1-dependent TLR4 signaling. By screening HMGB1 peptide libraries, we identified a tetramer (FSSE, designated P5779) as a specific MD-2 antagonist preventing MD-2–HMGB1 interaction and TLR4 signaling. P5779 does not interfere with lipopolysaccharide-induced cytokine/chemokine production, thus preserving PAMP-mediated TLR4–MD-2 responses. Furthermore, P5779 can protect mice against hepatic ischemia/reperfusion injury, chemical toxicity, and sepsis. These findings reveal a novel mechanism by which innate systems selectively recognize specific HMGB1 isoforms. The results may direct toward strategies aimed at attenuating DAMP-mediated inflammation while preserving antimicrobial immune responsiveness

Recombinant Human Endostatin Endostar Inhibits Tumor Growth and Metastasis in a Mouse Xenograft Model of Colon Cancer

To investigate the effects of recombinant human endostatin Endostar on metastasis and angiogenesis and lymphangiogenesis of colorectal cancer cells in a mouse xenograft model. Colon cancer cells SW620 were injected subcutaneously into the left hind flank of nude mice to establish mouse xenograft models. The mice were treated with normal saline or Endostar subcutaneously every other day. The growth and lymph node metastasis of tumor cells, angiogenesis and lymphangiogenesis in tumor tissue were detected. Apoptosis and cell cycle distribution were studied by flow cytometry. The expression of VEGF-A, -C, or -D in SW620 cells was determined by immunoblotting assays. Endostar inhibited tumor growth and the rate of lymph node metastasis (P < 0.01). The density of blood vessels in or around the tumor area was 12.27 ± 1.21 and 22.25 ± 2.69 per field in Endostar-treated mice and controls (P < 0.05), respectively. Endostar also decreased the density of lymphatic vessels in tumor tissues (7.84 ± 0.81 vs. 13.83 ± 1.08, P < 0.05). Endostar suppresses angiogenesis and lymphangiogenesis in the lymph nodes with metastases, simultaneously. The expression of VEGF-A, -C and -D in SW620 cells treated with Endostar was substantially lower than that of controls. Endostar inhibited growth and lymph node metastasis of colon cancer cells by inhibiting angiogenesis and lymphangiogenesis in a mouse xenograft model of colon cancer