Ethanol-induced yeast flocculation directed by the promoter of TPS1 encoding trehalose-6-phosphate synthase 1 for efficient ethanol production

Yeast flocculation is an important trait in the brewing industry as well as in ethanol production, through which biomass can be recovered by cost-effective sedimentation. However, mass transfer limitation may affect yeast growth and ethanol fermentation if the flocculation occurs earlier before fermentation is completed. In this article, a novel type of cell-cell flocculation induced by trehalose-6-phosphate synthase 1 (TPS1) promoter was presented. The linear cassette HO-P(TPS1)-FLO1(SPSC01)-KanMX4-HO was constructed to transform the non-flocculating industrial yeast S. cerevisiae 4126 by chromosome integration to obtain a new flocculating yeast strain, ZLH01, whose flocculation was induced by ethanol produced during fermentation. The experimental results illustrated that flocculation of ZLH01 was triggered by 3% (v/v) ethanol and enhanced as ethanol concentration increased till complete flocculation was achieved at ethanol concentration of 8% (v/v). Real time PCR analysis confirmed that the expression of FLO1(SPSC01) was dependent on ethanol concentration. The growth and ethanol fermentation of ZLH01 were improved significantly, compared with the constitutive flocculating yeast BHL01 engineered with the same FLO gene but directed by the constitutive 3-phosphoglycerate kinase promoter PGK1, particularly under high temperature conditions. These characteristics make the engineered yeast more suitable for ethanol production from industrial substrates under high gravity and temperature conditions. In addition, this strategy offers advantage in inducing differential expression of other genes for metabolic engineering applications of S. cerevisiae.     

Cell Therapy: A Safe and Efficacious Therapeutic Treatment for Alzheimer��s Disease in APP+PS1 Mice

Previously, our lab was the first to report the use of antigen-sensitized dendritic cells as a vaccine against Alzheimer’s disease (AD). In preparation of this vaccine, we sensitized the isolated dendritic cells ex vivo with Aβ peptide, and administered these sensitized dendritic cells as a therapeutic agent. This form of cell therapy has had success in preventing and/or slowing the rate of cognitive decline when administered prior to the appearance of Aβ plaques in PDAPP mice, but has not been tested in 2×Tg models. Herein, we test the efficacy and safety of this vaccine in halting and reversing Alzheimer’s pathology in 9-month-old APP+PS1 mice. The results showed that administration of this vaccine elicits a long-lasting antibody titer, which correlated well with a reduction of Aβ burden upon histological analysis. Cognitive function in transgenic responders to the vaccine was rescued to levels similar to those found in non-transgenic mice, indicating that the vaccine is capable of providing therapeutic benefit in APP+PS1 mice when administered after the onset of AD pathology. The vaccine also shows indications of circumventing past safety problems observed in AD immunotherapy, as Th1 pro-inflammatory cytokines were not elevated after long-term vaccine administration. Moreover, microhemorrhaging and T-cell infiltration into the brain are not observed in any of the treated subjects. All in all, this vaccine has many advantages over contemporary vaccines against Alzheimer’s disease, and may lead to a viable treatment for the disease in the future.     

Theoretical investigation of the radical scavenging activity of shikonin and acylshikonin derivatives

The radical scavenging activity of shikonin and acylshikonin derivatives was studied by using density functional theory. The hydrogen bond property of the studied structures was investigated using the atoms in molecules (AIM) theory. It turned out that the hydrogen bond is important for good radical scavenging activity. The hydrogen atom transfer for shikonin and acylshikonin derivatives is difficult to obtain because of the high bond dissociation energy (BDE). However, shikonin and acylshikonin derivatives appear to be good candidates for the one-electron-transfer. The introduction of acyl groups for shikonin decreases the ionization potential (IP) values compared with that of shikonin. The acylshikonin derivatives with 1H-pyrrole, furan, and thiophene groups are expected to be of the highest radical scavenging activity among the compounds investigated in this study. Taking this system as an example, we present an efficient method for the investigation of radical scavenging activity from theoretical point of view.     

Crucial Involvement of Tumor-Associated Neutrophils in the Regulation of Chronic Colitis-Associated Carcinogenesis in Mice

Ulcerative colitis (UC) is a major form of chronic inflammation that can frequently progress to colon cancer. Several studies have demonstrated massive infiltration of neutrophils and macrophages into the lamina propria and submucosa in the progression of UC-associated colon carcinogenesis. Macrophages contribute to the development of colitis-associated colon cancer (CAC). However, the role of neutrophils is not well understood. To better understand the involvement of tumor-associated neutrophils (TANs) in the regulation of CAC, we used a mouse CAC model produced by administering azoxymethane (AOM), followed by repeated dextran sulfate sodium (DSS) ingestion. This causes severe colonic inflammation and subsequent development of multiple tumors in mice colon. We observed that colorectal mucosal inflammation became increasingly severe with AOM and DSS treatment. Macrophages infiltrated the lamina propria and submucosa, together with a marked increase in neutrophil infiltration. The chemokine CXCL2 increased in the lamina propria and submucosal regions of the colons of the treated mice, together with the infiltration of neutrophils expressing CXCR2, a specific receptor for CXCL2. This process was followed by neoplastic transformation. After AOM and DSS treatment, the mice showed enhanced production of metalloproteinase (MMP)-9 and neutrophil elastase (NE), accompanied by excessive vessel generation and cell proliferation. Moreover, CXCL2 promoted neutrophil recruitment and induced neutrophils to express MMP-9 and NE in vitro. Furthermore, administration of neutrophil-neutralizing antibodies after the last DSS cycle markedly reduced the number and size of tumors and decreased the expression of CXCR2, CXCL2, MMP-9, and NE. These observations indicate a crucial role for TANs in the initiation and progression of CAC and suggest that the CXCL2–CXCR2 axis might be useful in reducing the risk of UC-associated colon cancer.     

Strong activation of cyclooxygenase I and II catalytic activity by dietary bioflavonoids

Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to prostaglandins (PGs), thromboxanes, and hydroxyeicosatetraenoic acids. In the present study, we investigated several dietary bioflavonoids for their ability to modulate the catalytic activity of COX I and II in vitro and also in cultured cells. We found that some of them are the most powerful direct stimulators of the catalytic activity of COX I and II known to date, increasing the formation of prostaglandin products in vitro by up to 11-fold over the controls. This stimulatory effect of bioflavonoids is enzyme specific because none of them stimulates the catalytic activity of a number of lipooxygenases tested. Compared with phenol, a prototypical COX stimulator commonly used in vitro, the naturally occurring bioflavonoids are up to 29 times more efficacious in stimulating the COX activity. Additional studies using intact cells in culture showed that some of the dietary compounds that were active in the biochemical assays also activated the formation of PGE(2) (a representative PG) when they were present at 0.01 to 1 muM concentrations. The stimulatory effect of dietary compounds on COX-mediated PG formation is far more potent in intact cells than in the in vitro assays. Mechanistically, bioflavonoids mainly acted to slow down the suicidal inactivation of the COX enzymes, but they did not appear to reactivate the inactivated enzymes. The finding of this study suggests that some of the bioflavonoids likely will serve as the naturally occurring cofactors for the COX enzymes in humans.     

Lineage-Specific Domain Fusion in the Evolution of Purine Nucleotide Cyclases in Cyanobacteria

Cyclic nucleotides (both cAMP and cGMP) play extremely important roles in cyanobacteria, such as regulating heterocyst formation, respiration, or gliding. Catalyzing the formation of cAMP and cGMP from ATP and GTP is a group of functionally important enzymes named adenylate cyclases and guanylate cyclases, respectively. To understand their evolutionary patterns, in this study, we presented a systematic analysis of all the cyclases in cyanobacterial genomes. We found that different cyanobacteria had various numbers of cyclases in view of their remarkable diversities in genome size and physiology. Most of these cyclases exhibited distinct domain architectures, which implies the versatile functions of cyanobacterial cyclases. Mapping the whole set of cyclase domain architectures from diverse prokaryotic organisms to their phylogenetic tree and detailed phylogenetic analysis of cyclase catalytic domains revealed that lineage-specific domain recruitment appeared to be the most prevailing pattern contributing to the great variability of cyanobacterial cyclase domain architectures. However, other scenarios, such as gene duplication, also occurred during the evolution of cyanobacterial cyclases. Sequence divergence seemed to contribute to the origin of putative guanylate cyclases which were found only in cyanobacteria. In conclusion, the comprehensive survey of cyclases in cyanobacteria provides novel insight into their potential evolutionary mechanisms and further functional implications.     

Matrix metalloproteinase 9 facilitates West Nile virus entry into the brain

West Nile virus (WNV) is the most-common cause of mosquito-borne encephalitis in the United States. Invasion of the brain by WNV is influenced by viral and host factors, and the molecular mechanism underlying disruption of the blood-brain barrier is likely multifactorial. Here we show that matrix metalloproteinase 9 (MMP9) is involved in WNV entry into the brain by enhancing blood-brain barrier permeability. Murine MMP9 expression was induced in the circulation shortly after WNV infection, and the protein levels remained high even when viremia subsided. In the murine brain, MMP9 expression and its enzymatic activity were upregulated and MMP9 was shown to partly localize to the blood vessels. Interestingly, we also found that cerebrospinal fluid from patients suffering from WNV contained increased MMP9 levels. The peripheral viremia and expression of host cytokines were not altered in MMP9(-/-) mice; however, these animals were protected from lethal WNV challenge. The resistance of MMP9(-/-) mice to WNV infection correlated with an intact blood-brain barrier since immunoglobulin G, Evans blue leakage into brain, and type IV collagen degradation were markedly reduced in the MMP9(-/-) mice compared with their levels in controls. Consistent with this, the brain viral loads, selected inflammatory cytokines, and leukocyte infiltrates were significantly reduced in the MMP9(-/-) mice compared to their levels in wild-type mice. These data suggest that MMP9 plays a role in mediating WNV entry into the central nervous system and that strategies to interrupt this process may influence the course of West Nile encephalitis.