Immunoregulation Effects of Bone Marrow-Derived Mesenchymal Stem Cells in Xenogeneic Acellular Nerve Grafts Transplant

This study evaluated whether bone marrow-derived mesenchymal stem cells (BM-MSCs) combined with xenogeneic acellular nerve grafts (xANGs) would reduce the inflammation reaction of xANGs transplantation. BM-MSCs were extracted, separated, purified, and cultured from the bone marrow of rats. Then BM-MSCs were seeded into 5 mm xANGs as experimental group, while xANGs group was chosen as control. Subcutaneous implantation and nerve grafts transplantation were done in this study. Walking-track tests, electrophysiological tests, H&E staining, and immunostaining of CD4, CD8, and CD68 of subcutaneous implantations, cytokine concentrations of IL-2, IL-10, IFN-γ and TNF-α in lymphocytes supernatants and serum of the two groups were evaluated. Walking-track tests and electrophysiological tests suggested the group of BM-MSCs with xANGs obtained better results than xANGs group (P < 0.05). H&E staining and immunostaining of CD4, CD8, and CD68 of subcutaneous implantations showed there were less inflammatory cells in the group of BM-MSCs when compared with the xANGs group. The cytokine concentrations of IL-2, IFN-γ, and TNF-α in BM-MSCs group were lower than xANGs group in lymphocytes supernatants and serum (P < 0.05). However, IL-10 concentrations in BM-MSCs group were higher than xANGs group (P < 0.05). xANG with BM-MSCs showed better nerve repair function when compared with xANG group. Furthermore, xANG with BM-MSCs showed less inflammatory reaction which might indicate the reason of its better nerve regeneration.     

The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells

The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals.     

Interaction Between Umami Peptide and Taste Receptor T1R1/T1R3

The umami taste receptor is a heterodimer composed of two members of the T1R taste receptor family: T1R1 and T1R3. The homology models of the ligand binding domains of the human umami receptor have been constructed based on crystallographic structures of the taste receptor of the central nervous system. Furthermore, the molecular simulations of the ligand binding domain show that the likely conformation was that T1R1 protein exists in the closed conformation, and T1R3 in the open conformation in the heterodimer. The molecular docking study of T1R1 and T1R3 in complex with four peptides, including Lys–Gly–Asp–Glu–Ser–Leu–Leu–Ala, Ser–Glu–Glu, G1u–Ser, and Asp–Glu–Ser, displayed that the amino acid residue of SER146 and Glu277 in T1R3 may play great roles in the synergism of umami taste. This docking result further validated the robustness of the model. In the paper, binding of umami peptide and the T1R1/T1R3 receptor was first described and the interaction is the base of umami activity theory.     

Wnt7a stimulates myogenic stem cell motility and engraftment resulting in improved muscle strength

Wnt7a/Fzd7 signaling stimulates skeletal muscle growth and repair by inducing the symmetric expansion of satellite stem cells through the planar cell polarity pathway and by activating the Akt/mTOR growth pathway in muscle fibers. Here we describe a third level of activity where Wnt7a/Fzd7 increases the polarity and directional migration of mouse satellite cells and human myogenic progenitors through activation of Dvl2 and the small GTPase Rac1. Importantly, these effects can be exploited to potentiate the outcome of myogenic cell transplantation into dystrophic muscles. We observed that a short Wnt7a treatment markedly stimulated tissue dispersal and engraftment, leading to significantly improved muscle function. Moreover, myofibers at distal sites that fused with Wnt7a-treated cells were hypertrophic, suggesting that the transplanted cells deliver activated Wnt7a/Fzd7 signaling complexes to recipient myofibers. Taken together, we describe a viable and effective ex vivo cell modulation process that profoundly enhances the efficacy of stem cell therapy for skeletal muscle.     

MicroRNA-135a Inhibits Cell Proliferation by Targeting Bmi1 in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal solid tumor due to the lack of reliable early detection markers and effective therapies. MicroRNAs (miRNAs), noncoding RNAs that regulate gene expression, are involved in tumorigenesis and have a remarkable potential for the diagnosis and treatment of malignancy. In this study, we investigated aberrantly expressed miRNAs involved in PDAC by comparing miRNA expression profiles in PDAC cell lines with a normal pancreas cell line and found that miR-135a was significantly down-regulated in the PDAC cell lines. The microarray results were validated by qRT-PCR in PDAC tissues, paired adjacent normal pancreatic tissues, PDAC cell lines, and a normal pancreas cell line. We then defined the tumor-suppressing significance and function of miR-135a by constructing a lentiviral vector to express miR-135a. The overexpression of miR-135a in PDAC cells decreased cell proliferation and clonogenicity and also induced G1 arrest and apoptosis. We predicted Bmi1 may be a target of miR-135a using bioinformatics tools and found that Bmi1 expression was markedly up-regulated in PDAC. Its expression was inversely correlated with miR-135a expression in PDAC. Furthermore, a luciferase activity assay revealed that miR-135a could directly target the 3''-untranslated region (3''-UTR) of Bmi1. Taken together, these results demonstrate that miR-135a targets Bmi1 in PDAC and functions as a tumor suppressor. miR-135a may offer a new perspective for the development of effective miRNA-based therapy for PDAC.     

Increased of exhaled breath condensate neutrophil chemotaxis in acute exacerbation of COPD

Background

Neutrophils have been involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Underlying mechanisms of neutrophil accumulation in the airways of stable and exacerbated COPD patients are poorly understood. The aim of this study was to assess exhaled breath condensate (EBC) neutrophil chemotactic activity, the level of two chemoattractants for neutrophils (GRO-α and LTB4) during the course of an acute exacerbation of COPD (AECOPD).

Methods

50 ex smoking COPD patients (33 with acute exacerbation and 17 in stable disease) and 20 matched ex smoking healthy controls were compared. EBC was collected by using a commercially available condenser (EcoScreen®). EBC neutrophil chemotactic activity (NCA) was assessed by using Boyden microchambers. Chemotactic index (CI) was used to evaluate cell migration. LTB₄ and GROα levels were measured by a specific enzyme immunoassay in EBC.

Results

Stable COPD and outpatients with AECOPD, but not hospitalized with AECOPD, had raised EBC NCA compared to healthy subjects (p < 0.05 and p < 0.01 respectively). In outpatients with AECOPD EBC NCA significantly decreased 6 weeks after the exacerbation. Overall EBC NCA was weakly correlated with sputum neutrophil counts (r = 0.26, p < 0.05).EBC LTB4 levels were increased in all groups of COPD compared to healthy subjects while GRO-α was only raised in patients with AECOPD. Furthermore, EBC LTB₄ and GRO-α significantly decreased after recovery of the acute exacerbation. Increasing concentrations (0.1 to 10 μg/mL) of anti- human GRO-α monoclonal antibody had no effect on EBC neutrophil chemotactic activity of 10 exacerbated COPD patients.

Conclusions

EBC NCA rose during acute exacerbation of COPD in ambulatory patients and decreased at recovery. While LTB4 seems to play a role both in stable and in exacerbated phase of the disease, the role of GRO-α as a chemotactic factor during AECOPD is not clearly established and needs further investigation.     

FastMG: a simple,fast, and accurate maximum likelihood procedure to estimate amino acid replacement rate matrices from large data sets

Background

Amino acid replacement rate matrices are a crucial component of many protein analysis systems such as sequence similarity search, sequence alignment, and phylogenetic inference. Ideally, the rate matrix reflects the mutational behavior of the actual data under study; however, estimating amino acid replacement rate matrices requires large protein alignments and is computationally expensive and complex. As a compromise, sub-optimal pre-calculated generic matrices are typically used for protein-based phylogeny. Sequence availability has now grown to a point where problem-specific rate matrices can often be calculated if the computational cost can be controlled.

Results

The most time consuming step in estimating rate matrices by maximum likelihood is building maximum likelihood phylogenetic trees from protein alignments. We propose a new procedure, called FastMG, to overcome this obstacle. The key innovation is the alignment-splitting algorithm that splits alignments with many sequences into non-overlapping sub-alignments prior to estimating amino acid replacement rates. Experiments with different large data sets showed that the FastMG procedure was an order of magnitude faster than without splitting. Importantly, there was no apparent loss in matrix quality if an appropriate splitting procedure is used.

Conclusions

FastMG is a simple, fast and accurate procedure to estimate amino acid replacement rate matrices from large data sets. It enables researchers to study the evolutionary relationships for specific groups of proteins or taxa with optimized, data-specific amino acid replacement rate matrices. The programs, data sets, and the new mammalian mitochondrial protein rate matrix are available at http://fastmg.codeplex.com.     

Metabolic Engineering of Escherichia coli for Efficient Conversion of Glycerol to Ethanol

Based on elementary mode analysis, an Escherichia coli strain was designed for efficient conversion of glycerol to ethanol. By using nine gene knockout mutations, the functional space of the central metabolism of E. coli was reduced from over 15,000 possible pathways to a total of 28 glycerol-utilizing pathways that support cell function. Among these pathways are eight aerobic and eight anaerobic pathways that do not support cell growth but convert glycerol into ethanol with a theoretical yield of 0.50 g ethanol/g glycerol. The remaining 12 pathways aerobically coproduce biomass and ethanol from glycerol. The optimal ethanol production depends on the oxygen availability that regulates the two competing pathways for biomass and ethanol production. The coupling between cell growth and ethanol production enabled metabolic evolution of the designed strain through serial dilution that resulted in strains with improved ethanol yields and productivities. In defined medium, the evolved strain can convert 40 g/liter of glycerol to ethanol in 48 h with 90% of the theoretical ethanol yield. The performance of the designed strain is predicted by the property space of remaining elementary modes.With the recent rising prices of fossil fuels, development of alternative renewable fuels, such as biodiesel, has become attractive. However, the increase in biodiesel production generates a surplus of crude glycerol since this compound is an inevitable waste by-product resulting directly from the transesterification of vegetable oils or animal fats. For every 3 mol of biodiesel produced, 1 mol of glycerol (about 5 to 10% weight equivalent of biodiesel) is generated. To maximize the full economic potential of the biodiesel process, it is important to convert crude glycerol into useful chemicals (9, 25).Both chemical conversion and biological conversion of crude glycerol into value-added products have been considered. For instance, chemical conversion based on the etherification of glycerol with either alcohols (methanol or ethanol) or alkenes (isobutene or 2-methylpropene) can produce useful fuels or solvents, or steam reforming of glycerol can result in methanol and hydrogen production (7). Biological conversion utilizes species belonging to the Enterobacteriaceae family, such as Klebsiella pneumoniae (11), Citrobacter freundii (5), Clostridium butyricum (4), and Pantoea agglomerans (3), to convert glycerol to 1,3-propanediol by fermentation.Even though Escherichia coli belongs to the family Enterobacteriaceae, it cannot ferment glycerol due to a lack of the dha regulon encoding glycerol dehydratase (dhaB) and 1,3-propanediol oxidoreductase (dhaT), which constitute the fermentative 1,3-propanediol-producing pathway (18). However, introduction of this pathway from K. pneumoniae into E. coli can facilitate glycerol fermentation (18), presumably because the redox potential is balanced. In fact, it is well documented that a wild-type E. coli strain cannot grow on glycerol anaerobically in defined medium except after addition of specific electron acceptors (18). Such electron acceptors can come from external sources, including nitrate, nitrite, fumarate, dimethyl sulfoxide, or trimethylalamine-N-oxide, or from internal sources through added fermentative pathways, such as the 1,3-propanediol-producing pathway.A recent study suggested the feasibility of fermenting glycerol into fuels and other reduced chemicals by inducing the dormant, native 1,2-propanediol fermentative pathway in E. coli without using external electron acceptors (10, 14). This approach, however, faces several critical challenges, such as low specific growth rates resulting in low chemical productivities and coproduction of unavoidable by-products, such as 1,2-propanediol. The reported specific growth rate appears to limit any practical application since a minimal doubling time of about 17 h results in consumption of only 8 to 10 g/liter glycerol after 110 h (10, 14). In addition, glycerol fermentation apparently worked only when complex components, such as yeast extract, tryptone, or amino acids that are required for biomass synthesis, were added to the medium (10, 14).Here, we took a different approach by employing oxygen as the electron acceptor in well-defined microaerobic growth conditions. We used elementary mode (EM) analysis to rationally design an E. coli cell with minimized metabolic functionality tailored to efficiently convert glycerol to ethanol under these microaerobic growth conditions. EM analysis is a metabolic pathway analysis tool that identifies all pathway options in a metabolic network (16). Knowledge of these pathway options allows rational implementation of only the efficient pathways of interest by removing the inefficient pathways, resulting in a cell with minimal but specialized functionality (20-22, 24). Furthermore, the cell developed is engineered to tightly couple cell growth and ethanol production. This unique characteristic facilitates metabolic evolution of the minimal cell to improve ethanol productivity because faster-growing cells also produce ethanol at a higher rate. We demonstrate here that the performance of the designed strain falls into the range defined by the EMs that are possible.     

Energy Taxis Drives Campylobacter jejuni toward the Most Favorable Conditions for Growth

Campylobacter jejuni is a serious food-borne bacterial pathogen in the developed world. Poultry is a major reservoir, and C. jejuni appears highly adapted to the gastrointestinal tract of birds. Several factors are important for chicken colonization and virulence, including a taxis mechanism for environmental navigation. To explore the mechanism of chemotaxis in C. jejuni, we constructed mutants with deletions of five putative mcp (methyl-accepting chemotaxis protein) genes (tlp1, tlp2, tlp3, docB, and docC). Surprisingly, the deletions did not affect the chemotactic behavior of the mutants compared to that of the parental strain. However, the tlp1, tlp3, docB, and docC mutant strains displayed a 10-fold decrease in the ability to invade human epithelial and chicken embryo cells, hence demonstrating that the corresponding proteins affect the host interaction. l-Asparagine, formate, d-lactate, and chicken mucus were identified as new attractants of C. jejuni, and we observed that chemical substances promoting tactic attraction are all known to support the growth of this organism. The attractants could be categorized as carbon sources and electron donors and acceptors, and we furthermore observed a correlation between an attractant''s potency and its efficiency as an energy source. The tactic attraction was inhibited by the respiratory inhibitors HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) and sodium azide, which significantly reduce energy production by oxidative phosphorylation. These findings strongly indicate that energy taxis is the primary force in environmental navigation by C. jejuni and that this mechanism drives the organism toward the optimal chemical conditions for energy generation and colonization.The food-borne pathogen Campylobacter jejuni is highly adapted to the environment of the avian gut, where the mucus-filled crypts of the lower gastrointestinal tract are the primary site of colonization (6). It has been speculated that C. jejuni bacteria apply chemotaxis to reach this particular milieu (10, 42). Chemotaxis allows motile bacteria to navigate according to the extracellular chemical composition. The bacteria are either attracted or repelled by chemicals sensed by trans-membrane methyl-accepting chemotaxis proteins (MCP), and the information is transmitted to the flagellum motor via the histidine kinase CheA and the response regulator CheY. In contrast to the classical, metabolism-independent chemotaxis, some bacteria, such as Azospirillum brasilense and Rhodobacter sphaeroides, display metabolism-dependent “energy taxis” (or redox taxis) in which the signal for navigation originates within the electron transport system (1, 37).C. jejuni NCTC11168 encodes 10 MCP-like proteins, termed Tlp (transducer-like proteins), and two proteins with homology to aerotaxis receptors (Aer) (31). A taxis mechanism is essential for C. jejuni colonization, since strains with mutations of the central histidine kinase, cheA, or the response regulator, cheY, are unable to establish colonization in mice, chickens, and ferrets (10, 20, 51). Furthermore, C. jejuni strains with mutations in docB (tlp10) and docC (tlp4) are severely impaired in establishing colonization in chickens, whereas none of the other putative receptors are required for colonization (however, tlp5 could not be mutated) (20). Mutants of aer2 (cetB) and tlp9 (cetA) displayed reduced migration in minimal medium supplemented with pyruvate or fumarate, which leads to the speculation that CetA and CetB mediate an energy taxis response of C. jejuni (19).C. jejuni is attracted to amino acids, organic acids, or mucus components, while it is repelled by bile components (23). However, specific Tlp proteins have not been matched to any of these substances. It is speculated that the attraction toward chicken mucus directs and retains C. jejuni in the optimal environment of the avian intestinal lumen and thus prevents direct interaction with epithelial cells. This notion is based on in vitro observations where chicken mucus inhibited C. jejuni invasion of primary human epithelial cells, while increased invasion was observed for mutants carrying deletions of either cheA or cheY (9, 44, 51).To explore the mechanism of C. jejuni chemotaxis and analyze the biological functions of individual MCP-like proteins, we have analyzed five mutants with deletions of tlp genes (tlp1, tlp2, tlp3, docB, and docC). Furthermore, we have explored whether C. jejuni is primarily driven by chemotaxis or energy taxis.