Exosomes surf on filopodia to enter cells at endocytic hot spots,traffic within endosomes,and are targeted to the ER

Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.     

Hydrolysis of polyethyleneterephthalate by p‐nitrobenzylesterase from Bacillus subtilis

From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p‐nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET‐hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2‐hydroxyethyl benzoate (HEB), and mono‐(2‐hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET‐hydrolase was isolated from non‐denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC‐MS/MS analysis. BsEstB was expressed in Escherichia coli with C‐terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p‐nitrophenyl acetate and 108 U/mg on p‐nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half‐life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2‐hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The k_cat values decreased with increasing complexity of the substrate from 6 and 8 (s?1) for p‐nitrophenyl‐acetate (4NPA) and p‐nitrophenyl‐butyrate (4NPB), respectively, to 0.14 (s?1) for bis(2‐hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water‐contact angle (WCA) from 68.2° ± 1.7° to 62.6° ± 1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2‐(bromomethyl)naphthalene. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Gaining knowledge from previously unexplained spectra-application of the PTM-Explorer software to detect PTM in HUPO BPP MS/MS data

A novel software tool named PTM-Explorer has been applied to LC-MS/MS datasets acquired within the Human Proteome Organisation (HUPO) Brain Proteome Project (BPP). PTM-Explorer enables automatic identification of peptide MS/MS spectra that were not explained in typical sequence database searches. The main focus was detection of PTMs, but PTM-Explorer detects also unspecific peptide cleavage, mass measurement errors, experimental modifications, amino acid substitutions, transpeptidation products and unknown mass shifts. To avoid a combinatorial problem the search is restricted to a set of selected protein sequences, which stem from previous protein identifications using a common sequence database search. Prior to application to the HUPO BPP data, PTM-Explorer was evaluated on excellently manually characterized and evaluated LC-MS/MS data sets from Alpha-A-Crystallin gel spots obtained from mouse eye lens. Besides various PTMs including phosphorylation, a wealth of experimental modifications and unspecific cleavage products were successfully detected, completing the primary structure information of the measured proteins. Our results indicate that a large amount of MS/MS spectra that currently remain unidentified in standard database searches contain valuable information that can only be elucidated using suitable software tools.     

Passive protective effect of egg-yolk antibodies against enterotoxigenic Escherichia coli K88+ infection in neonatal and early-weaned piglets

The protective effects of egg-yolk antibodies obtained from hens immunized with fimbrial antigens from a local strain (Escherichia coli K88+ MB, Manitoba, Canada) of K88+ piliated enterotoxigenic E. coli (ETEC) were evaluated in 3- and 21-day-old piglets in which ETEC diarrhea was induced and also in early-weaned piglets in a commercial farm. The results demonstrated that the E. coli K88+ MB-induced diarrhea in 3-day-old piglets was cured 24 h after treating with egg-yolk antibodies while those treated with egg-yolk powder from conventional hens continued to have diarrhea and 62.5% of them died of severe diarrhea. For 21-day-old weaned piglets, those fed egg-yolk antibodies had transient diarrhea, positive body weight gains and 100% survival during the period of the experiment, whereas control piglets that were treated with placebo had severe diarrhea and dehydration and some died within 48 h after infection. In the field trial, the incidence and severity of diarrhea of 14-18-day-old weaned piglets fed egg-yolk antibodies were much lower than in those fed a commercial diet containing an antibiotic. These results indicate that the neonatal and early-weaned piglets that received the egg-yolk antibodies were protected against ETEC infection.     

A novel transmembrane MSP-containing protein that plays a role in right ventricle development

We have identified and characterized a gene, Mospd3 on mouse chromosome 5 using gene trapping in ES cells. MOSPD3 is part of a family of proteins, including MOSPD1, which is defined by the presence of a major sperm protein (MSP) domain and two transmembrane domains. Interestingly Mospd3 is mammalian specific and highly conserved between mouse and man. Insertion of the gene trap vector at the Mospd3 locus is mutagenic and breeding to homozygosity results in a characteristic right ventricle defect and neonatal lethality in 50% of mice. The phenotypic defect is dependent on the genetic background, indicating the presence of genetic modifier loci. We speculate that the further characterization of Mospd3 will shed light on the complex genetic interactions involved in cardiac development and disease.     

High-level production of 1,3-propanediol from crude glycerol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> AKR102a

The aim of this study was to optimize a biotechnological process for the production of 1,3-propanediol (1,3-PD) based on low-quality crude glycerol derived from biodiesel production. Clostridium butyricum AKR102a was used in fed-batch fermentations in 1-L and 200-L scale. The newly discovered strain is characterized by rapid growth, high product tolerance, and the ability to use crude glycerol at the lowest purity directly gained from a biodiesel plant side stream. Using pure glycerol, the strain AKR102 reached 93.7 g/L 1,3-PD with an overall productivity of 3.3 g/(L*h). With crude glycerol under the same conditions, 76.2 g/L 1,3-PD was produced with a productivity of 2.3 g/(L*h). These are among the best results published so far for natural producers. The scale up to 200 L was possible. Due to the simpler process design, only 61.5 g/L 1,3-PD could be reached with a productivity of 2.1 g/(L*h).     

Individualistic responses of bird species to environmental change

We investigated how the population dynamics of the same bird species varied in different environments, and how the population dynamics of different species varied in the same environment, by calculating long-term population trends for 59 insectivorous songbird species in 22 regions or strata of eastern and central North America using data from the North American Breeding Bird Survey. Of the 47 species that occurred in more than one region 77% increased in some regions and declined in others. Of the 22 regions 91% had some species that increased and others that decreased. There were only slightly more significant correlations between strata in species trends and between species for stratum trends than would be expected by chance. Because of nonlinearities in the data, the actual patterns of population fluctuations of the same species in different regions and of different species in the same region were even more heterogeneous than suggested by our analyses of linear trends. We conclude that these bird species respond to spatial and temporal variation in their environment in a very individualistic fashion. These individualistic responses show that the extrapolation of population trends gained from a few local studies to a larger spatial scale, and the use of a few indicator species to monitor the status of a broader community, are suspect.     

Investigation of the potential for triterpene synthesis in rice through genome mining and metabolic engineering

The first committed step in sterol biosynthesis in plants involves the cyclization of 2,3-oxidosqualene by the oxidosqualene cyclase (OSC) enzyme cycloartenol synthase. 2,3-Oxidosqualene is also a precursor for triterpene synthesis. Antimicrobial triterpenes are common in dicots, but seldom found in monocots, with the notable exception of oat. Here, through genome mining and metabolic engineering, we investigate the potential for triterpene synthesis in rice. The first two steps in the oat triterpene pathway are catalysed by a divergent OSC (AsbAS1) and a cytochrome P450 (CYP51). The genes for these enzymes form part of a metabolic gene cluster. To investigate the origins of triterpene synthesis in monocots, we analysed systematically the OSC and CYP51 gene families in rice. We also engineered rice for elevated triterpene content. We discovered a total of 12 OSC and 12 CYP51 genes in rice and uncovered key events in the evolution of triterpene synthesis. We further showed that the expression of AsbAS1 in rice leads to the accumulation of the simple triterpene, β-amyrin. These findings provide new insights into the evolution of triterpene synthesis in monocots and open up opportunities for metabolic engineering for disease resistance in rice and other cereals.