Heritable Custom Genomic Modifications in Caenorhabditis elegans via a CRISPR–Cas9 System

We adapted the CRISPR–Cas9 system for template-mediated repair of targeted double-strand breaks via homologous recombination in Caenorhabditis elegans, enabling customized and efficient genome editing. This system can be used to create specific insertions, deletions, and base pair changes in the germline of C. elegans.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Evaluation and biosynthetic incorporation of chlorotyrosine into recombinant proteins

Recently, non-canonical amino acids (NCAA) incorporation was developed to enhance the functional properties of proteins. Incorporation of NCAA containing chlorine atom is conceptually an attractive approach to prepare pharmacologically active substances, which is a difficult task since chlorine is bulky atom. In this study, we evaluated the efficiency and extent of in vivo incorporation of tyrosine analogue 3-chlorotyrosine [(3-Cl)Tyr] into the recombinant proteins GFP and GFPHS (highly stable GFP). The incorporation of (3-Cl)Tyr into GFP leads to dramatic reduction in the expression level of protein. On the other hand, the incorporation of (3-Cl)Tyr into GFPHS was expressed well as a soluble form. In addition we used bioinformatics tools for the analysis to explore the possible constraints in micro-environment of each natural amino acid residue to be replaced with chlorine atom accommodation into GFPHS. In conclusion, our approaches are reliable and straightforward way to enhance the translation of chlorinated amino acids into proteins.     

<Emphasis Type="Italic">α</Emphasis>-Amylase inhibitor-1 gene from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> expressed in <Emphasis Type="Italic">Coffea arabica</Emphasis>plants inhibits α-amylases from the coffee berry borer pest

Background  

Coffee is an important crop and is crucial to the economy of many developing countries, generating around US70 billion per year. There are 115 species in the < i > Coffea < /i > genus, but only two, < i > C. arabica < /i > and < i > C. canephora < /i > , are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer ( < i > Hypotheneumus hampei < /i > ), is responsible for worldwide annual losses of around US70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.     

Optimisation of the azinobis-3-ethyl-benzothiazoline-6-sulphonic acid radical scavenging assay for physiological studies of total antioxidant activity in woody plant germplasm.

A robust spectroscopic method for determining total antioxidant activity in aqueous extractions has been applied to tissues from diverse woody plant species, including seeds of Coffea arabica and in vitro shoots from Ribes nigrum, Picea sitchensis and Shorea leprosula. The assay involves scavenging of an ABTS [2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid)] radical generated by the reaction of potassium persulphate with ABTS to produce an ABTS*(+) chromophore (lambda=734 nm). Antioxidants reduce ABTS*(+) back to ABTS with a concomitant decrease in absorbance. Aqueous extractions from C. arabica and S. leprosula had considerably higher (110-205 micromol Trolox eq. g(-1) FW) total antioxidant activities than P. sitchensis and R. nigrum (6-11 micromol Trolox eq. g(-1) FW). Further studies in two of these species showed that the inclusion of water-insoluble polyvinylpyrrolidone during aqueous tissue extraction enabled the combined phenolic and alkaloid antioxidant activity to be determined. These fractions accounted for 85% and 60% of total antioxidant activity for C. arabica seeds and R. nigrum shoots, respectively. The ABTS radical scavenging assay is presented herein as a robust method for determining total antioxidant activity in germplasm from diverse woody plant tissues and species. Its applicability to study oxidative stress in tissue cultures and germplasm employed in plant biotechnology, breeding and stress physiology programmes is discussed.     

Chemoproteomics reveals Toll-like receptor fatty acylation

Background

Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells.

Results

A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands.

Conclusions

This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. S-palmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.

     

Comparsion of staining characteristics of Toto bodies

Toto bodies are eosinophilic structures that resemble the cells of the superficial cell layer of the oral epithelium. Toto bodies commonly are associated with inflammatory gingival and other mucosal lesions including pyogenic granuloma, irritational fibroma, epulis fissuratum, peripheral giant cell granuloma and inflammatory hyperplastic gingivitis. We evaluated staining characteristics of Toto bodies to establish their origin and to identify their significance in lesions. We investigated pyogenic granuloma, fibroma and leukoplakia with epithelium that exhibited Toto bodies after hematoxylin and eosin (staining. Sections were stained with Alcian blue, periodic acid-Schiff and Ayoub-Shklar stains to evaluate staining intensity and distribution. More Toto bodies were found in pyogenic granuloma than in fibroma and leukoplakia. PAS and Alcian blue staining exhibited mild intensity and did not establish the origin of Toto bodies. High staining intensity and diffuse distribution of stain was observed using Ayoub-Shklar staining, which indicated that Toto bodies originate from keratin.     

Biosynthetic substitution of tyrosine in green fluorescent protein with its surrogate fluorotyrosine in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Introduction of a fluorine moiety into green fluorescent protein offers an interesting novel spectral variant. The calculated binding energy of fluorotyrosine (F-Tyr) (−8.42 kcal/mol) for tyrosyl tRNA synthetase was moderately higher than that of tyrosine (Tyr) (−8.36 kcal/mol). This result directly correlated with the expression level of F-Tyr containing GFP (38 mg/l), which was comparably higher than that of the parent GFP expression level (34 mg/l). Finally, we generated a model structure for GFP to assess possible interaction in the chromophore of the protein structure, which plays an important role in determining the spectral and folding behaviors of the F-Tyr incorporated GFP variant.