Use of fluorochrome-labeled rRNA targeted oligonucleotide probe and tyramide signal amplification to improve sensitivity of fluorescence in situ hybridization

A tyramide signal amplification (TSA) system was used in combination with a conventional fluorochrome-labeled 16S rRNA oligonucleotide probe to increase the sensitivity of fluorescence in situ hybridization. TSA was performed after hybridization resulted in a low fluorescence signal intensity. In contrast to the horseradish peroxidase-tyramide signal amplification (HRP-TSA) system and biotin-tyramide signal amplification (biotin-TSA) system, no additional expensive probe labeling was required. A whole cell hybridization technique was used to compare the fluorescence signal obtained using a monolabeled probe with that obtained using the TSA system. The fluorescence signal of the probe obtained using the TSA system was much higher than that obtained using the monolabeled probe. The technique was successfully applied to the in situ detection of microbial communities in anaerobic sludge. It was demonstrated that TSA resulted in an increased in sensitivity, as the fluorescence signal intensity was much higher than that obtained using a conventional probe.     

Towards Dynamic Drug Design: Identification and Optimization of β‐Galactosidase Inhibitors from a Dynamic Hemithioacetal System

A discovery strategy relying on the identification of fragments through resolution of a constitutional dynamic system, coupled to subsequent static ligand design and optimization, is demonstrated. The strategic design and synthesis of the best molecular fragments identified from a dynamic hemithioacetal system into static ligand structures yielded a range of β‐galactosidase inhibitors. Two series of structures mimicking the hemithioacetal motif were envisaged: thioglycosides and C‐glycosides. Inhibition studies provided important structural information for the two groups, and 1‐thiobenzyl‐β‐D ‐galactopyranoside demonstrated the best inhibitory effects.     

Preparation and characterization of modified telechelic natural rubber-based pressure-sensitive adhesive

Telechelic natural rubber (TNR) was prepared by the use of potassium persulfate and propanal at 70 °C and various degradation times from 0 to 30 h. These samples were then grafted by maleic anhydride (MA) in toluene solution before modification with 3-amino-1,2,4-triazole (ATA) to produce modified TNRs (AMTNRs). Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used to identify the chemical compositions. Carboxyl and hydroxyl groups of TNRs were clearly observed, due to chain scission, oxidation, and modified chain ends. The viscosities of TNRs were dropped greatly after 5 h and then decreased slowly as a function of degradation time. ATR-FTIR spectra of AMTNRs showed amide bonds between ATA and MA groups, and then the multiple hydrogen bonding arrays were formed. The glass transition temperatures (T_g) of AMTNRs were determined by differential scanning calorimetry. The T_g of AMTNR_0 moved to a higher temperature of –55 °C after modification by ATA, confirming the formation of multiple hydrogen bonding arrays. However, the T_g of AMTNR_5 to AMTNR_30 decreased slightly due to chain scission in the degradation process. The adhesive properties of AMTNR-based pressure-sensitive adhesive were evaluated by a Lloyd adhesion tester. The tack of AMTNRs depended on wettability whereas peel and shear strengths were responded by a combination between wettability and multiple hydrogen bonding arrays.     

Expression of the male reproduction‐related gene in spermatic ducts of the blue swimming crab,Portunus pelagicus,and transfer of modified protein to the sperm acrosome

Expression of a sex‐specific gene in Macrobrachium rosenbergii (Mr‐Mrr), encoding a male reproduction‐related (Mrr) protein, has been identified in the spermatic ducts (SDs) and postulated to be involved in sperm maturation processes. M. rosenbergii is the only decapod that the expression and fate of the Mrr protein has been studied. To determine that this protein was conserved in decapods, we firstly used cloning techniques to identify the Mrr gene in two crabs, Portunus pelagicus (Pp‐Mrr) and Scylla serrata (Ss‐Mrr). We then investigated expression of Pp‐Mrr by in situ hybridization, and immunolocalization, as well as phosphorylation and glycosylation modifications, and the fate of the protein in the male reproductive tract. Pp‐Mrr was shown to have 632 nucleotides, and a deduced protein of 110 amino acids, with an unmodified molecular weight of 11.79 kDa and a mature protein with molecular weight of 9.16 kDa. In situ hybridization showed that Pp‐Mrr is expressed in the epithelium of the proximal, middle, distal SDs, and ejaculatory ducts. In Western blotting, proteins of 10.9 and 17.2 kDa from SDs were all positive using anti‐Mrr, antiphosphoserine/threonine, and antiphosphotyrosine. PAS staining showed they were also glycosylated. Immunolocalization studies showed Pp‐Mrr in the SD epithelium, lumen, and on the acrosomes of spermatozoa. Immunofluorescence staining indicated the acrosome of spermatozoa contained the Mrr protein, which is phosphorylated with serine/threonine and tyrosine, and also glycosylated. The Mrr is likely to be involved in acrosomal activation during fertilization of eggs. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

A Dynamic Multicomponent Approach for One-Pot Synthesis of 3-Thioisoindolinones

A dynamic multicomponent reaction concept has been successfully applied to the syntheses of 3-thioisoindolinones and tricyclic γ-lactams. The reactions were efficiently designed and operated in the absence of any catalyst under mild reaction conditions, resulting in the convenient variation of substituents on the N- and S-positions of the target products.     

Asymmetric Synthesis of Substituted Thiolanes through Domino Thia‐Michael–Henry Dynamic Covalent Systemic Resolution using Lipase Catalysis

Dynamic systems based on consecutive thia‐Michael and Henry reactions were generated and transformed using lipase‐catalyzed asymmetric transformation. Substituted thiolane structures with three contiguous stereocenters were resolved in the process in high yields and high enantiomeric excesses.

     

Biosynthesis of xyrrolin, a new cytotoxic hybrid polyketide/non-ribosomal peptide pyrroline with anticancer potential, in Xylaria sp. BCC 1067

A gene from Xylaria sp. BCC 1067, pks3, that encodes a putative 3660-residue hybrid polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) was characterised by targeted gene disruption in combination with comprehensive product identification. Studies of the features of a corresponding mutant, YA3, allowed us to demonstrate that pks3 is responsible for the synthesis of a new pyrroline compound, named xyrrolin, in the wild-type Xylaria sp. BCC 1067. The structure of xyrrolin was established by extensive spectroscopic and spectrometric analyses, including low- and high-resolution MS, IR, (1)H NMR, (13)C NMR, (13)C NMR with Dept135, HMQC 2D NMR, HMBC 2D NMR and COSY 2D NMR. On the basis of the Pks3 domain organisation and the chemical structure of xyrrolin, we proposed that biosynthesis of this compound requires the condensation of a tetraketide and an L-serine unit, followed by Dieckmann or reductive cyclisation and enzymatic removal of ketone residue(s). Bioassays of the pure xyrrolin further displayed cytotoxicity against an oral cavity (KB) cancer cell line.     

Nylon fibers as supporting media in anaerobic hybrid reactors: it's effects on system's performance and microbial distribution

The performances of three anaerobic hybrid reactors with various nylon fiber densities per packed bed volume (33, 22, and 11 kg/m(3) in R1, R2, and R3, respectively) as supporting media were evaluated through their ability to remove organic compounds in cassava starch wastewater. In addition, the distributions of non-methanogenic and methanogenic population in the reactors were investigated. During a 6-month operation, the organic loading rate was increased in stepwise from 0.5 to 4.0 kg COD/m3/day and the hydraulic retention time (HRT) shortened to 5.4 days. The COD removal efficiency was more favorable in R1 (87%) and R2 (84%) than in R3 (70%). The total biomass in the reactors with greater nylon fiber densities was also higher and increased from 20.4 to 67.3 g VSS and to 57.5 g VSS in R1 and R2, respectively. When the HRT was further shortened to 3 days, however, the efficiency of both reactors demonstrated a declining trend and reached 74% in R1 and 61% in R2. The distribution of microbial populations involved in the reactors was determined using the Most Probable Number technique. The result showed the lowest number of methanogens in R3 which correlated well to its relatively low efficiency. The number of non-methanogens in all reactors was, nonetheless, comparable. By shortening the HRT to 3 days, the methanogenic population in R2 diminished in both attached and suspended biomass whereas a slight reduction was detected only in the attached biomass of R1.