Co-degradation of resorcinol and catechol in an UASB reactor

Co-degradation of resorcinol and catechol was studied in a catechol acclimated up flow anaerobic sludge blanket (UASB) reactor. Synthetic binary aqueous solution having a total concentration of 1000 mg/L with the resorcinol/catechol (R/C) ratio of 1/19, 1/9, 3/17, 1/4, 1/3, 3/7, 2/3 and then 1/3 was fed at various time intervals to the UASB reactor with a fixed organic loading rate of 5.7 kg COD/m(3) d and hydraulic retention time of 8h. The reactor was operated over a period of 145 days after its acclimation with catechol bearing synthetic wastewater at a constant feed rate of 1.2 L/h. When the resorcinol concentration was increased to have a R/C ratio of 1/4, the COD removal efficiency and the biogas production increased to the maximum levels. Pseudo steady state condition for COD removal was achieved at each of the stepped-up loading condition. An increase in the R/C ratio above 1/4 in the binary feed solution led to a decrease in the COD removal efficiency and the biogas production rate.     

Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells

The embryonic stem cell-specific cell cycle-regulating (ESCC) family of microRNAs (miRNAs) enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. Here we show that the human ESCC miRNA orthologs hsa-miR-302b and hsa-miR-372 promote human somatic cell reprogramming. Furthermore, these miRNAs repress multiple target genes, with downregulation of individual targets only partially recapitulating the total miRNA effects. These targets regulate various cellular processes, including cell cycle, epithelial-mesenchymal transition (EMT), epigenetic regulation and vesicular transport. ESCC miRNAs have a known role in regulating the unique embryonic stem cell cycle. We show that they also increase the kinetics of mesenchymal-epithelial transition during reprogramming and block TGFβ-induced EMT of human epithelial cells. These results demonstrate that the ESCC miRNAs promote dedifferentiation by acting on multiple downstream pathways. We propose that individual miRNAs generally act through numerous pathways that synergize to regulate and enforce cell fate decisions.     

Real-Time PCR Assays for the Specific Identification of Probiotic Strains Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242)

The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was < 4% for repeatability and < 8% for reproducibility. Both of the assays developed and validated in this study for the specific identification of Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains are specific, sensitive, and precise. These assays can be applied to evaluate and ensure compliance in probiotic products.

     

Location of binding sites in small molecule rescue of human carbonic anhydrase II

Small molecule rescue of mutant forms of human carbonic anhydrase II (HCA II) occurs by participation of exogenous donors/acceptors in the proton transfer pathway between the zinc-bound water and solution. To examine more thoroughly the energetics of this activation, we have constructed a mutant, H64W HCA II, which we have shown is activated by 4-methylimidazole (4-MI) by a mechanism involving the binding of 4-MI to the side chain of Trp-64 approximately 8 A from the zinc. A series of experiments are consistent with the activation of H64W HCA II by the interaction of imidazole and pyridine derivatives as exogenous proton donors with the indole ring of Trp-64; these experiments include pH profiles and H/D solvent isotope effects consistent with proton transfer, observation of approximately fourfold greater activation with the mutant containing Trp-64 compared with Gly-64, and the observation by x-ray crystallography of the binding of 4-MI associated with the indole side chain of Trp-64 in W5A-H64W HCA II. Proton donors bound at the less flexible side chain of Trp-64 in W5A-H64W HCA II do not show activation, but such donors bound at the more flexible Trp-64 of H64W HCA II do show activation, supporting suggestions that conformational mobility of the binding site is associated with more efficient proton transfer. Evaluation using Marcus theory showed that the activation of H64W HCA II by these proton donors was reflected in the work functions w(r) and w(p) rather than in the intrinsic Marcus barrier itself, consistent with the role of solvent reorganization in catalysis.     

A new enzymatic activity from elicitor‐treated pear cell cultures converting trans‐cinnamic acid to benzaldehyde

Cell cultures of Asian pear (Pyrus pyrifolia) are known to produce benzoate‐derived biphenyl phytoalexins upon elicitor treatment. Although the downstream pathway for biphenyl phytoalexin biosynthesis is almost known, the upstream route of benzoic acid biosynthesis in pear has not been completely elucidated. In the present work, we report benzaldehyde synthase (BS) activity from yeast extract‐treated cell suspension cultures of P. pyrifolia. BS catalyzes the in vitro conversion of trans‐cinnamic acid to benzaldehyde using a non‐oxidative C₂‐side chain cleavage mechanism. The enzyme activity was strictly dependent on the presence of a reducing agent, dithiothreitol being preferred. C₂‐side chain shortening of the cinnamic acid backbone resembled the mechanisms catalyzed by 4‐hydroxybenzaldehyde synthase (HBS) activity in Vanilla planifolia and salicylaldehyde synthase (SAS) activity in tobacco and apple cell cultures. A basal BS activity was also observed in the non‐elicited cell cultures. Upon yeast extract‐treatment, a 13‐fold increase in BS activity was observed when compared to the non‐treated control cells. Moreover, feeding of the cell cultures with trans‐cinnamic acid, the substrate for BS, resulted in an enhanced level of noraucuparin, a biphenyl phytoalexin. Comparable accumulation of noraucuparin was observed upon feeding of benzaldehyde, the BS product. The preferred substrate for BS was found to be trans‐cinnamic acid, for which the apparent K_m and V_max values were 0.5 mM and 50.7 pkat mg^?1 protein, respectively. Our observations indicate the contribution of BS to benzoic acid biosynthesis in Asian pear via the CoA‐independent and non‐β‐oxidative route.     

Thylakoid membrane dynamics and state transitions in Chlamydomonas reinhardtii under elevated temperature

Photosynthesis Research - Moderately elevated temperatures can induce state transitions in higher plants by phosphorylation of light-harvesting complex II (LHCII). In this study, we exposed...