Generation of diverse neural cell types through direct conversion

A characteristic of neurological disorders is the loss of critical populations of cells that the body is unable to replace,thus there has been much interest in identifying methods of generating clinically relevant numbers of cells to replace those that have been damaged or lost.The process of neural direct conversion,in which cells of one lineage are converted into cells of a neural lineage without first inducing pluripotency,shows great potential,with evidence of the generation of a range of functional neural cell types both in vitro and in vivo,through viral and non-viral delivery of exogenous factors,as well as chemical induction methods.Induced neural cells have been proposed as an attractive alternative to neural cells derived from embryonic or induced pluripotent stem cells,with prospective roles in the investigation of neurological disorders,including neurodegenerative disease modelling,drug screening,and cellular replacement for regenerative medicine applications,however further investigations into improving the efficacy and safety of these methods need to be performed before neural direct conversion becomes a clinically viable option.In this review,we describe the generation of diverse neural cell types via direct conversion of somatic cells,with comparison against stem cell-based approaches,as well as discussion of their potential research and clinical applications.     

Rapid detection of natural cells of Alexandrium tamarense and A. catenella (Dinophyceae) by fluorescence in situ hybridization

The marine toxic dinoflagellates Alexandrium tamarense (Lebor) Balech and A. catenella (Whedon and Kofoid) Taylor that cause paralytic shellfish poisoning (PSP) are identified on the basis of morphological features in routine monitoring. Rapid and simple identification is, however, often difficult because of the morphological similarity. Fluorescent in situ hybridization (FISH) using ribosomal RNA (rRNA)-targeted probes has been studied as a method of easily identifying and enumerating species responsible for harmful algal blooms (HABs). Its application to monitoring natural populations of HAB species, however, is limited. Here, we applied the FISH method to identify and enumerate cells of A. tamarense and A. catenella in natural plankton assemblages collected from Japanese coastal waters. A. tamarense-specific (Atm1) and A. catenella-specific (Act1) probes were established based on the D2 region of the large-subunit ribosomal RNA gene (28S rDNA). With these two probes, natural cells of A. tamarense or A. catenella in field samples could easily be identified when the following three conditions were met. First, cells should be concentrated by filtration, not centrifugation, in order to avoid the loss of cells. Second, autofluorescence should be minimized; acetone was an effective decolorization reagent. Third, samples should be stored at −20 or −80 °C for long-term preservation. The results indicate that FISH is a useful tool for the rapid identification of toxic Alexandrium spp. and can facilitate the analysis of numerous natural samples.     

Combining geometric morphometrics with molecular genetics to investigate a putative hybrid complex: a case study with barbels Barbus spp. (Teleostei: Cyprinidae)

This integrative study examined the morphological and genetic affinities of three endemic barbel species from Italy (brook barbel Barbus caninus, Italian barbel Barbus plebejus and horse barbel Barbus tyberinus) and of putative hybrid specimens to their species of origin. Two of the species frequently occur together with the non‐native barbel Barbus barbus. DNA barcoding indicates that mitochondrial (mt) haplotypes often do not match the species expected from morphology. Linear distance measurements and meristics are not informative for discrimination of the species and putative hybrids, but a discriminant analysis of principal components (DAPC) of geometric landmark data produces reassignments largely in congruence with mt and nuclear genetic data. Cyto‐nuclear conflicts confirm the presence of hybridization in B. plebejus and B. tyberinus and identify additional introgressed specimens. A comparison between mixed genotypes and their morphology‐based assignment reveals no predictable pattern. The finding that most individuals of the morphologically similar B. plebejus and B. tyberinus have very high assignment probabilities to their respective species suggests that the presented approach may serve as a valuable tool to distinguish morphologically very similar taxa.     

Fluorescence-based evaluation of shRNA efficacy

RNA interference is a cellular mechanism regulating levels of mRNAs. It has been widely exploited to knock down specific protein targets. The selected interfering RNA sequence greatly influences its ability to knock down the target. Here we present a method for constructing multiple testing plasmids which express small hairpin RNAs (shRNA) targeting different regions of an mRNA. A simple fluorescence test in cultured cells allows convenient evaluation of mRNA knockdown by many different shRNAs on 96-well plates. We show that software predicted shRNAs have varying efficacies and only 2 of the 7 tested shRNAs significantly knocked down their targets.     

Recent advances in biotechnological production of 2-phenylethanol

2-Phenylethanol (2-PE) is an important aromatic alcohol with a rose-like fragrance. It has been widely applied in the cosmetic, perfume, and food industries and is mainly produced by chemical synthesis. An alternative method for the production of natural flavors and fragrances is the microbial transformation process, which is attracting increasing attention because it is an environmentally friendly process and the products are considered “natural”. The production of 2-PE from L-phenylalanine by biotransformation is possible through the Ehrlich pathway and considerable progress has been made in the development of this process. The present report reviews recent advances in biotechnological production of 2-PE, with emphasis on the strategies used to increase production and the applications of in situ product removal techniques. Future research should focus on product scale-up and product recovery processes for the industrialization of microbial processes.     

Increased expression of polypyrimidine tract binding protein results in higher insulin mRNA levels

The aim of this study was to further elucidate the role of the polypyrimidine tract binding protein (PTB) in the control of insulin mRNA stability. We observed that the glucose- or interleukin-1beta-induced increase in insulin mRNA was paralleled by an increase in PTB mRNA. To further test the hypothesis that PTB controls insulin gene expression, betaTC-6 cells were treated with a PTB-specific siRNA to modify the beta-cell content of PTB. Surprisingly, we observed an increase in PTB mRNA and PTB protein levels in response to the siRNA treatment. In addition, the PTB-siRNA treatment also increased insulin mRNA. We conclude that expression of the PTB gene controls insulin production.     

Discovery of a new type inhibitor of human glyoxalase I by myricetin-based 4-point pharmacophore

The human glyoxalase I (hGLO I), which is a rate-limiting enzyme in the pathway for detoxification of apoptosis-inducible methylglyoxal (MG), has been expected as an attractive target for the development of new anti-cancer drugs. We have previously identified a natural compound myricetin as a substrate transition-state (Zn²⁺-bound MG-glutathione (GSH) hemithioacetal) mimetic inhibitor of hGLO I. Here, we constructed a hGLO I/inhibitor 4-point pharmacophore based on the binding mode of myricetin to hGLO I. Using this pharmacophore, in silico screening of chemical library was performed by docking study. Consequently, a new type of compound, which has a unique benzothiazole ring with a carboxyl group, named TLSC702, was found to inhibit hGLO I more effectively than S-p-bromobenzylglutathione (BBG), a well-known GSH analog inhibitor. The computational simulation of the binding mode indicates the contribution of Zn²⁺-chelating carboxyl group of TLSC702 to the hGLO I inhibitory activity. This implies an important scaffold-hopping of myricetin to TLSC702. Thus, TLSC702 may be a valuable seed compound for the generation of a new lead of anti-cancer pharmaceuticals targeting hGLO I.