Establishment of a transgenic zebrafish line for superficial skin ablation and functional validation of apoptosis modulators in vivo

Background

Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish.

Methodology/Principal Findings

This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)^cy17 (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR⁺ fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR⁺ signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR⁺ fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR⁺ fluorescent signaling.

Conclusion/Significance

The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.     

Life cycles,phenology and genetic structure of endangered Megacrania tsudai Shiraki (Phasmatodea: Phasmatidae): Male individuals from a geographic parthenogenesis species

Megacrania tsudai, a peripherally distributed member of Megacrania, requires conservation in Taiwan; it has limited distribution in Taiwan and its eastern offshore islands. It feeds on screw pines (Pandanus odoratissimus) in nature and has demonstrated a specific defensive mechanism involving actinidine secretion from the prothoracic gland. However, details of its distribution area, life cycle and developmental phenology remain largely unknown. In this study, a field survey and review of published works revealed M. tsudai distribution in coastal zones and along river shores near estuaries. At room temperature, the egg period was 128 days. The development of the first to sixth instars required 17, 26, 27, 26, 34 and 43 days, respectively, on average; and a generation cycle required approximately 204 days. The phenology of the mesonotal granules was recorded. Moreover, genetic analysis of the mitochondrial cytochrome oxidase I (COI), 16S ribosomal DNA (16S rDNA) and the nuclear ribosomal spacer indicated the occurrence of genetic drift. Therefore, the rearing procedures proposed in this study for the primary and last instars of M. tsudai can facilitate its conservation. Megacrania tsudai was previously recorded as parthenogenetic; however, two male individuals were fostered unexpectedly. The male body length was 91 mm, which is shorter than the female length (120 mm). During mating, the male climbs onto the female's back and protrudes its genitalia downward. Geographical parthenogenesis is likely the reproductive strategy among peripheral M. tsudai; however, the rarely found M. tsudai male could be an intermediate link of reproductive strategy in the transition from tychoparthenogenesis to parthenogenesis.     

Porphyromonas gingivalis GroEL Induces Osteoclastogenesis of Periodontal Ligament Cells and Enhances Alveolar Bone Resorption in Rats

Porphyromonas gingivalis is a major periodontal pathogen that contains a variety of virulence factors. The antibody titer to P. gingivalis GroEL, a homologue of HSP60, is significantly higher in periodontitis patients than in healthy control subjects, suggesting that P. gingivalis GroEL is a potential stimulator of periodontal disease. However, the specific role of GroEL in periodontal disease remains unclear. Here, we investigated the effect of P. gingivalis GroEL on human periodontal ligament (PDL) cells in vitro, as well as its effect on alveolar bone resorption in rats in vivo. First, we found that stimulation of PDL cells with recombinant GroEL increased the secretion of the bone resorption-associated cytokines interleukin (IL)-6 and IL-8, potentially via NF-κB activation. Furthermore, GroEL could effectively stimulate PDL cell migration, possibly through activation of integrin α1 and α2 mRNA expression as well as cytoskeletal reorganization. Additionally, GroEL may be involved in osteoclastogenesis via receptor activator of nuclear factor κ-B ligand (RANKL) activation and alkaline phosphatase (ALP) mRNA inhibition in PDL cells. Finally, we inoculated GroEL into rat gingiva, and the results of microcomputed tomography (micro-CT) and histomorphometric assays indicated that the administration of GroEL significantly increased inflammation and bone loss. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to osteoclastogenesis of PDL cells and resulting in periodontal disease with alveolar bone resorption.     

A population‐based temporal logic gate for timing and recording chemical events

Engineered bacterial sensors have potential applications in human health monitoring, environmental chemical detection, and materials biosynthesis. While such bacterial devices have long been engineered to differentiate between combinations of inputs, their potential to process signal timing and duration has been overlooked. In this work, we present a two‐input temporal logic gate that can sense and record the order of the inputs, the timing between inputs, and the duration of input pulses. Our temporal logic gate design relies on unidirectional DNA recombination mediated by bacteriophage integrases to detect and encode sequences of input events. For an E. coli strain engineered to contain our temporal logic gate, we compare predictions of Markov model simulations with laboratory measurements of final population distributions for both step and pulse inputs. Although single cells were engineered to have digital outputs, stochastic noise created heterogeneous single‐cell responses that translated into analog population responses. Furthermore, when single‐cell genetic states were aggregated into population‐level distributions, these distributions contained unique information not encoded in individual cells. Thus, final differentiated sub‐populations could be used to deduce order, timing, and duration of transient chemical events.     

The effect of different electrostatic potentials on docking accuracy: a case study using DOCK5.4

As a commonly used structure-based approach for virtual screening, molecular design and lead optimization, molecular docking can search the preferred orientation and conformation of a ligand for its optimal binding to a receptor or enzyme active site. In doing so, selecting an appropriate method to calculate the electrostatic potentials is critical. In the current report, nine different semi-empirical and empirical methods, including AM1, AM1-BCC, Del-Re, MMFF, Gasteiger, Hückel, Gasteiger-Hückel, Pullman and formal charges were investigated for their performance on the prediction of docking poses using the DOCK5.4 program. The results demonstrated that the AM1-BCC charges had the highest success rate.     

Optimal promising zone designs

Clinical trials with adaptive sample size reassessment based on an unblinded analysis of interim results are perhaps the most popular class of adaptive designs (see Elsäßer et al., 2007). Such trials are typically designed by prespecifying a zone for the interim test statistic, termed the promising zone, along with a decision rule for increasing the sample size within that zone. Mehta and Pocock (2011) provided some examples of promising zone designs and discussed several procedures for controlling their type‐1 error. They did not, however, address how to choose the promising zone or the corresponding sample size reassessment rule, and proposed instead that the operating characteristics of alternative promising zone designs could be compared by simulation. Jennison and Turnbull (2015) developed an approach based on maximizing expected utility whereby one could evaluate alternative promising zone designs relative to a gold‐standard optimal design. In this paper, we show how, by eliciting a few preferences from the trial sponsor, one can construct promising zone designs that are both intuitive and achieve the Jennison and Turnbull (2015) gold‐standard for optimality.