In vitro biocompatibility evaluation of a heat‐resistant 3D printing material for use in customized cell culture devices

Additive manufacturing (3D printing) enables the fabrication of highly customized and complex devices and is therefore increasingly used in the field of life sciences and biotechnology. However, the application of 3D‐printed parts in these fields requires not only their biocompatibility but also their sterility. The most common method for sterilizing 3D‐printed parts is heat steam sterilization—but most commercially available 3D printing materials cannot withstand high temperatures. In this study, a novel heat‐resistant polyacrylate material for high‐resolution 3D Multijet printing was evaluated for the first time for its resistance to heat steam sterilization and in vitro biocompatibility with mouse fibroblasts (L929), human embryonic kidney cells (HEK 293E), and yeast (Saccharomyces cerevisiae (S. cerevisiae)). Analysis of the growth and viability of L929 cells and the growth of S. cerevisiae confirmed that the extraction media obtained from 3D‐printed parts had no negative effect on the aforementioned cell types, while, in contrast, viability and growth of HEK 293E cells were affected. No different effects of the material on the cells were found when comparing heat steam sterilization and disinfection with ethanol (70%, v/v). In principle, the investigated material shows great potential for high‐resolution 3D printing of novel cell culture systems that are highly complex in design, customized and easily sterilizable—however, the biocompatibility of the material for other cell types needs to be re‐evaluated.     

Imputing supertrees and supernetworks from quartets

Inferring species phylogenies is an important part of understanding molecular evolution. Even so, it is well known that an accurate phylogenetic tree reconstruction for a single gene does not always necessarily correspond to the species phylogeny. One commonly accepted strategy to cope with this problem is to sequence many genes; the way in which to analyze the resulting collection of genes is somewhat more contentious. Supermatrix and supertree methods can be used, although these can suppress conflicts arising from true differences in the gene trees caused by processes such as lineage sorting, horizontal gene transfer, or gene duplication and loss. In 2004, Huson et al. (IEEE/ACM Trans. Comput. Biol. Bioinformatics 1:151-158) presented the Z-closure method that can circumvent this problem by generating a supernetwork as opposed to a supertree. Here we present an alternative way for generating supernetworks called Q-imputation. In particular, we describe a method that uses quartet information to add missing taxa into gene trees. The resulting trees are subsequently used to generate consensus networks, networks that generalize strict and majority-rule consensus trees. Through simulations and application to real data sets, we compare Q-imputation to the matrix representation with parsimony (MRP) supertree method and Z-closure, and demonstrate that it provides a useful complementary tool.     

Life and death of female gametes during oogenesis and folliculogenesis

The vertebrate ovary is an extremely dynamic organ in which excessive or defective follicles are rapidly and effectively eliminated early in ontogeny and thereafter continuously throughout reproductive life. More than 99% of follicles disappear, primarily due to apoptosis of granulosa cells, and only a minute fraction of the surviving follicles successfully complete the path to ovulation. The balance between signals for cell death and survival determines the destiny of the follicles. An abnormally high rate of cell death followed by atresia can negatively affect fertility and eventually lead irreversibly to premature ovarian failure. In this review we provide a short overview of the role of programmed cell death in prenatal differentiation of the primordial germ cells and in postnatal folliculogenesis. We also discuss the issue of neo-oogenesis. Next, we highlight molecules involved in regulation of granulosa cell apoptosis. We further discuss the potential use of scores for apoptosis in granulosa cells and characteristics of follicular fluid as prognostic markers for predicting the outcome of assisted reproduction. Potential therapeutic strategies for combating premature ovarian failure are also addressed.     

Electrophysiological and immunocytochemical characterization of DRG neurons on an organosilane surface in serum-free medium

We are attempting to recreate a stretch reflex circuit on a patterned Bio-MEMS (bio-microelectromechanical systems) chip with deflecting micro-cantilevers. The first steps to recreate this system is to be able to grow individual components of the circuit (sensory neuron, motoneuron, skeletal muscle, and muscle spindle) on a patternable, synthetic substrate coating the MEMS device. Sensory neurons represent the afferent portion of the stretch reflex arc and also play a significant role in transmitting the signal from the muscle spindle to the spinal cord motoneurons. We have utilized a synthetic silane substrate N-1[3-(trimethoxysilyl) propyl) diethylenetriamine (DETA) on which to grow and pattern the cells. DETA forms a self-assembled monolayer on a variety of silicon substrates, including glass, and can be patterned using photolithography. In this paper, we have evaluated the growth of sensory neurons on this synthetic silane substrate. We have investigated the immunocytochemical and electrophysiological properties of the sensory neurons on DETA and compared the resultant properties with a biological control substrate (ornithine/laminin). Immunocytochemical studies revealed the survival and growth of all three subtypes of sensory neurons: trkA, trkB, and trkC on both surfaces. Furthermore, whole-cell patch clamp recordings were used to study the electrophysiological properties of the sensory neurons on the two surfaces. There were no significant differences in the electrical properties of the neurons grown on either surface. This is the first study analyzing the immunocytochemical and electrophysiological properties of sensory neurons grown long-term in a completely defined environment and on a nonbiological substrate.     

Combined anaerobic ammonium and methane oxidation for nitrogen and methane removal

Anammox (anaerobic ammonium oxidation) is an environment-friendly and cost-efficient nitrogen-removal process currently applied to high-ammonium-loaded wastewaters such as anaerobic digester effluents. In these wastewaters, dissolved methane is also present and should be removed to prevent greenhouse gas emissions into the environment. Potentially, another recently discovered microbial pathway, n-damo (nitrite-dependent anaerobic methane oxidation) could be used for this purpose. In the present paper, we explore the feasibility of simultaneously removing methane and ammonium anaerobically, starting with granules from a full-scale anammox bioreactor. We describe the development of a co-culture of anammox and n-damo bacteria using a medium containing methane, ammonium and nitrite. The results are discussed in the context of other recent studies on the application of anaerobic methane- and ammonia-oxidizing bacteria for wastewater treatment.     

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.     

Tandem affinity purification of functional TAP-tagged proteins from human cells

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.     

Responses of Antarctic Iridaea cordata (Rhodophyta) tetraspores exposed to ultraviolet radiation

In Antarctica ozone depletion is highest during spring, coinciding with the reproduction of many seaweed species. Propagules are the life-stage of an alga most susceptible to environmental perturbations. Therefore, fertile thalli of Iridaea cordata (Turner) Bory (Rhodophyta) were collected in the eulittoral of King George Island (Antarctica) to examine spore susceptibility to ultraviolet radiation (UVR). In the laboratory, freshly released tetraspores were exposed to photosynthetically active radiation (PAR) (400–700 nm), PAR+UV-A (320–700 nm) or PAR+UV-A+UV-B (280–700 nm). Photosynthetic efficiency was measured during 1–8 h of exposure and after 48 h of recovery. Additionally, mycosporine-like amino acids (MAAs) and DNA damage were determined. Saturating irradiance of photosynthesis of freshly released tetraspores was 57 µmol photons m⁻² s⁻¹. Exposure to increasing fluence of PAR reduced photosynthetic efficiency. UVR further decreased the photosynthetic efficiencies of the tetraspores but spores were able to recover completely after UVR exposure and 2 days post-cultivation under low PAR. DNA damage was minimal and lesions were effectively repaired under photoreactivating light. Concentrations of the MAAs shinorine and palythine were higher in tetraspores treated with UVR than in spores only exposed to PAR. Generally, the tetraspores show a good UV tolerance. This flexible response of the tetraspores of this species to changing radiation conditions enables the alga to grow along a considerable depth gradient from the sublittoral to the eulittoral where they can be exposed to enhanced UVBR under conditions of stratospheric ozone depletion.     

Mapping the interactions between a RUN domain from DENND5/Rab6IP1 and sorting nexin 1

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses.     

Listening to Puns Elicits the Co-Activation of Alternative Homophone Meanings during Language Production

Recent evidence suggests that lexical-semantic activation spread during language production can be dynamically shaped by contextual factors. In this study we investigated whether semantic processing modes can also affect lexical-semantic activation during word production. Specifically, we tested whether the processing of linguistic ambiguities, presented in the form of puns, has an influence on the co-activation of unrelated meanings of homophones in a subsequent language production task. In a picture-word interference paradigm with word distractors that were semantically related or unrelated to the non-depicted meanings of homophones we found facilitation induced by related words only when participants listened to puns before object naming, but not when they heard jokes with unambiguous linguistic stimuli. This finding suggests that a semantic processing mode of ambiguity perception can induce the co-activation of alternative homophone meanings during speech planning.