Requirements for human‐induced pluripotent stem cells

‘Requirements for Human‐Induced Pluripotent Stem Cells’ is the first set of guidelines on human‐induced pluripotent stem cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, and instructions for use, labeling, packaging, storage, transportation, and waste handling for human‐induced pluripotent stem cells, which apply to the production and quality control of human‐induced pluripotent stem cells. It was released by the Chinese Society for Cell Biology on 9 January 2021 and came into effect on 9 April 2021. We hope that the publication of these guidelines will promote institutional establishment, acceptance, and execution of proper protocols and accelerate the international standardization of human‐induced pluripotent stem cells for applications.     

Structural basis for feedforward control in the PINK1/Parkin pathway

PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson’s disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin‐like (Ubl) domain of parkin. Here, we observed that phospho‐ubiquitin can bind to two distinct sites on parkin, a high‐affinity site on RING1 that controls parkin localization and a low‐affinity site on RING0 that releases parkin autoinhibition. Surprisingly, ubiquitin vinyl sulfone assays, ITC, and NMR titrations showed that the RING0 site has higher affinity for phospho‐ubiquitin than phosphorylated Ubl in trans. We observed parkin activation by micromolar concentrations of tetra‐phospho‐ubiquitin chains that mimic mitochondria bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1‐parkin pathway and likely represents an intermediate step in its evolutionary development.     

Combining two teaching techniques for young children on Aedes aegypti control: effects on entomological indices in western Mexico

A study of the effect of educating four- to six-year-old children in mosquito control was recently conducted in a city in the state of Jalisco, western Mexico. Four neighborhood districts were selected. Children attending one kindergarten in each of two experimental districts were taught mosquito control with a video from the American Mosquito Control Association (AMCA), joined to the use of the AMCA Touch Table Technique. The entomological indices monitored in the study decreased significantly (P<0.05) in houses in the experimental districts, apparently because parents acted on the comments and suggestions of the children and eliminated or monitored containers used as oviposition sites by mosquitoes. Based on these results, combining both techniques for teaching children mosquito control is a potentially useful tool for control efforts in Mexico and other places in Latin America.     

A novel environmental chamber for neuronal network multisite recordings

Environmental stability is a critical issue for neuronal networks in vitro. Hence, the ability to control the physical and chemical environment of cell cultures during electrophysiological measurements is an important requirement in the experimental design. In this work, we describe the development and the experimental verification of a closed chamber for multisite electrophysiology and optical monitoring. The chamber provides stable temperature, pH and humidity and guarantees cell viability comparable to standard incubators. Besides, it integrates the electronics for long‐term neuronal activity recording. The system is portable and adaptable for multiple network housings, which allows performing parallel experiments in the same environment. Our results show that this device can be a solution for long‐term electrophysiology, for dual network experiments and for coupled optical and electrical measurements. Biotechnol. Bioeng. 2012; 109: 2553–2566. © 2012 Wiley Periodicals, Inc.     

Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli

Availability, low price, and high degree of reduction have made glycerol a highly attractive and exploited carbon source for the production of fuels and reduced chemicals. Here we report the quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli through the use of kinetic modeling and metabolic control analysis (MCA) to gain a better understanding of glycerol fermentation and identify key targets for genetic manipulation that could enhance product synthesis. The kinetics of glycerol fermentation in a batch culture was simulated using a dynamic model consisting of mass balances for glycerol, ethanol, biomass, and 11 intracellular metabolites, along with the corresponding kinetic expressions for the metabolism of each species. The model was then used to calculate metabolic control coefficients and elucidate the control structure of the pathways involved in glycerol utilization and ethanol synthesis. The calculated flux control coefficients indicate that the glycolytic flux during glycerol fermentation is almost exclusively controlled by the enzymes glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM). In agreement with the MCA findings, overexpression of gldA and dhaKLM led to significant increase in glycerol utilization and ethanol synthesis fluxes. Moreover, overexpression of other enzymes involved in the pathways that mediate glycerol utilization and its conversion to ethanol had no significant impact on glycerol utilization and ethanol synthesis, further validating the MCA predictions. These findings were then applied as a means of increasing the production of ethanol: overexpression of glycerol dehyrdogenase and DHAK enabled the production of 20 g/L ethanol from crude glycerol, a by-product of biodiesel production, indicating the potential for industrial scale conversion of waste glycerol to ethanol under anaerobic conditions.     

Experimental field exclosure of birds and bats in agricultural systems — Methodological insights,potential improvements,and cost-benefit trade-offs

Experimental exclosure of birds and bats constitutes a powerful tool to study the impacts of wildlife on pests and crop yields in agricultural systems. Though widely utilized, exclosure experiments are not standardized across studies. Indeed, key differences surrounding the design, materials, and protocols for implementing field-based exclosure experiments of flying vertebrates increase heterogeneity across studies, and limit our understanding of biodiversity-friendly land use management. We reviewed the available literature on studies in which bird and bat exclosures were applied to study pest control in agricultural settings, and isolated 30 studies from both tropical and temperate land use systems, involving 12 crop types across 14 countries. Focusing on exclosure effects on crop yield, we analyzed effect detectability for a subset of suitable data. We then analyzed the potential of exclosure methods and possible extensions to improve our understanding of complex food webs and ecosystem services affecting the productivity of agricultural systems. While preferences exist in materials (e.g., nylon nets and bamboo frames), experimental exclosure studies of birds and bats differed greatly in their respective design, related costs, and effort — limiting the generalization and transferability of results at larger spatial scales. Most studies were based on experiments conducted in the United States and the Neotropics, mainly in coffee and cacao farms. A lack of preliminary or long-term data with repeated measurements makes it impossible to apply power analysis in most studies. Common constraints include, among other things, the choice of material and experimental duration, as well as the consideration of local versus landscape factors. We discuss such limitations, related common pitfalls, and options for optimization to inform improved planning, design, and execution of exclosure studies. By doing so, we aim to promote more comparable and transferable approaches in future field research on biodiversity-mediated ecosystem services.     

Pituitary adenylate cyclase‐activating polypeptide in the testis of the quail Coturnix coturnix: Expression,localization, and phylogenetic analysis

To evaluate the involvement of pituitary adenylate cyclase‐activating polypeptide (PACAP)/receptors system in the control of testis activity, we have investigated the expression and localization of PACAP and the distribution of its receptors in the testis of mature samples of quail Coturnix coturnix, and we have performed a phylogenetic analysis of PACAP in birds. Using histological, molecular, and bioinformatics tools, we demonstrated that (a) PACAP messenger RNA shows a high sequence identity with that reported in other birds studied so far and in other vertebrates. Furthermore, we showed that purifying selection acts on PACAP; (b) the PACAP peptide is present only in Leydig cells, whereas its receptors are localized within both Leydig and germ cells; (c) the synthesis of PACAP does not take place in seminiferous tubules. The role of PACAP in the control of spermatogenesis and steroidogenesis in birds is discussed. Finally, we talk about the phylogenetic and evolutionary relationships between PACAP in birds and in other vertebrates.