Evidence that Aspartate Aminotransferase Activity and Ketodicarboxylate Carrier Function Are Essential for Biosynthesis of Transmitter Glutamate

Based on the selective inhibition of glutamate release in cerebellar granule cells in primary cultures by the aspartate aminotransferase inhibitor, aminooxyacetic acid, and by the ketodicarboxylate carrier inhibitor, phenylsuccinate, a novel model for synthesis of transmitter glutamate is suggested: Glutamate is formed from glutamine in the mitochondrial intramembrane space by phosphate-activated glutaminase, transported across the inner membrane in exchange with aspartate, transaminated in the matrix to alpha-ketoglutarate, which via the ketodicarboxylate carrier is transferred to the cytoplasm, and transaminated to form transmitter glutamate. Such a mechanism would explain the functional role of aspartate aminotransferase in glutamatergic neurons.     

Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples

Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.     

Life cycle assessment of emerging technologies at the lab scale: The case of nanowire‐based solar cells

Nanomaterials are expected to play an important role in the development of sustainable products. The use of nanomaterials in solar cells has the potential to increase their conversion efficiency. In this study, we performed a life cycle assessment (LCA) for an emerging nanowire‐based solar technology. Two lab‐scale manufacturing routes for the production of nanowire‐based solar cells have been compared—the direct growth of GaInP nanowires on silicon substrate and the growth of InP nanowires on native substrate, peel off, and transfer to silicon substrate. The analysis revealed critical raw materials and processes of the current lab‐scale manufacturing routes such as the use of trifluoromethane (CHF₃), gold, and an InP wafer and a stamp, which are used and discarded. The environmental performance of the two production routes under different scenarios has been assessed. The scenarios include the use of an alternative process to reduce the gold requirements—electroplating instead of metallization, recovery of gold, and reuse of the InP wafer and the stamp. A number of suggestions, based on the LCA results—including minimization of the use of gold and further exploration for upscaling of the electroplating process, the increase in the lifetimes of the wafer and the stamp, and the use of fluorine‐free etching materials—have been communicated to the researchers in order to improve the environmental performance of the technology. Finally, the usefulness and limitations of lab‐scale LCA as a tool to guide the sustainable development of emerging technologies are discussed.     

New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re‐quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.     

Ex ante life cycle assessment of GaAs/Si nanowire–based tandem solar cells: a benchmark for industrialization

The International Journal of Life Cycle Assessment - The goal of this study is to perform an ex-ante life cycle assessment (LCA) of the emerging gallium-arsenide nanowire tandem solar cells on...     

Computational optimization of the SARS-CoV-2 receptor-binding-motif affinity for human ACE2

The coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the coronavirus disease 2019 pandemic, and the closely related SARS-CoV coronavirus enter cells by binding at the human angiotensin converting enzyme 2 (hACE2). The stronger hACE2 affinity of SARS-CoV-2 has been connected with its higher infectivity. In this work, we study hACE2 complexes with the receptor-binding domains (RBDs) of the human SARS-CoV-2 and human SARS-CoV viruses, using all-atom molecular dynamics simulations and computational protein design with a physics-based energy function. The molecular dynamics simulations identify charge-modifying substitutions between the CoV-2 and CoV RBDs, which either increase or decrease the hACE2 affinity of the SARS-CoV-2 RBD. The combined effect of these mutations is small, and the relative affinity is mainly determined by substitutions at residues in contact with hACE2. Many of these findings are in line and interpret recent experiments. Our computational protein design calculations redesign positions 455, 493, 494, and 501 of the SARS-CoV-2 receptor binding motif, which contact hACE2 in the complex and are important for ACE2 recognition. Sampling is enhanced by an adaptive importance sampling Monte Carlo method. Sequences with increased affinity replace CoV-2 glutamine by a negative residue at position 493; serine by a nonpolar or aromatic residue or an asparagine at position 494; and asparagine by valine or threonine at position 501. Substitutions at positions 455 and 501 have a smaller effect on affinity. Substitutions suggested by our design are seen in viral sequences encountered in other species, including bat and pangolin. Our results might be used to identify potential virus strains with higher human infectivity and assist in the design of peptide-based or peptidomimetic compounds with the potential to inhibit SARS-CoV-2 binding at hACE2.