Metabolic fuels along the nephron: Pathways and intracellular mechanisms of interaction

Substrates in large numbers are removed from the blood by the kidney in linear relationship to their arterial concentrations [1, 2]. At normal arterial blood levels, the kidney utilizes significant amounts of free fatty acids, lactate, glutamine, 3-hydroxybutyrate, and citrate. Furthermore, the kidney removes substrates like pyruvate, α-ketoglutarate, glycerol, proline, and some other amino acids of low arterial concentrations. However, when blood levels of these substances increase, their renal uptake rates likewise increase [1, 2]. Metabolic fates of these substrates in the kidney are related intimately to major functions of the kidney including excretion of waste materials, reabsorption of life conserving substances and water, and other important endocrine and metabolic functions.When studied in vitro, the capacity of renal tissue to take up substrates was shown to be far in excess of the rates occurring under in vivo conditions [2]. This indicates that saturation is not reached in vivo due to suboptimal substrate concentrations. For lactate, pyruvate, glutamine, proline, fatty acids, and ketone bodies, normal arterial levels are below or around the half-maximal concentration kinetically determined in in vitro uptake studies [3–8]. However, even under this nonsaturating condition, the rates of substrate uptake in vivo exceed the quantities of fuel needed to meet the energy demands of the kidney as calculated from oxygen uptake [1, 9]. Table 1 summarizes the calculated oxygen uptake and ATP formation rates for some substrates. From the theoretical and the experimental data on substrate uptake rates and measurements of oxygen consumption [1, 2], it becomes clear that the kidney takes up more substrates than could be accounted for by oxidation.The term “incomplete oxidation” was introduced by Cohen [1] to explain this phenomenon. For example, 3-hydroxybutyrate taken up by the kidney is partially released as acetoacetate [8]. On the other hand, no net product release was found for other substrates taken up in excess. From recent in vitro studies, it was concluded that the kidney can utilize substrates by metabolic pathways that do not lead to their oxidation [2–7]. Thus, lactate, glycerol, glutamine, and other substrates are in part converted to glucose through the gluconeogenic pathway, whereas fatty acids which cannot be converted to glucose are recovered mainly as triacylglycerol [5, 10].Two major questions may be raised at this point: (1) What nephron cells are responsible for the substrate uptake rates observed? (2) What are the mechanisms regulating intracellular interactions of various substrates?This review will briefly summarize some recent findings on intercellular heterogeneity and intracellular regulatory mechanisms that may help explain the metabolic balances observed in vivo

Biochemical and Structural Studies of Amyloid Proteins

Amyloidogenic neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) are an important health issue. However, the underlying molecular mechanisms of the disease-related protein aggregates, that are present in humans, are only understood partially. I have used and developed biophysical methods to study the structural and biological properties of individual aggregates of Amyloid β peptide and α-Synuclein, proteins whose aggregation is associated with the development of Alzheimer’s and Parkinson’s disease respectively. I expanded the single aggregate visualisation through enhancement (SAVE) technique, which is a method based on the fluorescent dye Thioflavin T (ThT) that reversibly bind to the aggregates and whose fluorescence increases upon binding. I firstly explored the use of other dyes for these experiments and found that a ThT dimer has higher affinity to α-Synuclein aggregates in vitro. I then applied the SAVE method to the cerebral spinal fluid (CSF) of a cohort of AD patients and control CSF and observed no clear difference in aggregate number. However, these experiments provided insights into how antibodies bind the aggregates in human CSF. I could show, that despite altering the Ca2+ influx into both cells and vesicles, the antibody did not measurably affect the aggregate structure. To study the size specific effects of the Amyloid β 42 (Aβ42) peptide in more detail, I used and optimised gradient ultracentrifugation combined with single aggregate imaging to study the structural properties of the isolated aggregates. This aggregation kinetic independent method allowed me to compare the properties of fluorescently labelled and unlabelled Aβ42 and characterize the size dependent properties of aggregates in a single experiment. Since I could measure the relative concentration of different size aggregates it was also possible to compare the properties of single aggregates of different sizes. I then used biological assays to examine the ability of aggregates to permeabilise membranes resulting in the entry of calcium ions, and their ability to induce TNFα production in microglia cells. Both processes are thought to play key roles in the development of AD. I found that small soluble oligomers are most potent at inducing Ca2+ influx, whereas longer protofilaments are the most potent inducers of TNFα production. My results suggest that the mechanism by which aggregates damage cells changes as aggregation proceeds, as longer aggregates with different structures are formed. Protofilaments with a diameter of 1 nm or less have a structure that could make them particularly potent at causing the signalling of toll-like receptors, providing a molecular basis for their ability to induce TNFα production.EPSR

Biochemical, Nutritional, and Clinical Parameters of Vitamin B12 Deficiency in Infants: A Systematic Review and Analysis of 292 Cases Published between 1962 and 2022

Pooled data from published reports on infants with clinically diagnosed vitamin B12 (B12) deficiency were analyzed with the purpose of describing the presentation, diagnostic approaches, and risk factors for the condition to inform prevention strategies. An electronic (PubMed database) and manual literature search following the PRISMA approach was conducted (preregistration with the Open Science Framework, accessed on 15 February 2023). Data were described and analyzed using correlation analyses, Chi-square tests, ANOVAs, and regression analyses, and 102 publications (292 cases) were analyzed. The mean age at first symptoms (anemia, various neurological symptoms) was four months; the mean time to diagnosis was 2.6 months. Maternal B12 at diagnosis, exclusive breastfeeding, and a maternal diet low in B12 predicted infant B12, methylmalonic acid, and total homocysteine. Infant B12 deficiency is still not easily diagnosed. Methylmalonic acid and total homocysteine are useful diagnostic parameters in addition to B12 levels. Since maternal B12 status predicts infant B12 status, it would probably be advantageous to target women in early pregnancy or even preconceptionally to prevent infant B12 deficiency, rather than to rely on newborn screening that often does not reliably identify high-risk children

The human CTF4-orthologue AND-1 interacts with DNA polymerase α/primase via its unique C-terminal HMG box

A dynamic multi-protein assembly known as the replisome is responsible for DNA synthesis in eukaryotic cells. In yeast, the hub protein Ctf4 bridges DNA helicase and DNA polymerase and recruits factors with roles in metabolic processes coupled to DNA replication. An important question in DNA replication is the extent to which the molecular architecture of the replisome is conserved between yeast and higher eukaryotes. Here we describe the biochemical basis for the interaction of the human CTF4-orthologue AND-1 with Pol α/primase, the replicative polymerase that initiates DNA synthesis. AND-1 has maintained the trimeric structure of yeast Ctf4, driven by its conserved SepB domain. However, the primary interaction of AND-1 with Pol α/primase is mediated by its C-terminal HMG box, unique to mammalian AND-1, which binds the B subunit, at the same site targeted by the SV40 T antigen for viral replication. In addition, we report a novel DNA-binding activity in AND-1, which might promote the correct positioning of Pol α/primase on the lagging-strand template at the replication fork. Our findings provide a biochemical basis for the specific interaction between two critical components of the human replisome, and indicate that important principles of replisome architecture have changed significantly in evolution.This work was supported by a Wellcome Trust investigator award to L.P. (104641/Z/14/Z), a Cambridge Gates PhD scholarship to A.C.S., a PhD fellowship of the Boehringer-Ingelheim Fonds and awards from the Janggen-Pöhn-Stiftung and the Swiss National Science Foundation to S.H

Perceptions and expectations of authorship: Towards development of an e-learning tool facilitating discussion and reflection between post-graduate supervisors and candidates

Michelle Picard, Kerry Wilkinson and Michelle Wirthensoh

Changes in fatty acid and tocopherol content during almond (Prunus dulcis, cv. Nonpareil) kernel development

Lipids are the major nutritional component of almonds and almond lipids comprise a range of fatty acids from C14 up to C20, including saturated, monounsaturated and polyunsaturated fatty acids, and oil soluble compounds such as plant sterols and tocopherols. This study investigated the change in fatty acid and tocopherol levels during almond kernel maturation, in the variety Nonpareil, grown in the Adelaide Plains of South Australia. The investigation was carried out between November 2012 and February 2013. The accumulation of lipids was determined over six timepoints, commencing at 74 days post-anthesis, and then at 20 day intervals. Almond lipid accumulation occurred rapidly between 95 and 115 days post-anthesis, i.e. at a rate of up to 1.83 g/day per 100 g fresh weight but then slowed. Tocopherols accumulated steadily and were positively correlated with lipid development; with α-tocopherol forming at the highest rate, being 0.58 mg/day in 100 g lipid, between the first two timepoints. The key timing for accumulation of the major fatty acid, oleic acid, was between 95 and 115 days post-anthesis, after which accumulation remained constant, at 0.57% of total lipids per day. In contrast, linoleic acid accumulated during the first two timepoints then declined to 23% of final lipid content. This study aimed to determine the timing of almond lipophilic antioxidant production, to inform almond orchard management practices, such as irrigation and fertilisation, which may impact kernel composition, and therefore, quality.Ying Zhu, Kerry L. Wilkinson, Michelle Wirthensoh

Molecular modeling of S-RNases involved in almond self-incompatibility

Gametophytic self-incompatibility (GSI) is a mechanism in flowering plants, to prevent inbreeding and promote outcrossing. GSI is under the control of a specific locus, known as the S-locus, which contains at least two genes, the RNase and the SFB. Active S-RNases in the style are essential for rejection of haploid pollen, when the pollen S-allele matches one of two S-alleles of the diploid pistil. However, the nature of their mutual interactions at genetic and biochemical levels remain unclear. Thus, detailed understanding of the protein structure involved in GSI may help in discovering how the proteins involved in GSI may function and how they fulfill their biological roles. To this end, 3D models of the SC (Sf) and two SI (S8 and S23) S-RNases of almond were constructed, using comparative modeling tools. The modeled structures consisted of mixed α and β folds, with six helices and six β-strands. However, the self-compatible (Sf) RNase contained an additional extended loop between the conserved domains RC4 and C5, which may be involved in the manifestation of self-compatibility in almond

Molecular modeling of RNases from almond involved in serlf-incompatibility

Gametophytic self-incompatibility (GSI) is a natural mechanism in flowering plants, including almond and other fruit tree species, to prevent inbreeding and promote outcrossing. It is typically under the control of a specific locus, known as the S-locus, which contains at least two genes. The first gene encodes glycoproteins with ribonuclease (S-RNase) activity in the pistils, and the second is a specific F-box gene (SFB) expressed in the pollen. In Solanaceae, Scrophulariaceae and Rosaceae, active S-RNases in the style are essential for rejection of haploid pollen, when the S-allele of pollen matches one of two S-alleles of the diploid pistil. The S-RNase was first identified in Prunus more than 20 years ago, whereas SFB was identified only recently. In spite of the knowledge of the genetic structure of the female and male determinants of GSI, the nature of their mutual interactions at genetic and biochemical levels remain unclear. Thus, detailed understanding of the protein structure involved in GSI may help in discovering how proteins involved in GSI function and fulfil their biological roles. To this aim, three-dimensional (3D) models of a self-compatible (Sf) and a self-incompatible (S8) S-RNase of almond have been constructed, using comparative modelling tools. The molecular models of Sf and S8 showed that 3D architectures of their folds had the same topology as typical members of the RNase T2 family. The modelled structures consisted of mixed α and β folds, with six helices and six beta-strands.Peer Reviewedalmondself-(in) compatibility3D modellingRNase T2Publishe

Structure of the archaeal chemotaxis protein CheY in a domain-swapped dimeric conformation

Archaea are motile by the rotation of the archaellum. The archaellum switches between clockwise and counterclockwise rotation, and movement along a chemical gradient is possible by modulation of the switching frequency. This modulation involves the response regulator CheY and the archaellum adaptor protein CheF. In this study, two new crystal forms and protein structures of CheY are reported. In both crystal forms, CheY is arranged in a domain-swapped conformation. CheF, the protein bridging the chemotaxis signal transduction system and the motility apparatus, was recombinantly expressed, purified and subjected to X-ray data collection