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Fe-S proteins are ubiquitous and control a wide variety of key biological processes; therefore, maintaining Fe-S cluster homeostasis is an essential task for all organisms. Here, we provide the first example of how a bacterium from the Deltaproteobacteria branch coordinates expression of two Fe-S cluster biogenesis machineries.
Spiking neural networks (SNN) offer a power efficient, biologically plausible learning paradigm by encoding information into spikes. The discovery of the memristor has accelerated the progress of spiking neuromorphic systems, as the intrinsic plasticity of the device makes it an ideal candidate to mimic a biological synapse. Despite providing a nanoscale form factor, non-volatility, and low-power operation, memristors suffer from device-level non-idealities, which impact system-level performance. To address these issues, this article presents a memristive crossbar-based neuromorphic system using unsupervised learning with twin-memristor synapses, fully digital pulse width modulated spike-timing-dependent plasticity, and homeostasis neurons. Additionally, the implemented single-layer SNN was applied to a pattern-recognition task of classifying handwritten-digits. The performance of the system was analyzed by varying design parameters such as number of training epochs, neurons, and capacitors. Furthermore, the impact of memristor device non-idealities, such as device-switching mismatch, aging, failure, and process variations, were investigated and the resilience of the proposed system was demonstrated.
Copper (Cu) is an essential cofactor required for redox enzymes in all domains of life. Because of its toxicity, tightly controlled mechanisms ensure Cu delivery for cuproenzyme biogenesis and simultaneously protect cells against toxic Cu. Many Gram-negative bacteria contain extracytoplasmic multicopper oxidases (MCOs), which are involved in periplasmic Cu detoxification. MCOs are unique cuproenzymes because their catalytic center contains multiple Cu atoms, which are required for the oxidation of Cu 1+ to the less toxic Cu 2+ . Hence, Cu is both substrate and essential cofactor of MCOs. Here, we investigated the maturation of Rhodobacter capsulatus MCO CutO and its role in periplasmic Cu detoxification. A survey of CutO activity of R. capsulatus mutants with known defects in Cu homeostasis and in the maturation of the cuproprotein cbb 3 -type cytochrome oxidase ( cbb 3 -Cox) was performed. This revealed that CutO activity is largely independent of the Cu-delivery pathway for cbb 3 -Cox biogenesis, except for the cupric reductase CcoG, which is required for full CutO activity. The most pronounced decrease of CutO activity was observed with strains lacking the cytoplasmic Cu chaperone CopZ, or the Cu-exporting ATPase CopA, indicating that CutO maturation is linked to the CopZ-CopA mediated Cu-detoxification pathway. Our data demonstrate that CutO is important for cellular Cu resistance under both aerobic and anaerobic growth conditions. CutO is encoded in the cutFOG operon, but only CutF, and not CutG, is essential for CutO activity. No CutO activity is detectable when cutF or its putative Cu-binding motif are mutated, suggesting that the cutF product serves as a Cu-binding component required for active CutO production. Bioinformatic analyses of CutF-like proteins support their widespread roles as putative Cu-binding proteins for several Cu-relay pathways. Our overall findings show that the cytoplasmic CopZ-CopA dependent Cu detoxification pathway contributes to providing Cu to CutO maturation, a process that strictly relies on cutF .
A bifunctional peroxidase enzyme, 4-coumarate 3-hydroxylase (C3H/APX), provides a parallel route to the shikimate shunt pathway for the conversion of 4-coumarate to caffeate in the early steps of lignin biosynthesis. Knockdown of C3H/APX (C3H/APX-KD) expression has been shown to reduce the lignin content in Brachypodium distachyon . However, like many other lignin-modified plants, C3H/APX-KDs show unpredictable pleiotropic phenotypes, including stunted growth, delayed senescence, and reduced seed yield. A system-wide level understanding of altered biological processes in lignin-modified plants can help pinpoint the lignin-modification associated growth defects to benefit future studies aiming to negate the yield penalty. Here, a multi-omic approach was used to characterize molecular changes resulting from C3H/APX-KD associated lignin modification and negative growth phenotype in Brachypodium distachyon . Our findings demonstrate that C3H/APX knockdown in Brachypodium stems substantially alters the abundance of enzymes implicated in the phenylpropanoid biosynthetic pathway and disrupt cellular redox homeostasis. Moreover, it elicits plant defense responses associated with intracellular kinases and phytohormone-based signaling to facilitate growth-defense trade-offs. A deeper understanding along with potential targets to mitigate the pleiotropic phenotypes identified in this study could aid to increase the economic feasibility of lignocellulosic biofuel production.
The ionome represents elemental composition in plant tissues and can be an indicator of nutrient status as well as overall plant performance. Thus, identifying genetic determinants governing elemental uptake and storage is an important goal for breeding and engineering biomass feedstocks with improved performance. In this study, we coupled high-throughput ionome characterization of leaf tissues with high-resolution genome-wide association studies (GWAS) to uncover genetic loci that modulate ionomic composition in leaves of poplar ( Populus trichocarpa ). Significant agreement was observed across the three ionomic profiling platforms tested: inductively coupled plasma-mass spectrometry (ICP-MS), neutron activation analysis (NAA) and laser-induced breakdown spectroscopy (LIBS). Relative quantification of 20 elements using ICP-MS across a population of 584 genotypes, revealed larger variation in micro-nutrients and trace elements content than for macro-nutrients across genotypes. The GWAS performed using a set of high-density (>8.2 million) single nucleotide polymorphisms, identified over 600 loci significantly associated with variations in these mineral elements, pointing to numerous uncharacterized candidate genes. A significant enrichment for genes related to ion homeostasis and transport was observed, including several members of the cation-proton antiporters (CPA) family and MATE efflux transporters, previously reported to be critical for plant growth and fitness in other species. Our results also included a polymorphic copy of the high-affinity molybdenum transporter MOT1 found directly associated to molybdenum content. For the first time in a perennial plant, our results provide evidence of genetic control of mineral content in a model tree species.
Among the rare earth elements (REEs), a crucial group of metals for high-technologies. Gadolinium (Gd) is the only REE intentionally injected to human patients. The use of Gd-based contrasting agents for magnetic resonance imaging (MRI) is the primary route for Gd direct exposure and accumulation in humans. Consequently, aquatic environments are increasingly exposed to Gd due to its excretion through the urinary tract of patients following an MRI examination. The increasing number of reports mentioning Gd toxicity, notably originating from medical applications of Gd, necessitates an improved risk–benefit assessment of Gd utilizations. To go beyond toxicological studies, unravelling the mechanistic impact of Gd on humans and the ecosystem requires the use of genome-wide approaches. We used functional deletomics, a robust method relying on the screening of a knock-out mutant library of Saccharomyces cerevisiae exposed to toxic concentrations of Gd. The analysis of Gd-resistant and -sensitive mutants highlighted the cell wall, endosomes and the vacuolar compartment as cellular hotspots involved in the Gd response. Furthermore, we identified endocytosis and vesicular trafficking pathways (ESCRT) as well as sphingolipids homeostasis as playing pivotal roles mediating Gd toxicity. Finally, tens of yeast genes with human orthologs linked to renal dysfunction were identified as Gd-responsive. Therefore, the molecular and cellular pathways involved in Gd toxicity and detoxification uncovered in this study underline the pleotropic consequences of the increasing exposure to this strategic metal.
Nature-inspired synthetic dissipative self-assemblies have attracted much attention recently. However, it remains a major challenge to achieve precise control over dissipative supramolecular assembly structures and functions of self-contained systems. Here we combine light and electricity as two clean, and spatiotemporally addressable fuels to provide precise control over the morphology for dissipative self-assembly of a perylene bisimide glycine (PBIg) building block in a self-contained solution. In this design, electrochemical oxidation provides the positive fuel to activate PBIg self-assembly while photoreduction supplies the negative fuel to deactivate the system for disassembly. Further, through programming the two counteracting fuels, we demonstrated the control of PBIg self-assembly into a variety of assembly morphologies in a self-contained system. In addition, by exerting light and electrical dual fuels simultaneously, we could create an active homeostasis exhibiting dynamic instability, leading to morphological change to asymmetric assemblies with curvatures. Such precise control over self-assembly of self-contained systems may find future applications in programming complex active materials as well as formulating pharmaceutical reagents with desired morphologies.
Plant organs and tissues contain multiple cell types, which are well organized in 3-dimensional structure to efficiently perform physiological functions such as homeostasis, response to environmental perturbation, pathogen infection. It is critically important to perform molecular measurements at the cell-type-specific level to discover mechanisms and unique features of cell populations that govern differentiation and respond to external perturbations. Although mass spectrometry-based proteomics has been demonstrated as an enabling discovery tool to study plant physiology, conventional approaches require millions of cells to generate robust biological conclusions. Such requirements mask the cell-to-cell heterogeneities and limit the comprehensive profiling of plant proteins at spatially resolved and cell-type-specific resolutions. This protocol describes a recently-developed proteomics workflow for studying a small number of plant cells by integrating laser capture microdissection, microfluidic nanodroplet-based sample preparation, with ultrasensitive liquid chromatography-mass spectrometry. Using poplar as a model tree species, we provide detailed protocols, including plant tissue harvest, tissue preparation, cryosectioning, laser microdissection, protein digestion, mass spectrometry measurement, and data analysis. We show the workflow enables the precise identification and quantification of thousands of proteins from hundreds of isolated plant root and leaf cells.
Mixed lineage kinase domain-like pseudokinase (MLKL), a key terminal effector of necroptosis, also plays a role in intracellular vesicle trafficking that is critical for regulating liver inflammation and injury in alcohol-associated liver disease (ALD). Although receptor interacting protein kinase 3 (Rip3) -/- mice are completely protected from ethanol-induced liver injury, Mlkl -/- mice are only partially protected. Therefore, we hypothesized that cell-specific functions of MLKL may contribute to ethanol-induced injury. Bone marrow transplants between Mlkl -/- mice and littermates were conducted to distinguish the role of myeloid versus nonmyeloid Mlkl in the Gao-binge model of ALD. Ethanol-induced hepatic injury, steatosis, and inflammation were exacerbated in Mlkl -/- →wild-type (WT) mice, whereas Mlkl deficiency in nonmyeloid cells (WT→Mlkl -/- ) had no effect on Gao-binge ethanol-induced injury. Importantly, Mlkl deficiency in myeloid cells exacerbated ethanol-mediated bacterial burden and accumulation of immune cells in livers. Mechanistically, challenging macrophages with lipopolysaccharide (LPS) induced signal transducer and activator of transcription 1–mediated expression and phosphorylation of MLKL, as well as translocation and oligomerization of MLKL to intracellular compartments, including phagosomes and lysosomes but not plasma membrane. Importantly, pharmacological or genetic inhibition of MLKL suppressed the phagocytic capability of primary mouse Kupffer cells (KCs) at baseline and in response to LPS with/without ethanol as well as peripheral monocytes isolated from both healthy controls and patients with alcohol-associated hepatitis. Further, in vivo studies revealed that KCs of Mlkl -/- mice phagocytosed fewer bioparticles than KCs of WT mice. Together, these data indicate that myeloid MLKL restricts ethanol-induced liver inflammation and injury by regulating hepatic immune cell homeostasis and macrophage phagocytosis.
Molecular oxygen levels vary during development and disease. Adaptations to decreased oxygen bioavailability (hypoxia) are mediated by hypoxia-inducible factor (HIF) transcription factors. HIFs are composed of an oxygen-dependent α subunit (HIF-α), of which there are two transcriptionally active isoforms (HIF-1α and HIF-2α), and a constitutively expressed β subunit (HIFβ). Under normoxic conditions, HIF-α is hydroxylated via prolyl hydroxylase domain (PHD) proteins and targeted for degradation via Von Hippel-Lindau (VHL). Under hypoxic conditions, hydroxylation via PHD is inhibited, allowing for HIF-α stabilization and induction of target transcriptional changes. Our previous studies showed that Vhl deletion in osteocytes (Dmp1-cre; Vhlf/f) resulted in HIF-α stabilization and generation of a high bone mass (HBM) phenotype. The skeletal impact of HIF-1α accumulation has been well characterized; however, the unique skeletal impacts of HIF-2α remain understudied. Because osteocytes orchestrate skeletal development and homeostasis, we investigated the role of osteocytic HIF-α isoforms in driving HBM phenotypes via osteocyte-specific loss-of-function and gain-of-function HIF-1α and HIF-2α mutations in C57BL/6 female mice. Deletion of Hif1a or Hif2a in osteocytes showed no effect on skeletal microarchitecture. Constitutively stable, degradation-resistant HIF-2α (HIF-2α cDR), but not HIF-1α cDR, generated dramatic increases in bone mass, enhanced osteoclast activity, and expansion of metaphyseal marrow stromal tissue at the expense of hematopoietic tissue. Our studies reveal a novel influence of osteocytic HIF-2α in driving HBM phenotypes that can potentially be harnessed pharmacologically to improve bone mass and reduce fracture risk.
ABSTRACT The development of Wnt-based osteoanabolic agents has progressed rapidly in recent years, given the potent effects of Wnt modulation on bone homeostasis. Simultaneous pharmacologic inhibition of the Wnt antagonists sclerostin and Dkk1 can be optimized to create potentiated effects in the cancellous bone compartment. We looked for other candidates that might be co-inhibited along with sclerostin to potentiate the effects in the cortical compartment. Sostdc1 (Wise), like sclerostin and Dkk1, also binds and inhibits Lrp5/6 coreceptors to impair canonical Wnt signaling, but Sostdc1 has greater effects in the cortical bone. To test this concept, we deleted Sostdc1 and Sost from mice and measured the skeletal effects in cortical and cancellous compartments individually. Sost deletion alone produced high bone mass in all compartments, whereas Sostdc1 deletion alone had no measurable effects on either envelope. Mice with codeletion of Sostdc1 and Sost had high bone mass and increased cortical properties (bone mass, formation rates, mechanical properties), but only among males. Combined administration of sclerostin antibody and Sostdc1 antibody in wild-type female mice produced potentiation of cortical bone gain despite no effect of Sostdc1 antibody alone. In conclusion, Sostdc1 inhibition/deletion can work in concert with sclerostin deficiency to improve cortical bone properties. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
Abstract The choroid plexus (CP) is part of the blood‐cerebrospinal fluid barrier (BCSFB) and was recently described as an important component of the circadian clock system. It is the principal source of cerebrospinal fluid (CSF) and responsible for the synthesis and secretion of various neuroprotective peptides including those involved in amyloid‐β (Aβ) transport/degradation, contributing to Aβ homeostasis. Inadequate Aβ metabolic clearance and transport across the BCSFB have been associated with circadian dysfunctions in Alzheimer's disease (AD) patients. To investigate whether AD pathology influences Aβ scavengers circadian expression, we collected CP at different time points from an AD mouse model (APP/PS1) (female and male animals, aged 6‐ and 12‐months‐old) and analyzed their mRNA expression by Real‐time RT‐PCR. Only angiotensin‐converting enzyme (Ace) expression in 6‐month‐old female wild‐type mice and transthyretin (Ttr) expression in 12‐month‐old female wild‐type mice presented significant rhythmicity. The circadian rhythmicity of Ace and Ttr, prompt us to analyze the involvement of circadian rhythm in Aβ uptake. A human CP papilloma (HIBCPP) cell line was incubated with Aβ‐488 and uptake was evaluated at different time points using flow cytometry. Aβ uptake displayed circadian rhythmicity. Our results suggest that AD might affect Aβ scavengers rhythmicity and that Aβ clearance is a rhythmic process possibly regulated by the rhythmic expression of Aβ scavengers.
Hypoxia-inducible factors (HIFs) are transcription factors stabilized under hypoxia and degraded under normoxia by the E3 ubiquitin ligase Von Hippel-Lindau (Vhl). In osteocytes, the central orchestrators of bone homeostasis, Vhl and HIFs promote an osteoanabolic transcriptional program. In this study, we assessed unique impacts of osteocytic Vhl deletion versus individual HIF-α paralog stabilization on bone structure, mineralization, and mechanics as a function of sex and skeletal region. Microcomputed tomography revealed that osteocytic Vhl deletion robustly increased trabecular bone mass in both sexes and at both axial and appendicular regions, while HIF-2α accumulation increased femoral metaphyseal bone mass in both sexes while enhancing vertebral bone mass only in males. Vhl deletion paradoxically reduced mineral heterogeneity in male vertebrae, despite raising peak and mean calcium content in both sexes. In contrast, HIF-2α stabilization impaired cortical mineralization and mechanics, especially in females. While cortical mineralization was disrupted in both genotypes, Vhl deletion improved whole bone mechanical properties, suggesting that enhanced geometry and mass offset compromised tissue quality. HIF-1α stabilization exerted negligible impacts on any outcome.
Abstract Membrane lipids serve as substrates for the generation of numerous signaling lipids when plants are exposed to environmental stresses, and jasmonic acid, an oxidized product of 18‐carbon unsaturated fatty acids (e.g., linolenic acid), has been recognized as the essential signal in wound‐induced gene expression. Yet, the contribution of individual membrane lipids in linolenic acid generation is ill‐defined. In this work, we performed spatial lipidomic experiments to track lipid changes that occur locally at the sight of leaf injury to better understand the potential origin of linolenic and linoleic acids from individual membrane lipids. The central veins of tomato leaflets were crushed using surgical forceps, leaves were cryosectioned and analyzed by two orthogonal matrix‐assisted laser desorption/ionization mass spectrometry imaging platforms for insight into lipid spatial distribution. Significant changes in lipid composition are only observed 30 min after wounding, while after 60 min lipidome homeostasis has been re‐established. Phosphatidylcholines exhibit a variable pattern of spatial behavior in individual plants. Among lysolipids, lysophosphatidylcholines strongly co‐localize with the injured zone of wounded leaflets, while, for example, lysophosphatidylglycerol (LPG) (16:1) accumulated preferentially toward the apex in the injured zone of wounded leaflets. In contrast, two other LPGs (LPG [18:3] and LPG [18:2]) are depleted in the injured zone. Our high‐resolution co‐localization imaging analyses suggest that linolenic acids are predominantly released from PCs with 16_18 fatty acid composition along the entire leaf, while it seems that in the apex zone PG (16:1_18:3) significantly contributes to the linolenic acid pool. These results also indicate distinct localization and/or substrate preferences of phospholipase isoforms in leaf tissue.
Abstract The tonoplast sucrose transporter PtaSUT4 is well expressed in leaves of Populus tremula × Populus alba (INRA 717‐IB4), and its inhibition by RNA‐interference (RNAi) alters leaf sucrose homeostasis. Whether sucrose partitioning between the vacuole and the cytosol is modulated by PtaSUT4 for specific physiological outcomes in Populus remains unexplored. In this study, partial defoliation was used to elicit compensatory increases in photosynthesis and transpiration by the remaining leaves in greenhouse‐grown poplar. Water uptake, leaf gas exchange properties, growth and nonstructural carbohydrate abundance in source and sink organs were then compared between wild‐type and SUT4 ‐RNAi lines. Partial defoliation increased maximum photosynthesis rates similarly in all lines. There was no indication that source leaf sugar levels changed differently between wild‐type and RNAi plants following partial defoliation. Sink levels of hexose (glucose and fructose) and starch decreased similarly in all lines. Interestingly, plant water uptake after partial defoliation was not as well sustained in RNAi as in wild‐type plants. While the compensatory increase in photosynthesis was similar between genotypes, leaf transpiration increased less robustly in RNAi than wild‐type plants. SUT4‐RNAi and wild‐type source leaves differed constitutively in their bulk modulus of elasticity, a measure of leaf turgor, and storage water capacitance. The data demonstrate that reduced sucrose partitioning due to PtaSUT4 ‐RNAi altered turgor control and compensatory transpiration capacity more strikingly than photosynthesis and sugar export. The results are consistent with the interpretation that SUT4 may control vacuolar turgor independently of sink carbon provisioning.
Abstract Cell walls surround all plant cells, and their composition and structure are tightly regulated to maintain cellular and organismal homeostasis. In response to wall damage, the cell wall integrity (CWI) system is engaged to ameliorate effects on plant growth. Despite the central role CWI plays in plant development, our current understanding of how this system functions at the molecular level is limited. Here, we investigated the transcriptomes of etiolated seedlings of mutants of Arabidopsis thaliana with defects in three major wall polysaccharides, pectin ( quasimodo2 ), cellulose ( cellulose synthase3 je5 ), and xyloglucan ( xyloglucan xylosyltransferase1 and 2 ), to probe whether changes in the expression of cell wall‐related genes occur and are similar or different when specific wall components are reduced or missing. Many changes occurred in the transcriptomes of pectin‐ and cellulose‐deficient plants, but fewer changes occurred in the transcriptomes of xyloglucan‐deficient plants. We hypothesize that this might be because pectins interact with other wall components and/or integrity sensors, whereas cellulose forms a major load‐bearing component of the wall; defects in either appear to trigger the expression of structural proteins to maintain wall cohesion in the absence of a major polysaccharide. This core set of genes functioning in CWI in plants represents an attractive target for future genetic engineering of robust and resilient cell walls.
Abstract In Populus and many other tree species, photoassimilate sucrose diffuses down a concentration gradient via symplastically connected mesophyll cells to minor vein phloem for long‐distance transport. There is no evidence for apoplastic phloem‐loading in Populus . However, plasma membrane sucrose transporters (SUT1 and SUT3) orthologous to those associated with apoplastic phloem loading are expressed in vascular tissues of poplar. While SUT3 functions in sucrose import into developing xylem, the role of SUT1 remains unclear. Here, we overexpressed PtaSUT1 in Populus tremula x P. alba to examine the effects on sucrose partitioning in transgenic plants. Overall leaf sucrose levels were similar between wild type and transgenic lines. Stem sucrose levels were not changed in bark but were significantly reduced in the adjacent xylem, suggesting hindered intercellular sucrose trafficking from the phloem to the developing xylem. Fully expanded leaves of transgenic plants deteriorated prematurely with declining photosynthesis prior to severe necrotic spotting. Necrotic spotting advanced most rapidly in the distal portion of mature leaves and was accompanied by sharp hexose increases and sharp sucrose decreases there. Leaf transcriptome profiling and network inference revealed the down‐regulation of copper proteins and elevated expression of copper microRNAs prior to noticeable leaf injury. Our results suggest ectopic expression of PtaSUT1 altered sucrose partitioning in stems with systemic effects on leaf health and copper homeostasis mediated in part by sucrose‐sensitive copper miRNAs.
The low-density lipoprotein receptor (LDLR) is a major apolipoprotein receptor that regulates cholesterol homeostasis. LDLR deficiency is associated with cognitive impairment by the induction of synaptopathy in the hippocampus. Despite the close relationship between LDLR and neurodegenerative disorders, proteomics research for protein profiling in the LDLR knockout (KO) model remains insufficient. Therefore, understanding LDLR KO-mediated differential protein expression within the hippocampus is crucial for elucidating a role of LDLR in neurodegenerative disorders. In this study, we conducted first-time proteomic profiling of hippocampus tissue from LDLR KO mice using tandem mass tag (TMT)-based MS analysis. LDLR deficiency induces changes in proteins associated with the transport of diverse molecules, and activity of kinase and catalyst within the hippocampus. Additionally, significant alterations in the expression of components in the major synaptic pathways were found. Furthermore, these synaptic effects were verified using a data-independent acquisition (DIA)-based proteomic method. In conclusion, our data will serve as a valuable resource for further studies to discover the molecular function of LDLR in neurodegenerative disorders.