Subunit Ratio Research Articles

FRI0006 ASSESSING PRO-INFLAMMATORY PROPERTIES OF H-FERRITIN BY EX VIVO AND IN VITRO OBSERVATIONS

Background:The concept of ‘hyperferritinemic syndrome’ has recently been proposed, suggesting high levels of ferritin as pathogenic pro-inflammatory mediator [1] Ferritin is an intracellular iron storage protein, comprising 24 subunits, heavy (H) and light (L) based on molecular weight. The H-/L subunits ratio may be different in tissues, since the ferritin enriched in L subunits (L-ferritin) and the ferritin enriched in H subunits (H-ferritin) may be observed in different tissues, according to pathophysiologic status [1].Objectives:We aimed to assess the presence of H- and L-ferritin as well as of CD68/H-ferritin and CD68/L-ferritin in bone marrow (BM) biopsies of adult macrophage activation syndrome (MAS) patients. In the same patients, we matched the findings of BM biopsies with sera to identify the main represented subunits of ferritin. Furthermore, we evaluated effects of ferritin, L-ferritin, and H-ferritin on human monocytes, assessing pro- and anti-inflammatory cytokines, and expression of NLRP3 inflammasome. Finally, we checked the ability of monocytes, which were treated with ferritin, to stimulate or not the proliferation of peripheral blood mononuclear cells (PBMCs).Methods:Immunofluorescence analysis was performed to investigate the tissue presence of L- and H-ferritin in BM biopsies as well as of CD68/H-ferritin and CD68/L-ferritin. Liquid chromatography mass spectrometry (LC-MS/MS) based proteomics was performed to identify L- and H-ferritin in sera proteins. Human monocytes were cultured with M-CSF for 7 days and, after that, treated with ferritin, H-ferritin, and L-ferritin at 10nM, for 120 and 240 minutes. After stimulation, IL-1β, IL-6, IL-10, IL-12, IFN-γ, TGF-β, TNF, and VEGF were assessed by RT-PCR and, in case of positive finding, evaluated by western blot. NLRP3 inflammasome was also assessed. Finally, the proliferation of PBMCs when co-cultured with ferritin-treated monocytes was tested by a specific proliferation assay.Results:Immunofluorescence showed an increased H-ferritin expression in the BMs of MAS patients, whereas L-ferritin did not. Conversely, LC-MS/MS identified that the L-ferritin was the dominant form, after stringent probability matching.In vitro, H-Ferritin induced a significant increased expression of IL-1β, IL-6, IL-12, and TNF after 240 minutes. Ferritin also induced a significant increased expression of IL-1β, IL-6, IL-12, and TNF after 240 minutes. Effects on pro-inflammatory cytokines were more marked with H-ferritin than ferritin. Conversely, no significant effects were retrieved analysing IFN-γ, IL-10, TGF-β, and VEGF after 240 minutes, after ferritin and H-ferritin stimulation. Furthermore, both ferritin and H-ferritin induced a direct effect on NLRP3 inflammasome. Finally, monocytes, which were treated with H-ferritin, stimulated the proliferation of co-cultured PBMCs.Conclusion:In our work, results showed the presence of H-ferritin and CD68/H-ferritin cells in BM biopsies of MAS patients, by immunofluorescence. Conversely, LC-MS/MS identified L-ferritin in sera proteins of those patients. Furthermore, pro-inflammatory effects of ferritin and, particularly, of H-ferritin on human monocytes were observedin vitro, increasing pro-inflammatory cytokines and NLRP3 inflammosome. Finally, H-ferritin-treated monocytes stimulated the proliferation of co-cultured PBMCs.

The distinctive assembly pattern of ε subunit in ternary α1β3ε and binary β3ε GABAA receptors

Among all GABAA receptor subunits, ε subunit is a more recent discovery. ε subunit-containing GABAA receptors exhibit spontaneous channel activity, rapid desensitisation, low sensitivity to Zn2+, smaller GABAmediated current amplitudes and an insensitivity to benzodiazepines, despite displaying an agonistic effect at higher benzodiazepine concentrations. The promiscuous role of the ε subunit, co-assembling with other subunits forming GABAARs, may add to the complexities of the pharmacological properties of GABAARs; however, these varying pharmacological responses can be used to distinguish varying subunit combinations of these receptors. Using two electrode voltage-clamped electrophysiology, we investigated the GABAA α1β3ε and β3ε receptors and explored the effects of different stoichiometries of these receptor subtypes by varying the relative ratios of α1/β3/ε (for α1β3ε receptors) and β3/ε (for β3ε receptors) subunit complementary RNA injections into Xenopus laevis oocytes. We discovered the existence of different populations of GABAA α1β3ε and β3ε receptors, due to subunit ratio variation, in which receptors formed at each injection ratio showed different level of GABA sensitivities, spontaneous current activities and Zn2+ mediated current inhibition. These unique pharmacological features are tightly associated with various subtypes of GABAA receptors contributed by the unique assembly pattern of ε subunit.

Unbiased Proteomics Supports a Key Role for Mitochondria in Skeletal Muscle of Highly Functioning Octogenarians

IntroductionSkeletal muscle mass and function decline with aging, and more precipitously after the age 75 y. To help provide insights into biologically relevant mechanisms for preserving muscle mass and function in advanced age, in this study we performed proteomics on skeletal muscle of world class octogenarian track and field athletes in comparison to healthy octogenarian non‐athletes.MethodsMuscle cross‐sectional area by MRI and a vastus lateralis muscle biopsy were performed in 15 octogenarian world class track and field athletes (8 of whom were world record holders in their discipline at the time of testing) and 14 non‐athlete age‐ and sex‐matched non‐athlete controls. From these subjects, a portion of muscle from a subset of 12 master athletes (MA mean age 81.19 ± 5.1 y) and 12 non‐athlete controls (NA mean age 80.94 ± 4.5 y) was used for liquid‐chromatography mass spectrometry to generate quantitative tandem mass tag proteomics data. In addition, we measured mtDNA copy number, COX/SDH histochemistry to identify respiratory compromised fibers, and Western blot of mitochondrial inner and outer membrane proteins.ResultsMuscle cross‐sectional area was higher in MA. Tandem mass spectrometry identified over 6000 proteins, and significant differences in abundance were found between NA controls and MA for more than 800 proteins. A pathway analysis revealed that pathways involved in mitochondria (e.g., TCA cycle, respiratory electron transport, cristae formation, sirtuins) were higher in MA, while proteins in pathways involved in the spliceosome and nuclear pore were downregulated in MA. Finally, 8 mtDNA‐encoded proteins that were included in the analysis were elevated in MA versus NA. These proteomics data are consistent with phenotypic data showing MA have higher mtDNA copy number, fewer respiratory chain compromised muscle fibers, and an increased ratio of ETC subunits (inner mitochondrial membrane proteins) relative to VDAC (outer mitochondrial membrane protein), suggesting an increase in cristae formation.ConclusionOur data underscore that mitochondrial pathways are key to maintaining a high level of physical function in advanced age. With the current study design we cannot determine the degree to which these differences are attributable to the physical activity habits of MA, but it is likely to play a role.Support or Funding InformationCIHR (MOP 125986 to RTH)NIA Intramural Research program

Modulation by neonicotinoids of honeybee α1/chicken β2 hybrid nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes

Neonicotinoids targeting insect nicotinic acetylcholine (ACh) receptors (insect nAChRs) are used for crop protection, but there is a concern about adverse effects on pollinators such as honeybees (Apis mellifera). Thus, we investigated the agonist actions of neonicotinoids (imidacloprid, thiacloprid and clothianidin) on A. mellifera α1 (Amα1)/chicken β2 hybrid nAChRs in Xenopus laevis oocytes according to the subunit stoichiometry of (Amα1)3(β2)2 and (Amα1)2(β2)3 using voltage-clamp electrophysiology. ACh activated (Amα1)3(β2)2 and (Amα1)2(β2)3 nAChRs with similar current amplitude. We investigated the agonist activity of imidacloprid, thiacloprid and clothianidin for the two hybrid nAChRs and found that: 1) imidacloprid showed higher affinity than clothianidin, whereas clothianidin showed higher efficacy than imidacloprid for the nAChRs; 2) Thiacloprid showed the highest agonist affinity and the lowest efficacy for the nAChRs. The Amα1/β2 subunit ratio influenced the efficacy of imidacloprid and thiacloprid, but hardly affected that of clothianidin. Hydrogen bond formation by the NH group in clothianidin with the main chain carbonyl of the loop B may account, at least in part, for the unique agonist actions of clothianidin on the hybrid nAChRs tested.

Eltrombopag Improves Erythroid Differentiation in a Human iPSC Model of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a congenital bone marrow (BM) failure syndrome primarily characterized by defective erythropoiesis. In most patients, pathogenic heterozygous mutations have been identified in genes encoding ribosomal proteins (RP). The resulting RP haploinsufficiency was recently shown to delay globin protein translation in erythroid cells, whereas synthesis of heme, the nonprotein iron-containing component of hemoglobin, proceeds normally (Yang et al., Sci Transl Med 2016). Because heme is first synthesized at or just before the proerythroblast stage, free heme is in excess of globin in these cells. High levels of free heme induce proerythroblast cell death, and erythroid differentiation thus halts at the earlier BFU-E/CFU-E progenitor stage. Consistent with these observations, inhibition of heme synthesis with succinylacetone was previously shown to improve erythroid differentiation of DBA marrow cells in vitro. In this study, we investigated whether eltrombopag (Epag), an FDA-approved mimetic of thrombopoietin that promotes trilineage hematopoiesis in subjects with acquired BM failure (Olnes et al., NEJM 2012; Townsley et al., NEJM 2017), could rescue erythropoiesis in DBA. We hypothesized that Epag might inhibit heme synthesis by restricting iron availability due to its robust intracellular iron chelating properties, leading to decrements in iron-induced reactive oxygen species (ROS) and increased proerythroblast survival and maturation. To test this possibility,we first established an induced pluripotent stem cell (iPSC) model of DBA by reprogramming mononuclear cells (MNCs) from a patient with inactivating mutations in RPS19, the most commonly mutated gene in DBA. We also generated a control isogenic iPSC line by CRISPR/Cas9-mediated correction of RPS19 point mutations in the established DBA iPSC line.RPS19 haploinsufficiency was confirmed by Western blot and the expected reduction in 40S/60S ribosomal subunit ratio was detected by polysome profiling of DBA iPSCs. This phenotype normalized in the isogenic iPSCs. Both DBA and isogenic iPSC lines, and iPSCs derived from a healthy donor, were then subjected to hematopoietic differentiation for 21 days using the STEMdiffTMmonolayer-based approach (Ruiz et al., BioRxiv 2019). Hematopoietic cells were harvested between day 19 and 21 of culture when maximum erythroid production is observed in this system. Normal and isogenic iPSCs efficiently gave rise to erythroid cells at various stages of maturation, including CD71+CD45+EPOR+primitive erythroid progenitors (P1), CD71+CD45loEPOR-proerythroblasts (P2), and more mature CD71+CD45-EPOR-erythroblasts (P3) (Figure). In contrast, the majority of erythroid cells detected after differentiation of DBA iPSCs were comprised within P1 with limited maturation to P2 and P3, consistent with a block in differentiation at the early erythroid progenitor stage (Figure). Furthermore, in colony forming unit (CFU) assays, DBA iPSCs generated numbers of myeloid colonies (CFU-G, CFU-M and CFU-GM) comparable to normal and isogenic iPSCs, but erythroid colonies (BFU-E and CFU-E) were undetectable, in keeping with DBA progenitor's inability to differentiate in vitro. Next, DBA iPSCs were differentiated in the presence of Epag 3 µg/mL from day 10 to 21 of culture. Addition of Epag improved late erythroid maturation, as indicated by reduced percentages of early progenitors (P1) and a concomitant increase in more mature P2 and P3 erythroblastic populations (Figure). Investigations are ongoing to confirm Epag-mediated iron restriction and decreased heme synthesis as the primary molecular mechanism underpinning the improved erythroid maturation observed in this study. Overall, our data indicate that directed differentiation of DBA iPSCs recapitulates early erythroid maturation defects in vitro, and erythropoiesis can be rescued in part by addition of Epag during culture. These results suggest that Epag may improve red blood cell production in patients with DBA. Figure Disclosures No relevant conflicts of interest to declare.

Protein-Level Evidence of Novel β-Type Hemocyanin and Heterogeneous Subunit Usage in the Pacific Whiteleg Shrimp, Litopenaeus vannamei

The functional diversity of crustacean hemocyanins is broad, encompassing O2 delivery, innate immune response, metabolite storage, and osmolyte balance, all in a heterogeneous protein structure. As such, the sequence diversity of this class of proteins and its subunit composition are the focus of many studies on crustacean adaptation to environmental challenges. Recent transcriptomic and genomic assemblies on the Pacific whiteleg shrimp Litopenaeus vannamei have identified unique isoforms of hemocyanin including an ancestral β-type subunit thought to be lost in penaeid shrimp. However, it is unknown the degree to which these isoforms are translated as proteins, and their abundances within the hemolymph. The present study uses proteomic approaches to characterize the protein-level abundance and organization of these hemocyanin isoforms within their native oligomeric structures. Fractions of each hemocyanin oligomeric form were purified by size-exclusion high performance liquid chromatography for identification of subunit isoforms using tandem mass spectrometry at <1% protein false discovery rate. Data are available via ProteomeXchange with identifier PXD014575. Relative abundances of hemocyanin oligomers and monomeric subunits from hemolymph and fractions were also quantified by polyacrylamide gel electrophoresis with and without denaturation for comparison of subunit heterogeneity. Hemocyanin subunits were organized primarily as hexamers (95-99% relative abundance) as opposed to dodecamers. Ten unique hemocyanins predicted by transcriptome and genome assemblies were identified by tandem mass spectrometry in both oligomer fractions including the first protein-level evidence of a β-type subunit in penaeid shrimp. Identified hemocyanins mapped to four genomic scaffolds and had two or three exons. A single small (75 kDa) subunit constituted half or more of all isoforms identified in each oligomer. The dodecameric fraction exhibited greater subunit heterogeneity utilizing a significantly lower ratio (1.04:1) of small subunit to large subunit compared to hexamers (2.05:1). One isoform, XP_027232115.1, appeared to be more dominant within dodecamers than hexamers. The ability to distinguish and quantify hemocyanin isoforms within oligomeric structures will aid future studies linking hemocyanin genes to transcripts to function and physiology as well as offer insight into the evolutionary history of crustaceans.

The Impact of Nylon-3 Copolymer Composition on the Efficiency of siRNA Delivery to Glioblastoma Cells.

Glioblastoma multiforme is a devastating disease that has attracted enormous attention due to poor prognosis and high recurrence. Small interfering RNA (siRNA) in principle offers a promising therapeutic approach by the downregulation of disease-related genes via RNA interference. For efficient siRNA delivery to target sites, cationic polymers are often used in preclinical studies for the protection of siRNA and complex formation based on electrostatic interactions. In an effort to develop biocompatible and efficient nanocarriers with a translational outlook for optimal gene silencing at reduced toxicity, we synthesized two sets of nylon-3 copolymers with variable cationic content (DM or NM monomer) and hydrophobic subunits (CP monomer) and evaluated their suitability for in vitro siRNA delivery into glioblastoma cells. DM0.4/CP0.6 and NM0.4/CP0.6 polymers with similar subunit ratios were synthesized to compare the effect of different cationic subunits. Additionally, we utilized NM0.2/CP0.8 polymers to evaluate the impact of the different hydrophobic content in the polymer chain. The siRNA condensation ability and polymer–siRNA complex stability was evaluated by unmodified and modified SYBR gold assays, respectively. Further physicochemical characteristics, e.g., particle size and surface charge, were evaluated by dynamic light scattering and laser Doppler anemometry, whereas a relatively new method for polyplex size distribution analysis—tunable resistive pulse sensing—was additionally developed and compared to DLS measurements. Transfection efficiencies, the route of cell internalization, and protein knockdown abilities in glioblastoma cells were investigated by flow cytometry. Furthermore, cellular tolerability was evaluated by MTT and LDH assays. All the polymers efficiently condensed siRNA at N/P ratios of three, whereas polymers with NM cationic subunits demonstrated smaller particle size and lower polyplex stability. Furthermore, NM0.2/CP0.8 polyplexes with the highest hydrophobic content displayed significantly higher cellular internalization in comparison to more cationic formulations and successful knockdown capabilities. Detailed investigations of the cellular uptake route demonstrated that these polyplexes mainly follow clathrin-mediated endocytotic uptake mechanisms, implying high interaction capacity with cellular membranes. Taken together with conducive toxicity profiles, highly hydrophobic nylon-3 polymers provide an appropriate siRNA delivery agent for the potential treatment of glioblastoma.

Changes in lignin structure during earlywood and latewood formation in Scots pine stems

Lignification of earlywood and latewood during annual ring formation in Scots pine stem in the season occurs with opposite dynamics. The subject of this study was to examine the structure of lignin deposited at successive stages of secondary cell wall maturation of these two wood types. Lignin specimens derived from developing cells by the thioglycolic acid were analyzed by the methods of alkaline oxidation, alkaline and acid hydrolysis and IR Fourier spectroscopy. The composition of lignin structural subunits was found to change at each stage of lignification in dependence of the forming wood type. The molar ratio of p-hydroxyphenyl, guaiacyl and syringyl subunits in polymer was modified in earlywood and latewood oppositely. In the course of earlywood cell maturation, syringyl subunits in lignin increased in parallel with p-hydroxyphenyl units. During latewood lignification, syringyl and p-hydroxyphenyl subunits in lignin structure decreased, whereas guaiacyl subunits increased as secondary cell wall maturation. In lignin isolated from both wood types, the ether-bound carbohydrates were more than by an order of magnitude of ester-bound carbohydrates. In early xylem, the content of ether-bound carbohydrates practically did not change during lignin deposition, whereas carbohydrates with ester linkages gradually decreased toward mature xylem. Lignification in latewood was accompanied by the decline of both ester-linked and ether-linked carbohydrates with a sharp increase in the latter in mature xylem. Hemicelluloses, included in such bonds, contained mainly the residues of xylose and arabinose. The composition and the content of hydroxycinnamic acids, taking part in these linkages, also changed in dependence of lignification steps and wood type. The data received are in agreement with the alterations in absorption IR spectra of lignins isolated from the cells at the beginning of lignification and mature xylem. The reasons for the differences in the lignin structure during earlywood and latewood development are discussed.

Response to IL-6 trans- and IL-6 classic signalling is determined by the ratio of the IL-6 receptor \u03b1 to gp130 expression: fusing experimental insights and dynamic modelling

BackgroundInterleukin-6 is a pleiotropic cytokine with high clinical relevance and an important mediator of cellular communication, orchestrating both pro- and anti-inflammatory processes. Interleukin-6-induced signalling is initiated by binding of IL-6 to the IL-6 receptor α and subsequent binding to the signal transducing receptor subunit gp130. This active receptor complex initiates signalling through the Janus kinase/signal transducer and activator of transcription pathway. Of note, IL-6 receptor α exists in a soluble and a transmembrane form. Binding of IL-6 to membrane-bound IL-6 receptor α induces anti-inflammatory classic signalling, whereas binding of IL-6 to soluble IL-6 receptor α induces pro-inflammatory trans-signalling. Trans-signalling has been described to be markedly stronger than classic signalling. Understanding the molecular mechanisms that drive differences between trans- and classic signalling is important for the design of trans-signalling-specific therapies. These differences will be addressed here using a combination of dynamic mathematical modelling and molecular biology.MethodsWe apply an iterative systems biology approach using set-based modelling and validation approaches combined with quantitative biochemical and cell biological analyses.ResultsThe combination of experimental analyses and dynamic modelling allows to relate the observed differences between IL-6-induced trans- and classic signalling to cell-type specific differences in the expression and ratios of the individual subunits of the IL-6 receptor complex. Canonical intracellular Jak/STAT signalling is indifferent in IL-6-induced trans- and classic signalling.ConclusionThis study contributes to the understanding of molecular mechanisms of IL-6 signal transduction and underlines the power of combined dynamical modelling, model-based validation and biological experiments. The opposing pro- and anti-inflammatory responses initiated by IL-6 trans- and classic signalling depend solely on the expression ratios of the subunits of the entire receptor complex. By pointing out the importance of the receptor expression ratio for the strength of IL-6 signalling this study lays a foundation for future precision medicine approaches that aim to selectively block pro-inflammatory trans-signalling. Furthermore, the derived models can be used for future therapy design.Graphical abstract

Prenatal Nutritional Intervention Reduces Autistic-Like Behavior Rates Among Mthfr-Deficient Mice.

The causes and contributing factors of autism spectrum disorders (ASD) are poorly understood. One gene associated with increased risk for ASD is methylenetetrahydrofolate-reductase (MTHFR), which encodes a key enzyme in one carbon (C1) metabolism. The MTHFR 677C > T polymorphism reduces the efficiency of methyl group production with possible adverse downstream effects on gene expression. In this study, the effects of prenatal and/or postnatal diets enriched in C1 nutrients on ASD-like behavior were evaluated in Mthfr-deficient mice. Differences in intermediate pathways between the mice with and without ASD-like behaviors were tested. The findings indicate that maternal and offspring Mthfr deficiency increased the risk for an ASD-like phenotype in the offspring. The risk of ASD-like behavior was reduced in Mthfr-deficient mice supplemented with C1 nutrients prenatally. Specifically, among offspring of Mthfr+/- dams, prenatal diet supplementation was protective against ASD-like symptomatic behavior compared to the control diet with an odds ratio of 0.18 (CI:0.035, 0.970). Changes in major C1 metabolites, such as the ratios between betaine/choline and SAM/SAH in the cerebral-cortex, were associated with ASD-like behavior. Symptomatic mice presenting ASD-like behavior showed decreased levels of GABA pathway proteins such as GAD65/67 and VGAT and altered ratios of the glutamate receptor subunits GluR1/GluR2 in males and NR2A/NR2B in females. The altered ratios, in turn, favor receptor subunits with higher sensitivity to neuronal activity. Our study suggests that MTHFR deficiency can increase the risk of ASD-like behavior in mice and that prenatal dietary intervention focused on MTHFR genotypes can reduce the risk of ASD-like behavior.

Concatenated γ-aminobutyric acid type A receptors revisited: Finding order in chaos.

γ-aminobutyric acid type A receptors (GABAARs), the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are arguably the most challenging member of the pentameric Cys-loop receptors to study due to their heteromeric structure. When two or more subunits are expressed together in heterologous systems, receptors of variable subunit type, ratio, and orientation can form, precluding accurate interpretation of data from functional studies. Subunit concatenation is a technique that involves the linking of individual subunits and in theory allows the precise control of the uniformity of expressed receptors. In reality, the resulting concatemers from widely used constructs are flexible in their orientation and may therefore assemble with themselves or free GABAAR subunits in unexpected ways. In this study, we examine functional responses of receptors from existing concatenated constructs and describe refinements necessary to allow expression of uniform receptor populations. We find that dimers from two commonly used concatenated constructs, β-23-α and α-10-β, assemble readily in both the clockwise and the counterclockwise orientations when coexpressed with free subunits. Furthermore, we show that concatemers formed from new tetrameric α-10-β-α-β and α-10-β-α-γ constructs also assemble in both orientations with free subunits to give canonical αβγ receptors. To restrict linker flexibility, we systematically shorten linker lengths of dimeric and pentameric constructs and find optimized constructs that direct the assembly of GABAARs only in one orientation, thus eliminating the ambiguity associated with previously described concatemers. Based on our data, we revisit some noncanonical GABAAR configurations proposed in recent years and explain how the use of some concatenated constructs may have led to wrong conclusions. Our results help clarify current contradictions in the literature regarding GABAAR subunit stoichiometry and arrangement. The lessons learned from this study may guide future efforts in understanding other related heteromeric receptors.

Nanosized and tunable design of biosilica particles using novel silica-forming peptide-modified chimeric ferritin templates

Biosilica materials can be generated by biomolecules under physiological or mild/ambient conditions. However, nanosized and tunable design method of biosilica particles has not yet been set up. Here, Kps, a silica forming peptide (KPSHHHHHTGAN) was introduced to N-terminus of ferritin via protein fusion method to generate new Kps-modified ferritin (Kps-Fn) for reliable formation of biosilica particles. Then, novel chimeric Kps-Fn was designed for controllable generation of biosilica nanoparticles (NPs). By changing the ratios of Kps-Fn and Fn subunits in the chimeric Kps-Fn templates, desired size of biosilica NPs (100–500nm) can be achieved. The low surface density of Kps on the chimeric template could lead to the small-sized biosilica NPs with the increased surface area/amount. The biosilica NPs developed here showed better DNA adsorption/elution performance than the commercially available silica NPs. This work is a new way to generate desirable biosilica NPs, which can be applied for the design of various biohybrid nanomaterials.

On the structure and chemistry of iron oxide cores in human heart and human spleen ferritins using graphene liquid cell electron microscopy.

Ferritin is a protein that regulates the iron ions in humans by storing them in the form of iron oxides. Despite extensive efforts to understand the ferritin iron oxide structures, it is still not clear how ferritin proteins with a distinct light (L) and heavy (H) chain subunit ratio impact the biomineralization process. In situ graphene liquid cell-transmission electron microscopy (GLC-TEM) provides an indispensable platform to study the atomic structure of ferritin mineral cores in their native liquid environment. In this study, we report differences in the iron oxide formation in human spleen ferritins (HSFs) and human heart ferritins (HHFs) using in situ GLC-TEM. Scanning transmission electron microscopy (STEM) along with selected area electron diffraction (SAED) of the mineral core and electron energy loss spectroscopy (EELS) analyses enabled the visualization of morphologies, crystal structures and the chemistry of iron oxide cores in HSFs and HHFs. Our study revealed the presence of metastable ferrihydrite (5Fe2O3·9H2O) as a dominant phase in hydrated HSFs and HHFs, while a stable hematite (α-Fe2O3) phase predominated in non-hydrated HSFs and HHFs. In addition, a higher Fe3+/Fe2+ ratio was found in HHFs in comparison with HSFs. This study provides new understanding on iron-oxide phases that exist in hydrated ferritin proteins from different human organs. Such new insights are needed to map ferritin biomineralization pathways and possible correlations with various iron-related disorders in humans.

Plane-strain fracture toughness of AISI 4140 steel austempered below MS

Ultrahigh-strength low alloy steels transformed below the martensite start temperature (MS) with a mixed microstructure consisting of lath martensite, lower bainite, and minor amounts of retained austenite are of interest in a variety of potential commercial applications as they exhibit good combinations of strength, ductility, and toughness. However, their use requires a knowledge of the resistance to crack propagation. Analysis of the microstructure and fractography show that it is possible to distinguish the fracture behavior of steels austempered below MS from those quenched and tempered. The sharp-notch plane-strain fracture toughness KIC has been determined by the notched tensile testing and modeled with different expressions from literature. Based on pioneer works of Irwin (1962) [20] and Naylor (1979) [22], KIC associated with austempering below MS has been modeled with the aspect ratio of the microstructural subunits and monotonic plastic zone size ahead of the crack tip. A comparison between existing expressions and the new one is conducted. It is found that modeling with the new expression delivers the best agreement with the experimental values.

Increased CO2 exacerbates the stress of ultraviolet radiation on photosystem II function in the diatom Thalassiosira weissflogii

Diatoms usually dominate phytoplankton community in coastal waters and experience rapid changes of underwater light. However, little is known regarding how increased CO2 would affect diatoms’ capacity in dealing with changing photosynthetically active radiation (PAR) and ultraviolet radiation (UVR). Here, we cultured a globally abundant diatom Thalassiosira weissflogii under two levels of CO2 (400, 1000 ppmv), and then analysed its PSII function during an increase in PAR and UVR to mimic an upward mixing event. UVR noticeably reduced photosystem II (PSII) activity (FV/FM) during the high light exposure, which was more significant for cells grown at the higher CO2 condition. The PsbA removal rate (KPsbA) was synergistically increased by high CO2 and UVR, while the PsbD removal rate (KPsbD) was decreased under higher CO2. Both CO2 and UVR had an inducible effect on sustained phase of nonphotochemical quenching (NPQs). The higher CO2 decreased the ratio of Rubisco large subunit (RbcL) to PsbA regardless of the radiation treatments. It seems that the increased NPQs and turnover of PsbA induced by higher CO2 were not enough to offset the stressful effect it brought about, particularly when higher CO2 was combined with UVR. These findings indicate that increased CO2 may exacerbate the harmful effect of UVR on PSII function in the T. weissflogii through reducing PsbD removal rate and the ratio of RbcL to PsbA during UVR exposure, and thus would affect its abundance and distribution in future ocean environment.

Recalcitrance Assessment of the Agro-industrial Residues from Five Agave Species: Ionic Liquid Pretreatment, Saccharification and Structural Characterization

Agave has recently shown its potential as a bioenergy feedstock with promising features such as higher biomass productivity than leading bioenergy feedstock while at the same time being drought-resistant with low water requirements and high sugar to ethanol conversion using ionic liquid (IL) pretreatment. IL pretreatment was studied to develop the first direct side-by-side comparative recalcitrance assessment of the agro-industrial residues from five Agave species [Agave americana (AME), A. angustifolia (ANG), A. fourcroydes (FOU), A. salmiana (SAL), and A. tequilana (TEQ)] using compositional analysis, X-ray diffraction, and the lignin syringyl/guaiacyl subunit ratio (S/G) by pyrolysis molecular beam mass spectrometry (PyMBMS). Prominent calcium oxalate peaks were found only in unpretreated AME, SAL, and TEQ. The S/G ratios of all five unpretreated Agave species were between 1.27 and 1.57 while the IL-pretreated samples were from 1.39 to 1.72. The highest overall sugar production was obtained with IL-pretreated FOU with 492 mg glucose/g biomass and 157 mg xylose/g biomass at 120 °C and 3 h using 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]). An estimated theoretical ethanol yield from the studied agro-industrial residues from the five Agave species was in the range of 1060 to 5800 L ethanol/ha/year. These comparison results demonstrate the potential of the Agave spp. as a suitable biofuel feedstock which can be employed within a biorefinery scheme.

HIPK2-Mediated Transcriptional Control of NMDA Receptor Subunit Expression Regulates Neuronal Survival and Cell Death.

NMDA receptors are critical for neuronal communication. Dysfunction in NMDA receptors has been implicated in neuropsychiatric diseases. While it is well recognized that the composition of NMDA receptors undergoes a GluN2B-to-GluN2A switch in early postnatal life, the mechanism regulating this switch remains unclear. Using transcriptomic and functional analyses in brain tissues from male and female Hipk2+/+ and Hipk2-/- mice, we showed that the HIPK2-JNK-c-Jun pathway is important in suppressing the transcription of Grin2a and Grin2c, which encodes the GluN2A and GluN2C subunits of the NMDA receptors, respectively. Loss of HIPK2 leads to a significant decrease in JNK-c-Jun signaling, which in turn derepresses the transcription of Grin2a and Grin2c mRNA and upregulates GluN2A and GluN2C protein levels. These changes result in a significant increase of GluN2A/GluN2B ratio in synapse and mitochondria, a persistent activation of the ERK-CREB pathway and the upregulation of synaptic activity-regulated genes, which collectively contribute to the resistance of Hipk2-/- neurons to cell death induced by mitochondrial toxins.SIGNIFICANCE STATEMENT We identify HIPK2-JNK-c-Jun signaling as a key mechanism that regulates the transcription of NMDA receptor subunits GluN2A and GluN2C in vivo Our results provide insights into a previously unrecognized molecular mechanism that control the switch of NMDA receptor subunits in early postnatal brain development. Furthermore, we provide evidence that changes in the ratio of NMDA subunits GluN2A/GluN2B can also be detected in the synapse and mitochondria, which contributes to a persistent activation of the prosurvival ERK-CREB pathway and its downstream target genes. Collectively, these changes protect HIPK2 deficient neurons from mitochondrial toxins.

Differential participation of calcium-activated potassium channel in endothelium-dependent hyperpolarization-type relaxation in superior mesenteric arteries of spontaneously hypertensive rats.

The purpose of this study was to determine the relationship of KCa channels to endothelium-dependent hyperpolarizing factor (EDHF)-mediated relaxation induced by acetylcholine (ACh) in the superior mesenteric arteries of 7-month-old spontaneously hypertensive rats (SHR). Upon inhibition of nitric oxide synthase and cyclooxygenase, ACh-induced EDHF-mediated relaxation was found to be weaker in SHR than in age-matched Wistar Kyoto rats (WKY). These relaxations in both group were attenuated by combined treatment with small-conductance and intermediate-conductance Ca2+-activated K+ channels (SKCa and IKCa) inhibitors, with the exception of relaxation resistant to inhibition of these channels in SHR (vs. WKY). Treatment with large-conductance Ca2+-activated K+ channels (BKCa) inhibitor specifically attenuated relaxation in SHR, but not in WKY. Protein expression of IKCa and SKCa in the arteries did not differ between the 2 groups, whereas ratio of sloβ1 subunit to α subunit of BKCa was increased in SHR (vs. WKY). These results suggest that EDHF-mediated relaxations in superior mesenteric arteries are impaired in SHR, and utilize components of BKCa in addition to SKCa/IKCa channel activities, that the increased participation of BKCa may be attributable to alterations in α and sloβ1 subunit ratio, and that components unrelated to KCa activity may also contribute to the difference between SHR and WKY arteries.

GluN2B Subunit of the NMDA Receptor: The Keystone of the Effects of Alcohol During Neurodevelopment.

The glutamatergic system plays a central role in both the acute and chronic effects of ethanol. Among all the glutamate receptors the ionotropic NMDA receptors are crucial because of their role in synaptic plasticity. A large body of evidences suggests that short-term and long-term effects of ethanol may change synaptic plasticity via an alteration of the expression of the GluN2B subunit, one constitutive element of the NMDA receptor. The present review is focusing on the role of the GluN2B subunit after ethanol exposure during early life (in utero and adolescence) and also at adulthood. The roles of other NMDA subunits are also discussed in the context of the increasing evidence that the ratio of the different subunits, such as GluN2A-to-GluN2B, seems to better reflect the effects of ethanol and to explain how ethanol exposure can have short lasting and long lasting effects on synaptic plasticity, cognitive processes and some of the ethanol-related behaviors.

Reprogramming the Activatable Peptide Display Function of Adeno-Associated Virus Nanoparticles.

We harnessed an intrinsic activatable peptide display behavior shared by several parvoviruses, including the adeno-associated virus (AAV), in order to design protein-based nanodevices that can carry out an exogenous functional output in response to stimulus detection. Specifically, we generated truncated viral capsid subunits that, when combined with native capsid components into mosaic capsids, can perform robust activatable peptide display. By modulating the ratio of subunits in the mosaic capsid, properties of the activatable peptide display function can be optimized. Interestingly, the truncated subunits can form homomeric capsids not observed in nature, but at the price of losing the ability to carry out activatable peptide display. Collectively, our results demonstrate the importance of capsid mosaicism when activatable peptide display is desired and help explain why the wild-type AAV capsid exists as a mosaic of different subunits. This proof-of-concept study illustrates a strategy for reprogramming a particular conformational output behavior of AAV in pursuit of the long-term vision of creating stimulus-responsive nanodevices.