1.
Tjondro, Harry C., et al.
(författare)
Hyper-truncated Asn355- And Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase
2021
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 296
Tidskriftsartikel (refereegranskat) abstract
Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure–biosynthesis–activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.
2.
Lindberg, Frida A., et al.
(författare)
SLC38A10 Deficiency in Mice Affects Plasma Levels of Threonine and Histidine in Males but Not in Females : A Preliminary Characterization Study of SLC38A10(-/-) Mice
2023
Ingår i: Genes. - : MDPI. - 2073-4425. ; 14:4
Tidskriftsartikel (refereegranskat) abstract
Solute carriers belong to the biggest group of transporters in the human genome, but more knowledge is needed to fully understand their function and possible role as therapeutic targets. SLC38A10, a poorly characterized solute carrier, is preliminary characterized here. By using a knockout mouse model, we studied the biological effects of SLC38A10 deficiency in vivo. We performed a transcriptomic analysis of the whole brain and found seven differentially expressed genes in SLC38A10-deficient mice (Gm48159, Nr4a1, Tuba1c, Lrrc56, mt-Tp, Hbb-bt and Snord116/9). By measuring amino acids in plasma, we found lower levels of threonine and histidine in knockout males, whereas no amino acid levels were affected in females, suggesting that SLC38A10(-/-) might affect sexes differently. Using RT-qPCR, we investigated the effect of SLC38A10 deficiency on mRNA expression of other SLC38 members, Mtor and Rps6kb1 in the brain, liver, lung, muscle, and kidney, but no differences were found. Relative telomere length measurement was also taken, as a marker for cellular age, but no differences were found between the genotypes. We conclude that SLC38A10 might be important for keeping amino acid homeostasis in plasma, at least in males, but no major effects were seen on transcriptomic expression or telomere length in the whole brain.
3.
Lindberg, Frida A., et al.
(författare)
SLC38A10 knockout mice display a decreased body weight and an increased risk-taking behavior in the open field test
2022
Ingår i: Frontiers in Behavioral Neuroscience. - : Frontiers Media S.A.. - 1662-5153. ; 16
Tidskriftsartikel (refereegranskat) abstract
The solute carrier 38 family (SLC38) is a family of 11 members. The most commonsubstrate among these are alanine and glutamine, and members are present in a widerange of tissues with important functions for several biological processes, such as liverand brain function. Some of these transporters are better characterized than others and,in this paper, a behavioral characterization of SLC38A10−/− mice was carried out. Abattery of tests for general activity, emotionality, motor function, and spatial memorywere used. Among these tests, the elevated plus maze, Y-maze, marble burying, andchallenging beamwalk have not been tested on the SLC38A10−/− mice previously, whilethe open field and the rotarod tests have been performed by the International MousePhenotyping Consortium (IMPC). Unlike the results from IMPC, the results from this studyshowed that SLC38A10−/− mice spend less time in the wall zone in the open field testthan WT mice, implying that SLC38A10-deficient mice have an increased explorativebehavior, which suggests an important function of SLC38A10 in brain. The present studyalso confirmed IMPC’s data regarding rotarod performance and weight, showing thatSLC38A10−/− mice do not have an affected motor coordination impairment and havea lower body weight than both SLC38A10+/− and SLC38A10+/+ mice. These resultsimply that a complete deficiency of the SLC38A10 protein might affect body weighthomeostasis, but the underlying mechanisms needs to be studied further.
4.
Lindberg, Frida A.
(författare)
The Biological Importance of the Amino Acid Transporter SLC38A10 : Characterization of a Knockout Mouse
2022
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
The biggest group of transporters, the solute carriers (SLCs), has more than 400 members, and about 30% of these are still orphan. In order to decipher their biological function and possible role in disease, there is a need for characterization of these. Around 25% of SLCs are estimated to have amino acids as substrates, including transporters belonging to the SLC38 family. The SLC38 members are sometimes referred to their alternative name: sodium-coupled neutral amino acid transporters (SNATs). One of these transporters, SNAT10 (or SLC38A10), has been characterized as a bidirectional transporter of glutamate, glutamine, alanine and aspartate, as well as having an efflux of serine, and is ubiquitously expressed in the body. However, its biological importance is not yet understood. The aim with this thesis was to characterize a mouse model deficient in SNAT10 protein in order to find the biological importance of this transporter. In paper I, this is done by using a series of behavioral tests, including the open field test, elevated plus maze, rotarod and Y-maze, among others. The SNAT10 knockout mouse was found to have an increased risk-taking behavior, but no motor or spatial working memory impairments. Furthermore, the knockout mouse was found to have a decreased body weight. In paper II, an additional behavioral characterization was performed by using the multivariate concentric square field™ (MCSF) test. The MCSF test is an arena with different zones associated to different behavioral traits, which generates a behavioral profile depending on where the mouse spends its time. The result from this test implies that the SNAT10 deficient mouse has a lower explorative behavior than its wild type littermates. In paper III, gene expression was studied in whole brain and some genes related to cell cycle regulation and p53 expression were found to be differentially expressed in the knockout brain. Additional gene expression was studied in kidney, liver, lung and muscle, but no changes were found. Plasma levels of histidine and threonine were altered in males, but no altered amino acid levels were found in knockout females, suggesting a possible sex-specific effect. These studies together imply that SNAT10 might be involved in processes related to risk-taking and explorative behavior in the open field and MCSF tests. SNAT10 deficiency also affected amino acid levels in plasma, indicating a disrupted amino acid homeostasis.
5.
Omer, Abubakr A. M., 1982-, et al.
(författare)
Plantaricin NC8 αβ rapidly and efficiently inhibits flaviviruses and SARS-CoV-2 by disrupting their envelopes
2022
Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 17:11
Tidskriftsartikel (refereegranskat) abstract
Potent broad-spectrum antiviral agents are urgently needed to combat existing and emerging viral infections. This is particularly important considering that vaccine development is a costly and time consuming process and that viruses constantly mutate and render the vaccine ineffective. Antimicrobial peptides (AMP), such as bacteriocins, are attractive candidates as antiviral agents against enveloped viruses. One of these bacteriocins is PLNC8 αβ, which consists of amphipathic peptides with positive net charges that display high affinity for negatively charged pathogen membrane structures, including phosphatidylserine rich lipid membranes of viral envelopes. Due to the morphological and physiological differences between viral envelopes and host cell plasma membranes, PLNC8 αβ is thought to have high safety profile by specifically targeting viral envelopes without effecting host cell membranes. In this study, we have tested the antiviral effects of PLNC8 αβ against the flaviviruses Langat and Kunjin, coronavirus SARS-CoV-2, influenza A virus (IAV), and human immunodeficiency virus-1 (HIV-1). The concentration of PLNC8 αβ that is required to eliminate all the infective virus particles is in the range of nanomolar (nM) to micromolar (μM), which is surprisingly efficient considering the high content of cholesterol (8–35%) in their lipid envelopes. We found that viruses replicating in the endoplasmic reticulum (ER)/Golgi complex, e.g. SARS-CoV-2 and flaviviruses, are considerably more susceptible to PLNC8 αβ, compared to viruses that acquire their lipid envelope from the plasma membrane, such as IAV and HIV-1. Development of novel broad-spectrum antiviral agents can significantly benefit human health by rapidly and efficiently eliminating infectious virions and thereby limit virus dissemination and spreading between individuals. PLNC8 αβ can potentially be developed into an effective and safe antiviral agent that targets the lipid compartments of viral envelopes of extracellular virions, more or less independent of virus antigenic mutations, which faces many antiviral drugs and vaccines.
6.
Landberg, Rikard, 1981, et al.
(författare)
Avenanthramides as lipoxygenase inhibitors
2020
Ingår i: Heliyon. - : Elsevier BV. - 2405-8440. ; 6:6
Tidskriftsartikel (refereegranskat) abstract
Avenanthramides (AVAs) present in oats are amides of anthranilic and cinnamic acids. AVAs are potent antioxidants and have anti-inflammatory properties. There are various potential mechanisms for their anti-inflammatory effects, including inhibition of lipoxygenases (LOX), which catalyse oxygenation of polyunsaturated fatty acids into potent signal molecules involved in inflammatory processes. In this study, AVAs were screened for LOX inhibition in vitro and structure-activity relationships were examined. Twelve different AVAs at 0.6 mM were tested as LOX inhibitors. The corresponding free cinnamic acids, the AVA analogue Tranilast® and the known LOX inhibitor trans-resveratrol were included for comparison. It was found that AVAs comprising caffeic or sinapic acid exhibited significant lipoxygenase inhibition (60–90%) (P < 0.05), whereas low or no inhibition was observed with AVAs containing p-coumaric or ferulic acid. No difference in inhibition was seen on comparing AVAs with their free corresponding cinnamic acids, which implies that the anthranilic acid part of the avenanthramide molecule does not affect inhibition. Trans-resveratrol showed inhibition, whereas no inhibition was seen for Tranilast® at the concentrations used in this study. This study suggests that aventahtramides comprising caffeic acid or sinapic acid partly exert their antioxidant and anti-inflammatory effects via lipoxygenase inhibition.
7.
Bally, Marta, 1981, et al.
(författare)
Physicochemical tools for studying virus interactions with targeted cell membranes in a molecular and spatiotemporally resolved context
2021
Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 413, s. 7157-7178
Tidskriftsartikel (refereegranskat) abstract
The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.
8.
Noborn, Fredrik, et al.
(författare)
Mapping the Human Chondroitin Sulfate Glycoproteome Reveals an Unexpected Correlation Between Glycan Sulfation and Attachment Site Characteristics. : Chondroitin Sulfation and Attachment Site Characteristics
2023
Ingår i: Molecular & cellular proteomics : MCP. - : Elsevier. - 1535-9484 .- 1535-9476. ; 22:8
Tidskriftsartikel (refereegranskat) abstract
Chondroitin sulfate proteoglycans (CSPGs) control key events in human health and disease and are composed of chondroitin sulfate (CS) polysaccharide(s) attached to different core proteins. Detailed information on the biological effects of site-specific CS structures is scarce as the polysaccharides are typically released from their core proteins prior to analysis. Here we present a novel glycoproteomic approach for site-specific sequencing of CS modifications from human urine. Software-assisted and manual analysis revealed that certain core proteins carried CS with abundant sulfate modifications, while others carried CS with lower levels of sulfation. Inspection of the amino acid sequences surrounding the attachment sites indicated that the acidity of the attachment site motifs increased the levels of CS sulfation, and statistical analysis confirmed this relationship. However, not only the acidity but also the sequence and characteristics of specific amino acids in the proximity of the serine glycosylation site correlated with the degree of sulfation. These results demonstrate attachment site-specific characteristics of CS polysaccharides of CSPGs in human urine and indicate that this novel method may assist in elucidating the biosynthesis and functional roles of CSPGs in cellular physiology.
9.
Singh, Vandana, 1985, et al.
(författare)
Quantification of single-strand DNA lesions caused by the topoisomerase II poison etoposide using single DNA molecule imaging
2022
Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 594, s. 57-62
Tidskriftsartikel (refereegranskat) abstract
DNA-damaging agents, such as radiation and chemotherapy, are common in cancer treatment, but the dosing has proven to be challenging, leading to severe side effects in some patients. Hence, to be able to personalize DNA-damaging chemotherapy, it is important to develop fast and reliable methods to measure the resulting DNA damage in patient cells. Here, we demonstrate how single DNA molecule imaging using fluorescence microscopy can quantify DNA-damage caused by the topoisomerase II (TopoII) poison etoposide. The assay uses an enzyme cocktail consisting of base excision repair (BER) enzymes to repair the DNA damage caused by etoposide and label the sites using a DNA polymerase and fluorescently labeled nucleotides. Using this DNA-damage detection assay we find a large variation in etoposide induced DNA-damage after in vitro treatment of blood cells from healthy individuals. We furthermore used the TopoII inhibitor ICRF-193 to show that the etoposide-induced damage in DNA was TopoII dependent. We discuss how our results support a potential future use of the assay for personalized dosing of chemotherapy.
10.
Kallas, Pawel, et al.
(författare)
Adhesion of Escherichia Coli to Nanostructured Surfaces and the Role of Type 1 Fimbriae
2020
Ingår i: Nanomaterials. - : MDPI AG. - 2079-4991. ; 10:11
Tidskriftsartikel (refereegranskat) abstract
Bacterial fimbriae are an important virulence factor mediating adhesion to both biotic and abiotic surfaces and facilitating biofilm formation. The expression of type 1 fimbriae of Escherichia coli is a key virulence factor for urinary tract infections and catheter-associated urinary tract infections, which represent the most common nosocomial infections. New strategies to reduce adhesion of bacteria to surfaces is therefore warranted. The aim of the present study was to investigate how surfaces with different nanotopography-influenced fimbriae-mediated adhesion. Surfaces with three different nanopattern surface coverages made in polycarbonate were fabricated by injection molding from electron beam lithography nanopatterned templates. The surfaces were constructed with features of approximately 40 nm width and 25 nm height with 100 nm, 250 nm, and 500 nm interspace distance, respectively. The role of fimbriae type 1-mediated adhesion was investigated using the E. coli wild type BW25113 and Delta fimA (with a knockout of major pilus protein FimA) and Delta fimH (with a knockout of minor protein FimH) mutants. For the surfaces with nanotopography, all strains adhered least to areas with the largest interpillar distance (500 nm). For the E. coli wild type, no difference in adhesion between surfaces without pillars and the largest interpillar distance was observed. For the deletion mutants, increased adhesion was observed for surfaces without pillars compared to surfaces with the largest interpillar distance. The presence of a fully functional type 1 fimbria decreased the bacterial adhesion to the nanopatterned surfaces in comparison to the mutants.
11.
Chen, Yu, 1990, et al.
(författare)
Yeast optimizes metal utilization based on metabolic network and enzyme kinetics
2021
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 118:12
Tidskriftsartikel (refereegranskat) abstract
Metal ions are vital to metabolism, as they can act as cofactors on enzymes and thus modulate individual enzymatic reactions. Although many enzymes have been reported to interact with metal ions, the quantitative relationships between metal ions and metabolism are lacking. Here, we reconstructed a genome-scale metabolic model of the yeast Saccharomyces cerevisiae to account for proteome constraints and enzyme cofactors such as metal ions, named CofactorYeast. The model is able to estimate abundances of metal ions binding on enzymes in cells under various conditions, which are comparable to measured metal ion contents in biomass. In addition, the model predicts distinct metabolic flux distributions in response to reduced levels of various metal ions in the medium. Specifically, the model reproduces changes upon iron deficiency in metabolic and gene expression levels, which could be interpreted by optimization principles (i.e., yeast optimizes iron utilization based on metabolic network and enzyme kinetics rather than preferentially targeting iron to specific enzymes or pathways). At last, we show the potential of using the model for understanding cell factories that harbor heterologous iron-containing enzymes to synthesize high-value compounds such as p-coumaric acid. Overall, the model demonstrates the dependence of enzymes on metal ions and links metal ions to metabolism on a genome scale.
12.
Ghirmai, Semhar, 1993, et al.
(författare)
Improving the Stability of Red Blood Cells in Rainbow Trout (Oncorhynchus mykiss) and Herring (Clupea harengus): Potential Solutions for Post-mortem Fish Handling to Minimize Lipid Oxidation
2020
Ingår i: Food and Bioprocess Technology. - : Springer Science and Business Media LLC. - 1935-5130 .- 1935-5149. ; 13, s. 1344-1355
Tidskriftsartikel (refereegranskat) abstract
This study aimed at limiting hemolysis of fish red blood cells (RBCs) as a strategy to limit hemoglobin (Hb)-induced lipid oxidation duringpost-mortemhandling and processing. Effects of varying temperature, salinity, and mechanical impact were studied using washed resuspended RBCs (wr-RBCs) and whole blood (WB) from rainbow trout (Oncorhynchus mykiss) and herring (Clupea harengus). The wr-RBCs were most stable avoiding mechanical stress, keeping isotonic conditions (0.9-1.3% NaCl) and low temperature 0-6 degrees C, with predicted minimum at 2.5 degrees C. When compared at the same salinity, it was found that hemolysis was more pronounced in herring than trout wr-RBCs. Furthermore, WB was more stable than wr-RBCs, showing protecting the effects of blood plasma. Studying individual plasma components, stabilizing effects were found from glucose, proteins, and ascorbic acid. This study indicates that small adjustments in the early handling and processing of fish such as changing salinity of storage and rinsing solutions could minimize Hb contamination of the fish muscle and thereby improve quality.
13.
Wu, Haizhou, 1987, et al.
(författare)
Effects of sodium chloride and sodium tripolyphosphate on the prooxidant properties of hemoglobin in washed turkey muscle system
2022
Ingår i: Food Chemistry: X. - : Elsevier BV. - 2590-1575. ; 16
Tidskriftsartikel (refereegranskat) abstract
This study examined the effects of sodium chloride (NaCl) and sodium tripolyphosphate (STPP) on lipid oxidation induced by oxyhemoglobin (oxyHb) in washed turkey muscle (WTM) model. To explore the reasons for observed effects, the pro-oxidant abilities of Hb derivatives (e.g., metHb, oxyHb, hemin, Fe2+, and Fe3+), pH change, and antioxidation of Hb in the presence of NaCl or STPP were also analyzed. The observed lipid oxidation capacity in WTM followed the order metHb > hemin > oxyHb > Fe2+ > Fe3+. Added Fe2+ accelerated auto-oxidation of oxyHb and oxyHb-mediated lipid oxidation. Hb auto-oxidation to metHb increased as the pH decreased from 6.6 to 5.0. NaCl promoted oxyHb-mediated lipid oxidation due to NaCl causing decreased pH value and increased formation of metHb. STPP inhibited oxyHb-mediated lipid oxidation and weakened the pro -oxidative effect of NaCl. This could be attributed to STPP increasing the pH, inactivating free iron, and inhibiting formation of metHb.
14.
McStay, Natasha, et al.
(författare)
Click and Cut: a click chemistry approach to developing oxidative DNA damaging agents
2021
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 49:18, s. 10289-10308
Tidskriftsartikel (refereegranskat) abstract
Metallodrugs provide important first-line treatment against various forms of human cancer. To overcome chemotherapeutic resistance and widen treatment possibilities, new agents with improved or alternative modes of action are highly sought after. Here, we present a click chemistry strategy for developing DNA damaging metallodrugs. The approach involves the development of a series of polyamine ligands where three primary, secondary or tertiary alkyne-amines were selected and 'clicked' using the copper-catalysed azide-alkyne cycloaddition reaction to a 1,3,5-azide mesitylene core to produce a family of compounds we call the 'Tri-Click' (TC) series. From the isolated library, one dominant ligand (TC1) emerged as a high-affinity copper(II) binding agent with potent DNA recognition and damaging properties. Using a range of in vitro biophysical and molecular techniques-including free radical scavengers, spin trapping antioxidants and base excision repair (BER) enzymes-the oxidative DNA damaging mechanism of copper-bound TC1 was elucidated. This activity was then compared to intracellular results obtained from peripheral blood mononuclear cells exposed to Cu(ll)-TC1 where use of BER enzymes and fluorescently modified dNTPs enabled the characterisation and quantification of genomic DNA lesions produced by the complex. The approach can serve as a new avenue for the design of DNA damaging agents with unique activity profiles.
15.
Venkatakrishnan, Vignesh, 1987, et al.
(författare)
Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery
2020
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 295:36, s. 12648-12660
Tidskriftsartikel (refereegranskat) abstract
Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called "targeting by timing." Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation, andN- andO-glycans present in each compartment were analyzed by LC-MS. We found abundant paucimannosidicN-glycans and lack ofO-glycans in the early-formed azurophil granules, whereas the later-formed specific and gelatinase granules and secretory vesicles contained complexN-andO-glycans with remarkably elongatedN-acetyllactosamine repeats with Lewis epitopes. Immunoblotting and histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils, and their complexity increased progressively from azurophil granules to specific granules and then to gelatinase granules, suggesting temporal changes in the glycosylation machinery indicative of "glycosylation by timing" during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step toward a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.
16.
Trigo, João Pedro, 1995, et al.
(författare)
Mild blanching prior to pH-shift processing of Saccharina latissima retains protein extraction yields and amino acid levels of extracts while minimizing iodine content
2023
Ingår i: Food Chemistry. - : Elsevier BV. - 0308-8146 .- 1873-7072. ; 404
Tidskriftsartikel (refereegranskat) abstract
The seaweed Saccharina latissima is often blanched to lower iodine levels, however, it is not known how blanching affects protein extraction. We assessed the effect of blanching or soaking (80/45/12 °C, 2 min) on protein yield and protein extract characteristics after pH-shift processing of S. latissima. Average protein yields and extract amino acid levels ranked treatments as follows: blanching-45 °C ∼ control > soaking ∼ blanching-80 °C. Although blanching-45 °C decreased protein solubilization yield at pH 12, it increased isoelectric protein precipitation yield at pH 2 (p < 0.05). The former could be explained by a higher ratio of large peptides/proteins in the blanched biomass as shown by HP-SEC, whereas the latter by blanching-induced lowering of ionic strength, as verified by a dialysis model. Moreover, blanching-45 °C yielded a protein extract with 49 % less iodine compared with the control extract. We recommend blanching-45 °C since it is effective at removing iodine and does not compromise total protein extraction yield.
17.
Esmaily, Mohsen, 1987, et al.
(författare)
Oxidation and electrical properties of chromium–iron alloys in a corrosive molten electrolyte environment
2020
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10, s. 1-19
Tidskriftsartikel (refereegranskat) abstract
Chromium–iron (CrFe) binary alloys have recently been proposed to serve as the “inert” anode for molten oxide electrolysis (MOE). Herein, the effects of anodic polarization on physical and functional properties of CrFe anodes in the corrosive environment of MOE are studied via empirical observations and theoretical calculations. The findings indicate that the alloys form an inner chromia–alumina solid-solution covered by an MgCr2O4 spinel layer. A survey into the electrical properties of the detected oxides suggests that the layered oxide scale function as an efficient conductor of electricity at elevated temperature. The formation mechanism of the oxides is also investigated.
18.
Gupta, Swarnim, 1984, et al.
(författare)
The development of a novel ferric phytate compound for iron fortification of bouillons (part I)
2020
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
Tidskriftsartikel (refereegranskat) abstract
In a series of two studies, we report the development (this study) and evaluation (part II) of a novel ferric phytate compound designed as a condiment iron fortificant. Condiments are used as iron fortification vehicles to reduce the prevalence of iron deficiency. The challenge for iron fortificants in e.g. a bouillon matrix is to avoid undesired sensory effects and to ensure a reasonable cost. We added phytic acid to chelate iron, and hydrolysed protein to counteract the inhibiting effect of phytic acid on iron bioaccessibility. We characterised four novel ferric phytate compounds, destabilised by hydrolysed plant protein or amino acids. Colour stability of fortified bouillons with ferric phytate compounds was superior to bouillons fortified with ferrous sulfate. The iron-phytate-hydrolysed corn protein compound (Fe-PAHCP) resulted in highest cellular ferritin induction in Caco-2 cells, in both vegetable (36.1 ± 13.40 ng/mg protein) and chicken (73.9 ± 19.93 ng/mg protein) bouillon matrices as observed in the human Caco-2/ HepG2 cell model. Iron uptake (as estimated by ferritin production) from the Fe-PA-HCP compound was about 55% (chicken bouillon) and 66% (vegetable bouillon) of the iron uptake from ferrous sulfate. Based on this study, the Fe-PA-HCP compound was chosen for further evaluation (part II).
19.
Steer, Philippe, et al.
(författare)
Earthquake statistics changed by typhoon-driven erosion
2020
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
Tidskriftsartikel (refereegranskat) abstract
Tectonics and climate-driven surface processes govern the evolution of Earth’s surface topography. Topographic change in turn influences lithospheric deformation, but the elementary scale at which this feedback can be effective is unclear. Here we show that it operates in a single weather-driven erosion event. In 2009, typhoon Morakot delivered ~ 3 m of precipitation in southern Taiwan, causing exceptional landsliding and erosion. This event was followed by a step increase in the shallow (< 15 km depth) earthquake frequency lasting at least 2.5 years. Also, the scaling of earthquake magnitude and frequency underwent a sudden increase in the area where mass wasting was most intense. These observations suggest that the progressive removal of landslide debris by rivers from southern Taiwan has acted to increase the crustal stress rate to the extent that earthquake activity was demonstrably affected. Our study offers the first evidence of the impact of a single weather-driven erosion event on tectonics.
20.
Visser, Dennis, et al.
(författare)
Embossed Mie resonator arrays composed of compacted TiO2 nanoparticles for broadband anti-reflection in solar cells
2020
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10
Tidskriftsartikel (refereegranskat) abstract
Mie resonator arrays formed by embossing titanium dioxide (TiO2) nanoparticles (NPs) from solution are investigated as optical coatings for anti-reflection applications. Compacted nanoparticle assemblies offer unique possibilities to tailor the effective refractive index (RI). Here, we demonstrate a simple table-top, low pressure, and low temperature method to fabricate structured optical coatings. TiO2 nanostructures in the form of nanodisks support Mie resonances in the visible wavelength spectrum and exhibit strong forward scattering into the high index substrates, making them suitable as broadband anti-reflection coatings for solar cells. TiO2 NP-based nanodisk arrays are designed, fabricated, and characterized regarding their anti-reflection properties on Si, GaAs, and InP substrates and solar cells. Detailed finite-difference time-domain simulations are performed to optimize the TiO2 NP-based Mie resonator arrays for the broadband anti-reflection as well as to explain the measured reflectance spectra. The solar-weighted reflectance is used as a figure of merit (FoM). TiO2 nanodisk arrays on Si show a FoM of ~ 7% in the 400–1,100 nm wavelength spectrum; similar values are obtained for GaAs and InP substrates. TiO2 nanodisk arrays embossed directly on prefabricated planar single-junction Si, GaAs, and InP solar cells result in an appreciable increase (~ 1.3 times) in the short-circuit current densities.
21.
Paul, Alexandra, 1988, et al.
(författare)
Effect of ambient temperature on respiratory tract cells exposed to SARS-CoV-2 viral mimicking nanospheres - An experimental study
2021
Ingår i: Biointerphases. - : American Vacuum Society. - 1559-4106 .- 1934-8630. ; 16:1
Tidskriftsartikel (refereegranskat) abstract
The novel coronavirus caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached more than 160 countries and has been declared a pandemic. SARS-CoV-2 infects host cells by binding to the angiotensin-converting enzyme 2 (ACE-2) surface receptor via the spike (S) receptor-binding protein (RBD) on the virus envelope. Global data on a similar infectious disease spread by SARS-CoV-1 in 2002 indicated improved stability of the virus at lower temperatures facilitating its high transmission in the community during colder months (December–February). Seasonal viral transmissions are strongly modulated by temperatures, which can impact viral trafficking into host cells; however, an experimental study of temperature-dependent activity of SARS-CoV-2 is still lacking. We mimicked SARS-CoV-2 with polymer beads coated with the SARS-CoV-2 S protein to study the effect of seasonal temperatures on the binding of virus-mimicking nanospheres to lung epithelia. The presence of the S protein RBD on nanosphere surfaces led to binding by Calu-3 airway epithelial cells via the ACE-2 receptor. Calu-3 and control fibroblast cells with S-RBD-coated nanospheres were incubated at 33 and 37 °C to mimic temperature fluctuations in the host respiratory tract, and we found no temperature dependence in contrast to nonspecific binding of bovine serum ablumin-coated nanospheres. Moreover, the ambient temperature changes from 4 to 40 °C had no effect on S-RBD-ACE-2 ligand-receptor binding and minimal effect on the S-RBD protein structure (up to 40 °C), though protein denaturing occurred at 51 °C. Our results suggest that ambient temperatures from 4 to 40 °C have little effect on the SARS-CoV-2-ACE-2 interaction in agreement with the infection data currently reported.
22.
Lindberg, Frida A, et al.
(författare)
Behavioral profiling of SLC38A10 knockout mice using the multivariate concentric square field™ test
2022
Ingår i: Frontiers in Behavioral Neuroscience. - : Frontiers Media S.A.. - 1662-5153. ; 16
Tidskriftsartikel (refereegranskat) abstract
Introduction: SLC38A10 is a gene that encodes the SLC38A10 protein, also known as SNAT10. The SLC38 family is evolutionary old, and SLC38A10 is one of the oldest members of the family. It is ubiquitously expressed, and its substrates are glutamine, glutamate, alanine, aspartate, and serine. However, little is known about its biological importance.Methods: In the current study, an SLC38A10 knockout mouse was run in the multivariate concentric square field (TM) (MCSF) test. The MCSF test gives the mouse a choice of areas to explore; sheltered areas, elevated and illuminated areas, or open spaces, and a behavioral profile is obtained. The multivariate data obtained were analyzed (i) for each parameter, (ii) parameters grouped into functional categories, and (iii) with a principal component analysis.Results: In the trend analysis, knockout mice had a decreased exploratory behavior compared to controls but did not show a distinct grouping in the principal component analysis.Discussion: There was not a pronounced difference in the behavioral profile in SLC38A10 knockout mice compared to their wild-type controls, although subtle alterations in zones associated with exploratory behavior and risk assessment in female and male knockout mice, respectively, could be observed. These results imply that a loss of function of the SLC38A10 protein in mice does not drastically alter behavior in the MSCF test.
23.
Konzock, Oliver, 1991, et al.
(författare)
Tolerance of Yarrowia lipolytica to inhibitors commonly found in lignocellulosic hydrolysates
2021
Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 21:1
Tidskriftsartikel (refereegranskat) abstract
Background Lignocellulosic material is a suitable renewable carbon and energy source for microbial cell factories, such as Yarrowia lipolytica. To be accessible for microorganisms, the constituent sugars need to be released in a hydrolysis step, which as a side effect leads to the formation of various inhibitory compounds. However, the effects of these inhibitory compounds on the growth of Y. lipolytica have not been thoroughly investigated. Results Here we show the individual and combined effect of six inhibitors from three major inhibitor groups on the growth of Y. lipolytica. We engineered a xylose consuming strain by overexpressing the three native genes XR, XDH, and XK and found that the inhibitor tolerance of Y. lipolytica is similar in glucose and in xylose. Aromatic compounds could be tolerated at high concentrations, while furfural linearly increased the lag phase of the cultivation, and hydroxymethylfurfural only inhibited growth partially. The furfural induced increase in lag phase can be overcome by an increased volume of inoculum. Formic acid only affected growth at concentrations above 25 mM. In a synthetic hydrolysate, formic acid, furfural, and coniferyl aldehyde were identified as the major growth inhibitors. Conclusion We showed the individual and combined effect of inhibitors found in hydrolysate on the growth of Y. lipolytica. Our study improves understanding of the growth limiting inhibitors found in hydrolysate and enables a more targeted engineering approach to increase the inhibitor tolerance of Y. lipolytica. This will help to improve the usage of Y. lipolytica as a sustainable microbial cell factory.
24.
Manaprasertsak, Auraya, et al.
(författare)
Imaging the distribution of DMPBD and terpinen-4-ol inclusion complexes with 2-hydroxypropyl-beta-cyclodextrin by using TOF-SIMS
2021
Ingår i: Analytical Methods. - : Royal Society of Chemistry (RSC). - 1759-9679 .- 1759-9660. ; 13:1, s. 84-89
Tidskriftsartikel (refereegranskat) abstract
The distribution of terpinen-4-ol (TP4ol) and DMPBD inclusion complexes with 2-hydroxypropyl-beta-cyclodextrin (HPbCD) in human skin has been investigated using the TOF-SIMS technique. TP4ol and DMPBD have been found to be major components of Zingiber cassumunar Roxb. (Plai) oil extracted by steam distillation. The results mainly show accumulation of TP4ol and DMPBD inclusion complexes with HPbCD in the epidermis and dermis whereas these two compounds without cyclodextrin cannot penetrate into the epidermis. This approach can be expanded for investigation of anti-inflammatory action and relief of muscle pain.
25.
Shah, Kinjal
(författare)
Mechanisms of therapy resistance in acute lymphoblastic leukemia
2021
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Acute lymphoblastic leukemia (ALL) is a highly aggressive pediatric cancer that can affect both B cells and T cells. The advent of new therapies has increased the cure rates for both B-ALL and T-ALL patients. However, some patients still experience relapse with a variable response to the treatment and display poor survival. Thus,identification of novel predictive biomarkers that can predict therapy resistance may help to stratify this group of patients. This could also aid in developing an effective treatment strategy.Glucocorticoids are widely used along with the chemotherapeutic regimens for treating ALL patients. The response to glucocorticoids can predict long-term remission outcome. To understand the mechanisms of resistance to glucocorticoids, such as dexamethasone, we generated dexamethasone-resistant B-ALL cell lines in paper I. One such resistant cell line was found to possess increased FLT3 expression levels with FLT3-ITD and FLT3-R845G mutations that led to the activation of oncogenic RTK signaling. Further, second-generation FLT3 inhibitors, such as AC220 and crenolanib, suppressed this signaling both in vitro and in vivo.We continued exploring the dexamethasone resistance mechanisms in paper II using a different approach. We observed that dexamethasone exposure caused upregulation of Aurora kinase and its various downstream effector kinases such as JAK, p38, mTOR, and S6K. These kinases lead to β-catenin stabilization through phosphorylation-dependent inactivation of GSK-3β either directly or indirectly. Indeed, we observed partial restoration of dexamethasone sensitivity with a combination of dexamethasone and inhibitors targeting either these kinases or β-catenin.The expression of BCL2 varies in T-ALL depending on its stage of maturation, thereby T-ALL displays a heterogenous response to the BCL2-specific inhibitor venetoclax. We thus studied the mechanisms of venetoclax resistance using a panel of T-ALL cell lines in paper III. We observed that all the venetoclax-resistant T-ALL cell lines displayed non-universal changes in the expression of BCL2 family members and cancer stem cell markers, along with specific enrichment of cytokine signaling pathways. However, further investigations are warranted to identify additional mechanisms of venetoclax resistance in T-ALL.Combination therapy is usually the choice of treatment to overcome monotherapy resistance. With this in mind, in paper IV we identified that inhibiting BCL2 by venetoclax synergizes with PLK1 inhibition by volasertib in T-ALL cell lines and PDX models. We observed that BCL2L13 and PMAIP1 genes get upregulated upon PLK1 inhibition, probably through transcriptional regulation by FOXOs in interaction with β-catenin. Thus, the pro-apoptotic functions exhibited by BCL2L13 and PMAIP1 probably synergize with BCL2 inhibition in T-ALL, with the help of sustained β-catenin levels. Moreover, we also identified upregulation of oxidative phosphorylation (OXPHOS) in T-ALL PDXs that didn’t display synergy, which could be treated with a combination of venetoclax and oligomycin. However, additional experiments will be required to verify the above results.
26.
Chen, Yu, 1990, et al.
(författare)
Yeast has evolved to minimize protein resource cost for synthesizing amino acids
2022
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 119:4
Tidskriftsartikel (refereegranskat) abstract
Proteins, as essential biomolecules, account for a large fraction of cell mass, and thus the synthesis of the complete set of proteins (i.e., the proteome) represents a substantial part of the cellular resource budget. Therefore, cells might be under selective pressures to optimize the resource costs for protein synthesis, particularly the biosynthesis of the 20 proteinogenic amino acids. Previous studies showed that less energetically costly amino acids are more abundant in the proteomes of bacteria that survive under energy-limited conditions, but the energy cost of synthesizing amino acids was reported to be weakly associated with the amino acid usage in Saccharomyces cerevisiae. Here we present a modeling framework to estimate the protein cost of synthesizing each amino acid (i.e., the protein mass required for supporting one unit of amino acid biosynthetic flux) and the glucose cost (i.e., the glucose consumed per amino acid synthesized). We show that the logarithms of the relative abundances of amino acids in S. cerevisiae's proteome correlate well with the protein costs of synthesizing amino acids (Pearson's r = 20.89), which is better than that with the glucose costs (Pearson's r = 20.5). Therefore, we demonstrate that S. cerevisiae tends to minimize protein resource, rather than glucose or energy, for synthesizing amino acids.
27.
Baumann, Leonie, et al.
(författare)
Transcriptomic response of Saccharomyces cerevisiae to octanoic acid production
2021
Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 21:2
Tidskriftsartikel (refereegranskat) abstract
The medium-chain fatty acid octanoic acid is an important platform compound widely used in industry. The microbial production from sugars in Saccharomyces cerevisiae is a promising alternative to current non-sustainable production methods, however, titers need to be further increased. To achieve this, it is essential to have in-depth knowledge about the cell physiology during octanoic acid production. To this end, we collected the first RNA-Seq data of an octanoic acid producer strain at three time points during fermentation. The strain produced higher levels of octanoic acid and increased levels of fatty acids of other chain lengths (C6-C18) but showed decreased growth compared to the reference. Furthermore, we show that the here analyzed transcriptomic response to internally produced octanoic acid is notably distinct from a wild type's response to externally supplied octanoic acid as reported in previous publications. By comparing the transcriptomic response of different sampling times, we identified several genes that we subsequently overexpressed and knocked out, respectively. Hereby we identified RPL40B, to date unknown to play a role in fatty acid biosynthesis or medium-chain fatty acid tolerance. Overexpression of RPL40B led to an increase in octanoic acid titers by 40%.
28.
Norling, Karin, 1988
(författare)
Liposomes for mucosal vaccine delivery: physicochemical characterization and biological application
2020
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Liposomes are attractive vaccine carriers due to their potential to act as adjuvants, and to the fact that their composition and characteristics are virtually endlessly customizable. However, the precise physicochemical profile of an ideal carrier liposome for mucosal vaccines is still widely unknown, and how different properties affect key steps in the acquisition of protective immunity remains to be elucidated. Additionally, there is no consensus in the field regarding characterization of vaccine formulations, often with incomplete reporting of properties as a result. The focus of this work is therefore twofold: i) to contribute to a better understanding of how the physicochemical profile of vaccine carrier liposomes impacts the development of protective immunity using models at different levels of complexity, and ii) to improve and simplify the physicochemical characterization of liposomes through development and use of new analytical methods. The work in the first area consists of, firstly, an in vivo characterization of the biological response to vaccine liposomes carrying a vaccine protein and characterized by varying surface hydrophilicity (PEGylation). This study showed that non-PEGylated vaccine liposomes more efficiently induced local cell- and antibody-mediated immune responses, as well as better protection against a lethal virus challenge than both PEGylated liposomes and free vaccine protein. Secondly, in vitro studies focused on how liposome stiffness influences dendritic cells, investigating effects on uptake, antigen presentation and cellular activation. These investigations demonstrated that stiff, gel phase liposomes were able to more efficiently activate dendritic cells and induce significantly higher levels of antigen presentation and co-stimulatory signaling compared to both soft, fluid phase liposomes, and free vaccine protein. The work in the second part comprises two studies: a surface plasmon resonance-based method to characterize the influence on liposome deformation from specific multivalent interactions with supported cell membrane mimics, and a waveguide microscopy technique for characterization of optical properties of individual liposomes. While the latter method might become valuable in the context of quantifying the efficiency of dye labelling of liposomes, the surface plasmon resonance study offered information on how liposome deformation depends on membrane stiffness and ligand-receptor pair density. Taken together, the work presented in this thesis demonstrate the value of multidisciplinary approaches to complex biological and medical challenges.
29.
Yang, Shen, et al.
(författare)
Ozone Initiates Human-Derived Emission of Nanocluster Aerosols
2021
Ingår i: Environmental Science and Technology. - : American Chemical Society (ACS). - 0013-936X .- 1520-5851. ; 55:21, s. 14536-14545
Tidskriftsartikel (refereegranskat) abstract
Nanocluster aerosols (NCAs, particles <3 nm) are important players in drivingclimate feedbacks and processes that impact human health. This study reports, for the first time, NCA formation when gas-phase ozone reacts with human surfaces. In an occupied climatecontrolledchamber, we detected NCA only when ozone was present.NCA emissions weredependent on clothing coverage, occupant age, air temperature, and humidity. Ozone-initiated chemistry with human skin lipids (particularly their primary surface reaction products) is thekey mechanism driving NCA emissions, as evidenced by positive correlations with squalene in human skin wipe samples and known gaseous products from ozonolysis of skin lipids. Oxidation by OH radicals, autoxidation reactions, and human-emitted NH3 may also play a role in NCA formation. Such chemical processesare anticipated to generate aerosols of the smallest size (1.18−1.55 nm), whereas larger clusters result from subsequent growth of the smaller aerosols. This study shows that whenever we encounter ozone indoors, where we spend most of our lives, NCAs will be produced in the air around us.
30.
Tang, Shi, et al.
(författare)
Aggregation-Induced Emission by Molecular Design: A Route to High-Performance Light-Emitting Electrochemical Cells
2023
Ingår i: Angewandte Chemie - International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 62:23
Tidskriftsartikel (refereegranskat) abstract
The emission efficiency of organic semiconductors (OSCs) often suffers from aggregation caused quenching (ACQ). An elegant solution is aggregation-induced emission (AIE), which constitutes the design of the OSC so that its morphology inhibits quenching π–π interactions and non-radiative motional deactivation. The light-emitting electrochemical cell (LEC) can be sustainably fabricated, but its function depends on motion of bulky ions in proximity of the OSC. It is therefore questionable whether the AIE morphology can be retained during LEC operation. Here, we synthesize two structurally similar OSCs, which are distinguished by that 1 features ACQ while 2 delivers AIE. Interestingly, we find that the AIE-LEC significantly outperforms the ACQ-LEC. We rationalize our finding by showing that the AIE morphology remains intact during LEC operation, and that it can feature appropriately sized free-volume voids for facile ion transport and suppressed non-radiative excitonic deactivation.
31.
Juul, Louise, et al.
(författare)
Ulva fenestrata protein – comparison of three extraction methods with respect to protein yield and protein quality
2021
Ingår i: Algal Research. - : Elsevier BV. - 2211-9264. ; 60
Tidskriftsartikel (refereegranskat) abstract
Seaweed is gaining attention as a possible alternative and sustainable source of proteins. This study investigates three protein extraction methods and their effect on protein yield and quality when applied to Ulva fenestrata . Two of the methods included alkaline extractions (pH-shifts); one version solubilizing the proteins at pH 8.5 and one solubilizing them at pH 8.5 followed by pH 12 (pH 8.5+12). The third method was a mechanical pressing, using a double screw press. All extraction methods were followed by isoelectric precipitation to concentrate the proteins. Extraction at pH 8.5 gave the significantly highest total protein yield after the isoelectric precipitation, followed by extraction at pH 8.5+12 and lastly mechanical extraction gave the lowest yield. Proteins extracted with both alkaline methods had a significantly higher solubility at pH 7 and pH 9, compared to proteins from the mechanical pressing. There were no significant differences between the three methods in total D/L-amino acid ratio. Amino acid cross-links measured as lysinoalanine (LAL) and lanthionine (LAN) where found in significantly higher amounts in alkali-extracted proteins compared to mechanically extracted, however not to a degree that expect to compromise functional or nutritional quality. Further, no significant difference in protein in vitro digestibility was found between extraction methods. In conclusion, results indicated that protein extraction at pH 8.5 can be recommended, especially regarding total protein yield and solubility of the final protein extract.
32.
Maertens, Jeroen, 1990, et al.
(författare)
Molecular-dynamics-simulation-guided membrane engineering allows the increase of membrane fatty acid chain length in Saccharomyces cerevisiae
2021
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
Tidskriftsartikel (refereegranskat) abstract
The use of lignocellulosic-based fermentation media will be a necessary part of the transition to a circular bio-economy. These media contain many inhibitors to microbial growth, including acetic acid. Under industrially relevant conditions, acetic acid enters the cell predominantly through passive diffusion across the plasma membrane. The lipid composition of the membrane determines the rate of uptake of acetic acid, and thicker, more rigid membranes impede passive diffusion. We hypothesized that the elongation of glycerophospholipid fatty acids would lead to thicker and more rigid membranes, reducing the influx of acetic acid. Molecular dynamics simulations were used to predict the changes in membrane properties. Heterologous expression of Arabidopsis thaliana genes fatty acid elongase 1 (FAE1) and glycerol-3-phosphate acyltransferase 5 (GPAT5) increased the average fatty acid chain length. However, this did not lead to a reduction in the net uptake rate of acetic acid. Despite successful strain engineering, the net uptake rate of acetic acid did not decrease. We suggest that changes in the relative abundance of certain membrane lipid headgroups could mitigate the effect of longer fatty acid chains, resulting in a higher net uptake rate of acetic acid.
33.
Obi, Ikenna, et al.
(författare)
Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication
2020
Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 48:19, s. 10998-11015
Tidskriftsartikel (refereegranskat) abstract
G-quadruplex (G4) structures are stable noncanonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancerassociated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.
34.
Abdollahi, Mehdi, 1985, et al.
(författare)
A novel cold biorefinery approach for isolation of high quality fish oil in parallel with gel-forming proteins
2020
Ingår i: Food Chemistry. - : Elsevier BV. - 0308-8146 .- 1873-7072. ; 332
Tidskriftsartikel (refereegranskat) abstract
The pH-shift process for isolation of gel-forming proteins from fish processing by-products was extended to allow parallel isolation of fish oil. Subjecting the floating emulsion layer formed during pH-shift processing of salmon by-products to pH-adjustment or freeze/thawing efficiently released the emulsified oil at 4 °C. However, for herring by-products higher temperature (10 °C) and a combination of the emulsion-breaking techniques was required for efficient oil release. Oil recovery yield using the adjusted pH-shift process was lower than with classic heat-induced oil isolation (90 °C/20 min), but pH-shift-produced oils had higher amounts of n-3 polyunsaturated fatty acids (n-3 PUFA). Also, alkaline pH-shift processing produced oils with remarkably less oxidation products and free fatty acids compared with acid pH-shift process or heat-induced isolation. Extending the pH-shift process with emulsion breaking techniques can thus be a promising biorefinery approach for parallel cold production of high-quality fish oil and gel-forming proteins from fish by-products.
35.
Abdollahi, Mehdi, 1985, et al.
(författare)
Minimizing lipid oxidation during pH-shift processing of fish by-products by cross-processing with lingonberry press cake, shrimp shells or brown seaweed
2020
Ingår i: Food Chemistry. - : Elsevier BV. - 0308-8146 .- 1873-7072. ; 327
Tidskriftsartikel (refereegranskat) abstract
The potential of cross-processing herring or salmon by-products with brown seaweed, shrimp peeling by-products and lingonberry press cake (called “helpers”) to minimize lipid oxidation during acid/alkaline pH-shift protein isolation was evaluated. Lingonberry press cake remarkably reduced levels of lipid hydroperoxides, malondialdehyde (MDA) and 4-hydroxy-trans2-hexenal (HHE) in both herring and salmon protein isolates (PI) while seaweed and shrimp by-product mitigated generation of MDA and HHE in herring PI. The salmon PI, however, by itself had very low oxidation levels. Cross-processing reduced protein yield compared with the classical pH-shift process by diminishing protein solubility, but still, the alkaline process version was promising. The color of the PIs varied greatly between different process combinations, reflecting partitioning of natural pigments into isolates. Altogether, cross-processing seems a promising tool for inhibition of lipid oxidation during pH-shift processing of sensitive fish by-products, with the alkaline version and lingonberry press cake being closest at hand for further studies.
36.
Masdeu Gámez, Gerard, 1990, et al.
(författare)
Synthesis of a precursor of D-fagomine by immobilized fructose-6-phosphate aldolase
2021
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 16:4 April 2021
Tidskriftsartikel (refereegranskat) abstract
Fructose-6-phosphate aldolase (FSA) is an important enzyme for the C-C bond-forming reactions in organic synthesis. The present work is focused on the synthesis of a precursor of D-fagomine catalyzed by a mutant FSA. The biocatalyst has been immobilized onto several supports: magnetic nanoparticle clusters (mNC), cobalt-chelated agarose (Co-IDA), amino-functionalized agarose (MANA-agarose) and glyoxal-agarose, obtaining a 29.0%, 93.8%, 89.7% and 53.9% of retained activity, respectively. Glyoxal-agarose FSA derivative stood up as the best option for the synthesis of the precursor of D-fagomine due to the high reaction rate, conversion, yield and operational stability achieved. FSA immobilized in glyoxal-agarose could be reused up to 6 reaction cycles reaching a 4-fold improvement in biocatalyst yield compared to the non-immobilized enzyme.
37.
Sörensen Ristinmaa, Amanda, 1992, et al.
(författare)
Biochemical characterization of three tannases from Clostridium butyricum
2021
Konferensbidrag (övrigt vetenskapligt/konstnärligt) abstract
Bark is the outer tissue of the tree, shielding the wood from microbial attack. The compounds present in bark which gives the bark this ability to hamper microbial attack are the secondary metabolites called extractives, such as tannins. Biochemical degradation of tannins is known to be conducted by tannase enzymes (EC 3.1.1.20), which are serine-esterases able to separate gallic acid from the aromatic-sugar complexes. In this study we investigated three tannases from the bacterium Clostridium butyricum, named CbTan1-3, both on model substrates but also on oak bark extract. The enzymes were tested on a range of galloyl ester model substrates and display different substrate preferences, with CbTan1-3 preferring galloyl moiety linked to glucose and CbTan2 not displaying preference towards any model substrate tested in this study. On oak bark extract, the three enzymes act on different molecules, with CbTan2 degrading all identified substrates and CbTan3 and CbTan1 able to degrade similar substrates, however, CbTan3 was also able to liberate additional glucose from the oak bark extract.
38.
Tokin, Radina, et al.
(författare)
Inhibition of lpmos by fermented persimmon juice
2021
Ingår i: Biomolecules. - : MDPI AG. - 2218-273X. ; 11:12
Tidskriftsartikel (refereegranskat) abstract
Fermented persimmon juice, Kakishibu, has traditionally been used for wood and paper protection. This protective effect stems at least partially from inhibition of microbial cellulose degrading enzymes. The inhibitory effect of Kakishibu on lytic polysaccharide monooxygenases (LPMOs) and on a cocktail of cellulose hydrolases was studied, using three different cellulosic substrates. Dose dependent inhibition of LPMO activity by a commercial Kakishibu product was assessed for the well-characterized LPMO from Thermoascus aurantiacus TaAA9A, and the inhibitory effect was confirmed on five additional microbial LPMOs. The model tannin compound, tannic acid exhibited a similar inhibitory effect on TaAA9A as Kakishibu. It was further shown that both polyethylene glycol and tannase can alleviate the inhibitory effect of Kakishibu and tannic acid, indicating a likely mechanism of inhibition caused by unspecific tannin–protein interactions.
39.
Vate, Naveen Kumar, 1988, et al.
(författare)
Resource efficient collagen extraction from common starfish with the aid of high shear mechanical homogenization and ultrasound
2022
Ingår i: Food Chemistry. - : Elsevier BV. - 0308-8146 .- 1873-7072. ; 393
Tidskriftsartikel (refereegranskat) abstract
Processes currently used for collagen extraction are complicated requiring a great deal of time and chemicals. Here, high shear mechanical homogenization (HSMH) and ultrasound (US) were integrated in the pretreatment step of collagen extraction from common starfish to reduce chemical use and time consumption. Effects of the assistant technologies on yield, structural integrity and functionality of collagen were also investigated. HSMH reduced the deproteinization time from 6 h to 5 min and its required amount of alkali 4 times, compared with classic methods. HSMH + US reduced the demineralization time from 24 h to 12 h and improved its efficiency in extraction of minerals. Collagen extraction with HSMH and HSMH + US resulted in similar yield as the classic method and did not affect triple helical structural integrity, polypeptide pattern, thermal stability or fibril-formation capacity of the collagens. Altogether, HSMH and US can effectively improve resource efficiency during collagen extraction without imposing negative effect on collagen quality.
40.
Wu, Haizhou, 1987, et al.
(författare)
Effect of recovery technique, antioxidant addition and compositional features on lipid oxidation in protein enriched products from cod- salmon and herring backbones
2021
Ingår i: Food Chemistry. - : Elsevier BV. - 0308-8146 .- 1873-7072. ; 360
Tidskriftsartikel (refereegranskat) abstract
The influence of recovery technique (pH-shift processing vs mechanical separation), antioxidant addition and endogenous factors on lipid oxidation in protein-enriched products from herring, salmon and cod backbones was investigated. Salmon-derived products were very stable during both ice and −20 °C storage. Contrary, peroxide value and TBA-reactive substances in herring- and cod-derived products increased rapidly during ice storage, with the pH-shift-produced protein isolates (PI) being most susceptible to oxidation in case of cod. Duralox MANC (0.5%) however largely increased the oxidation lag phase in both PI and mechanically separated meat (MSM); from <1 day to >15 days. At −20 °C, mainly the herring products oxidized, and particularly the MSM. Pearson correlation tests showed that endogenous levels of Hb, total Fe, ascorbic acid and α-tocopherol correlated significantly (p < 0.05) with lipid oxidation development. Evaluating the role of pre-processing storage indicated that fish co-products should be processed immediately after the filleting process unless antioxidants are added.
41.
Wu, Haizhou, 1987, et al.
(författare)
Impact of lipid composition and muscle microstructure on myoglobin-mediated lipid oxidation in washed cod and pig muscle
2021
Ingår i: Food Chemistry. - : Elsevier BV. - 0308-8146 .- 1873-7072. ; 336
Tidskriftsartikel (refereegranskat) abstract
The roles of lipid oxidation substrates and muscle microstructure in lipid oxidation were investigated in two muscle models (cod and pig). Added myoglobin (Mb) promoted lipid oxidation in washed cod muscle (WCM) but not in washed pig muscle (WPM). The differing microstructure of WCM e.g. more exposed fat cells or membrane of muscle cells compared to the “denseness” or “wrapped” structure of WPM, may have contributed to the better ability of Mb to facilitate lipid oxidation in the WCM. Added phospholipids with polyenoic indexes of 282 and 24 activated Mb as an oxidant similarly in WPM while added neutral lipids and added free fatty acids had little effect. It is suggested that muscle microstructure and accessibility of Mb to phospholipids play critical roles in relation to Mb-mediated lipid oxidation while the degree of unsaturation in the phospholipids was less important.
42.
Wu, Haizhou, 1987, et al.
(författare)
Lipid oxidation in sorted herring (Clupea harengus) filleting co-products from two seasons and its relationship to composition
2022
Ingår i: Food Chemistry. - : Elsevier BV. - 0308-8146 .- 1873-7072. ; 373
Tidskriftsartikel (refereegranskat) abstract
Lipid oxidation in ice-stored sorted herring fractions (head, backbone, viscera + belly flap, tail, fillet) from spring and fall, and its association with endogenous prooxidants, antioxidants and lipid substrates were investigated. Peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) had increased significantly in all fractions after 1 day, but for both seasons, the most rapid PV and TBARS development occurred in head, which also had highest hemoglobin (Hb) levels and lipoxygenases (LOX) activity. Viscera + belly flap was overall the most stable part, and also had the highest α-tocopherol content. Pearson correlation analyses across all five fractions confirmed a significant impact of Hb, LOX and α-tocopherol on the lipid oxidation susceptibility, while content of total iron, copper, lipids or polyunsaturated fatty acids provided no significant correlation. Overall, the study showed which pro-oxidants that should be inhibited or removed to succeed with value adding of herring filleting co-products and the fillet itself.
43.
Wu, Haizhou, 1987, et al.
(författare)
Mechanisms involved in the inhibitory effects of free fatty acids on lipid peroxidation in turkey muscle
2021
Ingår i: Food Chemistry. - : Elsevier BV. - 0308-8146 .- 1873-7072. ; 342
Tidskriftsartikel (refereegranskat) abstract
The pro-oxidant and anti-oxidant effects of free fatty acids (FFA) in meat remain ambiguous. To clarify the role of FFA in lipid oxidation of muscle food, the FFA was added into two systems (turkey mince and washed turkey muscle (WTM)) and lipid oxidation was investigated. A mixture of FFA inhibited lipid oxidation in both systems. The absorbance spectrum of oxyHb and metHb was determined in the presence of C18:1 and C18:2. Conversion to hemichrome spectra was observed and was particularly rapid when metHb was mixed with C18:2. C18:2 was also reacted with metHb followed by a chromatography step to remove unbound C18:2, and termed ‘modified Hb’. Electron spin resonance (ESR) spectra of the modified Hb was indicative of hemichrome formation. The modified Hb did not promote lipid oxidation in washed turkey muscle during storage. This suggested that hemichrome formation due to added FFA diminished the lipid oxidation capacity of Hb.
44.
Wu, Haizhou, 1987, et al.
(författare)
Pro-oxidative activity of trout and bovine hemoglobin during digestion using a static in vitro gastrointestinal model
2022
Ingår i: Food Chemistry. - : Elsevier BV. - 0308-8146 .- 1873-7072. ; 393:1
Tidskriftsartikel (refereegranskat) abstract
The degradation of trout and bovine hemoglobin (Hb) and their pro-oxidant activities in washed cod muscle mince (WCM) were studied using simple pH-shifts to simulate gastrointestinal (GI) conditions (pH 7 → 6 → 3 → 7), as well as full static in vitro GI digestion. Following gastric acidification to pH 6, metHb formation increased, especially for trout Hb. Subsequent acidification to pH 3 promoted Hb unfolding and partial or complete heme group-loss. During full GI digestion, polypeptide/peptide analyses revealed more extensive Hb-degradation in the gastric than duodenal phase, without any species-differences. When digesting WCM +/-Hb, both Hbs strongly promoted malondialdehyde (MDA), 4-hydroxy-2-hexenal (HHE), and 4-hydroxy-2-nonenal (HNE) formation, peaking at the end of the gastric phase. Trout-Hb stimulated MDA and HHE more than bovine Hb in the first gastric phase. Altogether, partially degraded Hb, and/or free hemin -both mammal and fish-derived- stimulated oxidation of PUFA-rich lipids under GI-conditions, especially gastric ones.
45.
Blomgren, Per-Ola, et al.
(författare)
Healthcare workers' perceptions and acceptance of an electronic reminder system for hand hygiene
2021
Ingår i: Journal of Hospital Infection. - : Elsevier. - 0195-6701 .- 1532-2939. ; 108, s. 197-204
Tidskriftsartikel (refereegranskat) abstract
Background:Healthcare-associated infections (HCAIs) have a large negative impact onmorbidity, mortality, and quality of life. Approximately 9% of all patients hospitalized inSweden suffer from HCAI. Hand hygiene plays a key role and is considered the single mostimportant measure to reduce HCAI. The hospital organization works actively to reduceHCAI. Implementing electronic systems to remind and/or notify healthcare workers raisesawareness of and adherence to hand hygiene. However, there is a paucity of studiesaddressing individuals’ perceptions of having such a system and how the organizationworks.Aim:To investigate healthcare workers’ perceptions of infection prevention in thehealthcare organization and perceptions and acceptance of an electronic reminder systemthat encourages good hand hygiene.Methods:Qualitative descriptive design with data collected in eight focus group inter-views including assistant nurses, nurses, and physicians (N¼38). Content analysis wasapplied and data were related to the Theory of Planned Behaviour.Findings:Healthcare workers perceive lack of feedback from the hospital organizationand are positive towards an electronic reminder system to increase adherence to handhygiene. The electronic reminder system should not register data at an individual levelsince it could be used as an instrument for control by the management that could bestressful for staff.Conclusion:In general, there is positive acceptance of the electronic reminder system,and the respondents perceived it as having the ability to change behaviour. However, theconcept has to be further developed to protect the individual’s integrity and needs to beused with feedback on a group level
46.
47.
Högdahl, Louise, et al.
(författare)
Internet-based cognitive behavioral therapy for bulimic eating disorders in a clinical setting : Results from a randomized trial with one-year follow-up
2023
Ingår i: Internet Interventions. - : Elsevier. - 2214-7829. ; 31
Tidskriftsartikel (refereegranskat) abstract
BackgroundThose who suffer from eating disorders often experience serious impairment in quality of life and the majority never receive treatment. Treatment availability may be increased by implementing methods that demand less resources and are more easy accessible such as internet-based treatments, but knowledge about their effects is still insufficient. The study evaluated effects of two types of internet-based cognitive behavioral therapy and a structured day patient program, the latter being a standard treatment at an eating disorder clinic at the time for the study.Methods150 participants with bulimic eating disorders randomized to two types of internet based treatments (one pure online treatment and one based on a self-help guide in book-format) or an intensive 16-week day patient program. The number of participants that started treatment was 120 of which 98 in internet treatment and 22 in the day program. Outcome assessments were carried out at baseline, post treatment, and at one-year follow-up.ResultsAll treatments were associated with significantly improved eating disorder pathology, self-image, and clinical impairment. Although the day program generally showed larger effects, only one significant difference found was in diagnostic remission post treatment; 51 % of the participant was in remission in internet treatment and 88 % in the day program. At one-year follow-up, participants in the internet treatments had continued to improve, whereas in the day patient program the effect sustained. Internet treatment had a 36 % drop out rate, there were no dropouts found in the day program.ConclusionsAll treatments were comparable in effect at follow-up, suggesting that internet treatment is a conceivable alternative to standard treatment. Internet treatment in a book-based format was also equally effective as a pure online format. Internet delivered cognitive behavioral treatment forms can make important contributions to achieve increased access to treatment for patients with bulimic eating disorders. Future research and clinical implications for internet delivered treatments in eating disorder services are discussed.
48.
Öberg, Linnéa, 1987-, et al.
(författare)
Non-Word Repetition and Vocabulary in Arabic-Swedish-Speaking 4–7-Year-Olds with and without Developmental Language Disorder
2022
Ingår i: Languages. - : MDPI. - 2226-471X. ; 7:3
Tidskriftsartikel (refereegranskat) abstract
The Arabic-speaking community in Sweden is large and diverse, yet linguistic reference data are lacking for Arabic-Swedish-speaking children. This study presents reference data from 99 TD children aged 4;0-7;11 on receptive and expressive vocabulary in the minority and the majority language, as well as for three types of non-word repetition (NWR) tasks. Vocabulary scores were investigated in relation to age, language exposure, and socio-economic status (SES). NWR performance was explored in relation to age, type of task, item properties, language exposure, and vocabulary. Eleven children with DLD were compared to the TD group. Age and language exposure were important predictors of vocabulary scores in both languages, but SES did not affect vocabulary scores in any language. Age and vocabulary size had a positive effect on NWR accuracy, whilst increasing item length and presence of clusters had an adverse effect. There was substantial overlap between the TD and DLD children for both vocabulary and NWR performance. Diagnostic accuracy was at best suggestive for NWR; no task or type of item was better at separating the two groups. Reports from parents and teachers on devleopmental history, language expoosure, and functional language skills emerged as important facotrs for correctly identifying DLD in bilinguals.
49.
Alsafadi, Hani N, et al.
(författare)
Applications and Approaches for Three-Dimensional Precision-Cut Lung Slices. Disease Modeling and Drug Discovery
2020
Ingår i: American Journal of Respiratory Cell and Molecular Biology. - 1044-1549. ; 62:6, s. 681-691
Forskningsöversikt (refereegranskat) abstract
Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.
50.
Lundmark, Fanny, et al.
(författare)
Reduction of renal activity retention of radiolabeled albumin binding domain-derived affinity proteins using a non-residualizing label strategy compared with a cleavable glycine-leucine-glycine-lysine-linker
2024
Ingår i: Molecular Medicine Reports. - : Spandidos Publications. - 1791-2997 .- 1791-3004. ; 29:2
Tidskriftsartikel (refereegranskat) abstract
The feasibility of targeted imaging and therapy using radiolabeled albumin-binding domain-derived affinity proteins (ADAPTs) has been demonstrated. However, high renal uptake of radioactivity limits the maximum tolerated dose. Successful reduction of renal retention of radiolabeled Fab fragments has been demonstrated by incorporating a cleavable linker between the targeting agent and the radiometal chelator. The present study investigated if the introduction of a glycine-leucine-glycine-lysine (GLGK)-linker would reduce the kidney uptake of radiolabeled ADAPT6 and also compared it with the non-residualizing [125I]I-[(4-hydroxyphenyl)ethyl]maleimide ([125I]I-HPEM) labeling strategy. GLGK was site-specifically coupled to human epidermal growth factor receptor 2 (HER2)-targeting ADAPT6. Conjugates without the cleavable linker were used as controls and all constructs were labeled with lutetium-177 (177Lu). [125I]I-HPEM was coupled to ADAPT6 at the C-terminus. Biodistribution of all constructs was evaluated in NMRI mice 4 h after injection. Specific binding to HER2-expressing cells in vitro was demonstrated for all constructs. No significant difference in kidney uptake was observed between the [177Lu]Lu-2,2 ',2",2"'-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid-GLGK-conjugates and the controls. The renal activity of [125I]I-HPEM-ADAPT6 was significantly lower compared with all other constructs. In conclusion, the incorporation of the cleavable GLGK-linker did not result in lower renal retention. Therefore, the present study emphasized that, in order to achieve a reduction of renal retention, alternative molecular design strategies may be required for different targeting agents.
