Showing papers on "Low protein published in 2020"

The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization

Abstract: Sturgeons seem to be frozen in time. The archaic characteristics of this ancient fish lineage place it in a key phylogenetic position at the base of the ~30,000 modern teleost fish species. Moreover, sturgeons are notoriously polyploid, providing unique opportunities to investigate the evolution of polyploid genomes. We assembled a high-quality chromosome-level reference genome for the sterlet, Acipenser ruthenus. Our analysis revealed a very low protein evolution rate that is at least as slow as in other deep branches of the vertebrate tree, such as that of the coelacanth. We uncovered a whole-genome duplication that occurred in the Jurassic, early in the evolution of the entire sturgeon lineage. Following this polyploidization, the rediploidization of the genome included the loss of whole chromosomes in a segmental deduplication process. While known adaptive processes helped conserve a high degree of structural and functional tetraploidy over more than 180 million years, the reduction of redundancy of the polyploid genome seems to have been remarkably random.

Controlled division of cell-sized vesicles by low densities of membrane-bound proteins

Abstract: The proliferation of life on earth is based on the ability of single cells to divide into two daughter cells. During cell division, the plasma membrane undergoes a series of morphological transformations which ultimately lead to membrane fission. Here, we show that analogous remodeling processes can be induced by low densities of proteins bound to the membranes of cell-sized lipid vesicles. Using His-tagged fluorescent proteins, we are able to precisely control the spontaneous curvature of the vesicle membranes. By fine-tuning this curvature, we obtain dumbbell-shaped vesicles with closed membrane necks as well as neck fission and complete vesicle division. Our results demonstrate that the spontaneous curvature generates constriction forces around the membrane necks and that these forces can easily cover the force range found in vivo. Our approach involves only one species of membrane-bound proteins at low densities, thereby providing a simple and extendible module for bottom-up synthetic biology. Membrane fission of a cell into two daughters is a core ability of cell-based life. Here the authors show that in artificial cells division can be controlled by regulating membrane curvature using low protein density.

Single Molecule Protein Detection with Attomolar Sensitivity Using Droplet Digital Enzyme-Linked Immunosorbent Assay

Abstract: Many proteins are present at low concentrations in biological samples, and therefore, techniques for ultrasensitive protein detection are necessary To overcome challenges with sensitivity, the digital enzyme-linked immunosorbent assay (ELISA) was developed, which is 1000× more sensitive than conventional ELISA and allows sub-femtomolar protein detection However, this sensitivity is still not sufficient to measure many proteins in various biological samples, thereby limiting our ability to detect and discover biomarkers To overcome this limitation, we developed droplet digital ELISA (ddELISA), a simple approach for detecting low protein levels using digital ELISA and droplet microfluidics ddELISA achieves maximal sensitivity by improving the sampling efficiency and counting more target molecules ddELISA can detect proteins in the low attomolar range and is up to 25-fold more sensitive than digital ELISA using Single Molecule Arrays (Simoa), the current gold standard tool for ultrasensitive protein detection Using ddELISA, we measured the LINE1/ORF1 protein, a potential cancer biomarker that has not been previously measured in serum Additionally, due to the simplicity of our device design, ddELISA is promising for point-of-care applications Thus, ddELISA will facilitate the discovery of biomarkers that have never been measured before for various clinical applications

PIP/TMC Interfacial Polymerization with Electrospray: Novel Loose Nanofiltration Membrane for Dye Wastewater Treatment

Abstract: A loose nanofiltration (NF) membrane with excellent dye rejection and high permeation of inorganic salt is required to fractionate dye/salt mixture in dye wastewater treatment. In this study, we fabricated the loose NF membrane by using the electrospray interfacial polymerization (EIP) method. It is a novel and facile interfacial polymerization method, which controls the thickness of the poly(piperazine-amide) (PPA) layer in nanometers (1 nm/min) and changes cross-linking degree of PPA layer and pore size by varying the electrospray time; consequently, water permeance and dye/salt rejection ratio can be handled. The fabricated EIP membrane with an optimized fabrication condition (M30, electrospray time was 30 min) possessed excellent pure water permeance (20.2 LMH/bar), high dye rejection (e.g., 99.6% for congo red (CR)), and low salt rejection (e.g., 6.3% for NaCl). Moreover, the EIP membrane exhibited enhanced antifouling property than commercial NF membrane (NF90) with a high flux recovery rate (FRR) of 87.1% and low irreversible fouling (Rir) of 12.9% after fouled by bovine serum albumin (BSA) due to its great smooth surface (average roughness (Ra) is 12.2 nm), hydrophilicity property, enhanced zeta potential, and low protein adsorption. The results indicate that the EIP loose NF membrane had a high potential for dye wastewater treatment.

Plant-Dominant Low-Protein Diet for Conservative Management of Chronic Kidney Disease.

Abstract: Chronic kidney disease (CKD) affects >10% of the adult population. Each year, approximately 120,000 Americans develop end-stage kidney disease and initiate dialysis, which is costly and associated with functional impairments, worse health-related quality of life, and high early-mortality rates, exceeding 20% in the first year. Recent declarations by the World Kidney Day and the U.S. Government Executive Order seek to implement strategies that reduce the burden of kidney failure by slowing CKD progression and controlling uremia without dialysis. Pragmatic dietary interventions may have a role in improving CKD outcomes and preventing or delaying dialysis initiation. Evidence suggests that a patient-centered plant-dominant low-protein diet (PLADO) of 0.6–0.8 g/kg/day composed of >50% plant-based sources, administered by dietitians trained in non-dialysis CKD care, is promising and consistent with the precision nutrition. The scientific premise of the PLADO stems from the observations that high protein diets with high meat intake not only result in higher cardiovascular disease risk but also higher CKD incidence and faster CKD progression due to increased intraglomerular pressure and glomerular hyperfiltration. Meat intake increases production of nitrogenous end-products, worsens uremia, and may increase the risk of constipation with resulting hyperkalemia from the typical low fiber intake. A plant-dominant, fiber-rich, low-protein diet may lead to favorable alterations in the gut microbiome, which can modulate uremic toxin generation and slow CKD progression, along with reducing cardiovascular risk. PLADO is a heart-healthy, safe, flexible, and feasible diet that could be the centerpiece of a conservative and preservative CKD-management strategy that challenges the prevailing dialysis-centered paradigm.

Rapid fabrication of precise high-throughput filters from membrane protein nanosheets.

Abstract: Biological membranes are ideal for separations as they provide high permeability while maintaining high solute selectivity due to the presence of specialized membrane protein (MP) channels. However, successful integration of MPs into manufactured membranes has remained a significant challenge. Here, we demonstrate a two-hour organic solvent method to develop 2D crystals and nanosheets of highly packed pore-forming MPs in block copolymers (BCPs). We then integrate these hybrid materials into scalable MP-BCP biomimetic membranes. These MP-BCP nanosheet membranes maintain the molecular selectivity of the three types of β-barrel MP channels used, with pore sizes of 0.8 nm, 1.3 nm, and 1.5 nm. These biomimetic membranes demonstrate water permeability that is 20–1,000 times greater than that of commercial membranes and 1.5–45 times greater than that of the latest research membranes with comparable molecular exclusion ratings. This approach could provide high performance alternatives in the challenging sub-nanometre to few-nanometre size range. Protein channels are highly selective, but application in membranes is limited due to low protein content. Here, protein channels are embedded into block copolymers to form nanosheets using rapid solvent casting, with better water permeability and similar molecular exclusions relative to other membrane systems.

The alarmones (p)ppGpp directly regulate translation initiation during entry into quiescence.

Abstract: Many bacteria exist in a state of metabolic quiescence where energy consumption must be minimized so as to maximize available resources over a potentially extended period of time As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be an appropriate target for down-regulation during the transition from growth to quiescence We observe that when Bacillus subtilis exits rapid growth, a subpopulation of cells emerges with very low protein synthetic activity This phenotypic heterogeneity requires the production of the nucleotides (p)ppGpp, which we show are sufficient to inhibit protein synthesis in vivo We then show that one of these molecules, ppGpp, inhibits protein synthesis by preventing the allosteric activation of the essential GTPase Initiation Factor 2 (IF2) during translation initiation Finally, we demonstrate that the observed attenuation of protein synthesis during the entry into quiescence is a consequence of the direct interaction of (p)ppGpp and IF2

Polyelectrolyte interactions enable rapid association and dissociation in high-affinity disordered protein complexes.

Abstract: Highly charged intrinsically disordered proteins can form complexes with very high affinity in which both binding partners fully retain their disorder and dynamics, exemplified by the positively charged linker histone H1.0 and its chaperone, the negatively charged prothymosin α. Their interaction exhibits another surprising feature: The association/dissociation kinetics switch from slow two-state-like exchange at low protein concentrations to fast exchange at higher, physiologically relevant concentrations. Here we show that this change in mechanism can be explained by the formation of transient ternary complexes favored at high protein concentrations that accelerate the exchange between bound and unbound populations by orders of magnitude. Molecular simulations show how the extreme disorder in such polyelectrolyte complexes facilitates (i) diffusion-limited binding, (ii) transient ternary complex formation, and (iii) fast exchange of monomers by competitive substitution, which together enable rapid kinetics. Biological polyelectrolytes thus have the potential to keep regulatory networks highly responsive even for interactions with extremely high affinities.

Single Extracellular Vesicle Protein Analysis Using Immuno‐Droplet Digital Polymerase Chain Reaction Amplification

Abstract: There is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding of heterogeneous EV populations, to allow identification of unique subpopulations, and to enable earlier and more sensitive disease detection. Because of the small size of EV and their low protein content, ultrahigh sensitivity technologies are required. Here, an immuno-droplet digital polymerase chain reaction (iddPCR) amplification method is described that allows multiplexed single EV protein profiling. Antibody-DNA conjugates are used to label EV, followed by stochastic microfluidic incorporation of single EV into droplets. In situ PCR with fluorescent reporter probes converts and amplifies the barcode signal for subsequent read-out by droplet imaging. In these proof-of-principle studies, it is shown that multiplex protein analysis is possible in single EV, opening the door for future analyses.

Physiological, Ecological, and Biochemical Implications in Tomato Plants of Two Plant Biostimulants: Arbuscular Mycorrhizal Fungi and Seaweed Extract

Abstract: The worldwide use of plant biostimulants (PBs) represents an environmentally friendly tool to increase crop yield and productivity. PBs include different substances, compounds, and growth-promoting microorganism formulations, such as those derived from arbuscular mycorrhizal fungi (AMF) or seaweed extracts (SEs), which are used to regulate or enhance physiological processes in plants. This study analyzed the physiological, ecological, and biochemical implications of the addition of two PBs, AMF or SE (both alone and in combination), on tomato plants (Solanum lycopersicum L. cv. "Rio Fuego"). The physiological responses evaluated were related to plant growth and photosynthetic performance. The ecological benefits were assessed based on the success of AMF colonization, flowering, resistance capacity, nonphotochemical quenching (NPQ), and polyphenol content. Biochemical effects were evaluated via protein, lipid, carbohydrate, nitrogen, and phosphorous content. Each PB was found to benefit tomato plants in a different but complementary manner. AMF resulted in an energetically expensive (high ETRMAX but low growth) but protective (high NPQ and polyphenol content) response. AMF + nutritive solution (NS) induced early floration but resulted in low protein, carbohydrate, and lipid content. Both AMF and AMF + NS favored foliar instead of root development. In contrast, SE and SE + NS favored protein content and root development and did not promote flowering. However, the combination of both PBs (AMF + SE) resulted in an additive effect, reflected in an increase in both foliar and root growth as well as protein and carbohydrate content. Moreover, a synergistic effect was also found, which was expressed in accelerated flowering and AMF colonization. We present evidence of benefits to plant performance (additive and synergistic) due to the interactive effects between microbial (AMF) and nonmicrobial (SEs) PBs and propose that the complementary modes of action of both PBs may be responsible for the observed positive effects due to the new and emerging properties of their components instead of exclusively being the result of known constituents. These results will be an important contribution to biostimulant research and to the development of a second generation of PBs in which combined and complementary mechanisms may be functionally designed.

The Urinary Exosomal miRNA Expression Profile is Predictive of Clinical Response in Lupus Nephritis.

Abstract: Data on exosomal-derived urinary miRNAs have identified several miRNAs associated with disease activity and fibrosis formation, but studies on prognosis are lacking. We conducted a qPCR array screening on urinary exosomes from 14 patients with biopsy-proven proliferative lupus glomerulonephritis with a renal outcome of clinical response (n = 7) and non-response (n = 7) following therapy. Validation studies were performed by qRT-PCR in a new lupus nephritis (LN) cohort (responders = 22 and non-responders = 21). Responder patients expressed significantly increased levels of miR-31, miR-107, and miR-135b-5p in urine and renal tissue compared to non-responders. MiR-135b exhibited the best predictive value to discriminate responder patients (area under the curve = 0.783). In vitro studies showed exosome-derived miR-31, miR-107, and miR-135b-5p expression to be mainly produced by tubular renal cells stimulated with inflammatory cytokines (e.g IL1, TNFα, IFNα and IL6). Uptake of urinary exosomes from responders by mesangial cells was superior compared to that from non-responders (90% vs. 50%, p < 0.0001). HIF1A was identified as a potential common target, and low protein levels were found in non-responder renal biopsies. HIF1A inhibition reduced mesangial proliferation and IL-8, CCL2, CCL3, and CXCL1 mesangial cell production and IL-6/VCAM-1 in endothelial cells. Urinary exosomal miR-135b-5p, miR-107, and miR-31 are promising novel markers for clinical outcomes, regulating LN renal recovery by HIF1A inhibition.

Accelerated Kidney Aging in Diabetes Mellitus.

Abstract: With aging, the kidney undergoes inexorable and progressive changes in structural and functional performance. These aging-related alterations are more obvious and serious in diabetes mellitus (DM). Renal accelerated aging under DM conditions is associated with multiple stresses such as accumulation of advanced glycation end products (AGEs), hypertension, oxidative stress, and inflammation. The main hallmarks of cellular senescence in diabetic kidneys include cyclin-dependent kinase inhibitors, telomere shortening, and diabetic nephropathy-associated secretory phenotype. Lysosome-dependent autophagy and antiaging proteins Klotho and Sirt1 play a fundamental role in the accelerated aging of kidneys in DM, among which the autophagy-lysosome system is the convergent mechanism of the multiple antiaging pathways involved in renal aging under DM conditions. Metformin and the inhibitor of sodium-glucose cotransporter 2 are recommended due to their antiaging effects independent of antihyperglycemia, besides angiotensin-converting enzyme inhibitors/angiotensin receptor blockers. Additionally, diet intervention including low protein and low AGEs with antioxidants are suggested for patients with diabetic nephropathy (DN). However, their long-term benefits still need further study. Exploring the interactive relationships among antiaging protein Klotho, Sirt1, and autophagy-lysosome system may provide insight into better satisfying the urgent medical needs of elderly patients with aging-related DN.

Plant-based diets for prevention and management of chronic kidney disease

Abstract: Purpose of reviewPlant-based diets have been used with growing popularity for the treatment of a wide range of lifestyle-related diseases, including diabetes, hypertension, and obesity. With the reinvigoration of the dietary management of chronic kidney disease (CKD) and the use of low protein diets

Defining the impact of dietary macronutrient balance on PCOS traits.

Abstract: Lifestyle, mainly dietary, interventions are first-line treatment for women with polycystic ovary syndrome (PCOS), but the optimal diet remains undefined. We combined a hyperandrogenized PCOS mouse model with a systematic macronutrient approach, to elucidate the impact of dietary macronutrients on the development of PCOS. We identify that an optimum dietary macronutrient balance of a low protein, medium carbohydrate and fat diet can ameliorate key PCOS reproductive traits. However, PCOS mice display a hindered ability for their metabolic system to respond to diet variations, and varying macronutrient balance did not have a beneficial effect on the development of metabolic PCOS traits. We reveal that PCOS traits in a hyperandrogenic PCOS mouse model are ameliorated selectively by diet, with reproductive traits displaying greater sensitivity than metabolic traits to dietary macronutrient balance. Hence, providing evidence to support the development of evidence-based dietary interventions as a promising strategy for the treatment of PCOS, especially reproductive traits. Lifestyle interventions are first-line treatment for women with polycystic ovary syndrome (PCOS), but the optimal diet remains undefined. Here the authors identify an optimum dietary macronutrient balance that can rectify PCOS reproductive traits in a mouse model of PCOS, while metabolic features were less sensitive to diet changes.

The RNA-Binding Ubiquitin Ligase MEX3A Affects Glioblastoma Tumorigenesis by Inducing Ubiquitylation and Degradation of RIG-I

Abstract: Glioblastoma multiforme (GB) is the most malignant primary brain tumor in humans, with an overall survival of approximatively 15 months. The molecular heterogeneity of GB, as well as its rapid progression, invasiveness and the occurrence of drug-resistant cancer stem cells, limits the efficacy of the current treatments. In order to develop an innovative therapeutic strategy, it is mandatory to identify and characterize new molecular players responsible for the GB malignant phenotype. In this study, the RNA-binding ubiquitin ligase MEX3A was selected from a gene expression analysis performed on publicly available datasets, to assess its biological and still-unknown activity in GB tumorigenesis. We find that MEX3A is strongly up-regulated in GB specimens, and this correlates with very low protein levels of RIG-I, a tumor suppressor involved in differentiation, apoptosis and innate immune response. We demonstrate that MEX3A binds RIG-I and induces its ubiquitylation and proteasome-dependent degradation. Further, the genetic depletion of MEX3A leads to an increase of RIG-I protein levels and results in the suppression of GB cell growth. Our findings unveil a novel molecular mechanism involved in GB tumorigenesis and suggest MEX3A and RIG-I as promising therapeutic targets in GB.

High-protein diet more effectively reduces hepatic fat than low-protein diet despite lower autophagy and FGF21 levels

Abstract: Background and aims Non-alcoholic fatty liver disease (NAFLD) is becoming increasingly prevalent and nutrition intervention remains the most important therapeutic approach for NAFLD. Our aim was to investigate whether low- (LP) or high-protein (HP) diets are more effective in reducing liver fat and reversing NAFLD and which mechanisms are involved. Methods 19 participants with morbid obesity undergoing bariatric surgery were randomized into two hypocaloric (1500-1600 kcal/day) diet groups, a low protein (10E% protein) and a high protein (30E% protein), for three weeks prior to surgery. Intrahepatic lipid levels (IHL) and serum fibroblast growth factor 21 (FGF21) were measured before and after the dietary intervention. Autophagy flux, histology, mitochondrial activity, and gene expression analyses were performed in liver samples collected during surgery. Results IHL levels decreased by 42.6% in the HP group, but were not significantly changed in the LP group despite similar weight loss. Hepatic autophagy flux and serum FGF21 increased by 66.7% and 42.2%, respectively, after 3 weeks in the LP group only. Expression levels of fat uptake and lipid biosynthesis genes were lower in the HP group compared with those in the LP group. RNA-seq analysis revealed lower activity of inflammatory pathways upon HP diet. Hepatic mitochondrial activity and expression of β-oxidation genes did not increase in the HP group. Conclusions HP diet more effectively reduces hepatic fat than LP diet despite of lower autophagy and FGF21. Our data suggest that liver fat reduction upon HP diets result primarily from suppression of fat uptake and lipid biosynthesis.

A Clinical Tool to Predict Low Serum Selenium in Patients with Worsening Heart Failure.

Abstract: Selenium is an essential micronutrient, and a low selenium concentration ( 80% of patients with low selenium (sensitivity of 44%, specificity of 80%). Given that selenium and iron overlap in their physiological roles, we evaluated the shared determinants and prognostic associates. Both deficiencies shared similar clinical characteristics, including the model risk factors and, in addition, a low protein intake and high levels of C-reactive protein. Low selenium was associated with a similar or worse prognosis compared to iron deficiency. In conclusion, although it is difficult to exclude low selenium based on clinical characteristics alone, we provide a prediction tool which identifies heart failure patients at higher risk of having a low selenium status.

Engineered Interactions with Mesoporous Silica Facilitate Intracellular Delivery of Proteins and Gene Editing.

Abstract: Intracellular delivery of functional proteins is a promising, but challenging, strategy for many therapeutic applications. Here, we report a new methodology that overcomes drawbacks of traditional mesoporous silica (MSi) particles for protein delivery. We hypothesize that engineering enhancement in interactions between proteins and delivery vehicles can facilitate efficient encapsulation and intracellular delivery. In this strategy, surface lysines in proteins were modified with a self-immolative linker containing a terminal boronic acid for stimulus-induced reversibility in functionalization. The boronic acid moiety serves to efficiently interact with amine-functionalized MSi through dative and electrostatic interactions. We show that proteins of different sizes and isoelectric points can be quantitatively encapsulated into MSi, even at low protein concentrations. We also show that the proteins can be efficiently delivered into cells with retention of activity. Utility of this approach is further demonstrated with gene editing in cells, through the delivery of a CRISPR/Cas9 complex.

Small protein number effects in stochastic models of autoregulated bursty gene expression

Abstract: A stochastic model of autoregulated bursty gene expression by Kumar et al. [Phys. Rev. Lett. 113, 268105 (2014)] has been exactly solved in steady-state conditions under the implicit assumption that protein numbers are sufficiently large such that fluctuations in protein numbers due to reversible protein–promoter binding can be ignored. Here, we derive an alternative model that takes into account these fluctuations and, hence, can be used to study low protein number effects. The exact steady-state protein number distribution is derived as a sum of Gaussian hypergeometric functions. We use the theory to study how promoter switching rates and the type of feedback influence the size of protein noise and noise-induced bistability. Furthermore, we show that our model predictions for the protein number distribution are significantly different from those of Kumar et al. when the protein mean is small, gene switching is fast, and protein binding to the gene is faster than the reverse unbinding reaction.

Association Between Macronutrients Intake and Depression in the United States and South Korea.

Abstract: Although the risk for depression appears to be related to daily dietary habits, how the proportion of major macronutrients affects the occurrence of depression remains largely unknown. This study aims to estimate the association between macronutrients (i.e., carbohydrate, protein, fat) and depression through national survey datasets from the United States and South Korea. Association between the prevalence of depression and each macronutrient was measured from 60,935 participants from the National Health and Nutrition Examination Survey (NHANES) and 15,700 participants from the South Korea NHANES (K-NHANES) databases. When the proportion of calories intake by protein increased by 10%, the prevalence of depression was significantly reduced both in the United States (Odds Ratio, OR [95% CI], 0.621 [0.530-0.728]) and South Korea (0.703 [0.397-0.994]). An association between carbohydrate intake and the prevalence of depression was seen in the United States (1.194 [1.116-1.277]), but not in South Korea. Fat intake was not significantly associated with depression in either country. Subsequent analysis showed that the low protein intake groups had significantly higher risk for depression than the normal protein intake groups in both the United States (1.648 [1.179-2.304]) and South Korea (3.169 [1.598-6.286]). In the daily diet of macronutrients, the proportion of protein intake is significantly associated with the prevalence of depression. These associations were more prominent in adults with insufficient protein intake, and the pattern of association between macronutrients and depression in Asian American and South Korean populations were similar. Our findings suggest that the proportion of macronutrients intake in everyday life may be related to the occurrence of depression.

Multiple Amino Acid Supplementations to Low-Protein Diets: Effect on Performance, Carcass Yield, Meat Quality and Nitrogen Excretion of Finishing Broilers under Hot Climate Conditions

Abstract: The objective of this study was to evaluate the effect of low-protein diets with amino acid supplementation on growth performance, carcass yield, meat quality and nitrogen excretion of broilers raised under hot climate conditions during the finisher period. In trial 1, broilers from 28 to 49 days of age were fed 18% crude protein (CP) as a positive control or 15% CP supplemented with (1) DL-methionine (Met) + L-lysine (Lys), (2) Met + Lys + L-Arginine (Arg), or (3) Met + Lys + L-Valine (Val). In trial 2, broilers from 30 to 45 days of age, were fed an 18% CP diet as a positive control or 15% CP supplemented with Met, Lys, Arg, Val, L-Isoleucine (Ile) or combination with glycine (Gly) and/or urea as nitrogen sources: (1) Met + Lys, (2) Met + Lys + Arg, (3) Met + Lys + Val, (4) Met + Lys + Ile, (5) Met + Lys + Arg +Val + Ile + Gly, and (6) Met+ Lys + Arg + Val + Ile + Gly + urea. Protein use was improved by feeding low-protein amino acid-supplemented diets as compared to the high-protein diet. Feeding 15% crude protein diet supplemented with only methionine and lysine had no negative effects on carcass yield, CP, total lipids and moisture% of breast meat while decreasing nitrogen excretion by 21%.