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Iron (Fe) is necessary for plant growth and development. The mechanism of uptake and translocation in Cadmium (Cd) is similar to iron, which shares iron transporters. Yellow stripe-like transporter (YSL) plays a pivotal role in transporting iron and other metal ions in plants. In this study, MsYSL6 and its promoter were cloned from leguminous forage alfalfa. The transient expression of MsYSL6-GFP indicated that MsYSL6 was localized to the plasma membrane and cytoplasm. The expression of MsYSL6 was induced in alfalfa by iron deficiency and Cd stress, which was further proved by GUS activity driven by the MsYSL6 promoter. To further identify the function of MsYSL6, it was heterologously overexpressed in tobacco. MsYSL6-overexpressed tobacco showed better growth and less oxidative damage than WT under Cd stress. MsYSL6 overexpression elevated Fe and Cd contents and induced a relatively high Fe translocation rate in tobacco under Cd stress. The results suggest that MsYSL6 might have a dual function in the absorption of Fe and Cd, playing a role in the competitive absorption between Fe and Cd. MsYSL6 might be a regulatory factor in plants to counter Cd stress. This study provides a novel gene for application in heavy metal enrichment or phytoremediation and new insights into plant tolerance to toxic metals. [ABSTRACT FROM AUTHOR]

Iron(Fe) is a trace metal element necessary for plant growth, but excess iron is harmful to plants. Natural resistance-associated macrophage proteins (NRAMPs) are important for divalent metal transport in plants. In this study, we isolated the MsNRAMP2 (MN_547960) gene from alfalfa, the perennial legume forage. The expression of MsNRAMP2 is specifically induced by iron excess. Overexpression of MsNRAMP2 conferred transgenic tobacco tolerance to iron excess, while it conferred yeast sensitivity to excess iron. Together with the MsNRAMP2 gene, MsMYB (MN_547959) expression is induced by excess iron. Y1H indicated that the MsMYB protein could bind to the "CTGTTG" cis element of the MsNRAMP2 promoter. The results indicated that MsNRAMP2 has a function in iron transport and its expression might be regulated by MsMYB. The excess iron tolerance ability enhancement of MsNRAMP2 may be involved in iron transport, sequestration, or redistribution. [ABSTRACT FROM AUTHOR]

Iron (Fe) and (Mn) are essential for the plant but are toxic when in excess. Vacuolar iron transporters (VITs) are involved in plant metal storage and detoxication. In this study, we screened two soybean cultivars (HN51 and SN37) with different responses to iron stress. From HN51 and SN37, we identified a new gene GmVIT1, for which expression is closely related to iron stress response by transcriptomic and quantitative analysis. We obtained GmVIT1 and GmVIT1 promoter from the iron deficiency-tolerant soybean variety Heinong51. Sequence analysis showed that GmVIT1 contained a conserved 170-residue VIT domain and localized at the tonoplast. Moreover, GmVIT1 is expressed in soybean leaves, stems, and roots. The expression of GmVIT1 was significantly induced by excessive Fe/Mn in leaves and stems. GUS assay showed that excess Fe/Mn enhanced GmVIT1 promoter activity. Furthermore, overexpression of GmVIT1 in Arabidopsis seedlings showed reduced phytotoxic effects induced by excess Fe/Mn stress, including yellowing in leaves, decreased chlorophyll content, and accumulated MDA. GmVIT1 overexpression in Arabidopsis showed relatively higher soluble sugar content and SOD, POD, and CAT activity. In addition, the ferric reductase activity in GmVIT1 overexpression in Arabidopsis decreased under excess Fe, while it increased under excess Mn. By integrating all these results, we found that GmVIT1 plays a vital role in plant response to excess Fe/Mn. The results showed that GmVIT1 was worthy of metal homeostasis mechanism research in plants and could be applied in the metal toxic-tolerance improvement in crops. [ABSTRACT FROM AUTHOR]

Subchondral bone lesions, as the crucial inducement for accelerating cartilage degeneration, have been considered as the initiating factor and the potential therapeutic target of knee osteoarthritis (KOA). Acupotomy, the biomechanical therapy guided by traditional Chinese meridians theory, alleviates cartilage deterioration by correcting abnormal mechanics. Whether this mechanical effect of acupotomy inhibits KOA subchondral bone lesions is indistinct. This study aimed to investigate the effects of acupotomy on inhibiting subchondral bone resorption and to define the possible mechanism in immobilization-induced KOA rabbits. After KOA modeling, 8 groups of rabbits (4w/6w acupotomy, 4w/6w electroacupuncture, 4w/6w model, and 4w/6w control groups) received the indicated intervention for 3 weeks. Histological and bone histomorphometry analyses revealed that acupotomy prevented both cartilage surface erosion and subchondral bone loss. Further, acupotomy suppressed osteoclast activity and enhanced osteoblast activity in KOA subchondral bone, showing a significantly decreased expression of tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinases-9 (MMP-9), and cathepsin K (Ctsk) and a significantly increased expression of osteocalcin (OCN); this regulation may be mediated by blocking the decrease in osteoprotegerin (OPG) and the increase in NF- κ B receptor activated protein ligand (RANKL). These findings indicated that acupotomy inhibited osteoclast activity and promoted osteoblast activity to ameliorate hyperactive subchondral bone resorption and cartilage degeneration in immobilization-induced KOA rabbits, which may be mediated by the OPG/RANKL signaling pathway. Taken together, our results indicate that acupotomy may have therapeutic potential in KOA by restoring the balance between bone formation and bone resorption to attenuate subchondral bone lesions. [ABSTRACT FROM AUTHOR]

Objective: To reveal the effect and mechanism of Jiaotai Pill (交泰丸, JTP) on insomniac rats. Methods: The insomniac model was established by intraperitoneal injection of p-chlorophenylalanine (PCPA). In behavioral experiments, rats were divided into control, insomniac model, JTP [3.3 g/(kg•d)], and diazepam [4 mg/(kg•d)] groups. The treatment effect of JTP was evaluated by weight measurement (increasement of body weight), open field test (number of crossings) and forced swimming test (immobility time). A high performance liquid chromatography-electrochemical detection (HPLC-ECD) method was built to determine the concentration of monoamine transmitters in hypothalamus and peripheral organs from normal, model, JTP, citalopram [30 mg/(kg•d)], maprotiline [40 mg/(kg•d)] and bupropion [40 mg/(kg•d)] groups. Expressions of serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET) were analyzed by quantitative polymerase chain reaction (qPCR) and Western blot in normal, model and JTP groups. A high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS/MS) method was established to determine the pharmacokinetics, urine cumulative excretion of metformin in vivo, and tissue slice uptake in vitro, which were applied to assess the activity of organic cation transporters (OCTs) in hypothalamus and peripheral organs. Results: Compared with the insomniac model group, the body weight and spontaneous locomotor were increased, and the immobility time was decreased after treatment with JTP (P<0.01). Both serotonin and dopamine contents in hypothalamus and peripheral organs were increased (P<0.01). The norepinephrine content was increased in peripheral organs and decreased in hypothalamus (P<0.05 or P<0.01). At the same time, SERT, DAT, OCT1, OCT2, and OCT3 were down-regulated in hypothalamus and peripheral organs (P<0.05). NET was down-regulated in peripheral organs and up-regulated in hypothalamus (P<0.05 or P<0.01). Moreover, the activity of OCTs in hypothalamus and peripheral organs was inhibited (P<0.05). Conclusion: JTP alleviates insomnia through regulation of monoaminergic system and OCTs in hypothalamus and peripheral organs. [ABSTRACT FROM AUTHOR]

Alcoholic liver disease (ALD) is caused by heavy alcohol consumption over a long period. Acetaldehyde-mediated toxicity, oxidative stress, and imbalance of lipid metabolism are generally considered involved in the initiation of ALD. There is an increasing requirement for alternative and natural medicine to treat ALD. Recently, aldose reductase (AR) has been reported to be involved in the development of ALD by affecting inflammatory cytokines, oxidative stress, and lipid metabolism. Here, we review the effect of plant-derived AR inhibitors on ALD in rodents. And we conclude that AR inhibitors of plant origin may enhance antioxidant capacity, inhibit lipid peroxidation and inflammatory cytokines expression, and activate AMP-activated protein kinase thereby subsequently suppressing alcohol-induced lipid synthesis in liver to achieve ALD protection. This review reveals that natural AR inhibitor may be potential therapeutic agent for ALD. [ABSTRACT FROM AUTHOR]

We recently reported that doxorubicin decreased the expression of calpain-1/2, while inhibition of calpain activity promoted doxorubicin-induced cardiac injury in mice. In this study, we investigated whether and how elevation of calpain-2 could affect doxorubicin-triggered cardiac injury. Transgenic mice with inducible cardiomyocyte-specific expression of calpain-2 were generated. An acute cardiotoxicity was induced in both transgenic mice and their relevant wild-type littermates by injection of a single dose of doxorubicin (20 mg/kg) and cardiac injury was analyzed 5 days after doxorubicin injection. Cardiomyocyte-specific up-regulation of calpain-2 did not induce any adverse cardiac phenotypes under physiological conditions by age 3 months, but significantly reduced myocardial injury and improved myocardial function in doxorubicin-treated mice. Cardiac protection of calpain-2 up-regulation was also observed in a mouse model of chronic doxorubicin cardiotoxicity. Up-regulation of calpain-2 increased the protein levels of mitogen activated protein kinase phosphatase-1 (MKP-1) in cultured mouse cardiomyocytes and heart tissues. Over-expression of MKP-1 prevented, whereas knockdown of MKP-1 augmented doxorubicin-induced apoptosis in cultured cardiomyocytes. Moreover, knockdown of MKP-1 offset calpain-2-elicited protective effects against doxorubicin-induced injury in cultured cardiomyocytes. Mechanistically, up-regulation of calpain-2 reduced the protein levels of phosphatase and tensin homolog and consequently promoted Akt activation, leading to increased MKP-1 protein steady-state levels by inhibiting its degradation. Collectively, this study reveals a new role of calpain-2 in promoting MKP-1 expression via phosphatase and tensin homolog/Akt signaling. This study also suggests that calpain-2/MKP-1 signaling may represent new therapeutic targets for doxorubicin-induced cardiac injury. [ABSTRACT FROM AUTHOR]

Experimental modeling to identify specific inhalation hazards for nanomaterials has in the main focused on in vivo approaches. However, these models suffer from uncertainties surrounding species-specific differences and cellular targets for biologic response. In terms of pulmonary exposure, approaches which combine ‘inhalation-like’ nanoparticulate aerosol deposition with relevant human cell and tissue air-liquid interface cultures are considered an important complement to in vivo work. In this study, we utilized such a model system to build on previous results from in vivo exposures, which highlighted the small airway epithelium as a target for silver nanoparticle (AgNP) deposition. RNA-SEQ was used to characterize alterations in mRNA and miRNA within the lung. Organotypic-reconstituted 3D human primary small airway epithelial cell cultures (SmallAir) were exposed to the same spark-generated AgNP and at the same dose used in vivo, in an aerosol-exposure air-liquid interface (AE-ALI) system. Adverse effects were characterized using lactate, LDH release and alterations in mRNA and miRNA. Modest toxicological effects were paralleled by significant regulation in gene expression, reflective mainly of specific inflammatory events. Importantly, there was a level of concordance between gene expression changes observed in vitro and in vivo. We also observed a significant correlation between AgNP and mass equivalent silver ion (Ag+) induced transcriptional changes in SmallAir cultures. In addition to key mechanistic information relevant for our understanding of the potential health risks associated with AgNP inhalation exposure, this work further highlights the small airway epithelium as an important target for adverse effects. [ABSTRACT FROM AUTHOR]

Gynura formosana Kitam. belongs to the Compositae family and has been traditionally used for the prevention of cancer, diabetes, and inflammation in China. Previous studies had indicated that the ethyl acetate extract of Gynura formosana Kitam. leaves (EAEG) exhibited antioxidant and anti-inflammatory activity. In this report, we demonstrated that EAEG possessed potent anticancer activity through autophagy-mediated inhibition of cell proliferation. EAEG induced a strong cytostatic effect towards HeLa cells and, to a lesser extent, HepG2 and MCF-7 cells. This cytostatic effect of EAEG was not a consequence of increased apoptosis, as neither DNA fragmentation nor change in protein expression level for a number of apoptosis-related genes including Bid, Bax, Bcl-2, and caspase-3 was observed after EAEG treatment, and the apoptosis inhibitor Z-VAD-FMK did not inhibit the EAEG-elicited cytostatic effect. On the other hand, EAEG induced autophagy in a dose-dependent fashion, as shown by increased GFP puncta formation, enhanced conversion of the microtubule-associated protein light chain LC3-I to LC3-II, and downregulation of the p62 protein. Treating the HeLa cells with EAEG together with Chloroquine (CQ) further accelerated LC3 conversion and p62 clearance, indicating that EAEG induced complete autophagy flux. Importantly, the autophagy inhibitor 3-methyladenine (3MA) significantly abrogated the cytostatic effect of EAEG, strongly suggesting that EAEG inhibited HeLa cell proliferation through the induction of autophagy rather than apoptosis. Our results provided a novel and interesting mechanistic insight into the anticancer action of EAEG, supporting the traditional use of this plant for the treatment of the cancer. [ABSTRACT FROM AUTHOR]

Objective. By observing the needle-knife of KOA rabbit morphology, knee joint cartilage p-FAK, p-PI3K, Aggrecan gene, and protein expression, to study the effect of needle-knife to promote cartilage cell synthesis metabolism mechanism. Method. 49 male New Zealand rabbits, randomly divided into normal group (Z), model group (M), model-inhibitors (MP), needle-knife group (D), needle-knife inhibitors group (DP), electroacupuncture group (E), and electroacupuncture inhibitors (EP). RT-PCR and Western Blot were used to test each animal cartilage p-FAK, p-PI3K, and Aggrecan gene and protein expression level. Results. Compared with N group, p-FAK and p-PI3K protein and mRNA expression of M group, D group, and E group increased (P < 0.05), while the protein and mRNA expression of Aggrecan reduced (P < 0.05). Compared with M group, p-FAK, p-PI3K, Aggrecan protein, and mRNA of E and D group increased (P < 0.05). Compared with E group, p-FAK, p-PI3K, Aggrecan protein, and mRNA expression of D group increased (P < 0.05); after adding inhibitors, p-FAK, p-PI3K, Aggrecan protein, and mRNA expression reduced (P < 0.05). Conclusion. Needle-knife therapy can promote the repairment of cartilage cells by activating FAK-PI3K signaling pathways, promoting the synthesis of cartilage cell metabolism. [ABSTRACT FROM AUTHOR]

Objective To establish the colonic expression quantitative trait locus map in Han Chinese population and provide a functional reference for interpreting genetic associations of diseases such as inflammatory bowel disease (IBD). Methods Colonic mucosal biopsies and peripheral blood samples were obtained from 48 Chinese Han individuals (24 ulcerative colitis patients and 24 healthy controls). Transcription profiling was performed using human whole genome expression array. Genotyping was done using a population-specific genotype array. Imputation was performed using IMPUTE2. Association between genotypes and gene expression was analyzed using a Matrix Expression Quantitative Trait Loci (eQTL) R package to identify eQTL. We used ChIPpeakAnno R package for annotation of the eQTL. Linkage disequilibrium between the eQTL and IBD risk loci was also investigated. Results We identified 6 377 single nucleotide polymorphism-transcript interactions ( cis-eQTL) in the colon of the Chinese participants. Most of the eQTL located near the transcription starting sites and overlapped with histone modification marks on the genome. A significant proportion of the eQTL were found to be within transcription factor-binding sites. Two IBD risk loci were found to be colon cis-eQTL in Chinese individuals, and 51 cis-eQTL were identified in another 18 IBD risk loci. Conclusions This study defined a population-specific catalogue of colon eQTL in the Chinese population. Potential functional variants of IBD association signals were identified. We provided a useful reference dataset for fine mapping IBD risk loci and identifying causal variants in the Chinese Han population. [ABSTRACT FROM AUTHOR]

Introduction To investigate the cardioprotective effect of Micro RNA-21 (miR-21) in murine myocardial infarction ( MI). Methods Forty C57 BL/6 male mice were divided into sham group, MI group, LV- GFP group, and miR-21 group. Mice in the MI group, LV- GFP group, and miR-21 group were subjected to MI by left anterior descending artery ( LAD) ligation, while chest was opened/closed without ligation in sham group. In MI group, expression of miR-21 in the MI area and its surrounding areas was detected at 1st, 2nd, and 4th week after experiment. Subsequently, lentivirus expressing miR-21 and lentivirus that did not express miR-21 were transfected into mice left ventricular cavity of miR-21 group and LV- GFP group, respectively. Cardiac function, MI size, miR-21 expression, collagen I level, fibronectin content, number of α- SMA-positive cells, number of apoptotic cells, apoptosis-related factors were compared between the three groups. Results Compared with sham group, miR-21 levels in MI group were significantly decreased in the 1st week and 2nd week, but were almost the same in the 4th week. Left ventricular fractional shortening ( LVFS) and left ventricular ejection fraction ( LVEF) in the miR-21 group improved compared to the LV- GFP group. In miR-21 group, myocardial infarct size reduced by 36.9% in comparison with LV- GFP group. Compared to sham group, miR-21 expression in the miR-21 group and LV- GFP group decreased significantly. In the miR-21 group, collagen I level, fibronectin content and number of α- SMA-positive cells of miR-21 decreased significantly compared to the LV- GFP group. The number of apoptotic cells in the MI areas of the miR-21 group was significantly less than the LV- GFP group. Compared with the LV- GFP group, Bcl-2 level and the ratio of Bcl-2 to Bax were significantly increased, and the levels of Bax and Caspase-3 decreased. Conclusions Our results suggest miR-21 is an important regulatory molecule in the pathophysiology of MI. [ABSTRACT FROM AUTHOR]

Paederus fuscipes is a medically and agriculturally important insect all over the world. P. fuscipes cause not only a skin condition but is also an aggressive predator in different agro-ecosystems. Prior to performing RT-qPCR under a variety of conditions to investigate the function of target genes in P. fuscipes , it is essential to screen reference genes. However, no P. fuscipes reference gene(s) has yet been discovered. Using the RefFinder software package, which includes ΔCt, geNorm, NormFinder, and BestKeeper, we evaluated the stability of seven housekeeping genes of P. fuscipes under five conditions (developmental stage, diet, temperature, tissue and sex). During the RT-qPCR analysis, geNorm software was used to evaluate pairwise variation in order to identify the most appropriate number of reference genes. The results revealed that for developmental stages RPL-13, EF1A and β-tubulin ; for sex-related experiments β-tubulin and Actin , for tissue-related experiments PRS-18, 18S and RPL-13 ; for temperature-related experiments β-tubulin and PRL-13 and diet-related experiments, β-tubulin and PRS-18 are suitable reference genes. Furthermore, the associated HSP-70 (as the reporter gene) expression patterns differed significantly when the three most stable and three least stable reference genes were selected in different temperature treatments. This is the first time that a complete list of standardized reference genes has been given for P. fuscipes to use in an RT-qPCR study, which could be helpful for potential functional studies of target genes in P. fuscipes. [ABSTRACT FROM AUTHOR]

Cardiac fibroblasts and cardiomyocytes are the main cells involved in the pathophysiology of myocarditis (MCD). These cells are especially sensitive to changes in iron homeostasis, which is extremely important for the optimal maintenance of crucial cellular processes. However, the exact role of iron status in the pathophysiology of MCD remains unknown. We cultured primary human cardiomyocytes (hCM) and cardiofibroblasts (hCF) with sera from acute MCD patients and healthy controls to mimic the effects of systemic inflammation on these cells. Next, we performed an initial small-scale (n = 3 per group) RNA sequencing experiment to investigate the global cellular response to the exposure on sera. In both cell lines, transcriptomic data analysis revealed many alterations in gene expression, which are related to disturbed canonical pathways and the progression of cardiac diseases. Moreover, hCM exhibited changes in the iron homeostasis pathway. To further investigate these alterations in sera-treated cells, we performed a larger-scale (n = 10 for controls, n = 18 for MCD) follow-up study and evaluated the expression of genes involved in iron metabolism. In both cell lines, we demonstrated an increased expression of transferrin receptor 1 (TFR1) and ferritin in MCD serum-treated cells as compared to controls, suggesting increased iron demand. Furthermore, we related TFR1 expression with the clinical profile of patients and showed that greater iron demand in sera-treated cells was associated with higher inflammation score (interleukin 6 (IL-6), C-reactive protein (CRP)) and advanced neurohormonal activation (NT-proBNP) in patients. Collectively, our data suggest that the malfunctioning of cardiomyocytes and cardiofibroblasts in the course of MCD might be related to alterations in the iron homeostasis. [ABSTRACT FROM AUTHOR]

Tamarixia radiata (Waterston) is a predominant parasitoid of the Asian citrus psyllid (ACP), a destructive citrus pest and vector of huanglongbing (HLB) disease in the fields of southern China. To explore the functioning of target genes in T. radiata, the screening of specific reference genes is critical for carrying out the reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) under different experimental conditions. However, no reference gene(s) for T. radiata has yet been reported. Here, we selected seven housekeeping genes of T. radiata and evaluated their stability under the six conditions (developmental stage, sex, tissue, population, temperature, diet) by using RefFinder software, which contains four different programs (geNorm, ΔCt, BestKeeper, and NormFinder). Pairwise variation was analyzed by geNorm software to determine the optimal number of reference genes during the RT-qPCR analysis. The results reveal better reference genes for differing research foci: 18S and EF1A for the developmental stage; PRS18 and EF1A for sex, PRS18 and RPL13 for different tissues (head, thorax, abdomen); EF1A and ArgK between two populations; β-tubulin and EF1A for different temperatures (5, 15, 25, 35 °C); and ArgK and PRS18 for different feeding diets. Furthermore, when the two optimal and two most inappropriate reference genes were chosen in different temperatures and tissue treatments, respectively, the corresponding expression patterns of HSP70 (as the reporter gene) differed substantially. Our study provides, for the first time, a more comprehensive list of optimal reference genes from T. radiata for use in RT-qPCR analysis, which should prove beneficial for subsequent functional investigations of target gene(s) in this natural enemy of ACP. [ABSTRACT FROM AUTHOR]

Recent studies have shown that myocardial ischemia/reperfusion (I/R)-induced necrosis can be controlled by multiple genes. In this study, we observed that both strands (5p and 3p) of miR- 223 were remarkably dysregulated in mouse hearts upon I/R. Precursor miR-223 (pre-miR-223) transgenic mouse hearts exhibited better recovery of contractile performance over reperfusion period and lesser degree of myocardial necrosis than wild type hearts upon ex vivo and in vivo myocardial ischemia. Conversely, pre-miR-223 knock-out (KO) mouse hearts displayed opposite effects. Furthermore, we found that the RIP1/ RIP3/MLKL necroptotic pathway and inflammatory response were suppressed in transgenic hearts, whereas they were activated in pre-miR-223 KO hearts upon I/R compared with wild type controls. Accordingly, treatment of pre-miR-223 KO mice with necrostatin-1s, a potent necroptosis inhibitor, significantly decreased I/R-triggered cardiac necroptosis, infarction size, and dysfunction. Mechanistically, we identified two critical cell death receptors, TNFR1 and DR6, as direct targets of miR-223- 5p, whereas miR-223-3p directly suppressed the expression of NLRP3 and IκB kinaseα, two important mediators known to be involved in I/R-induced inflammation and cell necroptosis. Our findings indicate that miR-223-5p/-3p duplex works together and cooperatively inhibits I/R-induced cardiac necroptosis at multiple layers. Thus, pre-miR-223 may constitute a new therapeutic agent for the treatment of ischemic heart disease. [ABSTRACT FROM AUTHOR]

Abstract: Stringent negative regulation of the transcription factor NF-κB is essential for maintaining cellular stress responses and homeostasis. However, the tight regulation mechanisms of IKKβ are still not clear. Here, we reported that nemo-like kinase (NLK) is a suppressor of tumor necrosis factor (TNFα)-induced NF-κB signaling by inhibiting the phosphorylation of IKKβ. Overexpression of NLK largely blocked TNFα-induced NF-κB activation, p65 nuclear localization and IκBα degradation; whereas genetic inactivation of NLK showed opposing results. Mechanistically, we identified that NLK interacted with IκB kinase (IKK)-associated complex, which in turn inhibited the assembly of the TAK1/IKKβ and thereby, diminished the IκB kinase phosphorylation. Our results indicate that NLK functions as a pivotal negative regulator in TNFα-induced activation of NF-κB via disrupting the interaction of TAK1 with IKKβ. [Copyright &y& Elsevier]