Antarctic krill (Euphausia superba) is a key component of the Southern Ocean food web. It supports a large number of upper trophic-level predators, and is also a major fishery resource. Understanding changes in krill abundance has long been a priority for research and conservation in the Southern Ocean. In this study, we performed stable isotope analyses on ancient Adélie penguin tissues and inferred relative krill abundance during the Holocene epoch from paleodiets of Adélie penguin (Pygoscelis adeliae), using inverse of δ15N (ratio of 15N/14N) value as a proxy. We find that variations in krill abundance during the Holocene are in accord with episodes of regional climate changes, showing greater krill abundance in cold periods. Moreover, the low δ15N values found in modern Adélie penguins indicate relatively high krill availability, which supports the hypothesis of krill surplus in modern ages due to recent hunt for krill-eating seals and whales by humans.
We investigated the locations of 5S and 45S rDNA in Gossypium diploid A, B, D, E, F, G genomes and tetraploid genome (AD) using multi-probe fluorescent in situ hybridization (FISH) for evolution analysis in Gossypium genus. The rDNA numbers and sizes, and synteny relationships between 5S and 45S were revealed using 5S and 45S as double-probe for all species, and the rDNA-bearing chromosomes were identified for A, D and AD genomes with one more probe that is single-chromosome-specific BAC clone from G. hirsutum (A1D1). Two to four 45S and one 5S loci were found in diploid-species except two 5S loci in G. incanum (E4), the same as that in tetraploid species. The 45S on the 7th and 9th chromosomes and the 5S on the 9th chromosomes seemed to be conserved in A, D and AD genomes. In the species of B, E, F and G genomes, the rDNA numbers, sizes, and synteny relationships were first reported in this paper. The rDNA pattern agrees with previously reported phylogenetic history with some disagreements. Combined with the whole-genome sequencing data from G. raimondii (D5) and the conserved cotton karyotype, it is suggested that the expansion, decrease and transposition of rDNA other than chromosome rearrangements might occur during the Gossypium evolution.
Background and Hypothesis
Klotho is a recently discovered anti-aging gene. The purpose of this study was to investigate if klotho gene transfer attenuates superoxide production and oxidative stress in rat aorta smooth muscle (RASM) cells.
Methods and Results
RASM cells were transfected with AAV plasmids carrying mouse klotho full-length cDNA (mKL) or LacZ as a control. Klotho gene transfer increased klotho expression in RASM cells. Notably, klotho gene expression decreased Nox2 NADPH oxidase protein expression but did not affect Nox2 mRNA expression, suggesting that the inhibition may occur at the post-transcriptional level. Klotho gene transfer decreased intracellular superoxide production and oxidative stress in RASM cells. Klotho gene expression also significantly attenuated the angiotensin II (AngII)-induced superoxide production, oxidative damage, and apoptosis. Interestingly, klotho gene delivery dose-dependently increased the intracellular cAMP level and PKA activity in RASM cells. Rp-cAMP, a competitive inhibitor of cAMP, abolished the klotho-induced increase in PKA activity, indicating that klotho activated PKA via cAMP. Notably, inhibition of cAMP-dependent PKA activity by RP-cAMP abolished klotho-induced inhibition of Nox2 protein expression, suggesting an important role of the cAMP-dependent PKA in this process.
The present finding revealed a previously unidentified role of klotho in regulating Nox2 protein expression in RASM cells. Klotho not only downregulated Nox2 protein expression and intracellular superoxide production but also attenuated AngII-induced superoxide production, oxidative damage, and apoptosis. The klotho-induced suppression of Nox2 protein expression may be mediated by the cAMP PKA pathway.
klotho; Nox2; NADPH oxidase; superoxide; cAMP; PKA; smooth muscle cell
KCNE1 associates with KCNQ1 to increase its current amplitude and slow the activation gating process, creating the slow delayed rectifier channel that functions as a “repolarization reserve” in human heart. The transmembrane domain (TMD) of KCNE1 plays a key role in modulating KCNQ1 pore conductance and gating kinetics, and the extracellular juxtamembrane (EJM) region plays a modulatory role by interacting with the extracellular surface of KCNQ1. KCNE2 is also expressed in human heart and can associate with KCNQ1 to suppress its current amplitude and slow the deactivation gating process. KCNE1 and KCNE2 share the transmembrane topology and a high degree of sequence homology in TMD and surrounding regions. The structural basis for their distinctly different effects on KCNQ1 is not clear. To address this question, we apply cysteine (Cys) scanning mutagenesis to TMDs and EJMs of KCNE1 and KCNE2. We analyze the patterns of functional perturbation to identify high impact positions, and probe disulfide formation between engineered Cys side chains on KCNE subunits and native Cys on KCNQ1. We also use methanethiosulfonate reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Our data suggest that the TMDs of both KCNE subunits are at about the same location but interact differently with KCNQ1. In particular, the much closer contact of KCNE2 TMD with KCNQ1, relative to that of KCNE1, is expected to impact the allosteric modulation of KCNQ1 pore conductance and may explain their differential effects on the KCNQ1 current amplitude. KCNE1 and KCNE2 also differ in the relationship between their EJMs and KCNQ1. Although the EJM of KCNE1 makes intimate contacts with KCNQ1, there appears to be a crevice between KCNQ1 and KCNE2. This putative crevice may perturb the electrical field around the voltage-sensing domain of KCNQ1, contributing to the differential effects of KCNE2 versus KCNE1 on KCNQ1 gating kinetics.
Identification of natural and anthropogenic events in the past is important for studying their patterns and mechanisms; and sensitive proxies in marine sediments are more reliable for identifying these events than those in terrestrial sediments, which are usually disturbed by human activities. Since the main source materials for the sediments in the Northern Yellow Sea Mud are transported by the Yellow River, sedimentary characteristics can be used to reconstruct the historical events that occurred in the Yellow River Valley. In the present study, by analyzing sorting coefficient of grain size in a 250-year sediment core from the Northern Yellow Sea Mud, we identified several major historical events: the Haiyuan Earthquake in AD 1920 and several times of relocation of the Yellow River estuary. The proxy has the potential of detecting and reconstructing historical events; in combination with historical archives, they also provide an accurate dating method.
The purpose of this study was to determine changes in klotho, endothelin (ET) receptors, and superoxide production in kidneys of aged rats and whether these changes are exacerbated in aged rats with cognitive impairment. Twenty aged rats (male, 27 months) were divided into an Old Impaired group (n = 9) and an Old Intact group (n = 11) according to a cognitive function test. A group of 12-month-old rats (n = 10) was used as a Young Intact group. Serum creatinine was increased significantly in the Old Impaired group, suggesting impaired renal function. Aged rats showed glomerulosclerosis and tubulointerstitialfibrosis. These pathological changes were markedly aggravated in the old cognitively impaired than in the old cognitively intact animals. Notably, aged rats demonstrated a significant decrease in klotho protein expression in renal cortex and medulla. Protein expression of IL-6, Nox2, ETa receptors and superoxide production were increased whereas mitochondrial SOD (MnSOD) and ETb receptors expression were decreased in kidneys of the aged rats. Interestingly, these changes were more pronounced in the old impaired than in the old intact rats. In conclusion, the aging-related kidney damage was exacerbated in aged rats with cognitive impairment. Klotho, ETB, and MnSOD were downregulated but ETa, IL-6, Nox2, and superoxide production were upregulated in the aging-related kidney damage. These changes were more pronounced in rats with cognitive impairment.
Aging; Klotho; Glomerusclerosis; ET receptor; Superoxide; Interleukin-6
We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3 and Cy5 labeled tRNAs. Pre-translocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G·GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the post-translocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA·EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.
Small-molecule compounds approved for use as drugs may be “repurposed” for new indications and studied to determine the mechanisms of their beneficial and adverse effects. A comprehensive collection of all small-molecule drugs approved for human use would be invaluable for systematic repurposing across human diseases, particularly for rare and neglected diseases, for which the cost and time required for development of a new chemical entity are often prohibitive. Previous efforts to build such a comprehensive collection have been limited by the complexities, redundancies, and semantic inconsistencies of drug naming within and among regulatory agencies worldwide; a lack of clear conceptualization of what constitutes a drug; and a lack of access to physical samples. We report here the creation of a definitive, complete, and nonredundant list of all approved molecular entities as a freely available electronic resource and a physical collection of small molecules amenable to high-throughput screening.
Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established.
Antarctic krill Euphausia superba is a predominant species in the Southern Ocean, it is very sensitive to climate change, and it supports large stocks of fishes, seabirds, seals and whales in Antarctic marine ecosystems. Modern krill stocks have been estimated directly by net hauls and acoustic surveys; the historical krill density especially the long-term one in the Southern Ocean, however, is unknown. Here we inferred the relative krill population changes along the West Antarctic Peninsula (WAP) over the 20th century from the trophic level change of Antarctic fur seal Arctocephalus gazella using stable carbon (δ13C) and nitrogen (δ15N) isotopes of archival seal hairs. Since Antarctic fur seals feed preferentially on krill, the variation of δ15N in seal hair indicates a change in the proportion of krill in the seal's diets and thus the krill availability in local seawater. For the past century, enriching fur seal δ15N values indicated decreasing krill availability. This is agreement with direct observation for the past ∼30 years and suggests that the recently documented decline in krill populations began in the early parts of the 20th century. This novel method makes it possible to infer past krill population changes from ancient tissues of krill predators.
We describe a novel algorithm, Grid algorithm, and the corresponding computer program for high throughput fitting of dose-response curves that are described by the four-parameter symmetric logistic dose-response model. The Grid algorithm searches through all points in a grid of four dimensions (parameters) and finds the optimum one that corresponds to the best fit. Using simulated dose-response curves, we examined the Grid program’s performance in reproducing the actual values that were used to generate the simulated data and compared it with the DRC package for the language and environment R and the XLfit add-in for Microsoft Excel. The Grid program was robust and consistently recovered the actual values for both complete and partial curves with or without noise. Both DRC and XLfit performed well on data without noise, but they were sensitive to and their performance degraded rapidly with increasing noise. The Grid program is automated and scalable to millions of dose-response curves, and it is able to process 100,000 dose-response curves from high throughput screening experiment per CPU hour. The Grid program has the potential of greatly increasing the productivity of large-scale dose-response data analysis and early drug discovery processes, and it is also applicable to many other curve fitting problems in chemical, biological, and medical sciences.
Curve fitting; Hill equation; Grid algorithm; Dose-response curve; High throughput screening.
Klotho is a recently discovered antiaging gene. The objective of this study was to test the hypothesis that klotho gene delivery attenuates the progression of spontaneous hypertension and renal damage in spontaneous hypertensive rats (SHRs). An adeno-associated virus (AAV) carrying mouse klotho full-length cDNA (AAV.mKL) was constructed for in vivo expression of klotho. Four groups of male SHRs and 1 group of sex- and age-matched Wistar-Kyoto rats (5 rats per group) were used. Blood pressure was measured twice in all of the animals before gene delivery. Four groups of SHRs received an IV injection of AAV.mKL, AAV.LacZ, AAV.GFP, and PBS, respectively. The Wistar-Kyoto group received PBS and served as a control. AAV.mKL stopped the further increase in blood pressure in SHRs, whereas blood pressures continued to increase in other SHR groups. One single dose of AAV.mKL prevented the progression of spontaneous hypertension for at least 12 weeks (length of the study). Klotho expression and production were suppressed in SHRs, which were reverted by AAV.mKL. AAV.mKL increased plasma interleukin 10 levels but decreased Nox2 expression, NADPH oxidase activity, and superoxide production in kidneys and aortas in SHRs. AAV.mKL abolished renal tubular atrophy and dilation, tubular deposition of proteinaceous material, glomerular collapse, and collagen deposition seen in SHRs, indicating that klotho gene delivery attenuated renal damage. Therefore, the suppressed klotho expression may play a role in the progression of spontaneous hypertension and renal damage in SHRs. AAV delivery of klotho may offer a new approach for the long-term control of hypertension and for renoprotection.
klotho; Nox2; blood pressure; adeno-associated virus; renoprotection
Clinical trials and epidemiological studies have suggested that dietary fish oil (FO) supplementation can provide an anti-arrhythmic benefit in some patient populations. The underlying mechanisms are not entirely clear. We wanted to understand how FO supplementation (for 4 weeks) affected the action potential configuration/duration of ventricular myocytes, and the ionic mechanism(s)/molecular basis for these effects. The experiments were conducted on adult rabbits, a widely used animal model for cardiac electrophysiology and pathophysiology. We used gas chromatography - mass spectroscopy to confirm that FO feeding produced a marked increase in the content of n-3 polyunsaturated fatty acids in the phospholipids of rabbit hearts. Left ventricular myocytes were used in current and voltage clamp experiments to monitor action potentials and ionic currents, respectively. Action potentials of myocytes from FO-fed rabbits exhibited much more positive plateau voltages and prolonged durations. These changes could be explained by an increase in the L-type Ca current (ICaL) and a decrease in the transient outward current (Ito) in these myocytes. FO feeding did not change the delayed rectifier or inward rectifier current. Immunoblot experiments showed that the FO-feeding induced changes in ICaL and Ito were associated with corresponding changes in the protein levels of major pore-forming subunits of these channels: increase in Cav1.2 and decrease in Kv4.2 and Kv1.4. There was no change in other channel subunits (Cav1.1, Kv4.3, KChIP2, and ERG1). We conclude that long-term fish oil supplementation can impact on cardiac electrical activity at least partially by changing channel subunit expression in cardiac myocytes.
Klotho is a new anti-aging gene. Genetic mutation of klotho causes multiple premature aging-like phenotypes and strikingly shortens lifespan. Overexpression of the klotho gene in mice suppresses aging and extends lifespan which may involve the mechanism of suppression of insulin signaling and oxidant stress. Klotho functions as a cofactor/coreceptor regulating fibroblast growth factor (FGF) 23 signaling. Klotho acts as a glucuronidase and activates ion channel TRPV5. Klotho protects against endothelial dysfunction and regulates the production of nitric oxide. Klotho also influences intracellular signaling pathways including p53/p21, cAMP, protein kinase C (PKC) and Wnt signaling pathways. The discovery of klotho has a great impact on aging research. The purpose of this review is to provide the recent progress and future directions of klotho research. Specifically, this review will cover: klotho and aging, structure and expression of the klotho gene, localization of klotho expression, source of circulating klotho, current understanding of klotho functions, and signaling pathways of klotho.
klotho; aging; insulin signaling; fibroblast growth factor; glucuronidase; vitamin D
Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS) experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 µL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC50s ranging from micromolar to the assay response limit (∼25 nM). This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump-start development of novel therapeutics for other neglected tropical diseases.
Schistosomiasis, also known as bilharzia, is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system, thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx2), have been recently identified and validated as targets for anti-schistosomiasis drug development. In search of inhibitors of this critical redox cascade, we optimized and performed a highly miniaturized automated screen of 71,028 compounds arrayed as 7- to 15-point dilution sets. We identified novel structural series of TGR inhibitors, several of which are highly potent and should serve both as mechanistic tools for probing redox pathways in S. mansoni and as starting points for developing much-needed new treatments for schistosomiasis. The paradigm presented here effectively bridges the gap between academic target identification and the first steps of drug development, and should be applicable to a variety of other important neglected diseases.
The relative importance of north–south migrations of the intertropical convergence zone (ITCZ) versus El Niño-Southern Oscillation and its associated Pacific Walker Circulation (PWC) variability for past hydrological change in the western tropical Pacific is unclear. Here we show that north–south ITCZ migration was not the only mechanism of tropical Pacific hydrologic variability during the last millennium, and that PWC variability profoundly influenced tropical Pacific hydrology. We present hydrological reconstructions from Cattle Pond, Dongdao Island of the South China Sea, where multi-decadal rainfall and downcore grain size variations are correlated to the Southern Oscillation Index during the instrumental era. Our downcore grain size reconstructions indicate that this site received less precipitation during relatively warm periods, AD 1000–1400 and AD 1850–2000, compared with the cool period (AD 1400–1850). Including our new reconstructions in a synthesis of tropical Pacific records results in a spatial pattern of hydrologic variability that implicates the PWC.
Tropical Pacific hydrology affects the global climate through the strength of the Pacific Walker Circulation. Yan et al. reconstruct variations in the Pacific Walker Circulation in the South China Sea over the last millennium and find that less precipitation fell during warmer and more rainfall during cool periods.
Gossypium mustelinum ((AD)4) is one of five disomic species in Gossypium. Three 45S ribosomal DNA (rDNA) loci were detected in (AD)4 with 45S rDNA as probe, and three pairs of brighter signals were detected with genomic DNA (gDNA) of Gossypium D genome species as probes. The size and the location of these brighter signals were the same as those detected with 45S rDNA as probe, and were named GISH-NOR. One of them was super-major, which accounted for the fact that about one-half of its chromosome at metaphase was located at chromosome 3, and other two were minor and located at chromosomes 5 and 9, respectively. All GISH-NORs were located in A sub-genome chromosomes, separate from the other four allopolyploid cotton species. GISH-NOR were detected with D genome species as probe, but not A. The greatly abnormal sizes and sites of (AD)4 NORs or GISH-NORs indicate a possible mechanism for 45S rDNA diversification following (AD)4 speciation. Comparisons of GISH intensities and GISH-NOR production with gDNA probes between A and D genomes show that the better relationship of (AD)4 is with A genome. The shortest two chromosomes of A sub-genome of G. mustelinum were shorter than the longest chromosome of D sub-genome chromosomes. Therefore, the longest 13 chromosomes of tetraploid cotton being classified as A sub-genome, while the shorter 13 chromosomes being classified as D sub-genome in traditional cytogenetic and karyotype analyses may not be entirely correct.
Wu Q, Liu F, Li S, Song G, Wang C, Zhang X, Wang Y, Stelly D, Wang K (2013) Uniqueness of the Gossypium mustelinum genome revealed by GISH and 45S rDNA FISH. J. Integr. Plant Biol. 55(7), 654–662.
Chromosomes; cotton; Gossypium mustelinum; in situ hybridization; nucleolar organizer region; genome