Satellite DNAs are among the most abundant repetitive DNAs found in eukaryote genomes, where they participate in a variety of biological roles, from being components of important chromosome ...structures to gene regulation. Experimental methodologies used before the genomic era were insufficient, too laborious and time-consuming to recover the collection of all satDNAs from a genome. Today, the availability of whole sequenced genomes combined with the development of specific bioinformatic tools are expected to foster the identification of virtually all the "satellitome" of a particular species. While whole genome assemblies are important to obtain a global view of genome organization, most of them are incomplete and lack repetitive regions. We applied short-read sequencing and similarity clustering in order to perform a de novo identification of the most abundant satellite families in two Drosophila species from the virilis group: Drosophila virilis and D. americana, using the Tandem Repeat Analyzer (TAREAN) and RepeatExplorer pipelines. These species were chosen because they have been used as models to understand satDNA biology since the early 70's. We combined the computational approach with data from the literature and chromosome mapping to obtain an overview of the major tandem repeat sequences of these species. The fact that all of the abundant tandem repeats (TRs) we detected were previously identified in the literature allowed us to evaluate the efficiency of TAREAN in correctly identifying true satDNAs. Our results indicate that raw sequencing reads can be efficiently used to detect satDNAs, but that abundant tandem repeats present in dispersed arrays or associated with transposable elements are frequent false positives. We demonstrate that TAREAN with its parent method RepeatExplorer may be used as resources to detect tandem repeats associated with transposable elements and also to reveal families of dispersed tandem repeats.
Diseases that occur during the transition period are exacerbated when cows are unable to cope with an increased pro-oxidant load that results in oxidative stress. Dairy cattle are routinely ...supplemented with the vitamin E analog α-tocopherol to mitigate the severity of oxidative stress. Nonetheless, oxidative stress remains a disease predisposing condition for many dairy cattle. A better method of optimizing the antioxidant functions of vitamin E is needed. α-Tocopherol is only 1 of 8 analogs of vitamin E, all of which have varying antioxidant properties in other mammals, albeit a shorter physiological half-life compared with α-tocopherol. A primary bovine mammary endothelial cell oxidant challenge model was used to determine functions of certain vitamin E analogs. The aim of this study was to determine if other analogs, namely γ-tocopherol or γ-tocotrienol, have antioxidative functions in bovine cells and if these functions may protect cellular viability and endothelial function from oxidant damage. Physiological (10 μM) and supraphysiological (50 μM) concentrations of γ-tocopherol and γ-tocotrienol had a greater capacity to reduce accumulated reactive oxygen species derived from a nitric oxide donating pro-oxidant antagonist, when compared with α-tocopherol, after 30 min to 6 h of treatment. Further, γ-tocotrienol (10 μM) decreased cell cytotoxicity to a greater amount than other analogs at like concentrations, whereas γ-tocopherol (10 μM) reduced lipid peroxidation and apoptosis more effectively than other analogs. Last, α-tocopherol (5 and 10 μM) and γ-tocopherol (5 and 10 μM) significantly slowed pro-oxidant induced loss of endothelial cell barrier integrity over a 48-h period using an electrical cell-substrate impedance sensing system. Concerningly, γ-tocotrienol drastically reduced the endothelial barrier integrity at only 5 μM despite no apparent effect on cellular viability at like concentrations. γ-Tocotrienol, however, was also the only analog to show significant cytotoxicity and reductions in viability at supraphysiological doses (25 and 50 μM). Our results suggest that γ-tocopherol has antioxidant activities that reduces cellular damage and loss of function due to oxidant challenge as effectively as α-tocopherol. These data set the foundation for further investigation into the antioxidant properties of vitamin E analogs in other bovine cells types or whole animal models.
Posttranslational modification of a protein, either alone or in combination with other modifications, can control properties of that protein, such as enzymatic activity, localization, stability, or ...interactions with other molecules.
-ε-Lysine acetylation is one such modification that has gained attention in recent years, with a prevalence and significance that rival those of phosphorylation. This review will discuss the current state of the field in bacteria and some of the work in archaea, focusing on both mechanisms of
-ε-lysine acetylation and methods to identify, quantify, and characterize specific acetyllysines. Bacterial
-ε-lysine acetylation depends on both enzymatic and nonenzymatic mechanisms of acetylation, and recent work has shed light into the regulation of both mechanisms. Technological advances in mass spectrometry have allowed researchers to gain insight with greater biological context by both (i) analyzing samples either with stable isotope labeling workflows or using label-free protocols and (ii) determining the true extent of acetylation on a protein population through stoichiometry measurements. Identification of acetylated lysines through these methods has led to studies that probe the biological significance of acetylation. General and diverse approaches used to determine the effect of acetylation on a specific lysine will be covered.
Map-based cloning of avirulence genes of the AvrLm1-2-6 cluster was recently undertaken in Leptosphaeria maculans and led to the identification of AvrLm1. The ensuing chromosome walk toward AvrLm6 ...resulted in the delineation of a 562-kb bacterial artificial chromosome (BAC) clone contig in an avirulent isolate. Following sequencing of the contig and sequence comparison with a virulent isolate, four AvrLm6 candidate genes were identified. Complementation of the virulent isolate with the four candidates was performed and one gene was found to fully restore the avirulent phenotype on Rlm6 oilseed rape genotypes. AvrLm6 was found to be located in the same genome context as AvrLm1, because it is a solo gene surrounded by 85 and 48 kb of degenerated repeats on its 5(prime) and 3(prime) sides, respectively. AvrLm6 is an orphan gene encoding a small, potentially secreted, cysteine-rich protein. Comparison of AvrLm1 and AvrLm6 expressions by quantitative reverse-transcription polymerase chain reaction revealed that both genes are highly overexpressed during primary leaf infection. Using RNA interference, decreasing expression of AvrLm6 was shown to result in virulence toward Rlm6 genotypes whenever the expression was reduced by more than 60% compared with the wild-type isolate.
Femtosecond x-ray laser pulses are used to probe the carbon monoxide (CO) oxidation reaction on ruthenium (Ru) initiated by an optical laser pulse. On a time scale of a few hundred femtoseconds, the ...optical laser pulse excites motions of CO and oxygen (O) on the surface, allowing the reactants to collide, and, with a transient close to a picosecond (ps), new electronic states appear in the O K-edge x-ray absorption spectrum. Density functional theory calculations indicate that these result from changes in the adsorption site and bond formation between CO and O with a distribution of OC–O bond lengths close to the transition state (TS). After 1 ps, 10% of the CO populate the TS region, which is consistent with predictions based on a quantum oscillator model.
During Martian solar days 57–100, the Mars Science Laboratory Curiosity rover acquired and processed a solid (sediment) sample and analyzed its mineralogy and geochemistry with the Chemistry and ...Mineralogy and Sample Analysis at Mars instruments. An aeolian deposit—herein referred to as the Rocknest sand shadow—was inferred to represent a global average soil composition and selected for study to facilitate integration of analytical results with observations from earlier missions. During first‐time activities, the Mars Hand Lens Imager (MAHLI) was used to support both science and engineering activities related to sample assessment, collection, and delivery. Here we report on MAHLI activities that directly supported sample analysis and provide MAHLI observations regarding the grain‐scale characteristics of the Rocknest sand shadow. MAHLI imaging confirms that the Rocknest sand shadow is one of a family of bimodal aeolian accumulations on Mars—similar to the coarse‐grained ripples interrogated by the Mars Exploration Rovers Spirit and Opportunity—in which a surface veneer of coarse‐grained sediment stabilizes predominantly fine‐grained sediment of the deposit interior. The similarity in grain size distribution of these geographically disparate deposits support the widespread occurrence of bimodal aeolian transport on Mars. We suggest that preservation of bimodal aeolian deposits may be characteristic of regions of active deflation, where winnowing of the fine‐sediment fraction results in a relatively low sediment load and a preferential increase in the coarse‐grained fraction of the sediment load. The compositional similarity of Martian aeolian deposits supports the potential for global redistribution of fine‐grained components, combined with potential local contributions.
Key Points
Curiosity acquired and examined its first solid sample at the Rocknest sand shadow
MAHLI images were critical in the science investigation of Rocknest materials
MAHLI images played a critical role supporting first‐time engineering activities
Dairy cattle are at the greatest risk of developing diseases around the time of calving because of compromised immune responses and the occurrence of oxidative stress. Both the development of ...compromised immunity and oxidative stress are influenced directly or indirectly by the metabolism of polyunsaturated fatty acids (PUFA) and fat-soluble vitamins. The cytochrome P450 (CYP450) family of enzymes is central to the metabolism of both classes of these compounds, but to date, the importance of CYP450 in the health of dairy cattle is underappreciated. As certain CYP450 isoforms metabolize both PUFA and fat-soluble vitamins, potential interactions may occur between PUFA and fat-soluble vitamins that are largely unexplored. For example, one CYP450 that generates anti-inflammatory oxylipids from arachidonic acid additionally contributes to the activation of vitamin D. Other potential substrate interactions between PUFA and vitamins A and E may exist as well. The intersection of PUFA and fat-soluble vitamin metabolism by CYP450 suggest that this enzyme system could provide an understanding of how immune function and oxidant status interconnect, resulting in increased postpartum disease occurrence. This review will detail the known contributions of bovine CYP450 to the regulation of oxylipids with a focus on enzymes that may also be involved in the metabolism of fat-soluble vitamins A, D, and E that contribute to antioxidant defenses. Although the activity of specific CYP450 is generally conserved among mammals, important differences exist in cattle, such as the isoforms primarily responsible for activation of vitamin D that makes their specific study in cattle of great importance. Additionally, a CYP450-driven inflammatory positive feedback loop is proposed, which may contribute to the dysfunctional inflammatory responses commonly found during the transition period. Establishing the individual enzyme isoform contributions to oxylipid biosynthesis and the regulation of vitamins A, D, and E may reveal how the CYP450 family of enzymes can affect inflammatory responses during times of increased susceptibility to disease. Determining the potential effect of each CYP450 on disease susceptibility or pathogenesis may allow for the targeted manipulation of the CYP450 pathways to influence specific immune responses and antioxidant defenses during times of increased risk for health disorders.
The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental ...models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti‐CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti‐human CD154 domain antibody (dAb, BMS‐986004). The anti‐CD154 dAb effectively blocked CD40‐CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti‐CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti‐CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti‐CD154 dAb treatment increased the frequency of CD4+CD25+Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti‐CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti‐CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.
An Fc‐silent anti‐CD154 domain antibody safely and effectively promotes long‐term kidney transplant survival in nonhuman primates without the risk of thromboembolism when combined with a clinically relevant conventional immunosuppression regimen. See the editorial from Thomson and Ezzelarab on page 1156.
Satellite DNA (satDNA) is a class of tandemly repeated non-protein coding DNA sequences which can be found in abundance in eukaryotic genomes. They can be functional, impact the genomic architecture ...in many ways, and their rapid evolution has consequences for species diversification. We took advantage of the recent availability of sequenced genomes from 23
species from the
group to study their satDNA landscape. For this purpose, we used publicly available whole-genome sequencing Illumina reads and the TAREAN (tandem repeat analyzer) pipeline. We provide the characterization of 101 non-homologous satDNA families in this group, 93 of which are described here for the first time. Their repeat units vary in size from 4 bp to 1897 bp, but most satDNAs show repeat units < 100 bp long and, among them, repeats ≤ 10 bp are the most frequent ones. The genomic contribution of the satDNAs ranges from ~1.4% to 21.6%. There is no significant correlation between satDNA content and genome sizes in the 23 species. We also found that at least one satDNA originated from an expansion of the central tandem repeats (CTRs) present inside a Helitron transposon. Finally, some satDNAs may be useful as taxonomic markers for the identification of species or subgroups within the group.
Nε-lysine acetylation was recently discovered on many bacterial proteins that function in diverse cellular processes. Thus, many questions remain unanswered. For example, what mechanisms regulate ...lysine acetylation? Does acetylation affect physiology? To help answer these questions, we studied the Escherichia coli response regulator and transcription factor RcsB, which is reported to be acetylated in vitro. To characterize RcsB acetylation, we monitored transcription from the rprA promoter, which requires RcsB. The conventional view is that RcsB is activated by phosphorylation through either the Rcs phosphorelay or acetyl phosphate. We affirmed that rprA transcription requires phosphorylated RcsB and showed that acetyl-phosphate (AcP) is a phosphoryl group donor to RcsB. However, a mutant that accumulates AcP (ackA) exhibited a reduction in rprA transcription instead of the predicted increase. rprA transcription also diminished in the cobB mutant, which lacks the only known E. coli protein deacetylase. This suggests the existence of an inhibitory mechanism that involves lysine acetylation, a supposition supported by the observation that RcsB isolated from the ackA or cobB mutant was hyperacetylated. Finally, we used a genetic approach to identify an AckA- and CobB-sensitive lysine (Lys-154) that controls RcsB activity. We propose that acetylation inhibits RcsB activity and that some of this inhibition acts through the acetylation of Lys-154.
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