Analysis of Polycerate Mutants Reveals the Evolutionary Co-option of HOXD1 for Horn Patterning in Bovidae.

In the course of evolution, pecorans (i.e., higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as "headgear," which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.

Analytical Validation of a Highly Sensitive, Multiplexed Chronic Myeloid Leukemia Monitoring System Targeting BCR-ABL1 RNA.

This study describes the analytical performance of the QuantideX qPCR BCR-ABL IS Kit, the first Food and Drug Administration-cleared assay designed to monitor breakpoint cluster region-Abelson tyrosine-protein kinase 1 (BCR-ABL1) fusion transcripts isolated from peripheral blood specimens from patients with chronic myeloid leukemia. This multiplex real-time quantitative RT-PCR assay amplifies both e13a2 and e14a2 Major BCR-ABL1 transcripts and the reference target ABL1. The test results are provided in international scale (IS) values by incorporating armored RNA-based calibrators that have defined IS values tied directly to the World Health Organization BCR-ABL1 Primary Reference Materials, without the necessity of determining and maintaining conversion factors. For each batch run, the integrated interpretive software evaluates run and specimen quality control metrics (including a sufficient amount of ABL1 control transcripts to ensure a minimal limit of detection) and calculates both molecular response (MR) and %IS values for each specimen. The test has a limit of detection of MR4.7 (0.002%IS) and a linear range from MR0.3 (50%IS) to MR4.7 (0.002%IS) for both Major transcripts. Single-site and multisite precision studies demonstrated a maximum SD of 0.13 MR (30% CV within the assay range between MR0.7 and MR3.7). The performance of this BCR-ABL1 monitoring test meets all of the clinical guideline recommendations for sensitivity and IS reporting for the management of chronic myeloid leukemia patients.

Establishment and validation of analytical reference panels for the standardization of quantitative BCR-ABL1 measurements on the international scale.

BACKGROUND: Current guidelines for managing Philadelphia-positive chronic myeloid leukemia include monitoring the expression of the BCR-ABL1 (breakpoint cluster region/c-abl oncogene 1, non-receptor tyrosine kinase) fusion gene by quantitative reverse-transcription PCR (RT-qPCR). Our goal was to establish and validate reference panels to mitigate the interlaboratory imprecision of quantitative BCR-ABL1 measurements and to facilitate global standardization on the international scale (IS). METHODS: Four-level secondary reference panels were manufactured under controlled and validated processes with synthetic Armored RNA Quant molecules (Asuragen) calibrated to reference standards from the WHO and the NIST. Performance was evaluated in IS reference laboratories and with non-IS-standardized RT-qPCR methods. RESULTS: For most methods, percent ratios for BCR-ABL1 e13a2 and e14a2 relative to ABL1 or BCR were robust at 4 different levels and linear over 3 logarithms, from 10% to 0.01% on the IS. The intraassay and interassay imprecision was <2-fold overall. Performance was stable across 3 consecutive lots, in multiple laboratories, and over a period of 18 months to date. International field trials demonstrated the commutability of the reagents and their accurate alignment to the IS within the intra- and interlaboratory imprecision of IS-standardized methods. CONCLUSIONS: The synthetic calibrator panels are robust, reproducibly manufactured, analytically calibrated to the WHO primary standards, and compatible with most BCR-ABL1 RT-qPCR assay designs. The broad availability of secondary reference reagents will further facilitate interlaboratory comparative studies and independent quality assessment programs, which are of paramount importance for worldwide standardization of BCR-ABL1 monitoring results and the optimization of current and new therapeutic approaches for chronic myeloid leukemia.

Marine biopolymer self-assembly: implications for carbon cycling in the ocean.

Dissolved organic matter is the largest reservoir of reduced carbon in the ocean and is primarily composed of small biopolymers. It is a critical substrate for the microbial community and plays a pivotal role in global carbon cycling.

Novel interaction of the 60S ribosomal subunit export adapter Nmd3 at the nuclear pore complex.

Nuclear export of the large (60S) ribosomal subunit depends on the adapter protein Nmd3 to provide a nuclear export signal (NES). The leucine-rich NES is recognized by the export receptor Crm1 to mediate export via interaction with the nuclear pore complex (NPC). Here, we show that certain mutant Nmd3 proteins that are impaired for binding to the 60S subunit accumulate at the nuclear envelope. These mutant proteins also show enhanced binding to Crm1, both in vivo and in vitro. Although their interaction with the NPC is dependent on recognition of the NES by Crm1, their interaction with Crm1 is not strictly dependent on RanGTP. Using a collection of GFP-tagged nucleoporin mutants, we identified several nucleoporins, including components of the Nup82 complex that copurified with the mutant Nmd3. The Nup82 complex is on the cytoplasmic face of the NPC and has previously been shown to be important as a terminal binding site for Crm1-mediated export. Mutations in the Nup82 complex led to accumulation of wild-type Nmd3 in the nucleoplasm, suggesting that the interaction of mutant Nmd3 with the Nup82 complex reflects a defect in the bona fide export pathway for the 60S subunit. These results suggest that in the absence of the ribosome, Nmd3 is not efficiently released from Crm1 at the NPC.

Mapping the functional domains of yeast NMD3, the nuclear export adapter for the 60 S ribosomal subunit.

Nuclear export of the large ribosomal subunit requires the adapter protein Nmd3p to provide a leucine-rich nuclear export signal that is recognized by the export receptor Crm1. Nmd3p binds to the pre-60 S subunit in the nucleus. After export to the cytoplasm, the release of Nmd3p depends on the ribosomal protein Rpl10p and the GTPase Lsg1p. Here, we have carried out a mutational analysis of Nmd3 to better define the domains responsible for nucleocytoplasmic shuttling and ribosome binding. We show that mutations in two regions of Nmd3p affect 60 S binding, suggesting that its binding to the subunit is multivalent.

Young organic matter as a source of carbon dioxide outgassing from Amazonian rivers.

Rivers are generally supersaturated with respect to carbon dioxide, resulting in large gas evasion fluxes that can be a significant component of regional net carbon budgets. Amazonian rivers were recently shown to outgas more than ten times the amount of carbon exported to the ocean in the form of total organic carbon or dissolved inorganic carbon. High carbon dioxide concentrations in rivers originate largely from in situ respiration of organic carbon, but little agreement exists about the sources or turnover times of this carbon. Here we present results of an extensive survey of the carbon isotope composition (13C and 14C) of dissolved inorganic carbon and three size-fractions of organic carbon across the Amazonian river system. We find that respiration of contemporary organic matter (less than five years old) originating on land and near rivers is the dominant source of excess carbon dioxide that drives outgassing in medium to large rivers, although we find that bulk organic carbon fractions transported by these rivers range from tens to thousands of years in age. We therefore suggest that a small, rapidly cycling pool of organic carbon is responsible for the large carbon fluxes from land to water to atmosphere in the humid tropics.

Photodegradation and photosensitization of mycosporine-like amino acids.

The photodegradation and photosensitization of several mycosporine-like amino acids (MAAs) were investigated. The photodegradation of the MAA, palythine, was tested with three photosensitizers: riboflavin, rose bengal and natural seawater. For comparison of degradation rates, the riboflavin-mediated photosensitization of six other MAAs was also examined. When riboflavin was used as a photosensitizer in distilled water, MAAs were undetectable after 1.5h. Palythine showed little photodegradation when rose bengal was added as the photosensitizer (k=0.12x10(-3)m(2)kJ(-1)). Palythine dissolved in natural seawater containing high nitrate concentrations also showed slow photodegradation rate constants (k=0.26x10(-3)m(2)kJ(-1)) over a 24-h period of constant irradiation. Similar experiments in deep seawater with porphyra-334 and shinorine resulted in 75% of the initial MAA remaining after 4h of irradiation and rates of 0.018 and 0.026x10(-3) m(2) kJ(-1), respectively. Experiments conducted in deep seawater with riboflavin additions resulted in photodegradation rate constants between 0.77x10(-3) and 1.19x10(-3)m(2)kJ(-1) for shinorine and porphyra-334, respectively. Photoproduct formation appeared to be minimal with the presence of a dehydration product of the cycloheximine ring structure indicated as well as the presence of amino acids. Evidence continues to build for the role of MAAs as potent and stable UV absorbers. This study further highlights the photostability of several MAAs in both distilled and seawater in the presence of photosensitizers.

Defining the order in which Nmd3p and Rpl10p load onto nascent 60S ribosomal subunits.

The large ribosomal subunit protein Rpl10p is required for subunit joining and 60S export in yeast. We have recently shown that Rpl10p as well as the cytoplasmic GTPase Lsg1p are required for releasing the 60S nuclear export adapter Nmd3p from subunits in the cytoplasm. Here, we more directly address the order of Nmd3p and Rpl10p recruitment to the subunit. We show that Nmd3p can bind subunits in the absence of Rpl10p. In addition, we examined the basis of the previously reported dominant negative growth phenotype caused by overexpression of C-terminally truncated Rpl10p and found that these Rpl10p fragments are not incorporated into subunits in the nucleus but instead sequester the WD-repeat protein Sqt1p. Sqt1p is an Rpl10p binding protein that is proposed to facilitate loading of Rpl10p into the 60S subunit. Although Sqt1p normally only transiently binds 60S subunits, the levels of Sqt1p that can be coimmunoprecipitated by the 60S-associated GTPase Lsg1p are significantly increased by a dominant mutation in the Walker A motif of Lsg1p. This mutant Lsg1 protein also leads to increased levels of Sqt1p in complexes that are coimmunoprecipitated with Nmd3p. Furthermore, the dominant LSG1 mutant also traps a mutant Rpl10 protein that does not normally bind stably to the subunit. These results support the idea that Sqt1p loads Rpl10p onto the Nmd3p-bound subunit after export to the cytoplasm and that Rpl10p loading involves the GTPase Lsg1p.

Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplasmic GTPase Lsg1p.

In eukaryotes, nuclear export of the large (60S) ribosomal subunit requires the adapter protein Nmd3p to provide the nuclear export signal. Here, we show that in yeast release of Nmd3p from 60S subunits in the cytoplasm requires the ribosomal protein Rpl10p and the G-protein, Lsg1p. Mutations in LSG1 or RPL10 blocked Nmd3-GFP shuttling into the nucleus and export of pre-60S subunits from the nucleus. Overexpression of NMD3 alleviated the export defect, indicating that the block in 60S export in lsg1 and rpl10 mutants results indirectly from failing to recycle Nmd3p. The defect in Nmd3p recycling and the block in 60S export in both lsg1 and rpl10 mutants was also suppressed by mutant Nmd3 proteins that showed reduced binding to 60S subunits in vitro. We propose that the correct loading of Rpl10p into 60S subunits is required for the release of Nmd3p from subunits by Lsg1p. These results suggest a coupling between recycling the 60S export adapter and activation of 60S subunits for translation.

Reburial of fossil organic carbon in marine sediments.

Marine sediments act as the ultimate sink for organic carbon, sequestering otherwise rapidly cycling carbon for geologic timescales. Sedimentary organic carbon burial appears to be controlled by oxygen exposure time in situ, and much research has focused on understanding the mechanisms of preservation of organic carbon. In this context, combustion-derived black carbon has received attention as a form of refractory organic carbon that may be preferentially preserved in soils and sediments. However, little is understood about the environmental roles, transport and distribution of black carbon. Here we apply isotopic analyses to graphitic black carbon samples isolated from pre-industrial marine and terrestrial sediments. We find that this material is terrestrially derived and almost entirely depleted of radiocarbon, suggesting that it is graphite weathered from rocks, rather than a combustion product. The widespread presence of fossil graphitic black carbon in sediments has therefore probably led to significant overestimates of burial of combustion-derived black carbon in marine sediments. It could be responsible for biasing radiocarbon dating of sedimentary organic carbon, and also reveals a closed loop in the carbon cycle. Depending on its susceptibility to oxidation, this recycled carbon may be locked away from the biologically mediated carbon cycle for many geologic cycles.

Electrospray ionization tandem mass spectrometric and electron impact mass spectrometric characterization of mycosporine-like amino acids.

Positive-ion mass spectral fragmentations of seven mycosporine-like amino acids (MAAs) are reported and discussed. The MAAs studied are small compounds composed of a cycloheximine ring substituted with amino acid or amino alcohol units. Techniques used include electron impact (EI) and electrospray ionization (ESI) with tandem mass spectrometry (MS/MS). ESI-MS/MS showed unusual small radical losses, generally resulting from the loss of a methyl group with the exception of shinorine and porphyra for which the initial losses were 30 and 44 Da, respectively. As expected from structural similarities, porphyra, shinorine and palythinol displayed similar fragmentation patterns, while palythenic acid and palythene fragmented in a similar manner. Overall, the ESI-MS/MS fragmentations at m/z <200 exhibited a distinctive pattern for all seven MAAs with characteristic ions at m/z 137, 168, 186, and 197 or 199. Several ions were observed for each of the MAAs analyzed, and together provide a useful and potentially diagnostic pattern for identification of MAAs and as an aid in structure elucidation of novel MAAs. For GC/EI-MS analysis, trimethylsilyl (TMS) derivatives were made. The EI-MS fragmentation patterns of TMS-MAAs showed many features typical of TMS-derivatized alpha-amines. The precursor TMS-MAA ion was not detected, but a [M-90](+ radical) ion was the highest-mass intense peak observed for palythine, palythinol and shinorine, while palythene gave a [M-116](+ radical) ion. Besides determining the number of acidic hydrogens, EI-MS of TMS-derivatized MAAs will aid in structure elucidation of novel MAAs.

The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively.

We characterized two essential putative GTPases, Nog1p and Lsg1p, that are found associated with free 60S ribosomal subunits affinity purified with the nuclear export adapter Nmd3p. Nog1p and Lsg1p are nucleolar and cytoplasmic, respectively, and are not simultaneously on the same particle, reflecting the path of Nmd3p shuttling in and out of the nucleus. Conditional mutants of both NOG1 and LSG1 are defective in 60S subunit biogenesis and display diminished levels of 60S subunits at restrictive temperature. Mutants of both genes also accumulate the 60S ribosomal reporter Rpl25-eGFP in the nucleolus, suggesting that both proteins are needed for subunit export from the nucleolus. Since Lsg1p is cytoplasmic, its role in nuclear export is likely to be indirect. We suggest that Lsg1p is needed to recycle an export factor(s) that shuttles from the nucleus associated with the nascent 60S subunit.