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As part of our study of the old (~2.5 Gyr) open cluster NGC 6819 in the Kepler field, we present photometric (Kepler and ground-based BVRcIc) and spectroscopic observations of the detached eclipsing binary WOCS 24009 (Auner 665; KIC 5023948) with a short orbital period of 3.6 days. WOCS 24009 is a triple-lined system, and we verify that the brightest star is physically orbiting the eclipsing binary using radial velocities and eclipse timing variations. The eclipsing binary components have masses M_B =1.090+/-0.010 Msun and M_C =1.075+/-0.013 Msun, and radii R_B =1.095+/-0.007 Rsun and R_C =1.057+/-0.008 Rsun. The bright non-eclipsing star resides at the cluster turnoff, and ultimately its mass will directly constrain the turnoff mass: our preliminary determination is M_A =1.251+/-0.057 Msun. A careful examination of the light curves indicates that the fainter star in the eclipsing binary undergoes a very brief period of total eclipse, which enables us to precisely decompose the light of the three stars and place them in the color-magnitude diagram. We also present improved analysis of two previously discussed detached eclipsing stars in NGC 6819 (WOCS 40007 and WOCS 23009) en route to a combined determination of the cluster's distance modulus (m-M)_V=12.38+/-0.04. Because this paper significantly increases the number of measured stars in the cluster, we can better constrain the age of the color-magnitude diagram to be 2.21+/-0.10+/-0.20 Gyr. Additionally, using all measured eclipsing binary star masses and radii, we constrain the age to 2.38+/-0.05+/-0.22 Gyr. The quoted uncertainties are estimates of measurement and systematic uncertainties (due to model physics differences and metal content), respectively., Comment: accepted to AJ, 13 figures, eclipse timings and radial velocity data will be available with the journal article

International audience; Forebrain circuits rely upon a relatively small but remarkably diverse population of GABAergic interneurons to bind and entrain large principal cell assemblies for network synchronization and rhythmogenesis. Despite the high degree of heterogeneity across cortical interneurons, members of a given subtype typically exhibit homogeneous developmental origins, neuromodulatory response profiles, morphological characteristics, neurochemical signatures and electrical features. Here we report a surprising divergence among hippocampal oriens-lacunosum moleculare (O-LM) projecting interneurons that have hitherto been considered a homogeneous cell population. Combined immunocytochemical, anatomical and electrophysiological interrogation of Htr3a-GFP and Nkx2-1-cre:RCE mice revealed that O-LM cells parse into a caudal ganglionic eminence-derived subpopulation expressing 5-HT3A receptors (5-HT(3A)Rs) and a medial ganglionic eminence-derived subpopulation lacking 5-HT(3A)Rs. These two cohorts differentially participate in network oscillations, with 5-HT3AR-containing O-LM cell recruitment dictated by serotonergic tone. Thus, members of a seemingly uniform interneuron population can exhibit unique circuit functions and neuromodulatory properties dictated by disparate developmental origins.

Giant reed was evaluated for enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) using a commercial cellulase/β-glucosidase and Scheffersomyces ( Pichia ) stipitis CBS 6054 for ethanol production following dilute-oxalic acid pretreatment. A response surface methodology with two input parameters – severity factor (SF) and oxalic acid concentration (OA) – was employed to optimize both enzymatic hydrolysis and SSF. Xylan content after dilute-OA pretreatment decreased with increasing SF and OA; almost complete hydrolysis was observed when the harsher pretreatment conditions were used. Glucan and lignin content showed an opposite trend with respect to xylan content after dilute-OA pretreatment. Accordingly, enzymatic hydrolysis and ethanol production reached 95% of glucan conversion and 18 g l −1 (75.3% of the maximum theoretical ethanol yield), respectively, with the pretreatment condition 4.05 SF and 5% OA w/w. Dilute-OA mediated pretreatment of giant reed followed by coupled saccharification and fermentation can be considered a promising methodology for second generation bioethanol production.

We present the discovery of the totally eclipsing long-period (P = 771.8 d) binary system WOCS 23009 in the old open cluster NGC 6819 that contains both an evolved star near central hydrogen exhaustion and a low-mass (0.45 Msun) star. This system was previously known to be a single-lined spectroscopic binary, but the discovery of an eclipse near apastron using data from the Kepler space telescope makes it clear that the system has an inclination that is very close to 90 degrees. Although the secondary star has not been identified in spectra, the mass of the primary star can be constrained using other eclipsing binaries in the cluster. The combination of total eclipses and a mass constraint for the primary star allows us to determine a reliable mass for the secondary star and radii for both stars, and to constrain the cluster age. Unlike well-measured stars of similar mass in field binaries, the low-mass secondary is not significantly inflated in radius compared to model predictions. The primary star characteristics, in combination with cluster photometry and masses from other cluster binaries, indicates a best age of 2.62+/-0.25 Gyr, although stellar model physics may introduce systematic uncertainties at the ~10% level. We find preliminary evidence that the asteroseismic predictions for red giant masses in this cluster are systematically too high by as much as 8%., 38 pages,11 figures, accepted for ApJ

Although vastly outnumbered, inhibitory interneurons critically pace and synchronize excitatory principal cell populations to coordinate cortical information processing. Precision in this control relies upon a remarkable diversity of interneurons primarily determined during embryogenesis by genetic restriction of neuronal potential at the progenitor stage. Like their neocortical counterparts, hippocampal interneurons arise from medial and caudal ganglionic eminence (MGE and CGE) precursors. However, while studies of the early specification of neocortical interneurons are rapidly advancing, similar lineage analyses of hippocampal interneurons have lagged. A "hippocampocentric" investigation is necessary as several hippocampal interneuron subtypes remain poorly represented in the neocortical literature. Thus, we investigated the spatiotemporal origins of hippocampal interneurons using transgenic mice that specifically report MGE- and CGE-derived interneurons either constitutively or inducibly. We found that hippocampal interneurons are produced in two neurogenic waves between E9-E12 and E12-E16 from MGE and CGE, respectively, and invade the hippocampus by E14. In the mature hippocampus, CGE-derived interneurons primarily localize to superficial layers in strata lacunosum moleculare and deep radiatum, while MGE-derived interneurons readily populate all layers with preference for strata pyramidale and oriens. Combined molecular, anatomical, and electrophysiological interrogation of MGE/CGE-derived interneurons revealed that MGE produces parvalbumin-, somatostatin-, and nitric oxide synthase-expressing interneurons including fast-spiking basket, bistratified, axo-axonic, oriens-lacunosum moleculare, neurogliaform, and ivy cells. In contrast, CGE-derived interneurons contain cholecystokinin, calretinin, vasoactive intestinal peptide, and reelin including non-fast-spiking basket, Schaffer collateral-associated, mossy fiber-associated, trilaminar, and additional neurogliaform cells. Our findings provide a basic blueprint of the developmental origins of hippocampal interneuron diversity.

Cellulosic biomass is an abundant and underused substrate for biofuel production. The inability of many microbes to metabolize the pentose sugars abundant within hemicellulose creates specific challenges for microbial biofuel production from cellulosic material. Although engineered strains of Saccharomyces cerevisiae can use the pentose xylose, the fermentative capacity pales in comparison with glucose, limiting the economic feasibility of industrial fermentations. To better understand xylose utilization for subsequent microbial engineering, we sequenced the genomes of two xylose-fermenting, beetle-associated fungi, Spathaspora passalidarum and Candida tenuis . To identify genes involved in xylose metabolism, we applied a comparative genomic approach across 14 Ascomycete genomes, mapping phenotypes and genotypes onto the fungal phylogeny, and measured genomic expression across five Hemiascomycete species with different xylose-consumption phenotypes. This approach implicated many genes and processes involved in xylose assimilation. Several of these genes significantly improved xylose utilization when engineered into S. cerevisiae , demonstrating the power of comparative methods in rapidly identifying genes for biomass conversion while reflecting on fungal ecology.

GABAergic interneurons critically regulate cortical computation through exquisite spatiotemporal control over excitatory networks. Precision of this inhibitory control requires a remarkable diversity within interneuron populations that is largely specified during embryogenesis. Although interneurons expressing the neuronal isoform of nitric oxide synthase (nNOS) constitute the largest hippocampal interneuron cohort their origin and specification remain unknown. Thus, as neurogliaform cells (NGC) and Ivy cells (IvC) represent the main nNOS+interneurons, we investigated their developmental origins. Although considered distinct interneuron subtypes, NGCs and IvCs exhibited similar neurochemical and electrophysiological signatures, including NPY expression and late spiking. Moreover, lineage analyses, including loss-of-function experiments and inducible fate-mapping, indicated that nNOS+IvCs and NGCs are both derived from medial ganglionic eminence (MGE) progenitors under control of the transcription factor Nkx2-1. Surprisingly, a subset of NGCs lacking nNOS arises from caudal ganglionic eminence (CGE) progenitors. Thus, while nNOS+NGCs and IvCs arise from MGE progenitors, a CGE origin distinguishes a discrete population of nNOS−NGCs.

Forward genetic mutational studies, adaptive evolution, and phenotypic screening are powerful tools for creating new variant organisms with desirable traits. However, mutations generated in the process cannot be easily identified with traditional genetic tools. We show that new high-throughput, massively parallel sequencing technologies can completely and accurately characterize a mutant genome relative to a previously sequenced parental (reference) strain. We studied a mutant strain of Pichia stipitis, a yeast capable of converting xylose to ethanol. This unusually efficient mutant strain was developed through repeated rounds of chemical mutagenesis, strain selection, transformation, and genetic manipulation over a period of seven years. We resequenced this strain on three different sequencing platforms. Surprisingly, we found fewer than a dozen mutations in open reading frames. All three sequencing technologies were able to identify each single nucleotide mutation given at least 10–15-fold nominal sequence coverage. Our results show that detecting mutations in evolved and engineered organisms is rapid and cost-effective at the whole-genome level using new sequencing technologies. Identification of specific mutations in strains with altered phenotypes will add insight into specific gene functions and guide further metabolic engineering efforts.

Native strains of Saccharomyces cerevisiae do not assimilate xylose. S. cerevisiae engineered for d -xylose utilization through the heterologous expression of genes for aldose reductase ( XYL1 ), xylitol dehydrogenase ( XYL2 ), and d -xylulokinase ( XYL3 or XKS1 ) produce only limited amounts of ethanol in xylose medium. In recombinant S. cerevisiae expressing XYL1 , XYL2 , and XYL3 , mRNA transcript levels for glycolytic, fermentative, and pentose phosphate enzymes did not change significantly on glucose or xylose under aeration or oxygen limitation. However, expression of genes encoding the tricarboxylic acid cycle, respiration enzymes ( HXK1 , ADH2 , COX13 , NDI1 , and NDE1 ), and regulatory proteins ( HAP4 and MTH1 ) increased significantly when cells were cultivated on xylose, and the genes for respiration were even more elevated under oxygen limitation. These results suggest that recombinant S. cerevisiae does not recognize xylose as a fermentable carbon source and that respiratory proteins are induced in response to cytosolic redox imbalance; however, lower sugar uptake and growth rates on xylose might also induce transcripts for respiration. A petite respiration-deficient mutant (ρ°) of the engineered strain produced more ethanol and accumulated less xylitol from xylose. It formed characteristic colonies on glucose, but it did not grow on xylose. These results are consistent with the higher respiratory activity of recombinant S. cerevisiae when growing on xylose and with its inability to grow on xylose under anaerobic conditions.

XYL3 , which encodes a d -xylulokinase (EC 2.7.1.17), was isolated from Pichia stipitis CBS 6054 genomic DNA by using primers designed against conserved motifs. Disruption of XYL3 eliminated d -xylulokinase activity, but d -ribulokinase activity was still present. Southern analysis of P. stipitis genomic DNA with XYL3 as a probe confirmed the disruption and did not reveal additional related genes. Disruption of XYL3 stopped ethanol production from xylose, but the resulting mutant still assimilated xylose slowly and formed xylitol and arabinitol. These results indicate that XYL3 is critical for ethanol production from xylose but that P. stipitis has another pathway for xylose assimilation. Expression of XYL3 using its P. stipitis promoter increased Saccharomyces cerevisiae d -xylulose consumption threefold and enabled the transformants to produce ethanol from a mixture of xylose and xylulose, whereas the parental strain only accumulated xylitol. In vitro, d -xylulokinase activity in recombinant S. cerevisiae was sixfold higher with a multicopy than with a single-copy XYL3 plasmid, but ethanol production decreased with increased copy number. These results confirmed the function of XYL3 in S. cerevisiae .

Two genes coding for isozymes of alcohol dehydrogenase (ADH); designated PsADH1 and PsADH2 , have been identified and isolated from Pichia stipitis CBS 6054 genomic DNA by Southern hybridization to Saccharomyces cerevisiae ADH genes, and their physiological roles have been characterized through disruption. The amino acid sequences of the PsADH1 and PsADH2 isozymes are 80.5% identical to one another and are 71.9 and 74.7% identical to the S. cerevisiae ADH1 protein. They also show a high level identity with the group I ADH proteins from Kluyveromyces lactis . The PsADH isozymes are presumably localized in the cytoplasm, as they do not possess the amino-terminal extension of mitochondrion-targeted ADHs. Gene disruption studies suggest that PsADH1 plays a major role in xylose fermentation because PsADH1 disruption results in a lower growth rate and profoundly greater accumulation of xylitol. Disruption of PsADH2 does not significantly affect ethanol production or aerobic growth on ethanol as long as PsADH1 is present. The PsADH1 and PsADH2 isozymes appear to be equivalent in the ability to convert ethanol to acetaldehyde, and either is sufficient to allow cell growth on ethanol. However, disruption of both genes blocks growth on ethanol. P. stipitis strains disrupted in either PsADH1 or PsADH2 still accumulate ethanol, although in different amounts, when grown on xylose under oxygen-limited conditions. The PsADH double disruptant, which is unable to grow on ethanol, still produces ethanol from xylose at about 13% of the rate seen in the parental strain. Thus, deletion of both PsADH1 and PsADH2 blocks ethanol respiration but not production, implying a separate path for fermentation.

Anabaena cylindrica sparged with argon gas produced H2 continuously for 30 days under limited light conditions (6.0 W/m2) and for 18 days under elevated light conditions (32 W/m2) in the absence of exogenous nitrogen. The efficiency of converting visible light energy (32 W/m2) into chemical energy that is trapped as H2 ranged between 0.35 and 0.85% (approximately 13 microliter of H2 per mg [drywt] per h). Ammonium additions (0.2 mM NH4+) at various times destabilized the system and eventually suppressed H2 production completely, as compared with the control. Cultures grown with 5.0 mg of Fe3+ per liter produced H2 at a rate about twice that of cultures with 0.5 mg of Fe3+ per liter. Cultures grown at pH 7.4 produced H2 at the same initial rates as cultures that were grown at pH 9.4; however, the latter cultures continued to produce H2 after CO2 deprivation.

Previous studies have shown that a lignin-degrading system appears in cultures of the white rot fungus Phanerochaete chrysosporium in response to nitrogen starvation, apparently as part of secondary metabolism. We examined the influence of limiting carbohydrate, sulfur, or phosphorus and the effect of varying the concentrations of four trace metals, Ca 2+ , and Mg 2+ . Limitation of carbohydrate or sulfur but not limitation of phosphorus triggered ligninolytic activity. When only carbohydrate was limiting, supplementary carbohydrate caused a transient repression of activity. In carbohydrate-limited cultures, ligninolytic activity appeared when the supplied carbohydrate was depleted, and this activity was associated with a decrease in mycelial dry weight. The amount of lignin degraded depended on the amount of carbohydrate provided, which determined the amount of mycelium produced during primary growth. Carbohydrate-limited cultures synthesized only small amounts of the secondary metabolite veratryl alcohol compared with nitrogen-limited cultures. l -Glutamate sharply repressed ligninolytic activity in carbohydrate-starved cultures, but NH 4 + did not. Ligninolytic activity was also triggered in cultures supplied with 37 μM sulfur as the only limiting nutrient. The balance of trace metals, Mg 2+ , and Ca 2+ was important for lignin degradation.