Patterns of in situ Mineral Colonization by Microorganisms in a ~60°C Deep Continental Subsurface Aquifer

The microbial ecology of the deep biosphere is difficult to characterize, owing in part to sampling challenges and poorly understood response mechanisms to environmental change. Pre-drilled wells, including oil wells or boreholes, offer convenient access, but sampling is frequently limited to the water alone, which may provide only a partial view of the native diversity. Mineral heterogeneity demonstrably affects colonization by deep biosphere microorganisms, but the connections between the mineral-associated and planktonic communities remain unclear. To understand the substrate effects on microbial colonization and the community response to changes in organic carbon, we conducted an 18-month series of in situ experiments in a warm (57°C), anoxic, fractured carbonate aquifer at 752 m depth using replicate open, screened cartridges containing different solid substrates, with a proteinaceous organic matter perturbation halfway through this series. Samples from these cartridges were analyzed microscopically and by Illumina (iTag) 16S rRNA gene libraries to characterize changes in mineralogy and the diversity of the colonizing microbial community. The substrate-attached and planktonic communities were significantly different in our data, with some taxa (e.g., Candidate Division KB-1) rare or undetectable in the first fraction and abundant in the other. The substrate-attached community composition also varied significantly with mineralogy, such as with two Rhodocyclaceae OTUs, one of which was abundant on carbonate minerals and the other on silicic substrates. Secondary sulfide mineral formation, including iron sulfide framboids, was observed on two sets of incubated carbonates. Notably, microorganisms were attached to the framboids, which were correlated with abundant Sulfurovum and Desulfotomaculum sp. sequences in our analysis. Upon organic matter perturbation, mineral-associated microbial diversity differences were temporarily masked by the dominance of putative heterotrophic taxa in all samples, including OTUs identified as Caulobacter, Methyloversatilis, and Pseudomonas. Subsequent experimental deployments included a methanogen-dominated stage (Methanobacteriales and Methanomicrobiales) 6 months after the perturbation and a return to an assemblage similar to the pre-perturbation community after 9 months. Substrate-associated community differences were again significant within these subsequent phases, however, demonstrating the value of in situ time course experiments to capture a fraction of the microbial assemblage that is frequently difficult to observe in pre-drilled wells

Pimavanserin and Lorcaserin Attenuate Measures of Binge Eating in Male Sprague-Dawley Rats

Binge eating disorder (BED) is characterized by dysregulated feeding and reward-related processes, and treatment is often challenged by limited therapeutic options. The serotonin (5-HT) 5-HT2A receptor (5-HT2AR) and 5-HT2CR are implicated in both feeding-related and reward-related behaviors and are thus poised to regulate BED-related behaviors. The purpose of this study was to assess the efficacy of the FDA-approved medications pimavanserin, a 5-HT2AR antagonist/inverse agonist, and lorcaserin, a 5-HT2CR agonist, in a rodent model of binge eating. The effects of pimavanserin (0.3–3.0 mg/kg), lorcaserin (0.25–1.0 mg/kg), and the lowest effective dose of pimavanserin (0.3 mg/kg) plus lorcaserin (1.0 mg/kg) were tested in a high-fat food (HFF) intermittent access binge eating model in adult male Sprague-Dawley rats (n = 64). We assessed three measures related to binge eating – binge episode occurrence, binge intake, and weight gain associated with HFF access. Pimavanserin decreased binge intake and weight gain associated with HFF access, but did not prevent binge episode occurrence. Lorcaserin decreased binge intake, but did not prevent binge episode occurrence or weight gain associated with HFF access. Combined pimavanserin plus lorcaserin prevented binge episode occurrence in addition to decreasing binge intake and weight gain associated with HFF access. These preclinical findings in male rats suggest that pimavanserin and lorcaserin may be effective in treating patients with BED. Our studies further indicate that administration of one or both drugs may be more effective in certain sub-populations of patients with BED because of the unique profile each treatment elicits. These data support future assessment in clinical populations with BED

Ghrelin receptor antagonist JMV2959 blunts cocaine and oxycodone drug-seeking, but not self-administration, in male rats

The drug overdose crisis has spawned serious health consequences, including the increased incidence of substance use disorders (SUDs), conditions manifested by escalating medical and psychological impairments. While medication management is a key adjunct in SUD treatment, this crisis has crystallized the need to develop additional therapeutics to facilitate extended recovery from SUDs. The “hunger hormone” ghrelin acts by binding to the growth hormone secretagogue receptor 1α (GHS1αR) to control homeostatic and hedonic aspects of food intake and has been implicated in the mechanisms underlying SUDs. Preclinical studies indicate that GHS1αR antagonists and inverse agonists suppress reward-related signaling associated with cocaine and opioids. In the present study, we found that the GHS1αR antagonist JMV2959 was efficacious to suppress both cue-reinforced cocaine and oxycodone drug-seeking, but not cocaine or oxycodone self-administration in male Sprague-Dawley rats. These data suggest a role of the ghrelin-GHS1αR axis in mediating overlapping reward-related aspects of cocaine and oxycodone and premises the possibility that a GHS1αR antagonist may be a valuable therapeutic strategy for relapse vulnerability in SUDs

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories

Membrane protein megahertz crystallography at the European XFEL

The world's first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.peerReviewe