Discrimination against major groove adducts by Y-family polymerases of the DinB subfamily

Y-family DNA polymerases bypass DNA adducts in a process known as translesion synthesis (TLS). Y-family polymerases make contacts with the minor groove side of the DNA substrate at the nascent base pair. The Y-family polymerases also contact the DNA major groove via the unique little finger domain, but they generally lack contacts with the major groove at the nascent base pair. Escherichia coli DinB efficiently and accurately copies certain minor groove guanosine adducts. In contrast, we previously showed that the presence in the DNA template of the major groove-modified base 1,3-diaza-2-oxophenothiazine (tC) inhibits the activity of E. coli DinB. Even when the DNA primer is extended up to three nucleotides beyond the site of the tC analog, DinB activity is strongly inhibited. These findings prompted us to investigate discrimination against other major groove modifications by DinB and its orthologs. We chose a set of pyrimidines and purines with modifications in the major groove and determined the activity of DinB and several orthologs with these substrates. DinB, human pol kappa, and Sulfolobus solfataricus Dpo4 show differing specificities for the major groove adducts pyrrolo-dC, dP, N⁶-furfuryl-dA, and etheno-dA. In general, DinB was least efficient for bypass of all of these major groove adducts, whereas Dpo4 was most efficient. DinB activity was essentially completely inhibited by the presence of etheno-dA, while pol kappa activity was strongly inhibited. All three of these DNA polymerases were able to bypass N⁶-furfuryl-dA with modest efficiency, with DinB being the least efficient. We also determined that the R35A variant of DinB enhances bypass of N⁶-furfuryl-dA but not etheno-dA. In sum, we find that whereas DinB is specific for bypass of minor groove adducts, it is specifically inhibited by major groove DNA modifications.     

An Investigation of Fecal Volatile Organic Metabolites in Irritable Bowel Syndrome

Diagnosing irritable bowel syndrome (IBS) can be a challenge; many clinicians resort to invasive investigations in order to rule out other diseases and reassure their patients. Volatile organic metabolites (VOMs) are emitted from feces; understanding changes in the patterns of these VOMs could aid our understanding of the etiology of the disease and the development of biomarkers, which can assist in the diagnosis of IBS. We report the first comprehensive study of the fecal VOMs patterns in patients with diarrhea-predominant IBS (IBS-D), active Crohn''s disease (CD), ulcerative colitis (UC) and healthy controls. 30 patients with IBS-D, 62 with CD, 48 with UC and 109 healthy controls were studied. Diagnosis of IBS-D was made using the Manning criteria and all patients with CD and UC met endoscopic, histologic and/or radiologic criteria. Fecal VOMs were extracted by solid phase microextraction (SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS). 240 VOMs were identified. Univariate analysis showed that esters of short chain fatty acids, cyclohexanecarboxylic acid and its ester derivatives were associated with IBS-D (p<0.05), while aldehydes were more abundant in IBD (p<0.05). A predictive model, developed by multivariate analysis, separated IBS-D from active CD, UC and healthy controls with a sensitivity of 94%, 96% and 90%; and a specificity of 82%, 80% and 80% respectively (p<0.05). The understanding of the derivation of these VOMs may cast light on the etiology of IBS-D and IBD. These data show that fecal VOMs analyses could contribute to the diagnosis of IBS-D, for which there is no laboratory test, as well as IBD.     

A Fungal Pathogen of Amphibians,Batrachochytrium dendrobatidis,Attenuates in Pathogenicity with In Vitro Passages

Laboratory investigations into the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), have accelerated recently, given the pathogen’s role in causing the global decline and extinction of amphibians. Studies in which host animals were exposed to Bd have largely assumed that lab-maintained pathogen cultures retained the infective and pathogenic properties of wild isolates. Attenuated pathogenicity is common in artificially maintained cultures of other pathogenic fungi, but to date, it is unknown whether, and to what degree, Bd might change in culture. We compared zoospore production over time in two samples of a single Bd isolate having different passage histories: one maintained in artificial media for more than six years (JEL427-P39), and one recently thawed from cryopreserved stock (JEL427-P9). In a common garden experiment, we then exposed two different amphibian species, Eleutherodactylus coqui and Atelopus zeteki, to both cultures to test whether Bd attenuates in pathogenicity with in vitro passages. The culture with the shorter passage history, JEL427-P9, had significantly greater zoospore densities over time compared to JEL427-P39. This difference in zoospore production was associated with a difference in pathogenicity for a susceptible amphibian species, indicating that fecundity may be an important virulence factor for Bd. In the 130-day experiment, Atelopus zeteki frogs exposed to the JEL427-P9 culture experienced higher average infection intensity and 100% mortality, compared with 60% mortality for frogs exposed to JEL427-P39. This effect was not observed with Eleutherodactylus coqui, which was able to clear infection. We hypothesize that the differences in phenotypic performance observed with Atelopus zeteki are rooted in changes of the Bd genome. Future investigations enabled by this study will focus on the underlying mechanisms of Bd pathogenicity.     

Fluorescent multiplexing of 3D spheroids: Analysis of biomarkers using automated immunohistochemistry staining platform and multispectral imaging

In preclinical cancer studies, three-dimensional (3D) cell spheroids and aggregates are preferred over monolayer cell cultures due to their architectural and functional similarity to solid tumors. We performed a proof-of-concept study to generate physiologically relevant and predictive preclinical models using non–small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line-derived 3D spheroids and aggregates. Distinct panels were designed to determine the expression profiles of frequently studied biomarkers of the two cancer subtypes. The lung adenocarcinoma panel included ALK, EGFR, TTF-1, and CK7 biomarkers, and the colon and colorectal adenocarcinoma panel included BRAF V600E, MSH2, MSH6, and CK20. Recent advances in immunofluorescence (IF) multiplexing and imaging technology enable simultaneous detection and quantification of multiple biomarkers on a single slide. In this study, we performed IF staining of multiple biomarkers per section on formalin-fixed paraffin-embedded 3D spheroids and aggregates. We optimized protocol parameters for automated IF and demonstrated staining concordance with automated chromogenic immunohistochemistry performed with validated protocols. Next, post-acquisition spectral unmixing of the captured fluorescent signals were utilized to delineate four differently stained biomarkers within a single multiplex IF image, followed by automated quantification of the expressed markers. This workflow has the potential to be adapted to preclinical high-throughput screening and drug efficacy studies utilizing 3D spheroids from cancer cell lines and patient-derived organoids. The process allows for cost, time, and resource savings through concurrent staining of several biomarkers on a single slide, the ability to study the interactions of multiple expressed proteins within a single region of interest, and enable quantitative assessment of biomarkers in cancer cells.     

Polymerase manager protein UmuD directly regulates Escherichia coli DNA polymerase III α binding to ssDNA

Replication by Escherichia coli DNA polymerase III is disrupted on encountering DNA damage. Consequently, specialized Y-family DNA polymerases are used to bypass DNA damage. The protein UmuD is extensively involved in modulating cellular responses to DNA damage and may play a role in DNA polymerase exchange for damage tolerance. In the absence of DNA, UmuD interacts with the α subunit of DNA polymerase III at two distinct binding sites, one of which is adjacent to the single-stranded DNA-binding site of α. Here, we use single molecule DNA stretching experiments to demonstrate that UmuD specifically inhibits binding of α to ssDNA. We predict using molecular modeling that UmuD residues D91 and G92 are involved in this interaction and demonstrate that mutation of these residues disrupts the interaction. Our results suggest that competition between UmuD and ssDNA for α binding is a new mechanism for polymerase exchange.     

16 Mechanical energy,protein motion,and the possible origin of life between mica sheets

The origins of life require reliable energy sources. One feasible energy source has not been considered until recently. This is mechanical energy-work (Hansma, 2010, 2012). The spaces between moving muscovite mica sheets are the environment in which mechanical energy is hypothesized to have been involved in the origins of life. Mechanical energy from moving mica sheets has two main sources: (1) The open-and-shut motions of mica sheets in response to water movements in and out between the sheets, and (2) Thermal cycles of day and night acting on bubble ‘defects’ between mica sheets. This mechanical energy is hypothesized to have been involved in the formation (and breaking) of covalent bonds, the rearrangement of polymers and molecular aggregates, and the budding-off of protocells, in the earliest form of cell division. Furthermore, it is hypothesized that the mechanical energy from mica sheets moving open-and-shut is the source of the common open-and-shut motions of enzymes, originating from a protobiotic era when mechanical energy was plentiful and chemical energy was not yet available.     

Induction of a ricinosomal-protease and programmed cell death in tomato endosperm by gibberellic acid

Several examples of programmed cell death (PCD) in plants utilize ricinosomes, organelles that appear prior to cell death and store inactive KDEL-tailed cysteine proteinases. Upon cell death, the contents of ricinosomes are released into the cell corpse where the proteinases are activated and proceed to degrade any remaining protein for use in adjacent cells or, in the case of nutritive seed tissues, by the growing seedling. Ricinosomes containing pro-SlCysEP have been observed in anther tissues prior to PCD and ricinosome-like structures have been observed in imbibed seeds within endosperm cells of tomato. The present study confirms that the structures in tomato endosperm cells contain pro-SlCysEP making them bona fide ricinosomes. The relative abundance of pro- versus mature SlCysEP is suggested to be a useful indicator of the degree of PCD that has occurred in tomato endosperm, and is supported by biochemical and structural data. This diagnostic tool is used to demonstrate that a sub-region of the micropylar endosperm surrounding the emerged radical is relatively long-lived and may serve to prevent loss of mobilized reserves from the lateral endosperm. We also demonstrate that GA-induced reserve mobilization, SlCysEP accumulation and processing, and PCD in tomato endosperm are antagonized by ABA.     

Mass spectrometry images acylcarnitines,phosphatidylcholines, and sphingomyelin in MDA-MB-231 breast tumor models

The lipid compositions of different breast tumor microenvironments are largely unknown due to limitations in lipid imaging techniques. Imaging lipid distributions would enhance our understanding of processes occurring inside growing tumors, such as cancer cell proliferation, invasion, and metastasis. Recent developments in MALDI mass spectrometry imaging (MSI) enable rapid and specific detection of lipids directly from thin tissue sections. In this study, we performed multimodal imaging of acylcarnitines, phosphatidylcholines (PC), a lysophosphatidylcholine (LPC), and a sphingomyelin (SM) from different microenvironments of breast tumor xenograft models, which carried tdTomato red fluorescent protein as a hypoxia-response element-driven reporter gene. The MSI molecular lipid images revealed spatially heterogeneous lipid distributions within tumor tissue. Four of the most-abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1), and PC(18:0/18:1), were localized in viable tumor regions, whereas LPC(16:0/0:0) was detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoylcarnitine, stearoylcarnitine, PC(16:0/22:1), and SM(d18:1/16:0) sodium adduct, which colocalized primarily with hypoxic tumor regions. For the first time, we have applied a multimodal imaging approach that has combined optical imaging and MALDI-MSI with ion mobility separation to spatially localize and structurally identify acylcarnitines and a variety of lipid species present in breast tumor xenograft models.     

Characterising the seasonal cycle of dissolved organic nitrogen using Cefas SmartBuoy high-resolution time-series samples from the southern North Sea

The Cefas SmartBuoy network provides a unique insight into the biogeochemical dynamics of the Northern European shelf seas, particularly the North Sea, through high-resolution automated offshore water sampling. We present total dissolved nitrogen and dissolved organic nitrogen (DON) from the Dowsing SmartBuoy site (53.531° N, 1.053° E) from January to October 2010, the first high resolution seasonal (winter-autumn) cycle of DON from the open North Sea. On top of a refractory background DON concentration of approximately 5 μM, a rapid increase in DON of a further ～5 μM is observed over the course of the spring bloom. This rapidly produced DON declines at an estimated net decay rate of between 0.6 and 1.8 μM month^?1. The slow decay suggests that the majority of the additional DON produced during the spring bloom is of semi-labile nature and has a lifetime of weeks to months. The dataset allows us to tightly constrain the budget for water column nitrogen over the winter, spring and summer of 2010 and clearly demonstrates the ‘sawtooth’ nature of the seasonal cycle of DON in the open North Sea, which has been impossible to resolve with a more traditional ship-based mode of operation. This work highlights the importance of autonomous sampling approaches in better understanding shelf sea biogeochemistry in the future.