85 research outputs found
Genetic Engineering of the Rock Inhabitant Knufia petricola Provides Insight Into the Biology of Extremotolerant Black Fungi
Black microcolonial fungi (Ascomycetes from Arthonio-, Dothideo-, and Eurotiomycetes) are stress-tolerant and persistent dwellers of natural and anthropogenic extreme habitats. They exhibit slow yeast-like or meristematic growth, do not form specialized reproduction structures and accumulate the black pigment 1,8-dihydroxynaphthalene (DHN) melanin in the multilayered cell walls. To understand how black fungi live, survive, colonize mineral substrates, and interact with phototrophs genetic methods are needed to test these functions and interactions. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a model for developing methods for genetic manipulation. Here, we report on the expansion of the genetic toolkit by more efficient multiplex CRISPR/Cas9 using a plasmid-based system for expression of Cas9 and multiple sgRNAs and the implementation of the three resistance selection markers genR (geneticin/nptII), baR (glufosinate/bar), and suR (chlorimuron ethyl/sur). The targeted integration of expression constructs by replacement of essential genes for pigment synthesis allows for an additional color screening of the transformants. The black-pink screening due to the elimination of pks1 (melanin) was applied for promoter studies using GFP fluorescence as reporter. The black-white screening due to the concurrent elimination of pks1 and phs1 (carotenoids) allows to identify transformants that contain the two expression constructs for co-localization or bimolecular fluorescence complementation (BiFC) studies. The co-localization and interaction of the two K. petricola White Collar orthologs were demonstrated. Two intergenic regions (igr1, igr2) were identified in which expression constructs can be inserted without causing obvious phenotypes. Plasmids of the pNXR-XXX series and new compatible entry plasmids were used for fast and easy generation of expression constructs and are suitable for a broad implementation in other fungi. This variety of genetic tools is opening a completely new perspective for mechanistic and very detailed study of expression, functioning and regulation of the genes/proteins encoded by the genomes of black fungi
Seabed corrugations beneath an Antarctic ice shelf revealed by autonomous underwater vehicle survey: Origin and implications for the history of Pine Island Glacier
Ice shelves are critical features in the debate about West Antarctic ice sheet change and sea level rise, both because they limit ice discharge and because they are sensitive to change in the surrounding ocean. The Pine Island Glacier ice shelf has been thinning rapidly since at least the early 1990s, which has caused its trunk to accelerate and retreat. Although the ice shelf front has remained stable for the past six decades, past periods of ice shelf collapse have been inferred from relict seabed "corrugations" (corrugated ridges), preserved 340 km from the glacier in Pine Island Trough. Here we present high-resolution bathymetry gathered by an autonomous underwater vehicle operating beneath an Antarctic ice shelf, which provides evidence of long-term change in Pine Island Glacier. Corrugations and ploughmarks on a sub-ice shelf ridge that was a former grounding line closely resemble those observed offshore, interpreted previously as the result of iceberg grounding. The same interpretation here would indicate a significantly reduced ice shelf extent within the last 11 kyr, implying Holocene glacier retreat beyond present limits, or a past tidewater glacier regime different from today. The alternative, that corrugations were not formed in open water, would question ice shelf collapse events interpreted from the geological record, revealing detail of another bed-shaping process occurring at glacier margins. We assess hypotheses for corrugation formation and suggest periodic grounding of ice shelf keels during glacier unpinning as a viable origin. This interpretation requires neither loss of the ice shelf nor glacier retreat and is consistent with a "stable" grounding-line configuration throughout the Holocene
An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola
The role of apoptosis repressor with a CARD domain (ARC) in the therapeutic resistance of renal cell carcinoma (RCC): the crucial role of ARC in the inhibition of extrinsic and intrinsic apoptotic signalling
Background: Renal cell carcinomas (RCCs) display broad resistance against conventional radio- and chemotherapies, which is due at least in part to impairments in both extrinsic and intrinsic apoptotic pathways. One important anti-apoptotic factor that is strongly overexpressed in RCCs and known to inhibit both apoptotic pathways is ARC (apoptosis repressor with a CARD domain). Methods: Expression and subcellular distribution of ARC in RCC tissue samples and RCC cell lines were determined by immunohistochemistry and fluorescent immunohistochemistry, respectively. Extrinsic and intrinsic apoptosis signalling were induced by TRAIL (TNF-related apoptosis-inducing ligand), ABT-263 or topotecan. ARC knock-down was performed in clearCa-12 cells using lentiviral transduction of pGIPZ. shRNAmir constructs. Extrinsic respectively intrinsic apoptosis were induced by TRAIL (TNF-related apoptosis-inducing ligand), ABT263 or topotecan. Potential synergistic effects were tested by pre-treatment with topotecan and subsequent treatment with ABT263. Activation of different caspases and mitochondrial depolarisation (JC-1 staining) were analysed by flow cytometry. Protein expression of Bcl-2 family members and ARC in RCC cell lines was measured by Western blotting. Statistical analysis was performed by Student’s t-test. Results: Regarding the extrinsic pathway, ARC knockdown strongly enhanced TRAIL-induced apoptosis by increasing the activation level of caspase-8. Regarding the intrinsic pathway, ARC, which was only weakly expressed in the nuclei of RCCs in vivo, exerted its anti-apoptotic effect by impairing mitochondrial activation rather than inhibiting p53. Topotecan- and ABT-263-induced apoptosis was strongly enhanced following ARC knockdown in RCC cell lines. In addition, topotecan pre-treatment enhanced ABT-263-induced apoptosis and this effect was amplified in ARC-knockdown cells. Conclusion: Taken together, our results are the first to demonstrate the importance of ARC protein in the inhibition of both the extrinsic and intrinsic pathways of apoptosis in RCCs. In this context, ARC cooperates with anti-apoptotic Bcl-2 family members to exert its strong anti-apoptotic effects and is therefore an important factor not only in the therapeutic resistance but also in future therapy strategies (i.e., Bcl-2 inhibitors) in RCC. In sum, targeting of ARC may enhance the therapeutic response in combination therapy protocols
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Lightest Isotope of Bh Produced Via the 209Bi(52Cr,n)260BhReaction
The lightest isotope of Bh known was produced in the new {sup 209}Bi({sup 52}Cr,n){sup 260}Bh reaction at the Lawrence Berkeley National Laboratory's 88-Inch Cyclotron. Positive identification was made by observation of eight correlated alpha particle decay chains in the focal plane detector of the Berkeley Gas-Filled Separator. {sup 260}Bh decays with a 35{sub -9}{sup +19} ms half-life by alpha particle emission mainly by a group at 10.16 MeV. The measured cross section of 59{sub -20}{sup +29} pb is approximately a factor of four larger than compared to recent model predictions. The influences of the N = 152 and Z = 108 shells on alpha decay properties are discussed
Alteration of the murine gut microbiota during infection with the parasitic helminth Heligmosomoides polygyrus
Background: In a murine model of inflammatory bowel disease (IBD), treatment of colitis in IL-10 gene-deficient mice with the parasitic helminth Heligmosomoides polygyrus ameliorates colonic inflammation. The cellular and molecular mechanisms driving this therapeutic host response are being studied vigorously. One proposed mechanism is that H. polygyrus infection favors the outgrowth or suppression of certain bacteria, which in turn help modulate host immunity. Methods: To quantify the effect of H. polygyrus infection on the composition of the gastrointestinal (GI) tract microbiota, we conducted two independent microbial ecology analyses of C57BL/6 mice. We obtained and analyzed 3,353 bacterial 16S rRNA encoding gene sequences from the ileum and cecum of infected and uninfected mice as well as incective H. polygyrus larvae at the outset of the second experiment and adult worms taken directly from the mouse duodenum at the end of the second experiment. Results: We found that a significant shift in the abundance and relative distribution of bacterial species in the ileum of mice is associated with H. polygyrus infection. Members of the bacterial family Lactobacillaceae significantly increased in abundance in the ileum of infected mice reproducibly in two independent experiments despite having different microbiotas present at the outset of each experiment. Conclusions: These data support the concept that helminth infection shifts the composition of intestinal bacteria. The clinical consequences of these shifts in intestinal flora are yet to be explored. (Inflamm Bowel Dis 2010)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78230/1/21299_ftp.pd
Study protocol : Minimum effective low dose: anti-human thymocyte globulin (MELD-ATG): phase II, dose ranging, efficacy study of antithymocyte globulin (ATG) within 6 weeks of diagnosis of type 1 diabetes
Introduction Type 1 diabetes (T1D) is a chronic autoimmune disease, characterised by progressive destruction of the insulin-producing beta cells of the pancreas. One immunosuppressive agent that has recently shown promise in the treatment of new-onset T1D subjects aged 12-45 years is antithymocyte globulin (ATG), Thymoglobuline, encouraging further exploration in lower age groups. Methods and analysis Minimal effective low dose (MELD)-ATG is a phase 2, multicentre, randomised, double-blind, placebo-controlled, multiarm parallel-group trial in participants 5-25 years diagnosed with T1D within 3-9 weeks of planned treatment day 1. A total of 114 participants will be recruited sequentially into seven different cohorts with the first cohort of 30 participants being randomised to placebo, 2.5 mg/kg, 1.5 mg/kg, 0.5 mg/kg and 0.1 mg/kg ATG total dose in a 1:1:1:1:1 allocation ratio. The next six cohorts of 12-15 participants will be randomised to placebo, 2.5 mg/kg, and one or two selected middle ATG total doses in a 1:1:1:1 or 1:1:1 allocation ratio, as dependent on the number of middle doses, given intravenously over two consecutive days. The primary objective will be to determine the changes in stimulated C-peptide response over the first 2 hours of a mixed meal tolerance test at 12 months for 2.5 mg/kg ATG arm vs the placebo. Conditional on finding a significant difference at 2.5 mg/kg, a minimally effective dose will be sought. Secondary objectives include the determination of the effects of a particular ATG treatment dose on (1) stimulated C-peptide, (2) glycated haemoglobin, (3) daily insulin dose, (4) time in range by intermittent continuous glucose monitoring measures, (5) fasting and stimulated dry blood spot (DBS) C-peptide measurements. Ethics and dissemination MELD-ATG received first regulatory and ethical approvals in Belgium in September 2020 and from the German and UK regulators as of February 2021. The publication policy is set in the INNODIA (An innovative approach towards understanding and arresting Type 1 diabetes consortium) grant agreement (www.innodia.eu).Peer reviewe
Training in the practice of noninvasive brain stimulation: Recommendations from an IFCN committee
© 2020 As the field of noninvasive brain stimulation (NIBS) expands, there is a growing need for comprehensive guidelines on training practitioners in the safe and effective administration of NIBS techniques in their various research and clinical applications. This article provides recommendations on the structure and content of this training. Three different types of practitioners are considered (Technicians, Clinicians, and Scientists), to attempt to cover the range of education and responsibilities of practitioners in NIBS from the laboratory to the clinic. Basic or core competencies and more advanced knowledge and skills are discussed, and recommendations offered regarding didactic and practical curricular components. We encourage individual licensing and governing bodies to implement these guidelines
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Comparison of reactions for the production of 258,257Db: 208Pb(51V,xn) and 209Bi(50Ti,xn)
Excitation functions for the 1n and 2n exit channels of the 208Pb(51V,xn)259-xDb reaction were measured. A maximum cross section of the 1n exit channel of 2070+1100/-760 pb was measured at an excitation energy of 16.0 +- 1.8 MeV. For the 2n exit channel, a maximum cross section of 1660+450/-370 pb was measured at 22.0 +- 1.8 MeV excitation energy. The 1n excitation function for the 209Bi(50Ti,n)258Db reaction was remeasured, resulting in a cross section of 5480+1750/-1370 pb at an excitation energy of 16.0 +- 1.6 MeV, in agreement with previous values [F. P. Hebberger, et al., Eur. Phys. J. A 12, 57 (2001)]. Differences in cross section maxima are discussed in terms of the fusion probability below the barrier
[Avian cytogenetics goes functional] Third report on chicken genes and chromosomes 2015
High-density gridded libraries of large-insert clones using bacterial artificial chromosome (BAC) and other vectors are essential tools for genetic and genomic research in chicken and other avian species... Taken together, these studies demonstrate that applications of large-insert clones and BAC libraries derived from birds are, and will continue to be, effective tools to aid high-throughput and state-of-the-art genomic efforts and the important biological insight that arises from them
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