Diversity and novelty of the gut microbial community of an herbivorous rodent (Neotoma bryanti)

Mammalian herbivores host diverse microbial communities to aid in fermentation and potentially detoxification of dietary compounds. However, the microbial ecology of herbivorous rodents, especially within the largest superfamily of mammals (Muroidea) has received little attention. We conducted a preliminary inventory of the intestinal microbial community of Bryant’s woodrat (Neotoma bryanti), an herbivorous Muroidea rodent. We collected woodrat feces, generated 16S rDNA clone libraries, and obtained sequences from 171 clones. Our results demonstrate that the woodrat gut hosts a large number of novel microorganisms, with 96% of the total microbial sequences representing novel species. These include several microbial genera that have previously been implicated in the metabolism of plant toxins. Interestingly, a comparison of the community structure of the woodrat gut with that of other mammals revealed that woodrats have a microbial community more similar to foregut rather than hindgut fermenters. Moreover, their microbial community was different to that of previously studied herbivorous rodents. Therefore, the woodrat gut may represent a useful resource for the identification of novel microbial genes involved in cellulolytic or detoxification processes.     

The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions

The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell.     

In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.     

Selected contribution: axial stretch increases spontaneous pacemaker activity in rabbit isolated sinoatrial node cells.

Isolated, spontaneously beating rabbit sinoatrial node cells were subjected to longitudinal stretch, using carbon fibers attached to both ends of the cell. Their electrical behavior was studied simultaneously in current-clamp or voltage-clamp mode using the perforated patch configuration. Moderate stretch ( approximately 7%) caused an increase in spontaneous beating rate (by approximately 5%) and a reduction in maximum diastolic and systolic potentials (by approximately 2.5%), as seen in multicellular preparations. Mathematical modeling of the stretch intervention showed the experimental results to be compatible with stretch activation of cation nonselective ion channels, similar to those found in other cardiac cell populations. Voltage-clamp experiments validated the presence of a stretch-induced current component with a reversal potential near -11 mV. These data confirm, for the first time, that the positive chronotropic response of the heart to stretch is, at least in part, encoded on the level of individual sinoatrial node pacemaker cells; all reported data are in agreement with a major contribution of stretch-activated cation nonselective channels to this response.     

Structural basis for altered soybean agglutinin lectin binding between a murine metastatic lymphoma and an adhesive low malignant variant

By selection for plastic adhesiveness we have previously established a variant tumor line (ESb-MP) from the metastatic murine lymphoma ESb. In contrast to the parental line, the adhesion variant is significantly decreased in malignancy and is altered in the capacity to bind soybean agglutinin (SBA) lectin. Here we show biochemically that the major SBA-binding cell-surface component of ESb-MP cells is the T200 glycoprotein. In ESb cells, T200 antigens bind SBA only after sialidase treatment. Enzymatic studies suggested that glycans detected by the lectin with or without sialidase treatment are different. Inhibition of N-glycosylation by tunicamycin and biosynthetic labeling revealed two T200 chains for ESb-MP cells that were larger in size than the single chain detected in ESb cells. Studies on the biosynthesis revealed that ESb-MP cells expressed two precursor chains for T200 whereas ESb cells displayed only one. There was no size difference detectable in the mature T200 molecules of ESb and ESb-MP cells. Our data suggest that the molecules differ in expression of O-linked glycans that can be recognized by SBA. Additional O-linked sugars on ESb-MP T200 molecules seem to be expressed in particular after trimming of the second T200 precursor chain.     

The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

Selecting appropriate methods of knowledge synthesis to inform biodiversity policy

Responding to different questions generated by biodiversity and ecosystem services policy or management requires different forms of knowledge (e.g. scientific, experiential) and knowledge synthesis. Additionally, synthesis methods need to be appropriate to policy context (e.g. question types, budget, timeframe, output type, required scientific rigour). In this paper we present a range of different methods that could potentially be used to conduct a knowledge synthesis in response to questions arising from knowledge needs of decision makers on biodiversity and ecosystem services policy and management. Through a series of workshops attended by natural and social scientists and decision makers we compiled a range of question types, different policy contexts and potential methodological approaches to knowledge synthesis. Methods are derived from both natural and social sciences fields and reflect the range of question and study types that may be relevant for syntheses. Knowledge can be available either in qualitative or quantitative form and in some cases also mixed. All methods have their strengths and weaknesses and we discuss a sample of these to illustrate the need for diversity and importance of appropriate selection. To summarize this collection, we present a table that identifies potential methods matched to different combinations of question types and policy contexts, aimed at assisting teams undertaking knowledge syntheses to select appropriate methods.     

Human Rod Monochromacy: Linkage Analysis and Mapping of a Cone Photoreceptor Expressed Candidate Gene on Chromosome 2q11

We have performed linkage analysis in eight families with rod monochromacy, an autosomal recessively inherited condition with complete color blindness. Significant linkage was found with markers located at the pericentromeric region of chromosome 2. A maximum lod score of 5.36 was obtained for marker D2S2333 at θ = 0.00. Mapping of meiotic breakpoints localized the disease gene between markers D2S2187 and D2S2229. Homozygosity for a number of subsequent markers indicating identity by descent was found in two families and provides evidence for a further refinement of the locus proximal to D2S373. This defines an interval of ≈3 cM covering theACHM2locus for rod monochromacy. Radiation hybrid mapping of theCNGA3gene encoding the α-subunit of the cGMP gated cation channel in human cone photoreceptors resulted in a maximum lod score of 16.1 with marker D2S2311 combined with a calculated physical distance of 6.19cR_10,000. Screening of the CEPH YAC library and subsequent STS mapping indicated the physical order cen–D2S2222–D2S2175–(D2S2187/D2S2311)–qtel ofmarkers on 2q11 and showed that theCNGA3gene maps most closely to D2S2187 and D2S2311. These data indicate that theCNGA3gene maps within the critical interval of theACHM2locus for rod monochromacy and thus is a candidate gene for this disease.     

Physiological consequences of mowing and burning of Phragmites australisstands for rhizome ventilation and amino acid metabolism

Mowing and burning of Phragmitesaustralis-stands have been recommended in the recentliterature as management tools for both protection andcontrol, and both favourable and detrimental effectsof these treatments were actually observed. This studyaims to clarify this apparent contradiction using anew physiological approach. Reed stands in thebiosphere reserves of Trebon (Czech Republic) and theDanube Delta (Romania) were investigated usingparameters of convective ventilation and amino acidpatterns. Flooded mown reed and unflooded burned reedwas compared to unmanaged control stands withcomparable hydrology and trophic level. Managementtook place in winter. The elimination of old culmsthrough mowing resulted in a lower ventilationefficiency due to a high counterpressure of rhizomes.The corresponding gas flow rates were reduced to 38%of the value in control stands, indicating a stronglyimpaired oxygen supply to basal and below-ground plantparts after mowing. Concomitantly, significantlyincreased levels of alanine and gamma-aminobutyricacid in basal culm internodes of shoots were measuredas signs of a metabolic shift due to hypoxic stress.Conversely, shoot loss by burning (without flooding)did not diminish ventilation efficiency and gas flowrate, i.e. oxygen supply to buried organs wasunaffected. Correspondingly, the level ofhypoxia-indicating amino acids (alanine,gamma-aminobutyric acid) did not indicate more severeoxygen deficiency in basal and below-ground plantparts of burned reed. It is concluded that the impactof mowing and burning on P. australisstronglydepends on the water level and on whether or notflooding occurs after the treatment. The mechanismresponsible for detrimental effects is probablyimpaired convective ventilation followed by hypoxia inbasal plant parts. This aspect should be taken intoaccount when mowing or burning in winter are used asmanagement tools for wetlands.