Early midcell localization of Escherichia coli PBP4 supports the function of peptidoglycan amidases

Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. Since most bacteria carry multiple enzymes carrying the same type of PG hydrolytic activity, we know little about the specific function of given enzymes. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that PBP4 interacts with NlpI and the FtsEX-interacting protein EnvC, an activator of amidases AmiA and AmiB, which are needed to generate denuded glycan strands to recruit the initiator of septal PG synthesis, FtsN. The domain 3 of PBP4 is needed for the interaction with NlpI and EnvC, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that the interaction of PBP4 with EnvC, whilst not absolutely necessary for mid-cell recruitment of either protein, coordinates the activities of PBP4 and the amidases, which affects the formation of denuded glycan strands that attract FtsN. Consistent with this model, we found that the divisome assembly at midcell was premature in cells lacking PBP4, illustrating how the complexity of interactions affect the timing of cell division initiation.     

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.     

Influence of promoter choice and trichostatin A treatment on expression of baculovirus delivered genes in mammalian cells

The expression of recombinant proteins following transduction of CHO cells with recombinant baculoviruses containing a mammalian expression cassette with the CMV-promoter is enhanced by the addition of trichostatin A (TSA), a specific histone deacetylase inhibitor. To further investigate the effect of TSA treatment on protein production following BacMam transduction, viruses containing various viral promoters (SV40, CMV, and RSV) and one cellular promoter (human ubiquitin C) were compared with regard to expression level of a gfp-luciferase fusion protein following transduction of CHO, COS-1, and HEK293 cells. The overall effect on expression appears to be cell specific, indicating that different mechanisms are active within different cell lines. Further, COS cells transfected with naked viral DNA, plasmids, and baculovirus particles were compared in regard to TSA treatment. The increase in reporter gene expression observed following BacMam transduction and TSA treatment were greater than those for transfection of either naked viral DNA or plasmid DNA.     

Modeling denitrification in aquatic sediments

Sediment denitrification is a major pathway of fixed nitrogen loss from aquatic systems. Due to technical difficulties in measuring this process and its spatial and temporal variability, estimates of local, regional and global denitrification have to rely on a combination of measurements and models. Here we review approaches to describing denitrification in aquatic sediments, ranging from mechanistic diagenetic models to empirical parameterizations of nitrogen fluxes across the sediment-water interface. We also present a compilation of denitrification measurements and ancillary data for different aquatic systems, ranging from freshwater to marine. Based on this data compilation we reevaluate published parameterizations of denitrification. We recommend that future models of denitrification use (1) a combination of mechanistic diagenetic models and measurements where bottom-waters are temporally hypoxic or anoxic, and (2) the much simpler correlations between denitrification and sediment oxygen consumption for oxic bottom waters. For our data set, inclusion of bottom water oxygen and nitrate concentrations in a multivariate regression did not improve the statistical fit.     

Exosomes surf on filopodia to enter cells at endocytic hot spots,traffic within endosomes,and are targeted to the ER

Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.     

Chlamydomonas raudensis (UWO 241), Chlorophyceae,exhibits the capacity for rapid D1 repair in response to chronic photoinhibition at low temperature1

Maximum photosynthetic capacity indicates that the Antarctic psychrophile Chlamydomonas raudensis H. Ettl UWO 241 is photosynthetically adapted to low temperature. Despite this finding, C. raudensis UWO 241 exhibited greater sensitivity to low‐temperature photoinhibition of PSII than the mesophile Chlamydomonas reinhardtii P. A. Dang. However, in contrast with results for C. reinhardtii, the quantum requirement to induce 50% photoinhibition of PSII in C. raudensis UWO 241 (50 μmol photons) was comparable at either 8°C or 29°C. To our knowledge, this is the first report of a photoautotroph whose susceptibility to photoinhibition is temperature independent. In contrast, the capacity of the psychrophile to recover from photoinhibition of PSII was sensitive to temperature and inhibited at 29°C. The maximum rate of recovery from photoinhibition of the psychrophile at 8°C was comparable to the maximum rate of recovery of the mesophile at 29°C. We provide evidence that photoinhibition in C. raudensis UWO 241 is chronic rather than dynamic. The photoinhibition‐induced decrease in the D1 content in C. raudensis recovered within 30 min at 8°C. Both the recovery of the D1 content as well as the initial fast phase of the recovery of F_v/F_m at 8°C were inhibited by lincomycin, a chloroplast protein synthesis inhibitor. We conclude that the susceptibility of C. raudensis UWO 241 to low‐temperature photoinhibition reflects its adaptation to low growth irradiance, whereas the unusually rapid rate of recovery at low temperature exhibited by this psychrophile is due to a novel D1 repair cycle that is adapted to and is maximally operative at low temperature.     

ATM requirement in gene expression responses to ionizing radiation in human lymphoblasts and fibroblasts

The heritable disorder ataxia telangiectasia (AT) is caused by mutations in the AT-mutated (ATM) gene with manifestations that include predisposition to lymphoproliferative cancers and hypersensitivity to ionizing radiation (IR). We investigated gene expression changes in response to IR in human lymphoblasts and fibroblasts from seven normal and seven AT-affected individuals. Both cell types displayed ATM-dependent gene expression changes after IR, with some responses shared and some responses varying with cell type and dose. Interestingly, after 5 Gy IR, lymphoblasts displayed ATM-independent responses not seen in the fibroblasts at this dose, which likely reflect signaling through ATM-related kinases, e.g., ATR, in the absence of ATM function.     

Diets of giants: the nutritional value of sauropod diet during the Mesozoic

A major uncertainty in estimating energy budgets and population densities of extinct animals is the carrying capacity of their ecosystems, constrained by net primary productivity (NPP) and its digestible energy content. The hypothesis that increases in NPP due to elevated atmospheric CO₂ contributed to the unparalleled size of the sauropods has recently been rejected, based on modern studies on herbivorous insects that imply a general, negative correlation of diet quality and increasing CO₂. However, the nutritional value of plants grown under elevated CO₂ levels might be very different for vertebrate megaherbivores than for insects. Here we show plant species‐specific responses in metabolizable energy and nitrogen content, equivalent to a two‐fold variation in daily food intake estimates for a typical sauropod, for dinosaur food plant analogues grown under CO₂ concentrations spanning estimates for Mesozoic atmospheric concentrations. Our results potentially rebut the hypothesis that constraints on sauropod diet quality were driven by Mesozoic CO₂ concentration.