Imaging local diffusion in microstructures using NV-based pulsed field gradient NMR

Understanding diffusion in microstructures plays a crucial role in many scientific fields, including neuroscience, cancer or energy research. While magnetic resonance (MR) methods are the gold standard for diffusion measurements, spatial encoding in MR imaging has limitations. Here, we introduce nitrogen-vacancy (NV) center based nuclear magnetic resonance (NMR) spectroscopy as a powerful tool to probe diffusion with an optical readouts. We have developed an experimental scheme combining pulsed gradient spin echo (PGSE) with optically detected NV-NMR spectroscopy, which allows for the local quantification of molecular diffusion and flow within microscopic sample volumes. We demonstrate correlated optical imaging with spatially resolved PGSE NV-NMR experiments probing anisotropic water diffusion within a model microstructure. Our optically detected PGSE NV-NMR technique opens up prospects for extending the current capabilities of investigating diffusion processes with the future potential of probing single cells, tissue microstructures, or ion mobility in thin film materials for battery applications.Comment: 37 pages, 5 figures, 2 table

Reduced mRNA Secondary-Structure Stability Near the Start Codon Indicates Functional Genes in Prokaryotes

Several recent studies have found that selection acts on synonymous mutations at the beginning of genes to reduce mRNA secondary-structure stability, presumably to aid in translation initiation. This observation suggests that a metric of relative mRNA secondary-structure stability, ZΔG, could be used to test whether putative genes are likely to be functionally important. Using the Escherichia coli genome, we compared the mean ZΔG of genes with known functions, genes with known orthologs, genes where function and orthology are unknown, and pseudogenes. Genes in the first two categories demonstrated similar levels of selection for reduced stability (increased ZΔG), whereas for pseudogenes stability did not differ from our null expectation. Surprisingly, genes where function and orthology were unknown were also not different from the null expectation, suggesting that many of these open reading frames are not functionally important. We extended our analysis by constructing a Bayesian phylogenetic mixed model based on data from 145 prokaryotic genomes. As in E. coli, genes with no known function had consistently lower ZΔG, even though we expect that many of the currently unannotated genes will ultimately have their functional utility discovered. Our findings suggest that functional genes tend to evolve increased ZΔG, whereas nonfunctional ones do not. Therefore, ZΔG may be a useful metric for identifying genes of potentially important function and could be used to target genes for further functional study

Expression Screening of Fusion Partners from an E. coli Genome for Soluble Expression of Recombinant Proteins in a Cell-Free Protein Synthesis System

While access to soluble recombinant proteins is essential for a number of proteome studies, preparation of purified functional proteins is often limited by the protein solubility. In this study, potent solubility-enhancing fusion partners were screened from the repertoire of endogenous E. coli proteins. Based on the presumed correlation between the intracellular abundance and folding efficiency of proteins, PCR-amplified ORFs of a series of highly abundant E. coli proteins were fused with aggregation-prone heterologous proteins and then directly expressed for quantitative estimation of the expression efficiency of soluble translation products. Through two-step screening procedures involving the expression of 552 fusion constructs targeted against a series of cytokine proteins, we were able to discover a number of endogenous E. coli proteins that dramatically enhanced the soluble expression of the target proteins. This strategy of cell-free expression screening can be extended to quantitative, global analysis of genomic resources for various purposes.National Research Foundation of KoreaKorea (South). Ministry of Education, Science and Technology (MEST) (grant 2011K000841)Korea (South). Ministry of Education, Science and Technology (MEST) (grant 2011-0027901

Perceptions of involvement in advance care planning and emotional functioning in patients with advanced cancer

Purpose: Advance Care Planning (ACP) is positively associated with the quality of care, but its impact on emotional functioning is ambiguous. This study investigated the association between perceptions of ACP involvement and emotional functioning in patients with advanced cancer. Methods: This study analyzed baseline data of 1,001 patients of the eQuiPe study, a prospective, longitudinal, multicenter, observational study on quality of care and quality of life in patients with advanced cancer in the Netherlands. Patients with metastatic solid cancer were asked to participate between November 2017 and January 2020. Patients’ perceptions of ACP involvement were measured by three self-administered statements. Emotional functioning was measured by the EORTC-QLQ-C30. A linear multivariable regression analysis was performed while taking gender, age, migrant background, education, marital status, and symptom burden into account. Results: The majority of patients (87%) reported that they were as much involved as they wanted to be in decisions about their future medical treatment and care. Most patients felt that their relatives (81%) and physicians (75%) were familiar with their preferences for future medical treatment and care. A positive association was found between patients’ perceptions of ACP involvement and their emotional functioning (b=0.162, p<0.001, 95%CI[0.095;0.229]) while controlling for relevant confounders. Conclusions: Perceptions of involvement in ACP are positively associated with emotional functioning in patients with advanced cancer. Future studies are needed to further investigate the effect of ACP on emotional functioning. Trial registration number: NTR6584 Date of registration: 30 June 2017 Implications for Cancer Survivors: Patients’ emotional functioning might improve from routine discussions regarding goals of future care. Therefore, integration of ACP into palliative might be promising

Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages

notes: PMCID: PMC4006850types: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov'tCandida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.Deutsche ForschungsgemeinschaftNational Institutes for HealthWellcome TrustBBSR

Programmable Ligand Detection System in Plants through a Synthetic Signal Transduction Pathway

There is an unmet need to monitor human and natural environments for substances that are intentionally or unintentionally introduced. A long-sought goal is to adapt plants to sense and respond to specific substances for use as environmental monitors. Computationally re-designed periplasmic binding proteins (PBPs) provide a means to design highly sensitive and specific ligand sensing capabilities in receptors. Input from these proteins can be linked to gene expression through histidine kinase (HK) mediated signaling. Components of HK signaling systems are evolutionarily conserved between bacteria and plants. We previously reported that in response to cytokinin-mediated HK activation in plants, the bacterial response regulator PhoB translocates to the nucleus and activates transcription. Also, we previously described a plant visual response system, the de-greening circuit, a threshold sensitive reporter system that produces a visual response which is remotely detectable and quantifiable.We describe assembly and function of a complete synthetic signal transduction pathway in plants that links input from computationally re-designed PBPs to a visual response. To sense extracellular ligands, we targeted the computational re-designed PBPs to the apoplast. PBPs bind the ligand and develop affinity for the extracellular domain of a chemotactic protein, Trg. We experimentally developed Trg fusions proteins, which bind the ligand-PBP complex, and activate intracellular PhoR, the HK cognate of PhoB. We then adapted Trg-PhoR fusions for function in plants showing that in the presence of an external ligand PhoB translocates to the nucleus and activates transcription. We linked this input to the de-greening circuit creating a detector plant.Our system is modular and PBPs can theoretically be designed to bind most small molecules. Hence our system, with improvements, may allow plants to serve as a simple and inexpensive means to monitor human surroundings for substances such as pollutants, explosives, or chemical agents