Exonuclease-enhanced prime editors.

Prime editing (PE) is a powerful gene-editing technique based on targeted gRNA-templated reverse transcription and integration of the de novo synthesized single-stranded DNA. To circumvent one of the main bottlenecks of the method, the competition of the reverse-transcribed 3' flap with the original 5' flap DNA, we generated an enhanced fluorescence-activated cell sorting reporter cell line to develop an exonuclease-enhanced PE strategy ('Exo-PE') composed of an improved PE complex and an aptamer-recruited DNA-exonuclease to remove the 5' original DNA flap. Exo-PE achieved better overall editing efficacy than the reference PE2 strategy for insertions ≥30 base pairs in several endogenous loci and cell lines while maintaining the high editing precision of PE2. By enabling the precise incorporation of larger insertions, Exo-PE complements the growing palette of different PE tools and spurs additional refinements of the PE machinery.

Hybrid Plasmonic Nanomaterials for Hydrogen Generation and Carbon Dioxide Reduction.

The successful development of artificial photosynthesis requires finding new materials able to efficiently harvest sunlight and catalyze hydrogen generation and carbon dioxide reduction reactions. Plasmonic nanoparticles are promising candidates for these tasks, due to their ability to confine solar energy into molecular regions. Here, we review recent developments in hybrid plasmonic photocatalysis, including the combination of plasmonic nanomaterials with catalytic metals, semiconductors, perovskites, 2D materials, metal-organic frameworks, and electrochemical cells. We perform a quantitative comparison of the demonstrated activity and selectivity of these materials for solar fuel generation in the liquid phase. In this way, we critically assess the state-of-the-art of hybrid plasmonic photocatalysts for solar fuel production, allowing its benchmarking against other existing heterogeneous catalysts. Our analysis allows the identification of the best performing plasmonic systems, useful to design a new generation of plasmonic catalysts.

Extensive regulation of enzyme activity by phosphorylation in Escherichia coli.

Protein serine/threonine/tyrosine (S/T/Y) phosphorylation is an essential and frequent post-translational modification in eukaryotes, but historically has been considered less prevalent in bacteria because fewer proteins were found to be phosphorylated and most proteins were modified to a lower degree. Recent proteomics studies greatly expanded the phosphoproteome of Escherichia coli to more than 2000 phosphorylation sites (phosphosites), yet mechanisms of action were proposed for only six phosphosites and fitness effects were described for 38 phosphosites upon perturbation. By systematically characterizing functional relevance of S/T/Y phosphorylation in E. coli metabolism, we found 44 of the 52 mutated phosphosites to be functional based on growth phenotypes and intracellular metabolome profiles. By effectively doubling the number of known functional phosphosites, we provide evidence that protein phosphorylation is a major regulation process in bacterial metabolism. Combining in vitro and in vivo experiments, we demonstrate how single phosphosites modulate enzymatic activity and regulate metabolic fluxes in glycolysis, methylglyoxal bypass, acetate metabolism and the split between pentose phosphate and Entner-Doudoroff pathways through mechanisms that include shielding the substrate binding site, limiting structural dynamics, and disrupting interactions relevant for activity in vivo.

Non-invasive and high-throughput interrogation of exon-specific isoform expression.

Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

Conservation of metabolic regulation by phosphorylation and non-covalent small-molecule interactions.

Here, we review extant observations of protein phosphorylation and small-molecule interactions in metabolism and ask which of their specific regulatory functions are conserved in Escherichia coli and Homo sapiens. While the number of phosphosites is dramatically higher in humans, the number of metabolite-protein interactions remains largely constant. Moreover, we found the regulatory logic of metabolite-protein interactions, and in many cases also the effector molecules, to be conserved. Post-translational regulation through phosphorylation does not appear to replace this regulation in human but rather seems to add additional opportunities for fine-tuning and more complex responses. The abundance of metabolite-protein interactions in metabolism, their conserved cross-species abundance, and the apparent conservation of regulatory logic across enormous phylogenetic distance demonstrate their relevance for maintaining cellular homeostasis in these ancient biological processes.

An Electrically Conducting Three-Dimensional Iron-Catecholate Porous Framework.

We report the synthesis of a unique cubic metal-organic framework (MOF), Fe-HHTP-MOF, comprising hexahydroxytriphenylene (HHTP) supertetrahedral units and FeIII ions, arranged in a diamond topology. The MOF is synthesized under solvothermal conditions, yielding a highly crystalline, deep black powder, with crystallites of 300-500 nm size and tetrahedral morphology. Nitrogen sorption analysis indicates a highly porous material with a surface area exceeding 1400 m2 g-1 . Furthermore, Fe-HHTP-MOF shows broadband absorption from 475 up to 1900 nm with excellent absorption capability of 98.5 % of the incoming light over the visible spectral region. Electrical conductivity measurements of pressed pellets reveal a high intrinsic electrical conductivity of up to 10-3  S cm-1 . Quantum mechanical calculations predict Fe-HHTP-MOF to be an efficient electron conductor, exhibiting continuous charge-carrier pathways throughout the structure.

Nonradiative Energy Transfer between Thickness-Controlled Halide Perovskite Nanoplatelets.

Despite showing great promise for optoelectronics, the commercialization of halide perovskite nanostructure-based devices is hampered by inefficient electrical excitation and strong exciton binding energies. While transport of excitons in an energy-tailored system via Förster resonance energy transfer (FRET) could be an efficient alternative, halide ion migration makes the realization of cascaded structures difficult. Here, we show how these could be obtained by exploiting the pronounced quantum confinement effect in two-dimensional CsPbBr3-based nanoplatelets (NPls). In thin films of NPls of two predetermined thicknesses, we observe an enhanced acceptor photoluminescence (PL) emission and a decreased donor PL lifetime. This indicates a FRET-mediated process, benefitted by the structural parameters of the NPls. We determine corresponding transfer rates up to k FRET = 0.99 ns-1 and efficiencies of nearly ηFRET = 70%. We also show FRET to occur between perovskite NPls of other thicknesses. Consequently, this strategy could lead to tailored energy cascade nanostructures for improved optoelectronic devices.

Further investigations of the IgE response to tetanus and diphtheria following covaccination with acellular rather than cellular Bordetella pertussis.

BACKGROUND: It has previously been shown in an uncontrolled study that the IgE response to vaccine antigens is downregulated by co-vaccination with cellular Bordetella pertussis vaccine. METHODS: In the present study, we compared in a controlled trial the humoral immune response to diphtheria toxoid (D) and tetanus toxoid (T) in relation to co-vaccinated cellular or acellular B pertussis vaccine. IgE, IgG4, and IgG to D and T were analyzed at 2, 7, and 12 months of age in sera of children vaccinated with D and T (DT, N = 68), cellular (DTPw, N = 68), 2- or 5-component acellular B pertussis vaccine (DTPa2, N = 64; DTPa5, N = 65). RESULTS: One month after vaccination, D-IgE was detected in 10% sera of DTPw-vaccinated children, whereas vaccination in the absence of whole-cell pertussis resulted in 50%-60% IgE positivity. Six months after vaccination, the IgE antibody levels were found to be more persistent than the IgG antibodies. These diphtheria findings were mirrored by those for tetanus. Only minor differences between vaccine groups were found with regard to D-IgG and T-IgG. No immediate-type allergic reactions were observed. CONCLUSION: Cellular (but not acellular) B pertussis vaccine downregulates IgE to co-vaccinated antigens in infants. We assume that the absence of immediate-type allergic reactions is due to the high levels of IgG antibodies competing with IgE antibodies.

Fungicide resistance toward fludioxonil conferred by overexpression of the phosphatase gene MoPTP2 in Magnaporthe oryzae.

The fungicide fludioxonil causes hyperactivation of the Hog1p MAPK within the high-osmolarity glycerol signaling pathway essential for osmoregulation in pathogenic fungi. The molecular regulation of MoHog1p phosphorylation is not completely understood in pathogenic fungi. Thus, we identified and characterized the putative MoHog1p-interacting phosphatase gene MoPTP2 in the filamentous rice pathogen Magnaporthe oryzae. We found overexpression of MoPTP2 conferred fludioxonil resistance in M. oryzae, whereas the 'loss of function' mutant ΔMoptp2 was more susceptible toward the fungicide. Additionally, quantitative phosphoproteome profiling of MoHog1p phosphorylation revealed lower phosphorylation levels of MoHog1p in the MoPtp2p overexpression mutant compared to the wild-type strain, whereas MoHog1p phosphorylation increased in the ΔMoptp2 mutant. Furthermore, we identified a set of MoHog1p-dependent genes regulated by the MoPtp2p expression level. Our results indicate that the phosphatase MoPtp2p is involved in the regulation of MoHog1p phosphorylation and that overexpression of the gene MoPTP2 is a novel molecular mechanism of fungicide resistance.

Thermal Disproportionation of Oxo-Functionalized Graphene.

Graphene production by wet chemistry is an ongoing scientific challenge. Controlled oxidation of graphite introduces oxo functional groups; this material can be processed and converted back to graphene by reductive defunctionalization. Although thermal processing yields conductive carbon, a ruptured and undefined carbon lattice is produced as a consequence of CO2 formation. This thermal process is not understood, but it is believed that graphene is not accessible. Here, we thermally process oxo-functionalized graphene (oxo-G) with a low (4-6 %) and high degree of functionalization (50-60 %) and find on the basis of Raman spectroscopy and transmission electron microscopy performed at atomic resolution (HRTEM) that thermal processing leads predominantly to an intact carbon framework with a density of lattice defects as low as 0.8 %. We attribute this finding to reorganization effects of oxo groups. This finding holds out the prospect of thermal graphene formation from oxo-G derivatives.

Identification of novel metabolic interactions controlling carbon flux from xylose to ethanol in natural and recombinant yeasts.

BACKGROUND: Unlike xylose-converting natural yeasts, recombinant strains of Saccharomyces cerevisiae expressing the same xylose assimilation pathway produce under anaerobic conditions xylitol rather than ethanol from xylose at low specific xylose conversion rates. Despite intense research efforts over the last two decades, differences in these phenotypes cannot be explained by current metabolic and kinetic models. To improve our understanding how metabolic flux of xylose carbon to ethanol is controlled, we developed a novel kinetic model based on enzyme mechanisms and applied quantitative metabolite profiling together with enzyme activity analysis to study xylose-to-ethanol metabolisms of Candida tenuis CBS4435 (q xylose = 0.10 g/gdc/h, 25 °C; Y ethanol = 0.44 g/g; Y xylitol = 0.09 g/g) and the recombinant S. cerevisiae strain BP000 (q xylose = 0.07 g/gdc/h, 30 °C; Y ethanol = 0.24 g/g; Y xylitol = 0.43 g/g), both expressing the same xylose reductase (XR), comprehensively. RESULTS: Results from strain-to-strain metabolic control analysis indicated that activity levels of XR and the maximal flux capacity of the upper glycolysis (UG; both ≥ tenfold higher in CBS4435) contributed predominantly to phenotype differentiation while reactions from the oxidative pentose phosphate pathway played minor roles. Intracellular metabolite profiles supported results obtained from kinetic modeling and indicated a positive correlation between pool sizes of UG metabolites and carbon flux through the UG. For CBS4435, fast carbon flux through the UG could be associated with an allosteric control of 6-phosphofructokinase (PFK) activity by fructose 6-phosphate. The ability of CBS4435 to keep UG metabolites at high levels could be explained by low glycerol 3-phosphate phosphatase (GPP, 17-fold lower in CBS4435) and high XR activities. CONCLUSIONS: By applying a systems biology approach in which we combined results obtained from metabolic control analysis based on kinetic modeling with data obtained from quantitative metabolite profiling and enzyme activity analyses, we could provide new insights into metabolic and kinetic interactions contributing to the control of carbon flux from xylose to ethanol. Supported by evidences presented two new targets, PFK and GPP, could be identified that aside from XR play pivotal roles in phenotype differentiation. Design of efficient and fast microbial ethanol producers in the future can certainly benefit from results presented in this study.

Similar Occurrence of Febrile Episodes Reported in Non-Atopic Children at Three to Five Years of Age after Prebiotics Supplemented Infant Formula.

This is a follow up study of a multicenter randomised placebo-controlled trial in seven centres in five West European countries. The RCT assessed the effect of infant formula supplemented with a mixture of prebiotics (with neutral short-chain and long-chain oligosaccharides and pectin-derived acidic oligosaccharides) during infancy in term-born children (n=1130). In the follow-up study 672 children (60% of the study population) participated: 232 (56%) from the prebiotics group (PG), 243 (58%) from the control group (CG), and 197 (66%) from the non-randomised breast-fed group (BG). The primary outcome was the occurrence of febrile episodes at three to five years of age prospectively documented by the parents: in the PG 1.17 (interquartile range 0.50-2.08) episodes per year versus 1.20 (0.52-2.57) in the CG; and 1.48 (0.65-2.60) in the BG. This specific prebiotics mixture given during infancy in healthy non-atopic subjects does not decrease febrile episodes and therefore seems not to prevent infection between their third and fifth birthday.

Density Functional Investigation of the Adsorption of Isooctane, Ethanol, and Acetic Acid on a Water-Covered Fe(100) Surface.

The presence of water in biofuels poses the question of how it affects the frictional performance of additives in fuels containing organic substances. To investigate the effect of water on the adsorption of molecules present in fuel and its additives we simulated within the framework of density functional theory the adsorption of ethanol, isooctane (2,2,4-trimethylpentane), and acetic acid on a bare and a water-covered Fe(100) surface. Van der Waals interactions are taken into account in our computations. In those molecules, where dispersion forces contribute significantly to the binding mechanism, the water layer has a stronger screening effect. Additionally, this effect can be enhanced by the presence of polar functional groups in the molecule. Thus, with the introduction of a water layer, the adsorption energy of isooctane and ethanol is reduced but it is increased in the case of the acetic acid. The adsorption configuration of ethanol is changed, while the one of acetic acid is moderately, and for isooctane only very slightly altered. Therefore, the effect of a water layer in the adsorption of organic molecules on an Fe(100) surface strongly depends on the type of bond and consequently, so do the tribological properties.

Harnessing Candida tenuis and Pichia stipitis in whole-cell bioreductions of o-chloroacetophenone: stereoselectivity, cell activity, in situ substrate supply and product removal.

Generally, recombinant and native microorganisms can be employed as whole-cell catalysts. The application of native hosts, however, shortens the process development time by avoiding multiple steps of strain construction. Herein, we studied the NAD(P)H-dependent reduction of o-chloroacetophenone by isolated xylose reductases and their native hosts Candida tenuis and Pichia stipitis. The natural hosts were benchmarked against Escherichia coli strains co-expressing xylose reductase and a dehydrogenase for co-enzyme recycling. Xylose-grown cells of C. tenuis and P. stipitis displayed specific o-chloroacetophenone reductase activities of 366 and 90 U gCDW (-1) , respectively, in the cell-free extracts. Fresh biomass was employed in batch reductions of 100 mM o-chloroacetophenone using glucose as co-substrate. Reaction stops at a product concentration of about 15 mM, which suggests sensitivity of the catalyst towards the formed product. In situ substrate supply and product removal by the addition of 40% hexane increased catalyst stability. Optimisation of the aqueous phase led to a (S)-1-(2-chlorophenyl)ethanol concentration of 71 mM (ee > 99.9%) obtained with 44 gCDW L(-1) of C. tenuis. The final difference in productivities between native C. tenuis and recombinant E. coli was < 1.7-fold. The optically pure product is a required key intermediate in the synthesis of a new class of chemotherapeutic substances (polo-like kinase 1 inhibitors).

Antibiotic use in infants in the first year of life in five European countries.

AIM: To assess in infants the number of illness episodes treated with antibiotics and prescription rates in five European countries. METHODS: This study was embedded in a multicenter nutritional intervention study and was conducted in five European countries. Infants were followed until 1 year of age. Illness episodes and prescriptions of systemic antibiotics were recorded by the parents. RESULTS: Illness episodes were caused by upper respiratory tract infections (URTIs) in 55-64% and by otitis media (OM) in 2-6.8%. URTIs were statistically significant and more frequently treated with antibiotics in Italy (18.8%), and less frequently in Switzerland (1.4%). OM was statistically significant and less frequently treated with antibiotics in the Netherlands (55%) when compared to Italy (82%). The antibiotic prescription rate varied between countries, ranging from 0.2 to 1.3 prescriptions per infant per year. CONCLUSIONS: As the frequency of illness episodes did not differ between countries, other factors, such as physician's attitude, parental pressure or other socio-economic determinants, most likely play a role in antibiotic prescribing habits in the first year of life.

Serum retinoic acid and atopy among children of different ethnic origin living in Germany.

We hypothesized that higher provitamin A carotenoid serum levels may be associated with higher concentrations of all-trans retinoic acid (ATRA) and atopy. Concentration of ATRA was measured by high-performance liquid chromatography in sera from German domestic and Turkish migrants' children. ATRA serum levels were significantly higher in German children if compared with Turkish children and correlated with those of ß-carotene (rs = 0.692) and other provitamin A carotenoids. They did not differ significantly between atopic and nonatopic individuals. Serum levels of ATRA are related to those of provitamin A carotenoids but are not directly related to atopy in the present study.

Somatoform respiratory disorders in children and adolescents-proposals for a practical approach to definition and classification.

Somatoform respiratory disorders represent conditions with dysfunctional breathing unexplained by structural abnormalities. This heterogeneous group includes disorders with neural dysregulation of respiration (vocal cord dysfunction) or with dysregulation of the respiratory pattern (hyperventilation, sighing dyspnea), psychogenic disorders such as unjustified anxiety of suffocation, and stereotype conditions such as throat clearing or habit cough. Many symptoms are nonspecific and largely overlap with respiratory disease symptoms of somatic etiology. Most patients will present in a nonspecialized clinical setting. This article provides symptom-based criteria for the definition of somatoform respiratory disorders and their differentiation from somatic disease. Emphasis is put on clinical criteria which can be easily integrated in a routine setting. Owing to the multifaceted etiology of somatoform respiratory disorders therapeutic approaches integrating somatic medicine, respiratory therapy and psychology are crucial. The introduction of defined clinical criteria may facilitate the discrimination of somatoform respiratory disorders from somatic disorders in routine patient encounters and avoid therapeutic detours.