Identification and characterization of secreted and pathogenesis-related proteins in Ustilago maydis

Interactions between plants and fungal pathogens require a complex interplay at the plant–fungus interface. Extracellular effector proteins are thought to play a crucial role in establishing a successful infection. To identify pathogenesis-related proteins in Ustilago maydis we combined the isolation of secreted proteins using a signal sequence trap approach with bioinformatic analyses and the subsequent characterization of knock-out mutants. We identified 29 secreted proteins including hydrophobins and proteins with a repetitive structure similar to the repellent protein Rep1. Hum3, a protein containing both, a hydrophobin domain and a repetitive Rep1-like region, is shown to be processed during passage through the secretory pathway. While single knock-outs of hydrophobin or repellent-like genes did not affect pathogenicity, we found a strong effect of a double knock-out of hum3 and the repetitive rsp1. Yeast-like growth, mating, aerial hyphae formation and surface hydrophobicity were unaffected in this double mutant. However, pathogenic development in planta stops early after penetration leading to a complete loss of pathogenicity. This indicates that Hum3 and Rsp1 are pathogenicity proteins that share an essential function in early stages of the infection. Our results demonstrate that focusing on secreted proteins is a promising way to discover novel pathogenicity proteins that might be broadly applied to a variety of fungal pathogens

Steering Evolution with Sequential Therapy to Prevent the Emergence of Bacterial Antibiotic Resistance

The increasing rate of antibiotic resistance and slowing discovery of novel antibiotic treatments presents a growing threat to public health. Here, we consider a simple model of evolution in asexually reproducing populations which considers adaptation as a biased random walk on a fitness landscape. This model associates the global properties of the fitness landscape with the algebraic properties of a Markov chain transition matrix and allows us to derive general results on the non-commutativity and irreversibility of natural selection as well as antibiotic cycling strategies. Using this formalism, we analyze 15 empirical fitness landscapes of E. coli under selection by different β-lactam antibiotics and demonstrate that the emergence of resistance to a given antibiotic can be either hindered or promoted by different sequences of drug application. Specifically, we demonstrate that the majority, approximately 70%, of sequential drug treatments with 2–4 drugs promote resistance to the final antibiotic. Further, we derive optimal drug application sequences with which we can probabilistically ‘steer’ the population through genotype space to avoid the emergence of resistance. This suggests a new strategy in the war against antibiotic–resistant organisms: drug sequencing to shepherd evolution through genotype space to states from which resistance cannot emerge and by which to maximize the chance of successful therapy

Differentiation and vascularization of human preadipocytes in a polyurethane scaffold in vivo

In der Arbeit wurden humane Präadipozyten isoliert, amplifiziert und in Polyurethangerüste eingebracht. Diese wurden um die Leistenarterie von Nacktmäusen implantiert und nach definierten Zeitpunkten explantiert. Als Ergebnis wurde die Eignung von Polyurethan als Scaffold bestätigt, das Überleben der Präadipozyten nachgewiesen, und die Neovaskularisierung durch das femorale Gefäßbündel dargestellt. Die Differenzierung der Präadipozyten zu reifen Fettzellen war größtenteils negativ.In this study human preadipocytes were isolated, amplificated and brought into polyurethane scaffolds. These were implanted around die femoral vessels of nude mice and harvested after specific time periods. As results the applicability of polyurethane as a scaffold was prooved, the survival of preadipocytes was shown and the neovascularization of the femoral pedicle was presented. The differentiation of the adipose precursor cells into mature fat cells was mostly negativ

Acoustofluidic medium exchange for preparation of electrocompetent bacteria using channel wall trapping

Comprehensive integration of process steps into a miniaturised version of synthetic biology workflows remains a crucial task in automating the design of biosystems. However, each of these process steps has specific demands with respect to the environmental conditions, including in particular the composition of the surrounding fluid, which makes integration cumbersome. As a case in point, transformation, i.e. reprogramming of bacteria by delivering exogenous genetic material (such as DNA) into the cytoplasm, is a key process in molecular engineering and modern biotechnology in general. Transformation is often performed by electroporation, i.e. creating pores in the membrane using electric shocks in a low conductivity environment. However, cell preparation for electroporation can be cumbersome as it requires the exchange of growth medium (high-conductivity) for low-conductivity medium, typically performed via multiple time-intensive centrifugation steps. To simplify and miniaturise this step, we developed an acoustofluidic device capable of trapping the bacterium Escherichia coli non-invasively for subsequent exchange of medium, which is challenging in acoustofluidic devices due to detrimental acoustic streaming effects. With an improved etching process, we were able to produce a thin wall between two microfluidic channels, which, upon excitation, can generate streaming fields that complement the acoustic radiation force and therefore can be utilised for trapping of bacteria. Our novel design robustly traps Escherichia coli at a flow rate of 10 μL min−1 and has a cell recovery performance of 47 ± 3% after washing the trapped cells. To verify that the performance of the medium exchange device is sufficient, we tested the electrocompetence of the recovered cells in a standard transformation procedure and found a transformation efficiency of 8 × 105 CFU per μg of plasmid DNA. Our device is a low-volume alternative to centrifugation-based methods and opens the door for miniaturisation of a plethora of microbiological and molecular engineering protocols.ISSN:1473-0197ISSN:1473-018

Engineering of vascularized adipose constructs

Adipose tissue engineering offers a promising alternative to the current surgical techniques for the treatment of soft tissue defects. It is a challenge to find the appropriate scaffold that not only represents a suitable environment for cells but also allows fabrication of customized tissue constructs, particularly in breast surgery. We investigated two different scaffolds for their potential use in adipose tissue regeneration. Sponge-like polyurethane scaffolds were prepared by mold casting with methylal as foaming agent, whereas polycaprolactone scaffolds with highly regular stacked-fiber architecture were fabricated with fused deposition modeling. Both scaffold types were seeded with human adipose tissuederived precursor cells, cultured and implanted in nude mice using a femoral arteriovenous flow-through vessel loop for angiogenesis. In vitro, cells attached to both scaffolds and differentiated into adipocytes. In vivo, angiogenesis and adipose tissue formation were observed throughout both constructs after 2 and 4 weeks, with angiogenesis being comparable in seeded and unseeded constructs. Fibrous tissue formation and adipogenesis were more pronounced on polyurethane foam scaffolds than on polycaprolactone prototyped scaffolds. In conclusion, both scaffold designs can be effectively used for adipose tissue engineering

Variability of care practices for extremely early deliveries

Objectives: Assess temporal changes, intercenter variability, and birthing person (BP) factors relating to interventions for extremely early deliveries. Methods: Retrospective study of BPs and newborns delivered from 22-24 completed weeks at 13 US centers from 2011-2020. Rates of neonatology consultation, antenatal corticosteroids, cesarean delivery, live birth, attempted resuscitation (AR), and survival were assessed by epoch, center, and gestational age. Results: 2028 BPs delivering 2327 newborns were included. Rates increased in epoch 2-at 22 weeks: neonatology consultation (37.6 vs 64.3%, P \u3c .001), corticosteroids (11.4 vs 29.5%, P \u3c .001), live birth (66.2 vs 78.6%, P \u3c .001), AR (20.1 vs 36.9%, P \u3c .001), overall survival (3.0 vs 8.9%, P = .005); and at 23 weeks: neonatology consultation (73.0 vs 80.5%, P = .02), corticosteroids (63.7 vs 83.7%, P \u3c .001), cesarean delivery (28.0 vs 44.7%, P \u3c .001), live birth (88.1 vs 95.1%, P \u3c .001), AR (67.7 vs 85.2%, P \u3c .001), survival (28.8 vs 41.6%, P \u3c .001). Over time, intercenter variability increased at 22 weeks for corticosteroids (interquartile range 18.0 vs 42.0, P = .014) and decreased at 23 for neonatology consultation (interquartile range 23.0 vs 5.2, P = .045). In BP-level multivariate analysis, AR was associated with increasing gestational age and birth weight, Black BP race, previous premature delivery, and delivery center. Conclusions: Intervention rates for extremely early newborns increased and intercenter variability changed over time. In BP-level analysis, factors significantly associated with AR included Black BP race, previous premature delivery, and center

Large-Scale Assessment of Binding Free Energy Calculations in Active Drug Discovery Projects

Here we present an evaluation of the binding affinity prediction accuracy of the free energy calculation method FEP+ on internal active drug discovery projects and on a large new public benchmark set.<br /

Discovery of Cycloalkyl[<i>c</i>]thiophenes as Novel Scaffolds for Hypoxia-Inducible Factor-2α Inhibitors

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors induced in diverse pathophysiological settings. Inhibition of HIF-2α has become a strategy for cancer treatment since the discovery that small molecules, upon binding into a small cavity of the HIF-2α PAS B domain, can alter its conformation and disturb the activity of the HIF dimer complex. Herein, the design, synthesis, and systematic SAR exploration of cycloalkyl[c]thiophenes as novel HIF-2α inhibitors are described, providing the first chemotype featuring an alkoxy–aryl scaffold. X-ray data confirmed the ability of these inhibitors to induce perturbation of key amino acids by appropriately presenting key pharmacophoric elements in the hydrophobic cavity. Selected compounds showed inhibition of VEGF-A secretion in cancer cells and prevention of Arg1 expression and activity in IL4-stimulated macrophages. Moreover, in vivo target gene modulation was demonstrated with compound 35r. Thus, the disclosed HIF-2α inhibitors represent valuable tools for investigating selective HIF-2α inhibition and its effect on tumor biology