Computational Characterization of the Dish-In-A-Dish, A High Yield Culture Platform for Endothelial Shear Stress Studies on the Orbital Shaker

Endothelial cells sense and respond to shear stress. Different in vitro model systems have been used to study the cellular responses to shear stress, but these platforms do not allow studies on high numbers of cells under uniform and controllable shear stress. The annular dish, or dish-in-a-dish (DiaD), on the orbital shaker has been proposed as an accessible system to overcome these challenges. However, the influence of the DiaD design and the experimental parameters on the shear stress patterns is not known. In this study, we characterize different designs and experimental parameters (orbit size, speed and fluid height) using computational fluid dynamics. We optimize the DiaD for an atheroprotective flow, combining high shear stress levels with a low oscillatory shear index (OSI). We find that orbit size determines the DiaD design and parameters. The shear stress levels increase with increasing rotational speed and fluid height. Based on our optimization, we experimentally compare the 134/56 DiaD with regular dishes for cellular alignment and KLF2, eNOS, CDH2 and MCP1 expression. The calculated OSI has a strong impact on alignment and gene expression, emphasizing the importance of characterizing shear profiles in orbital setups

MEK inhibition is a promising therapeutic strategy for MLL-rearranged infant acute lymphoblastic leukemia patients carrying RAS mutations

Acute lymphoblastic leukemia (ALL) in infants is an aggressive malignancy with a poor clinical outcome, and is characterized by translocations of the Mixed Lineage Leukemia (MLL) gene. Previously, we identified RAS mutations in 14-24% of infant ALL patients, and showed that the presence of a RAS mutation decreased the survival chances even further. We hypothesized that targeting the RAS signaling pathway could be a therapeutic strategy for RAS-mutant infant ALL patients. Here we show that the MEK inhibitors Trametinib, Selumetinib and MEK162 severely impair primary RAS-mutant MLL-rearranged infant ALL cells in vitro. While all RAS-mutant samples were sensitive to MEK inhibitors, we found both sensitive and resistant samples among RAS-wildtype cases. We confirmed enhanced RAS pathway signaling in RASmutant samples, but found no apparent downstream over-activation in the wildtype samples. However, we did confirm that MEK inhibitors reduced p-ERK levels, and induced apoptosis in the RAS-mutant MLL-rearranged ALL cells. Finally, we show that MEK inhibition synergistically enhances prednisolone sensitivity,

Construction of a secondary metabolite deficient penicillium chrysogenum strain as a generic production host for secondary metabolites

Secondary metabolism of the filamentous fungus Penicillium chrysogenum has been intensively explored to relate specific secondary metabolites to their respective biosynthetic gene clusters. We have removed the three main biosynthetic gene clusters that specify the antibiotic penicillin, the mycotoxin roquefortine and the yellow pigment chrysogine, in order to generate a secondary metabolite deficient strain. This strain produces increased levels of other secondary metabolites some of which have not been detected before. The strain and its biosynthetic potential will now be further investigated for the expression of novel enzymes and biosynthetic pathways to make the synthesis of antibiotics and other secondary metabolites more specific and efficient. Using structure guided protein engineering new enzymes will be further designed and optimized for the construction of a newly designed biosynthetic pathway into a novel platform strain

Genomic mutational analysis of the impact of the classical strain improvement program on β-lactam producing Penicillium chrysogenum

BACKGROUND: Penicillium chrysogenum is a filamentous fungus that is employed as an industrial producer of β-lactams. The high β-lactam titers of current strains is the result of a classical strain improvement program (CSI) starting with a wild-type like strain more than six decades ago. This involved extensive mutagenesis and strain selection for improved β-lactam titers and growth characteristics. However, the impact of the CSI on the secondary metabolism in general remains unknown. RESULTS: To examine the impact of CSI on secondary metabolism, a comparative genomic analysis of β-lactam producing strains was carried out by genome sequencing of three P. chrysogenum strains that are part of a lineage of the CSI, i.e., strains NRRL1951, Wisconsin 54-1255, DS17690, and the derived penicillin biosynthesis cluster free strain DS68530. CSI has resulted in a wide spread of mutations, that statistically did not result in an over- or underrepresentation of specific gene classes. However, in this set of mutations, 8 out of 31 secondary metabolite genes (20 polyketide synthases and 11 non-ribosomal peptide synthetases) were targeted with a corresponding and progressive loss in the production of a range of secondary metabolites unrelated to β-lactam production. Additionally, key Velvet complex proteins (LeaA and VelA) involved in global regulation of secondary metabolism have been repeatedly targeted for mutagenesis during CSI. Using comparative metabolic profiling, the polyketide synthetase gene cluster was identified that is responsible for sorbicillinoid biosynthesis, a group of yellow-colored metabolites that are abundantly produced by early production strains of P. chrysogenum. CONCLUSIONS: The classical industrial strain improvement of P. chrysogenum has had a broad mutagenic impact on metabolism and has resulted in silencing of specific secondary metabolite genes with the concomitant diversion of metabolism towards the production of β-lactams

Serendipitous discovery of radio flaring behaviour from a nearby M dwarf with MeerKAT

We report on the detection of MKT J174641.0$-$321404, a new radio transient found in untargeted searches of wide-field MeerKAT radio images centred on the black hole X-ray binary H1743$-$322. MKT J174641.0$-$321404 is highly variable at 1.3 GHz and was detected three times during 11 observations of the field in late 2018, reaching a maximum flux density of 590 $\pm$ 60 $\mu$Jy. We associate this radio transient with a high proper motion, M dwarf star SCR~1746$-$3214 12 pc away from the Sun. Multiwavelength observations of this M dwarf indicate flaring activity across the electromagnetic spectrum, consistent with emission expected from dMe stars, and providing upper limits on quiescent brightness in both the radio and X-ray regimes. \textit{TESS} photometry reveals a rotational period for SCR~1746$-$3214 of $0.2292 \pm 0.0025$ days, which at its estimated radius makes the star a rapid rotator, comparable to other low mass systems. Dedicated spectroscopic follow up confirms the star as a mid-late spectral M dwarf with clear magnetic activity indicated by strong H$\alpha$ emission. This transient's serendipitous discovery by MeerKAT, along with multiwavelength characterisation, make it a prime demonstration of both the capabilities of the current generation of radio interferometers and the value of simultaneous observations by optical facilities such as MeerLICHT. Our results build upon the literature of of M dwarfs' flaring behaviour, particularly relevant to the habitability of their planetary systems.Comment: Accepted to MNRAS, 11 pages, 9 figure

Identification of genes transcriptionally responsive to the loss of MLL fusions in MLL-rearranged acute lymphoblastic leukemia

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year) is characterized by high relapse rates and a dismal prognosis. To facilitate the discovery of novel therapeutic targets, we here searched for genes directly influenced by the repression of various MLL fusions. Methods For this, we performed gene expression profiling after siRNA-mediated repression of MLLAF4, MLL-ENL, and AF4-MLL in MLL -rearranged ALL cell line models. The obtained results were compared with various already established gene signatures including those consisting of known MLL-AF4 target genes, or those associated with primary MLL-rearranged infant ALL samples. Results Genes that were down-regulated in response to the repression of MLL-AF4 and MLL-ENL appeared characteristically expressed in primary MLL-rearranged infant ALL samples, and often represented known MLL-AF4 targets genes. Genes that were up-regulated in response to the repression of MLL-AF4 and MLL-ENL often represented genes typically silenced by promoter hypermethylation in MLL-rearranged infant ALL. Genes that were aff

Evaluation of pliable bioresorbable, elastomeric aortic valve prostheses in sheep during 12 months post implantation

Pliable microfibrous, bioresorbable elastomeric heart valve prostheses are investigated in search of sustainable heart valve replacement. These cell-free implants recruit cells and trigger tissue formation on the valves in situ. Our aim is to investigate the behaviour of these heart valve prostheses when exposed to the high-pressure circulation. We conducted a 12-month follow-up study in sheep to evaluate the in vivo functionality and neo-tissue formation of these valves in the aortic position. All valves remained free from endocarditis, thrombotic complications and macroscopic calcifications. Cell colonisation in the leaflets was mainly restricted to the hinge area, while resorption of synthetic fibers was limited. Most valves were pliable and structurally intact (10/15), however, other valves (5/15) showed cusp thickening, retraction or holes in the leaflets. Further research is needed to assess whether in-situ heart valve tissue engineering in the aortic position is possible or whether non-resorbable synthetic pliable prostheses are preferred.</p

FRB 20210405I: a nearby Fast Radio Burst localised to sub-arcsecond precision with MeerKAT

We present the first sub-arcsecond localised Fast Radio Burst (FRB) detected using MeerKAT. FRB 20210405I was detected in the incoherent beam using the MeerTRAP pipeline on 2021 April 05 with a signal to noise ratio of 140.8 and a dispersion measure of 565.17 pc cm$^{-3}$. It was detected while MeerTRAP was observing commensally with the ThunderKAT large survey project, and was sufficiently bright that we could use the ThunderKAT 8s images to localise the FRB. Two different models of the dispersion measure in the Milky Way and halo suggest that the source is either right at the edge of the Galaxy, or outside. This highlights the uncertainty in the Milky Way dispersion measure models, particularly in the Galactic Plane, and the uncertainty of Milky Way halo models. Further investigation and modelling of these uncertainties will be facilitated by future detections and localisations of nearby FRBs. We use the combined localisation, dispersion measure, scattering, specific luminosity and chance coincidence probability information to find that the origin is most likely extra-galactic and identify the likely host galaxy of the FRB: 2MASS J1701249$-$4932475. Using SALT spectroscopy and archival observations of the field, we find that the host is a disk/spiral galaxy at a redshift of $z=0.066$.Comment: 15 pages, 4 tables, 10 figures. Accepted to MNRA