Fusion of SpCas9 to E.coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells

Mammalian cells repair double-strand DNA breaks (DSB) by a range of different pathways following DSB induction by the engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9. While CRISPR-Cas9 thus enables predesigned modifications of the genome, applications of CRISPR-Cas9-mediated genome-editing are frequently hampered by the unpredictable and varying pathways for DSB repair in mammalian cells. Here we present a strategy of fusing Cas9 to recombinant proteins for fine-tuning of the DSB repair preferences in mammalian cells. By fusing Streptococcus Pyogenes Cas9 (SpCas9) to the recombinant protein A (Rec A, NP_417179.1) from E. coli, we create a recombinant Cas9 protein (rSpCas9) which enhances the generation of indel mutations at DSB sites in mammalian cells, increases the frequency of DSB repair by homology-directed single-strand annealing (SSA), and represses homology-directed gene conversion by approximately 33%. Our study thus proves for the first time that fusing SpCas9 to recombinant proteins can influence the balance between DSB repair pathways in mammalian cells. This approach may form the basis for further investigations of the applications of recombinant Cas9 proteins to fine-tuning DSB repair pathways in eukaryotic cells

The most vagile host as the main determinant of population connectivity in marine macroparasites

Although molecular ecology of macroparasites is still in its infancy, general patterns are beginning to emerge, e.g. that the most vagile host in a complex life cycle is the main determinantof the population genetic structure of their parasites. This insight stems from the observation that populations of parasites with only freshwater hosts are more structured than those with terrestrial or airborne hosts. Until now, the same has not been tested for marine systems, where, in theory, a fully marine life cycle might sustain high dispersal rates because of the absence of Obvious physical barriers in the sea. Here, we tested whether a marine trematode parasite that utilises migratory birds exhibited weaker population genetic structure than those whose life cycle utilises marine fish as the vagile host. Part of the mitochondrial cytochrome c oxidase 1 (COI) gene wassequenced from individual sporocysts from populations along the Atlantic coast of Europe and North Africa. Strong population structure (Φ-ST = 0.25, p < 0.0001) was found in the fully marinetrematode Bucephalus minimus (hosted by fish), while no significant structure (Φ-ST = 0.015, p = 0.19257) was detected in Gymnophallus choledochus (hosted by birds). However, demographicmodels indicate recent colonisation rather than high dispersal as an alternative explanation of the low levels of structure observed in G. choledochus. Our study is the first to identify significant genetic population structure in a marine autogenic parasite, suggesting that connectivity between populations of marine parasites can be limited despite the general potential for high dispersal of their hosts in the marine environment

Bipolar querying of valid-time intervals subject to uncertainty

Databases model parts of reality by containing data representing properties of real-world objects or concepts. Often, some of these properties are time-related. Thus, databases often contain data representing time-related information. However, as they may be produced by humans, such data or information may contain imperfections like uncertainties. An important purpose of databases is to allow their data to be queried, to allow access to the information these data represent. Users may do this using queries, in which they describe their preferences concerning the data they are (not) interested in. Because users may have both positive and negative such preferences, they may want to query databases in a bipolar way. Such preferences may also have a temporal nature, but, traditionally, temporal query conditions are handled specifically. In this paper, a novel technique is presented to query a valid-time relation containing uncertain valid-time data in a bipolar way, which allows the query to have a single bipolar temporal query condition

Bacterial chemoreceptor arrays are hexagonally packed trimers of receptor dimers networked by rings of kinase and coupling proteins

FWN – Publicaties zonder aanstelling Universiteit Leide

The ClpX chaperone controls autolytic splitting of Staphylococcus aureus daughter cells, but is bypassed by β-lactam antibiotics or inhibitors of WTA biosynthesis.

β-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of β-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of β-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with β-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by β-lactams or by inhibiting an early step in WTA biosynthesis. The finding that β-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that β-lactams do not kill S. aureus simply by weakening the cell wall

Diagnosing the Clumpy Protoplanetary Disk of the UXor Type Young Star GM Cephei

UX Orionis stars (UXors) are Herbig Ae/Be or T Tauri stars exhibiting sporadic occultation of stellar light by circumstellar dust. GM\,Cephei is such a UXor in the young ($\sim4$~Myr) open cluster Trumpler\,37, showing prominent infrared excess, emission-line spectra, and flare activity. Our photometric monitoring (2008--2018) detects (1)~an $\sim$3.43~day period, likely arising from rotational modulation by surface starspots, (2)~sporadic brightening on time scales of days due to accretion, (3)~irregular minor flux drops due to circumstellar dust extinction, and (4)~major flux drops, each lasting for a couple of months with a recurrence time, though not exactly periodic, of about two years. The star experiences normal reddening by large grains, i.e., redder when dimmer, but exhibits an unusual "blueing" phenomenon in that the star turns blue near brightness minima. The maximum extinction during relatively short (lasting $\leq 50$~days) events, is proportional to the duration, a consequence of varying clump sizes. For longer events, the extinction is independent of duration, suggestive of a transverse string distribution of clumps. Polarization monitoring indicates an optical polarization varying $\sim3\%$--8$\%$, with the level anticorrelated with the slow brightness change. Temporal variation of the unpolarized and polarized light sets constraints on the size and orbital distance of the circumstellar clumps in the interplay with the young star and scattering envelope. These transiting clumps are edge-on manifestations of the ring- or spiral-like structures found recently in young stars with imaging in infrared of scattered light, or in submillimeter of thermalized dust emission.Comment: 20 pages, 9 figure

Scaling predictions for radii of weakly bound triatomic molecules

The mean-square radii of the molecules $^4$He$_3$, $^4$He$_2-^6$Li, $^4$He$_2-^7$Li and $^4$He$_2-^{23}$Na are calculated using a three-body model with contact interactions. They are obtained from a universal scaling function calculated within a renormalized scheme for three particles interacting through pairwise Dirac-delta interaction. The root-mean-square distance between two atoms of mass $m_A$ in a triatomic molecule are estimated to be of de order of ${\cal C}\sqrt{\hbar^2/[m_A(E_3-E_2)]}$, where $E_2$ is the dimer and $E_3$ the trimer binding energies, and ${\cal C}$ is a constant (varying from $\sim 0.6$ to $\sim 1$) that depends on the ratio between $E_2$ and $E_3$. Considering previous estimates for the trimer energies, we also predict the sizes of Rubidium and Sodium trimers in atomic traps.Comment: 7 pages, 2 figure

General and abdominal adiposity and risk of death in Europe

BACKGROUND Previous studies have relied predominantly on the body-mass index (BMI, the weight in kilograms divided by the square of the height in meters) to assess the association of adiposity with the risk of death, but few have examined whether the distribution of body fat contributes to the prediction of death. METHODS We examined the association of BMI, waist circumference, and waist-to-hip ratio with the risk of death among 359,387 participants from nine countries in the European Prospective Investigation into Cancer and Nutrition (EPIC). We used a Cox regression analysis, with age as the time variable, and stratified the models according to study center and age at recruitment, with further adjustment for educational level, smoking status, alcohol consumption, physical activity, and height. RESULTS During a mean follow-up of 9.7 years, 14,723 participants died. The lowest risks of death related to BMI were observed at a BMI of 25.3 for men and 24.3 for women. After adjustment for BMI, waist circumference and waist-to-hip ratio were strongly associated with the risk of death. Relative risks among men and women in the highest quintile of waist circumference were 2.05 (95% confidence interval [CI], 1.80 to 2.33) and 1.78 (95% CI, 1.56 to 2.04), respectively, and in the highest quintile of waist-to-hip ratio, the relative risks were 1.68 (95% CI, 1.53 to 1.84) and 1.51 (95% CI, 1.37 to 1.66), respectively. BMI remained significantly associated with the risk of death in models that included waist circumference or waist-to-hip ratio (P<0.001). CONCLUSIONS These data suggest that both general adiposity and abdominal adiposity are associated with the risk of death and support the use of waist circumference or waist-tohip ratio in addition to BMI in assessing the risk of death