Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones <it>via </it>recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, <it>oriV</it>, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate <it>trans</it>-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer <it>oriV</it>-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a <it>P</it>_BAD-driven <it>trfA </it>gene generating strain MW005 that supports, independently, both recombineering and copy-number induction.</p> <p>Results</p> <p>The <it>P</it>_BAD-driven copy of <it>cre </it>in EL350 was replaced seamlessly with a copy of <it>trfA</it>, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based <it>Caenorhabditis elegans </it>genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation.</p> <p>Conclusions</p> <p>By retrofitting EL350, an established 'recombineering' <it>E. coli </it>strain, with a tightly regulated copy of <it>trfA </it>we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of <it>oriV</it>-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for many downstream purposes.</p

Reference values for low muscle mass and myosteatosis using tomographic muscle measurements in living kidney donors

Low muscle mass and myosteatosis are associated with poor clinical outcomes. Computed tomography (CT) imaging is an objective method for muscle mass and quality assessment; however consensus on cut-off values is lacking. This study assessed age-, sex-, and body mass index (BMI)-specific reference values of skeletal muscle parameters and correlated muscle mass with 24-h urinary creatinine excretion (24-h UCE). In total, 960 healthy subjects were included in this study. Muscle mass and quality were determined using axial CT slices at the vertebral level L3. The muscle area was indexed for height (skeletal muscle index [SMI]). The mean age was 53 ± 11 years, and 50% were male. The SMI reference values for low muscle mass in males were 38.8 cm2/m2 (20–29 years), 39.2 (30–39 years), 39.9 (40–49 years), 39.0 (50–59 years), 37.0 (60–69 years), and 36.8 (70–79 years). For females, these reference values were 37.5 cm2/m2 (20–29 years), 35.5 (30–39 years), 32.8 (40–49 years), 33.2 (50–59 years), 31.2 (60–69 years), and 31.5 (70–79 years). 24-h UCE and SMI were significantly correlated (r = 0.54, p &lt; 0.001) without bias between the two methods of assessing muscle mass. This study provides age-, sex-, and BMI-specific reference values for skeletal muscle parameters that will support clinical decision making.</p

Ventricular dyssynchrony assessed by gated myocardial perfusion SPECT using a geometrical approach: a feasibility study

Cardiovascular Aspects of Radiolog

C. elegans flavin-containing monooxygenase-4 is essential for osmoregulation in hypotonic stress

Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell. Thus, FMO-4 plays a crucial osmoregulatory role by promoting clearance of excess water that enters during hypotonicity, perhaps by synthesizing an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells. C. elegans FMO-4 contains a C-terminal extension conserved in all nematode FMO-4s. The coincidently numbered human FMO4 also contains an extended C-terminus with features similar to those of FMO-4. Although these shared sequence characteristics suggest potential orthology, human FMO4 was unable to rescue the fmo-4 osmoregulatory defect. Intriguingly, however, mammalian FMO4 is expressed predominantly in the kidney – an appropriate site if it too is, or once was, involved in osmoregulation

Functional Role of the Cytoplasmic Tail Domain of the Major Envelope Fusion Protein of Group II Baculoviruses

F proteins from baculovirus nucleopolyhedrovirus (NPV) group II members are the major budded virus (BV) viral envelope fusion proteins. They undergo furin-like proteolysis processing in order to be functional. F proteins from different baculovirus species have a long cytoplasmic tail domain (CTD), ranging from 48 (Spodoptera litura multicapsid NPV [MNPV]) to 78 (Adoxophyes honmai NPV) amino acid (aa) residues, with a nonassigned function. This CTD is much longer than the CTD of GP64-like envelope fusion proteins (7 aa), which appear to be nonessential for BV infectivity. Here we have investigated the functional role of the CTD of Helicoverpa armigera single-capsid NPV (HearNPV), a group II NPV. We combined a newly constructed HearNPV f-null bacmid knockout-repair system and an Autographa californica MNPV (AcMNPV) gp64-null bacmid knockout-pseudotype system with mutation and rescue experiments to study the functional role of the baculovirus F protein CTD. We show that except for the 16 C-terminal aa, the HearNPV F CTD is essential for virus spread from cell to cell. In addition, the CTD of HearNPV F is involved in BV production in a length-dependent manner and is essential for BV infectivity. The tyrosine residue Y658, located 16 aa from the C terminus, seems to be critical. However, HearNPV F without a CTD still rescues the infectivity of gp64-null AcMNPV BV, indicating that the CTD is not involved in processing and fusogenicity. Altogether, our results indicate that the F protein is essential for baculovirus BV infectivity and that the CTD is important for F protein incorporation into BV

The large Gunnera's (G. tinctoria and G. manicata) in Europe in relation to EU regulation 1143/2014.

Incorrect labelling of plants in the horticultural trade and misidentification is widespread. For the inspection services of the EU member states, correct identification of G. tinctoria has become important since the species was added to the List of Union concern in accordance with EU regulation 1143/2014 in August 2017. In the horticultural trade Gunnera plants are generally of modest dimensions and rarely flowering, so that the major distinguishing morphological characters for the identification of the two large species, G. tinctoria and G. manicata, are missing. As G. tinctoria is included in the EU regulation, its trade is prohibited, although the closely related species, G. manicata is not included on the list. Given that it is often difficult to distinguish between these two large herbaceous species using morphological attributes we used standard chloroplast DNA barcode markers, supplemented at a later stage by ITS markers. Plant material of putative G. tinctoria or G. manicata was obtained from the native and introduced range, both from "wild" sources, botanical gardens, and the horticultural trade. In western Europe plants circulating in the horticultural trade turned out to be predominantly G. tinctoria, with only one plant in cultivation identified as true G. manicata and the G. manicata found in botanical gardens was a hybrid recently described as G. x cryptica