In recent years the generation of antibodies by recombinant methods, such as phage display technology, has increased the speed by which antibodies can be obtained. However, in some cases when recombinant antibodies have to be validated, expression in E. coli can be problematic. This primarily occurs when codon usage or protein folding of specific antibody fragments is incompatible with the E. coli translation and folding machinery, for instance when recombinant antibody formats that include the Fc-region are needed. In such cases other expression systems can be used, including the protozoan parasite Leishmania tarentolae (L. tarentolae). This novel host for recombinant protein expression has recently shown promising properties for the expression of single-chain antibody fragments. We have utilised the L. tarentolae T7-TR system to achieve expression and secretion of two scFvs fused to the Fc-region of rabbit immunoglobulin G (IgG).
Based on the commercial vector pLEXSY_IE-blecherry4 (Jena Bioscience; Cat. No. EGE-255), we generated a vector containing the Fragment Crystallisable (Fc) region of rabbit IgG allowing insertions of single chain antibody fragments (scFvs) in frame via Nco l/Not l cloning (pMJ_LEXSY-rFc). For the expression of rabbit Fc-fusion scFvs (scFv-rFc) we cloned two scFvs binding to human vimentin (LOB7 scFv) and murine laminin (A10 scFv) respectively, into the modified vector. The LOB7-rFc and A10-rFc fusions expressed at levels up to 2.95 mg/L in L. tarentolae T7-TR. Both scFv-rFcs were purified from the culture supernatants using protein A affinity chromatography. Additionally, we expressed three different scFvs without the rFc regions using a similar expression cassette, obtaining yields up to 1.00 mg/L.
To our knowledge, this is the first time that antibody fragments with intact Fc-region of immunoglobulin have been produced in L. tarentolae. Using the plasmid pMJ_LEXSY-rFc, L. tarentolae T7-TR can be applied as an efficient tool for expression of rFc fusion antibody fragments, allowing easy purification from the growth medium. This system provides an alternative in cases where antibody constructs express poorly in standard prokaryotic systems. Furthermore, in cases where bivalent Fc-fused antibody constructs are needed, using L. tarentolae for expression provides an efficient alternative to mammalian expression.
Antibodies are applied in both basic research and diagnostics, and represent an increasingly important class of therapeutics. Monoclonal antibodies is the largest and fastest growing class of protein pharmaceuticals . In the discovery and development of these antibodies, antibody fragments such as the antigen binding fragment (Fab), the single-chain variable fragment (scFv), and the single variable domains (VH and VL, collectively sdAb) are often employed . The present recombinant antibody discovery platforms, such as ribosome and phage display , enable easy screening and selection of antibody fragments against virtually any antigen . Based on the initial screening or selection, a number of candidate antibodies are obtained . Often, these recombinant antibody candidates can be expressed in E. coli. However, although the field of recombinant protein expression in E. coli is developed and expanded [5, 6], the codon usage and folding dynamics of some recombinant antibody clones are incompatible with the bacterial expression machinery [7, 8]. In addition, for further evaluation of an antibody fragment it can be necessary to test additional formats, including the Fc-fusion format; such formats are inherently unsuitable for (but not outright incompatible with) prokaryotic expression [7, 9].
Modifying a Fab, scFv, or sdAb by fusing them to the Fc-region will produce a bivalent antibody format similar to the canonical antibody [10, 11]. The bivalent format increases the apparent affinity due to avidity, provided that multiple epitopes are available.
A further benefit of the Fc-fusion that potentially can be imparted to some antibody fragments is a decrease in their propensity to aggregate [1, 12]. At the same time the molecular sizes of the Fc-fused antibodies increase from approximately 12, 25, or 50 kDa (sdAb, scFv, and Fab respectively) to approximately 75, 100, or 150 kDa. An increase of molecular size in this range will greatly increase the serum half-life of a recombinant antibody, by putting it beyond the cut-off for renal clearance. For example native IgG1 of approximately 150 kDa has a serum half-life of around 21 days, whereas the serum half-lives of sdAb and scFv are in the area of 0.05 and 0.1 days respectively . The longer serum half-life of native IgG and of some of the larger recombinant formats is only partly attributed to their molecular size. The longer serum half-life is furthermore a consequence of the interactions of the Fc-region with the neonatal Fc receptor (FcRn). The interaction with FcRn salvages the antibodies from endosomes and returns them to circulation, rather than letting them enter the lysosomal degradation pathway [13, 14]. On the other hand, increasing size in general reduces the ability of the antibody fusion to penetrate tissue. The aspects of and need for prolonging the half-life of popular small antibody formats are reviewed in Kontermann 2009 .
Besides extending the serum half-life of potential protein therapeutics the Fc-region also confer other useful properties with regard to purification and immunochemistry. In protein purification, the Fc-region allows binding to protein A and protein G, hence supporting effective one step purification by affinity chromatography [16, 17]. With respect to the immunochemistry the presence of the Fc-region facilitates detection using many common secondary antibodies . Other obvious tags for detection and purification such as His-tag and C-myc tag are also present in our vector. One should nonetheless also consider protein L for purification of those antibodies holding a kappa light chain. We thus present a vector construct, which allows for versatile strategies of purification and immunochemistry.
The current method of choice for experimental scale expression of full-length antibody, and for formats including the Fc-region, is transient expression in mammalian hosts, such as Chinese hamster ovary cells (CHO) or Human Embryonic Kidney cells (HEK) [19, 20]. In such systems, high expression levels can be obtained reaching triple-digit mg/L levels. The drawbacks of mammalian cell expression systems are the need for dedicated labs and equipment, the labour intensive handling, and the economic considerations such as the price of reagents, culture medium, and labware .
Expression of recombinant proteins is one of the most central disciplines in molecular biology and medical research, and innovative systems with unique advantages are thus constantly being explored . Among emerging systems is the one based on L. tarentolae, a protozoan parasite infecting the gecko Tarentolae annularis. The unicellular eukaryote L. tarentolae is an extensively studied model for the disease leishmaniasis . Their unique transcriptional and translational machinery has enabled the generation of novel expression systems [23, 24] based on the use of exogenous RNA polymerases . Properties like non-laborious handling, post-translational modifications similar to those of mammalian systems, episomal vector maintenance, and effective secretion of recombinant proteins, are some of the strengths of the L. tarentolae systems. Recent studies aimed at addressing the importance of the signal peptide cleavage site have shown that expression of scFv antibodies (3.83 mg/L culture medium) can be obtained using this system .
In order to validate the system for expression of Fc-fused antibody fragments, we have created a vector allowing expression and secretion of scFvs fused with the Fc-region of rabbit IgG (rFc) using L. tarentolae T7-TR (Jena Bioscience). The rFc is a convenient choice when the fusion proteins are to be used in immunochemical analysis of cells (ICC) and tissues (IHC) of human or mice. This is essential to the research performed at laboratories, in which cells and tissues are of human and mice origin, are used. Using the rFc region decreases the interaction, if any, between the endogenous Fc-receptors and the rabbit Fc part. The use of this rFc construct thus reduces the need to block the endogenous Fc receptors of human and mouse tissue .
In summary, we here report a system based on L. tarentolae T7-TR, for the production of rFc-fused scFv antibodies. The resulting recombinant antibodies are ideal for IHC or ICT on human or rodent material, have a higher apparent affinity for multivalent antigens due to avidity, and are compatible with convenient purification methods. The expression host is characterised by non-laborious handling requirements, eukaryotic post-translational modification of expressed proteins with near-mammalian N-glycan structures, and effective secretion of recombinant protein to the culture medium .
The commercial episomal expression system based on L. tarentolae T7-TR (Jena Bioscience) is appealing as the target protein can be isolated directly from the culture supernatant, enabling convenient one-step affinity purification. In addition, expression in L. tarentolae facilitates post-translational modifications of proteins from higher mammals . However, the vector pLEXSY_IE-blecherry4 does not allow for cloning via Nco I/Not I, a routinely used restriction enzyme combination for antibody fragments , as this will remove the secretory signal peptide and the polyhistidine stretch (His-tag). To render Nco I/Not I cloning possible we replaced the existing expression cassette with a cassette from a modified version of the pF4SPImsapX1.4sat vector using Bgl II and Mlu I (own unpublished work). The expression cassette from this vector comprises an Nco I/Not I cloning site downstream from the signal peptide of L. mexicana secreted acid phosphatase 1 (LMSAP1) and a C-terminal His-tag for purification followed by a c-Myc-tag for detection. Replacement of the expression cassette was done without changing the untranslated regions of the parent vector; pLEXSY_IE-blecherry4. The new vector was named pMJ-LEXSY. Moreover, for production of Fc-fusion antibodies, we integrated the rabbit IgG Fc-encoding region (hinge region and CH2-CH3) into the pMJ-LEXSY vector whereby the pMJ-LEXSY-rFc vector was generated (Figure 1). The rFc-encoding region was inserted into the Not I site. The insert was prepared using sticky PCR at one end of the insert, resulting in deletion of the plus strand 3′ Not I site proximal to the tags upon insertion of the rFc-encoding sequence . Thus the unique Not I restriction site was retained, allowing Nco I/Not I insertion of antibody fragments.
Description of vector pMJ-LEXSY-rFc. Top: DNA and protein sequence of cloning site and tag region of pMJ-LEXSY-rFc. Bottom: Vectormap of pMJ-LEXSY-rFc. The T7/Tet region (blue) designates the T7 promoter under control of a Tet operator. LMSAP (black) is the secretory leader-peptide of Secreted Acid Phosphatase 1 found in Leishmania Mexicana. The scFv A10 (red) is inserted via Nco I and Not I. The Fc-encoding region of rabbit IgG (turquoise) including the IgG linker (green) is placed just downstream of Not I followed by a Tev restriction site (black), a His-tag (yellow), and a c-Myc-tag (pink) encoding region. The bleomycin resistance gene is fused with a cherry gene, hence designated BleCherry. This combination allows for selection of recombinants with bleomycin and subsequent screening of the best expressing clones by monitoring the cherry fluorescence. The Pci I restriction site is used for linearization and is placed in a telomere region (LTE + RTE), which upon transformation stabilises the linear episome. Finally, the vector holds a tandem T7 transcription terminator (2xT7), a bacterial origin of replication (PBR322 ori), and the Bla ampicillin resistance marker.
Expressions (80–100 mL in 250 mL flasks) of the two scFv-rFc constructs were carried out for 68-73 hours to explore the level of expression at small-scale. In addition, we explored the capacity of the expression system to produce classical scFvs using the vector pMJ-LEXSY. Three different scFvs, A10, LOB7, and Y4A were expressed together with A10-rFc and LOB7-rFc in L. tarentolae T7-TR (Figure 2). The antibody A10 is an scFv derived from the Tomlinson I library . It binds to murine laminin from Engelbreth-Holm-Swarm tumour cells. The antibody LOB7 in an scFv derived from the Tomlinson J library . This antibody binds to human vimentin. The Y4A antibody is an scFv with lambda light chain derived from the YAMO library . This antibody recognises C5a anaphylatoxin. These antibodies were chosen as model antibody fragments, as we had significant experience in expressing them as scFvs in E. coli.
Antibodies expressed and purified from T7-TR. Coomasie from the left: Molecular weight marker (SeeBlue plus2; Invitrogen), A10 scFv, LOB7 scFv, Y4A scFv, A10 scFv-rFc, and LOB7 scFv-rFc. Western blot from the left: LOB7-rFc reduced (r), and LOB7-rFc non-reduced (n-r). All antibodies seen in the Coomasie stain were separated on SDS in reducing conditions.
Expression and subsequent purification by affinity chromatography established that recombinant antibody fragments could be obtained from all supernatants (Figure 2). Purifications were carried out using Protein A chromatography for the rFc constructs and Ni-NTA Protein Purification for the scFvs. The expression levels observed for the various clones ranged from 0.3 mg/L to 1 mg/L for the scFvs and from 0.6 mg/L to 2.95 mg/L for the rFc-scFvs. Recently, Jäger et al. have shown that yields up to 600 mg/mL of scFv-Fc fusion proteins can be obtained by optimised transient expression in HEK 293 cells . Although the yields in our work is significantly lower, we still consider expression in L. tarentolae beneficial due to the few requirements of implementing this system into the laboratory and the non-laborious handling of L. tarentolae.
When the purified proteins were analysed by SDS-PAGE two bands were detected. This was also confirmed by the western blot analysis (Figure 2) in which the LOB7-rFc was detected with an HRP conjugated anti-rabbit antibody. Additionally, the western blot analysis could also confirm that the rFc-conjugated antibody assembles into a bivalent structure in non-reducing conditions. The same trend was seen for the A10-rFc antibody (Additional file 1). The apparent heterogeneity of size can be difficult to see for the non-reduced band in the western blot. This is due to the fact that the size difference of the two bands is relatively smaller to the total mass of the assembled bivalent antibody, hence giving a lower separation. This apparent heterogeneity of size was seen for all constructs; scFvs (28 kDa) and scfv-rFcs (55 kDa) (Figure 2). To investigate this issue we utilised that the pMJ-LEXSY-rFc vector (Figure 1) enables the removal of the His-tag and the c-Myc-tag by TEV protease digestion. By removing the c-terminal tags with TEV Protease it was possible to assess whether the source of size heterogeneity was placed in the tag-region. SDS-PAGE analysis of LOB7-rFc digested with TEV Protease showed that one band appeared on the gel as opposed to two bands from non-digested sample, confirming that the apparent heterogeneity of size was due to unpredicted modifications of the tag-region (Additional file 2: Figure S1). Furthermore, western blot analysis and mass spectrometry on the Y4A-scFv strongly suggest that the heterogeneity of size emerges from a truncation of the c-Myc tag in the lower molecular-weight protein (Additional file 2: Figure S2-S4). The truncation of the c-Myc tag has however no obvious influence on the intended application of the scFv-rFcs in IHC and ICC using secondary antibodies targeting the rFc-region.
To assess the binding activity of the scFv-rFcs, ELISAs were performed (Figure 3) with A10-rFc and LOB7-rFc against their respective antigens (mouse laminin and human vimentin). The antigens were coated on ELISA plates, targeted by the scFv-rFcs, and detected using polyclonal swine anti-rabbit immunoglobulin HRP.
ELISA; specific binding of rFc fusion scFvs against their cognate antigens. To test if the scFv-rFc antibodies bind their cognate antigens ELISA plates were coated with laminin (A10 scFv-rFc) and vimentin (LOB7 scFv-rFc). For both antibody constructs specific binding is seen.
The rFc-constructs show specific binding to their cognate antigens. Furthermore, the scFv-rFcs were detectable using HRP-conjugated anti-rabbit immunoglobulin. The detection with the secondary antibody shows that the rFc-regions were functionally intact, in line with the fact that the antibodies can be purified using protein A affinity chromatography . On this basis we conclude that our system is suitable for the production of rFc-fused antibody fragments with uncompromised folding. To further establish the capacity of this system for production of antibodies intended for immunochemistry, LOB7-rFc was used for immunocytochemical staining of fixed and permeabilised human adult skin fibroblasts (ASF-2) . A commercial mouse monoclonal antibody (V9) directed against vimentin was included as a benchmark for detection fidelity (Figure 4).
Using LOB7-rFc and V9 antibody for Immunocytochemistry. The cell line ASF-2 were stained with LOB7 scFv-rFc (A, B) and V9 (D, E) and detected with Goat-anti-Rabbit Alexa Fluor 488 (green) and Goat-anti-Mouse Alexa Fluor 546 (red) respectively. Negative controls (C, F) were done leaving out primary antibodies. The cell nuclei were stained with DAPI (blue). Both LOB7 and V9 detect vimentin, a cytoplasmic intermediate filament. Both give excellent staining. (Scale bar, 20 μm).
Both antibodies target vimentin, a cytoplasmic intermediate filament. As can be seen, LOB7-rFc and commercial V9 antibody produces similar labelling patterns when binding to vimentin.
Antibody fragments fused to the Fc-region of IgG have previously been produced in several organisms [35–39], but to our knowledge this is the first time such constructs have been expressed in L. tarentolae. The L.tarentolae system offers a system capable of making N-glycosylations and O-glycosylations. Breitling et al. reported the production of biological active biantennary homogenously N-glycosylated hEPO in L. tarentolae . The N-glycosylations performed in L. tarentolae are of higher mammal resemblance than those performed in yeast and insect cells. Moreover, studies by Klatt et al.  have shown that ssAlpha expressed in L. tarentolae are O-glycosylated at the same sites as in mammal cells and furthermore displayed an increased resistance to degradation compared to ssAplpha expressed in E. coli.
At the same time L. tarentolae expression cultures can be handled under standard laboratory conditions, similar to the conditions used for propagation of E. coli cultures. For the production of antibody fragments resulting from high throughput screening, the system we describe here presents substantial benefits: Firstly, the system can provide a rescue avenue for antibody fragments that express poorly in E. coli. Secondly, the system can provide recombinant antibodies in a bivalent format incorporating a native Fc-region; a format ideally suited for use in immunostaining of cells and tissues.
All restriction enzymes, polymerases, and chemicals used for cloning and PCR were purchased from Fermentas (Thermo Scientific Molecular Biology). Oligonucleotides used are listed in Table 1, and were purchased from Sigma Aldrich. To make the pLEXSY_IE-blecherry4 applicable for Nco I/Not I cloning of scFv antibodies for secretion an expression cassette from the vector pF4NAF was cloned into pLEXSY_IE-blecherry4 via Bgl I and Mlu I. The pF4NAF expression cassette had been constructed previously using components of two vectors; pIT2  and pF4SPImsapX1.4sat (Jena Bioscience; Cat. No. EGE-211). In this previous work, the vector pF4SPImsapX1.4sat (an early version of pLEXSY-sat2, Jena Bioscience; Cat. No. EGE-234) was modified by deleting the Nco I with primer Z and primer X, by use of PCR amplification of the region between Bgl II and Not I. The PCR product, now deleted for Nco I, was subsequently digested with Bgl II and Not I and re-inserted into the pF4SPImsapX1.4sat. Next, the expression cassette from the vector pIT2 was PCR amplified using primer A and primer B, followed by a digestion of the PCR product with Kas I (5′ digestion) and Bsp120 I (3′ digestion). Bsp120 I produces overhangs compatible with Not I. The PCR amplified expression cassette of pIT2 was then inserted into pF4SPImsapX1.4sat via Kas I and Not I hereby creating the pF4NAF cassette. In the ligation of the Bsp120 I overhang to the Not I overhang the recognition sequence for both was destroyed. This cassette was inserted into pLEXSY_IE-blecherry4 via Bgl I and Mlu I, resulting in pMJ-LEXSY. The ligated DNA was used to transform electrocompetent XL1-Blue, and the cells were plated on TYE agar-plates containing 100 μg/mL ampicillin (Sigma Aldrich). The plates were incubated overnight at 30°C and colonies were picked for sequence analysis. To construct the rFc-fusion vector pMJ-LEXSY-rFc, the Fc encoding region of rabbit IgG (hinge region and CH2-CH3) was amplified by two rounds of PCR amplification using the vector pFUSE-rIgG-Fc2 as template . The two PCR amplifications were performed applying primer pairs c/d and c/e. respectively. The rFc-encoding region was prepared for cloning using sticky end PCR as earlier described  and inserted into the Not I site of pMJ-LEXSY. The Ligations were electroporated into XL1-Blue and plated on agar-plates holding 100 μg/mL ampicillin. Colony PCR was used to identify positive clones and correct orientation of DNA inserts were verified by sequencing (Eurofins MWG).
Before transformation, XL1-Blue cells were made electrocompetent roughly as described for Pseudomonas Putida in , but without adding sucrose to the storage medium. Batches of competent E. coli were frozen in liquid nitrogen and stored at -80°C. Electroporation was carried out in 2 mm pre-chilled cuvettes holding 20-30 ng of vector and 50 μL cells. The cells were pulsed at 2500 V using an Electroporator 2510 (Eppendorf) and plated on TYE agar-plates with 100 μg/mL ampicillin. For plasmid propagation, XL1-Blue were grown at 30°C and 200 rpm in baffled Erlenmeyer flasks containing PDM medium  with 100 μg/mL ampicillin.
Manipulation of T7-TR was performed in accordance with Jena Bioscience protocols (Cat. No. EGE-1420blecherry). In brief, T7-TR were cultivated and maintained in tissue culture flasks (TC25) as static suspensions at 26°C. The flasks were placed upright, in the dark, and with the ventilating cap in the open position. Non-transformed Leishmania tarentolae T7-TR were grown in 10–15 mL LEXSY BHI medium per flask. The cells were passaged twice weekly in ratios of 1:50 or 1:20 on Mondays and Fridays respectively. For transformation, 10 μg of linearized DNA in 50 μL of water was incubated with 350 μL densely suspended Leishmania tarentolae T7-TR (OD600 > 2). Electroporation was carried out in pre-chilled 2 mm cuvettes. Cells were pulsed in a GENEPULSER Xcell (BIORAD) at 450 V and 450 μF, obtaining a pulse time in ranges of 5-6.5 ms. The cells were kept on ice for exactly 10 minutes after electroporation. Immediately hereafter the cells were transferred to a T25 flask and grown for 20 hours in 10 mL of non-selective LEXSY BHI medium. Cells were subsequently gently dispensed onto selective LEXSY BHI agar plates and grown for 10 days at 26°C in the dark. The plates for clonal selection contained 100 μg/mL Zeocin (Invitrogen). All colonies visible after 10 days were picked from the plates, and each clone was then cultivated in selective LEXSY BHI medium for one day in a single well of a 24-well plate. This cultivation was performed in 1 mL selective LEXSY BHI medium with 100 μg/mL zeocin (Invitrogen). The cell density and condition was assessed in the microscope and clones with low growth were cultivated for further 1–2 days before they were transferred to a larger volume. Clones exhibiting acceptable motility, cell shape, and growth were transferred to 5 mL selective LEXSY BHI medium and grown to OD 1.4. Assessment of expression levels for each clone was conducted in 96-well plates by inducing the expression with 100 μg/mL tetracycline. The cherry fluorescence of each clone was measured at 584 nm excitation/612 nm emission in a POLARstar OPTIMA fluorimeter (BMG Labtech).
Clones displaying the highest level of cherry fluorescence were chosen for the further work. Expression was carried out in 100 mL LEXSY BHI medium containing 100 μg/mL tetracycline (Sigma Aldrich) and 100 μg/mL Zeocin (Invitrogen). Transformed L. tarentolae T7-TR (OD600 1.4-2) were innoculated 1:10 into 100 mL cultures. The culturing was performed in the dark at 26°C for 72 hours in 250 mL baffled Erlenmeyer flasks with agitation (120 rpm). The cultures were centrifuged at 2700 × g for 30 min to precipitate cells and the proteins present in the supernatant precipitated by using 30% m/v ammonium sulphate. The precipitated proteins were then pelleted at 5250 × g for 45 min and re-suspended in 0.5 × PBS. Finally, affinity purification using a Protein A HP spinTrap column (GE healthcare) was used for recovery of the scFv-rFc antibodies. The scFv antibodies were purified using a Maxwell 16 instrument in combination with a Maxwell 16 Polyhistidine Protein Purification Kit (Promega). Protein concentrations were estimated by absorption at 280 nm using a NanoDrop 1000 instrument (Thermo Scientific) and applying the protein specific molecular weights and molar extinction coefficients. A repeated and up-scaled expression of LOB7-rFc was performed as described above, but this time as 5 times 80 mL cultures incubated for 72 hours in 250 mL baffled Erlenmeyer flasks. Supernatants from all cultures were pooled before the proteins were precipitated. The ammonium sulphate precipitated proteins were then resuspended in 20 mM sodium phosphate and purified by using a 1 mL HiTrap Protein A HP column. Protein A purification was carried out as outlined by the manufacturer (GE Healthcare).
Maxisorp 96-well flat bottom plates (Nunc) were coated with 30 μL of the relevant antigens at 20 μg/mL (in 2% BSA-PBS) for both vimentin and laminin. The protein was adsorbed to the plates during storage at 4°C overnight. Each well was washed with three times 200 μL PBS using a multi-channel pipette, before the plates were blocked with 300 μL 2% BSA in PBS (BSA-PBS) for 1 hour at room temperature under gentle agitation. The washing step was then repeated, followed by the addition of serial dilutions of scFv-rFcs in 100 μL 2% BSA-PBS. The antibodies were incubated in the antigen-coated wells for 1.5 hour at room temperature, and subsequently the plates were washed three times with PBS, each time for 5 min. Detection of bound scFv-rFcs was performed by incubating the plates with polyclonal swine anti-Rabbit Immunoglobulins (Dako) in a 1:2000 dilution in 2% BSA-PBS for 1 hour. Finally, the plates were washed three times with PBS, each time for 5 minutes. The amount of bound antibody was visualised by the addition of 80 uL TMB single solution (Sigma Aldrich), and the colour reaction was terminated after 7 minutes by the addition of 50 μL 1 M H2SO4 to each well. Absorbance was measured at 450 nm and corrected for at 655 nm. The absorbance was conducted in a microplate reader; Model 550 (Bio Rad).
ASF-2 cells were grown in DMEM (Lonza) with 10% fetal bovine serum (Thermo Scientific), 100 U/mL penicillin and streptomycin (Lonza) at 37°C, 5% CO2 and 95% humidity. ASF-2 cells of passage 10 were detached from a tissue culture flask by trypsination with Trypsin EDTA (Lonza) and spun down at 600 g for 6 minutes. Cells were then resuspended in growth medium and 15.000 cells/well were seeded out in an ibiTreat μ-Slide VI 0.4 (Ibidi) and grown overnight. The next day the cells were rinsed with PBS, fixed in 4% PFA for 15 min., permeabilised with 0.025% Triton X100 (Sigma-Aldrich) for 10 min. and blocked with 2% BSA in PBS (BSA-PBS) for 1 hour at room temperature. The cells were incubated with 5 μg of purified LOB7 in 2% BSA-PBS or 100 μl of a 1:100 dilution of V9 antibody in 2% BSA-PBS (Sigma Aldrich) pr. well for 1 hour at room temperature. Visualisation of LOB7 was accomplished by incubation with a 1:100 dilution of Goat-anti-Rabbit Alexa Fluor 488 (Invitrogen, USA). V9 was visualised by a 1:100 dilution of Goat-anti-Mouse Alexa Fluor 546 (Invitrogen). Cell nuclei were stained with Vectashield Mounting Medium with DAPI (Vector Labs). Fluorescent images were obtained with a Leica DMI3000 B inverted microscope (Leica Microsystems).
Mathias Lindh Jørgensen, Niels Anton Friis contributed equally to this work.
This work was supported by grants from Eva & Henry Frænkels Mindefond, The Danish Council for Independent Research—Technology and Production Sciences grant number 09–065063 and 10–081681.
MLJ, NAF and PK contributed to the design of the study and MLJ and NAF performed the majority of the experiments. JJ performed immunocytochemistry, PM participated in the manipulation of L. Tarentolae. SVP performed the mass spectrometry. All authors participated in writing and critical review of the manuscript.


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Want to know how to simplify expressions including rational exponents in algebra? Learn how with this free video lesson. From Ramanujan to calculus co-creator Gottfried Leibniz, many of the world's best and brightest mathematical minds have belonged to autodidacts. And, thanks to the Internet, it's easier than ever to follow in their footsteps (or just finish your homework or study for that next big test).
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text:
Musa hirta is a tropical Asian species of plant in the banana family native to Sarawak on the island of Borneo, in Malaysia. It is one of fourteen species of Musa endemic to the island of Borneo.  It is placed in section Callimusa (now including the former section Australimusa), having a diploid chromosome number of 2n = 20.

References

hirta
Endemic flora of Borneo
Flora of Sarawak
Plants described in 1902
Taxa named by Odoardo Beccari

redpajama_set_name: RedPajamaWikipedia


Greg DeVillers was a top biotech executive, and Kristen Rossum was embarking on a career in toxicology at the San Diego Medical Examiner's office. They seemed to be happily married, living the American dream. But only months shy of their second anniversary, Kristen found her handsome husband dead from a drug overdose,his corpse sprinkled with rose petals. By his side was their wedding photo. The scene was reminiscent of American Beauty, one of Kristen's favorite movies. Authorities deemed it a suicide.
Until they discovered that the rare poison found in Greg's body was the same poison missing from Kristen's office. Until they discovered the truth about Kristen's lurid affair, about her own long-time drug addiction, and about the personal and professional secrets she would kill to keep hidden-secrets that would ultimately expose the beautiful blonde as the deadly beauty she really was.
Once more John Glatt tells this story in the same easy writing I have grown to expect from him.If you have read one of John Glatt's book and you find that you enjoyed it then I can tell you that you will enjoy any John Glatt story.The same true crime story can sometimes be written by a few true crime writers.I have reached the stage where I will only buy the John Glatt version of a certain crime story.I know that I will not be disappointed.
This book Deadly American Beauty was,if I remember correctly, the first book I read by John Glatt.I have also since then watched a program on this story on the Crime & Investigation network and I would still advise on buying the book as it contains really interesting information that is not covered by the TV program.This is especially true of the background and childhood details of the main characters,Kristin Rossum and Greg de Villers.
This book covers background and childhood details including the way the couple met and some of their married life up to the day of the crime,the investigation and the court case.There is no graphic details of the crime.It is a book I would recommend to any of my readers.
I read this book in October 2007 and it also has 8 pages of photographs relevant to this true crime story.The title "Deadly American Beauty" is so very appropriate as she was indeed a great beauty.I was a bit diapppointed in the fact that there wasn't more information on trying to avoid the same trap Kristin Rossum fell in when she became addicted to drugs but the story is still extremely insightful and I was able to come to a few conclusions of my own,regardless.
Hi Yvette! I got my Kindle on the 26th and have read a book on it already. It was A Breed Apart (medical thriller.) I didn't have any trouble like you did in the begining so everything went smoothly when I got it. I downloaed some free books, mostly classics, bought a couple and charged it up. The charge lasted for 6 days, which I thought was pretty good since I played with it a lot and read a lot. I liked the feel of it while reading. It was easy to use.
How are you liking yours? Hope it is working out for you! Let me know.
I am loving mine.It took so long to get here that I ordered "real" books in the meantime so I have to read those first.I have already bought 5 books on Kindle which I have read.Wonderful best thing since sliced bread!!
You're ahead of me in the reading on the Kindle department. I have so many books on my TBR pile plus the ones I'm still checking out from the library. I'm glad you're liking yours!


It's almost traditional. Today's my birthday and my present from my husband is a book I've already got. Own goals in previous years have included THE BRIDGE OF THE SAN LUIS REY and THE CURIOUS INCIDENT OF THE DOG IN THE NIGHT-TIME. This year it is THE VIRAGO BOOK OF GHOST STORIES. I know exactly how this happens – he is standing there in the book shop, the minutes ticking away, and his eye lights on a book he thinks I would like. And I do like it – I like it so much that I've already bought it and read it and it's on the shelf at home. Some years I forsee this and give him a list, but I'm so busy at this time of year that I usually forget. What I wanted this year was Jenny Uglow's biography of Thomas Bewick (or a bottle of Chanel No 19). Still it's not too late for Christmas.


April 22, 2019The exotic and exclusive automobile club recently announced their next upcoming event to take place in May. The Rally will begin early morning May 4th at the founder's places of business, CNG Digital Marketing and SiO Digital of Sorrento Valley, and continue on to Viejas Casino and Resort for the weekend getaway.
The World's Top 10 Black and White Photographers of 2019 Have Been Revealed!
April 11, 2019Lead-in toPPA's International Photographic Competitionaccepting images now through May 8.
April 10, 2019ARCIS' management team broadens expertise to drive future growth.
April 08, 2019More than an integration, Frame.io is now a native feature set, tightly woven into the fabric of DaVinci Resolve 16 Studio.
April 08, 2019Photographer.org is celebrating the official launch of Photographer.org to the worldwide photography community with a month long giveaway, including a grand prize of a Sony a7 III camera.
April 03, 2019Morguefile.com now has over 400,000 free images available for download.
April 01, 2019Production has wrapped on the high-profile movie, '20 MINUTES' set to hit theaters in March 2020.


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Animals Endangered Species
Why African Wild Dogs Are Endangered and What We Can Do to Help Save Them
Katherine Gallagher
Katherine Gallagher is a writer and sustainability expert. She holds a B.A. in English Literature from Chapman University and a Sustainable Tourism certificate from the GSTC.
Elizabeth MacLennan
Fact checked by Elizabeth MacLennan
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Learn about our fact checking process
Michael Schwab / Getty Images
Known for its brightly colored, spotted coat and large, bat-like ears, the African wild dog is one of the planet's most endangered mammals.
The species has been endangered with decreasing numbers since 1990, and according to the IUCN, the global population is estimated at 6,600 adults. However, African wild dogs have a concrete social structure in which only one alpha female in each pack is reproductively active. So, out of that 6,600, only about 1,409 can produce offspring.
The largest wild dog populations remain limited to southern Africa and the southern part of East Africa, with more condensed communities found in Tanzania and northern Mozambique.
These unique animals are rarely seen, so many population estimates are based on observational data rather than systematic monitoring.
Mint Images / Getty Images
Despite their elusiveness, the various causes of these large canines' decline are relatively understood.
As opportunistic predators who can reach impressive speeds of up to 44 miles per hour, African wild dogs require ample space within short-grass plains, semi-desert, savannas, or upland forests in which to hunt and roam. As a result, they are highly susceptible to habitat fragmentation and conflict with livestock farmers, which can also foster other issues like prey scarcity and disease.
Habitat Loss and Fragmentation
Habitat fragmentation (which can be caused by both human and natural processes) divides larger and more contiguous wild dog habitats into smaller, more isolated patches of habitat.
According to a 2015 study published in the Journal of Mammalogy, packs of African wild dogs in the Okavango Delta had average range sizes of about 285 square miles and moved over three square miles each day. Breaking up that necessary range can lead to inbreeding and starvation. Moreover, less access to appropriate habitat can also increase their contact with humans and domestic animals, leading to the transmission of infectious diseases and opportunities for human-wildlife conflict.
When the animals are only given a chance to populate in smaller numbers, it makes them more vulnerable to catastrophic events (since larger populations have a greater probability of recovery) and predation by larger animals.
Human Conflict
As available habitats decrease and human settlements expand, African wild dogs are more likely to come into contact with people whose livelihoods depend on farming livestock. Often, they're killed by farmers who view them as a threat.
They can also be caught in poaching snares set for bushmeat and be subject to mortality on roads in more highly populated areas.
A 2021 study analyzing mortality patterns in radio-collared African wild dogs in Kenya, Botswana, and Zimbabwe discovered a connection between high ambient temperatures and dogs being killed by people. According to the research, African wild dogs shift the timing of their hunts and habitat choice when the weather is hotter, which could bring them closer to developed areas (and isn't exactly good news considering the progressively increasing temperatures from climate change). Between 2002 and 2017, a combination of killing by humans and disease spread by domestic dogs accounted for 44% of all African wild dog deaths.
Pack animals are typically more susceptible to viral diseases like rabies, canine distemper, and canine parvovirus, and the African wild dog is no exception. Members of the species are so closely connected to one another that they've even been observed communicating through sneezes.
Infectious diseases aren't limited to animals in the wild either. In December of 2000, a wave of canine distemper virus spread through a captive breeding ground of African wild dogs in Tanzania, killing 49 of the 52 individuals within two months.
Prey Scarcity
Ablestock.com / Getty Images
There's a lot of competition in Africa's savannas. African wild dogs share a limited supply of prey species—such as antelope, warthogs, and birds—with other, faster hunters like spotted hyenas and lions.
In Tanzania's Serengeti National Park, the African wild dog population disappeared entirely in 1991 after a gradual decline. Scientists believed that a viral disease was to blame—specifically one caused by human handling in a radio-collaring program—but it wasn't until a 2018 study published in Ecology and Evolution that the actual reason behind the pack loss was discovered. According to the study, the population never went extinct within the broader region but purposefully left the area because of other predator competition from hyenas. During the same Serengeti wild dog decline period, the spotted hyena population increased by 150%.
As with many endangered species, African wild dogs may require a little help from science to avoid extinction.
Researchers from James Cook University have developed a sperm freezing technique explicitly geared towards the species to solve some of the problems presented by population management and captive breeding programs.
African wild dogs have a complex social hierarchy, in which packs are led by a single dominant pair of an alpha male and female, so introducing new animals to an existing pack (for the sake of genetic diversity, for example) is rarely successful. The technique from James Cook will help develop a global sperm bank for the species.
Reintroduction projects have also shown significant progress and could help repopulate some of the regions where the species has already been extinct. For example, a 28-month study following a wild dog reintroduction project in Gorongosa National Park, Mozambique, showed a 73% survival rate and no deaths from unnatural causes.
Community engagement programs that educate locals who live in the same regions as African wild dogs can help relieve negative misconceptions and encourage tolerance.
In Kenya, installing "predator-proof" fencing around small reserves has succeeded in keeping wild dogs within protected areas and preventing conflicts with humans. Still, these types of band-aid solutions are certainly not 100% effective, and studies show that poorly constructed fences can lead to packs or parts of packs becoming trapped.
The African Wildlife Foundation works with communities to construct livestock enclosures but also employs scouts from neighboring communities in the Samburu landscape to monitor wild dog populations and learn about their movements; that way, they can alert local herders when wild dogs are present. The program combines conservation and economic opportunity to create an incentive to protect the species.
Establishing protected areas and wildlife corridors may help reduce conflicts with humans even more.
Save the African Wild Dog
Symbolically adopt an African wild dog with the World Wildlife Fund.
Learn more about African wild dogs with resources from the Range Wide Conservation Program for Cheetah and African Wild Dogs.
Support Painted Dog Conservation, a nonprofit (and partner of the Wildlife Conservation Network) that develops projects in Zimbabwe to protect African wild dogs specifically.
"African Wild Dog." World Wildlife Fund.
Woodroffe, R. and C. Sillero-Zubiri. "African Wild Dog." International Union for Conservation of Nature's Red List of Threatened Species, 2012., doi:10.2305/IUCN.UK.2020-1.RLTS.T12436A166502262.en
Pomilia, Matthew A., et al. "Ecological Predictors of African Wild Dog Ranging Patterns in Northern Botswana." Journal of Mammalogy, vol. 96, no. 6, 2015, pp. 1214-1223., doi:10.1093/jmammal/gyv130
Rabaiotti, Daniella, et al. "High Temperatures and Human Pressures Interact to Influence Mortality in an African Carnivore." Ecology and Evolution, vol. 11, no. 13, 2021, pp. 8495-8506., doi:10.1002/ece3.7601
Walker, Reena H., et al. "Sneeze to Leave: African Wild Dogs (Lycaon pictus) Use Variable Quorum Thresholds Facilitated by Sneezes in Collective Decisions." Proceedings of the Royal Society B, vol. 284, no. 1862, 2017., doi:10.1098/rspb.2017.0347
Reja, Salim, et al. "Investigation and Control Strategy for Canine Distemper Disease on Endangered Wild Dog Species: A Model-Based Approach." SN Applied Sciences, vol. 4, 2022, pp. 176., doi:10.1007/s42452-022-05053-5
Jackson, Craig R., et al. "No Evidence of Handling-Induced Mortality in Serengeti's African Wild Dog Population." Ecology and Evolution, vol. 9, no. 3, 2019, pp. 1110-1118., doi:10.1002/ece3.4798
Van den Berge, Femke, et al. "A Two-Step Dilution Tri-Egg Yolk Extender Containing Equex STM Significantly Improves Sperm Cryopreservation in the African Wild Dog (Lycaon pictus)." Cryobiology, vol. 80, 2018, pp. 18-25., doi:10.1016/j.cryobiol.2017.12.095
Bouley, Paola, et al. "The Successful Reintroduction of African Wild Dogs (Lycaon pictus) to Gorongosa National Park, Mozambique." PLOS ONE, vol. 16, no. 4, 2021., doi:10.1371/journal.pone.0249860
O'Neill, Helen M.K., et al. "Fencing Affects African Wild Dog Movement Patterns and Population Dynamics." Oryx, vol. 56, no. 1, 2021, pp. 128-136., doi:10.1017/S0030605320000320
Why the Tasmanian Devil Is Endangered
Why Chimpanzees Are Disappearing and What We Can Do
Why the Saola Is Endangered and What We Can Do
Why Amur Leopards Are Endangered and What We Can Do
Are Wolves Endangered? Conservation Status and Threats
25 of the Most Dangerous Animals in the World
16 of the World's Most Endangered Vulture Species
African Wild Dogs 'Vote' by Sneezing
Why Red Pandas Are Endangered
Are Cheetahs Racing Towards Extinction?
Africa's Rarest Carnivores Face Threats From Disease-Carrying Dogs
Why Indian Elephants Are Endangered
Why Bonobos Are Endangered and What We Can Do
Why the Snow Leopard Population Is Decreasing
The World's Smallest Tiger Is Inching Towards Extinction
13 Amazing and Critically Endangered Frogs

redpajama_set_name: RedPajamaCommonCrawl


Did you like brussels spouts growing up? Or do you really even like them now? Me neither. They were and still are in some ways the worst. They have a strange taste and are really bland on their own. I've tried a bunch of different seasonings and sauces to make them taste better but nothing seems to do the trick. However, in comes their only saving grace – bacon (shocking I know). Bacon can make an old shoe taste amazing so I knew it would work on these little guys. And now, I can't get enough of these little bulbous wonders.
Step 1: Cook your bacon. You can honestly never have enough bacon in your life.
Roughly chop and set aside.
Step 2: Prep your veggies. If your sprouts came on a stalk like this, begin by trimming off the bulbs with a paring knife. I like to cut my brussels sprouts in half as well but as long as they're roughly the same size you should be fine. You just want everything to cook evenly.
Step 3: Combine your veggies in a large mixing bowl and add in the honey, balsamic vinegar, olive oil, bacon, lemon juice, zest, salt and pepper and give everything a gentle toss to ensure that everything is evenly coated.
Step 4: Place on a baking sheet and bake for 25-30 min at 350 degrees or until they are fork tender.
And there you have it! Brussels sprouts that even your kids (or man-child) will enjoy. The bacon helps provide flavor and balance to the dish while the lemon juice and honey helps to keep things light. It's surprising how delicious these little guys can be with the right ingredients.
And with that, Happy Friday and thanks for reading!


Director Wong Kar Wai's latest movie, The Grandmasters, recently released its second trailer. Tony Leung Chiu Wai stars as the late wing chun master Ip Man. It's the 3rd films about the legendary martial artist, whose students included Bruce Lee. Other a stellar cast of the movie includes Zhang Ziyi, South Korean star Song Hye Kyo, comedian Zhao BenShan and Xiao ShenYang. The movie is set against the backdrop of the martial art world during the Republican Period. There will be other kung fu masters and schools making appearances in the movie.
This $25 million Mandarin-language biopic of Ip Man began filming back in December 2009. However Wong Kar Wai's attention to even the tiniest detail meant it has stopped and started again numerous times with the delays frustrating many. Tony Leung Chiu Wai even took a break to take the lead in another movie, The Great Magician, starring with Zhou Xun. Yuen Woo Ping is handling the fighting and choreography. No specific release date has been set.


Home / Latest News / Could the Fiat 500X be set for an Abarth makeover?
For a while Abarth has been evaluating which Fiat model will be next in line for a sporty revamp and after the success of the Abarth 500 range, it's been a difficult decision for the car brand to make. After a while of speculating, Abarth's next focus could be on a high performing Abarth 500X crossover which will join the 124 Spider in a new range of sports cars.
If the Abarth 500X gets the go ahead, it will help the rapidly growing car brand expand into the crossover market which is a completely different kettle of fish for Abarth. The model would take a slice in the crossover sector, it would also make strong mark in the performance crossover market, which to date is underdeveloped with competitors only being the MINI Country Man JCW and the Nissan Juke Nismo RS.
Various images have been released, and they give us an interesting glimpse of what we could be expecting from the future Abarth 500X. In the images, the 500X model is sporting a lowered stance, deeper side skirts, larger wheels and bulkier bumpers all contributing to the models mean and striking style.
To finish off the new style, the image shows the car with a larger rear spoiler, double tailpipes and an under-bumper diffuser. Inside we could expect even more fancy gauges and dials, low seated front seats and a wealth of 'Scorpion' badges both inside and out!
From the Abarth 500X, we could potentially expect the same engine as the 1.75- litre turbo Alfa Romeo 4C which is also in the books for an Abarth makeover. Unlike the two-seat, the 500X engine will be detuned to roughly around 200bhp.
Until production of the future model is confirmed, Abarth fanatics will have to sit patiently and wait for more details. Enthusiasts will not have to wait long however, and they can expect to get a fresh taste of what Abarth will be bringing the table next year with more performance driven Abarth models promised.


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De Chenghuangtempel van Taiwansheng is een taoïstische tempel die gewijd is aan de taoïstische god Chenghuang. Chenghuang is hier de god van de provincie Taiwan (Taiwansheng).

De tempel staat in Taipei en is niet de enige Chenghuangtempel van de stad. De Chenghuang die de provinciehoofdstad Taipei beheert, wordt vereerd in de Chenghuangtempel van Taipeifu. De Chenghuangtempel van Taiwanfu in Taiwan heeft dezelfde status als tempel waar de god van de provinciehoofdstad wordt vereerd.

Het huidige gebouw van de Chenghuangtempel van Taiwansheng in Taipei werd in november 1947 gebouwd. Het oorspronkelijke gebouw werd door de Japanners verwoest tijdens de Japanse bezetting van Taiwan. Gelovigen werden gedwongen te bidden in de Chenghuangtempel van Taipeifu. Na de oorlog startte men meteen met het werven van fondsen voor de bouw van de Chenghuangtempel van Taiwansheng.

In de tempel wordt behalve de god van de provincie, ook Guanyin en Jigong vereerd.

Taoïstische tempel in Taiwan


I love Microsoft Teams. There I said it, bias fully disclosed. I've always loved playing with new toys, and when Microsoft Teams came along I was excited! This article is about my own history working with Skype for Business, where Microsoft Teams fit into the future and my advice to those confused about which road they should take.
I'd been hanging out with Skype for Business and its previous incarnations for the last 10+ years, and was getting bored! (only a little). Don't get me wrong, I still love Skype for Business, and have had, and will continue to have, a great time helping customers get the most out of a great Unified Communications solution. Skype for Busines still has a tonne of mileage left in it, but at the same time, I am excited about a future with Microsoft Teams.
Skype for Business was the beginning of a new era in the way we would communicate. When Skype for Business was released with Enterprise Voice functionality way back in 2007 (a.k.a Office Communications Server), it was some serious blending edge stuff. There was little to no documentation on how to make Enterprise Voice work, SIP Trunking was in its very early days (at least here in New Zealand), and generally speaking, nobody thought a software solution would ever replace the good old trusty PABX. At the time I was fortunate enough to have an I.T Manager who loved bleeding edge on a budget. This was great news for me because I love to solve problems; the harder the better! And so my love for Skype for Business began. Thankfully for me, I picked the right horse; turns out Microsoft did a great job building a software solution that could outsmart the PABX.
Over the next 10 years, Skype for Business chipped away at the PABX market at an ever-increasing rate.
Now here we are at another turning point. Skype for Business is now set to be outsmarted by the Cloud, and in particular, by Microsoft Teams which leverages its technology. That doesn't mean it's going to disappear overnight or that you shouldn't invest in it; its still an excellent Unified Communication solution, and remember PABX's still exist today, and likewise, Skype for Business will continue to serve you for the foreseeable future. Case and point Skype for Business 2019 is due for release soon, and that comes with a renewed supportable lifespan of 5 years, and I believe we will see another server version before that expiry.
But one day, just like that PABX, Skype for Business may disappear. On-premises solutions are becoming less and less of a priority for Microsoft. Not so long ago Microsoft tried to retire SharePoint Server; long story short the decision was reversed, however, it does show their intent, and in my option, they are doing the right thing; simply put, Microsoft cannot compete with "born in the cloud" solution providers such as Google and Amazon. On-premises solutions with 3-year release cycles do not work these days! Cloud providers can deliver fixes, features, and enhancements too fast, and it's not practical to deliver the same to on-premises.
Microsoft Teams is not a replacement for Skype for Business specifically, its the beginning of a new era, and a different way of working. As I mentioned Skype for Business Server is sticking around for the foreseeable future. Skype for Business Online is in the same boat, however, Microsoft has signaled that at some point all online users will need to move to Teams. Microsoft Teams is pretty close to at feature parity with Skype for Business Online already, even so, Microsoft won't be forcing you to move any time soon. What they are doing, however, is working on providing interoperability and migration paths that will allow your Skype for Business users (Online or On-premises) to communicate with your Microsoft Teams users and vice versa, and give you a simple way to "upgrade" or migrate from Skype for Business to Microsoft Teams when you are ready; that might be now, 2 years from now or sometime further in the future. Microsoft Teams is only going to improve, and at some point, the benefits of moving will be a no-brainer.
Sure there are some teething problems, but on the whole, I am very happy with my move to Microsoft Teams, and I am looking forward to helping my customers embrace this new era of "Intelligent Communications".
Thanks for reading, over and out.
Hi Andrew. Is It ok for you to have your team appearing as an option at Outlook for mail sending? If so, How did you deal with It?
Hey Leandro, could you please elaborate on your question? I am not 100% sure I understand what you are asking.


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Brookfield Residential Properties Inc Stock Upgraded (BRP)
Brookfield Residential Properties (NYSE:BRP) has been upgraded by TheStreet Ratings from a hold to buy.
TheStreet Wire
Editor's Note: TheStreet ratings do not represent the views of TheStreet's staff or its contributors. Ratings are established by computer based on metrics for performance (which includes growth, stock performance, efficiency and valuation) and risk (volatility and solvency). Companies with poor cash flow or high debt levels tend to earn lower ratings in our model.
NEW YORK ( TheStreet) -- Brookfield Residential Properties (NYSE: BRP) has been upgraded by TheStreet Ratings from hold to buy. The company's strengths can be seen in multiple areas, such as its robust revenue growth, largely solid financial position with reasonable debt levels by most measures, solid stock price performance, good cash flow from operations and impressive record of earnings per share growth. We feel these strengths outweigh the fact that the company shows low profit margins.
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Highlights from the ratings report include:
BRP's very impressive revenue growth greatly exceeded the industry average of 27.0%. Since the same quarter one year prior, revenues leaped by 96.2%. Growth in the company's revenue appears to have helped boost the earnings per share.
The debt-to-equity ratio is somewhat low, currently at 0.82, and is less than that of the industry average, implying that there has been a relatively successful effort in the management of debt levels.
Powered by its strong earnings growth of 108.00% and other important driving factors, this stock has surged by 95.61% over the past year, outperforming the rise in the S&P 500 Index during the same period. Turning to the future, naturally, any stock can fall in a major bear market. However, in almost any other environment, the stock should continue to move higher despite the fact that it has already enjoyed nice gains in the past year.
Net operating cash flow has significantly increased by 394.58% to $156.03 million when compared to the same quarter last year. In addition, BROOKFIELD RESIDENTIAL PPTYS has also vastly surpassed the industry average cash flow growth rate of 41.66%.
BROOKFIELD RESIDENTIAL PPTYS reported significant earnings per share improvement in the most recent quarter compared to the same quarter a year ago. The company has demonstrated a pattern of positive earnings per share growth over the past year. During the past fiscal year, BROOKFIELD RESIDENTIAL PPTYS increased its bottom line by earning $0.90 versus $0.07 in the prior year.
Brookfield Residential Properties Inc. operates as a land developer and homebuilder in North America. The company engages in the entitlement and development of land; design, construction, and marketing of single-family and multi-family homes; and sale of lots to homebuilders and third parties. The company has a P/E ratio of 23.1, above the S&P 500 P/E ratio of 17.7. Brookfield has a market cap of $2.3 billion and is part of the financial sector and real estate industry. Shares are up 16.9% year to date as of the close of trading on Tuesday.
You can view the full Brookfield Ratings Report or get investment ideas from our investment research center.
-- Written by a member of TheStreet Ratings Staff
It's Official: Action Alerts PLUS beats the S&P 500 with Dividends Reinvested! Cramer and Link were up 16.72% in 2012. Were you? See what they are trading for 14-days FREE.
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When we arrived at the Victory Primary School, the students were taking a nap. There are 2 small buildings in the school. With yellow walls, the colorful One Playground seemed striking in the sun. Looking into the classrooms on the 1st floor, some of the students were sleeping at their desks. But most of them had not fallen asleep and they looked at us. At the end of nap time, the children cheered up and ran out of their classrooms. They were running toward One Playground. Some of them were playing with the wheel and some of them were playing on the slide. Obviously, the children are fond of this small green playground.
We asked some questions about the management of the playground. There were some aspects that need to be improved. We reached an agreement for the management of the playground. Students were elected to help with the management. Class leaders from each class make up different groups and each group is responsible for supervision. Hopefully this method will promote self-management and self-monitoring among the students. Student supervision also means that One Playground can better accomplish its full potential.
It's May Day. The children are growing up and have started to become involved in the planting work we do at the Home. On May 4th children from the Home came to the fields nearby and learned to transplant rice seedlings. The work is not always easy but learning is an important life skill. Happily, the children were able to find a lot of fun in the fields together.
A traditional Chinese festival called the Dragon Boat Festival is coming. Our Home has been very busy in preparation for the festival. The teachers bought materials and the children helped make Zongzi. Then everyone shared the results and agreed that they tasted great. It was a happy Dragon Boat Festival.
People with leprosy are sent to isolated villages where they are abandoned and forgotten by society. The Shalom Leprosy Compassion Ministry has a special place in its heart for these people and organizes groups of volunteers to travel to villages plagued with leprosy in order to provide medical, living, and psychological assistance. The Shalom Leprosy Compassion Ministry is located in Kunming, Yunnan.
May is a rainy season and Liang Ping, the person in charge, is about to take an examination, so the visit to the leprosy village has not been organized yet. Currently our work is focused on a solar energy project at the Yangjuangou leprosy village. We are trying to complete it as soon as possible, so that the rehabilitation clients can use it earlier.
We have also been working on installing lights and are waiting for the wiring to be finalized. All of this was organized by Andre, an American volunteer, who was assisted by Liang Ping. After the relevant work had been done, Liang Ping and Andre examined the project on May 23rd. They then installed a special-made pump for the clients.
Andre is about to install the new pump. It's easy for him, but he is working on it seriously.
After installation, he plugs in and checks if it works well and to ensure its safe and normal use.
Checking water temperature and pressure in the bath room.
Yunmeng Volunteers Association, Huberi Province works with poor students, which aims to verify the students' situation and find sponsors to help them with living expenses, psychological counseling, etc.
Before the Dragon Boat Festival, we visited Chen Biyu, a poor student in the Daodian township. Wu Fang bought snacks for her. Shu Jiamei bought pork ribs for her to help her grow. When we saw her, Wu gave her 200RMB, urging her to study hard and call us if she faces any difficulties. We showed our respect to Wu. We told her to take action hand-in-hand with us.
Shuai Zhenyu, one of our volunteers, was also concerned about the poor students. She decided to visit the students despite the hot weather in the afternoon. She gathered other volunteers up and visited two poor students in Daodian town. They brought the children fruit and stationary. They also asked detailed questions about their studies and their lives. It is volunteers like this that we respect so much and also learn from.
Tianlai Speech Therapy Center of Ganzhou, Jiangxi Province aims to provide speech rehabilitation services to the children with hearing impairment and early special education, instructing the parents to master the skills of rehabilitation for the children with hearing impairment. It's a professional rehabilitation organization for the deaf children, integration of education, rehabilitation and research.
In 2013, a national cochlear implant project helped 17 children from the Tianlai Speech Therapy Center. After 3 months of training, they developed quickly. Now they can understand simple sentences. Their parents were very happy and became more confident about their children's future.
Parents also play a key role in speech rehabilitation for children with hearing impairments, so we organized a parent's meeting after school every Friday. Teachers reported on the children's weekly performance to their parents, pointing out their developments and weaknesses. Meanwhile, we instructed the parents on how to consolidate and expand training in the family. The parents also gave feedback and asked questions.
In the one-hour meeting, the parents learned a lot. During the rehab process, not only the children, but the parents as well, are changing. The parents become positive and more willing to make an effort. This is a big change from the embarrassment and desperation they often feel after they learn about their child's problem.
Two East Villagers interns, Sarah Chang and Janna Wang, have just left the US to work for Transparent Fish Fund in China for the summer. They will both be living in Gansu working primarily with our partner Little Red Scarf. This summer they will have the opportunity to work with the TFish staff in Gansu in and out of the hospital, visiting and talking with the children and learning about the Little Red Scarf company. They will also have the opportunity to learn more about TFish and the process local NGOs go through to become a member. We look forward to hearing about their experiences!
Sarah is a junior studying philosophy at Stanford University.
Janna will be attending UCLA this coming fall.
Swing by to say hi and check out artwork done by interns and partners and catch up with our TFish Team.
Forward to all your friends!
At 9:00 in the morning, volunteers prepared gifts of rice and oil for the elderly.The distribution of goods went well. When we arrived at a blind man's home, he was having supper. We learned that he gets about 1200RMB a month from the government. He lives alone, so it covers almost all his expenses for a month. He is confident and optimistic about his life. The person we visited who lived the farthest away was Ban Jizhen. We had to walk 5 kilometers on a gravel road to reach her home. She was not at home so we put 2 bags of rice and 1 bottle of oil on her window. When we arrived it was lunch time and we were hungry so we ate some bread and cucumber. After lunch, we visited Meng Meilian. When we saw her, she had bare feet. She was not wearing the shoes we gave her last time. She said it was more comfortable to keep her feet bare. Then we visited 2 more people, but they were not at home, so we left the rice and oil at their neighbors' houses. The last person we visited was Wei Baoyuan. He lives in an old house. He does not have any income, so he has to rely on his 300RMB allowance from the government.
According to our interviews, all government allowances were in fact real and helping to sustain the lives of each of the old men, however their spirits were left unattended. They have no one to talk with. They are greatly moved whenever we visit. They even cook for us. They are kind and nice. We appreciate them for their attitude towards life, which encourages us all to accept the sorrow and happiness that life brings to us.


B-B-Q. Need we say more? This state is full of some of the most delicious BBQ known to humankind, and plenty of friendly folks to share a plate with. That is why Netchex is proud to expand our successful, service driven HCM solution in Kentucky. See for yourself how great it is to have innovative technology and friendly, personal support rolled into one.
Organizations throughout the state already love our powerful HR capabilities and seasoned industry experts. Now, we're expanding our presence with a local shop right in the heart of The Bluegrass State.


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Home » New London, CT Steamer WALLACE Explosion, June 1872
New London, CT Steamer WALLACE Explosion, June 1872
Connecticut |
Ship Disasters |
ANOTHER STEAMBOAT EXPLOSION.
FEARFUL DISASTER AT NEW LONDON, CONN.
A GOVERNMENT LIGHTER BLOWN UP.
THREE MEN KILLED AND THE OFFICERS ALL INJURED.
New London, Conn., June 24, 1872.
A fearful catastrophe occurred here today, which shades every other calamity at this place since the burning of the steamer City of New London. Terrible as the latter disaster was in most respects, there was at least an opportunity afforded for escape to a number of persons on board the ill-fated vessel; but in the present instance the suddenness of the accident almost precluded the possibility of escape.
The Explosion.
At about a quarter past six A.M. the steam-lighter Wallace, Captain THOMAS A. SCOTT, of Jersey City, started with a load of stone from the government wharf, back of the Custom House, for Race Rock, where the foundation for the new lighthouse is now being laid. When about twenty rods from the dock she exploded her boilers with a tremendous concussion, scattering the fragments in all directions, and dealing death and fearful injuries upon the unfortunate crew. There were on board at the time thirteen men, all of whom, excepting the captain, were more or less injured -- several fatally.
Captain SCOTT leaped overboard immediately and escaped with little or no injury. Two of the crew are missing, and one, at least, is known to be killed. There is a hope that the other was not on board. Fragments of the boilers were hurled into the air and carried a hundred rods from the scene of the explosion. The tug sunk immediately, and boats put off from the shore to the wreck to render whatever assistance was possible. The injured of the crew were brought ashore and taken into the Custom House, which Collector George H. Marshal converted into a hospital, and where the best medical aid of the town was summoned to the aid of the unfortunate men. The character of the wounds inflicted is frightful, the hot steam and fragments of the exploded boiler making dreadful work.
List Of Killed, Missing And Injured.
The following is a list of the crew, and their injuries as far as ascertained:
DANIEL ALLISON, fireman, killed; and as yet no traces of him have been found. He was probably blown to pieces, as he was in the engine room at the time. He belonged in Manahawken, N.J.
LAWRENCE BLACK, missing. He is also supposed to have been killed, but there is a possibility that he was not on board.
JOSEPH WATERS, steward, Sandy Hill, N.J.; badly scalded and otherwise injured by contusions.
J. W. CRAMMER, Manahawken, N.J.; leg broken in two places, badly bruised, but no scalds.
NAPOLEON CRAMMER, from the same place; badly scalded and bruised.
GEORGE BRICKLEY, spine injured and badly cut about the legs.
HARRY PASCO, New York; legs hurt.
WILLIAM WHITLER, engineer, Jersey City, fatally injured, with scalds and bruises.
Other members of the crew escaped with comparatively slight injury, and none of them are supposed to be dangerously hurt. Everything was done for the injured that could be, and during the forenoon all but the engineer were removed from the Custom House to more quiet quarters. The injuries of the unfortunate engineer are of such a character that his removal was not attempted.
Among those rendering valuable aid were Collector Marshal and the medical fraternity of New London. Captain SCOTT also did everything in his power for his unfortunate crew. A boat from the cutter James Campbell, Captain Warner, went to the assistance of the sufferers immediately after the accident and rendered valuable assistance. The remains of the killed and missing men have not been recovered, and the services of a diver will be needed to recover them. The Wallace was what is called a boom derrick lighter, and had two locomotive boilers on board, both of which blew up.
There is not cause assigned for the explosion.
New York Herald New York 1872-06-25
Connecticut Disasters
Air Disasters (9)
Animal Accidents (3)
Auto Accidents (21)
Bridge Collapses (4)
Building Collapses (3)
Cave Ins (2)
Collapses other (3)
Floods (19)
Horse and Buggy Accidents (8)
Ship Disasters (17)
Storms & Lightning (14)
Tornadoes (11)
Train Wrecks and Accidents (86)
South Hadley, CT Tow-Boat GREENFIELD Explosion, May 1840
New London, CT Fishers Island Steamer ATLANTIC Wreck, Nov 1846
New Haven, CT Steamer ELM CITY Fire, Jul 1891
Toronto, ON Steamer ROBERT LOWE Wreck, Nov 1872
Jewett City, CT Fires, 1863-1872


text:
Art from an interior design perspective - an interview with Jilly Greene
Art has the power to transform a room. It can command attention as a dramatic focal point, or it can gently complement the mood and style of the space.
But the way a room is designed also greatly influences the way art is received and appreciated. Certain colours, shapes, and textures can be used and manipulated to perfectly capture an artwork's story.
Art and interior design marry together to create awe-inspiring places. I chat to interior designer Jilly Greene about how her depth of artistic knowledge influences the way she creates a room.
Art and interior design: where did it all begin?
As a university student in Wellington looking for some extra income, Jilly began working for an art gallery and found herself spellbound by the art world.
Soon she was starting a gallery with a friend at Auckland International Airport, showcasing artwork that captured a cross-section of New Zealand art, from traditional designs to pieces with a modern edge.
After eighteen years Jilly embarked on her own interior design journey with her business, Stage My Home. Jilly creates awe-inspiring spaces that are enhanced by the business' impressive portfolio of New Zealand art.
"This kind of work keeps me on my toes because you have to allow the furniture and style they have already to shine through your work"
Seeing art from an interior design perspective: How do the two come together when you create a space?
Jilly says her job is to find the soul of a room, and it's often through art that this soul manifests. While furniture and fittings may be chosen for comfort or design, art is chosen for deeper personal reasons – and that is what makes it special.
In her work, Jilly says the most important thing is to identify what the client needs.
This can be a tough task, she explains, as often a client can't describe or explain what they want until you provide options – or until they disagree with your ideas!
Explaining the process: what comes first, the art or the space?
Jilly is a rarity in the interior design world because she found art first, whereas usually an interior designer first encounters art when they begin working.
This unique perspective lets her see both the art and interior design in a natural way.
Although Jilly visualises the different pieces, artists, and textures that would best fit in her mind, ultimately it's about what the client wants.
But often a client needs to truly get the feel of a piece before they know whether it's right for them, Jilly tells me.
"They take it home and live with it for a night, and that can be huge. People sometimes don't understand art in space like they see furniture, but as soon as you get art into their place it becomes a room."
But occasionally Jilly has designed a space so powerfully that the client falls in love with a piece of art they at first rejected. In one instance this resulted in a client purchasing the artwork Jilly used.
"They initially thought the colours would fight but in the end the room was brought together. They ended up being so drawn to the artwork that it brought them to tears."
While the client's needs must always come first, awe-inspiring spaces can be created when a client puts their trust in such an art-oriented interior designer.
Do you get inspired by the subject or colours in a piece or do you just find art that works within a room?
Again, the customer's vision has to come first. Jilly explains that while colours and textures are important, you have to approach the interior with a piece of art that the client loves.
"It won't feel as good if you approach it like painting-by-numbers."
Jilly reminds us that fusing art and interior design can put too many heroes in a room, creating unnecessary conflict. Keeping it simple is a philosophy she follows.
What's the first thing you'd tell someone looking to use art in your space?
"The core thing is that they need to love the artwork."
Drawing together what the client wants, Jilly says, is a journey in itself that comes with many challenges. Often it takes a few meetings of going through options to figure out whether art is important to the client, then it's sometimes about compromising between a client and their partner.
It's a process that is often harder when she has to find art for a room rather than create a room for a piece, but Jilly is always surprised when clients come back to her raving about an artwork she would never have chosen herself.
What about specific spaces, such as the lounge and bedroom, does that influence the artwork you choose or how you craft a space?
Jilly imagines the mood each room should provide, such as the peaceful haven of a bedroom, which she must take care to create as she works.
"Each room has its own place in the house."
But sometimes an interior designer faces a balancing act of form and function. Jilly talks about a recent challenge she faced designing an open-plan living space. The clients wanted to maintain the openness of the area whilst also clearly identifying a dining space within the room.
Commenting on the progression of interior design in the last 20 years, Jilly notes the growing importance of art in the client's mind.
"Art has become a part of interior design, as opposed to an afterthought. People go in with an idea of the art and the space."
What do you love about what you do?
"I love marrying furniture with art, and it puts me on a high at the end of every day when I finish up a room. I love artists that have variety and experiment with many different stories, styles, and genres."
Perhaps it is this appreciation of the stories within art that allows Jilly to create such inspiring spaces. Walking the line between two creative worlds, Jilly shows us what can be achieved by combining different artistic mediums.
Looking to bring your space to life with art? Discover fine art prints and paintings.


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You can find more Lee Davis alumni who participated in activities at Classmates.com®. Click here to join Classmates.com® for free and to view LDHS profiles.
Choose an activity that students at LDHS could particpate during their time in high school in Mechanicsville to view the names of the students who participated in that sport or activity. You can also add yourself to the list.	The main activities at Lee Davis that have members on this site were Art Club, Band, Baseball, Basketball, Cheerleading, Cross Country, Dance, Drama, Football, Future Business Leaders of America, Gymnastics, Music, Newspaper, Soccer, Softball, Student Council, Swimming, Tennis, Track, Wrestling and Yearbook.


text:
Google Stadia promises 10 timed-exclusives in the next six months
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Hurry: Apple AirPods 2 Are on Sale for $129, a $30 Discount
IBM Tops US Patent List for 2019
OnePlus teases 120Hz motion smoothing for its new Fluid Display
There's probably going to be a 'Bad Boys 4'
Dating apps Grindr, OkCupid and Tinder leak personal data, Norwegian group says
Google Is Planning to Bring Steam To...Chromebooks
UAE- Apple buys edge-based firm for $200m
Starlink is Getting Star Fox Content Exclusively on Switch
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This is the third in a series of eight blog posts detailing Signal's New Year's predictions for digital marketing. As outlined in the recently published report, "How Digital Marketing Will Change in 2016 – What Marketers Need to Know Now," Signal's distinguished panel of in-house digital experts and thought leaders addressed the challenges that marketers will face and the emerging trends in marketing and technology that can help to overcome them. This post focuses on the widening gap between consumers' mobile activities and an advertiser's ability to engage with them on the go.
By the beginning of 2016, mobile ad spending in the US will reach $42 billion, accounting for two-thirds of all digital ad spending. And that's no surprise. There are now more mobile-only consumers than desktop-only consumers. But just because mobile is where consumers are, doesn't mean that it's easy for marketers to reach them.
Marketers have yet to nail the challenges of recognizing customers on smartphones, tablets, wearables, and beacons, and then connecting that data with the rest of consumer journey in real-time to deliver relevant, in- the-moment customer experiences. And with 50 billion connected products anticipated to exist by 2020, the need for marketers to close this gap has never been more urgent.
In 2016, expect to see marketers get serious about addressing these gaps by exploring new and different solutions. Marketers will rethink their technology investments and consider where they might be missing information about the customer journey – and missing opportunities to interact with their customers.
Server-based technology that uses API connections and the always-on cloud to collect customer data from all devices and touchpoints, creating persistent profiles that continue to get richer and deeper over time.
Deterministic matching, which is considered the industry gold standard and the only method for targeting known customers.
The ability to harness live intent signals, rather than relying on batched processes, campaigns, or decaying cookies, to recognize customers immediately across any channel and respond in real-time.
By implementing solutions that fulfill these requirements, marketers will be on the path to closing this gap and achieving true people-based marketing in 2016 and beyond.
Signal offers a solution that ties consumer profiles to persistent cloud-based identifiers that are server-based, rather than tied to decaying third party cookies. Offline and online identifiers are linked at the profile level to power cross-channel engagement. Over time, these persistent profiles are enriched by each consumer engagement point and available to leverage for a lifetime of targeting across all channels and devices.