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This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.
Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease's pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.
Since the advent of antiretroviral therapy, there has been a substantial decrease in deaths related to HIV-1 monoinfection. However, liver disease has emerged as a common cause of morbidity in HIV-infected patients1,2. Coinfections of hepatitis viruses with HIV-1 infection are more common, accounting for 10% - 30% of HIV-infected persons in the United States3,4,5.
The host-specificity of HIV-1 and hepatitis viruses limits the utility of small animal models to study human-specific infectious diseases or to investigate multiple aspects of HIV-1-associated liver pathogenesis. Immunodeficient mice that permit the engraftment of human cells and/or tissues (termed humanized mouse models) are acceptable animal models for preclinical studies6,7,8. Since the introduction of humanized mice in the early 2000s, multiple preclinical studies of cholestatic human liver toxicity, human-specific pathogens, including HIV-1 and HIV-associated neurocognitive disorders, Epstein Barr virus, hepatitis, and other infectious diseases, have been investigated in these mice6,9,10,11. Multiple mouse models for CD34+ HSPCs and/or human hepatocyte transplantation have long been developed and have improved over time to study the disease pathogenesis of Hepatitis B virus (HBV)-associated liver disease12,13,14. Several models for HSPC and human hepatocyte (HEP) transplantation are based on strains, known as NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac)8,13, NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ)15, Balb/C-Rag2-/- γc-/- (Rag2tm1.1Flv Il2rgtm1.1Flv/J)12, and fah-/- NOD rag1-/-il2rγnull mouse16. However, each model has its own advantages and limitations; for example, AFC8 dual humanized mice for HEPs and human stem cells (HSCs) on a Balb/C-Rag2-/- γc-/- background enables the successful engraftment of immune cells and HSCs, but there is an absence of an antigen-specific T- and B-cell response in this model12. The major concerns in reconstituting double humanized mice include suboptimal engraftment, a lack of suitable models to support different tissues, mismatched conditions, immune rejection, or graft-versus-host disease (GVHD), and technical difficulties, such as risky manipulations with newborns and high mortality rates due to metabolic abnormalities13.
Although humanized mice have been used for HIV research for many years17,18,19, the use of humanized mice to study liver damage caused by HIV-1 has been limited20. We previously reported the establishment of a dual humanized TK-NOG mouse model and its application in HIV-associated liver disease8. This model shows the robust engraftment of liver and immune cells and recapitulates HIV infection pathogenesis. This discussion presents a detailed protocol, including the most critical steps in the transplantation of human hepatocytes. A description of the HSPCs required for a successful engraftment of HEPs and the establishment of a functional immune system in TK-NOG mice is also presented. The use of these mice to study HIV-associated liver immunopathogenesis is detailed. TK-NOG male mice carrying a liver-specific herpes simplex virus type 1 thymidine kinase (HSV-TK) transgene are used. Mouse liver cells expressing this transgene can easily be ablated after a brief exposure to a nontoxic dose of GCV. Transplanted human liver cells are stably maintained within the mouse liver without exogenous drugs21. The mice are also preconditioned with nonmyeloablative doses of treosulfan to create a niche in the mouse bone marrow for human cells8. Immunodeficient TK-NOG mice are intrasplenically injected with HEPs and multipotent HSPCs. The mice are then regularly monitored for blood and liver reconstitution by blood immunophenotyping and measurements of serum human-albumin levels, respectively. Mice with a successful reconstitution of more than 15% for both human immune cells and HEPs are intraperitoneally injected with HIV-1. The effect of HIV on the liver can be assessed as early as 4 - 5 weeks postinfection. It is critical to note that, because HIV-1 is used, all necessary precautions must be taken while handling the virus and injecting it into mice.
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This protocol has been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Nebraska Medical Center.
NOTE: Obtain approval from the local IACUC before performing experiments on animals.
1. Processing of Umbilical Cord Blood and the Isolation of Human HSPCs
- Perform all steps of the protocol under sterile conditions in laminar flow cabinets.
- Take umbilical cord blood (CB) collected in heparinized tubes and make the volume up to 35 mL by adding phosphate-buffered saline (PBS). Layer the sample on top of the lymphocytes separation medium (LSM) as illustrated in Figure 1 and centrifuge the LSM with the layered CB at 400 x g for 35 min at 4 °C with no brakes.
NOTE: Dilute the blood carefully and gently to avoid mixing at the interface.
- Remove the top LSM and plasma layer carefully and transfer the white buffy coat interface to a new tube using a transfer pipette.
- Resuspend the buffy coat in 30 - 40 mL of ice-cold buffer (PBS + 0.5% bovine serum albumin [BSA] + 2 mM ethylenediaminetetraacetic acid [EDTA]). Using a pipette, combine 20 µL of the cell suspension with 20 µL of 0.4% trypan blue and pipette 10 µL of the mixture into the outer opening of either of the two chambers of a counting slide and insert the slide in an automated cell counter to count the cells.
NOTE: Use ice-cold buffer in all steps, as it helps keep the cells viable.
- Centrifuge the cells at 300 x g for 10 min at 4 °C and aspirate the supernatant carefully. Then, add 300 µL of ice-cold buffer.
- Add 100 µL of human Fc receptor blocking reagent and 100 µL of monoclonal mouse anti-human CD34 antibody-conjugated microbeads for up to 1 x 108 cells (see the Table of Materials). Incubate for 30 min at 4 °C, add 10 mL of ice-cold buffer to wash the cells, and centrifuge at 300 x g for 10 min at 4 °C.
NOTE: Scale this up according to the cell number if more than 1 x 108 cells are present.
- Carefully remove the supernatant, resuspend the pellet in 500 µL of buffer, and proceed with the magnetic separation step to enrich HSPCs.
- Place a positive selection LS column (see the Table of Materials) in the magnetic-activated cell sorting field and pass it through with 3 mL of buffer.
- Load the sample to the LS column that can entrap microbeads bound to human CD34+ in samples and allow it to flow under the influence of gravity into the collection tube.
- Wash the column 3x with buffer and collect the elute in the same collection tube of the CD34- fraction of cells.
- Plunge the column with 5 mL of buffer to elute the CD34+ cells into a new collection tube. Repeat the procedure to achieve a purity of >90%.
- Count the eluted CD34+ cells using trypan blue dye in a hemocytometer. After counting, centrifuge the CD34+ cells at 300 x g for 5 min and discard the supernatant.
- Resuspend the CD34+ cells in 25 µL of PBS for an injection to be used immediately in transplantation or cryopreserve the cells at a concentration of 1-2 million/mL in freezing medium (Roswell Park Memorial Institute medium [RPMI 1640 medium] + 50% fetal bovine serum (FBS) + 10% dimethyl sulfoxide [DMSO]) for further use in transplantation.
NOTE: Always recount viable cells before using them in transplantation.
- To check the purity of the CD34+ elute, take 50 µL of the suspension and incubate it with 10 µL of PE-conjugated anti-human CD34 antibody for 30 min at 4 °C. After the antibody incubation, wash the stained cells with PBS, resuspend them in 100 µL of PBS, and then, proceed to perform flow cytometry. Add an additional tube of cells with no antibody to design the gate in the flow cytometer.
- After acquisition, analyze the data by selecting the region of interest on a forward scatter (FSC) and side scatter (SSC) plot, followed by gating for single cells on FSC-area and FSC-height plots. Gate CD34-positive cells on single cells in the PE channel and SSC-area plot.
2. Preparation of Human Hepatocytes for Transplantation
- Remove the cryopreserved hepatocytes from the liquid nitrogen, quickly submerge the vial in the water bath, and thaw for approximately 90 - 120 s.
- Remove the vial cap and pour the thawed hepatocytes into the 50 mL conical tube of the warmed thawing medium.
- Suspend the cells by rocking the 50 mL tube by hand for a few seconds.
NOTE: Do not vortex the tube.
- Pellet the cells at 100 x g for 8 min at room temperature.Wash the pelleted cells in PBS with 0.1% BSA and pool them with either fresh or thawed HSPCs (ratio 10:1) in PBS in a final volume of 80 µL/mouse.
3. Animal Handling, Screening, Genotyping, and Treatment for Human HSPC and Hepatocyte Transplantation
- Animal handling
- As a result of severe immunodeficiency, breed, house, and handle TK-NOG mice under aseptic conditions.
- Always wear a lab coat, gloves, shoe covers, and a face mask to prevent infection with potentially pathogenic microorganisms.
- Use sterile gloves and instruments for surgery and handle the animals aseptically throughout the surgery.
- Selecting TK-NOG mice for the experiment
- Maintain the TK-NOG strain colony by breeding female TK-NOG mice with male non-TK-NOG littermates and select transgenic offspring by genotyping.
NOTE: Perform genotyping (see step 3.3) to determine the presence or absence of the transgene in newborn male and female mice at the time of weaning.
- Select males at 6 - 8 weeks of age for transplantation due to their high sensitivity to the GCV-mediated depletion of HSV-TK transgene-expressing hepatocytes21.
- Ear-tag the mice at the time of weaning or surgery to ease identification. Note down the weight and health status of the animals.
- Maintain the TK-NOG strain colony by breeding female TK-NOG mice with male non-TK-NOG littermates and select transgenic offspring by genotyping.
- Genotyping for the presence of the HSV-TK transgene using quantitative real-time polymerase chain reaction
- Perform genotyping at the time of weaning (usually at 3 - 4 weeks of age). For genotyping, cut a piece of the mouse ear in a laminar flow biological safety cabinet to maintain sterility and extract genomic DNA by using a genomic DNA isolation kit.
- Amplify genomic DNA extracted from ear piece in a 20 µL reaction mixture to screen for HSV-TK transgene under control of human albumin promoter by adding 1 µL of forward primer 5'-CCATGCACGTCTTTATCCTGG-3', 1 µL of reverse primer 5'-TAAGTTGCAGCAGGGCGTC-3', 0.5 µL of FAM probe 5′-FAM-AATCGCCCGCCGGCTGC-MGB-3', and 10 µL of master mix on a real-time polymerase chain reaction (PCR) instrument22.
- Set the real-time PCR settings as follows: 60 °C for 30 s (preread stage), 95 °C for 10 min (hold stage), 40 cycles of 95 °C for 15 s and 60 °C for 1 min (PCR stage), and 60 °C for 30 s (postread stage).
NOTE: A cycle of threshold (Ct) below 22 is considered positive for HSV-TK transgene.
- Treatment using ganciclovir and treosulfan
- Using 27 G needle, inject the TK-NOG mice with intraperitoneal GCV injections (6 mg/kg) 2x a day at day 7 and at day 5 in 100 µL of saline before surgery to deplete the mice's transgenic parenchymal cells (as shown in the experimental strategy in Figure 2)23.
- On days 3, 2, and 1 before the surgery, precondition mice with nonmyeloablative intraperitoneal doses of treosulfan (1.5 g/kg/day) in 100 µL of saline, using a 27 G needle.
- One day before the surgery, draw two to three drops (~100 µL) of blood from the submandibular vein by pricking it with a 5 mm lancet, and isolate the serum by centrifuging (1,500 x g for 10 min at 4 °C) for the alanine aminotransferase (ALT) assay to assess the degree of liver damage.
- Preparation for the surgery
- Use clippers to shave the mouse's fur surrounding the incision site (at the left of the peritoneal wall) before surgery.
- Adjust the oxygen flow to 1 L/min and the isoflurane flow to 3% - 5% in an induction chamber using a mouse anesthesia machine. Place one mouse at a time in the induction chamber for anesthesia.
- Attach the one end of a sterile extension tube (with a holding capacity of 550 µL of suspension; see the Table of Materials for specifications) to a 30 G needle and the other end to a 1 mL syringe.
- Fill the syringe with the suspension (80 µL/mouse) of pooled HEPs and HSPCs (see section 2), fit the syringe in the notch of a repetitive dispensing pipette, and adjust the dispenser to dispense 10 µL in each press.
- Once the mice are anesthetized (usually after 3 - 4 min), switch the isoflurane flow to the nose cone and reduce the isoflurane flow rate to 2% - 3%.
- Intrasplenic transplantation of human HSPCs and hepatocytes in mice
- Perform all surgery steps in a laminar flow cabinet under sterile conditions.
- Place a clean sterile drape over the working surface and scrub the left side of the body of each mouse with 10 % povidone-iodine followed by 70 % isopropyl alcohol, before making an incision.
- Make a small incision (~1 - 1.5 cm in length and 5 mm deep) in the skin, muscle, and peritoneum at the left of the peritoneal wall with Vanna's type scissors to enter the peritoneal cavity approximately 5 mm below the lower edge of the rib cage.
- Locate the spleen, pull it slightly with forceps to the operating area for easy access, and insert the 30 G needle into the lower pole of the spleen.
- Unlock the plunger of the dispensing pipette and dispense 10 µL of the volume at a time, with a limit of 60 - 80 µL per spleen. Retract the needle slowly and clip the spleen with ligating clips using a ligation applier.
- Push the spleen back into the body cavity with cotton-tipped applicators wetted with sterile PBS.
- Close the muscle layer of the abdominal wall using an absorbable suture interrupted suture pattern (not continuous). Accomplish skin closure with an interrupted suture pattern using non-absorbable sutures.
- Use warm water circulating pads to protect the mice from hypothermia after surgery.
- Remove non-absorbable sutures 10-14 days after surgery.
- Postoperative care
- When the transplanted animal wakens, inject analgesic buprenorphine (0.1 mg/kg) intraperitoneally, 2x a day for a consecutive 3 days.
- Observe the animals at least 1x a day until they return to normal physical conditions.
NOTE: Check each animal's body weight, since some mice may lose weight postsurgery. Mice typically regain their original weight in 1 to 2 weeks.
4. Engraftment Validation of the Human Liver by ELISA and the Human Immune System by Flow Cytometry
NOTE: Evaluate the reconstitution of the human liver and immune system monthly, starting 1 month posttransplantation by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively.
- Collect blood samples from the submandibular vein using lancets in EDTA tubes, and centrifuge at 1,500 x g for 10 min at 4 °C. Isolate serum to check human albumin levels to assess the engraftment efficiency of the mouse liver for transplanted human hepatocytes, using a human albumin ELISA quantitation set (see the Table of Materials) by following the manufacturer's instructions.
NOTE: Do not discard the pellet and use the pelleted cells for a flow cytometry analysis to evaluate the human immune system reconstitution.
- Resuspend the cell pellet without serum in 35 µL of FACS buffer (PBS + 2% FBS) and stained with 5 µL of mouse-specific CD45 (concentration 0.5 mg/mL), 5 µL each of human-specific antibodies CD45 (0.1 mg/mL), CD3 (0.2 mg/mL), CD8 (0.1 mg/mL), and CD19 (0.5 mg/mL), and 20 µL of CD4 (0.25 mg/mL) and CD14 (0.25 mg/mL) each for 30 min at 4 °C, to check the development of a functional immune system from CD34+ HSPCs.
NOTE: Consider adding one additional tube of unstained cells to determine the gating of the stained cells.
- After incubation, transfer the stained suspension (~100 µL) in a polystyrene round-bottom flow cytometry tube and use 2 mL of 1x lysis buffer (see the Table of Materials) by diluting 1 part of 10x lysis buffer with 9 parts of distilled water and incubating 10 - 15 min to lyse red blood cells.
NOTE: Observe turbidity to evaluate the red blood cell lysis. Once the sample becomes clear, the lysis is complete.
- After the lysis, add 3 mL of the FACS buffer in the tube and centrifuge at 300 x g at 4 °C for 5 min to get a pellet. Repeat the washing by adding 3 mL of FACS buffer to the pellet and centrifuge at 300 x g at 4 °C for 5 min.
- Fix the cells in freshly made 1% paraformaldehyde (PFA) and acquire stained cells on the flow cytometer, analyzed with flow software.
- For the analysis, select lymphocytes gating on an FSC/SSC plot, followed by single-cell gating on FSC-area/FSC-height.
- Further, gate for human-specific CD45 (hCD45) on the single-cell population and include mouse-specific CD45 (mCD45) for the exclusion of cells of murine origin. Strategize gating of the stained population based on the gating of unstained cells.
- Gate hCD45+ cells to determine the frequency of CD3+ T cells and CD19+ B cells. Gate T cells to determine CD4 and CD8 subsets. To evaluate monocytes, gate on hCD45 to determine CD14+ monocytes.
5. HIV Infection of TK-NOG Mice and Its Effect on the Human Liver and Immune System
- Handle the HIV-1 virus and all infected mice in a designated biosafety level 2 facility.
CAUTION: Autoclave and discard all HIV-infected wastes in double biohazard bags. For safety reasons, wear cut-resistant gloves while handing HIV-infected mice.
- Wear personal protection equipment (PPE), including a disposable coverall gown, shoe covers, a face mask, and double gloves at all times while working with the virus.
- Select mice with a reconstitution of more than 15% of human CD45+ cells (tested in step 4.7) and with a presence of human albumin in the serum for HIV-1 infection (tested in step 4.1).
- Inject the mice with 1 x 103 to 1 x 104 tissue culture infectious doses 50 (TCID50) HIV-1ADA in a volume of 100 - 200 µL per mouse, intraperitoneally.
- Euthanize the HIV-infected mice 5 weeks postinfection by using isoflurane (with an isoflurane vapor concentration of >5%).
- After euthanizing the mice, collect blood in EDTA tubes by a cardiac puncture for the isolation of serum, to see the effect of HIV-1 on the liver by evaluating human-specific albumin levels by ELISA (see step 4.1) and blood cells to check for changes in the human immune cells using flow cytometry (see steps 4.2 - 4.8).
NOTE: Assess the peripheral viral load 5 weeks postinfection on a bioanalyzer to confirm if the mice are infected.
- After drawing blood, excise the liver from the euthanized mice.
- For the liver excision, expose the abdominal cavity by making an incision of 1.5 - 2 cm long and 0.5 cm deep in the skin, muscles, and peritoneum, from the xyphoid. Make a cut perpendicular to the spine between the liver and the diaphragm. Lift the liver and sever any membranes attaching it to the stomach and intestines.
- Collect and fix the liver in 4% paraformaldehyde overnight and follow a standard immunohistochemical protocol to evaluate the effect of HIV on CK18+ hepatocytes by using human-specific CK18 antibody8.
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Representative Results
The establishment of a dual humanized mouse model with human liver and immune cells can be easily monitored at each step with very simple ELISA and flow cytometry, respectively. Flow cytometry is regularly performed to evaluate the development of a functional immune system and to see the effect of HIV infection on immune cells. In dual humanized mice, the development of functional immune cells can range from 15% to 90% of the lymphocyte gate. Representative subsets of immune cells are shown in dot plots (Figure 3). For the evaluation of the engraftment of human hepatocytes, ELISA for human-specific albumin levels is performed monthly on mouse serum. Mice engrafted with both HSPCs and HEPs show human-specific albumin levels ranging from ~7 µg/mL to 377 µg/mL at one month, continuing to grow over the time of observation (6 months) (Figure 4). The effect of HIV infection on human immune cells in the blood of dual humanized mice is monitored by flow cytometry and on HEPs in the liver by human-specific albumin ELISA. By 5 weeks, HIV-1 causes a decrease in human albumin levels in the serum, as assessed by ELISA, and there is a depletion of human CK18+ hepatocytes in the liver sections of dual humanized mice, as evaluated by immunohistochemistry (Figure 5). A lower ratio of CD4:CD8 is typically observed, by flow cytometry, in the blood and liver of HIV-infected mice, compared to levels noted in the same mouse before infection (Figure 6). All reagents and materials important for the protocol are discussed in the Table of Materials.
Figure 1: Schematic of the enrichment of CD34+ cells from cord blood. (A) Cord blood is layered on lymphocytes separation medium (LSM) and centrifuged to isolate buffy coat. (B) LS columns are placed on a magnetic stand and rinsed with BSA buffer, followed by adding buffy coat. Cells positive for CD34 are trapped in the columns, and CD34- cells are eluted in separate tubes. Trapped CD34+ cells in column resins are plunged with a plunger, and the cells are collected in a new tube. Please click here to view a larger version of this figure.
Figure 2: Schematic view of the experimental design for the dual reconstitution of humanized liver and immune system mice, followed by HIV-1 infection. TK-NOG mice are injected with ganciclovir (GCV) at a dose of 6 mg/kg, 2x a day, on day -7 and day -5, followed by a treosulfan injection on days -3, -2 and -1. To screen the mice for the transplantation (Tx), an alanine aminotransferase (ALT) assay is performed one day before the surgery, and mice with ALT levels of >200 and <600 U/L are selected. After transplantation, the mice are checked for a reconstitution of the human immune system by flow cytometry (FACS) and for liver reconstitution by assessing their albumin level using ELISA. The mice are infected with HIV-1 5 weeks before they are sacrificed. Please click here to view a larger version of this figure.
Figure 3: Flow cytometry analysis gating strategy for the human cell distribution of blood. (A) First, lymphocytes are gated on whole blood based on FSC-A and SSC-A. (B) Single cells are gated on lymphocytes. (C) Human CD45+ leukocytes are gated on single cells using mouse CD45 and human CD45. (D) CD3+ T cells and CD19+ B cells are identified on gated CD45+ human leukocytes. (E) CD4+ T helper cells and CD8+ cytotoxic T cells are identified in gated CD3+ T cells. (F) CD14+ monocytes are gated from human CD45+ leukocytes. The results represented here are from one mouse transplanted with dual human hepatocytes and HSPCs. Please click here to view a larger version of this figure.
Figure 4: Albumin concentration is measured by ELISA in the serum of dual humanized mice. The mice are transplanted with both human hepatocytes (HEPs) and CD34+ hematopoietic stem/progenitor cells (HSPCs) (n = 11). Serum is collected at different times at 1, 4, and 6 months posttransplantation, and dilutions are made to adjust the unknown sample concentrations in the range of standards. Each symbol represents an individual mouse value. The results represent the median, as well as individual values. * P < 0.05, by one-way ANOVA. This figure has been modified from Dagur et al.8. Please click here to view a larger version of this figure.
Figure 5: Effect on HIV-1 on albumin levels in serum and the depletion of CK18+ human hepatocytes in the liver of dual humanized mice. (A) Albumin concentrations are monitored in uninfected mice (n = 9) transplanted with both human HEPs and HSPCs at 1 and 4 months. The mice are infected (n = 10) with HIV at 4 - 5 months posttransplantation and sacrificed 5 weeks postinfection. Each symbol represents an individual mouse value. The results represent the median, as well as individual values. * P < 0.05, by one-way ANOVA. This figure has been modified from Dagur et al.8. (B) Five-micron liver sections from uninfected (HEPs + HSPCs, left panel) and HIV-infected TK-NOG mice (HEPs + HSPCs + HIV, right panel) are fixed, paraffin embedded, and stained for anti-human cytokeratin-18 (CK18) antibody. HIV-1 causing a depletion of CK18+ hepatocytes is evidenced by a less occupied area by the CK18+ human cells. The results represented here are from one uninfected and one HIV-infected mouse transplanted with dual human hepatocytes and HSPCs. Scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 6: Ratio of CD4+ cells to CD8+ T cells in peripheral blood and in the liver of dual reconstituted uninfected and HIV-1-infected mice. For dual reconstituted uninfected mice: closed circle; HEPs + HSPCs; blood n = 7; liver n = 6. For HIV-1 infected mice: open circles; HEPs + HSPCs + HIV; blood n = 10; liver n = 11. The results represent the median, as well as individual values. * P < 0.05, by one-way ANOVA test between HIV-infected and uninfected mice. This figure has been modified from Dagur et al.8. Please click here to view a larger version of this figure.
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The liver is compromised and damaged in HIV-infected patients24. Experimental small animal models for studying human liver diseases in the presence of HIV-1 is extremely limited, despite the availability of a few cotransplanted animal models with CD34+ HSPCs and hepatocytes7,12,25. In in vitro experiments, hepatocytes are shown to have low-level HIV-1 infection26. Humanized mice that carry both types of human cells are a desirable model. The liver of mice reconstituted with only human immune system has been shown to be affected by HIV infection under the experimental depletion of human regulatory T cells20,27. However, the difference in immune and functional properties of mouse and human hepatocytes may underline the differences in their responses to HIV-1 and immune cells. In this review, a protocol is described to reconstitute both human immune system and liver and to address HIV-1-associated liver immunopathology, as observed in HIV-1-infected patients. TK-NOG male mice were selected due to their liver-selective high mRNA expression of HSV-TK transgene and the susceptibility of GCV toxicity to mouse transgenic liver21. Moreover, they can be maintained for long periods after transplantation without the use of exogenous drugs and do not develop spontaneous systemic diseases28. To establish human immune system and liver reconstitution, ablation of the mouse immune system and damage to mouse-specific liver cells are required and achieved using nonmyeloablative doses of treosulfan and GCV, as shown previously in TK-NOG male mice13,23. Mice are injected with GCV and treosulfan at the age of 6 - 8 weeks, as the expression of transgene and GCV-induced hepatic injury as assessed by ALT levels are optimal, then, for providing niche-to-transplanted human cells21. Mice showing ALT levels of >200 U/L, but <600 U/L, are usually selected for transplantation. Mice showing ALT levels of >600 U/L are at a greater risk of death as human hepatocytes are not able to rescue the damaged mouse liver function.
Currently, dual humanization is shown by the transplantation of human CD34+ HSPCs and fetal liver cells; however, the manipulation of newborn animals creates technical problems13,14. HSPCs can be derived or isolated from multiple sources, such as fetal liver cells (FLCs), embryonic stem cells (ESCs), and CB. However, ethical issues constrain the use of ESCs and FLCs. CB has no such restriction and is a most useful alternative to obtain HSPCs, as well as being a precious source of primitive hematopoietic stem and progenitor cells that can reconstitute the functional immune system. Cord blood should not be older than one day when used to isolate HSPCs, as the yield of HSPCs is highly affected by age. The purity of the isolated HSPCs needs to be checked before cryopreserving the cells. The cross-contamination of CD3+ T cells is avoided, as it may lead to systemic mouse graft-versus-host disease and acute allorejection of HEPs while transplanting with mismatched cells.
Commercially available hepatocytes were used as a source for liver reconstitution8,13. Adult hepatocytes are preferred for establishing liver reconstitution due to their increased efficiency in engraftment and sustainability for a long period of time29.
The presence of human immune system in a mouse model increased ALB levels, as shown previously30,31. However, the efficiency of hepatocytes and immune system reconstitution may vary with different sources of donor cells and depend on the recipient mouse. So, each mouse needs to be assessed for engraftment, and the most critical part is to utilize the antibodies or reagent that are human-specific and do not cross-react with mouse cells. The human-specific reagents and antibodies used in the study presented here are detailed in the Table of Materials. If antibodies other than provided in the Table of Materials are used for the study, be sure to check for the human specificity.
The optimal condition would be the transplantation of syngeneic cells; however, that is technically difficult to achieve. Wherever possible, HSPCs and hepatocytes should be pooled from donors with partially matched human leukocyte class-1 antigens (like HLA-A2).
To screen mice for HIV studies, blood is drawn at multiple time points to determine the optimal immune and liver reconstitution; flow cytometry and ELISA are preferred as they can be performed with only a little amount of blood. Blood cells and serum from the same sample could be used for flow cytometry and ELISA, respectively. It is important to make proper dilutions of serum at each time point (1,000 - 40,000 range) to evaluate ALB levels so that the unknown concentrations can be brought within the range of standard concentrations (kit range: 6.25 - 400 ng/mL).
Proinflammatory cytokines in response to HIV-1 infection in the presence of human immune system can also be useful in addressing the interaction of hepatocytes and immune cells. The model is useful for showing the immunopathogenesis of HIV-1-induced liver disease, given that it recapitulates liver damage in the same manner as in humans, evidenced by a low ratio of CD4:CD8, a decrease in ALB levels, human hepatocyte death, and liver immune activation. The model also has some limitations, such as a low level of cytotoxic T cells activity and impaired immunoglobulin class switching. Due to the presence of both human immune system and liver, the model presented here is promising for studi coinfections of HIV-1 and hepatitis viruses, chronic hepatitis infection (to clarify the mechanisms of the anti-hepatitis immune response), and as a cirrhosis model.
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The authors have nothing to disclose.
This work was supported by the National Institute of Health grant R24OD018546 (to L.Y.P. and S.G.). The authors would like to thank Weizhe Li, Ph.D., for the help in surgical procedures, Amanda Branch Woods, B.S., Yan Cheng for immunohistology, UNMC flow cytometry research facility members Director Phillip Hexley, Ph.D., Victoria B. Smith, B.S., and Samantha Wall, B.S., UNMC advanced microscopy core facility members Janice A. Taylor, B.S., and James R. Talaska, B.S., for the technical support. The authors acknowledge Drs. Mamoru Ito and Hiroshi Suemizu from CIEA for providing TK-NOG mice and Dr. Joachim Baumgart for providing treosulfan. The authors thank Dr. Adrian Koesters, UNMC, for her editorial contribution to the manuscript.
|Catalog Number
|27 G1/2" needles
|BD biosciences
|30 G1/2" needles
|BD biosciences
|5 mL polystyrene round-bottom tube 12 mm x 75 mm style
|BD 1 mL Tuberculin Syringe Without Needle
|BD biosciences
|BD FACS array bioanalyzer
|BD Biosciences
|For purity check of eluted CD34+ cells
|BD FACS array software
|BD Biosciences
|Software to analysis acquired CD34+ cell on FACS array
|BD FACS lysing solution
|BD Biosciences
|To lyse red blood cells
|BD Biosciences
|Instrument for acquisiton of flow cytometry samples
|BD Vacutainer Plastic Blood Collection Tube
|BD biosciences
|To collect Cord blood
|Bovine Serum Albumin
|Controlled substance and pain-killer
|BD Biosciences
|Specific to human
|BD Biosciences
|Specific to human
|CD34 MicroBead Kit, human
|Miltenyi Biotec
|For isoation of CD34+ HSPC
|CD34-PE, human
|Miltenyi Biotec
|Antibody used for purity check of eluted CD34+ cells
|BD Biosciences
|Specific to human
|BD Biosciences
|Specific to human
|BD Biosciences
|Specific to human
|BD Biosciences
|Specific to human
|Cell counting slides
|ChargeSwitch gDNA Mini Tissue Kit
|Thermofisher scientific
|for extraction of genomic DNA from ear piece
|Cobas Amplicor system v1.5
|Roche Molecular Diagnostics
|bioanalyzer to measure viral load
|Cotton-tipped applicators
|Cytokeratin-18 (CK18)
|Specific to human
|DMSO (Dimethyl sulfoxide)
|Extension set Microbore Slide Clamp(s) Fixed Male Luer Lock. L: 60 in L: 152 cm PV: 0.55 mL Fluid Path Sterile
|BD biosciences
|Attached to dispensing pippet and to load with HSPC and HEP suspesion
|FACS Diva version 6
|BD Biosciences
|flow cytometer software required for acqusition of sample
|Fetal Bovine Serum (FBS)
|FLOWJO analysis software
|flow cytometry analysis software
|APP Pharmaceuticals, Inc.
|Prescripition drug
|Greiner MiniCollect EDTA Tubes
|Greiner bio-one
|Hepatocytes thawing medium
|Triangle Research Labs
|Horizon Open Ligating Clip Appliers
|To hold the ligating clips
|Hospira Sterile Water for Injection
|ACE surgical supply co. Inc.
|For dilution of Buprenorphine (pain-killer)
|Human Albumin ELISA Quantitation Set
|Bethyl laboratories
|For assesing human albumin levels in mouse serum
|Human hepatocyte
|Triangle Research Labs
|Cryopreserved human hepatocytes, induction qualified
|Iris Scissors, Straight
|Ted Pella, Inc.
|Goldenrod 5 mm
|LS columns
|Miltenyi Biotec
|Used to entrap CD34+ microbeads (positive selection)
|Lymphocyte Separation Medium (LSM)
|MP Biomedicals
|For isoation of lymphocytes from peripheral blood
|MACS MultiStand
|Miltenyi Biotec
|holds Qudro MACS seperator and LS columns
|McPherson-Vannas Micro Dissecting Spring Scissors
|Roboz Surgical Instrument Co.
|Used to make an incision on skin to expose spleen
|Micro Dissecting Forceps
|Roboz Surgical Instrument Co.
|to hold and pull out spleen from peritoneal cavity
|mouse CD45-FITC
|BD Biosciences
|PBS (Phosphate Buffered Saline)
|Qudro MACS separator
|Miltenyi Biotec
|holds four LS columns
|RPMI 1640 medium
|StepOne Plus Real Time PCR
|Applied Biosystems
|Instrument used to genotype
|Stepper Series Repetitive Dispensing Pipette 1ml
|Used to dispense HSPC and HEP supension in controlled manner
|Suturevet PGA synthetic absorbale suture
|Henry Schein Animal Health
|Suturing of skin and peritoneum
|TaqMan Gene Expression Master Mix
|Thermofisher scientific
|TC20 automated cell counter
|TK-NOG mice
|Provided by the Central Institute for Experimental Animals (CIEA, Japan; Drs. Mamoru Ito and Hiroshi Suemizu)
|Medac GmbH
|Provided by Dr. Joachim Baumgart (medac GmbH)
|Trypan Blue
|Vannas-type Micro Scissors, Straight, 80mm L
|Ted Pella, Inc.
|Used to make an incision on skin to expose spleen
|Weck hemoclip traditional titanium ligating clips
|To ligate the spleen post-injection
- Smith, C., et al. Factors associated with specific causes of death amongst HIV-positive individuals in the D:A:D Study. AIDS. 24, (10), 1537-1548 (2010).
- Puoti, M., et al. Mortality for liver disease in patients with HIV infection: a cohort study. Journal of Acquired Immune Deficiency Syndromes. 24, (3), 211-217 (2000).
- Rodriguez-Mendez, M. L., Gonzalez-Quintela, A., Aguilera, A., Barrio, E. Prevalence, patterns, and course of past hepatitis B virus infection in intravenous drug users with HIV-1 infection. The American Journal of Gastroenterology. 95, (5), 1316-1322 (2000).
- Scharschmidt, B. F., et al. Hepatitis B in patients with HIV infection: relationship to AIDS and patient survival. Annals of Internal Medicine. 117, (10), 837-838 (1992).
- Lacombe, K., Rockstroh, J. HIV and viral hepatitis coinfections: advances and challenges. Gut. 61, Suppl 1 47-58 (2012).
- Brehm, M. A., Jouvet, N., Greiner, D. L., Shultz, L. D. Humanized mice for the study of infectious diseases. Current Opinion in Immunology. 25, (4), 428-435 (2013).
- Billerbeck, E., et al. Humanized mice efficiently engrafted with fetal hepatoblasts and syngeneic immune cells develop human monocytes and NK cells. The Journal of Hepatology. 65, (2), 334-343 (2016).
- Dagur, R. S., et al. Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice. Biology Open. 7, (2), (2018).
- Gaska, J. M., Ploss, A. Study of viral pathogenesis in humanized mice. Current Opinion in Virology. 11, 14-20 (2015).
- Gorantla, S., Poluektova, L., Gendelman, H. E. Rodent models for HIV-associated neurocognitive disorders. Trends in Neurosciences. 35, (3), 197-208 (2012).
- Xu, D., et al. Chimeric TK-NOG mice: a predictive model for cholestatic human liver toxicity. The Journal of Pharmacology and Experimental Therapeutics. 352, (2), 274-280 (2015).
- Washburn, M. L., et al. A humanized mouse model to study hepatitis C virus infection, immune response, and liver disease. Gastroenterology. 140, (4), 1334-1344 (2011).
- Gutti, T. L., et al. Human hepatocytes and hematolymphoid dual reconstitution in treosulfan-conditioned uPA-NOG mice. The American Journal of Pathology. 184, (1), 101-109 (2014).
- Strick-Marchand, H., et al. A novel mouse model for stable engraftment of a human immune system and human hepatocytes. PLoS One. 10, (3), 0119820 (2015).
- Keng, C. T., et al. Characterisation of liver pathogenesis, human immune responses and drug testing in a humanised mouse model of HCV infection. Gut. 65, (10), 1744-1753 (2016).
- Li, F., Nio, K., Yasui, F., Murphy, C. M., Su, L. Studying HBV Infection and Therapy in Immune-Deficient NOD-Rag1-/-IL2RgammaC-null (NRG) Fumarylacetoacetate Hydrolase (Fah) Knockout Mice Transplanted with Human Hepatocytes. Methods in Molecular Biology. 1540, 267-276 (2017).
- Poluektova, L. Y., Garcia, J. V., Koyanagi, Y., Manz, M. G., Tager, A. M. Humanized Mice for HIV Research. Springer-Verlag New York. New York, NY. (2014).
- Cheng, L., Ma, J., Li, G., Su, L. Humanized Mice Engrafted With Human HSC Only or HSC and Thymus Support Comparable HIV-1 Replication, Immunopathology, and Responses to ART and Immune Therapy. Frontiers in Immunology. 9, 817 (2018).
- Zhang, L., Su, L. HIV-1 immunopathogenesis in humanized mouse models. Cellular & Molecular Immunology. 9, (3), 237-244 (2012).
- Nunoya, J., Washburn, M. L., Kovalev, G. I., Su, L. Regulatory T cells prevent liver fibrosis during HIV type 1 infection in a humanized mouse model. The Journal of Infectious Diseases. 209, (7), 1039-1044 (2014).
- Hasegawa, M., et al. The reconstituted 'humanized liver' in TK-NOG mice is mature and functional. Biochemical and Biophysical Research Communications. 405, (3), 405-410 (2011).
- Higuchi, Y., et al. The human hepatic cell line HepaRG as a possible cell source for the generation of humanized liver TK-NOG mice. Xenobiotica. 44, (2), 146-153 (2014).
- Kosaka, K., et al. A novel TK-NOG based humanized mouse model for the study of HBV and HCV infections. Biochemical and Biophysical Research Communications. 441, (1), 230-235 (2013).
- Crane, M., Iser, D., Lewin, S. R. Human immunodeficiency virus infection and the liver. World Journal of Hepatology. 4, (3), 91-98 (2012).
- Bility, M. T., Li, F., Cheng, L., Su, L. Liver immune-pathogenesis and therapy of human liver tropic virus infection in humanized mouse models. Journal of Gastroenterology and Hepatology. 28, Suppl 1 120-124 (2013).
- Kong, L., et al. Low-level HIV infection of hepatocytes. Virology Journal. 9, 1-7 (2012).
- Dash, P. K., et al. Long-acting nanoformulated antiretroviral therapy elicits potent antiretroviral and neuroprotective responses in HIV-1-infected humanized mice. AIDS. 26, (17), 2135-2144 (2012).
- Sun, S., Li, J. Humanized chimeric mouse models of hepatitis B virus infection. International Journal of Infectious Diseases. 59, 131-136 (2017).
- Shafritz, D. A., Oertel, M. Model systems and experimental conditions that lead to effective repopulation of the liver by transplanted cells. The International Journal of Biochemistry & Cell Biology. 43, (2), 198-213 (2011).
- Almeida-Porada, G., Porada, C. D., Chamberlain, J., Torabi, A., Zanjani, E. D. Formation of human hepatocytes by human hematopoietic stem cells in sheep. Blood. 104, (8), 2582-2590 (2004).
- Streetz, K. L., et al. Hepatic parenchymal replacement in mice by transplanted allogeneic hepatocytes is facilitated by bone marrow transplantation and mediated by CD4 cells. Hepatology. 47, (2), 706-718 (2008).


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We will notify you when this product becomes available.
A poly/cotton sleeveless shirt for every day wear, double stitched in all the right places for comfort and durability! Adorned with glistening rhinestones, this shirt is sure to make your pet stand out!
NOTE: Unfolded, these shirts look just like the : Don't Worry be Hippy, Ball is Life, Bitches Love Bowties, Set Sail and Wild Child shirts in this collection.

subset: commoncrawl


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Digital TV does not sacrifice sound quality
Many people were forced to buy or upgrade for a better word, to a digital TV set to adjust to the digital merging of CableTV providers. TV manufacturers have lured many customers in with their "thin" Flat Screens, which produce amazing pictures. In doing this, the audio gets a major slap in the face. It is hard to squeeze a good sounding speaker set into a 1" thick tv.
The term "expense" for many consumers when in relation to "surround sound" means an elaborate amount of money. This is where they are wrong. If guided in the right way, people with budgets can still enjoy the benefit of having a better sounding system without spending large quantities of money to have an elaborate freestanding or in ceiling/wall surround system to produce better sound.
Many consumers are just not aware of the different solutions and options that are out there. A simple pair of bookshelf speakers connected to a receiver and a subwoofer can dramatically make your system sound better and not break the bank in doing it.
Consumers that aren't familiar with technology and the many varieties to choose from can often find themselves wandering through their local electronics superstores aimlessly. Checking out price tags, and not knowing the differences between them. There is a solution for this. Call the Wire Monkeys Team! We will help you every step of the way in selecting the best equipment within your budget.
Giving your integrator the information they need to put you into your price point really helps us provide you with different options within your budget. This is about home entertainment, and it was meant to be fun, not a headache! We can make that happen for YOU.


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Mayanka.com special offer: Lancome quality beauty products, for the best price!
Inspired through the new trendy Rouge for each other lip collection, Vernis for each other is really a high potency nail lacquer perfectly designed to enhance every woman's mood and elegance. With ultimate brilliance, intense color, along with a mistake-free application, your nails will like the lasting shine and pop of color that stays put for several days.Benefits and features:- Exclusive brush: The grooved stem enables the polish circulation straight to the middle, as the elongated, round base distributes uniform color in one stroke.- Lengthy-put on formula: The multi-polymer complex supplies a film that safeguards color and keeps nails perfectly polished.- Extreme shine: Reflects sun light for any high-shine finish.- Quick dry: Express-dry solvents accelerate drying out time. Color(s): 165n very for each other, 171b lovered, 343b rose bonheur, 362b peach appeal, 010m cristal quarta movement, 105b corail for each other, 112b rouge for each other, 147m rouge valentine, 154m miss coquelicot, 179m madame tulipe, 185 crimson fiction, 220m jolis matin, 224m beige bachelorette, 240m beige dentelle, 270n chocolat mordore, 300m rose plumeti, 311m jolie rosalie, 343b rose pitimini, 350m rose the, 375b rose boudoir, 385 black sepia, 407n gris angora, 419b violette coquette, 435b lilac love, 441n night time rose, 473n rouge reglisse, 485 gray lumiere, 501b aquamarine, 521m miss porcelaine, 533b i actually do blue, 537m gem gray, 585n noir angora, aqua bleu, beige poudre, bleu p flore, cosmopolitan kaki, flirtiny red-colored, ginger root ritzini, givre magique, infusion p prune, peach melodie, plunge pool, rose macaron, serendipity gold, simply aqua, sugar rose, ultramarine eco-friendly. Brand: Lancome. Style Title: Lancome 'Vernis in Love' Fade Resistant Gloss Shine Nail Polish. Style Number: 518690. Manufacturer part number L27015
Buy wide array of quality Lancome items online. By Lancome Nail Treatments, Hands & Nails by Lancome


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Sat 8 Dec 2007
Need a Bun to keep you warm on the cold winter nights?Posted by Erica under Uncategorized
1 Comment
Basil is a real cuddle bunny. He loves attention. So much so, that he jumps onto your lap on the couch to be pet. He would be great with children too. Here is a picture of one of the many times he's jumped onto my lap. Such a cutie pie!
~Erica, HRN fostermom
One Response to "Need a Bun to keep you warm on the cold winter nights?"
Emily Says:
December 12th, 2007 at 1:14 am
I love Basil!! Andy is still talking about him. Basil is the most friendly, cuddly bunny I have ever met.


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$3,987,781 Base price: GBP3100500
A luxury Turkish house for sale in the desirable area of Istinye, Istanbul. On a complex set in a private garden with a walk-in swimming pool.
Turkey Homes are delighted to offer this prestigious 5 bedroom villa for sale in Istanbul, found in the upmarket area of Istinye.
This prestigious home is perfect for families looking for a rich and luxury lifestyle in a desirable area, that has good city centre connections and close to all amenities.
Sitting in a boutique style complex with private landscaped gardens. Each villa also has a private walk-in plunge pool, surrounded by a wooden decked pool terrace and seating areas.
Villa Features
The villa has been designed with two living rooms, the first one has been furnished with a formal style with traditional furniture, chandelier style light fittings and a formal dining table. This one also offers two access points to the garden and pool area via large double glazed patio doors. The second is located on one of the upper floors and is more casually furnished with comfortable soft leather sofa's.
The kitchen is a modern design with quality white units and granite work tops, with a casual dining area that houses extra storage. The kitchen further patio doors leading again to the gardens and pool area.
With the villa designed across three floors, the bedrooms are located on the two top floors. Three on the first floor, one having an en-suite bathroom the other two sharing a separate family bathroom. The final two bedrooms are located on the top floor, which is attic space with gentle sloping ceilings, both having the luxury of en-suite bathrooms.
The villa is being offered for sale fully furnished to include all modern units, with white goods and air conditioning units being part of the deal.
Call Turkey Homes today and speak to one of our advisors, who can talk you through the full buying process and all the legal elements of buying property in Turkey.
Istinye is an area belonging of the Sariyer district on the European side of Istanbul. It sits on the northern shores of the Bosphorus and is one of the most attractive Bosphorus shoreline areas, with cafes and wonderful seafood restaurants lining the shores.
Istinye is also home to one of the most prestigious shopping malls in Istanbul 'Istinye Park', where stores are aimed at the higher end of the market, with many designer shops, world famous branded stores and a food court with many international fast food outlets.
It has also been home to the Istanbul Stock Exchange since 1995.
We appreciate your interest in Turkey Homes. One of our consultants will get back to you as soon as possible.
In the meantime if you require any assistance please call us on + 44 (0) 207 272 0002 or email us at firstname.lastname@example.com.


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Champion fight 3D for Android
Champion fight 3D - gather a team of legendary warriors and masters of martial arts. Fight against various opponents in the city streets and other arenas. Get ready for dynamic 3x3 battles in this Android game. Gather a squad of the strongest fighters you have at your disposal. Each character has strong and weak sides, unique battle style and appearance. Apply all abilities of the selected hero and crush opponents. Use incredible combat maneuvers and powerful combos. Add new heroes to your collection.
Game features:
Game features:
- 24 unique characters
- Over 48 levels
- Fight against other players
- Good graphics
0 votes
Download Champion fight 3D
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Raku is a centuries old Japanese tradition of making pottery. This tiny urn was hand thrown on a potter's wheel by Ronald Franklin, an award winning raku artist. The pure white crackle finish is accented with a dark calligraphic design. A very tactile piece, it is a pleasure to hold. This is the actual urn you will receive. Once it has been sold, there will be no more exactly like it. This original one-of-a-kind urn is signed by the artist.
The top opening lid may be sealed with glue (included).
Dimensions: 2.75" High x 4" Diameter, 8 Cubic Inches


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Wednesday, June 29, 2016
There are many things that can go wrong with an outright distribution:
Judgment creditor can seize a beneficiary's inheritance
Bankruptcy court can seize a beneficiary's inheritance
An incapacitated beneficiary can squander an inheritance before anyone can step in to help him.
A divorce court can award some of the beneficiary's inheritance to an ex-spouse
If the beneficiary doesn't plan properly himself, his spouse's family can receive your money
A lifetime discretionary trust, set up either during your life or through a Will, can mitigate against some of these risks. Some of the benefits of a lifetime discretionary trust include:
Protection from beneficiary's liabilities
Protection from beneficiary's divorce
Protection during beneficiary's incapacity
Protection from beneficiary's profligacy
Talk to an estate planning attorney to see if setting up a lifetime discretionary trust may be beneficial for your family.
Disclaimer: This article only offers general information. Each situation is unique.Read more . . .
Tuesday, June 21, 2016
B.B. King acknowledged 15 children from 15 different women during his life. 11 of the children survived him. Yet the executor of B.Read more . . .
Monday, June 13, 2016
You may have completed a Will and a Power of Attorney (all by yourself). You may think that your estate plan is in order and you can rest easily. However, you are likely less than half way done!
In general, over half of all assets in the United States pass outside of probate. These include assets that are jointly titled (i.e.Read more . . .
Wednesday, April 13, 2016
Income Tax Planning: In general, non-U.S. residents are taxed only on U.S. sourced income. If the income is considered to be effectively connected with a U.S. trade or business ("effectively connected income" or "ECI") then that income is taxed at graduated rates on a net income basis. If, instead, the income is "fixed, determinable, annual or periodic" ("FDAP") then it is subject to a flat 30% tax on gross income (or lower if there is an income tax treaty). FDAP income usually consists of interest, dividends, rents and royalties. Interest on U.S. bank deposit is exempt from U.S. tax for non-U.S. residents.
Estate and Gift Tax Planning.
Assets subject to gift and estate tax: For U.S. residents the tax applies to property and assets situated everywhere in the world. In contrast, non-U.S. residents are subject to a gift and estate tax only on U.S. real and tangible personal property.
Gift Tax Exclusions: Similar to a U.S. resident, a non-U.S. resident can make a tax-free annual gift up to $14,000, can make unlimited charitable gifts, and can make unlimited gifts on behalf of donees directly to educational or medical institutions.
Gifts to spouse. The amount of gift tax exclusion depends on the citizenship of the donee spouse. A citizen spouse may receive unlimited gifts of U.S. assets from his spouse. A non-citizen spouse can only receive $148,000 per year prior to a tax being imposed.
Estate Tax. U.S. citizens and residents have a $5.45MM estate tax exemption from federal taxes. In contrast, a non-U.S. resident is permitted only a $60,000 exemption. All property situated in the United States and owned at the death of a non-U.S. resident is included in the U.S. taxable estate, including retirement assets and stocks. Some assets, such as bank accounts and life insurance proceeds, are excluded.
Disclaimer: This article only offers general information. Each situation is unique. It is always helpful to talk to a specialized attorney, to figure out your various options and ramifications of actions. As every case has subtle differences, please do not use this article for legal advice. Only a signed engagement letter will create an attorney-client relationship. ATTORNEY ADVERTISING
Monday, April 4, 2016
The very first question to determine during planning is whether a non-citizen individual is considered a U.S. resident for income tax purposes and for estate tax purposes.
For income tax purposes, a non-citizen is considered a U.S. resident if the individual meets any one of these tests: (1) green card test or (2) the substantial presence test (present in US for at least 31 days during the current year and at least 183 days for three prior years using a weighted average calculation). If an individual is considered a U.S. resident for income tax purposes, he will be taxed on the worldwide income.
For estate and gift taxes, a non-citizen is considered a U.S. resident if the individual intends to establish a domicile in the United States. A domicile is a person's permanent place of abode in which the person intends to remain indefinitely or to which the person intends to return. One can have multiple residences, but only one domicile. This question is a subjective inquiry where many factors are considered, including income tax filing status, jurisdiction of the driver's license, visa status, and location of person's family and friends. The burden of proof is on the taxpayer to establish his domicile.
The difference in the outcome between U.S. residents and non-U.S. residents is huge: U.S. residents have an estate and gift tax exemption of $5.45MM, while non-U.S. residents have an estate tax exemption of only $60,000.
Wednesday, March 23, 2016
There are many reasons why one would want to withdraw money from an investment real estate. Some of these reasons include: no longer willing to manage the property, no longer needing the income tax benefit, desiring liquidity or desiring diversification in one's investments.
1. The easiest way of monetizing a real estate property is selling it. However, with a sale come a host of costs. These costs include brokerage fees and income taxes (both federal and state). Depending on the level of appreciation and on prior depreciation deductions, the gain can be quite substantial and may result in a net amount received that is significantly less than the sale price.
2. There are methods of minimizing the income taxes on the sale of the property. These methods include:
a. An installment sale. This is a method of sale where at least one payment occurs after the year in which the disposition took place. Under this method, gain is not taxed when the disposition occurs, gain is recognized gradually as the payments are received.
b. Borrowing against the property. If one wants to create liquidity while retaining ownership of the property, one can borrow against it. There are no tax consequences to this method. Cash can be used for other purposes.
c. Like-kind exchange under 1031. This method provides a tax-deferral mechanism. No federal gain or loss is recognized where a real estate property held for use in a trade or business or for investment is exchanged for another "like-kind" property. There are several specific steps that must be taken to qualify for the exemption under section 1031.
d. Contributing property to a Charitable Remainder Trust ("CRT"). If one is at least somewhat charitably inclined, one can contribute property to this trust, where specified payments are made to a non-charitable beneficiary for a number of years and the remainder goes to charity. There are many tax advantages to this transaction, the main one being that upon a sale of the property by the CRT, no federal income taxes are due.
Monday, March 14, 2016
Now that the "file and suspend" loophole has been eliminated (effective April 30, 2016), what other strategies remain for maximizing one's social security payments?
1. The main strategy is, of course, to delay the receipt of social security. At full retirement age, a worker is entitled to receive 100% of his Social Security retirement benefits. However, for each year that the worker delays the receipt of his benefits, he will receive an 8% delayed retirement credit. At age 70, however, one cannot delay any further and is obligated to receive the income. As a result, if the worker delays the receipt of his benefits until age 70 his benefit will increase by a total of 32%.
Of course, the downside of this strategy is that during the years that the worker delayed the receipt of his benefit, he was not receiving any payments from Social Security. As a result, this strategy only works for people who have an alternative source of income during these years of deferment, either through continued employment or through savings.
Furthermore, this strategy works only for workers who are in relatively good health and expected to live for a long period in retirement. If the worker develops an unexpected illness and dies sooner than he expected, the overall receipt of money will be significantly less than if he had chosen to receive full retirement benefits at full retirement age.
2. Another strategy involves survivor benefits. Depending on the survivor's work record, the survivor has a choice. She can either (i) receive full survivor benefits at age 60 and delay the receipt of her own larger benefit at the age of 70 or (ii) take her own benefits at the age of 62 and then switch over to survivor benefits at full retirement age.
3. Yet another strategy involves benefits for divorced spouse. As long as the spouses were married for at least 10 years prior to divorce, and the individual has not remarried, the ex-spouse may claim spousal benefits based on an ex-spouse's earning records. This strategy may be very beneficial, as the ex-spouse may claim her spousal benefits at full retirement age and delay the receipt of her own retirement benefits until the age of 70.
Friday, March 4, 2016
Approximately 50% of life insurance policies owned by seniors are allowed to lapse without any benefits being paid out. There are many reasons for this lapse: some can no longer afford the premiums while others feel that they no longer need the proceeds in the event of death.
A recent charitable organization called "Insuring a Better World Fund" (IABWF) was formed to aggregate and administer insurance policies for the benefit of charities. IABWF does not purchase the policies. However, the consumer will be entitled to a charitable deduction based on the fair market value of the donated policy.
There are several criteria which must be complied with before IABWF accepts the policy: the consumer must be above 65, information about health must be provided, policy must have a death benefit value of above $400,000 and the policy must have been purchased more than 3 years prior.
Once the policy is transferred, IABWF will pay the premiums and administer the policy. After the consumer's death, and after the premiums and expenses have been reimbursed, the death benefit will be distributed to the charities selected by the consumer.
The end result is that the charity receives a substantial amount of money, while the consumer receives a charitable deduction.
Wednesday, February 24, 2016
Basis is the cost of purchasing the asset. For real estate, it is the cost, plus any closing costs and improvements made later. Basis is important, because upon the sale of the property, capital gains liability is calculated based on the difference between the sale price and the basis.
Carry Over Basis: If the asset is gifted from the donor to the donee during the donor's life, the donee inherits the basis of the donor. Imagine a house that was purchased for $100,000 40 years ago, and is now worth $1.5MM. If the owner gifts the house outright to her son, the son will inherit the mother's basis of $100,000, and when he later sells it, he will have to pay capital gains taxes on $1.4MM.
Step Up Basis: property that transfers at death is stepped up to the fair market value of the property on the date of death. If the house above was transferred at death to the son, the son can later sell it for $1.5MM without paying any capital gains taxes.
Flexibility is Crucial: there are multiple methods of giving up outright possession of the asset, while retaining some powers that enable the asset to "step up" at death. One method is a life estate (in a real estate property). Another method is keeping a testamentary power of appointment in a trust. Yet another method is retaining the right to income from the asset.
Sunday, February 14, 2016
A researcher at Ohio State University found that Americans who receive an inheritance save about half of it and spend, donate or lose the rest. However, almost 30% of Americans who receive the inheritance had negative savings rate within 2 years of receiving the inheritance, meaning that they spent it all.
There are strong similarities in these spending habits with people who receive lottery winnings. Apparently, lottery winners save only 16 cents of every dollar won and have dramatically higher bankruptcy rates within 5 years after winning.
Of course, the inheritance that people receive may not be a large amount of money. The median inheritance for the past 30 years was $11,340. For those people who inherit $100,000 or more, the percentage of people who spent it all within two years dropped to 19%.
If you receive an inheritance (or a lottery winning) – be careful. I've seen bad financial advisors who have only their own interests at heart. I've also seen bad "friends" who convince unsophisticated people to invest in their 'brilliant' business schemes. There is no perfect answer about what to do with the money. But not spending it immediately would probably be the best advice!
Friday, February 5, 2016
Property partnership may end for a variety of reasons. One common category of circumstances when real estate litigation occurs involves siblings who inherit property from their parents and have different wishes.
Multiple problems can arise. One sibling may want to live in the property while the other one wants to rent it out. Alternatively, one sibling may want to cash out and sell the property while the other may want to keep it. The sibling who wants to keep the property may not have the money to buy the other sibling out. Furthermore, the siblings may not agree on the property's value.
One alternative for establishing the value of the property is to hire three appraisers and take the average of their given values. Of course, the appraisers cost money.
The better alternative is to structure your bequest in a way that avoids potential conflicts amongst the siblings. No parent wants to believe that their children will fight after their death, but unfortunately it happens all the time. First, Wills and Trusts can be written such that the real estate will be given to one child while the other assets will be given to the other. Another alternative is to provide in your Will or a Trust that real estate should be sold within a year of death and proceeds should be distributed equally amongst the beneficiaries.
Previous Posts
Social Security changes coming in 2020
What is a healthcare proxy?
What happens if there are not enough assets in the Estate to pay all claims
Disinheriting a Child
Account Closures and Cancellations
Basics of Planning a Funeral
Planning for Step-Children
A Good Plan Avoids Big Problems
My recent interview on Serene Home: mistakes, challenges and costs of estate planning.
KonMari consultant explains the organizing / cleaning method that took the world by storm!
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Kystvejen 12
9370 Hals
Mobile: 60 52 55 38
If you have not yet tried the ice cream from Vebbestrup, it is definitely time! Enjoy the ice and the view of the marina in Hou. Hou Waffel House & Beach Café is open during the spring and summer period.
Hou Waffel House offers:
- homemade waffles
- ice cream
- cold beer and water
... and much more.
Kystvejen 12
9370 Hals


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Have a look at this Used Mezzanine Floor that we supplied & installed for a Kitchen & Bedroom manufacturer this week.
Finding their warehouse becoming less spacious through time, this company required a larger space so they could continue to operate and grow their business effectively.
They approached us for a quote on a Mezzanine Floor as this was the most cost-effective option to create more space for their business.
We were able to supply and fit this bespoke Mezzanine Floor with a set of stairs and handrail to the exact requirements of their space while also keeping the price within their budget.
Another happy customer.
Does this Used Mezzanine Floor look like a storage solution that your business could benefit from?
Have a look at another Mezzanine Floor Installation we completed for one of our other clients, that helped them create a more spacious and dynamic showroom.
Book a Free Site Visit today and find out how we can help you get the most out of your space.


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IDGB Capital is working with the Better Food Project (BFP) investment firm that seeks to create a next generation food platform by building a collection of nutritious and healthy food brands and growing them in an ethical manner. We believe nutrition, together with a more active life style, are keys to a long and healthy life. We consult the latest science, not advertising, to determine if a product is healthy.
BFP's management team and board of directors consist of consumer packaged goods industry veterans, including former senior level executives of Kraft Foods, Dreyer's and Nature's Variety. Its long-term plan would be to create a platform that would employ a roll-up M&A strategy of companies in the Better for You Food Category that own complimentary assets and any financial backers may choose to be part of the platform or only be part of the Grow acquisition.
Acquisition Criteria and Select BFP Food Verticals
Size: $5MM to $50MM in revenues.
Provides a nutritional or health benefit and strives to use environmentally friendly, natural ingredients: Non-GMO, Organic and not high glycemic.
Majority ownership with a preference for full buyouts.
Polaris is a full-service real estate investment and management firm focused on value-added real estate transactions in the U.S. Polaris specializes in repurposing and repositioning existing income producing properties, mainly multi family assets and student housing properties. Polaris' investment strategy provides superior risk adjusted returns in asset classes that are widely regarded as the safest in real estate.
Polaris' Team brings an impressive rolodex of investors and equity partners ranging from ultra high net worth individuals and family offices through institutional investors.
In each local market, Polaris is partnering with a strong local property management partner with access to off-market deals and deep knowledge of the market.


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Autodesk has recently published Advance Steel product plans at BIM and Beam blog. Below you can see all of the planned and delivered improvements: Features and workflow enhancements: Quality improvements: If you're an active Advance Steel user, and would like to influence developer plans, head over to the Advance Steel Ideas forum to post your suggestions.Read More
Sneak peek into Dynamo 2.0
It's been a while since the last "major" Dynamo update (Dynamo 1.0 was released way back in 2016). And if you are tired waiting for something new, you can download and install one of the latest Dynamo 2.0 daily builds from the Dynamo development builds webpage. Starting from this year's daily builds, Dynamo team has changed…Read More
Autodesk Building Performance Analysis Certification Retirement
Today is the last day to finish your Autodesk Building Performance Analysis Course and earn the certificate before the entire course gets retired. If you are currently enrolled in the course, you'll have time until December 15th, 2017 to finish it. Any non-completed courses will be de-activated together with the training portal after December 15th, 2017. But…Read More
Autodesk Revit roadmap updated
Now that the fall has come, it's time to check out an updated Revit roadmap, which highlights the future of Revit. There's a lot of cool stuff in there, so go ahead and see this article at Autodesk blogsRead More
Revit railing enhancements with Dynamo
Lately I've been working on some Revit railing enhancements. This is still work in progress, but some of the Dynamo nodes are already available online in my updated Zhukoven.com (rev.2017.8.17) package. Below you can see available railing Dynamo nodes (please note that currently all of these nodes support only one railing instance at a time):…Read More
Revit 2018.1 update comes with enhanced Dynamo Player
Good news, everyone! Revit 2018.1 update is now available for download from the Autodesk Account portal. It comes with some cool new features (see this article at Autodesk blogs for the full info), including enhancements to Dynamo Player. Now it finally supports changing input values directly through the Player UI, which is super handy: As…Read More
WhatsApp Dynamo package is now available for download
Today I released my tiny WhatsApp Dynamo package, and it's now available for download and testing @ dynamopackages.com. WhatsAppDynamo provides integration with WhatsApp Web platform, allowing you to send text data (like URLs, file paths, parameter names, etc.) using Dynamo. Note that due to the platform limitations, images and files are not currently supported – they could…Read More


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the me 读后感1000字 平凡的世界读后感 三国演义的读后感 狗猫鼠读后感
My favourite festival is the Spring Festival. Why? Because I have lots of fun at the Spring Festival.
The Spring Festival is a popular holiday in our country. It is in January or February. Our family get together. We have a big dinner at my grandparents' home. I always eat a lot of delicious food. My cousin and me both get red packets from our grandparents. My parents usually go shopping with me. We buy some nice clothes for ourselves. We often watch TV at home and sometimes read books or surf the Internet. Sometimes, we greet other people on the phone.
I'm always happy at the Spring Festival. Which is your favourite festival?


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About MeLitcul Official
Litcul Official Aug 29 97 views 0 Comments 0 Likes 0 Reviews
In this article we are going to give a detailed analysis of Restoration Period or Age of Dryden and discuss on the progress of English Literature during this period.
Restoration Period or Age of Dryden
The period from 1660 to 1700 is known as the Restoration Period or the Age of Dryden. The English people were fed up with the restraints of the Puritan regime which denied to them all entertainments even of the most innocent kind. Prince Charles who had sought shelter in France, was invited to occupy the throne of his father. He was restored to the English throne in 1660 and became king Charles II of England. The Restoration of Charles II marked the beginning of a new era both in life send literature. Charles II brought with him refinement and Culture of the French, as well as the immortality and profligacy of the French Court. He also brought with him French literary influence. It marked the beginning of that Neo-Classicism or Pseudo-Classicism which was to dominate English Literature for next one hundred and fifty years.
The literature of the period reflected the spirit of the Age. Corruption, Moral Laxity, Rampant during this period are reflected in literature. The following tendencies distinguish the literature of the period :
The Restoration marked a complete break with the past. The people believed in the present, the real and the material. Ideas of conduct were set up in accordance with reason and common sense.
Rules and literary conventions became more important than the depth and seriousness of subject matter to the writers of this period. They expressed superficial manners and customs of the urban aristocratic life and did not delve deep into the mysteries of human mind and hearts. They turned to the ancient writers for guidance and inspiration. Thus, grew up the Neo-Classical school of poetry. The Neo-Classicist could not sear to great heights of imagination, instead directed attention to slavish imitation.
The Restoration era was an era of France influence both in literature and social life. The famous French Writers - Corneille, Racine, and Boileau were imitated. Boileau's 'Good Sense' ideal became very popular. English writers learned to look for guidance of the French writers and their styles like - lucidity, veracity.
The literary works of the Restoration Period were imitative and of limited quality. They lacked creativity and fight of imagination. They abandoned freedom of expression and slavishly followed the rules which can be summarized as 'Be Correct'. Correctness means avoidance of enthusiasm, moderate opinions, strict care and accuracy in poetic techniques.
Restoration literature is realistic and urban. It was mainly concerned with life in London and details of dress, fashion and manners. The Restoration writers sought to point realistic pictures of corrupt court and society, and emphasized vices rather than virtues.
The Restoration writers eschewed all extravagances of thought and language and aimed at achieving directness and simplicity of expression. To reform English prose, the Royal Society get rid of 'Swellings of style' and adopted the use of naked, natural way of speaking.
In Conclusion, imitation of the ancient, imitation of the French, the correct school, Realism, directness and simplicity of expression marked the salient features of the Restoration Literature.
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FS2004 exclusively Sopwith Camel This is a new model for FS2004. Model includes virtual cockpit, rotary type engine, smoking engine and 3 texture choices. Model by David Eckert, Flight Dynamics by Jerry Beckwith. Additional textures by John Terrell. Panel, instruments gauges and sound by Microsoft.
Users Reviews
FS2004 exclusively Sopwith Camel a new model. Rating: 9.56 of 10 over a total of 9 reviews.
FS2004 exclusively Sopwith Camel a new model. Rating: 9.56 of 10 over a total of 9 reviews.
nickkrating: 10
June 3, 2011
this plane is so awsome download it totaly
pilot555rating: 10
September 1, 2007
10 This is the best world war I aircraft out there it verry realistic, everything from colors to flight dynamics oh yeah, and has a automatic instalation.
Martin04rating: 9
January 1, 2007
Great to fly, good smoke effects, feels like the real thing. The only difficulty is taxiing as it almost impossible turn. Does anyone have a way round this
nimrod1rating: 10
July 22, 2006
nyce. finally a world war one download that doesn t look like it s made out of cheese.
CHESSBOARDERrating: 8
March 19, 2006
Good work
kuuhakerating: 10
March 18, 2006
Excellent I love flying with this beautie Nice camel.
purpleponcarating: 10
October 26, 2005
excellent addon to my collection one of the best bi planes in fleet many thanks and be looking for more like this one, download with confidence, well designed aircraft Happy Flying
shavnyakrating: 9
September 30, 2005
What a great bird This one is joy to fly. And yes, nice touch on the fuel cut off...
terrydwyerrating: 10
September 20, 2005
Excellent Flies really well. I especially like the way that engine stops in negative G. can see now why Snoopy made it his plane of choice


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Good Afternoon -
I am really interested in upgading my existing Rainbird controller (unknown model). I have been unable to identify the model, but was able to open it up, and it seems fairly straightforward. From the documentation online it seems that the wires on the right go into the zones, white on the lower left is common, but the mystery to me are the three in the upper left (red, yellow, blue)…
any help would be greatly appreciated.
Thanks, and have a great one.
Good Afternoon -
Looks similar to PC-106: http://www.rainbird.com/documents/diy/man_pc100series.pdf
These are power input terminals, can be disregarded / disconnected. Rachio comes with its own supply.
Hi Gene -
That is it! I can't believe I missed this. Thank you so much for the quick reply! I think I'm going to go for it!
Thanks again and have a great one!
No worries, the battery threw me for a loop, wanted to make sure it was for memory backup rather then running a DC controller. Switch over should be pretty straightforward process. Welcome to the club


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Adding small wind turbine to existing solar
miah1320 Registered Users Posts: 1 ✭
edited May 2017 in General Solar Power Topics #1
I have a 100w panel going to a 30amp pwm solar controller and 35ah 12v battery I use mostly to run a modem/router that pulls 15w 24/7...I got a very small 2amp max 12v wind turbine to keep a lil charge at night...the seller claims it can be hooked up through the controller with no need for a load dump...could I hook it inline with the panel if so would it be as simple as getting a y connector and hooking it to the panel?...heres a link to the turbine
Look at this on eBay http://www.ebay.com/itm/142363334811
Look at this on eBay http://www.ebay.com/itm/142363334811
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It is a small turbine and with so many blades will probably not over speed in high winds if "unloaded" (put through a series charge controller).
You could have an issue with the solar panel and PWM controller maximum input voltage ratings--It depends on how high of voltage the little wind turbine generates when unloaded... In high winds, it is possible that it could generate 48 volts or more--That could be enough to ruin your paralleled solar panel and/or the PWM controller.
In practice, most small wind turbines produce almost no useful energy. And therefore may not cause you any problems with the array+controller.
One alternative would be to connect the wind turbine (through a blocking diode) directly to the battery. And install a switch to turn off the turbine (or install between turbine and blocking diode, short out the turbine) and simply turn off when high winds (stormy weather) is forecast.
Realistically--Your 35 AH battery and solar panel are too small to "reliably" support a 15 Watt draw 24x7. A quick set of math would suggest:
Battery sizing:
- 15 watts / 12 volts = 1.25 Amp draw
- 1.25 amps * 24 hours per day = 30 AH per day
- 30 AH per day * 2 days stored power (no sun) * 1/0.50 max discharge (for longer battery life) = 120 AH battery minimum
- 120 AH battery * 14.5 volts charging * 0.13 rate of charge = 226 Watts charging
- 226 Watts charging * 1/0.77 panel+controller derating = 294 Watt array "cost effective" maximum
Des MoinesMeasured in kWh/m2/day onto a solar panel set at a 33° angle from vertical:
Average Solar Insolation figures
(Optimal winter settings)
So, 15 watts 24 hours per day for Des Moines Iowa:
Jan Feb Mar Apr May Jun 3.46
Jul Aug Sep Oct Nov Dec 4.59
- 15 watts * 24 hours per day * 1/0.80 flooded cell battery eff * 1/0.77 panel+controller derating * 1/3.17 hours of sun Dec avg * 1/0.66 full time energy usage fudge factor = 279 Watt array "minimum" (with fudge factor for days with poor sun)
You might look at a pair of "golf cart" batteries (6 volt @ ~200 AH) wired in series (12 volts @ ~200 AH)... They are relatively cheap (~$100 or less each) and rugged. You just need to monitor water levels once a month and check state of charge with a hydrometer... And you could support a larger solar array:
- 200 AH * 14.5 volts charging * 0.13 charging * 1/0.77 panel+controller derating = 490 Watt cost effective maximum array
Anyway--Just some ideas. If you have the ability to run a trench and 120 VAC to the location--It might be a better idea.
For the wind turbine, generally suggest 60 feet in the air and 20 feet above any nearby trees and buildings. May not be worht putting a 2 amp wind turbine on such a tower. Also, is the turbine weather proof (water, ice) and do you have lightning in the area (30-60 foot tower on hill==lightning magnet).
If the installation is on a hill in lightning area--Lightning rods, grounding, and surge suppressors are probably highly desirable too (whether wind, solar, or AC powered).
Near San Francisco California: 3.5kWatt Grid Tied Solar power system+small backup genset0 · Share on Twitter


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Disclosure: We may earn a small commission when you click on our affiliate links and make a purchase.
For a limited time or while supplies last, get the Crux 2.2-quart Touchscreen Air Convection Fryer for just $56.99! This air fryer has a 2.2-quart capacity and includes a frying basket. It heats up quick and cooks food evenly and crisply without any added oil. It features a touchscreen control panel and automatic timer shutoff.
Originally $114.99, the air fryer is now on sale at Macy's with free shipping.
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Expedition Leader &
Professional Sportsman
Yesterday, December 12, a team led by Sergey Kofanov left the assault camp (3,900 m) to climb the summit. Alexander Abramov left the low camp (2,900 m) in the same direction to reinforce the team. One of the team members, Alexander Orlov, had to turn back without reaching the summit due to a health condition. Abramov accompanied his descent.
The remaining team members: Vladimir Aristarkhov, Sergey Lavrov, Yaroslav Sabyrbayev, Konstantin Morozov, led by Sergey Kofanov, successfully scaled the summit. Then the team descended to the high camp, folded the camp and continued the descent to 2,900 meters, where they caught up with Abramov and Orlov. From there they guys made a call saying they intended to continue descending to the base camp and try to leave as soon as possible due to the bad weather forecasts for the nearest future.
Congratulations to the guys on the successful ascent to the highest peak of Antarctica. Congratulation to Sergey Kofanov on the successful completion of the Seven Summits Project!!!


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This kit is specific for the 2007-14 SHELBY GT500 passenger side.
Includes factory PCV hose for easy plug and play installation.
Most of the oil will come from the passenger side, so if only getting one, this is the one to get!
During the process of venting crankcase pressure back through your intake track, large amounts of oil, in the form of vapors, can coat your blower rotors, intercooler fins and even dilute your gas, lowering its octane level. This happens on all engines and is even worse when supercharged as crankcase pressures are increased.
To combat this, we have developed an oil separator of our own design. Milled from solid billet aluminum this is a rock solid piece. It has a knurled tank base for easy removal to drain the spent oil and an O ring seal to prevent any leaks. We took this unit a step further and added a metal mesh filter to the intake side that collects the vapors, allowing them to grow into larger oil droplets and drains back into the tank. The tank holds 3 oz of fluid, while that may not sound like a lot, you shouldn't see that much oil in your PCV system. Drain it every 2000-2500 miles and you will never come close to filling it.
Easy plug and play kits
Super easy to check and empty
Many kits use OEM hoses or come complete with all hoses, custom brackets and hardware
Retain stock hose if you ever return to stock
No hoses running all over your engine compartment
OEM look and quality
Perfect size, not too small and not huge by any means


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Batman v Superman: Dawn of Justice was a polarising movie when it was released this time last year. Personally, I really enjoyed elements of it but found others lacking. Justice League looks to be a vast improvement, the first trailer for the film has just been revealed and it is exciting to say the least.
In the first team-up of the DC cinematic universe, Batman was something of an anti-hero. The biggest difference between Affleck's Batman and every other iteration was that he kills his enemies. This had fans in uproar as the comic book version was completely opposed to killing.
The events of Batman v Superman made him see the error of his ways to some extent. Superman's sacrifice at the hands of Doomsday gave him a change of heart. In an interview with USA Today, director Zack Snyder noted that Batman would continue down this path in Justice League.
Snyder Commented that in his first appearance, Affleck's Batman is at his lowest ebb in life, resulting in his brutality. He continued: "That was always our intention, that Justice League would be this continuation for Batman to evolve into a more complete hero".
Source: USA Today


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Siena Elizabeth
Siena Elizabeth
|ACS Registration Number
|Year Registered
This 15-year-old C. japonica chance seedling of 'Tinker Bell' first bloomed in 2013. It was originated, registered and propagated by Don Bergamini of Martinez, California.
Bloom: The small sized 3" x 1.5" (treated 3.5" x 2") anemone to semi-double form flower is lavender pink with darker pink and lavender markings. It has yellow anthers and white filaments. This profuse bloomer falls in one piece and is long lasting with heavy petal texture and consistent form.
Plant: The plant habit is upright. The growth rate is average. The plant is suitable for landscaping and exhibition uses.
Leaf: The medium green leaves average 3" x 2" with low serration.
Bloom season: Midseason to late.


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The yearly grant is designed to encourage, recognise and reward educators across Education City who wish to implement creative and innovative pedagogical approaches in the classroom.
The girl, whose name was not released, was booked at a juvenile detention facility on a charge of negligent discharge of a firearm, said Officer Tony Im of the Los Angeles Police Department.
Investigators said the boy, thought to be 15 or 16, carried out the attack in a class of children aged around 12 or 13 in the city of Ulan-Ude near the Mongolian border.
The incident unfolded at the Banksia Road Public School on Tuesday when the car ploughed into a wooden classroom building with 24 students inside.
The tragedy occurred at the Banksia Road Public School when the car smashed into a wooden classroom building not long after lessons started.
For the first time in Qatar, Augmented Reality (AR) has been used in a classroom training environment at College of the North Atlantic - Qatar (CNA-Q) to improve quality control for industrial maintenance.


text: #Travel Aug 23, 2019 15:35 GMT The Guardian made an article about holiday in Jyväskylä Region being one of the best lake holidays in Europe. Read more about the article here. Photo: Julia Kivelä FacebookTwitterВконтактеGoogle+ Leave a Reply Cancel reply Your email address will not be published. Required fields are marked *Comment Name * Email * Website


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240 Publications
- A96 Dualling Inverness to Aberdeen (9)
- Forth Replacement Crossing (8)
- A9 Luncarty to Pass of Birnam (8)
- Aberdeen Western Peripheral Route / Balmedie to Tipperty (6)
- A9 Dualling Perth to Inverness (5)
- Borders Railway (5)
- A96 East of Huntly to Aberdeen (4)
- A9 Pass of Birnam to Tay Crossing (3)
- M74 Completion (3)
- A876 Upper Forth Crossing at Kincardine (3)
Mode Of Transport
A96 Online Dualling at Inverurie Supplementary Study October 202030 Oct 2020
A supplementary study investigating varying standards of online dualling through Inverurie to determine impacts and performance in relation to the scheme objectives.
Corporate Procurement Strategy 202029 Oct 2020
Transport Scotland's Procurement Strategy is aligned to its Corporate plan and sets out the strategic direction of Transport Scotland's procurement activity for 2020.
Lessons Learned from the Forth Replacement Crossing Project 2007 to 201716 Oct 2020
Noting the extent and scale of the work required to complete each stage of the Forth Replacement Crossing project.
The Disabled Persons' Parking Places (Scotland) Act 2009 Annual Report On Local Authorities Functions: 2019-202030 Sep 2020
The Disabled Persons' Parking Places (Scotland) Act 2009 places a duty on local authorities and the Scottish Ministers to publish annual performance reports.
Scottish Safety Camera Programme - Annual Progress Report 2019/2023 Sep 2020
As part of the performance management responsibilities, this annual report is informed largely by information submitted by the three regional Safety Camera Units
Development of Scotland's 2030 Road Safety Casualty Targets and Key Performance Indicators8 Sep 2020
The report makes recommendations for road casualty targets and key performance indicators to help accurately monitor progress against the Road Safety Framework to 2030 outcomes
Transport Scotland Section 70 return - 2019-2026 Aug 2020
Ministers are required to report to Parliament on the expenditure incurred each year under section 70 of the Transport Scotland Act 2001
Project newsletter - Summer 2020 - Luncarty to Pass of Birnam - A9 Dualling28 Jul 2020
The latest project newsletter for the Luncarty to Pass of Birnam section of the A9 Dualling programme.
Project newsletter - Spring 2020 - Luncarty to Pass of Birnam - A9 Dualling19 May 2020
The latest project newsletter for the Luncarty to Pass of Birnam section of the A9 Dualling programme
SQUIRE - Caledonian Sleeper - Q4 2019-20 percentage performance figures1 May 2020
SQUIRE reports provide a general overview of rail performance for the year


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Glass Door Display Cabinet Rustic Off White Small Bookcases Console Table Buffet. The Better Homes and Gardens Bedford 2 Door Cabinet has a distinctive and warm style that blends beautifully into a variety of spaces.This 2 door storage cabinet features a solid colored body with a weathered, wood grain textured top. Tempered glass doors accented by metal handles open to reveal inner shelves for organized storage.
Pair with other items from the Better Homes and Gardens Bedford Collection. Weathered top with wood grain texture. Choose from gray or ivory finishes. Assembled dimensions: 31.75"W x 14"D x 32H. Part of the better homes and gardens bedford collection.Brand: Better Homes & Gardens. Assembled Product Dimensions (L x W x H): 31.75 x 14.00 x 32.00 Inches. 1 x Glass Door Display Cabinet. We maintain high standards of excellence and your satisfaction is our number one priority.
The item "Glass Door Display Cabinet Rustic Off White Small Bookcases Console Table Buffet" is in sale since Saturday, October 20, 2018. This item is in the category "Home & Garden\Furniture\Bookcases". The seller is "us-plaza" and is located in Miami, Florida.This item can be shipped to United States.


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from £150.24 each (ex. VAT) with free delivery
from £150.24 each (ex. VAT) with free delivery
H x Dia (mm)
(Ex. VAT)
|1000 x 485
Back in stock est. 23/11/2020
Chat with us about - E321770
Victorian style lockable litter bin complete with liner.
The build up of wrappers and other waste around buildings and entrances can not only ruin the appearance of the surrounding landscape but it can also harm a business' image in the eyes of visitors. So why not encourage staff and visitors to dispose of their waste properly? The solution can be easily achieved and economical.
The traditional appearance of the Victorian outdoor litter bin blends seamlessly into almost any setting, this robust litter bin is designed to stand the test of time and exposure to the elements. The smooth hooded design requires minimal maintenance, the surfaces can be wiped over to clean and the hooded top helps protect against the elements. For easy emptying the lockable body can be removed granting easy access to the liner within, each Victorian litter bin has a 75 litre capacity and is also supplied with a base.
Concrete ballasts and floor fixing plates can also be supplied at additional cost.
Ordered Friday afternoon, delivered Tuesday, can't ask for more than that. Best prices too.
Lawrence Randall-Kattner - 08-Oct-2020
E321770 - A good quality product...
E321770 - Product is what I wanted, price could have been a little cheaper.
E321770 - Poor quality product .......
Free Delivery - the price and lead time you see is to UK mainland destinations (excluding HS, KW, ZE, IV, AB, DD, PH, PA, GY, JE, IM, BT, Isle of Anglesey, Isle of Wight and Eire) with the exception of bulky products requiring a crane or hiab offload which if required may be charged for.
To ensure we offer the best individual product prices, orders below £45 will incur a charge of £4.50 (automatically applied to your basket until carriage paid limit reached).
Anonymous User - 16 Feb 2015
A. It is a medium duty polyethelene
Company Reg No: 1279230 - UK VAT No: 287 8262 10
Phone number: 0808 250 0780
*FREE UK mainland delivery on all orders above £45 Ex. VAT