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CXCR4-associated depletion of bone marrow CD34+ cells following CCR5-tropic HIV-1 infection of humanized NOD/SCID/JAK3null mice and partial protection of those cells by a promotor-targeting shRNA
View ORCID ProfileTetsuo Tsukamoto
Tetsuo Tsukamoto
1The Kirby Institute for infection and immunity in society, University of New South Wales, Sydney, NSW, Australia
2Center for AIDS Research, Kumamoto University, Kumamoto, Japan
3Department of Immunology, Kindai University Faculty of Medicine, Osaka, Japan
ORCID record for Tetsuo Tsukamoto
Objectives Hematological abnormalities that include changes in bone marrow, such as in anemia and pancytopenia, are common among human immunodeficiency virus (HIV)-infected patients, particularly in the advanced stage of disease. This study aimed to provide better experimental evidence of such manifestations in animal models.
Design NOD/SCID/JAK3null (NOJ) mice were transplanted with human cord-derived CD34+ cells with or without transduction with a lentiviral vector expressing a promoter-targeting shRNA called PromA.
Methods At 16 weeks after transplantation, mice engrafted with CD34+ cells were infected with CCR5-tropic HIV-1JRFL.
Results At week 2 post infection, HIV replication was observed in peripheral blood mononuclear cells and splenocytes. In mice transplanted with unmanipulated CD34+ cells, viral replication was accompanied by a loss of peripheral/spleen CD4+CCR5+ T cells. Interestingly, bone marrow CD34+ cells in HIV-infected mice were also depleted, but in a CXCR4-associated manner. Conversely, the lentiviral transfer of PromA in CD34+ cells prior to transplantation rendered the humanized NOJ mice resistant to HIV replication in CD4+ T cells, resulting in better preservation of peripheral/spleen CD4+CCR5+ T cells and bone marrow CD34+ cells at two weeks after infection.
Conclusions These results implicate the importance of evaluating hematopoietic stem/progenitor cell pools in addition to peripheral CD4+ T-cell counts to assess the early stage of HIV infection. Moreover, stable gene transfer of PromA to hematopoietic stem cells not only limited HIV replication but also led to preservation of different subsets of hematopoietic cells, including bone marrow stem/progenitor cells.
Human immunodeficiency virus type 1 (HIV-1) infection induces hematological changes [1]. Although antiretroviral therapy (ART) is effective for viremia control and acquired immunodeficiency syndrome (AIDS) treatment, some patients fail to achieve sufficient restoration of T-cell immune function despite being aviremic during treatment [2]. Such immunological nonresponsiveness has been associated with immune activation and bone marrow impairment [3, 4]. Bone marrow abnormalities, such as dysplasia and abnormal hematopoietic cell development, are frequently observed in HIV-infected individuals [5]. Therefore, it is desirable to design a method to protect the whole hematopoietic cell population from HIV infection-associated immunological and hematological disorders.
Transcriptional gene silencing (TGS) occurs through small non-coding RNAs that direct epigenetic changes, such as DNA methylation and heterochromatin formation. TGS was first described in plants [6] and has since been adapted in humans for the development of new therapeutic agents [7]. For example, previous studies have reported sustained, profound, and highly specific suppression of viral replication by small interfering RNA (siRNA)- and short hairpin RNA (shRNA)-induced TGS of HIV-1 and simian immunodeficiency virus in various in vitro models via a mechanism that results in chromatin compaction [8-14]. HIV-1 has identical long terminal repeats (LTRs) at the 5' and 3' ends of the integrated virus; therefore, any promoter-targeted siRNA can potentially act through post-transcriptional gene silencing (PTGS). However, the use of a lead candidate, PromA, showed that the contribution of PTGS is limited [8].
In a previous collaborative study including in vivo animal experiments, a lentiviral delivery system was used to express the previously described shRNA PromA targeting the HIV-1 promoter region to transduce human peripheral blood mononuclear cells (PBMCs), whereby an antiviral effect of TGS produced by this construct on HIV-1 infection in vivo was demonstrated using NOD/SCID/Jak3null (NOJ) mice [15] transplanted with the lentivirally transduced PBMCs [13]. In the present study, we tested whether TGS using shRNA (shPromA) can be applied to more profound gene therapies, such as gene-modified hematopoietic stem cell transplantation to replace infected immune cells with HIV-resistant cells. Humanized mouse models have been characterized for tissue distribution and HIV infection of human hematopoietic cells [16]. To better utilize the NOJ-based humanized mouse model, irradiated newborn NOJ mice were transplanted with human cord-derived CD34+ cells, as previously described [15]. It is demonstrated in the present study that the generated hematopoietic/immune cells, particularly CD4+ T cells, were resistant to replication of the challenged HIV-1JRFL in vivo and that bone marrow CD34+ cells expressing shPromA may be better protected and preserved after HIV infection.
Virus stocks
HIV-1JRFL stocks were produced by 293T-cell transfection with the molecular DNA clone pJRFL. After transfection, the culture supernatant was collected and viral titers were determined using an HIV p24 Enzyme-Linked ImmunoSorbent Assay (ELISA) kit (Zeptometrix Corporation, Buffalo NY, USA).
Umbilical cord blood samples were collected from healthy newborn infants at Fukuda Hospital (Kumamoto, Japan) after obtaining informed consent. Cord blood mononuclear cells were isolated using Pancoll separation solution (PAN-Biotech GmbH, Aidenbach, Germany) and density gradient centrifugation. Labelled CD34+ cells were isolated using human CD34 micro beads and LS columns (Miltenyi Biotec, Macquarie Park, NSW, Australia). The purity of the CD34+ cells was consistently >92% by flow cytometric analysis.
Lentiviral production
Self-inactivating lentiviral vectors expressing shRNA PromA (shPromA) or PromA M2 (shPromA-M2) (Fig. 1) were constructed, as previously described [13]. Vesicular stomatitis virus-G pseudotyped lentiviral vectors were prepared by plasmid DNA transduction into 293T cells using Lipofectamin 2000 reagent (Thermo Fisher Scientific, Scoresby, VIC, Australia). The viruses were concentrated from supernatant, as previously described [17, 18], and stocks were titrated on 293T cells based on green fluorescent protein (GFP) expression.
Fig 1.
Design and plan of the study. (A) A schematic representation of the siRNAs PromA (JRFL) and PromA-M2. The two NF-kB binding sites in the HIV-1JRFL proviral DNA sequence are underlined. The three mismatched bases in PromA-M2 compared with PromAJRFL are indicated with italic letters. (B) Three mice were sacrificed without HIV challenge 15 weeks after transplantation with shPromA-transduced cells. Splenocytes and bone marrow cells were collected from three mice, and different major subsets of human CD45+ hematopoietic cells were screened for GFP expression. Representative plots showing both GFP+ and GFP- cells in all of the subsets of human hematopoietic cells tested. (C) Comparison of human CD45+ frequencies between the CD34 group (transplanted with unmanipulated CD34+ cells) and the PromA group (transplanted with shPromA-transduced cells) mice that were included in the following HIV challenge experiment. (D) The mean shRNA-expressing (GFP+) percentages in the different subsets of cells in the spleen. Statistical analysis was performed with the Mann–Whitney U-test. (E) The in vivo study plan. Newborn NOJ mice were irradiated and transplanted with PromA-transudced or unmanipulated CD34+ cells. At 14 weeks after treatment, blood was collected from the retro-orbital sinus and the PBMCs were tested for human CD45 expression. Two weeks later, mice were intraperitoneally challenged with HIV-1JRFL. Blood was collected at weeks 1 and 2 post challenge. Mice were sacrificed at week 2 for the collection of splenocytes and bone marrow cells.
CD34+ cell transduction with lentiviruses
After culturing in X-VIVO 10 media (Lonza, Sydney, NSW, Australia), fresh cord-derived CD34+ cells were transduced with a lentiviral vector at a multiplicity of infection (MOI) of 10 following pre-stimulation for 24 h with 50 ng/mL each of stem cell factor (SCF), FMS-related tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin (TPO) (R&D Systems, Inc., Minneapolis, MN, USA). The cells were then collected and cryopreserved. A small portion of cells was further incubated for 48 h to check the transduction rates by measuring GFP expression levels by flow cytometry.
Cord-derived human CD34+ cell transplantation into NOJ mice and HIV-1 infection
Humanized NOJ mice were generated, as previously described [15]. Briefly, newborn NOJ mice were irradiated (1.0 Gy) and then intrahepatically infused with shPromA-transduced or unmanipulated CD34+ cells (2 × 105) that were resuspended in phosphate-buffered saline (0.1 mL). At week 14 after transplantation, peripheral blood was collected from the retro-orbital sinus for flow cytometric analysis of PBMCs to check the engraftment of human hematopoietic cells (Fig. 3). At week 16 after transplantation, each humanized mouse was intraperitoneally inoculated with 200 ng of HIV-1JRFL p24, as determined with the HIV-1 p24 antigen ELISA (Fig. 3). Blood was collected at weeks 1 and 2 after infection to obtain plasma and PBMCs. At week 2, the mice were sacrificed, and the splenocytes and bone marrow cells were collected from the left femur. All animal experiments were performed in accordance with the guidelines of the Kumamoto University Graduate School of Medical Science.
Bone marrow cell counts and percentages in humanized mice. Cells were collected from the left femur at week 18 post transplantation for uninfected mice or at week 2 post infection, counted, and analyzed by flow cytometry. (A) Representative plots showing the CD34/CXCR4 expression patterns in untransduced uninfected (left), untransduced infected (middle), and shPromA-transduced infected (right) mice. (B) Bone marrow cell counts. (C) Bone marrow CD34+ cell counts. (D) Percentages of CD34+ cells in the population of bone marrow CD45+ cells. (E) Percentages of CXCR4+ cells in the population of bone marrow CD34+ cells. (F) Comparison of shPromA+ frequencies in CD45+ and CD34+ bone marrow cells of mice in the PromA group. Statistical analyses were performed by the nonparametric multiple comparison analysis with Dunn's method (B-E) or the Wilcoxon matched-pairs signed rank test (F).
PCR/RT-PCR analysis
Cellular DNA was extracted using the Kaneka Easy DNA Extraction Kit (Kaneka Corporation, Osaka, Japan). Cellular RNA was extracted using the High Pure RNA Tissue Kit (Roche Diagnostics, Tokyo, Japan). Extracted DNA and RNA were amplified by PCR/RT-PCR using an HIV gag primer set (sense: 5'-AGTGGGGGGACATCAAGCAGCCATGCAAAT-3', antisense: 5'-TACTAGTAGTTCCTGCTATGTCACTTCC-3'), as previously described [9]. Both HIV DNA and RNA levels were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All quantitative PCR/RT-PCR analyses were performed using a syber green method with an ABI PRISM Genetic Analyzer (Thermo Fisher Scientific).
Flow cytometric analysis of CD4+/CD8+ T cells and intracellular p24-expressing cells
Blood samples of HIV-infected NOJ mice were treated with red cell lysing buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA) for 15 min and then resuspended in staining media containing 2% fetal bovine serum (FBS). Cells were stained with Live/Dead Fixable Near-IR Dead Cell Stain dye (Thermo Fisher Scientific) and monoclonal antibodies against mouse CD45 and human CD45, CD3, CD4, CD8, and CCR5 for 15 min and then analyzed using a FACS LSR II Flow Cytometer (BD Biosciences, San Jose, CA, USA). The flow cytometry data were further analyzed using FlowJo v10.8 software (FlowJo, LLC, Ashland, OR, USA).
The 5' LTR region of the viral RNA was amplified by PCR using a viral RNA-specific primer set (sense: 5'-GACCATCAAGCGGCTATGCAGATT-3', antisense: 5'-GTAAATGTTGCCTACTGGTATGGG-3'), followed by sequence analysis of the region around the two NF-κB binding sites using the BigDye™ Terminator v3.1 Cycle Sequencing Kit and a sequence analyzer (Thermo Fisher Scientific).
In vitro generation and HIV-1JRFL infection of macrophages
After transduction with either shPromA or shPromA-M2, CD34+ cells were stimulated and expanded for two or three weeks in 12-well plates in the same X-VIVO 10 media supplemented with 4% FBS, 50 ng/mL SCF, 15 ng/mL TPO, 30 ng/mL Flt-3L, and 30 ng/mL IL-3 [19]. The cells were then washed and further cultured for 4–7 days in Roswell Park Memorial Institute 1640 media supplemented with 20% FBS, 25 ng/mL SCF, 30 ng/mL Flt-3L, 30 ng/mL IL-3, and 30 ng/mL macrophage colony-stimulating factor. Development of shPromA-expressing macrophages was detected under a fluorescence microscope as GFP+ cells with typical fried-egg shapes (Fig. 6A). Cells were then seeded at 1 × 105 per well in a 96-well plate and infected with 50 ng (p24) of HIV-1JRFL. Cells were collected five days after infection, stained for surface markers and intracellular HIV p24, and analyzed by flow cytometry.
Antibodies and reagents for flow cytometry
Anti-human CD34 APC and anti-human CD38 PE (BD Biosciences) were used for the analysis of magnetically separated CD34+ cells. The following antibodies were used for the flow cytometric analysis of NOJ mouse PBMCs, splenocytes, and bone marrow cells after human CD34+ cell transplantation: anti-mouse CD45 PerCP-Cy5.5, anti-mouse CD45 PE, anti-human CD45 Brilliant Violet (BV) 510, anti-human CCR5 PE-CF594, anti-human CD56 PerCP-Cy5.5, anti-human CD11c BV510, anti-human CD19 PE-CF594, anti-human CD33 PE-Cy7, anti-human CD66b Alexa Fluor 647 (AF647) (BD Biosciences), anti-human CD3 PE-Cy7, anti-human CD4 BV421, anti-human CD8 AF647, anti-human CD14 PerCP-Cy5.5 (BioLegend, San Diego, CA, USA), anti-human CD123 PE (Thermo Fisher Scientific), and anti-HIV-1 p24 PE (Beckman Coulter, Tokyo, Japan). In addition, the following antibodies were used to stain in vitro-generated macrophages: anti-human CD4 APC, anti-human CD11b BV421, anti-HLA-DR BV510 (BD Biosciences), anti-human CD14 PE-Cy7, anti-human CD16 AF700, and anti-human CD163 PerCP-Cy5.5 (BioLegend). Dead cells stained with the Live/Dead Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) were excluded from analysis.
Statistical analysis was performed using GraphPad Prism software version 7 (GraphPad Software, Inc., La Jolla, CA, USA). A probability (p) value of <0.05 was considered statistically significant. The unpaired Mann–Whitney test or Wilcoxon matched-pairs signed rank test was used for comparisons between two samples. Nonparametric multiple comparison analysis was performed using Dunn's method.
Cord-derived CD34+ cell transduction with lentiviruses expressing shPromA customized for HIV-1JRFL
The siRNAs PromA and PromA M2 are described previously [13]. There was a one-base mismatch in the PromA region between HIV-1NL4-3 and HIV-1JRFL; therefore, the present study utilized a version of PromA customized for HIV-1JRFL, which is simply referred to as PromA in the following text. The sequences of HIV-1JRFL, PromA, and PromA-M2 are described in Fig. 1A. The lentiviral vectors expressing PromA and PromA M2 in the shRNA forms (shPromA and shPromA-M2, respectively) were prepared for CD34+ cell transduction.
The volumes of the collected cord blood samples ranged from 20 to 130 mL. Each sample contained approximately 5 × 106–2 × 108 mononuclear cells with CD34+ cells accounting for about 0.1%–6%, as determined by flow cytometric analysis. After mononuclear cell isolation and CD34+ microbead selection, approximately 5 × 104–2 × 106 CD34+ cells were obtained. The average purity of the CD34+ cells after selection was 96.7%, whereas the average CD38low frequency was 25.6% (Fig. S1A and C). The CD34+ cell batches with >4 × 105 cells were used for lentiviral shPromA transduction. Fresh CD34+ cells were pre-stimulated in X-VIVO 10 media containing SCF, Flt-3L, and TPO for 24 h and then transduced with lentiviruses expressing shPromA at an MOI of 10. Cells were cryopreserved 24 h after transduction. A small portion of cells was cultured for another 48 h to evaluate transduction efficiency, which was expressed as the percentage of GFP+ cells in the culture. The average GFP+ frequency was 65.1% (Fig. S1B and C). The PromA-transduced CD34+ cells exhibited relevant in vitro colony formation capacity compared with untransduced CD34+ cells (Fig S2).
Fig. S1.
Fresh cord-derived CD34+ cells were isolated and transduced with a lentivirus vector expressing the shRNA PromA adapted to HIV-1JRFL (shPromAJRFL or simply shPromA). (A) A representative plot showing the CD34/CD38 expression pattern in freshly isolated CD34+ cells. (B) Representative plots showing the lentiviral transduction efficiency measured by GFP expression. (D) Summary of the CD34+ cell isolation results (upper and middle bars) and the efficiency of the following lentiviral transduction (lower bar).
PromA-transduced CD34+ cells showed equivalent colony forming capability to mock-transduced CD34+ cells. In vitro differentiation of shPromA-transduced CD34+ cells to erythrocytes, granulocytes, or monocytes were assessed in vitro by a colony formation assay. Cells were diluted to 500 /mL in MethoCult Optimum methylcellulose media (STEMCELL Technologies, VIC, Australia) containing recombinant human cytokines including SCF, granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), interleukin 3 (IL-3), and erythropoietin. Cells were seeded in 6-well plate (Corning, VIC, Australia) using a syringe and BD blunt plastic cannula (BD Biosciences, NSW, Australia). Colony-forming units per 1,000 CD34+ cells were counted after 14 days of culture. (A) Different types of GFP+ colonies were found. (B) PromA-transduced CD34+ cells showed no significant difference to mock-transduced cells in colony forming units of all the different colony types tested. (C) Colony forming cells were collected together for flow cytometric analysis. CD14/CD33 expression levels were compared between mock-transduced, shPromA-transduced GFP-, and PromA-tranduced GFP+ cells.
Engraftment of NOJ mice with shPromA-expressing cells
Newborn NOJ mice were irradiated and intrahepatically transplanted with CD34+ cells. At 12–14 weeks after transplantation, peripheral blood was obtained from the retro-orbital sinus. PBMCs were isolated and analyzed by flow cytometry. Twenty mice transplanted with shPromA-transduced cells (PromA group) and 12 transplanted with unmanipulated CD34+ cells (CD34 group) were alive during engraftment assessment. After excluding poorly engrafted mice with low human CD45+ frequencies (<3.0%) or poorly differentiated CD3+ cells, the percentages of human CD45+ cells were compared between the PromA and CD34 groups, but no significant difference was detected (Fig. 1C). These mice were included in the following HIV challenge experiment (Fig. 1E). The average GFP+ frequency of human CD45+ cells was 40.8% in the PromA group (Fig. S3A), which was lower than the average transduction efficiency measured prior to transplantation (65.1%, Fig. S1C). Three of the mice transplanted with shPromA-transduced cells were sacrificed at 15 weeks after transplantation, without HIV challenge. Splenocytes and bone marrow cells were collected and tested for GFP expression, as a marker of shPromA. Cells were also checked for the expression of CD45 (hematopoietic cells), CD3 (T cells), CD4, CD8, CD19 (B cells), CD56 (subset of NK cells), CD123 (subset of dendritic cells), CD33 (myeloid cells), CD11c, CD66b (granulocytes), and CD14 (monocytes). There was no obvious skew in GFP expression among all phenotypically unique cells tested (Fig. 1B and D).
Engraftment of human cells in three transplanted NOJ mice was tested 15 weeks after transplantation. (A) PromA-expressing frequencies in human CD45+ cells, expressed as GFP+ frequencies, in group PromA are shown. These were tested in mouse PBMC (prechallenge, week 1, week 2), splenocytes (week 2) and bone marrow cells (week 2) (B) The mean GFP+ percentages in the different subsets of cells in bone marrow.
HIV-1JRFL challenge of NOJ mice transplanted with shRNA PromA-expressing CD34+ cells
The two mice groups (PromA and CD34) were challenged with HIV-1JRFL at 14–16 weeks after transplantation (Fig. 1E). Peripheral blood was collected at weeks 1 and 2 post challenge. Mice were sacrificed at week 2, and splenocytes and bone marrow cells (from the left femur) were collected for further analysis (Fig. 1E). The frequencies of human CD45+ cells and their subsets were analyzed using PBMCs (weeks 1 and 2), splenocytes (week 2), and bone marrow cells (week 2) (Fig. S4, Table 1). In PBMCs, the percentage of human CD45+ cells was significantly lower in the PromA group than in the CD34 group (Fig. S4A). There was also a significant difference in counts of peripheral blood human CD45+ cells at week 1 (Fig. S4B). However, there was no significant difference in blood CD45+ cell counts at week 2 between the two groups (Fig. S4B). Moreover, the CD45+ cell counts in the spleen and bone marrow were much higher in the PromA group than that in the CD34 group (Table 1). The reasons for these discrepancies remain unclear.
Blood CD45+ frequencies were analyzed after HIV-1 infection of humanized mice. (A) Human CD45+ percentages in PBMC at prechallenge, week 1, and week 2. (B) Human CD45+ cell counts in blood (/ml) at prechallenge, week 1, and week 2. Comparison was done by the Mann-Whitney test. **: P<0.01, ***: P<0.001.
Table 1. Comparison of spleen/bone marrow cells among the animal groups untransduced (CD34) uninfected, untransduced (CD34) infected, PromA-transduced uninfected, and PromA-transduced infected.
Values are presented as mean ± standard deviation. Bone marrow cells were collected from the left femur of each animal. Samples were collected at week 15–18 post transplantation or at week 2 post challenge for HIV-infected animals (Fig. 3). N.D., not determined.
CD4+CCR5+ T cell dynamics after HIV-1JRFL challenge of humanized NOJ mice
Intracellular HIV Gag p24 expression levels were measured in PBMCs (weeks 1 and 2), splenocytes (week 2), and bone marrow cells (week 2) after HIV challenge. There were significant differences in HIV p24+ frequencies in CD3+CD8‒ T cells at weeks 1 and 2, indicating the impact of PromA on suppression of HIV replication (Fig. 2A and B, Fig. S5). Cell-associated HIV DNA/RNA copy numbers in splenocytes (week 2) relative to human GAPDH were measured by quantitative PCR/RT-PCR. Compared with those in the CD34 group, the average relative RNA copy number was low, whereas the relative DNA copy numbers were significantly high in the PromA group, (Fig. 2C left and middle). The ratios of HIV RNA to DNA levels were calculated. The average RNA/DNA ratio in the PromA group was >10-fold lower than in the CD34 group (Fig. 2C right), showing suppression of HIV RNA, including transcripts, compared with DNA. There was no mutation in the PromA region of the HIV RNA (data not shown).
Intracellular HIV p24+ frequencies in CD3+CD8- T cells were compared between group CD34 and PromA of HIV-infected humanized mice. The analysis was done using PBMC obtained at weeks 1 and 2 post challenge. Comparison was done by the Mann-Whitney test. ***: P<0.001.
PromA suppressed viral replication and protected CD4+ T cells from depletion in NOJ mice. (A) Representative plots showing suppression of intracellular HIV p24 expression in PromA-transduced mice. (B) Intracellular HIV p24+ frequencies in CD3+CD8-T cells were compared between the CD34 and PromA groups. The analysis was done using splenocytes (left) and bone marrow cells (right) obtained at week 2 post challenge. (C) Cell-associated RNA (left) and DNA (middle) copies were normalized to GAPDH. Relative RNA copies per DNA were calculated from these data (right). Statistical analysis was performed using the Mann–Whitney U-test. (D) Representative plots showing preserved CD4+CCR5+ cells in the PromA-transduced mice compared with the control mice. (E) CCR5+ frequencies of CD4+ T cells collected from the spleen at week 2 post challenge. Statistical analysis was performed by the multiple comparison test with Dunn's method.
There was a marked decrease in the numbers of CD4+CCR5+ splenocytes in the CD34 group at week 2, compared with the uninfected control group (Fig. 2D and E). Conversely, splenic CD4+CCR5+ T-cell frequencies were preserved in the PromA group, supporting the protective effect of PromA against challenge with the CCR5-tropic strain of HIV-1 (Fig. 2D and E). Analysis of PBMCs sampled at week 2 post challenge showed similar results (Fig. S6).
CCR5+ T-cell frequencies of CD4+ T cells in PBMCs were analyzed at weeks 1 and 2 post HIV challenge of humanized mice. Comparison was done by the Mann-Whitney test. ***: P<0.001.
Bone marrow CD34+ cell dynamics in humanized NOJ mice after challenge with CCR5-tropic HIV-1JRFL
To better clarify the protective effect of PromA on human hematopoietic cells in HIV infection, bone marrow CD34+ cells were analyzed (Fig. 3). At week 2 post challenge, the mean percentage of bone marrow CD34+ cells was severely decreased in the CD34 group, compared with the uninfected control group (2.7% vs. 14.6%, respectively) (Fig. 3A–D). Further analysis revealed preferential depletion of CD34+CXCR4+ cells, as shown by the drop in the mean percentage of CXCR4+ cells in the CD34+ cell population from 73.8% (HIV-) to 23.2% (CD34) (Fig 3A–D). The mean percentage of CD34+ cells in the PromA group was significantly greater than in the CD34 group (10.1% vs. 2.7%, respectively, Fig. 3D). This finding indicated that shPromA was associated with partial protection of bone marrow CD34+ cells, including the CD34+CXCR4+ cell population (Fig 3A–D). The expression levels of shPromA (GFP) in the bone marrow cells of mice in the PromA group were further analyzed. Interestingly, a greater percentage of CD34+ cells were positive for GFP compared with CD45+ cells (Fig. 3F), implicating a possible role of shPromA in the protection of CD34+ cells from HIV infection.
In vitro-generated PromA-expressing macrophages were resistant to HIV-1JRFL replication
In parallel with the HIV challenge experiment using the humanized mice, the effect of shPromA on suppression of HIV replication was tested with macrophages generated in vitro from cord-derived CD34+ cells (Fig. 4A). The average percentage of GFP+ cells in CD11b+ cell populations was 42.6%, and most CD11b+ cells were CD14+/CD16+/CD4+/CCR5+ (Fig. 4B). Five days after infection of these macrophages with HIV-1JRFL, viral replication levels, measured as intracellular HIV p24+ percentages, were compared among untransduced, shPromA-M2-transduced, and shPromA-transduced macrophages. As shown in Fig 4C and D, HIV replication was significantly suppressed in shPromA-transduced macrophages, compared with autologous control samples. Similar results were obtained with PM1-CCR5 cells transduced with shPromA or shPromA-M2 and infected with HIV-1JRFL (Fig. S7).
The efficacy of shPromA to suppress HIV infection was tested in vitro using the PM1-CCR5 cells1. Cells were infected with HIV-1JRFL at an MOI of 0.01. Twenty-four hours later, cells were seeded in RetroNectin-coated 48-well plate at the concentration of 5,000 cells/well and transduced with the lentivirus expressing shPromA at an MOI of 1 or 10. Cells were analyzed at days 3, 6, and 9 post-transduction for the intracellular expression of HIV p24+. (A) Transduction rates as determined by the expression of GFP analyzed by flow cytometry. The lentiviral MOIs were indicated. (B) Intracellular HIV p24+ percentages monitored at days 3, 6, and 9 post-transduction. (C) Percentages of suppression of HIV replication relative to the untransduced control at day 9 post-transduction were calculated by the data shown in B.
Freshly isolated CD34+ cells were transduced with shPromA and differentiated to macrophages in vitro. Cells were then infected with HIV-1JRFL. HIV replication levels were compared to autologous mock-transduced or shPromA-M2-transduced macrophages on day 5 post infection. (A) PromA-expressing (GFP+) macrophages with the typical fried-egg shapes were observed by fluorescence microscopy. (B) Representative plots showing expression patterns of GFP (PromA)/CD11b (left) in live cells as well as expression patterns of the CD14/CD16 (middle) and CD4/CCR5 (right) subets in the population of CD11b+ cells. (C) Representative plots showing intracellular HIV p24+ frequencies in mock-transduced, shPromA M2-transduced, and shPromA-transduced macrophages on day 5 post infection. (D) HIV replication was significantly lower in the shPromA-transduced samples than in mock-transduced samples. Nonparametric multiple comparison analysis was performed with the Dunn's method. (E) Fold suppression of HIV replication relative to mock-transduced samples was calculated for PromA M2-transduced and shPromA-transduced samples. The shPromA-transduced macrophages showed a significantly higher suppressive capacity. Comparisons were performed with the Wilcoxon matched-pairs signed rank test or Dunn's method of multiple comparison analysis.
One of the challenges with standard HIV therapies is the lack of an effective method to repair the compromised immune system during treatment initiation, regardless of AIDS manifestation [20]. Gene therapy with hematopoietic stem/progenitor cells (HSPCs) has been of great interest because this can possibly recover the loss of the naïve/memory T-cell repertoire, which then would enable re-immunization of the individual against various pathogens, including HIV [21]. Thus far, treatments using knockout or knockdown of CCR5, a coreceptor used by strains of HIV-1 that preferentially infect memory CD4+ T cells, are the most successful [22]. In addition, other potential host and non-host targets to render the host cells more resistant to HIV replication have been investigated [23]. The PromA region in the HIV genome has proved to be among the most safe and powerful non-host targets [24].
In the present study, newborn NOJ mice were transplanted with lentivirally transduced CD34+ cells expressing shPromA, which resulted in shPromA-expressing CD34+ cell engraftment and their differentiation to most major subtypes of myeloid and lymphoid cells in vivo, including CD4+/CD8+ T cells, CD19+ B cells, CD56+ NK cells, and CD11c+/CD14+/CD66b+/CD123+ myeloid cells (Fig. 1B–D, Table 1). These findings suggested that the off-target effects associated with PromA expression did not interfere with hematopoietic cell differentiation in vivo [13]. The method tested in this study is thus promising and also in line with previous reports showing that retroviral transduction does not alter immune function [25].
The results of the HIV-1JRFL challenge experiment showed that the HIV-infected mice were depleted of CD4+CCR5+ cells and bone marrow CD34+ cells. Interestingly, depletion of the latter occurred in a CXCR4-associated manner (Fig. 3A–D), which may implicate increased CD34+ cell turnover mediated by the CXCR4/SDF-1 signaling pathway, compensating for the increased CD4+ cell turnover following HIV infection. Although CXCR4-tropic HIV-1 infects HSPCs more efficiently than CCR5-tropic HIV-1 [26], CCR5-tropic HIV-1 alone may be sufficient to cause bone marrow dysfunction associated with immunological nonresponsiveness and/or pancytopenia [3, 4, 27]. In addition, in the CD34 group, bone marrow CD34+ cell depletion was more profound than spleen/bone marrow CD45+ cell depletion (Table 1). This may suggest early involvement of bone marrow dysfunction in disease progression, such as impaired CD4+ T-cell production, rather than being only a determinant of the immune reconstitution rates in the chronic phase.
Conversely, HIV replication was suppressed (Fig. 2A-C) and CD4+CCR5+ T cells were preserved (Fig. 2D and E, Fig. S6) in mice engrafted with shPromA-transduced cells; this is relevant to the previous in vivo results using adult NOJ mice transplanted with shPromA-transduced PBMCs [13]. Mice in the PromA group also showed partial preservation of bone marrow CD34+ cells (Fig. 3), which implies that the anti-HIV gene therapy of CD34+ HSPCs may be more advantageous than equivalent gene therapy of PBMCs [20, 28]. Multiple types of hematopoietic progenitor cells may harbor CCR5-or CXCR4-tropic viruses for extended periods [29]; therefore, the expression of an anti-HIV modality in these cell types may be highly effective to protect the hematopoietic system of HIV-infected patients.
Limitations of this study include the lack of an empty lentivirus vector for the patented shPromA lentivirus vector. Instead, mice transplanted with unmanipulated CD34+ cells were included as a control group. The number of animals was too limited to create another group of mice transplanted with CD34+ cells expressing shPromA-M2 with a three-base mismatch, compared with shPromA. To partly compensate for this limitation, in vitro viral suppression assays with macrophages (Fig. 4) were conducted using cells either untransduced or transduced with shPromA-M2 as controls. Another limitation is that the HIV challenge was conducted as a 2-week acute infection experiment with injection of a high dose of HIV-1JRFL to each animal. A chronic infection study with an injection of a low dose of HIV-1JRFL may be useful to determine whether PromA is able to support the preservation and function of bone marrow CD34+ cells for prolonged periods [30].
In summary, the present in vivo study of humanized mice demonstrated that infection of CCR5-tropic HIV-1 alone resulted in depletion of bone marrow CD34+ stem/progenitor cells in a CXCR4-associated manner. Gene therapy of HSPCs with shPromA targeting a non-host, non-coding gene sequence may be beneficial to preserve the hematopoietic potential of the bone marrow [16], which would be a substantial step toward the clinical application of TGS (e.g., combination of PromA, targeting of CCR5, and antiretroviral therapies).
I thank Drs. Kazuo Matsui and Shoichi Kawakami of Fukuda Hospital, Kumamoto, Japan, for their assistance with cord blood sampling. I thank Ms. Michelle Millington, Ms. Maureen Boyd, and Dr. Geoff Symonds for providing lentiviral vector constructs and the transduction protocol. I thank Prof. Anthony Kelleher, Dr. Kazuo Suzuki, and Ms. Katherine Marks of the Kirby Institute, UNSW, Australia, for their support and help with preparation of the shRNA-expressing lentivirus vectors. I thank Prof. Seiji Okada of the Center for AIDS Research, Kumamoto University, and his staff for their technical assistance with the animal experiments. I thank Enago (www.enago.jp) for the English language review. This work was supported by grants from the National Health and Medical Research Council, Australia (Project grant), the Japan Agency for Medical Research and Development (Research program on HIV/AIDS, no. 16fk0410108h0001), and the Ministry of Education, Culture, Sports, Science and Technology, Japan (Grants-in-Aid for Science Research, no. 25114711).
↵1 Yusa, K., Maeda, Y, Fujioka, A., Monde, K., Harada, S., 2005. Isolation of TAK-779-resistant HIV-1 from an R5 HIV-1 GP120 V3 loop library. J Biol Chem 280, 30083-30090.
Parinitha S, Kulkarni M. Haematological changes in HIV infection with correlation to CD4 cell count. Australas Med J 2012; 5:157–162.
Corbeau P, Reynes J. Immune reconstitution under antiretroviral therapy: the new challenge in HIV-1 infection. Blood 2011; 117:5582–5590.
Hunt PW, Martin JN, Sinclair E, Bredt B, Hagos E, Lampiris H, et al. T cell activation is associated with lower CD4+ T cell gains in human immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral therapy. J Infect Dis 2003; 187:1534–1543.
Isgro A, Leti W, De Santis W, Marziali M, Esposito A, Fimiani C, et al. Altered clonogenic capability and stromal cell function characterize bone marrow of HIV-infected subjects with low CD4+ T cell counts despite viral suppression during HAART. Clin Infect Dis 2008; 46:1902–1910.
Tripathi AK, Misra R, Kalra P, Gupta N, Ahmad R. Bone marrow abnormalities in HIV disease. J Assoc Physicians India 2005; 53:705–710.
Matzke MA, Primig M, Trnovsky J, Matzke AJ. Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J 1989; 8:643–649.
Weinberg MS, Morris KV. Transcriptional gene silencing in humans. Nucleic Acids Res 2016; 44:6505–6517.
Suzuki K, Juelich T, Lim H, Ishida T, Watanebe T, Cooper DA, et al. Closed chromatin architecture is induced by an RNA duplex targeting the HIV-1 promoter region. J Biol Chem 2008; 283:23353–23363.
Suzuki K, Shijuuku T, Fukamachi T, Zaunders J, Guillemin G, Cooper D, et al. Prolonged transcriptional silencing and CpG methylation induced by siRNAs targeted to the HIV-1 promoter region. J RNAi Gene Silencing 2005; 1:66–78.
Lim HG, Suzuki K, Cooper DA, Kelleher AD. Promoter-targeted siRNAs induce gene silencing of simian immunodeficiency virus (SIV) infection in vitro. Mol Ther 2008; 16:565–570.
Yamagishi M, Ishida T, Miyake A, Cooper DA, Kelleher AD, Suzuki K, et al. Retroviral delivery of promoter-targeted shRNA induces long-term silencing of HIV-1 transcription. Microbes Infect 2009; 11:500–508.
Suzuki K, Ishida T, Yamagishi M, Ahlenstiel C, Swaminathan S, Marks K, et al. Transcriptional gene silencing of HIV-1 through promoter targeted RNA is highly specific. RNA Biol 2011; 8:1035–1046.
Suzuki K, Hattori S, Marks K, Ahlenstiel C, Maeda Y, Ishida T, et al. Promoter Targeting shRNA Suppresses HIV-1 Infection In vivo Through Transcriptional Gene Silencing. Mol Ther Nucleic Acids 2013; 2:e137.
Ahlenstiel C, Mendez C, Lim ST, Marks K, Turville S, Cooper DA, et al. Novel RNA Duplex Locks HIV-1 in a Latent State via Chromatin-mediated Transcriptional Silencing. Mol Ther Nucleic Acids 2015; 4:e261.
Okada S, Harada H, Ito T, Saito T, Suzu S. Early development of human hematopoietic and acquired immune systems in new born NOD/Scid/Jak3null mice intrahepatic engrafted with cord blood-derived CD34 + cells. Int J Hematol 2008; 88:476–482.
Arainga M, Su H, Poluektova LY, Gorantla S, Gendelman HE. HIV-1 cellular and tissue replication patterns in infected humanized mice. Sci Rep 2016; 6:23513.
Miyoshi H, Takahashi M, Gage FH, Verma IM. Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proceedings of the National Academy of Sciences of the United States of America 1997; 94:10319–10323.
Zhang B, Xia HQ, Cleghorn G, Gobe G, West M, Wei MQ. A highly efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors. Gene Ther 2001; 8:1745–1751.
Stec M, Weglarczyk K, Baran J, Zuba E, Mytar B, Pryjma J, et al. Expansion and differentiation of CD14+CD16(-) and CD14+ +CD16+ human monocyte subsets from cord blood CD34+ hematopoietic progenitors. J Leukoc Biol 2007; 82:594–602.
Tsukamoto T, Yamamoto H, Okada S, Matano T. Recursion-based depletion of human immunodeficiency virus-specific naive CD4(+) T cells may facilitate persistent viral replication and chronic viraemia leading to acquired immunodeficiency syndrome. Med Hypotheses 2016; 94:81–85.
Rosenzweig M, Marks DF, Hempel D, Heusch M, Kraus G, Wong-Staal F, et al. Intracellular immunization of rhesus CD34+ hematopoietic progenitor cells with a hairpin ribozyme protects T cells and macrophages from simian immunodeficiency virus infection. Blood 1997; 90:4822–4831.
Myburgh R, Ivic S, Pepper MS, Gers-Huber G, Li D, Audige A, et al. Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice. J Virol 2015; 89:6761–6772.
Kitchen SG, Shimizu S, An DS. Stem cell-based anti-HIV gene therapy. Virology 2011; 411:260–272.
Ahlenstiel CL, Suzuki K, Marks K, Symonds GP, Kelleher AD. Controlling HIV-1: Non-Coding RNA Gene Therapy Approaches to a Functional Cure. Front Immunol 2015; 6:474.
Duarte RF, Chen FE, Lowdell MW, Potter MN, Lamana ML, Prentice HG, et al. Functional impairment of human T-lymphocytes following PHA-induced expansion and retroviral transduction: implications for gene therapy. Gene Ther 2002; 9:1359–1368.
Carter CC, McNamara LA, Onafuwa-Nuga A, Shackleton M, Riddell Jt, Bixby D, et al. HIV-1 utilizes the CXCR4 chemokine receptor to infect multipotent hematopoietic stem and progenitor cells. Cell Host Microbe 2011; 9:223–234.
Dhurve SA, Dhurve AS. Bone Marrow Abnormalities in HIV Disease. Mediterr J Hematol Infect Dis 2013; 5:e2013033.
Tsukamoto T. HIV disrupts CD34+ progenitors involved in T-cell differentiation. bioRxiv 2017:142539.
Sebastian NT, Zaikos TD, Terry V, Taschuk F, McNamara LA, Onafuwa-Nuga A, et al. CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo. PLoS Pathog 2017; 13:e1006509.
Terahara K, Ishige M, Ikeno S, Mitsuki YY, Okada S, Kobayashi K, et al. Expansion of activated memory CD4+ T cells affects infectivity of CCR5-tropic HIV-1 in humanized NOD/SCID/JAK3null mice. PLoS One 2013; 8:e53495.
You are going to email the following CXCR4-associated depletion of bone marrow CD34+ cells following CCR5-tropic HIV-1 infection of humanized NOD/SCID/JAK3null mice and partial protection of those cells by a promotor-targeting shRNA
bioRxiv 190702; doi: https://doi.org/10.1101/190702


Stereogram Recordings are delighted to announce the addition of Dundee based alternative rock outfit STOOR to their ever increasing roster. 'Witchfinder General', their debut download single, finds them in characteristic post-punk/sci-fi mode, merging insistent riffs and melodies with dark, mysterious lyrics. The title itself comes from a Hammer Horror movie, so that should give you some idea of where they're coming from. Echoes of bands as diverse as Pere Ubu, The Dead Kennedys and even late 90s sensations Ultrasound abound as the songs buries itself deep within your psyche. This is what Andy Wood of IsThisMusic? had to say about it: 'Witchfinder General' is, on the surface, a lighter counterpoint, but still has a nervy, anxious feel to it and the lyrics and delivery seem to warn of human folly and the danger of Messiah's: "Brothers and Sisters gather/To meet the latest hazard/Hear the word" Formed from the fumes of the original West Port bar and distilled from earlier Dundee punk/New Wave bands, STOOR have been a presence on the Scottish music scene since the 1990s. The nuclear core of the band (Scott Mckinlay, drums; Stef Murray, bass and vocals; Ross Matheson, guitar) fused over their love of punk, film and TV themes and the Kinks to name but three influences. Over the years other members have come and gone with the present fourth member being Davie Youngblood on guitar. Sporadically gigging like a Time Lord on a sabbatical, STOOR have been a rare band to witness (which could be of interest to Pokemon-Go players). Recent years has seen a flurry of activity with many sightings of their musical Tardis in venues around Scotland and the North of England.


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Home » Ferrero opens distribution center in Pennsylvania
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Ferrero opens distribution center in Pennsylvania
Facility to create 375 jobs by the end of 2020.
From left: Luca Robbiano, supply chain senior v.p., Ferrero North America; Paul Chibe, president & CEO, Ferrero North America; Congressman Daniel P. Meuser; and Franklin Littleton, president, consumer, DHL Supply Chain North America mark the official opening of Ferrero's new distribution center in Jonestown, Pa.
KEYWORDS distribution / Ferrero Group / Ferrero U.S.A. / Pennsylvania
Ferrero has officially opened a new distribution center in Jonestown, Pa. that's designed to support the company's organic growth and integration of new brands.
The 730,000-sq.-ft. facility will add 225 local jobs with 75 new positions in 2019 and an additional 150 jobs by the end of 2020.
"The new Jonestown distribution center will play an important role in our U.S. and North American growth plans," said Paul Chibe, president and CEO, Ferrero North America.
The new facility adds capacity for Ferrero North America to distribute its growing portfolio of brands, including Baby Ruth, Butterfinger, and Crunch. Over time, the facility, which is managed by DHL Supply Chain, will also distribute other Ferrero brands.
"We are thrilled to be an active business partner in Jonestown and Union Township,' Chibe said. "These communities' commitment to economic growth was an integral part of the decision to select this location for our new distribution center. In DHL, we have the perfect partner whose best-in-class experience and technology will ensure this facility helps power Ferrero's continued growth for years to come."
Joining the ribbon-ceremony was Franklin Littleton, president, consumer, DHL Supply Chain North America, which provides warehouse services for Ferrero's new distribution center, including shipment consolidation, packaging, distribution, and inventory management.
With digitalization being a priority for DHL Supply Chain, the facility will utilize lithium powered lifts as an energy and cost savings initiative. The center also features an energy-saving HVAC system and zero-waste-to-landfill practices to reduce environmental impact.
The facility will also use JDA (NTV) warehouse management system and DHL's proprietary MySupplyChain that provides visibility across the entire supply chain.
"We were immediately attracted to Ferrero's vision for future growth and believed our proven processes to consistently deliver predictability and cost certainty uniquely positioned us to help them increase capacity and achieve that vision," Littleton said. "The addition of this new facility is an exciting and critical step in continuing to deliver on Ferrero's growth strategy as it will bring its products to the market with even greater speed and efficiency."
Also in attendance at the ribbon-cutting were Congressman Daniel P. Meuser, representatives from Union Township, Jonestown and the Pennsylvania Congressional Delegation, as well as other Ferrero and DHL special guests.
Ferrero Group commits to making all packaging sustainable by 2025
Ferrero launches Tic Tac Coca-Cola
Ferrero to host speakeasy pop-up to celebrate U.S. launch of Kinder Bueno
Nutella brand to welcome three superfans to Hotella Nutella
Kettle Awards 2020

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Hempel Special Metals has large stocks of semi-finished products including sheets, plates, rods, tubes and wires in titanium. These are manufactured by leading titanium producers certified to ISO or ASTM standards.
Full traceability - an increasingly important issue - can be guaranteed at all times. Our certification according to ISO 13485 is regularly confirmed by audits by notified institutions as well as by long-standing customers!


I don't know about you, but we probably spend more time in our backyard enjoying this beautiful fall weather than we do in the summer.
Enjoy it for another month and then it'll be all ready for a full season next year.
Go with a light color of granite if possible. The darker colors get HOT, especially if they have the sun hitting them directly.
Darker colors are better for areas that have a lot of freeze/thaw though. This is because the darker colors of granite are more dense.
If possible, have the countertops under a cover of some sort. Whether it's an awning or canopy or whatever. The less rain, snow and dirt on the countertops the better.
Use 3 cm (or thicker) granite if possible and don't laminate the edge. The countertops will just hold up better/ longer. If you want a laminated edge though just be sure that your fabricator uses a glue for the laminations that's specifically made for outdoor applications.
Cover your countertops during the winter months.
Use a reputable fabricator. You don't want to waste your money paying for sub-par fabrication and/or installation.


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Records In My Life
Northern Transmissions is a music website started for music lovers, by music lovers. We feature interviews, album and live reviews from today's most influential independent bands and artists. Northern Transmissions also features music news from around the world everyday.
The End of Music/Universal Music Enterprises
Montage of Heck: The Home Recordings
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This summer, a Michigan bookstore called Brilliant Books issued refunds to customers who were disappointed by author Harper Lee's recently released Go Set a Watchman, an archival draft of a novel that was was written prior to Lee's 1960 classic To Kill a Mocking Bird. In a statement, the store warned prospective buyers that Go Set a Watchman should be considered "as an academic insight rather than as a nice summer novel."
Similar logic can be applied to Kurt Cobain's Montage of Heck: The Home Recordings. This is not a proper album, and it shouldn't be regarding as the debut solo effort from the late Nirvana frontman. Rather, it's a collection of oddments found in Cobain's private collection of homemade cassettes during the making of Brett Morgen's recent documentary Montage of Heck. These tapes weren't made for public consumption — in most cases, they probably weren't intended to be heard by anyone other than their maker — meaning that this is a bit like peeking into an artist's sketchbook or a writer's journal.
As such, The Home Recordings isn't designed to be enjoyed in the same way as Nirvana's brilliant (and sadly small) catalogue. Rather, it's a glimpse into the subconscious of the '90s most iconic rock star, and it provides interesting moments for those seeking to understand his artistic process. The preliminary solo version of Nirvana's "Been a Son," for example, consists of gibberish lyrics, and Cobain finishes the demo by separately performing the bassline as a guide for future reference. "Something in the Way" appears as a distorted electric dirge in a medley of unfinished material, "Sappy" is a chilling ballad based around stark arpeggios, and "Frances Farmer Will Have Her Revenge on Seattle" is delivered with a slowed-down acoustic chug.
The collection's tracklist is padded with a few abstract pieces: Cobain speaks in tongue-in-cheek accents and sings in a country bumpkin moan on "The Yodel Song," fucks around with wah-wah and a fuzz pedal on "Reverb Experiment," and sets an uncharacteristically jaunty mood with the instrumental sketches "The Happy Guitar" and "Letters to Frances."
All told, the album's 13-track standard version contains only a couple of proper unheard songs. "She Only Lies" is a bass-based composition that doesn't make much of an impression beyond its spiteful lyrics ("I really hate her / And I know that you will hate her too"). The collection's most intriguing inclusion is the ramshackle folk-grunge song "Desire"; Cobain sings this in a strangled, scratchy whisper that's a little too harsh to be enjoyable, but the simple refrain and sweet pop melodies sound like an "In Bloom"-sized anthem in the making. It's fun — although a little frustrating — to imagine how good the song could have been in Nirvana ever got a hold of it.
Montage of Heck is as messy and intimate as the documentary of the same name (large parts of which were made up of raw home movie footage), but it's thankfully much less harrowing. While the film traced his tragic downfall, these demos capture Cobain doing what he loved: creating music and goofing around. Even darker moments, like the ghostly cover of the Beatles' "And I Love Her," are intriguing rather than upsetting.
Whether or not this amounts to something enjoyable depends entirely on how you approach it. These recordings don't reveal Cobain's genius, and they don't lift the lid on any undiscovered treasures. Rather, this is a fans-only curiosity that needs to be considered within the context of his legacy, and in that regard, it's a captivating study. To once again quote Brilliant Books and its comments on Go Set a Watchman, this is "a rough beginning for a classic, but only that."
Alex Hudson
Cub Scout Bowling Pins
Heaven Beats Iowa
Blame Game EP
Pearl Charles
Dilip & Otxhello
Late Year
Goat Girl Debut Video For "Badibaba"
TRZTN Collaborates With Karen O
Sharon Van Etten joins Local Natives On Kimmel
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Cub Scout Bowling Pins Heaven Beats Iowa
Bully Covers Orville Peck And Nirvana
Mark Lanegan Is Not Taking The Easy Ride
Spare Parts for Broken Hearts Debut Single
SXSW: Kurt Cobain — Montage of Heck
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St Patrick's Day may be over for this year, but that doesn't mean a good old Irish saying can't be used when the right situation arises.
Whether you are living in Ireland or among the Irish diaspora around the world there is always a time and place to quote from the motherland. A major influence in any Irish person's life is family, love them or hate them we all have to bear with them.
Keeping that in mind IrishCentral has decided to put together a list of the best Irish sayings about family and sayings only a parent would have the honesty to say to their child. Don't worry there won't be any wooden spoons involved, we don't want to stir up any haunting childhood memories.
Let us know what your favorite Irish quote about family is in the comments below.


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This story was written by the ILO Newsroom For official ILO statements and speeches, please visit our "Statements and Speeches" section.
ILO Global Commission homes in on key opportunities and challenges ...
Global Commission on the Future of Work
ILO Global Commission homes in on key opportunities and challenges surrounding the future of work
Press release | 23 October 2017
GENEVA (ILO News) – The Global Commission on the Future of Work wrapped up its first meeting on Sunday 22 October following an in-depth discussion of the main forces shaping the world of work, and the opportunities to be leveraged and challenges to be addressed for a future of work with social justice to be secured.
The 28 member Commission looked at the so-called "mega drivers of change" – technology, demography, climate change and globalization – and also focused on a number of more specific issues, including the changing role of women in the workforce, the importance of education and skills, the relationships between poverty, inequality and informality in the world of work, the prospects for youth, the role of businesses, employers and trade unions, and the evolving values and preferences in societies that will have an impact on the world of work.
The Commission emphasized the need to generate action and impact on key world of work issues which are at the centre of societal concerns around the world. In developing its recommendations the Commission plans to engage with relevant actors and stakeholders.
Co-Chair President Ameenah Gurib-Fakim stated that: "The globalized world that we are living in calls for a global solidarity for addressing those issues which are no longer regional, no longer national but of global nature." Co-Chair Prime Minister Stefan Löfven of Sweden commented that, "We're talking about the need for a global economy that works for everybody, not just for a few."
The Commission's report will be submitted to the ILO's Centenary International Labour Conference in 2019.
The next meeting of the Commission is scheduled to take place in February 2018 in Geneva.
Note: The Global Commission was set up to assess the transformative changes taking place in the world of work and to make recommendations on the way forward. It marks the second stage of the ILO's Future of Work Initiative, launched by ILO Director-General Guy Ryder in 2013, following the first phase during which ILO member States convened national and sub-regional dialogues to provide their perspectives on the future of work.
Tags: future of work
ILO Global Commission tackles the changes needed for a fair future of work for everyone
ILO portal on the Future of Work
newsroom@ilo.org


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The Leading Man is an album by pianist Harold Mabern. It was released by DIW Records in 1993. Columbia Records released an album with the same title and some of the same tracks two years later.

Recording and music
The core personnel are Harold Mabern (piano), Ron Carter (bass), and Jack DeJohnette (drums). They play on all of the tracks except "Mercury Retro", which is performed by Mabern alone. The album was produced by James Williams.

Release and reception

The Leading Man was released in 1993 by DIW Records. An album of the same title was issued by Columbia Records in March 1995. Some of the tracks on this release are the same as for the original album, and the other tracks feature some different personnel.

The AllMusic reviewer commented that the album "is considered a classic, both for its selection of material and the performances of the various ensembles Mabern assembled for the date", and that Mabern's "playing, while always inspired, is revelatory in its sense of full orchestration and the shifting timbres of his solos". The American Visions reviewer wrote that: "Out of the crucible of Art Blakey's band, Mabern held his own for many years and carried some neophytes along the way. Here's a chance to hear him in his terribleness with some real giants."

Track listing

DIW release
"Yes or No"
"Save the Best for Last"
"Full House"
"She/Mr. Lucky"
"Alone Together"
"The Man from Hyde Park"
"B&B"
"T-Bone Steak"
"Mercury Retro"

Columbia release
"Look on the Bright Side"
"Save the Best for Last"
"Full House"
"Alone Together"
"It's a Lonesome Old Town"
"Yes and No"
"Moment's Notice"
"Au Privave"
"B and B"
"Mercury Retro"

Source:

Personnel

DIW release
Harold Mabern – piano
Ron Carter – bass (tracks 1–8)
Jack DeJohnette – drums (tracks 1–8)
Bill Mobley – trumpet, flugelhorn
Bill Easley – alto sax
Kevin Eubanks – guitar
Pamela Baskin-Watson – vocals

Columbia release
Harold Mabern – piano
Ron Carter – bass (tracks 2–4, 6, 9)
Christian McBride – bass (tracks 1, 5, 7, 8)
Jack DeJohnette – drums (tracks 1–9)
Bill Mobley – trumpet, flugelhorn (tracks 6, 9)
Bill Easley – alto sax (track 2)
Kevin Eubanks – guitar (track 3)

Source:

References

1993 albums
DIW Records albums
Harold Mabern albums

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FYI on the new Build Initiative to integrate libraries and museums in early learning efforts.
To view this message in a browser, please click here<http://www.idevmail.net/message.aspx?d=73&m=2255&e=reishk@michigan.gov&r=1248483>.
Learn more about how IMLS is partnering with the BUILD Initiative to meet critical early learning needs.
Read full post online at http://blog.imls.gov/?p=5114<http://www.idevmail.net/link.aspx?l=2&d=73&mid=363882&m=2255>.
The Institute of Museum and Library Services is the primary source of federal support for the nation's 123,000 libraries and 35,000 museums. Our mission is to inspire libraries and museums to advance innovation, lifelong learning, and cultural and civic engagement. Our grant making, policy development, and research help libraries and museums deliver valuable services that make it possible for communities and individuals to thrive. To learn more, visit www.imls.gov<http://www.idevmail.net/link.aspx?l=3&d=73&mid=363882&m=2255> and follow us on Facebook<http://www.idevmail.net/link.aspx?l=4&d=73&mid=363882&m=2255> and Twitter<http://www.idevmail.net/link.aspx?l=5&d=73&mid=363882&m=2255>.


On Friday Marvel Studios added five new films to their release slate leading up to 2019. These unannounced titles add to a schedule that already has two unknowns, and with Marvel boss Kevin Feige confirming that Marvel know's exactly what all these features are going to be, we can expect to see most, if not, all of Phase 3 laid out this Saturday during the Marvel Studios panel at Comic-Con 2014.
What we know so far is that Ant-Man begins shooting next month for a July 2015 release - two months after Phase 2 concludes with Joss Whedon's The Avengers: Age of Ultron. After that, directors Anthony and Joe Russo return with Captain America 3 (no official title yet). With Scott Derrickson already announced to direct Doctor Strange, it's a probable release for 2016 as well, taking the July date Marvel set last summer.
Toss in Thor 3 which already has writers as another absolute somewhere in (the likely extended) Phase 3, and that leaves us with 5 unknowns. Let's take a quick look at some possibilities, a few of which are more hopeful rather than realistic. We'll even throw in a curve ball.
Update 1: Benedict Cumberbatch downplays Doctor Strange Rumors.
Update 2: Joaquin Phoenix is rumored to be in talks for Doctor Strange.
Ultron is coming to the Marvel Cinematic Universe next year in The Avengers sequel and in it, he'll be on a quest to continually evolve by upgrading himself. If that plot point is anything like the comics, and we suspect it is, then Ultron may be headed to the high-tech Marvel Comics African nation known as Wakanda, home of Black Panther.
Given what we heard a while back about the status of the Black Panther screenplay draft, this story is unlikely to happen as a solo adventure. Feige tells Collider that part of the juggling act of continually adding new properties is deciding which gets sequels and which get introduced, and so he hints that Black Panther could be a part of their efforts to "introduce new ones within the body of the films." So, Black Panther guest appearance instead? Still, we can hope.
While Marvel Studios president of production Kevin Feige says they don't currently have plans for a leading lady solo movie and that they won't be "swayed by any backlash," we hope their bigger picture story plans for Phase 3 of the MCU involve Carol Danvers a.k.a. Ms. Marvel - who eventually takes on the mantle of Captain Marvel.
Guardians of the Galaxy 2 is practically a guarantee at this point even though the first has yet to hit theaters. Early reactions are positive and Marvel is banking greatly on the Marvel cosmic side which GOTG introduces. Director James Gunn even said he's a lock to direct it if the first does "well" which it will. He told us this weekend that he prefers to stick to the cosmos for his next Marvel feature, and while that probably means more Guardians, he also said in the past he hopes his first Marvel effort expands into other things (spinoffs) so he has some ideas in mind. Of the many cosmic Marvel characters Guardians of the Galaxy introduces and teases, the most notable featured in his movie are the Nova Corps, the galactic police force of the Marvel universe.
With the more practical design of the Nova Corps officers featured in Guardians of the Galaxy, a super-powered Nova Centurion like the titular "Nova" characters from the comics may not necessarily be a fit for the Marvel Cinematic Universe, but with the introduction of Nova Prime Irani Rael (Glenn Close) we can only hope that we see a Nova officer head to earth and deputize young Earth kid Richard Rider. Picture a galactic war in Phase 4 where Rider is the sole surviving Nova Centurion after the planet Xandar (introduced in GOTG) is ambushed and has to embrace all the power of the Nova Corps. The tie-ins to the Marvel cosmic universe, future Guardians of the Galaxy stories, Thanos and Earth-based teams are ripe for the picking. Guardians of the Galaxy 2 could feasibly serve as a Nova introduction.


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No. 294 - St Andrew's at Lefroy - "The Name of Our Church is St Andrew's"
The township of Lefroy lies 15 km east of George Town on the north east coast of Tasmania. Lefroy is now a virtual ghost town having at its peak a population of about 5000 that has now declined to 77 (2016 Census).
It was originally called Nine Mile Springs, a reference to the distance to George Town and the existence of springs in the vicinity. The George Town and District Historical Society's booklet on Lefroy provides the following information:
"The settlement moved to its present site in the mid 1870s following the development of the Native Youth reef. Known by the locals as Excelsior, after the original mine on the site, it was named Lefroy in 1881 after the visit by the Acting Governor, Sir Henry Lefroy. It was a bustling town which is said to have contained 5,000 people in its peak boom period of 1890-95. It was the fourth largest town in Tasmania. It had a race track, rifle club, cricket club and brass band. There were six hotels, three churches, a state and private grammar school, a masonic lodge and mechanics institute. The town had several shops, two butchers and a cordial factory. In 1907 the headquarters of the George Town Municipality was located there, remaining there until the 1930s. Once commercial mining ceased Lefroy slowly declined, its school and last church closing in 1954. Many of the houses were removed to George Town and Beaconsfield".
Before the establishment of an Anglican church at Lefroy in 1881, services were conducted by Reverend John Fereday from St Mary Magdalene at George Town. In 1872, Fereday met a tragic death on the road to George Town, returning from a service at Lefroy:
"The Ven. Archdeacon Browne and Mr Fereday left Nine Mile Springs about 4 p.m. on Tuesday, in a gig to return to George Town. As Mr Fereday was very near sighted the Archdeacon undertook to drive. They had got near to Quin's farm, when a wheel of the gig came in contact with one of the numerous stumps, which render driving on the portion of the George Town Road from within two or three miles this side the Mount so perilous. The collision was so forcible that the Archdeacon was thrown violently out, the reins were jerked out of his hand, and the horse taking fright bolted with Mr Fereday in the gig. When the Archdeacon was able to get up from where he was thrown, he could see neither horse, gig, nor Mr Fereday. He ran on towards George Town, and at last found Mr Fereday lying on the road bleeding, and quite unconscious".
Fereday never regained consciousness and his death left Lefroy without an Anglican minister for 8 years. The absence of a minister may have provided the catalyst for building a church in the town and in August 1880, the foundation stone for St Andrew's was laid.
The church was completed in the following year and was opened by Bishop Bromby on the 10th of December 1881. The church was described in a report in the Launceston Examiner:
"A neat and substantial wooden structure, lined inside, and painted throughout, and while its cost has been about £500, only £80 of the debt remains on it, the balance having been nearly all collected locally. The committee contemplate not only wiping the debt off shortly, but adding a chancel to the present building".
Another report from the Hobart Mercury's Lefroy correspondent provides considerable detail about the opening service and subsequent celebrations. I have included a substantial part of this as it provides a rich insight into life at Lefroy.
"Since my last report we have had a lively time here, on the occasion of opening the new Church of England by our highly esteemed Lord Bishop of Tasmania, Archdeacon Hales accompanying His Lordship. It was announced that morning service was to be held at 11 o'clock. You may wonder at me stating this, Mr. Editor, but allow me to inform you that it is the first morning service held by the Church of England folk at Lefroy for the past eight years. However, the new bell tolled for the worshippers to assemble together, and after a short time the Archdeacon and His Lordship took their seat at the communion table…. In the latter part of his sermon he earnestly entreated the people to
prepare for the Master's coming, stating that He was still calling for them. Very many in the church were weeping, and I trust much good was done.
At half-past 2 in the afternoon His Lordship addressed the Sunday school children, and by way of text explained to them the meaning of the words, sowing the seed of eternal life in their hearts. The children listened attentively to the explanation, which was plain and simple. Holy Baptism was administered to the infant daughter of Mr. C. Tregurths, of George Town…
On the morrow, being Monday, a tea was held at half-put 4 o'clock, the room being crowded to excess. Before partaking His Lordship pronounced the grace, and justice was done to the good things prepared by Mr. W.B. Dean, of Launceston, which I must say were a credit to him, and could not be surpassed for quantity and quality. About six or seven hundred partook of the good things; and after the tea was over the ladies were busily engaged clearing away, and at the same time giving the bachelors some good hints, as they were allowed to take tea by themselves; some good jokes followed, and all seemed to enjoy themselves heartily, and, allow me to state, without a single murmur. At 8 o'clock the sacred concert commenced. His Lordship, who was in the chair, gave an eloquent address at great length; the singing was good, and more especially that of the Welsh singers, who gave entire satisfaction, singing beautifully in their mother tongue.
… I was almost forgetting to state that the Lefroy Brass Band was in attendance, playing some well regulated pieces, and considering the short time they have been practicing they play exceedingly well, His Lordship and Lady, the Archdeacon, and others being highly delighted with the band. The name of our new Church is St. Andrew's".
By the close of the 19th century, gold mining at Lefroy had passed its peak. The last of the larger mines closed in 1908 and commercial development of the goldfields ended in 1914. With the end of mining the population of the town moved away and the Methodist, Catholic and Anglican churches closed and the buildings were removed. An indication of the size of this former bustling town is its large and beautifully maintained cemetery, where the dead now outnumber the living by a ratio of three to one.
An undated photograph of St Andrew's. Source: https://eheritage.libraries.tas.gov.au/ (Object Number GTH_HS0197)
Lefroy in the 1890's. Source: George Town Pictorial History FaceBook group (posted 12 March 2017) Original source bot indicated.
A view of the Lefroy gold mine in 1890. Source Queen Victoria Museum (QVM: 1994:P:0768
The General Cemetery at Lefroy
The Tasmanian, Saturday 15 April 1871, page 12
The Mercury, Saturday 3 December 1881, page 2
The Examiner, Wednesday 14 December 1881, page 3
The Mercury, Friday 16 December 1881, page 3
The Tasmanian, Saturday 17 Dec 1881
The Mercury, Thursday 19 July 1883, page 4
The Examiner, Monday 23 July 1883, page 3
The Examiner, Friday 16 March 1928, page 11
http://www.gtdhs.com/html/lefroy.html (George Town Historical Society)
http://www.ozburials.com/CemsTas/lefroy.htm
1880-1889 Anglican North East Tasmania


Are you looking for a fresh start, a career move, or just the next step? Why not pop up to St Elizabeth's on our Recruitment Open Day!
On the day there will be the chance to meet our friendly HR team, have an informal chat about a potential career with us and the current vacancies we have on offer. There will also be a chance to have a tour of the Centre, and enjoy our Summer Fayre!
No need to book - just pop in between 12pm-4pm for a friendly chat.


This is a slow pace safari at a stunning time of year, cooler by night and sunny altitudes by day, with views to die for. We stay in simple as well as honeymoon destinations with all the romance of the bush.
The views and countryside we paint on this trip are incredible. Dramatic mountains rise from theplains. Sleek animals graze contentedly. Liwonde National Park, Thyolo Tea Estates near Mount Mulanje, Zomba Mountain and an idyllic tropical island on Lake Malawi form the backdrop for a focus on landscape painting and photography.
For anyone who wants to experience the life and wonder of Africa, then Malawi is a wonderful country to visit. It's impossible not to smile in Malawi, the Malawians make it a very happy place to be painting. There is stunning wildlife at this time of year, and we spend time in the heart of local communities and national parks.
We float on the River Shire painting hippos and eagles; we linger at the waterhole close to herds of elephant, sable, eland, hartebeest; we enjoy the frolics of the baby warthogs, and at all times keep our eyes open for lion, rhino, buffalo and leopard.
There is lots of time for fun and freedom. We can spend time with local people in schools, villages, farms, galleries, paper-making and textile projects.
This is the country where Art Safari began in 1999, and Mary-Anne is a Malawi expert. Over the years many people have travelled and painted with her in Malawi, enjoying the warmth with which the Art Safari groups are welcomed.
Our prices include: – All accommodation, all meals, safari guide and art safari tutor, all game activities as indicated, all park entrance fees, all transport Lilongwe return.
Why not extend your stay in Africa and combine two tours. Art Safari in Zambia 29th September to 9th October 2014 with Large Grey and Unmistakable with Mary-Anne Bartlett. Or why not take a few days/weeks elsewhere in Zambia or Malawi? Visit the splendour of Victoria Falls or relax on Lake Malawi, go hiking/riding/kayaking. Talk to us about the possibilities.
We are happy to arrange your flights for you but offer you the option of sourcing them yourself. We can also offer advice on your personal travel insurance.
Further information about what art materials and clothing to take with you, medical and insurance requirements etc will be given to you later.
Please feel free to discuss your own private safari, with dates to suit you and with an itinerary to suit you.


Q&A Should I Have the Same Anger towards My Affair Partner That My Spouse Has?
My Spouse has strong anger/hatred toward my Affair Partner for the damage she did to us while at the same time holding me 100% responsible for my actions. While I have no desire to have anything to do with her and am totally committed to restoring with my wife, I don't think I feel the same level of anger/hatred toward my AP. My wife feels that lack of anger/hatred indicated that I am protecting my AP and if I was truly over her, I would feel such strong emotions. Am I missing feelings that I am Supposed to have? How do I deal with this?
Because of continual serial lying I secretly recorded my husbands conversations w/ his accountability partner after finding out about his multi decade addictions and sexual sin, and discovered via the recordings that he thought I was obsessed w/ her (the AP), that I was mentally unhealthy and stalking her (I looked at her FB page and he called that stalking) Etc., He was very agitated and angry speaking about me on the recording but defensive and seemingly protective of "her". Putting 2+2 together it doesn't take a rocket scientist to suspect there are residual emotions. -Signed a "mentally unhealthy" betrayed spouse because she "cared" enough to want to know who her husband was seeking to have sexual relations with for almost 5 years..
Q&A What are the Issues Related to Being a F.O.B.A.D.?
Q&A What Does it Mean to Transform Our Pain?
Q&A How Can I Combat My Bitterness Towards My Wife and Her Affair Partner?


text:
An emotional Amanda Knox after court decision: 'I'm so grateful to have my life back'
Posted 2:46 PM, March 27, 2015, by Associated Press and CNN Wire, Updated at 09:10PM, March 27, 2015
ROME (AP) -- Italy's highest court on Friday overturned the murder conviction against Seattle's Amanda Knox and her Italian ex-boyfriend, bringing a definitive end to the high-profile case.
The decision by the supreme Court of Cassation is the final ruling in the case, ending the long legal battle waged by Knox and co-defendant Raffaele Sollecito.
Knox , 27, of Seattle, was convicted in 2009 for the killing of Meredith Kercher, who shared an apartment with her in the Italian university town of Perugia. Her boyfriend at the time, Sollecito, was found guilty, too.
Since then, their cases have been a series of back-and-forths.
"I am tremendously relieved and grateful for the decision of the Supreme Court of Italy," Knox said in a written statement. "The knowledge of my innocence has given me strength in the darkest times of this ordeal.
"And throughout this ordeal, I have received invaluable support from family,
friends, and strangers. To them, I say: Thank you from the bottom of my heart. Your
kindness has sustained me. I only wish that I could thank each and every one of you in person."
In an emotional appearance before cameras outside her mother's West Seattle home Friday night, Knox said, "I'm incredibly grateful for what has happened, for the justice I have received, for the support I have had from everyone. You saved my life."
Knox said she is still "absorbing" all the news. "I'm so grateful to have my life back," she said.
Asked what she would say to the Kercher family, Knox said, "Meredith was my friend. She deserved so much in this life."
Earlier, Knox's family and friends could be heard yelling 'freedom' inside the family's West Seattle home after she was acquitted by Italian judges. Sounds of cheers rang out at Knox's mother's house as news of the overturned verdict broke. Knox's friends and family could later be seen celebrating in the garden.
Both Knox, who was awaiting the verdict in her hometown of Seattle, and Sollecito have long maintained their innocence.
The supreme Court of Cassation on Friday overturned last year's convictions by a Florence appeals court, and declined to order another trial.
The decision means the judges, after thoroughly examining the case, concluded that a conviction could not be supported by the evidence. Their reasoning will be released within 90 days.
After their initial convictions, both Knox and Sollecito were acquitted in 2011 on appeal, and Knox returned to the United States. Two years later, they were retried and their acquittals overturned. Knox was sentenced in absentia to 28 ½ years in prison. Her ex-boyfriend got 25 years.
When the Supreme Court rendered its verdict Friday, Knox was in Seattle.
In addition to her statement, the Knox family released this written statement:
"We want to express our profound gratitude to all of those who have supported Amanda and our family. Countless people – from world-renowned DNA experts, to former FBI agents, to everyday citizens committed to justice – have spoken about her innocence. We are thrilled with and grateful for today's decision from the Supreme Court of Italy. And we are grateful beyond measure for all that so many of you have done for her."
The lawyer for Kercher's family, Francesco Maresca, has expressed his disappointment with the verdict.
"I think that it's a defeat for the Italian justice system," he told the Associated Press news agency.
Kercher's mother was quoted by the AFP news agency as saying that she is "surprised and very shocked" by the acquittals.
Leave a Reply to I'm offended Cancel reply
"peety"
Can this end now so the Kutcher family can have some peace?
big john stankus
Conclusive proof that the Italian justice system is totally inept. Innocent people do NOT change their story – they tell the truth. Knox changed her story 3 times. And yes, there was enough evidence too. Her nickname should now be "O.J.", because she got away with murder just like he did.
So you saw all the evidence and attended the trial and heard all the testimony?
since I saw that online interview she volunteered to do from UW I have never believed her since! Her story had evolved and change over the years in her own interviews. This is just one of those situations that will be handled in the life after this one
Frank D.
Her and OJ ought to hook up and look for the killers.
I'm offended
The more stories I see like this and other examples, I've learned that if you're a good looking white woman you can get away with just about anything ! (murder, child rape, domestic violence, etc.). Is this what feminist refer to as white privilage?
ben kolder
I'd be happy too, if I learned I had just gotten away with murder. Which is what she just did.
Good, it's over!! "The decision means the judges, after thoroughly examining the case, concluded that a conviction could not be supported by the evidence." End of story, I wish her well.
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