wMel Wolbachia genome remains stable after 7 years in Australian Aedes aegypti field populations.

Infection of wMel Wolbachia in Aedes aegypti imparts two signature features that enable its application for biocontrol of dengue. First, the susceptibility of mosquitoes to viruses such as dengue and Zika is reduced. Second, a reproductive manipulation is caused that enables wMel introgression into wild-type mosquito populations. The long-term success of this method relies, in part, on evolution of the wMel genome not compromising the critical features that make it an attractive biocontrol tool. This study compared the wMel Wolbachia genome at the time of initial releases and 1-7 years post-release in Cairns, Australia. Our results show the wMel genome remains highly conserved up to 7 years post-release in gene sequence, content, synteny and structure. This work suggests the wMel genome is stable in its new mosquito host and, therefore, provides reassurance on the potential for wMel to deliver long-term public-health impacts

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequencespecificity of four VapC proteins from two different organisms (VapCPAE0151 and VapCPAE2754 from Pyrobaculum aerophilum, and VapCRv0065 and VapCRv0617 from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms. Published by Cold Spring Harbor Laboratory Press

Dasatinib Targets B-Lineage Cells but Does Not Provide an Effective Therapy for Myeloproliferative Disease in c-Cbl RING Finger Mutant Mice

<div><p>This study aimed to determine whether the multi-kinase inhibitor dasatinib would provide an effective therapy for myeloproliferative diseases (MPDs) involving c-Cbl mutations. These mutations, which occur in the RING finger and linker domains, abolish the ability of c-Cbl to function as an E3 ubiquitin ligase and downregulate activated protein tyrosine kinases. Here we analyzed the effects of dasatinib in a c-Cbl RING finger mutant mouse that develops an MPD with a phenotype similar to the human MPDs. The mice are characterized by enhanced tyrosine kinase signaling resulting in an expansion of hematopoietic stem cells, multipotent progenitors and cells within the myeloid lineage. Since c-Cbl is a negative regulator of c-Kit and Src signaling we reasoned that dasatinib, which targets these kinases, would be an effective therapy. Furthermore, two recent studies showed dasatinib to be effective in inhibiting the <i>in vitro</i> growth of cells from leukemia patients with c-Cbl RING finger and linker domain mutations. Surprisingly we found that dasatinib did not provide an effective therapy for c-Cbl RING finger mutant mice since it did not suppress any of the hematopoietic lineages that promote MPD development. Thus we conclude that dasatinib may not be an appropriate therapy for leukemia patients with c-Cbl mutations. We did however find that dasatinib caused a marked reduction of pre-B cells and immature B cells which correlated with a loss of Src activity. This study is therefore the first to provide a detailed characterization of <i>in vivo</i> effects of dasatinib in a hematopoietic disorder that is driven by protein tyrosine kinases other than BCR-ABL.</p></div

High dose dasatinib treatment markedly reduces the numbers of B-lineage cells.

<p>c-Cbl RING finger mutant mice aged 8–9 months were dosed daily with 30 mg/kg (am) +50 mg/kg (pm) of dasatinib or vehicle, and analyzed after 4 weeks. (A) Numbers of nucleated bone marrow cells from each group. (B) Bone marrow cells analyzed by flow cytometry to determine the percentage of CD11b⁺ myeloid-lineage cells; CD19⁺ B-lineage cells; lineage negative cells (Lin⁻); c-Kit⁺ lineage negative (LK) cells; Lin⁻ Sca-1⁺ c-Kit⁺ (LSK) cells; and multi-potent progenitors (MPPs). (C) Spleen weights from each treatment group. (D) Spleen cells analyzed by flow cytometry to determine percentages of CD11b⁺, CD19⁺ and T cell receptor⁺ (TCR) cells. All of the above data are from 4 dasatinib and 4 vehicle treated mice. The results are expressed as means ± standard errors. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 using the unpaired Student’s <i>t</i> test. (E) H&E stained sections of spleens showing the effects of dasatinib in markedly reducing the size of the follicles in the white pulp. The images were acquired at room temperature using an Olympus BX51 microscope. Photomicrographs were taken at 40× and 200× magnification with 4×/0.16 and 20×/0.70 objective lenses using an Olympus DP70 digital camera. Scale bars are 2 mm and 200 μm respectively.</p

Dasatinib targets immature and germinal center B cells.

<p>B6.CD45.1 mice repopulated with bone marrow cells from a c-Cbl^A/− mouse were dosed twice daily with 15 mg/kg of dasatinib for 16 days. WBCs and spleen cells were analyzed by flow cytometry to identify B cell populations that are most sensitive to dasatinib. Shown are representative flow cytometry profiles and data from 3 vehicle and 3 dasatinib treated mice. (A) B220⁺ WBCs and (B) B220⁺ spleen cells were analyzed for the expression of IgM and IgD to identify immature and mature B cells. The gates show immature IgM⁺ IgD⁻ B cells. (C) B220⁺ spleen cells analyzed for the expression of GL7 and CD95 to identify germinal center (GC) B cells. The results are expressed as means ± standard errors. *<i>P</i><0.05, **<i>P</i><0.01 using the unpaired Student’s <i>t</i> test.</p

Dosing c-Cbl RING finger mutant mice with dasatinib results in a reduction of lymphocytes and a corresponding increase in neutrophils.

<p>c-Cbl^A/− mice aged 8–9 months were bled 6 days before dosing (Pre-Tx), and after 2 and 4 weeks of daily dosing with 15 mg/kg of dasatinib or vehicle. (A) Numbers of white blood cells (WBC), lymphocytes, neutrophils and monocytes. The counts are expressed as means ± standard errors. *<i>P</i><0.05, **<i>P</i><0.01, using unpaired Student’s <i>t</i> test. (B) WBC lysates prepared from mice dosed for 4 weeks with vehicle or dasatinib were immunoblotted with the indicated antibodies. (C) Analysis of mice after 4 weeks of dosing showing the numbers of nucleated bone marrow cells, (D) the proportion of CD11b⁺; CD19⁺; lineage negative (Lin⁻); Lin⁻, c-Kit⁺ (LK); Lin⁻, Sca-1⁺, c-Kit⁺ (LSK) cells; and multi-potent progenitors (MPPs: defined as FLT3⁺ LSK cells), expressed as percentages of the indicated populations. (E) Megakaryocyte erythroid progenitors (MEPs) and common lymphoid progenitors (CLPs), and (F) common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) expressed as percentages of Lin⁻ bone marrow cells. The bone marrow data is from 4 dasatinib and 4 vehicle treated mice, and the results are expressed as means ± standard errors. ***<i>P</i><0.001 using the unpaired Student’s <i>t</i> test. (G) Spleen weights from 4 vehicle and 4 dasatinib treated mice after 4 weeks of dosing.</p

High dose dasatinib treatment results in a significant reduction of B lymphocytes in the blood.

<p>c-Cbl RING finger mutant mice aged 8–9 months were dosed daily with 30 mg/kg (am) +50 mg/kg (pm) of dasatinib or vehicle, and bled before treatment (Pre-Tx), and after 2 and 4 weeks of treatment. Differential blood counts from 9 vehicle and 7 dasatinib treated mice were determined by Hemavet analysis. Shown are (A) total WBC numbers and (B) lymphocyte numbers. (C) WBCs were analyzed by flow cytometry to determine the percentage of B-lineage cells, by anti-CD19 staining, and the percentage of T cells, by anti-T cell receptor staining. (D) Numbers of neutrophils and (E) monocytes. (F) Blood films from 2 vehicle and 2 dasatinib treated mice following 4 weeks of dosing illustrate the loss of lymphocytes in the dasatinib-treated mice. Lymphocytes are indicated by red arrows, and myeloid cells by black arrows, in the blood films from the two vehicle treated mice. The images were acquired at room temperature using an Olympus BX51 microscope with a 60×/0.09 objective and photographed with a SIS 3VCU Olympus digital camera. Scale bar = 50 μm. Lymphocyte numbers (G) and percentages (H) in the blood return to pre-treatment levels 4 weeks after ceasing dasatinib treatment. Neutrophil numbers remain unaltered (I) but the high percentages (J) returned to normal 4 weeks after dasatinib dosing ceases. Mice were bled from the tail vein before treatment (Pre-Tx), after 2 and 4 weeks of treatment, and following 4 weeks without treatment (i.e. post-Tx). Numbers and percentages were determined by Hemavet differential counting, and are from 4 vehicle and 3 dasatinib treated mice. The results are expressed as means ± standard errors. **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001 using unpaired Student’s <i>t</i> test.</p

High dose dasatinib treatment markedly reduces the numbers of B-lineage cells.

<p>c-Cbl RING finger mutant mice aged 8–9 months were dosed daily with 30 mg/kg (am) +50 mg/kg (pm) of dasatinib or vehicle, and analyzed after 4 weeks. (A) Numbers of nucleated bone marrow cells from each group. (B) Bone marrow cells analyzed by flow cytometry to determine the percentage of CD11b⁺ myeloid-lineage cells; CD19⁺ B-lineage cells; lineage negative cells (Lin⁻); c-Kit⁺ lineage negative (LK) cells; Lin⁻ Sca-1⁺ c-Kit⁺ (LSK) cells; and multi-potent progenitors (MPPs). (C) Spleen weights from each treatment group. (D) Spleen cells analyzed by flow cytometry to determine percentages of CD11b⁺, CD19⁺ and T cell receptor⁺ (TCR) cells. All of the above data are from 4 dasatinib and 4 vehicle treated mice. The results are expressed as means ± standard errors. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 using the unpaired Student’s <i>t</i> test. (E) H&E stained sections of spleens showing the effects of dasatinib in markedly reducing the size of the follicles in the white pulp. The images were acquired at room temperature using an Olympus BX51 microscope. Photomicrographs were taken at 40× and 200× magnification with 4×/0.16 and 20×/0.70 objective lenses using an Olympus DP70 digital camera. Scale bars are 2 mm and 200 μm respectively.</p

Assessment of fitness and vector competence of a New Caledonia <i>wMel Aedes aegypti</i> strain before field-release.

BACKGROUND: Biological control programs involving Wolbachia-infected Aedes aegypti are currently deployed in different epidemiological settings. New Caledonia (NC) is an ideal location for the implementation and evaluation of such a strategy as the only proven vector for dengue virus (DENV) is Ae. aegypti and dengue outbreaks frequency and severity are increasing. We report the generation of a NC Wolbachia-infected Ae. aegypti strain and the results of experiments to assess the vector competence and fitness of this strain for future implementation as a disease control strategy in Noumea, NC. METHODS/PRINCIPAL FINDINGS: The NC Wolbachia strain (NC-wMel) was obtained by backcrossing Australian AUS-wMel females with New Caledonian Wild-Type (NC-WT) males. Blocking of DENV, chikungunya (CHIKV), and Zika (ZIKV) viruses were evaluated via mosquito oral feeding experiments and intrathoracic DENV challenge. Significant reduction in infection rates were observed for NC-wMel Ae. aegypti compared to WT Ae. aegypti. No transmission was observed for NC-wMel Ae. aegypti. Maternal transmission, cytoplasmic incompatibility, fertility, fecundity, wing length, and insecticide resistance were also assessed in laboratory experiments. Ae. aegypti NC-wMel showed complete cytoplasmic incompatibility and a strong maternal transmission. Ae. aegypti NC-wMel fitness seemed to be reduced compared to NC-WT Ae. aegypti and AUS-wMel Ae. aegypti regarding fertility and fecundity. However further experiments are required to assess it accurately. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated that the NC-wMel Ae. aegypti strain is a strong inhibitor of DENV, CHIKV, and ZIKV infection and prevents transmission of infectious viral particles in mosquito saliva. Furthermore, our NC-wMel Ae. aegypti strain induces reproductive cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, supporting field-releases in Noumea, NC