Taxonomic notes and new species of <i>Burmomiles </i>and <i>Sanaungulus</i> (Coleoptera, Cantharidae) from northern Myanmar during the late Mesozoic

In this study, Poinarelektronmiles Fanti & Damgaard, 2020 is considered as a junior synonym of Burmomiles Fanti, Damgaard & Ellenberger, 2018, since no generic diagnostic differences can be found in their type species except for the elytral length, which is a yet unstable and more ecology-related character. The two hitherto known species of Poinarelektronmiles are transferred to Burmomiles or Sanaungulus Fanti, Damgaard & Ellenberger, 2018, including B. ellenbergeri (Fanti & Damgaard, 2020) comb. nov. and S. cuaroni (Bramanti & Fanti, 2022) comb. nov. Meanwhile, B. dominikiweissbachi (Fanti & Müller, 2022) comb. nov., B. kachinensis (Fanti & Müller, 2022) comb. nov. and B. lethi (Fanti & Damgaard, 2020) comb. nov. are transferred from Sanaungulus. Six Sanaungulus species are suggested to be placed in Cantharidae incertae sedis, including S. electrum Fanti & Müller, 2022, S. franziskaeweissbachae Fanti & Müller, 2022, S. nalae Fanti & Müller, 2022, S. morellii Fanti & Damgaard, 2020, S. rosenzweigi Fanti & Damgaard, 2020 and S. ruicheni (Hsiao & Huang, 2018), due to their absence of antennal appendages in males. The gender identity for S. kirstenaeweissbachae Fanti & Müller, 2022 and S. cuaroni originally defined as females are corrected into males, according to their pectinate antennae. Additionally, four new species, S. marginalis sp. nov., S. longicornis sp. nov., S. elongaticollis sp. nov., and S. undecimus sp. nov., are described and illustrated. These results will significantly complement and expand our knowledge on the Burmite cantharid diversity.</p

<i>Ex vivo</i> expansion of CB CD34⁺ cells over 14 days.

<p>The effect of <i>ex vivo</i> expansion of HIF1α/KD cells on CB CD34⁺ cells was assayed according to the methods described above.</p>*<p>p<0. 05 versus CTRL cells (<i>n</i> = 6) (Student’s <i>t</i>-test).</p

<i>Ex vivo</i> expansion of CB CD34⁺ cells over 14 days.

<p>The cells were stained with PE-conjugated mAb to CD34 and FITC-conjugated mAb to CD38, and analyzed by flow cytometry. Values indicate the fold increase compared with the initial number of cells (2.0×10⁴/well).The results are given as mean ± standard deviation (SD) (<i>n</i> = 6). ^*p<0. 05 versus the CTRL cells (Student’s <i>t</i>-test).</p

The effect of <i>miR-17</i> modulation in FBMOB-hTERT cells on CB CD34⁺ cells.

<p><b>A:</b> The effect of <i>miR-17</i> modulation in FBMOB-hTERT cells on long-term culture initiating cells activity of CB CD34⁺ cells. 1.0×10⁴ CD34⁺ CB cells were co-cultured with FBMOB-hTERT cells after transduced with vectors for <i>miR-17</i> overexpression (17/OE), <i>miR-17</i> knockdown (17/KD), or control (CTRL) for 5-8 weeks and then subject to colony-forming-unit (CFU) assay. After 14-16 days of culture, the colonies, including CFU-Mixs, CFU-E, and BFU-E with greater than 50 cells were counted. The results are expressed as mean ± SD (n = 6). *p<0.05, compared between 17/OE or 17/KD, and CTRL group (Student’s <i>t</i>-test). <b>B:</b> Effect of <i>miR-17</i> modulation in FBMOB-hTERT cells on repopulation of CB CD34⁺ cells in non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. 5.0×10⁴CB CD34⁺ cells were co-cultured with 17/OE, 17/KD or CTRL, harvested at 4 weeks of culture and then injected intravenously into the sublethally irradiated NOD/SCID mice (n = 6 per group). The mice were sacrificed 12 weeks after transplantation and the mononuclear cells from bone marrow were analyzed for human cells composed of CD45⁺, CD45^–CD36⁺ and CD36^–GPA⁺ cells and CD45⁺CD34⁺ population by flow cytometry. The level of total human cell engraftment was shown in the left panel. P = 0.031 or 0.046, compared between the NOD/SCID mice injected with CD34⁺ cells co-cultured with 17/OE or 17/KD and those injected with CD34⁺ cells co-cultured with CTRL (Student’s <i>t</i>-test). The fraction of CD45⁺, erythroid (CD45⁻CD36⁺ and CD36⁻GPA⁺) and CD45⁺CD34⁺ population among the engrafted human cells was shown in right panel. *p<0.05, compared between the NOD/SCID mice injected with CD34⁺ cells co-cultured with 17/OE and those injected with CD34⁺ cells co-cultured with CTRL (Student’s <i>t</i>-test). The significant difference was only analyzed between the mice injected with CD34⁺ cells co-cultured with 17/OE or 17/KD and those injected with CD34⁺ cells co-cultured with CTRL.</p

Effect of <i>miR-17</i> modulation on repopulation of CD34⁺ cells in primary and secondary NOD/SCID mice.

<p>A: Flow cytometry analysis of the human CB CD34⁺ cell repopulation in a representative primary NOD/SCID mouse after co-cultured with 17/OE, 17/KD or CTRL cells. Fresh CD34⁺ cells were served as controls. The mononuclear cells from bone marrow harvested from the engrafted NOD/SCID mice were examined by flow cytometry for the assessment of human cells composed of CD45⁺ cells (R1) and erythroid cells including CD45^–CD36⁺ (R2) and CD36^–GPA⁺ (R3) population and CD45⁺CD34⁺ cells (R4). <b>B:</b> The bone marrow mononuclear cells (MNCs) containing the different percentage of human cells (lanes 2-5) from the primary representative engrafted mice were analyzed for human-specific 17α-satellite DNA by PCR. The human-specific 17α-satellite gene was detected when the human cells were over 0.70% (lanes 3–6) whereas it was indetectable at a percentage of 0.12% (lane 2). Lane 1, one mouse without transplants; lane 2, one mouse receiving transplants of fresh CD34⁺ cells; lane 3, one mouse receiving transplants of CD34⁺ cells co-cultured with 17/KD cells; lane 4, one mouse receiving transplants of CD34⁺ cells co-cultured with CTRL cells; lane 5, one mouse receiving transplants of CD34⁺ cells co-cultured with 17/OE cells; lane 6 indicates positive control (human peripheral blood (PB) MNCs). <b>C:</b> Effect of <i>miR-17</i> modulation in FBMOB-hTERT cells on repopulation of CB CD34⁺ cells in secondary NOD/SCID mice. Human cells were analyzed in secondary mice 12 weeks after intravenous injection of bone marrow cells from primary mice, which were injected with CB CD34⁺ cells co-cultured with 17/OE or CTRL for 4 weeks and sacrificed 8 weeks after transplantation. P = 0.026, compared between 1 and 2 (1, bone marrow mononuclear cells in secondary mice from primary mice injected with CB CD34⁺ cells co-cultured with 17/OE; 2, bone marrow mononuclear cells in secondary mice from primary mice injected with CB CD34⁺ cells co-cultured with CTRL) (Student’s <i>t</i>-test).</p

<i>MiR-17 </i>up-regulates <i>HIF-1α</i> expression upon interaction with CB CD34⁺ cells.

<p><b>A:</b> 1.0×10⁶ 17/OE or CTRL cells were co-cultured with CB CD34⁺ cells (1.0×10⁴/well) in six-well plates for 24 hours, and then washed twice to remove loosely adherent and non-adherent cells. The adherent cells were harvested and analyzed by real-time RT-PCR for transcripts of the niche associated genes: HIF-1α, SDF-1, KL and EPO. The results are expressed as mean ± SD (n = 3). *p<0.05, compared between 17/OE cells and CTRL cells (Student’s <i>t</i>-test). <b>B:</b> HIF-1α protein levels were measured by western blotting in 17/OE, 17/KD or CTRL cells after interaction with CB CD34⁺ cells for 24 hours. <b>C:</b> The transcripts of the niche associated genes: <i>HIF-1α</i>, <i>SDF-1</i>, <i>KL</i> and <i>EPO</i> were analyzed by real-time RT-PCR in 17/OE, 17/KD or CTRL cells. The results are expressed as mean ± SD (n = 3). Without CB CD34⁺ cell existing, the expression of the indicated niche associated genes was not changed significantly in 17/OE or 17/KD cells comparing with that in CTRL cells. <b>D.</b> The transcripts of the niche associated genes were analyzed by real-time RT-PCR in bone marrow stromal cells (BMSCs) with over-expressed <i>miR-17</i> and the control cells upon interaction with CB CD34⁺ cells. The expression of <i>HIF-1α</i> was up-regulated after <i>miR-17 </i>over-expressed whereas the expression of <i>SDF-1</i>, <i>KL</i> and <i>EPO</i> did not changed obviously. The results are expressed as mean ± SD (n = 3). <b>E:</b> Luciferase reporter assays to check whether <i>miR-17</i> directly target <i>HIF-1α</i> in FBMOB-hTERT. The luciferase activities were not significantly decreased in 17/OE cells compared with that in CTRL cells. The results are given as mean ± standard deviation (SD) (<i>n</i> = 3).</p

HIF-1α knockdown partially abrogates the hematopoietic supporting ability of osteoblastic <i>miR-17</i>.

<p><b>A:</b> Western blotting was performed to evaluate the expression level of HIF-1α protein in 17/OE cells after transduced with vectors for HIF1α knockdown (HIF1α/KD), or control (CTRL). <b>B:</b> The effect of the HIF1α/KD cells on long-term culture initiating cells activity of CB CD34⁺ cells. 1.0×10⁴ CD34⁺ CB cells were co-cultured with HIF1α/KD cells or CTRL for 5-8 weeks and then subject to CFU assay. After 14–16 days of culture, the colonies, including CFU-Mixs, CFU-E, and BFU-E with greater than 50 cells were counted. The results are expressed as mean ± SD (n = 6). *p<0.05, compared between HIF1α/KD and CTRL group (Student’s <i>t</i>-test). <b>C:</b> Effect of HIF1α/KD cells on repopulation of CB CD34⁺ cells in non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. 5.0×10⁴CB CD34⁺ cells were co-cultured with HIF1α/KD or CTRL cells, harvested at 4 weeks of culture and then injected intravenously into the sublethally irradiated NOD/SCID mice (n = 6 per group). The mice were sacrificed 12 weeks after transplantation and the mononuclear cells from bone marrow were analyzed for human cells composed of CD45⁺, CD45^–CD36⁺ and CD36^–GPA⁺ cells and CD45⁺CD34⁺ population by flow cytometry. The level of total human cell engraftment was shown in the left panel. p = 0.046, compared between the mice injected with CD34⁺ cells co-cultured with HIF1α/KD and those injected with CD34⁺ cells co-cultured with CTRL (Student’s <i>t</i>-test). The fraction of CD45⁺, erythroid (CD45⁻CD36⁺ and CD45⁻ CD36⁻GPA⁺) and CD45⁺CD34⁺ cells among the engrafted human cells was shown in the right panel. *p<0.05, compared between the mice injected with CD34⁺ cells co-cultured with HIF1α/KD and those injected with CD34⁺ cells co-cultured with CTRL (Student’s <i>t</i>-test). The significant difference was only analyzed between the mice injected with CD34⁺ cells co-cultured with HIF1α/KD and those injected with CD34⁺ cells co-cultured with CTRL.</p

The expression of <i>miR-17</i> in FBMOB-hTERT cells.

<p><b>A:</b> The expression level of <i>miR-17</i> in FBMOB-hTERT cells was evaluated by real-time RT-PCR. Each reaction was performed in triplicate. The data are presented as the ratio of <i>miR-17</i> levels (relative to U6) in FBMOB-hTERT to that in bone marrow stromal cells (BMSCs). <b>B:</b> Real-time RT-PCR was performed to evaluate the expression level of <i>miR-17</i> in FBMOB-hTERT cells after retrovirally transduced with vectors for <i>miR-17</i> overexpression (17/OE), <i>miR-17</i> knockdown (17/KD or 17/KD1), or control (CTRL). The data are presented as the ratio of <i>miR-17</i> levels (relative to U6) in 17/OE, FBMOB-hTERT, 17/KD or 17/KD1 to that in CTRL. Each reaction was performed in triplicate.</p

Repopulation of CD34⁺ cells after co-cultured with HIF1α/KD in primary and secondary NOD/SCID mice.

<p><b>A:</b> Flow cytometry analysis of the human CD34⁺ cell repopulation in a representative primary NOD/SCID mouse after co-cultured with HIF1α/KD or CTRL. Fresh CD34⁺ cells were served as controls. The mononuclear cells from bone marrow harvested from the primary injected NOD/SCID mice were examined by flow cytometry for the assessment of human cells composed of CD45⁺ cells (R1) and erythroid cells including CD45^–CD36⁺ (R2) and CD36^–GPA⁺ population (R3) and CD45⁺CD34⁺ cells (R4). <b>B:</b> The bone marrow mononuclear cells (MNCs) containing the different percentage of human cells (lanes 2-5) from the primary representative engrafted mice were analyzed for human-specific 17α-satellite DNA by PCR. The human-specific 17α-satellite gene was detected when the human cells were over 0.50% (lanes 3-5), whereas it was indetectable at a percentage of 0.12% (lanes 2). Lane 1, one mouse without transplants; lanes 2-3, two mice receiving transplants of fresh CD34⁺ cells; lane 4, one mouse receiving transplants of CD34⁺ cells co-cultured with HIF1α/KD cells; lane 5, one mouse receiving transplants of CD34⁺ cells co-cultured with CTRL cells. <b>C:</b> Effect of HIF-1α knockdown in 17/OE cells on repopulation of CB CD34⁺ cells in secondary NOD/SCID mice. Human cells were analyzed in secondary mice 12 weeks after intravenous injection of bone marrow cells from primary mice, which were injected with CB CD34⁺ cells co-cultured with HIF1α/KD or CTRL for 4 weeks and sacrificed 8 weeks after transplantation. The percentage of total human cell or erythroid cells in the secondary BM from the primary recipient transplanted with CD34⁺ cells co-cultured with HIF1α/KD was significant lower than that from the primary recipient transplanted with CD34⁺ cells co-cultured with CTRL (p = 0.032 and 0.038 respectively) (Student’s <i>t</i>-test).</p

Additional file 2: Figure S2. of miR-17 promotes expansion and adhesion of human cord blood CD34+ cells in vitro

(A) Flow cytometry analysis of the expression of GFP on CB CD34+CD38−/CD38+cells upon miR-17 modulation after culturing for 20 days (red line) and control cells (black line). (B). The expression of N-cadherin and β1-integrin on CB CD34+ cells after miR-17 knockdown (17/KD) or control cells (CTRL) was analyzed by flow cytometry (left panels). The results are expressed as mean ± SD from multiple independent experiments (right panels). (TIFF 318 kb