New method for arbuscular mycorrhizal fungus spore separation using a microfluidic device based on manual temporary flow diversion.

Arbuscular mycorrhizal fungi are beneficial components often included in biofertilizers. Studies of the biology and utilization of these fungi are key to their successful use in the biofertilizer industry. The acquisition of isolated spores is a required step in these studies; however, spore quality control and spore separation are bottlenecks. Filtered and centrifuged spores have to be hand-picked under a microscope. The conventional procedure is skill-demanding, labor-intensive, and time-consuming. Here, we developed a microfluidic device to aid manual separation of spores from a filtered and centrifuged suspension. The device is a single spore streamer equipped with a manual temporary flow diversion (MTFD) mechanism to select single spores. Users can press a switch to generate MTFD when the spore arrives at the selection site. The targeted spore flows in a stream to the collection chamber via temporary cross flow. Using the device, spore purity, the percentage of spore numbers against the total number of particles counted in the collecting chamber reached 96.62% (median, n = 10) which is greater than the spore purity obtained from the conventional method (88.89% (median, n = 10)).

Gain-of-function mutation in PTPN11 in histiocytic sarcomas of Bernese Mountain Dogs.

Histiocytic sarcoma (HS) is an aggressive malignant neoplasm of dendritic cell origin that is common in certain breeds of dogs. High prevalence of fatal, disseminated HS has been described in Bernese Mountain Dogs (BMDs). Support for genetic predisposition to develop HS has been presented in several studies, but to date, causative genetic events have not been reported. In addition, no driver mutations have been identified in tumours. Recently, E76K gain-of-function mutation in SHP2 encoded by the PTPN11 gene has been described in human histiocytic malignancies. In our study, we identified the PTPN11E76K in HS of BMDs. Amplification of exon 3 of the PTPN11 gene followed by Sanger sequencing was used to detect the mutation and estimate the prevalence in HS from 30 BMDs, 13 Golden Retrievers and 10 other dog breeds. The overall prevalence of PTPN11E76K in HS of BMDs was 36.67% compared with 8.69% in other breeds. No mutation was identified in normal tissues from 10 BMDs with HS that carried the mutation and 12 control dogs with no neoplastic disease, including 6 BMDs. Increased immunoreactivity for AKT, phosphorylated ERK1/2 and phosphorylated AKT in a small subset of BMDs with PTPN11E76K suggests that a gain-of-function might be mediated by the ERK and AKT pathways. These data suggest PTPN11E76K as an important driver mutation of HS in BMDs. This information may not only aid in unravelling the tumourigenic events associated with HS in BMDs, but also help in identifying more promising therapeutic strategies.

AE1/AE3, vimentin and p63 immunolocalization in canine mammary gland tumours: roles in differentiation between luminal epithelial and myoepithelial lineages.

Mammary tumors are by far the most common tumors in female dogs and effective treatment relies on prompt and accurate diagnostic procedures. Canine mammary tumors may originate from various cell types, such as luminal epithelial, myoepithelial and stromal cells. This study aimed to differentiate luminal epithelial and myoepithelial lineages, using specific markers including AE1/AE3, Vimentin, and p63. Such data can be useful for prognosis. Canine mammary tumors were collected by surgical resection and tissue samples were investigated using the avidin-biotin-immunoperoxidase method with used primary antibodies against AE1/AE3, vimentin, and p63. Luminal epithelial-origin tumors were found to be immunoreactive with AE1/AE3 and vimentin monoclonal antibody, while myoepithelial-origin tumors were positive for p63 and vimentin . In addition, canine mixed tumors showed reactivity with all three antibodies. In summary, AE1/AE3, p63 and vimentin can be used as specific immunohistochemical markers to distinguish lumino-epithelial and myoepithelial lineages of canine mammary tumors.