Mitochondrial Genome Sequence of Echinostoma revolutum from Red-Crowned Crane (Grus japonensis)

Echinostoma revolutum is a zoonotic food-borne intestinal trematode that can cause intestinal bleeding, enteritis, and diarrhea in human and birds. To identify a suspected E. revolutum trematode from a red-crowned crane (Grus japonensis) and to reveal the genetic characteristics of its mitochondrial (mt) genome, the internal transcribed spacer (ITS) and complete mt genome sequence of this trematode were amplified. The results identified the trematode as E. revolutum. Its entire mt genome sequence was 15,714 bp in length, including 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and one non-coding region (NCR), with 61.73% A+T base content and a significant AT preference. The length of the 22 tRNA genes ranged from 59 bp to 70 bp, and their secondary structure showed the typical cloverleaf and D-loop structure. The length of the large subunit of rRNA (rrnL) and the small subunit of rRNA (rrnS) gene was 1,011 bp and 742 bp, respectively. Phylogenetic trees showed that E. revolutum and E. miyagawai clustered together, belonging to Echinostomatidae with Hypoderaeum conoideum. This study may enrich the mitochondrial gene database of Echinostoma trematodes and provide valuable data for studying the molecular identification and phylogeny of some digenean trematodes.

Establishment of a T(m)-shift Method for Detection of Cat-Derived Hookworms

Melting temperature shift (T(m)-shift) is a new detection method that analyze the melting curve on real-time PCR thermocycler using SYBR Green I fluorescent dye. To establish a T(m)-shift method for the detection of Ancylostoma ceylanicum and A. tubaeforme in cats, specific primers, with GC tail of unequal length attached to their 5′ end, were designed based on 2 SNP loci (ITS101 and ITS296) of the internal transcribed spacer 1 (ITS1) sequences. The standard curve of T(m)-shift was established using the standard plasmids of A. ceylanicum (AceP) and A. tubaeforme (AtuP). The T(m)-shift method stability, sensitivity, and accuracy were tested with reference to the standard curve, and clinical fecal samples were also examined. The results demonstrated that the 2 sets of primers based on the 2 SNPs could accurately distinguish between A. ceylanicum and A. tubaeforme. The coefficient of variation (CV) of T(m)-values of AceP and AtuP was 0.07% and 0.06% in ITS101 and was 0.06% and 0.08% in ITS296, respectively. The minimum detectable DNA concentration was 5.22×10⁻⁶ and 5.28×10⁻⁶ ng/μl samples of AceP and AtuP, respectively. The accuracy of T(m)-shift method reached 100% based on examination of 10 hookworm DNA samples with known species. In the clinical detection of hookworm in 69 stray cat fecal sample, the T(m)-shift detection results were consistent with the microscopic examination and successfully differentiated between the 2-hookworm species. In conclusion, the developed method is a rapid, sensitive and accurate technique and can provide a promising tool for clinical detection and epidemiological investigation of cat-derived hookworms.

PRDM1 expression and its relationship with PI3K/AKT pathway activation in extranodal NK/T cell lymphoma-nasal type / 中华血液学杂志

Objective@#To investigate the expression of PRDM1 and its relationship with PI3K/AKT pathway activation in extranodal NK/T cell lymphoma-nasal type.@*Methods@#Immunocytochemistry and Western blot were used to detect the expression of PRDM1 and p-AKT in 10 EN-NK/T-NT tissue or 3 cell lines (PRDM1-positive YT cell line, PRDM1-negative NKL and NK92 cell lines). Nanostring gene expression profiling technique was used to detect the activation of the PI3K/AKT pathway in normal nasal mucosa, PRDM1-negative and positive EN-NK/T-NT tissue. MTS was used to detect cell proliferation, and flow cytometry was used to detect cell cycle and apoptosis.@*Results@#Nanostring gene expression profiling revealed that genes associated with PI3K/AKT signaling pathway (eg, IL-7, BRCA1, ITGA8, IL2RB, FASLG, CDK2, COL27A1, CSF3R, KITLG and IL-6) were highly expressed in EN-NK/T-NT cases (P<0.05). Also, we found that p-AKT was highly expressed in YT cell line, but lower or not expressed in NK92 and NKL cells. In addition, LY294002, a PI3K/AKT pathway inhibitor, increased PRDM1 and PTEN expression in a dose dependent manner in YT cells. More importantly, YT cell were treated with 20 μmol/L LY294002 48 h, the proliferation rate was significantly decreasing (58.18% vs 100.00%, t=12.770, P=0.006), and the proportion of cells in G1 phase was significantly increased (30.05% vs 76.93%, t=11.570, P<0.001). However, there was no significant difference in cell proliferation and cell cycle between NKL cells and control group (P>0.05).@*Conclusion@#The activation of PI3K/AKT pathway is positive associated with the expression of PRDM1 in EN-NK/T-NT, and inhibition of PI3K/AKT pathway may have significant therapeutic potential for PRDM1-positive EN-NK/T-NT.