Regulation of PI3K signaling in T-cell acute lymphoblastic leukemia: a novel PTEN/Ikaros/miR-26b mechanism reveals a critical targetable role for PIK3CD.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy, and T-ALL patients are prone to early disease relapse and suffer from poor outcomes. The PTEN, PI3K/AKT and Notch pathways are frequently altered in T-ALL. PTEN is a tumor suppressor that inactivates the PI3K pathway. We profiled miRNAs in Pten-deficient mouse T-ALL and identified miR-26b as a potentially dysregulated gene. We validated decreased expression levels of miR-26b in mouse and human T-ALL cells. In addition, expression of exogenous miR-26b reduced proliferation and promoted apoptosis of T-ALL cells in vitro, and hindered progression of T-ALL in vivo. Furthermore, miR-26b inhibited the PI3K/AKT pathway by directly targeting PIK3CD, the gene encoding PI3Kδ, in human T-ALL cell lines. ShRNA for PIK3CD and CAL-101, a PIK3CD inhibitor, reduced the growth and increased apoptosis of T-ALL cells. Finally, we showed that PTEN induced miR-26b expression by regulating the differential expression of Ikaros isoforms that are transcriptional regulators of miR-26b. These results suggest that miR-26b functions as a tumor suppressor in the development of T-ALL. Further characterization of targets and regulators of miR-26b may be promising for the development of novel therapies.

First Report of Ascochyta Blight Caused by QoI-Resistant Isolates of Ascochyta rabiei in Chickpea Fields of Nebraska and South Dakota.

Ascochyta blight, caused by Ascochyta rabiei, is a serious disease of chickpea (Cicer arietinum) and fungicide applications are used to manage the disease in the North Central plains (4). During the 2010 growing season, a commercial field near Stanley, SD was treated with pyraclostrobin (Headline, BASF, NC) and called a management failure by the grower. Similarly, limited efficacy of pyraclostrobin was observed in an ascochyta research trial near Scott's Bluff, NE. In both locations, symptoms and signs consistent with A. rabiei infection existed on leaves, stems, and pods; namely, circular brown lesions with concentric rings of dark brown pycnidia. Symptomatic samples were collected, disinfected with 95% ethanol for 1 min, rinsed with sterile water, placed in 0.5% NaOCl for 1 min, and rinsed again with sterile water for 1 min (4). Samples were air dried, placed on potato dextrose agar (PDA) plates for 3 to 7 days, and colonies with morphological characteristics typical of A. rabiei were single-spored and transferred to new PDA plates and incubated for 7 to 14 days. Three and six putative A. rabiei isolates were obtained from South Dakota and Nebraska samples, respectively. Morphological characteristics were consistent with A. rabiei; cultures were brown with concentric rings of dark, pear-shaped pycnidia with an ostiole, and conidia were hyaline, single-celled, and oval-shaped (2). Comparison of the internal transcribed spacer (ITS) region amplified from the genomic DNA of 3-day-old liquid cultures using ITS4/ITS5 primers by BLASTN searches using the nr database in GenBank (Accession Number FJ032643) also confirmed isolates to be A. rabiei. Mismatch amplification mutation assay (MAMA) PCR was used for detection of sensitive and resistant isolates to QoI fungicides (1). Confirmation of the presence of the G143A mutation was carried out by cloning an mRNA fragment of the cytochrome b gene using cDNA synthesized from total RNA of A. rabiei and CBF1/CBR2 (1,3). Total RNA was extracted from 3-day-old liquid cultures and it was used instead of genomic DNA for this PCR to avoid large intronic regions commonly present in mitochondrial genes. The G143A mutation has previously been correlated with resistance to QoI fungicides in other fungal plant pathogens (3). Also, these isolates were determined to be QoI-resistant in vitro by PDA amended with a discriminatory dose of 1 µg/ml of azoxystrobin (4). To our knowledge, this is the first report of QoIresistant A. rabiei isolates causing infections on chickpeas in South Dakota and Nebraska. QoI-resistant isolates were reported in North Dakota and Montana in 2005 and 2007, respectively (4). Of nearly 300 isolates collected from these states from 2005 and 2007, approximately 65% were determined to be QoI resistant (4). The widespread occurrence of QoIresistant isolates and reduction of fungicide performance in fields led the North Dakota State University Cooperative Extension Service to actively discourage the use of QoI fungicides on chickpeas in North Dakota and Montana (4). It is likely that similar recommendations will need to be adopted in South Dakota and Nebraska for profitable chickpea production. References: (1) J. A. Delgado, 2012 Ph.D. Diss. Department of Plant Pathology, North Dakota State University. (2) R. M. Harveson et al. 2011. Online. Plant Health Progress doi:10.1094/PHP-2011-0103-01-DG. (3) Z. Ma et al. Pestic. Biochem. Physiol. 77:66, 2003. (4) K. A. Wise et al. Plant Dis. 93:528, 2009.

Characterization and Pathogenicity of Rhizoctonia solani Isolates Affecting Pisum sativum in North Dakota.

Acreage of dry field pea (Pisum sativum) in North Dakota has increased approximately eightfold from the late 1990s to the late 2000s to over 200,000 ha annually. A coincidental increase in losses to root rots has also been observed. Root rot in dry field pea is commonly caused by a complex of pathogens which included Fusarium spp. and Rhizoctonia solani. R. solani isolates were obtained from roots sampled at the three- to five-node growth stage from North Dakota pea fields and from symptomatic samples received at the Plant Diagnostic Lab at North Dakota State University in 2008 and 2009. Using Bayesian inference and maximum likelihood analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), 17 R. solani pea isolates were determined to belong to anastomosis group (AG)-4 homogenous group (HG)-II and two isolates to AG-5. Pathogenicity of select pea isolates was determined on field pea and two rotation hosts, soybean and dry bean. All isolates caused disease on all hosts; however, the median disease ratings were higher on green pea, dry bean, and soybean cultivars when inoculated with pea isolate AG-4 HG-II. Identification of R. solani AGs and subgroups on field pea and determination of relative pathogenicity on rotational hosts is important for effective resistance breeding and appropriate rotation strategies.

First Report of Ascochyta Blight of Field Pea Caused by Ascochyta pisi in South Dakota.

Tan lesions approximately 1.7 × 0.8 cm with distinct dark brown margins and small pycnidia were observed on leaves of field peas (Pisum sativum L. 'Agassiz') growing in Campbell County, South Dakota (45°45.62'N, 100°9.13'W) in July 2008. Small pieces of symptomatic leaves were surface sterilized (10% NaOCl for 1 min, 70% EtOH for 1 min, and sterile distilled H2O for 2 min) and placed on potato dextrose agar (PDA) for 7 days under fluorescent lights with a 12-h photoperiod to induce sporulation. A pure culture was established by streaking a conidial suspension on PDA and isolating a single germinated spore 3 days later. The culture was grown on clarified V8 media for 10 days. Conidia were 10 to 16 × 3 to 4.5 µm and uniseptate with a slightly constricted septum, similar to those of Ascochyta pisi Lib. The exuding spore mass from pycnidia growing on the medium was carrot red. No chlamydospores or pseudothecia were observed (1,2). To confirm the identity of A. pisi, DNA was extracted from the lyophilized mycelium of the 10-day-old culture with the DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Internal transcribed spacer (ITS) regions I and II were amplified with PCR primers ITS 5 and ITS 4 (3). PCR amplicons were cleaned and directly sequenced in both directions using the primers. A BLASTN search against the NCBI nonredundant nucleotide database was performed using the consensus sequence generated by alignment of the forward and reverse sequences for this region. The consensus sequence (GenBank Accession No. GU722316) most closely matched A. pisi var. pisi strain (GenBank Accession No. EU167557). These observations confirm the identity of the fungus as A. pisi. A suspension of 1 × 106 conidia/ml of the isolate was spray inoculated to runoff on 10 replicate plants of 2-week-old, susceptible green field pea 'Sterling'. Plants were incubated in a dew chamber for 48 h at 18°C and moved to the greenhouse bench where they were maintained at 20 to 25°C with a 12-h photoperiod for 1 week. Tan lesions with dark margins appeared 7 days after inoculation and disease was assessed after 10 days (4). No symptoms were observed on water-treated control plants. A. pisi was reisolated from lesions and confirmed by DNA sequencing of the ITS region, fulfilling Koch's postulates. Currently, states bordering South Dakota (North Dakota and Montana) lead the United States in field pea production. Although acreage is limited in South Dakota, the identification of A. pisi in this region is serious. The disease is yield limiting and foliar fungicides are used for disease management (1). To our knowledge, this is the first report of Ascochyta blight on P. sativum caused by A. pisi occurring in South Dakota and the MonDak production region (the Dakotas and Montana). References: (1) T. W. Bretag et al. Aust. J. Agric. Res. 57:88, 2006. (2) A. S. Lawyer. Page 11 in: The Compendium of Pea Diseases. D. J. Hagedorn, ed. The American Phytopathological Society, St Paul, MN, 1984. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990. (4) J. M. Wroth. Can. J. Bot. 76:1955, 1998.

Spinal hamartoma in a neonate: Unusual presentation and MR imaging findings.

We report an extremely rare case of a congenital spinal cord hamartoma in a male neonate who presented with upper extremity weakness and a port wine stain on the right upper extremity and chest. MR imaging findings are described, and the importance of localizing the lesion in the spinal canal with respect to the dura and its impact on neurosurgical management is stressed.