Identification of Fungi in the Botryosphaeriaceae Family Associated with Stem Blight of Vaccinium spp. in the Southeastern United States

Stem blight is a major disease of blueberry caused by Botryosphaeriaceae fungi. Chemical and cultural management options are limited, putting emphasis on breeding efforts to identify sources of resistance. The efficacy and durability of host resistance could be impacted by the species composition of the pathogen population in a region and by the isolates employed in the screenings used to identify the resistance. Samples (365) were collected from southern highbush (SHB) and rabbiteye blueberry (REB) cultivars from 28 sites in the southeastern US (AL, FL, GA, NC, and SC). Colony morphology identified 86% of the isolates as Botryosphaeriaceae. Conidia morphology and Maximum Likelihood analysis of the Internal Transcribed Spacer rDNA regions (ITS), translation elongation factor one alpha (tef1-α), and β-tubulin were used to identify isolates at genera or species level. A PCR-restriction fragment length polymorphism (PCR-RFLP) test was used to identify isolates to genus. Neofusicoccum and Lasiodiplodia were the predominant genera. N. kwambonambiense, N. ribis, L. theobromae and L. pseudotheobromae were the most common species isolated. Phylogenies conducted with a limited number of isolates indicated non-clonal and potentially diverse populations occur on blueberry that warrant additional study. Botryosphaeria corticis, B. dothidea, and Diplodia seriata were isolated infrequently.     

Genetic Control of Variegated KIR Gene Expression: Polymorphisms of the Bi-Directional KIR3DL1 Promoter Are Associated with Distinct Frequencies of Gene Expression

Natural killer (NK) cells play an important role in the detection and elimination of tumors and virus-infected cells by the innate immune system. Human NK cells use cell surface receptors (KIR) for class I MHC to sense alterations of class I on potential target cells. Individual NK cells only express a subset of the available KIR genes, generating specialized NK cells that can specifically detect alteration of a particular class I molecule or group of molecules. The probabilistic behavior of human KIR bi-directional promoters is proposed to control the frequency of expression of these variegated genes. Analysis of a panel of donors has revealed the presence of several functionally relevant promoter polymorphisms clustered mainly in the inhibitory KIR family members, especially the KIR3DL1 alleles. We demonstrate for the first time that promoter polymorphisms affecting the strength of competing sense and antisense promoters largely explain the differential frequency of expression of KIR3DL1 allotypes on NK cells. KIR3DL1/S1 subtypes have distinct biological activity and coding region variants of the KIR3DL1/S1 gene strongly influence pathogenesis of HIV/AIDS and other human diseases. We propose that the polymorphisms shown in this study to regulate the frequency of KIR3DL1/S1 subtype expression on NK cells contribute substantially to the phenotypic variation across allotypes with respect to disease resistance.     

Changes in the Expression of miR-381 and miR-495 Are Inversely Associated with the Expression of the MDR1 Gene and Development of Multi-Drug Resistance

Multidrug resistance (MDR) frequently develops in cancer patients exposed to chemotherapeutic agents and is usually brought about by over-expression of P-glycoprotein (P-gp) which acts as a drug efflux pump to reduce the intracellular concentration of the drug(s). Thus, inhibiting P-gp expression might assist in overcoming MDR in cancer chemotherapy. MiRNAome profiling using next-generation sequencing identified differentially expressed microRNAs (miRs) between parental K562 cells and MDR K562 cells (K562/ADM) induced by adriamycin treatment. Two miRs, miR-381 and miR-495, that were strongly down-regulated in K562/ADM cells, are validated to target the 3’-UTR of the MDR1 gene. These miRs are located within a miR cluster located at chromosome region 14q32.31, and all miRs in this cluster appear to be down-regulated in K562/ADM cells. Functional analysis indicated that restoring expression of miR-381 or miR-495 in K562/ADM cells was correlated with reduced expression of the MDR1 gene and its protein product, P-gp, and increased drug uptake by the cells. Thus, we have demonstrated that changing the levels of certain miR species modulates the MDR phenotype in leukemia cells, and propose further exploration of the use of miR-based therapies to overcome MDR.     

Effects of Caste on the Expression of Genes Associated with Septic Injury and Xenobiotic Exposure in the Formosan Subterranean Termite

As social insects, termites live in densely populated colonies with specialized castes under conditions conducive to microbial growth and transmission. Furthermore, termites are exposed to xenobiotics in soil and their lignocellulose diet. Therefore, termites are valuable models for studying gene expression involved in response to septic injury, immunity and detoxification in relation to caste membership. In this study, workers and soldiers of the Formosan subterranean termite, Coptotermes formosanus, were challenged by bacterial injection or by no-choice feeding with a sublethal concentration (0.5%) of phenobarbital. Constitutive and induced expression of six putative immune response genes (two encoding for lectin-like proteins, one for a ficolin-precursor, one for the Down syndrome cell adhesion molecule, one for a chitin binding protein, and one for the gram-negative binding protein 2) and four putative detoxification genes (two encoding for cytochrome P450s, one for glutathione S-transferase, and one for the multi antimicrobial extrusion protein), were measured via quantitative real time polymerase chain reaction and compared within and among 1) colonies, 2) treatment types and 3) castes via ANOVA. Eight genes were inducible by septic injury, feeding with phenobarbital or both. Colony origin had no effect on inducibility or differential gene expression. However, treatment type showed significant effects on the expression of the eight inducible genes. Caste effects on expression levels were significant in five of the eight inducible genes with constitutive and induced expression of most target genes being higher in workers than in soldiers.     

Loss of NAC1 Expression Is Associated with Defective Bony Patterning in the Murine Vertebral Axis

NAC1 encoded by NACC1 is a member of the BTB/POZ family of proteins and participates in several pathobiological processes. However, its function during tissue development has not been elucidated. In this study, we compared homozygous null mutant Nacc1^-/- and wild type Nacc1^+/+ mice to determine the consequences of diminished NAC1 expression. The most remarkable change in Nacc1^-/- mice was a vertebral patterning defect in which most knockout animals exhibited a morphological transformation of the sixth lumbar vertebra (L6) into a sacral identity; thus, the total number of pre-sacral vertebrae was decreased by one (to 25) in Nacc1^-/- mice. Heterozygous Nacc1^+/- mice had an increased tendency to adopt an intermediate phenotype in which L6 underwent partial sacralization. Nacc1^-/- mice also exhibited non-closure of the dorsal aspects of thoracic vertebrae T10-T12. Chondrocytes from Nacc1^+/+ mice expressed abundant NAC1 while Nacc1^-/- chondrocytes had undetectable levels. Loss of NAC1 in Nacc1^-/- mice was associated with significantly reduced chondrocyte migratory potential as well as decreased expression of matrilin-3 and matrilin-4, two cartilage-associated extracellular matrix proteins with roles in the development and homeostasis of cartilage and bone. These data suggest that NAC1 participates in the motility and differentiation of developing chondrocytes and cartilaginous tissues, and its expression is necessary to maintain normal axial patterning of murine skeleton.     

Differential Tropism and Replication Kinetics of Human Immunodeficiency Virus Type 1 Isolates in Thymocytes: Coreceptor Expression Allows Viral Entry, but Productive Infection of Distinct Subsets Is Determined at the Postentry Level

Human thymocytes are readily infected with human immunodeficiency virus type 1 (HIV-1) in vivo and in vitro. In this study, we found that the kinetics of replication and cytopathic effects of two molecular isolates, NL4-3 and JR-CSF, in postnatal thymocytes are best explained by the distribution of chemokine receptors used for viral entry. CXCR4 was expressed at high levels on most thymocytes, whereas CCR5 expression was restricted to only 0.1 to 2% of thymocytes. The difference in the amount of proviral DNA detected after infection of fresh thymocytes with NL4-3 or JR-CSF correlated with the levels of CXCR4 and CCR5 surface expression. Anti-CCR5 blocking studies showed that low levels of CCR5 were necessary and sufficient for JR-CSF entry in thymocytes. Interleukin-2 (IL-2), IL-4, and IL-7, cytokines normally present in the thymus, influenced the expression of CXCR4 and CCR5 on thymocytes and thus increased the infectivity and spread of both NL4-3 and JR-CSF in culture. NL4-3 was produced by both immature and mature thymocytes, whereas JR-CSF production was restricted to the mature CD1⁻/CD69⁺ population. Although CXCR4 and CCR5 distribution readily explained viral entry in mature CD69⁺ and immature CD69⁻ cells, and correlated with proviral DNA distribution, we found that viral production was favored in CD69⁺ cells. Therefore, while expression of CD4 and appropriate coreceptors are essential determinants of viral entry, factors related to activation and stage-specific maturation contribute to HIV-1 replication in thymocyte subsets. These results have direct implications for HIV-1 pathogenesis in pediatric patients.     

Increased Expression of the Spindle Checkpoint Protein BubR1 is Associated with High Cell Proliferation in Primary Gastrointestinal Diffuse Large B Cell Lymphoma

Primary gastrointestinal diffuse large B cell lymphoma (PGI-DLBCL) is a relatively rare malignancy with limited results on clinical characteristics and carcinogenesis. Chromosome instability (CIN) is a hallmark of cancer cells, including lymphoma. As a key component of spindle assembly checkpoint (SAC), BubR1 plays a crucial role in maintaining genome stability. To elucidate the roles of BubR1 in the pathogenesis of PGI-DLBCL, and its relationship to cell proliferation, Helicobacter pylori infection, and BCL-6 gene translocation, we examined the possible alterations of BubR1 protein expression in PGI-DLBCL patients. Paraffin-embedded cancer tissues from 20 PGI-DLBCL patients were evaluated for BubR1 and H. pylori expression by immunohistochemistry, as well as cell proliferative activity measured by Ki-67 proliferation index (PI). BCL-6 gene rearrangement was assessed by fluorescence in situ hybridization (FISH) and BubR1 expression status was compared with clinicopathological parameters in PGI-DLBCL patients. Overexpression of BubR1 was observed in the majority of PGI-DLBCL patients. The mean expression level of BubR1 was 57.33 ± 23.27 % in PGI-DLBCL, which was higher than normal gastrointestinal tissue (10.18 ± 5.65 %) and reactive lymph node (26.74 ± 8.60 %) (P < 0.01), while it was comparable to nodal lymphoma (54.32 ± 21.28 %) (P > 0.05). BubR1 overexpression had a positive correlation with Ki-67 proliferation index (PI) (r = 0.51, P < 0.01) in PGI-DLBCL, but had no relationship to H. pylori infection and BCL-6 gene translocation. In addition, no correlation was found between BubR1 expression levels and overall survivals (P > 0.05). BubR1 overexpression was associated with cell proliferation and may play a role in the carcinogenesis of PGI-DLBCL. Aberrant BubR1 expression may potentially be a biomarker for estimating biologic characteristics of PGI-DLBCL.     

Distinct subfamilies of primate L1Gg retroposons, with some elements carrying tandem repeats in the 5'' region.

Two subfamilies of L1 elements, differing dramatically in the first 1.2 kb of sequence at their 5' ends, were identified in the prosimian primate, Galago garnetti. Interesting patterns of sequence similarity were observed between the galago subfamilies, and with the L1s from human and from another prosimian, the slow loris. Furthermore, members of one of the subfamilies have six to eight tandemly repeated units of 73 bp, starting about 730 bp from their 5' ends. Such tandem repeats have not been reported in other primate L1s, but a striking sequence similarity was found between the galago tandem repeats and those previously described at the 5' termini of some mouse L1s [Loeb, D. D. et al. Mol. Cell. Biol. 6, 168-182, 1986]. Although the similar sequence indicates a shared, conserved function, the galago repeats are sub-terminal and therefore cannot serve as portable RNA polymerase II promoters, as has been suggested for the mouse tandem repeats.