FOXO3 is a latent tumor suppressor for FOXO3-positive and cytoplasmic-type gastric cancer cells

FOXO3 is a member of the FOXO transcription factors thought to play a tumor-suppressor role in gastrointestinal cancer, while tumor-promoting function of FOXO3 has also been reported. These results suggest a context-dependent function of FOXO3 in tumor development. However, the relationship between the FOXO3 expression pattern and its role in tumorigenesis has not been elucidated. We examined the FOXO3 expression in 65 human primary gastric cancer and patient-derived xenograft tissues by immunohistochemistry and identified three subtypes according to subcellular localization: FOXO3-nuclear accumulated (FOXO3-Nuc), FOXO3-nuclear/cytoplasmic or cytoplasmic distributed (FOXO3-Cyt), and FOXO3-negative. In the FOXO3-Cyt gastric cancer cells, the expression of the constitutive active mutant FOXO3 (Act-ER FOXO3) induced the nuclear accumulation of FOXO3 and significantly suppressed colony formation and proliferation. The inhibition of the PI3K-AKT pathway by inhibitor treatment also suppressed the proliferation of FOXO3-Cyt gastric cancer cells, which was associated with the nuclear accumulation of endogenous FOXO3. Furthermore, the expression of Act-ER FOXO3 by an endogenous promoter significantly suppressed gastric tumorigenesis in Gan mice, a model of gastric cancer. Finally, treatment of FOXO3-Cyt human gastric cancer-derived organoids with an AKT inhibitor significantly suppressed the survival and proliferation. These results indicate that FOXO3 is a latent tumor suppressor for FOXO3-Cyt-type gastric cancer cells and that activation of the PI3K-AKT pathway protects this type of gastric cancer cell from FOXO3-mediated growth suppression via constitutive nuclear export. Thus, the inhibition of the PI3K-AKT pathway and nuclear translocation of endogenous FOXO3 may have therapeutic applications in the treatment of FOXO3-positive and cytoplasmic-type gastric cancer.

Physicochemical and biological compatibility of insecticide mixtures with acaricide in the management of Brevipalpus yothersi

Citrus leprosis, caused by Citrus leprosis virus cytoplasmic type (CiLV-C), is one of the major citrus diseases. Such disease is mainly managed by controlling the vector, which is a mite of the genus Brevipalpus (Acari: Tenuipalpidae). To increase pest control spectrum and reduce costs, citrus growers often prefer to tank mix pesticides. However, the effect of pesticide combinations made up of insecticides and acaricides is little known yet. Therefore, our goal was to evaluate the physicochemical compatibility of spray mixtures with acaricides (spirodiclofen, propargite, and cyflumetofen) and the most commonly used insecticides in citrus orchards, as well as to evaluate their biological effect on Brevipalpus yothersi (Baker). Mixing insecticides with the acaricides such as spirodiclofen, propargite, and cyflumetofen had no interfere with the physicochemical stability of tank mixtures. However, the combination of imidacloprid, bifenthrin, cypermethrin, and phosmet with spirodiclofen reduced acaricide control efficiency in 20.9%, 18.9%, 9.7%, and 21.9%, respectively. These mixtures are not recommended for B. yothersi control.

KI-67 INDEX AND TYPE OF HSP70 EXPRESSION IN THE FORMATION OF PROGNOSTIC GROUPS OF PATIENTS WITH PAPILLARY THYROID CANCER

The aim of our work was to determine the possibility of stratification of papillary thyroid cancer (PTC) patients into prognosis groups depending on the Ki-67 index and the type of HSP70 expression. The study included 89 patients with a known prognosis for the period of 2005-2017, treated for PTC over the period of 2005-2013. The immunohistochemical study was carried out with Ki-67 and HSP70 antibodies on the Ventana XT Steiner device using standard protocols. The method of tissue matrices was used to standardize the study. Different variants of PTC, depending on the level of Ki-67 expression in combination with the HSP70 response allowed identifying 3 groups of PTC prognosis. The favorable prognosis group included patients with samples with cytoplasmic type of HSP70 expression and Ki-67 response < 6.3%, with mixed type of HSP70 expression and Ki-67 response < 6.3%, with cytoplasmic HSP70 expression and Ki-67 response > 6.3%. The intermediate prognosis group consisted of patients with tumors with mixed type of HSP70 expression and Ki-67 response > 6.3%, with nuclear HSP70 expression and Ki-67 response < 6.3%. The group of poor prognosis included patients with nuclear HSP70 expression and Ki-67 response > 6.3%. In the group of favorable prognosis, the 10-year survival rate was 89.6 ± 3.5%. In the intermediate prognosis group, the 10-year survival rate was 45 ± 14.5% and in the unfavorable prognosis group, no patient survives 6 years.

History and Diversity of Citrus leprosis virus Recorded in Herbarium Specimens

Leprosis refers to two diseases of citrus that present similar necrotic local lesions, often surrounded by chlorotic haloes on citrus. Two distinct viruses are associated with this disease, one that produces particles primarily in the nucleus of infected plant cells (Citrus leprosis virus nuclear type [CiLV-N]; Dichorhavirus) and another type that produces particles in the cytoplasm of infected plant cells (Citrus leprosis virus cytoplasmic type [CiLV-C]; Cilevirus). Both forms are transmitted by Brevipalpid mites and have bipartite, single-stranded, RNA genomes. CiLV-C and CiLV-N are present in South and Central America and as far north as parts of Mexico. Although leprosis disease was originally described from Florida, it disappeared from there in the 1960s. The United States Department of Agriculture–Agricultural Research Service maintains preserved citrus specimens identified at inspection stations 50 or more years ago with symptoms of citrus leprosis. We isolated RNA from these samples and performed degradome sequencing. We obtained nearly full-length genome sequences of both a typical CiLV-C isolate intercepted from Argentina in 1967 and a distinct CiLV-N isolate obtained in Florida in 1948. The latter is a novel form of CiLV-N, not known to exist anywhere in the world today. We have also documented the previously unreported presence of CiLV-N in Mexico in the mid-20th century.

First Report of Citrus leprosis virus Nuclear Type in Sweet Orange in Colombia

Colombia is ranked 18th in the world in citrus production and contributed 0.9% of the total world share. Among four important citrus-producing regions of Colombia, the Orinoco region (3 to 6°N, 68 to 74°W) consists of two citrus-producing states, Meta and Casanare. Citrus leprosis is the most important viral disease of citrus in Colombia (1,3). Three types of Citrus leprosis virus (CiLV) infect citrus, producing leprosis-like lesion symptoms. Two of the three CiLV species, Citrus leprosis virus cytoplasmic type (CiLV-C) and cytoplasmic type 2 (CiLV-C2), produce particles only in the cytoplasm (3). The other species, Citrus leprosis virus nuclear type (CiLV-N), produces particles in both the cytoplasm and nucleus (4). CiLV-C is more prevalent and destructive while CiLV-N has been reported only in Brazil, Panama, and Mexico (4). Interestingly, both CiLV-C and -C2 were reported from the same regions of Meta and Casanare States in Colombia in 2004 and 2012 (1,3). CiLV-C lesions are usually rounded (initially 2 to 3 mm in diameter and extending up to 30 mm), have dark-brown or greenish central chlorotic spots, and are surrounded by yellow halos. CiLV-N lesions have been described as smaller in size and form three well-defined regions including a necrotic center with an intermediate orange color halo and an outer chlorotic halo (2). In 2013, ‘Valencia’ sweet orange (Citrus sinensis L.) leaves with suspected CiLV-N symptoms were collected from 8 plants in Casanare State and shipped under permit to the USDA-APHIS-PPQ-CPHST, Beltsville, MD. Total RNA from symptomatic and healthy sweet orange leaves were extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). RT-PCR primers specific to CiLV-C, CiLV-C2 (3), and CiLV-N nucleocapsid (N) (CiLV-N-NPF: 5′-ATGGCTAACCCAAGTGAGATCGATTA-3′; CiLV-N-NPR: 5′-AGTTGCCTTGAGATCATCACATTGGT-3′) and putative matrix protein (M) genes (CiLV-N-MF: 5′-ATGTCTAAACAGATTAATATGTGCACTGTG-3′; CiLV-N-MR: 5′-CTAACCACTGGGTCCCGC-3′) were utilized to identify the CiLV associated with the leprosis-affected leaf samples from Casanare. RT-PCR with CiLV-C primers failed to produce any amplicon, but CiLV-N primers successfully amplified the partial N gene (681 bp) and entire M gene (552 nt) amplicons from multiple leaves of all leprosis samples. In addition, a 795-bp amplicon specific to CiLV-C2 also was amplified from the CiLV-N suspected samples. Similar results were obtained when the vector, flat spider mite (Brevipalpus spp.) total RNA was used as template for RT-PCR. For further confirmation, each amplicon was cloned and sequenced. Sequencing of the N and M gene amplicons of CiLV-N (accession nos. KJ195893 and KJ195894) and coat protein gene of CiLV-C2 showed 97 to 99% nucleotide sequence identity with the CiLV-N M2345 isolate sequence (KF209275) from Mexico (4) and CiLV-C2 L147V1 isolate sequence (JX000024) from Colombia (3), respectively. Phylogenetic analyses of these N and M protein gene sequences confirmed a mixed infection of the same plant with two viruses, one from an unassigned new genus Dichorhavirus (CiLV-N) and another from genus Cilevirus (CiLV-C2). This is the first report of CiLV-N in Colombia, and also the first report of an occurrence of CiLV-N in mixed infection with CiLV-C2. All three known species of CiLV occur in the Orinoco region of Colombia. References: (1) M. G. León et al. Plant Dis. 90: 682, 2006. (2) J. P. R. Marques et al. Anais da Academia Brasileira de Ciências 82:501, 2010. (3) A. Roy et al. Phytopathology 103:488, 2013. (4) A. Roy et al. Genome Announc. 1(4): e00519-13, 2013.

In vivolocalization of antibodies raised againstEimeria maximawall forming bodies during sexual intracellular development

SUMMARYApicomplexan parasites cause devastating diseases in humans and livestock. Previously we demonstrated that antibodies targeting transmissible forms of the apicomplexan parasite,Eimeria, are effective at reducing parasite shedding thus preventing the transmission of the disease. However, the mechanisms responsible have not been fully defined. Moreover, there is no direct evidence that the parasite-specific IgG antibodies can reach the parasite developing in the enterocytes of the infected chicken host. This study summarizes our efforts using host immunity, parasite proteomics and 3D microscopy to provide a step forward in our understanding of how this immune response works.Eimeria maximais an important pathogen of poultry and used as a surrogate for a number of human pathogens includingToxoplasmaandPlasmodium. Our studies demonstrate that immunization with the purified wall forming bodies (WFBs) results in a production of parasite-specific IgG antibodies, which have the ability to reachin situgametocytes in the intestinal lumen and permeate the enterocyte/parasite membranes in order to bind to the cytoplasmic Type 1 and Type 2 WFBs. This raises the intriguing possibility that via this process antibodies block the development ofEimeria maxima in vivo.

Common Bean: Experimental Indicator Plant for Citrus leprosis virus C and Some Other Cytoplasmic-Type Brevipalpus-Transmitted Viruses

Citrus leprosis (CL) caused by Citrus leprosis virus C (CiLV-C) is present in Latin America from Mexico to Argentina, where citrus plants are grown. CiLV-C is transmitted by the tenuipalpid mite, Brevipalpus phoenicis, causing localized lesions on citrus leaves, fruit, and stems. One limitation to study of the virus–vector–host relationship in this pathosystem is the lack of a suitable assay plant. On Citrus spp. used as susceptible hosts, symptoms may take weeks or months to appear after experimental inoculation by viruliferous mites. Common bean (Phaseolus vulgaris) was found to respond with localized necrotic lesions after inoculation with viruliferous B. phoenicis in 5 days. Thus far, 113 tested common bean varieties and lines and some recent accessions of varied genetic background behaved in a similar way. Black bean ‘IAC Una’ was adopted as a standard test variety. When inoculated leaves were left at 28 to 30°C, the period for the lesion appearance was reduced to only 2 days. Confirmation that the lesions on common bean leaves are caused by CiLV-C were made by transmission electron microscopy, immunofluorescence, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction specific for CiLV-C. Common bean plants mite-inoculated with some other cytoplasmic-type Brevipalpus-transmitted viruses (BrTVs) (Passion fruit green spot virus, Solanum violaefolium ringspot virus, Ligustrum ringspot virus, and Hibiscus green spot virus) also responded with necrotic local lesions and may serve as test plants for these viruses. Two nuclear types of BrTV (Coffee ringspot virus and Clerodendrum chlorotic spot virus) were unable to produce symptoms on common bean.

Comparative morpho-anatomical studies of the lesions caused by citrus leprosis virus on sweet orange

The leprosis disease shows a viral etiology and the citrus leprosis virus is considered its etiologic agent. The disease may show two types of cytopatologic symptom caused by two virus: nuclear (CiLV-N) and cytoplasmic (CiLV-C) types. The aim of this study was to compare the morpho-anatomical differences in the lesions caused by leprosis virus-cytoplasmic and nuclear types in Citrus sinensis (L.) Osbeck 'Pêra'. Leaf and fruit lesions were collected in Piracicaba/São Paulo (cytoplasmic type) and Monte Alegre do Sul/São Paulo and Amparo/São Paulo (nuclear type). The lesions were photographed and then fixed in Karnovsky solution, dehydrated in a graded ethylic series, embedded in hydroxy-ethyl methacrylate resin (Leica Historesin), sectioned (5 μm thick), stained and mounted in synthetic resin. The digital images were acquired in a microscope with digital video camera. Leaf and fruit lesions caused by the two viruses were morphologically distinct. Only the lesion caused by CiLV-N virus presented three well-defined regions. In both lesions there was the accumulation of lipidic substances in necrotic areas that were surrounded by cells with amorphous or droplets protein. Only leaf and fruit lesions caused by CiLV-N virus exhibited traumatic gum ducts in the vascular bundles.

A new source of cytoplasmic male sterility found in wild beet and its relationship to other CMS types

We found a number of male-sterile plants in a wild beet ( Beta vulgaris L. subsp. maritima) accession line, FR4-31. The inheritance study of the male sterility indicated the trait to be of the cytoplasmic type. The mitochondrial genome of FR4-31 proved to lack the male-sterility-associated genes preSatp6 and orf129, which are characteristic of the Owen CMS and I-12CMS(3) cytoplasms of beets, respectively. Instead, the truncated cox2 gene involved in G CMS originating from wild beets was present in the FR4-31 mitochondrial genome. In Southern hybridization using four mitochondrial gene probes, the FR4-31 cytoplasm showed patterns similar to those typical of the G cytoplasm. It is thus likely that the FR4-31 cytoplasm has a different CMS mechanism from both Owen CMS and I-12CMS(3), and that the FR4-31 and G cytoplasms resemble each other closely. A restriction map of the FR4-31 mitochondrial DNA was generated and aligned with those published for the Owen and normal fertile cytoplasms. The FR4-31 mitochondrial genome was revealed to differ extensively in arrangement from the Owen and normal genomes, and the male-sterile Owen and FR4-31 genomes seem to be derived independently from an ancestral genome.