Corn360: a method for quantification of corn kernels.

BACKGROUND: The rapidly advancing corn breeding field calls for high-throughput methods to phenotype corn kernel traits to estimate yield and to study their genetic inheritance. Most of the existing methods are reliant on sophisticated setup, expertise in statistical models and programming skills for image capturing and analysis. RESULTS: We demonstrated a portable, easily accessible, affordable, panoramic imaging capturing system called Corn360, followed by image analysis using freely available software, to characterize total kernel count and different patterned kernel counts of a corn ear. The software we used did not require programming skills and utilized Artificial Intelligence to train a model and to segment the images of mixed patterned corn ears. For homogeneously patterned corn ears, our results showed accuracies of 93.7% of total kernel count compared to manual counting. Our method allowed to save an average of 3 min 40 s per image. For mixed patterned corn ears, our results showed accuracies of 84.8% or 61.8% of segmented kernel counts. Our method has the potential to greatly decrease counting time per image as the number of images increases. We also demonstrated a case of using Corn360 to count different categories of kernels on a mixed patterned corn ear resulting from a cross of sweet corn and sticky corn and showed that starch:sweet:sticky segregated in a 9:4:3 ratio in its F2 population. CONCLUSIONS: The panoramic Corn360 approach enables for a portable low-cost high-throughput kernel quantification. This includes total kernel quantification and quantification of different patterned kernels. This can allow for quick estimate of yield component and for categorization of different patterned kernels to study the inheritance of genes controlling color and texture. We demonstrated that using the samples resulting from a sweet × sticky cross, the starchiness, sweetness and stickiness in this case were controlled by two genes with epistatic effects. Our achieved results indicate Corn360 can be used to effectively quantify corn kernels in a portable and cost-efficient way that is easily accessible with or without programming skills.

Comparison of meiotic transcriptomes of three maize inbreds with different origins reveals differences in cell cycle and recombination.

BACKGROUND: Cellular events during meiosis can differ between inbred lines in maize. Substantial differences in the average numbers of chiasmata and double-strand breaks (DSBs) per meiotic cell have been documented among diverse inbred lines of maize: CML228, a tropical maize inbred line, B73 and Mo17, temperate maize lines. To determine if gene expression might explain these observed differences, an RNA-Seq experiment was performed on CML228 male meiocytes which was compared to B73 and Mo17 male meiocytes, where plants were grown in the same controlled environment. RESULTS: We found that a few DSB-repair/meiotic genes which promote class I crossovers (COs) and the Zyp1 gene which limits newly formed class I COs were up-regulated, whereas Mus81 homolog 2 which promotes class II COs was down-regulated in CML228. Although we did not find enriched gene ontology (GO) categories directly related to meiosis, we found that GO categories in membrane, localization, proteolysis, energy processes were up-regulated in CML228, while chromatin remodeling, epigenetic regulation, and cell cycle related processes including meiosis related cell cycle processes were down-regulated in CML228. The degree of similarity in expression patterns between the three maize lines reflect their genetic relatedness: B73 and Mo17 had similar meiotic expressions and CML228 had a more distinct expression profile. CONCLUSIONS: We found that meiotic related genes were mostly conserved among the three maize inbreds except for a few DSB-repair/meiotic genes. The findings that the molecular players in limiting class I CO formation (once CO assurance is achieved) were up-regulated and those involved in promoting class II CO formation were down-regulated in CML228 agree with the lower chiasmata number observed in CML228 previously. In addition, epigenetics such as chromatin remodeling and histone modification might play a role. Transport and energy-related processes was up-regulated and Cyclin13 was down-regulated in CML228. The direction of gene expression of these processes agree with that previously found in meiotic tissues compared with vegetative tissues. In summary, we used different natural maize inbred lines from different climatic conditions and have shown their differences in expression landscape in male meiocytes.

High-resolution crossover mapping reveals similarities and differences of male and female recombination in maize.

Meiotic crossovers (COs) are not uniformly distributed across the genome. Factors affecting this phenomenon are not well understood. Although many species exhibit large differences in CO numbers between sexes, sex-specific aspects of CO landscape are particularly poorly elucidated. Here, we conduct high-resolution CO mapping in maize. Our results show that CO numbers as well as their overall distribution are similar in male and female meioses. There are, nevertheless, dissimilarities at local scale. Male and female COs differ in their locations relative to transcription start sites in gene promoters and chromatin marks, including nucleosome occupancy and tri-methylation of lysine 4 of histone H3 (H3K4me3). Our data suggest that sex-specific factors not only affect male-female CO number disparities but also cause fine differences in CO positions. Differences between male and female CO landscapes indicate that recombination has distinct implications for population structure and gene evolution in male and in female meioses.

Genomic features shaping the landscape of meiotic double-strand-break hotspots in maize.

Meiotic recombination is the most important source of genetic variation in higher eukaryotes. It is initiated by formation of double-strand breaks (DSBs) in chromosomal DNA in early meiotic prophase. The DSBs are subsequently repaired, resulting in crossovers (COs) and noncrossovers (NCOs). Recombination events are not distributed evenly along chromosomes but cluster at recombination hotspots. How specific sites become hotspots is poorly understood. Studies in yeast and mammals linked initiation of meiotic recombination to active chromatin features present upstream from genes, such as absence of nucleosomes and presence of trimethylation of lysine 4 in histone H3 (H3K4me3). Core recombination components are conserved among eukaryotes, but it is unclear whether this conservation results in universal characteristics of recombination landscapes shared by a wide range of species. To address this question, we mapped meiotic DSBs in maize, a higher eukaryote with a large genome that is rich in repetitive DNA. We found DSBs in maize to be frequent in all chromosome regions, including sites lacking COs, such as centromeres and pericentromeric regions. Furthermore, most DSBs are formed in repetitive DNA, predominantly Gypsy retrotransposons, and only one-quarter of DSB hotspots are near genes. Genic and nongenic hotspots differ in several characteristics, and only genic DSBs contribute to crossover formation. Maize hotspots overlap regions of low nucleosome occupancy but show only limited association with H3K4me3 sites. Overall, maize DSB hotspots exhibit distribution patterns and characteristics not reported previously in other species. Understanding recombination patterns in maize will shed light on mechanisms affecting dynamics of the plant genome.

Can the Otago falls prevention program be delivered by video? A feasibility study.

OBJECTIVES: We assessed the feasibility of delivering the Otago Exercise Programme (OEP) via an interactive DVD (ie, OEP-DVD) in combination with monthly physical therapist phone calls to older adults. DESIGN: This pre-post (baseline and 6-month follow-up) study included an intervention group (n=61) based in a rural location and a control group (n=21) based in a city. SETTING: Sechelt and Vancouver, British Columbia. PARTICIPANTS: 82 community-dwelling adults ≥75 years. INTERVENTION: Individuals in the intervention group received the OEP-DVD and were instructed to do the exercises 3 times a week after their initial home physical therapist visit for 6 months. PRIMARY AND SECONDARY OUTCOMES: Feasibility was ascertained by withdrawal rate and compliance to the OEP-DVD. The number of participants and the frequency (ie, number of times weekly) they performed the OEP exercises and walking were used to estimate compliance. The potential benefit of the OEP-DVD on falls risk profile (Physiological Profile Assessment (PPA)) and mobility were examined by comparing the change in the intervention group compared with the control group. Self-reported compliance to the exercise programme was assessed by monthly returned diary. RESULTS: Of the 82 participants, 2 withdrew from the OEP-DVD group and none withdrew from the control group. We obtained compliance data on 72% of participants in the intervention group. The mean OEP-DVD compliance was 87% and the mean walking compliance was 166%. After adjusting for baseline PPA, baseline age, sex, baseline comorbidities, baseline cognitive status and baseline falls-related self-efficacy, there was a significant between-group improvement in the overall PPA score (OEP group pre-PPA to post-PPA score: 0.79±1.2 to 0.7±0.9; p<0.05) at study completion. CONCLUSIONS: Although the OEP-DVD resulted in significant reductions in falls risk among community-dwelling older adults, there was a notable loss to follow-up limiting the feasibility of this approach.

Dissecting Plant Chromosomes by the Use of Ionizing Radiation.

Radiation treatment of genomes is used to generate chromosome breaks for numerous applications. This protocol describes the preparation of seeds and the determination of the optimal level of irradiation dosage for the creation of a radiation hybrid (RH) population. These RH lines can be used to generate high-resolution physical maps for the assembly of sequenced genomes as well as the fine mapping of genes. This procedure can also be used for mutation breeding and forward/reverse genetics.

The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015.

Since the year 2000, a concerted campaign against malaria has led to unprecedented levels of intervention coverage across sub-Saharan Africa. Understanding the effect of this control effort is vital to inform future control planning. However, the effect of malaria interventions across the varied epidemiological settings of Africa remains poorly understood owing to the absence of reliable surveillance data and the simplistic approaches underlying current disease estimates. Here we link a large database of malaria field surveys with detailed reconstructions of changing intervention coverage to directly evaluate trends from 2000 to 2015, and quantify the attributable effect of malaria disease control efforts. We found that Plasmodium falciparum infection prevalence in endemic Africa halved and the incidence of clinical disease fell by 40% between 2000 and 2015. We estimate that interventions have averted 663 (542-753 credible interval) million clinical cases since 2000. Insecticide-treated nets, the most widespread intervention, were by far the largest contributor (68% of cases averted). Although still below target levels, current malaria interventions have substantially reduced malaria disease incidence across the continent. Increasing access to these interventions, and maintaining their effectiveness in the face of insecticide and drug resistance, should form a cornerstone of post-2015 control strategies.

Mitochondrial dynamics and the cell cycle.

Nuclear-mitochondrial (NM) communication impacts many aspects of plant development including vigor, sterility, and viability. Dynamic changes in mitochondrial number, shape, size, and cellular location takes place during the cell cycle possibly impacting the process itself and leading to distribution of this organelle into daughter cells. The genes that underlie these changes are beginning to be identified in model plants such as Arabidopsis. In animals disruption of the drp1 gene, a homolog to the plant drp3A and drp3B, delays mitochondrial division. This mutation results in increased aneuploidy due to chromosome mis-segregation. It remains to be discovered if a similar outcome is observed in plants. Alloplasmic lines provide an opportunity to understand the communication between the cytoplasmic organelles and the nucleus. Examples of studies in these lines, especially from the extensive collection in wheat, point to the role of mitochondria in chromosome movement, pollen fertility and other aspects of development.

Accelerated evolution of the mitochondrial genome in an alloplasmic line of durum wheat.

BACKGROUND: Wheat is an excellent plant species for nuclear mitochondrial interaction studies due to availability of large collection of alloplasmic lines. These lines exhibit different vegetative and physiological properties than their parents. To investigate the level of sequence changes introduced into the mitochondrial genome under the alloplasmic condition, three mitochondrial genomes of the Triticum-Aegilops species were sequenced: 1) durum alloplasmic line with the Ae. longissima cytoplasm that carries the T. turgidum nucleus designated as (lo) durum, 2) the cytoplasmic donor line, and 3) the nuclear donor line. RESULTS: The mitochondrial genome of the T. turgidum was 451,678 bp in length with high structural and nucleotide identity to the previously characterized T. aestivum genome. The assembled mitochondrial genome of the (lo) durum and the Ae. longissima were 431,959 bp and 399,005 bp in size, respectively. The high sequence coverage for all three genomes allowed analysis of heteroplasmy within each genome. The mitochondrial genome structure in the alloplasmic line was genetically distant from both maternal and paternal genomes. The alloplasmic durum and the Ae. longissima carry the same versions of atp6, nad6, rps19-p, cob and cox2 exon 2 which are different from the T. turgidum parent. Evidence of paternal leakage was also observed by analyzing nad9 and orf359 among all three lines. Nucleotide search identified a number of open reading frames, of which 27 were specific to the (lo) durum line. CONCLUSIONS: Several heteroplasmic regions were observed within genes and intergenic regions of the mitochondrial genomes of all three lines. The number of rearrangements and nucleotide changes in the mitochondrial genome of the alloplasmic line that have occurred in less than half a century was significant considering the high sequence conservation between the T. turgidum and the T. aestivum that diverged from each other 10,000 years ago. We showed that the changes in genes were not limited to paternal leakage but were sufficiently significant to suggest that other mechanisms, such as recombination and mutation, were responsible. The newly formed ORFs, differences in gene sequences and copy numbers, heteroplasmy, and substoichiometric changes show the potential of the alloplasmic condition to accelerate evolution towards forming new mitochondrial genomes.

Physical mapping resources for large plant genomes: radiation hybrids for wheat D-genome progenitor Aegilops tauschii.

BACKGROUND: Development of a high quality reference sequence is a daunting task in crops like wheat with large (~17Gb), highly repetitive (>80%) and polyploid genome. To achieve complete sequence assembly of such genomes, development of a high quality physical map is a necessary first step. However, due to the lack of recombination in certain regions of the chromosomes, genetic mapping, which uses recombination frequency to map marker loci, alone is not sufficient to develop high quality marker scaffolds for a sequence ready physical map. Radiation hybrid (RH) mapping, which uses radiation induced chromosomal breaks, has proven to be a successful approach for developing marker scaffolds for sequence assembly in animal systems. Here, the development and characterization of a RH panel for the mapping of D-genome of wheat progenitor Aegilops tauschii is reported. RESULTS: Radiation dosages of 350 and 450 Gy were optimized for seed irradiation of a synthetic hexaploid (AABBDD) wheat with the D-genome of Ae. tauschii accession AL8/78. The surviving plants after irradiation were crossed to durum wheat (AABB), to produce pentaploid RH1s (AABBD), which allows the simultaneous mapping of the whole D-genome. A panel of 1,510 RH1 plants was obtained, of which 592 plants were generated from the mature RH1 seeds, and 918 plants were rescued through embryo culture due to poor germination (<3%) of mature RH1 seeds. This panel showed a homogenous marker loss (2.1%) after screening with SSR markers uniformly covering all the D-genome chromosomes. Different marker systems mostly detected different lines with deletions. Using markers covering known distances, the mapping resolution of this RH panel was estimated to be <140kb. Analysis of only 16 RH lines carrying deletions on chromosome 2D resulted in a physical map with cM/cR ratio of 1:5.2 and 15 distinct bins. Additionally, with this small set of lines, almost all the tested ESTs could be mapped. A set of 399 most informative RH lines with an average deletion frequency of ~10% were identified for developing high density marker scaffolds of the D-genome. CONCLUSIONS: The RH panel reported here is the first developed for any wild ancestor of a major cultivated plant species. The results provided insight into various aspects of RH mapping in plants, including the genetically effective cell number for wheat (for the first time) and the potential implementation of this technique in other plant species. This RH panel will be an invaluable resource for mapping gene based markers, developing a complete marker scaffold for the whole genome sequence assembly, fine mapping of markers and functional characterization of genes and gene networks present on the D-genome.

Formal feasibility studies in palliative care: why they are important and how to conduct them.

The concept of clinical trial feasibility is of great interest to the community of palliative care researchers, clinicians, and granting agencies. Significant allocation of resources is required in the form of funding, time, intellect, and motivation to carry out clinical research, and understandably, clinical investigators, institutions, and granting agencies are disappointed when funded trials are unsuccessfully conducted. We argue that for many trials conducted in palliative care, the feasibility of conducting the proposed trial should be formally explored before implementation. There is substantial information available within the literature on the topic of study feasibility but no singular guide on how one can pragmatically apply this advice in the palliative care setting. We suggest that a Formal Feasibility Study for palliative care trials should be commonly conducted before development of a larger pivotal trial, to prospectively identify barriers to research, develop strategies to address these barriers, and predict whether the larger study is feasible. If a Formal Feasibility Study is not required, elements of feasibility can be specifically tested before launching clinical trials. The purpose of this article is to offer a draft framework for the design and conduct of a Formal Feasibility Study that, if implemented, could concretely support successful completion of high-quality research in a timely fashion. Additionally, we hope to foster dialogue within the palliative care research community regarding the relevance of establishing feasibility before initiation of definitive trials in the palliative care population.

[Transmission dynamics and cost-effectiveness of rabies control in dogs and humans in an African city]. / Dynamique de transmission et coût de la lutte contre la rage chez les chiens et les hommes dans une ville Africaine.

Control of human rabies in developing countries depends on prevention in dogs. The purpose of this study was to evaluate the cost-saving potential for the public health sector of intervention to control rabies in animal-host reservoirs. An existing deterministic model was adapted to allow study of dog-to-human rabies transmission. Model parameters were fitted to data from routine weekly reports on the number of rabid dogs and human rabies exposures in N'Djamena, Chad. At the onset of study, the estimated effective reproductive ratio (Re) was 1.01 indicating stable low-level endemic rabies transmission. Simulations were performed to determine what effects mass vaccination and culling of dogs would have on the incidence of human rabies. Findings showed that a mass campaign allowing single parenteral vaccination of at least 70% of the canine population would be sufficient to interrupt transmission of rabies to humans for at least 6 years. The cost-effectiveness of mass dog vaccination was compared to that of "postexposure prophylaxis" (PEP) which would not reduce future human exposure. Results showed that a sustained 5-year PEP program together with a dog-vaccination campaign would be as cost-effective as PEP alone. Beyond a time-frame of 7 years, combining parenteral dog vaccination campaigns with human PEP appeared to be more cost-effective than human PEP alone.

Transmission dynamics of highly pathogenic avian influenza at Lake Constance (Europe) during the outbreak of winter 2005-2006.

Highly pathogenic avian influenza virus (HPAI) H5N1 poses a serious threat to domestic animals. Despite the large number of studies on influenza A virus in waterbirds, little is still known about the transmission dynamics, including prevalence, behavior, and spread of these viruses in the wild waterbird population. From January to April 2006, the HPAI H5N1 virus was confirmed in 82 dead wild waterbirds at the shores of Lake Constance. In this study, we present simple mathematical models to examine this outbreak and to investigate the transmission dynamics of HPAI in wild waterbirds. The population dynamics model of wintering birds was best represented by a sinusoidal function. This model was considered the most adequate to represent the susceptible compartment of the SIR model. The three transmission models predict a basic reproduction ratio (R (0)) with value of approximately 1.6, indicating a small epidemic, which ended with the migration of susceptible wild waterbirds at the end of the winter. With this study, we quantify for the first time the transmission of HPAI H5N1 virus at Lake Constance during the outbreak of winter 2005-2006. It is a step toward the improvement of the knowledge of transmission of the virus among wild waterbirds.

Transmission dynamics and economics of rabies control in dogs and humans in an African city.

Human rabies in developing countries can be prevented through interventions directed at dogs. Potential cost-savings for the public health sector of interventions aimed at animal-host reservoirs should be assessed. Available deterministic models of rabies transmission between dogs were extended to include dog-to-human rabies transmission. Model parameters were fitted to routine weekly rabid-dog and exposed-human cases reported in N'Djaména, the capital of Chad. The estimated transmission rates between dogs (beta(d)) were 0.0807 km2/(dogs x week) and between dogs and humans (beta(dh)) 0.0002 km2/(dogs x week). The effective reproductive ratio (R(e)) at the onset of our observations was estimated at 1.01, indicating low-level endemic stability of rabies transmission. Human rabies incidence depended critically on dog-related transmission parameters. We simulated the effects of mass dog vaccination and the culling of a percentage of the dog population on human rabies incidence. A single parenteral dog rabies-mass vaccination campaign achieving a coverage of least 70% appears to be sufficient to interrupt transmission of rabies to humans for at least 6 years. The cost-effectiveness of mass dog vaccination was compared to postexposure prophylaxis (PEP), which is the current practice in Chad. PEP does not reduce future human exposure. Its cost-effectiveness is estimated at US $46 per disability adjusted life-years averted. Cost-effectiveness for PEP, together with a dog-vaccination campaign, breaks even with cost-effectiveness of PEP alone after almost 5 years. Beyond a time-frame of 7 years, it appears to be more cost-effective to combine parenteral dog-vaccination campaigns with human PEP compared to human PEP alone.

Efficacy and safety evaluation of human reovirus type 3 in immunocompetent animals: racine and nonhuman primates.

PURPOSE: Human reovirus type 3 has been proposed to kill cancer cells with an activated Ras signaling pathway. The purpose of this study was to investigate the efficacy of reovirus in immunocompetent glioma animal models and safety/toxicity in immunocompetent animals, including nonhuman primates. EXPERIMENTAL DESIGN: Racine glioma cells 9L and RG2 were implanted s.c. or intracranially in Fisher 344 rats with or without reovirus antibodies, followed by treatment of reovirus. To study whether reovirus kills contralateral tumors in the brain and to determine viral distribution, we established an in situ dual tumor model followed by reovirus intratumoral inoculation only into the ipsilateral tumor. To evaluate neurotoxicity/safety of reovirus, Cynomolgus monkeys and immunocompetent rats were given intracranially with reovirus, and pathological examination and/or behavioral studies were done. Viral shedding and clinical biochemistry were systematically studied in monkeys. RESULTS: Intratumorally given reovirus significantly suppressed the growth of both s.c. and intracranially tumors and significantly prolonged survival. The presence of reovirus-neutralizing antibodies did not abort the reovirus' antitumor effect. Reovirus inhibited glioma growth intracranially in the ipsilateral but not the contralateral tumors; viral load in ipsilateral tumors was 15 to 330-fold higher than the contralateral tumors. No encephalitis or behavioral abnormalities were found in monkeys and rats given reovirus intracranially. No treatment-related clinical biochemistry changes or diffuse histopathological abnormality were found in monkeys inoculated intracranially with Good Manufacturing Practice prepared reovirus. Microscopic changes were confined to the region of viral inoculation and were dose related, suggesting reovirus intracranially was well tolerated in nonhuman primates. CONCLUSIONS: These data show the efficacy and safety of reovirus when it is used in the treatment of gliomas in immunocompetent hosts. Inoculation of reovirus into the brain of nonhuman primates did not produce significant toxicities.

A retrospective study on the use of post-operative colonoscopy following potentially curative surgery for colorectal cancer in a Canadian province.

BACKGROUND: Surveillance colonoscopy is commonly recommended following potentially curative surgery for colorectal cancer. We determined factors associated with patients undergoing a least one colonoscopy within five years of surgery. METHODS: In this historical cohort study, data on 3918 patients age 30 years or older residing in Alberta, Canada, who had undergone a potentially curative surgical resection for local or regional stage colorectal cancer between 1983 and 1995 were obtained from the provincial cancer registry, ministry of health and cancer clinic charts. Kaplan-Meier estimates of the probability of undergoing a post-operative colonoscopy were calculated for patient, tumor and treatment-related variables of interest. RESULTS: A colonoscopy was performed within five years of surgery in 1979 patients. The probability of undergoing a colonoscopy for those diagnosed in the 1990s was greater than for those diagnosed earlier (0.65 vs 0.55, P < 0.0001). The majority of the difference was seen at one-year following surgery, consistent with changes in surveillance practices. Those most likely to undergo a colonoscopy were those under age 70 (0.74 vs 0.50 for those age 70-79, P < 0.0001), who underwent a pre-operative colonoscopy (0.69 vs 0.54, P < 0.0001), and who underwent a resection with reanastomosis (0.62 vs 0.47 for abdominoperineal resection, P < 0.0001) by a surgeon who performs colonoscopies (0.68 vs 0.54, P < 0.0001). CONCLUSIONS: The majority of patients undergo colonoscopy following colorectal cancer surgery. However, there are important variations in surveillance practices across different patient and treatment characteristics.

Altered subcellular localization and low frequency of mutations of ING1 in human brain tumors.

PURPOSE: Clinical and experimental evidence suggest that the p33ING1b candidate tumor suppressor functionally cooperates with p53 in controlling biochemical and biological functions. Because p53 is frequently mutated in brain tumors and the ING1 locus maps to a site of which the loss is associated with gliomas, we analyzed the mutation and expression profiles of ING1B in human brain tumors. Here we present the first report of ING1 expression and mutation analyses in human brain tumor samples and malignant glioma cell lines. EXPERIMENTAL DESIGN: Expression and mutation analyses of ING1B together with subcellular localization studies of ING1 proteins were performed on 29 brain tumor specimens and 6 human glioma cell lines. RESULTS: A single point mutation (3.5%) was detected in the 29 brain tumor specimens analyzed. This missense mutation occurred in a sequence reported previously to confer nuclear translocation properties to p33ING1b. Interestingly, overexpression and subcellular mislocalization of p33ING1b were observed in all 29 of the brain tumor specimens and some glioma cell lines. In tumor samples, ING1 proteins aberrantly localized to the cytoplasm, and to a lesser extent, to the nucleus of glioma cells. CONCLUSIONS: Our data indicate that although mutations of ING1 seem to be infrequent in human brain tumors, deregulated expression and mislocalization of ING1 proteins, particularly the p33ING1b isoform, are common events in gliomas and glioblastomas.

Reovirus prolongs survival and reduces the frequency of spinal and leptomeningeal metastases from medulloblastoma.

Medulloblastoma (MB), the most common pediatric brain tumor, is a highly malignant disease with a 5-year survival rate of only 60%. Tumor cells invade surrounding tissue and disseminate through cerebral spinal fluid, making treatment difficult. Human reovirus type 3 exploits an activated Ras pathway in tumor cells to support productive infection as an oncolytic virus. Here, we examined the ability of human reovirus to kill MB cells lines and surgical specimens in vitro and inhibit tumor growth/metastases in vivo. Most human MB cell lines tested (five of seven = 71.4%), two MB cell lines derived from spontaneously arising tumors in Patched-1(+/-) mice (two of two = 100%) and three MB primary cultures derived from surgical specimens, were susceptible to reovirus infection. Reovirus was internalized and transcribed in both susceptible and resistant cell lines. However, viral protein synthesis was restricted to cell lines with higher levels of activated Ras, suggesting that Ras plays a critical role in reovirus oncolysis in MB. Using an in vivo Daoy orthotopic animal model, we found that a single i.t. injection of reovirus dramatically prolonged survival compared with controls (160 versus 70 days, respectively; P = 0.0003). Repeating this experiment with GFP-labeled Daoy cells and multiple i.t. administrations of reovirus, we again found prolonged survival and a dramatic reduction in spinal and leptomeningeal metastases (66.7% in control injections versus 0.0% in the live virus group). These data suggest that this oncolytic virus may be a potentially effective novel therapy against human MB. Its ability to reduce metastases to the spinal cord could allow a reduction in the dose/field of total neuroaxis cerebral-spinal radiotherapy currently used to treat/prevent cerebral spinal fluid dissemination.

Investigation of laser cervical cone biopsies negative for premalignancy or malignancy.

OBJECTIVE: To measure the rate of carbon dioxide, laser cone biopsies negative for premalignancy or malignancy and determine whether the clinical indications were appropriate or the pathology evaluations were correct. MATERIALS AND METHODS: The patient charts of 95 negative cone biopsies were reviewed by one of the authors to determine the indications for the procedure. All of the slide reviews were done by two of the authors. Following a review of the cone biopsy slides, three deeper sections of the tissue blocks were examined in specimens that were still negative or equivocal for premalignancy. Thereafter, for those still negative the preconization, referral Pap tests, and colposcopic directed tissue samples were reviewed. RESULTS: The overall negative rate of laser cone biopsy was 28% (95/341) and 68% (65/95) were done to investigate high-grade squamous intraepithelial lesions (HGSIL) (cervical intraepithelial neoplasia [CIN] 2,3). There were 25 false negative cone biopsy specimens because of misinterpretation of the original slides or discovery of pathology in additional sections. False positive reporting of some preconization Pap tests or tissue specimens as premalignant when none were seen on review likely resulted in 11 unnecessary conizations. The number of negative cones would thereby be reduced by 36 for a rate of 17% (59/341). CONCLUSIONS: The negative rate could be reduced by 11% with routine deeper sectioning of the tissue blocks of the cone biopsy specimen and improved accuracy of pathological interpretation.

Exclusive amplification of cDNA template (EXACT) RT-PCR to avoid amplifying contaminating genomic pseudogenes.

Genomic DNA contamination within RNA samples has important implications for RT-PCR, particularly if there is a pseudogene related to the gene under investigation, because amplification from pseudogenes and reverse-transcribed cDNA can be very difficult to distinguish. Methods to remove DNA contamination cannot guarantee the absolute absence of DNA from the sample without a loss of RNA quantity or quality, which can be crucial for small amounts of RNA or for the investigation of transcripts with a low level of expression. Here, we describe a general technique for RT-PCR that applies a sequence to the 5' tail of reverse-transcribed cDNA that is not present in genomic DNA and uses this for annealing the reverse PCR primer to exclude genomic DNA amplification in unmodified RNA samples.