Computer-Generated Ovaries to Assist Follicle Counting Experiments

Precise estimation of the number of follicles in ovaries is of key importance in the field of reproductive biology, both from a developmental point of view, where follicle numbers are determined at specific time points, as well as from a therapeutic perspective, determining the adverse effects of environmental toxins and cancer chemotherapeutics on the reproductive system. The two main factors affecting follicle number estimates are the sampling method and the variation in follicle numbers within animals of the same strain, due to biological variability. This study aims at assessing the effect of these two factors, when estimating ovarian follicle numbers of neonatal mice. We developed computer algorithms, which generate models of neonatal mouse ovaries (simulated ovaries), with characteristics derived from experimental measurements already available in the published literature. The simulated ovaries are used to reproduce in-silico counting experiments based on unbiased stereological techniques; the proposed approach provides the necessary number of ovaries and sampling frequency to be used in the experiments given a specific biological variability and a desirable degree of accuracy. The simulated ovary is a novel, versatile tool which can be used in the planning phase of experiments to estimate the expected number of animals and workload, ensuring appropriate statistical power of the resulting measurements. Moreover, the idea of the simulated ovary can be applied to other organs made up of large numbers of individual functional units

Suppression of Jasmonic Acid-Dependent Defense in Cotton Plant by the Mealybug Phenacoccus solenopsis

The solenopsis mealybug, Phenacoccus solenopsis, has been recently recognized as an aggressively invasive pest in China, and is now becoming a serious threat to the cotton industry in the country. Thus, it is necessary to investigate the molecular mechanisms employed by cotton for defending against P. solenopsis before the pest populations reach epidemic levels. Here, we examined the effects of exogenous jasmonic acid (JA), salicylic acid (SA), and herbivory treatments on feeding behavior and on development of female P. solenopsis. Further, we compared the volatile emissions of cotton plants upon JA, SA, and herbivory treatments, as well as the time-related changes in gossypol production and defense-related genes. Female adult P. solenopsis were repelled by leaves from JA-treated plant, but were not repelled by leaves from SA-treated plants. In contrast, females were attracted by leaves from plants pre-infested by P. solenopsis. The diverse feeding responses by P. solenopsis were due to the difference in volatile emission of plants from different treatments. Furthermore, we show that JA-treated plants slowed P. solenopsis development, but plants pre-infested by P. solenopsis accelerated its development. We also show that P. solenopsis feeding inhibited the JA-regulated gossypol production, and prevented the induction of JA-related genes. We conclude that P. solenopsis is able to prevent the activation of JA-dependent defenses associated with basal resistance to mealybugs

Large-Scale Phylogenetic Analysis of Emerging Infectious Diseases

Microorganisms that cause infectious diseases present critical issues of national security, public health, and economic welfare.  For example, in recent years, highly pathogenic strains of avian influenza have emerged in Asia, spread through Eastern Europe and threaten to become pandemic. As demonstrated by the coordinated response to Severe Acute Respiratory Syndrome (SARS) and influenza, agents of infectious disease are being addressed via large-scale genomic sequencing.  The goal of genomic sequencing projects are to rapidly put large amounts of data in the public domain to accelerate research on disease surveillance, treatment, and prevention. However, our ability to derive information from large comparative genomic datasets lags far behind acquisition.  Here we review the computational challenges of comparative genomic analyses, specifically sequence alignment and reconstruction of phylogenetic trees.  We present novel analytical results on from two important infectious diseases, Severe Acute Respiratory Syndrome (SARS) and influenza.SARS and influenza have similarities and important differences both as biological and comparative genomic analysis problems.  Influenza viruses (Orthymxyoviridae) are RNA based.  Current evidence indicates that influenza viruses originate in aquatic birds from wild populations. Influenza has been studied for decades via well-coordinated international efforts.  These efforts center on surveillance via antibody characterization of the hemagglutinin (HA) and neuraminidase (N) proteins of the circulating strains to inform vaccine design. However we still do not have a clear understanding of: 1) various transmission pathways such as the role of intermediate hosts such as swine and domestic birds and 2) the key mutation and genomic recombination events that underlie periodic pandemics of influenza.  In the past 30 years, sequence data from HA and N loci has become an important data type. In the past year, full genomic data has become prominent.  These data present exciting opportunities to address unanswered questions in influenza pandemics.SARS is caused by a previously unrecognized lineage of coronavirus, SARS-CoV, which like influenza has an RNA based genome.  Although SARS-CoV is widely believed to have originated in animals there remains disagreement over the candidate animal source that lead to the original outbreak of SARS.  In contrast to the long history of the study of influenza, SARS was only recognized in late 2002 and the virus that causes SARS has been documented primarily by genomic sequencing.In the past, most studies of influenza were performed on a limited number of isolates and genes suited to a particular problem.  Major goals in science today are to understand emerging diseases in broad geographic, environmental, societal, biological, and genomic contexts. Synthesizing diverse information brought together by various researchers is important to find out what can be done to prevent future outbreaks {JON03}.  Thus comprehensive means to organize and analyze large amounts of diverse information are critical.  For example, the relationships of isolates and patterns of genomic change observed in large datasets might not be consistent with hypotheses formed on partial data.  Moreover when researchers rely on partial datasets, they restrict the range of possible discoveries.Phylogenetics is well suited to the complex task of understanding emerging infectious disease. Phylogenetic analyses can test many hypotheses by comparing diverse isolates collected from various hosts, environments, and points in time and organizing these data into various evolutionary scenarios.  The products of a phylogenetic analysis are a graphical tree of ancestor-descendent relationships and an inferred summary of mutations, recombination events, host shifts, geographic, and temporal spread of the viruses.  However, this synthesis comes at a price.  The cost of computation of phylogenetic analysis expands combinatorially as the number of isolates considered increases. Thus, large datasets like those currently produced are commonly considered intractable.  We address this problem with synergistic development of heuristics tree search strategies and parallel computing.Fil: Janies, D.. Ohio State University; Estados UnidosFil: Pol, Diego. Ohio State University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Randomized trial of achieving healthy lifestyles in psychiatric rehabilitation: the ACHIEVE trial

<p>Abstract</p> <p>Background</p> <p>Overweight and obesity are highly prevalent among persons with serious mental illness. These conditions likely contribute to premature cardiovascular disease and a 20 to 30 percent shortened life expectancy in this vulnerable population. Persons with serious mental illness need effective, appropriately tailored behavioral interventions to achieve and maintain weight loss. Psychiatric rehabilitation day programs provide logical intervention settings because mental health consumers often attend regularly and exercise can take place on-site. This paper describes the Randomized Trial of Achieving Healthy Lifestyles in Psychiatric Rehabilitation (ACHIEVE). The goal of the study is to determine the effectiveness of a behavioral weight loss intervention among persons with serious mental illness that attend psychiatric rehabilitation programs. Participants randomized to the intervention arm of the study are hypothesized to have greater weight loss than the control group.</p> <p>Methods/Design</p> <p>A targeted 320 men and women with serious mental illness and overweight or obesity (body mass index ≥ 25.0 kg/m²) will be recruited from 10 psychiatric rehabilitation programs across Maryland. The core design is a randomized, two-arm, parallel, multi-site clinical trial to compare the effectiveness of an 18-month behavioral weight loss intervention to usual care. Active intervention participants receive weight management sessions and physical activity classes on-site led by study interventionists. The intervention incorporates cognitive adaptations for persons with serious mental illness attending psychiatric rehabilitation programs. The initial intensive intervention period is six months, followed by a twelve-month maintenance period in which trained rehabilitation program staff assume responsibility for delivering parts of the intervention. Primary outcomes are weight loss at six and 18 months.</p> <p>Discussion</p> <p>Evidence-based approaches to the high burden of obesity and cardiovascular disease risk in person with serious mental illness are urgently needed. The ACHIEVE Trial is tailored to persons with serious mental illness in community settings. This multi-site randomized clinical trial will provide a rigorous evaluation of a practical behavioral intervention designed to accomplish and sustain weight loss in persons with serious mental illness.</p> <p>Trial Registration</p> <p>Clinical Trials.gov NCT00902694</p

SIVagm Infection in Wild African Green Monkeys from South Africa: Epidemiology, Natural History, and Evolutionary Considerations

Pathogenesis studies of SIV infection have not been performed to date in wild monkeys due to difficulty in collecting and storing samples on site and the lack of analytical reagents covering the extensive SIV diversity. We performed a large scale study of molecular epidemiology and natural history of SIVagm infection in 225 free-ranging AGMs from multiple locations in South Africa. SIV prevalence (established by sequencing pol, env, and gag) varied dramatically between infant/juvenile (7%) and adult animals (68%) (p<0.0001), and between adult females (78%) and males (57%). Phylogenetic analyses revealed an extensive genetic diversity, including frequent recombination events. Some AGMs harbored epidemiologically linked viruses. Viruses infecting AGMs in the Free State, which are separated from those on the coastal side by the Drakensberg Mountains, formed a separate cluster in the phylogenetic trees; this observation supports a long standing presence of SIV in AGMs, at least from the time of their speciation to their Plio-Pleistocene migration. Specific primers/probes were synthesized based on the pol sequence data and viral loads (VLs) were quantified. VLs were of 104-106 RNA copies/ml, in the range of those observed in experimentally-infected monkeys, validating the experimental approaches in natural hosts. VLs were significantly higher (107-108 RNA copies/ml) in 10 AGMs diagnosed as acutely infected based on SIV seronegativity (Fiebig II), which suggests a very active transmission of SIVagm in the wild. Neither cytokine levels (as biomarkers of immune activation) nor sCD14 levels (a biomarker of microbial translocation) were different between SIV-infected and SIV-uninfected monkeys. This complex algorithm combining sequencing and phylogeny, VL quantification, serology, and testing of surrogate markers of microbial translocation and immune activation permits a systematic investigation of the epidemiology, viral diversity and natural history of SIV infection in wild African natural hosts. © 2013 Ma et al

Targeting the ERG oncogene with splice-switching oligonucleotides as a novel therapeutic strategy in prostate cancer

This is the author accepted manuscript. The final version is available from Springer Nature via the DOI in this recordBackground The ERG oncogene, a member of the ETS family of transcription factor encoding genes, is a genetic driver of prostate cancer. It is activated through a fusion with the androgen-responsive TMPRSS2 promoter in 50% of cases. There is therefore significant interest in developing novel therapeutic agents that target ERG. We have taken an antisense approach and designed morpholino-based oligonucleotides that target ERG by inducing skipping of its constitutive exon 4. Methods We designed antisense morpholino oligonucleotides (splice-switching oligonucleotides, SSOs) that target both the 5′ and 3′ splice sites of ERG’s exon 4. We tested their efficacy in terms of inducing exon 4 skipping in two ERG-positive cell lines, VCaP prostate cancer cells and MG63 osteosarcoma cells. We measured their effect on cell proliferation, migration and apoptosis. We also tested their effect on xenograft tumour growth in mice and on ERG protein expression in a human prostate cancer radical prostatectomy sample ex vivo. Results In VCaP cells, both SSOs were effective at inducing exon 4 skipping, which resulted in a reduction of overall ERG protein levels up to 96 h following a single transfection. SSO-induced ERG reduction decreased cell proliferation, cell migration and significantly increased apoptosis. We observed a concomitant reduction in protein levels for cyclin D1, c-Myc and the Wnt signalling pathway member β-catenin as well as a marker of activated Wnt signalling, p-LRP6. We tested the 3′ splice site SSO in MG63 xenografts in mice and observed a reduction in tumour growth. We also demonstrated that the 3′ splice site SSO caused a reduction in ERG expression in a patient-derived prostate tumour tissue cultured ex vivo. Conclusions We have successfully designed and tested morpholino-based SSOs that cause a marked reduction in ERG expression, resulting in decreased cell proliferation, a reduced migratory phenotype and increased apoptosis. Our initial tests on mouse xenografts and a human prostate cancer radical prostatectomy specimen indicate that SSOs can be effective for oncogene targeting in vivo. As such, this study encourages further in vivo therapeutic studies using SSOs targeting the ERG oncogene.Prostate Cancer U

Limiting damage during infection:lessons from infection tolerance for novel therapeutics

The distinction between pathogen elimination and damage limitation during infection is beginning to change perspectives on infectious disease control, and has recently led to the development of novel therapies that focus on reducing the illness caused by pathogens ("damage limitation") rather than reducing pathogen burdens directly ("pathogen elimination"). While beneficial at the individual host level, the population consequences of these interventions remain unclear. To address this issue, we present a simple conceptual framework for damage limitation during infection that distinguishes between therapies that are either host-centric (pro-tolerance) or pathogen-centric (anti-virulence). We then draw on recent developments from the evolutionary ecology of disease tolerance to highlight some potential epidemiological and evolutionary responses of pathogens to medical interventions that target the symptoms of infection. Just as pathogens are known to evolve in response to antimicrobial and vaccination therapies, we caution that claims of "evolution-proof" anti-virulence interventions may be premature, and further, that in infections where virulence and transmission are linked, reducing illness without reducing pathogen burden could have non-trivial epidemiological and evolutionary consequences that require careful examination

Neurotoxicant-induced inflammatory response in three-dimensional brain cell cultures.

Brain inflammatory response is triggered by the activation of microglial cells and astrocytes in response to various types of CNS injury, including neurotoxic insults. Its outcome is determined by cellular interactions, inflammatory mediators, as well as trophic and/or cytotoxic signals, and depends on many additional factors such as the intensity and duration of the insult, the extent of both the primary neuronal damage and glial reactivity and the developmental stage of the brain. Depending on particular circumstances, the brain inflammatory response can promote neuroprotection, regeneration or neurodegeneration. Glial reactivity, regarded as the central phenomenon of brain inflammation, has also been used as an early marker of neurotoxicity. To study the mechanisms underlying the glial reactivity, serum-free aggregating brain cell cultures were used as an in vitro model to test the effects of conventional neurotoxicants such as organophosphate pesticides, heavy metals, excitotoxins and mycotoxins. This approach was found to be relevant and justified by the complex cell-cell interactions involved in the brain inflammatory response, the variability of the glial reactions and the multitude of mediators involved. All these variables need to be considered for the elucidation of the specific cellular and molecular reactions and their consequences caused by a given chemical insult