In this work, we report the use of iodine-contrast microCT to perform high-throughput 3D morphological analysis of mouse embryos and neonates between embryonic day 8.5 to postnatal day 3, with high ...spatial resolution up to 3µm/voxel. We show that mouse embryos at early stages can be imaged either within extra embryonic tissues such as the yolk sac or the decidua without physically disturbing the embryos. This method enables a full, undisturbed analysis of embryo turning, allantois development, vitelline vessels remodeling, yolk sac and early placenta development, which provides increased insights into early embryonic lethality in mutant lines. Moreover, these methods are inexpensive, simple to learn and do not require substantial processing time, making them ideal for high throughput analysis of mouse mutants with embryonic and early postnatal lethality.
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•MicroCT is suitable for 3D imaging of mouse development from early post-implantation to early postnatal development.•Comparable resolution of E12.5 and E9.5 embryos acquired on microCT to OPT.•Preserve extra-embryonic connections by imaging within yolk sac and decidua.
Sildenafil citrate revolutionized the practice of sexual medicine upon its federal regulatory agency approval approximately 15 years ago as the prototypical phosphodiesterase type 5 inhibitor ...indicated for the treatment of male erectile dysfunction. We now provide scientific support for its alternative use in the management of priapism, a clinical disorder of prolonged and uncontrolled penile erection. Sildenafil administered continuously to sickle cell mice, which show a priapism phenotype, reverses oxidative/nitrosative stress effects in the penis, mainly via reversion of uncoupled endothelial nitric oxide synthase to the functional coupled state of the enzyme, which in turn corrects aberrant signaling and function of the nitric oxide/cyclic GMP/protein kinase G/phosphodiesterase type 5 cascade. Priapism tendencies in these mice are reverted partially toward normal neurostimulated erection frequencies and durations after sildenafil treatment in association with normalized cyclic GMP concentration, protein kinase G activity and phosphodiesterase type 5 activity in the penis. Thus, sildenafil exerts pleiotropic effects in the penis that extend to diverse erection disorders.
The International Mouse Phenotyping Consortium (IMPC) continues to expand the catalogue of mammalian gene function by conducting genome and phenome-wide phenotyping on knockout mouse lines. The ...extensive and standardized phenotype screens allow the identification of new potential models for human disease through cross-species comparison by computing the similarity between the phenotypes observed in the mutant mice and the human phenotypes associated to their orthologous loci in Mendelian disease. Here, we present an update on the novel disease models available from the most recent data release (DR10.0), with 5861 mouse genes fully or partially phenotyped and a total number of 69,982 phenotype calls reported. With approximately one-third of human Mendelian genes with orthologous null mouse phenotypes described, the range of available models relevant for human diseases keeps increasing. Among the breadth of new data, we identify previously uncharacterized disease genes in the mouse and additional phenotypes for genes with existing mutant lines mimicking the associated disorder. The automated and unbiased discovery of relevant models for all types of rare diseases implemented by the IMPC constitutes a powerful tool for human genetics and precision medicine.
In metagenomic studies, a process called binning is necessary to assign contigs that belong to multiple species to their respective phylogenetic groups. Most of the current methods of binning, such ...as BLAST, k-mer and PhyloPythia, involve assigning sequence fragments by comparing sequence similarity or sequence composition with already-sequenced genomes that are still far from comprehensive. We propose a semi-supervised seeding method for binning that does not depend on knowledge of completed genomes. Instead, it extracts the flanking sequences of highly conserved 16S rRNA from the metagenome and uses them as seeds (labels) to assign other reads based on their compositional similarity.
The proposed seeding method is implemented on an unsupervised Growing Self-Organising Map (GSOM), and called Seeded GSOM (S-GSOM). We compared it with four well-known semi-supervised learning methods in a preliminary test, separating random-length prokaryotic sequence fragments sampled from the NCBI genome database. We identified the flanking sequences of the highly conserved 16S rRNA as suitable seeds that could be used to group the sequence fragments according to their species. S-GSOM showed superior performance compared to the semi-supervised methods tested. Additionally, S-GSOM may also be used to visually identify some species that do not have seeds. The proposed method was then applied to simulated metagenomic datasets using two different confidence threshold settings and compared with PhyloPythia, k-mer and BLAST. At the reference taxonomic level Order, S-GSOM outperformed all k-mer and BLAST results and showed comparable results with PhyloPythia for each of the corresponding confidence settings, where S-GSOM performed better than PhyloPythia in the >/= 10 reads datasets and comparable in the > or = 8 kb benchmark tests.
In the task of binning using semi-supervised learning methods, results indicate S-GSOM to be the best of the methods tested. Most importantly, the proposed method does not require knowledge from known genomes and uses only very few labels (one per species is sufficient in most cases), which are extracted from the metagenome itself. These advantages make it a very attractive binning method. S-GSOM outperformed the binning methods that depend on already-sequenced genomes, and compares well to the current most advanced binning method, PhyloPythia.
Prior retrospective and prospective studies suggest improved survival with the use of stereotactic radiosurgery (SRS) and bevacizumab in the treatment of limited-volume glioblastoma (GBM) ...recurrences.
We retrospectively reviewed our experience with gamma knife SRS in combination with bevacizumab for the treatment of focal GBM recurrence during 2009–2015. Outcomes include overall survival, progression free survival (PFS), and radiation-related adverse events. Kaplan–Meier methods and multivariable Cox proportional hazards models were performed for survival analysis.
Within a median of 13.7 months after diagnosis, a total of 45 patients with GBM underwent gamma knife SRS and bevacizumab treatment. Median age was 57 years (range: 20–78 years) and 63.3% were women. The median Karnofsky Performance Score (KPS) at recurrence was 80 (range: 40–100). Sixty-four percent of patients had single radiosurgery target (range: 1–4) and median target volume and margin dose were 2.2 cm3 (range: 0.1–25.2 cm3) and 17.0 gray (Gy) (range: 13–24 Gy), respectively. Median PFS and overall survival were 9.3, 31.0 months following diagnosis, and 5.2, 13.3 months after SRS, respectively. Factors associated with poor outcomes were KPS ≤70, SRS dose <18 Gy, and use of <2 chemotherapy agents prior to SRS. No radiation-related adverse events occurred.
SRS in combination with bevacizumab can be safely used to treat focal GBM recurrence. KPS, radiation dose, and multi-agent chemotherapy usage prior to SRS demonstrated significant impact on PFS. Bevacizumab may provide clinically relevant radioprotection.
Priapism is a poorly understood disease process with little information on the etiology and pathophysiology of this erectile disorder. One group of patients with a high prevalence of priapism is men ...with sickle-cell disease.
Establish an in vivo transgenic sickle-cell mouse model to study the pathophysiology of sickle-cell disease-associated priapism.
Transgenic sickle-cell disease mice, expressing human sickle hemoglobin, were utilized. Three groups of mice were used: (i) wild type (WT), (ii) sickle-cell heterozygotes (Hemi), and (ii) sickle-cell homozygotes (Sickle). Two age groups of each cohort of mice were utilized: young adult (4–6 months) and aged (18–22 months).
Histological (trichrome stain to measure ratio of collagen to smooth muscle), penile hydroxyproline content (collagen content), and transmission electron microscopic analysis of WT, Hemi, and Sickle mice penes, as well as in vivo erectile responses change in intracavernous pressure (ICP) to cavernous nerve stimulation (CNS), were determined. The frequency of erectile responses (erections/hour) pre- and poststimulation was also measured in each of the experimental groups.
Sickle mice had increased (P < 0.05) collagen to smooth muscle ratio and hydroxyproline content in the penis when compared with WT and Hemi mice penes. Transmission electron microscopy demonstrated thickened smooth muscle cell bundles, disruption of the endothelial lining of the corporal sinusoids, and increased (P < 0.05) caveolae number. Sickle mice had significantly (P < 0.05) higher ICP to CNS and increased (P < 0.05) frequency of erections pre- and post-CNS when compared with WT and Hemi mice erectile responses. Sickle mice did develop ED (change in ICP in response to CNS) with increasing age.
The morphometric changes of the penis and exaggerated in vivo erectile responses support the use of this transgenic sickle-cell disease animal model to study the pathophysiological mechanisms involved in sickle-cell disease-associated priapism. Bivalacqua TJ, Musicki B, Hsu LL, Gladwin MT, Burnett AL, and Champion HC. Establishment of a transgenic sickle-cell mouse model to study the pathophysiology of priapism. J Sex Med 2009;6:2494–2504.
Glioblastoma multiforme (GBM) is a CNS (central nervous system) malignancy with a low cure rate. Median time to progression after standard treatment is 7 months and median overall survival is 15 ...months 1. Post-treatment vasculogenesis promoted by recruitment of bone marrow derived cells (BMDCs, CD11b+ myelomonocytes) is one of main mechanisms of GBM resistance to initial chemoradiotherapy treatment 2. Local secretion of SDF-1, cognate ligand of BMDCs CXCR4 receptors attracts BMDCs to the post-radiation tumor site.3. This SDF-1 hypoxia-dependent effect can be blocked by AMD3100 (plerixafor) 4. We report a GBM case treated after chemo- radiotherapy with plerixafor and a combination of an mTOR, a Sirt1 and an EGFRvIII inhibitor. After one year temozolomide and the EGFRvIII inhibitor were stopped. Plerixafor, and the MTOR and Sirt-1 inhibitors were continued. He is in clinical and radiologic remission 30 months from the initiation of his adjuvant treatment. To our knowledge, this is the first report of a patient treated for over two years with a CXCR4 inhibitor (plerixafor), as part of his adjuvant treatment. We believe there is sufficient experimental evidence to consider AMD3100 (plerixafor) part of the adjuvant treatment of GBM.
The adjuvant inhibition of GBM vasculogenesis(a process different from local angiogenesis) by specifically blocking the migration of BMDCs to the primary tumor site with inhibitors of the CXCR4/SDF-1 axis represents a potential novel therapeutic approach to GBM. There is significant pre-clinical evidence and validation for its use as demonstrated in a patient derived tumor xenograft model of GBM. Together with other specific anti-tumoral therapies, the active inhibition of vasculogenesis in the adjuvant treatment of GBM is deserving of further exploration.
Novel venom gene discovery in the platypus Whittington, Camilla M; Papenfuss, Anthony T; Locke, Devin P ...
Genome biology,
09/2010, Letnik:
11, Številka:
9
Journal Article
Recenzirano
Odprti dostop
To date, few peptides in the complex mixture of platypus venom have been identified and sequenced, in part due to the limited amounts of platypus venom available to study. We have constructed and ...sequenced a cDNA library from an active platypus venom gland to identify the remaining components.
We identified 83 novel putative platypus venom genes from 13 toxin families, which are homologous to known toxins from a wide range of vertebrates (fish, reptiles, insectivores) and invertebrates (spiders, sea anemones, starfish). A number of these are expressed in tissues other than the venom gland, and at least three of these families (those with homology to toxins from distant invertebrates) may play non-toxin roles. Thus, further functional testing is required to confirm venom activity. However, the presence of similar putative toxins in such widely divergent species provides further evidence for the hypothesis that there are certain protein families that are selected preferentially during evolution to become venom peptides. We have also used homology with known proteins to speculate on the contributions of each venom component to the symptoms of platypus envenomation.
This study represents a step towards fully characterizing the first mammal venom transcriptome. We have found similarities between putative platypus toxins and those of a number of unrelated species, providing insight into the evolution of mammalian venom.
Recent research suggests that priapism in sickle cell disease (SCD) is due to dysregulation of penile erection homeostasis including alteration of nitric oxide synthase (NOS) and phosphodiesterase ...type 5 (PDE5) activities by excessive levels of reactive oxygen species (ROS) released during hemolysis. It is unknown if subacute exposure to hemolysis is sufficient or if chronic reconditioning of erectile tissues is required for perturbation of homeostatic pathways and whether PDE5 inhibitor (PDE5I) treatment can restore erectile homeostasis in the subacute setting.
The aim of this study was to investigate the effects of subacute hemolysis (3‐month exposure) on priapism and NO pathway regulation.
Mice underwent bone marrow transplantation with either SCD (BM‐SS) or wild‐type (WT) bone marrow. BM‐SS mice were treated with sildenafil 100 mg/kg/day. We measured intracavernous pressure (ICP) measurements with or without cavernous nerve stimulation following bone marrow transplantation to assess for priapism.
ICP and frequency of erections were assessed. Penile tissues were analyzed for NOS, protein kinase G (PKG), PDE5, and ROS activities.
BM‐SS mice demonstrated a priapism phenotype. PDE5I treatment reduced the frequency of erections in BM‐SS mice (1.7 ± 1.1 vs. 5.5 ± 2.8 erections per hour, P < 0.05). Penile tissues from BM‐SS mice demonstrated decreased NOS, PKG, PDE5 and elevated ROS activities compared with that of control mice. PDE5I treatment increased NOS (11.6 ± 1.3% vs. 7.8 ± 2.3%, P < 0.05) and PDE5 (76.3 ± 9.8% vs. 52.3 ± 11.1%, P < 0.05) activities and decreased ROS activity (137.8 ± 12.1% vs. 199.1 ± 11.3%, P < 0.05) compared with non‐PDE5I treated BM‐SS mice. PKG activity was increased beyond control levels with PDE5I treatment (158.4 ± 10.3%, P < 0.05).
Short‐term hemolysis is sufficient to establish a priapism phenotype and results in loss of erectile function. PDE5I treatment ameliorates priapism, in part, because of restored NO balance with decreased ROS generation and increased PDE5 activity. Sopko NA, Matsui H, Hannan JL, Berkowitz D, Champion HC, Hsu LL, Musicki B, Burnett AL, and Bivalacqua TJ. Subacute Hemolysis in Sickle Cell Mice Causes Priapism Secondary to NO Imbalance and PDE5 Dysregulation. J Sex Med 2015;12:1878–1885.
Retinal function relies on precisely organized neurons and synapses and a properly patterned vasculature to support them. Alterations in these features can result in vision loss. However, our ...understanding of retinal organization pathways remains incomplete because of a lack of methods to rapidly identify neuron and vasculature regulators in mammals. Here we developed a pipeline for the identification of neural and synaptic integrity genes by high-throughput retinal screening (INSiGHT) that analyzes candidate expression, vascular patterning, cellular organization, and synaptic arrangement. Using this system, we examined 102 mutant mouse lines and identified 16 unique retinal regulatory genes. Fifteen of these candidates are identified as novel retina regulators, and many (9 of 16) are associated with human neural diseases. These results expand the genetic landscape involved in retinal circuit organization and provide a road map for continued discovery of mammalian retinal regulators and disease-causing alleles.
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•Developed pipeline for identifying neural and synaptic integrity genes in the retina•Identified 16 genes that regulate diverse aspects of retinal organization•Candidates play distinct roles in neural, synaptic, and vascular patterning•Data expand the genetic landscape involved in retinal circuit function
Vision requires an organized retina, but many retina regulatory genes remain unknown. Albrecht et al. develop a pipeline for identifying neural and synaptic integrity genes by high-throughput retinal screening. They examine 102 mutant mouse lines and uncover 16 genes responsible for distinct aspects of retina organization.
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