The effect of preoperative therapy on the histologic features of Wilms' tumor. An analysis of cases from the Third National Wilms' Tumor Study.
Preoperative chemotherapy is being used with increasing frequency in the US for the management of selected children with Wilms' tumor, despite relatively limited knowledge as to the effects of such therapy on tumor histologic features. The authors reviewed pathologic material from all preoperatively treated unilateral Wilms' tumors registered on the third National Wilms' Tumor Study. Preoperative therapy was associated with increased ambiguity of tumor stage. The prevalence of anaplasia in the 83 evaluable specimens was similar to, although slightly increased over, that in comparable high-stage untreated Wilms' tumors (11% versus 5% to 8%), and it appeared to retain its adverse connotations. These data also suggest that alterations of tumor character and distribution by therapy provide useful prognostic information. All 17 children whose residual viable tumor was limited to intrarenal sites at the time of resection were alive and disease-free at last follow-up. Extensive (greater than 90%) tumor necrosis, low mitotic activity, and high degrees of differentiation of residual tumor were also associated with favorable outcomes. Although careful utilization of preoperative chemotherapy can be of value in children with otherwise difficult-to-manage Wilms' tumors, the potential benefits of preoperative tumor bulk reduction must be weighed against the risk of overtreatment or undertreatment as a result of increased ambiguity of tumor stage.


Human skeletal muscle insulin receptor substrate-1. Characterization of the cDNA, gene, and chromosomal localization.
Insulin receptor substrate-1 is a major substrate of insulin receptor Tyr kinase. We have now cloned the IRS-1 cDNA from human skeletal muscle, one of the most important target tissues of insulin action, localized and cloned the human IRS-1 gene, and studied the expression of the protein in Chinese hamster ovary cells. Human IRS-1 cDNA encodes a 1242 amino acid sequence that is 88% identical with rat liver IRS-1. The 14 potential Tyr phosphorylation sites include 6 Tyr-Met-X-Met motifs and 3 Tyr-X-X-Met motifs that are completely conserved in human IRS-1. Human IRS-1 has > 50 possible Ser/Thr phosphorylation sites and one potential ATP-binding site close to the NH2-terminal. The human IRS-1 gene contains the entire 5'-untranslated region and protein coding region in a single exon and was localized on chromosome 2 q36-37 by in situ hybridization. By Northern blot analysis, IRS-1 mRNA is rare and consists of two species of 6.9 and 6 kilobase. By using quantitative polymerase chain reaction after reverse transcription of total RNA from human fetal tissues, IRS-1 mRNA could be identified in all tissues. When human IRS-1 cDNA was expressed in Chinese hamster ovary cells, the protein migrated between 170,000-180,000 M(r) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was rapidly Tyr phosphorylated upon insulin stimulation. Thus, IRS-1 is widely expressed and highly conserved across species and tissues.(ABSTRACT TRUNCATED AT 250 WORDS)


Surgical Class III treatment: long-term stability and patient perceptions of treatment outcome.
To evaluate long-term changes after surgical correction of skeletal Class III deformity, postoperative cephalometric radiographs at 1 year and 2 or more years postsurgery were digitized for 92 patients who had received either a bilateral sagittal split osteotomy for mandibular setback, Le Fort I maxillary advancement, or a combination of the two procedures. Patients' perceptions of treatment were determined by four self-administered questionnaires: satisfaction, postsurgical perception of occlusion and function, problems with facial sensation, and postsurgical perceptions. From 1 year to longest follow-up, there were almost no mean changes in landmark positions for the maxillary advancement group and minimal mean changes in the mandibular setback and two-jaw groups. In all three groups, more than 90% of the patients showed no clinically significant long-term changes, which suggests that long-term changes are less likely after Class III than Class II treatment. At long-term recall, 89% of the patients expressed satisfaction with their treatment and would recommend it to others, 74% reported improved social interaction, and 63% said their appearance changed as they expected. The predominant problems reported were altered facial sensation for 67% of the patients and surprise at the length of recovery for 52%.


Antiangiogenesis and antioxidant activity of ethanol extracts of Pithecellobium jiringa.
Angiogenesis plays a critical role in embryonic development and various physiological processes. However, excessive angiogenesis is associated with several pathological conditions including cancer. Pithecellobium jiringa (Jack) Prain is a traditional medicinal plant from the family Leguminosae. It is native to the Southeast Asia, where it has been used traditionally for treatment of various ailments such as hypertension and diabetes. The present work is aimed to study antioxidant and antiangiogenesis activities of P. jiringa ethanol extracts. P. jiringa fruit rinds were extracted with ethanol and 50% ethanol. The antioxidant property was analysed using, 1,1-diphenyl-2-picryl-hydrazyl free radical scavenging assay. Phytochemical analysis was performed using thin layer chromatography and colorimetric methods. Then, cell growth inhibition was studied against a panel of human cell lines by MTT test. In vitro inhibition of angiogenesis was studied by the following assays: isolated rat aortic rings cell viability, colony formation, endothelial cell migration, endothelial tube formation on matrigel, and expression of vascular endothelial growth factor by endothelial cells. In vivo antiangiogenesis effect was studied by utilising fertilised chick embryos assay. The results were statistically analysed by analysis of variance. Ethanolic and 50% hydro-ethanolic extracts showed relatively high concentration of total phenolics associated with potent antioxidant activity. The rat aortic rings study conducted showed potent inhibition of the microvessels outgrowth with IC50s 5.27 ± 0.81 μg/ml (ethanolic) and 4.45 ± 0.63 μg/ml (50% hydro-ethanolic). Both extracts arrested the growth of human endothelial cells via down-regulation of VEGF expression, leading to inhibition of other angiogenesis cascades including migration of endothelial cells, and formation of capillary network on matrigel matrix. The extracts also inhibited the neovascularisation of chick embryo chorioallantoic membrane. P. jiringa extracts inhibit angiogenesis by blocking the VEGF expression thus inhibiting endothelial cells proliferation, migration and differentiation most likely due to presence of the antioxidant phenolics.


Current status and future of relapsed primary central nervous system lymphoma (PCNSL).
The treatment of primary central nervous system lymphoma (PCNSL) has centered around high-dose methotrexate and radiotherapy (RT). Methotrexate administered intra-arterially (IA) with blood-brain barrier disruption (BBBD) and without RT, has been a highly effective treatment with a 5 year survival of 42% without cognitive loss. The purpose of this analysis is to determine responses for patients with relapsed PCNSL treated with second line IA carboplatin-based chemotherapy with BBBD. Between February 1991 and April 2000, 37 relapsed PCNSL patients, most who failed front line therapy with methotrexate based chemotherapy, were treated at Oregon Health & Science University (OHSU) and Hadassah Hebrew University Hospital (HHUH) with IA carboplatin-based chemotherapy with BBBD. Nine patients had prior RT. The mean age was 57.5 years, and all but 1 patient were treated within 8 months after relapse. The median time for survival from first IA carboplatin/BBBD treatment was 6.8 months;however, 7 out of 37 patients survived > or = 27 months. Nine patients had radiographic complete response (CR), 4 patients had radiographic partial response (PR), 12 had stable disease (SD), 10 had progressive disease (PD), and 2 were non-evaluable. The median time to failure for patients with CR and PR was 9.1 months. One long-term survivor is alive at 91.0 months from first carboplatin/BBBD treatment. In conclusion, we show that relapsed PCNSL has shown sensitivity to second line IA carboplatin-based chemotherapy with BBBD. We have developed a new protocol using i.v. rituximab prior to BBBD with IA carboplatin, i.v. cyclophosphamide and i.v. etoposide phosphate. The long-term program goal is to consolidate dose-intensive chemotherapy with monoclonal antibody directed radiation. Because patients with recurrent PCNSL commonly continue to relapse even after obtaining a complete response to enhanced chemotherapy treatment, patients w ho complete or fail the above carboplatin/BBBD treatment regimen will be offered consolidation with radioimmunotherapy using zevalin (Ibritumomab tiuxetan), IDEC-2B8 conjugated with yttrium-90 (90Y).


Geometric visual hallucinations, Euclidean symmetry and the functional architecture of striate cortex.
This paper is concerned with a striking visual experience: that of seeing geometric visual hallucinations. Hallucinatory images were classified by Klüver into four groups called form constants comprising (i) gratings, lattices, fretworks, filigrees, honeycombs and chequer-boards, (ii) cobwebs, (iii) tunnels, funnels, alleys, cones and vessels, and (iv) spirals. This paper describes a mathematical investigation of their origin based on the assumption that the patterns of connection between retina and striate cortex (henceforth referred to as V1)-the retinocortical map-and of neuronal circuits in V1, both local and lateral, determine their geometry. In the first part of the paper we show that form constants, when viewed in V1 coordinates, essentially correspond to combinations of plane waves, the wavelengths of which are integral multiples of the width of a human Hubel-Wiesel hypercolumn, ca. 1.33-2 mm. We next introduce a mathematical description of the large-scale dynamics of V1 in terms of the continuum limit of a lattice of interconnected hypercolumns, each of which itself comprises a number of interconnected iso-orientation columns. We then show that the patterns of interconnection in V1 exhibit a very interesting symmetry, i.e. they are invariant under the action of the planar Euclidean group E(2)-the group of rigid motions in the plane-rotations, reflections and translations. What is novel is that the lateral connectivity of V1 is such that a new group action is needed to represent its properties: by virtue of its anisotropy it is invariant with respect to certain shifts and twists of the plane. It is this shift-twist invariance that generates new representations of E(2). Assuming that the strength of lateral connections is weak compared with that of local connections, we next calculate the eigenvalues and eigenfunctions of the cortical dynamics, using Rayleigh-Schrödinger perturbation theory. The result is that in the absence of lateral connections, the eigenfunctions are degenerate, comprising both even and odd combinations of sinusoids in straight phi, the cortical label for orientation preference, and plane waves in r, the cortical position coordinate. 'Switching-on' the lateral interactions breaks the degeneracy and either even or else odd eigenfunctions are selected. These results can be shown to follow directly from the Euclidean symmetry we have imposed. In the second part of the paper we study the nature of various even and odd combinations of eigenfunctions or planforms, the symmetries of which are such that they remain invariant under the particular action of E(2) we have imposed. These symmetries correspond to certain subgroups of E(2), the so-called axial subgroups. Axial subgroups are important in that the equivariant branching lemma indicates that when a symmetrical dynamical system becomes unstable, new solutions emerge which have symmetries corresponding to the axial subgroups of the underlying symmetry group. This is precisely the case studied in this paper. Thus we study the various planforms that emerge when our model V1 dynamics become unstable under the presumed action of hallucinogens or flickering lights. We show that the planforms correspond to the axial subgroups of E(2), under the shift-twist action. We then compute what such planforms would look like in the visual field, given an extension of the retinocortical map to include its action on local edges and contours. What is most interesting is that, given our interpretation of the correspondence between V1 planforms and perceived patterns, the set of planforms generates representatives of all the form constants. It is also noteworthy that the planforms derived from our continuum model naturally divide V1 into what are called linear regions, in which the pattern has a near constant orientation, reminiscent of the iso-orientation patches constructed via optical imaging. The boundaries of such regions form fractures whose points of intersection correspond to the well-known 'pinwheels'. To complete the study we then investigate the stability of the planforms, using methods of nonlinear stability analysis, including Liapunov-Schmidt reduction and Poincaré-Lindstedt perturbation theory. We find a close correspondence between stable planforms and form constants. The results are sensitive to the detailed specification of the lateral connectivity and suggest an interesting possibility, that the cortical mechanisms by which geometric visual hallucinations are generated, if sited mainly in V1, are closely related to those involved in the processing of edges and contours.


Low prevalence of Listeria monocytogenes in human stool.
Listeria monocytogenes is a foodborne pathogen that is found widely in the environment and in a variety of ready-to-eat foods, yet human invasive infection is relatively rare (five cases per million people annually in the United States). Despite wide exposure to this organism, little is known about the prevalence of L. monocytogenes in human stool, and it is not known whether human fecal dispersal contributes to human foodborne transmission. We cultured 827 stool specimens (well formed and loose-watery) from individuals from four large metropolitan areas of New York state for L. monocytogenes and found only 1 (0.12%) positive specimen. L. monocytogenes was also isolated from the blood of the person with the single positive specimen, and the two isolates were indistinguishable by molecular subtyping (both were ribotype DUP-1042B). This provides further evidence that human L. monocytogenes fecal carriage among persons with and without diarrheal disease is remarkably low. Unlike the case for other foodborne pathogens (e.g., Salmonella), human shedders probably do not contribute significantly to L. monocytogenes contamination of foods. However, we observed a single individual with invasive listeriosis that shed the pathogen in feces, indicating the potential for fecal dispersal of L. monocytogenes from persons with listeriosis.


Polymorphism rs4263535 in GABRA1 intron 4 was related to deeper sedation by intravenous midazolam.
To evaluate whether polymorphisms in the gamma-aminobutyric acid A receptor α1 subunit (GABRA1) gene influence sleep induction time, bispectral index score (BIS) during sleep induction and the total dose of midazolam required to reach a Ramsay Sedation Assessment Scale (RSAS) score of 4. Patients scheduled for elective orthopaedic surgery were enrolled. All patients received initial doses of 0.02 mg/kg intravenous midazolam. If the RSAS score did not reach 4, an additional 1-mg dose of midazolam was administered. Results were compared among groups of patients with five single-nucleotide polymorphisms (SNPs) in GABRA1: rs4263535, rs980791, rs6556562, rs998754 and rs2279020. A total of 104 patients were evaluated. Polymorphism rs4263535 was associated with the lowest BIS during sedation induction. Multinomial logistic regression analysis demonstrated that polymorphism rs4263535 was significantly associated with the total dose of midazolam required for sedation induction. Polymorphism rs4263535 in GABRA1 intron 4 was associated with deeper sedation by intravenous midazolam. Patients with the A/A rs4263535 genotype required a smaller dose of midazolam.


Femtosecond laser induced robust periodic nanoripple structured mesh for highly efficient oil-water separation.
Marine oil spills have induced severe water pollution and threatened sea ecosystems, which also result in a loss of energy resources. To deal with this problem, much work has been done for using superhydrophobic or superhydrophilic mesh for oil-water separation. Nevertheless, there are still great challenges in the rapid fabrication of extremely durable mesh with superwetting properties, particularly considering the highly efficient oil-water separation. In this study, we present a simple, efficient method to fabricate superhydrophilic and underwater superoleophobic stainless steel mesh surfaces with one-step femtosecond laser induced periodic nanoripple structures. The as-prepared mesh shows high separation efficiency, which is higher than 99% for various oil-water mixtures. More importantly, the wettability and the separation efficiency of the fabricated mesh show no obvious change after the abrasion tests and corrosion tests, indicating that the as-prepared samples possess robust stability. This study provides an efficient route for constructing durable and highly efficient separation mesh, which can be applied in the cleanup of large-scale oil spills in the near future.


Telomerase Gene (hTERT) and Survival: Results From Two Swedish Cohorts of Older Adults.
Telomere length has been associated with longevity. As telomere length is partly determined by the human telomerase reverse transcriptase (hTERT), we investigated the association between an hTERT polymorphism located in its promoter region ((-) (1327)T/C) and longevity in two cohorts of older adults. Participants from the Kungsholmen project (KP; n = 1,205) and the Swedish National study of Aging and Care in Kungsholmen (SNAC-K; n = 2,764) were followed for an average period of 7.5 years. The main outcomes were hazard ratios (HR) of mortality and median age at death. In both cohorts, mortality was lower in female T/T carriers, aged 75+ years in KP (HR = 0.8, 95% CI: 0.5-0.9) and 78+ years in SNAC-K (HR = 0.6, 95% CI: 0.4-0.8) compared with female C/C carriers. T/T carriers died 1.8-3 years later than the C/C carriers. This effect was not present in men, neither in SNAC-K women aged 60-72 years. The association was not modified by presence of cancer, cardiovascular diseases, number of chronic diseases, or markers of inflammation, and did not interact with APOE genotype or estrogen replacement therapy. The gender-specific increased survival in T/T carriers can be due to a synergistic effect between genetic background and the life-long exposure to endogenous estrogen.


Development of a five-year mortality model in systemic sclerosis patients by different analytical approaches.
Systemic sclerosis (SSc) is a multiorgan disease with high mortality rates. Several clinical features have been associated with poor survival in different populations of SSc patients, but no clear and reproducible prognostic model to assess individual survival prediction in scleroderma patients has ever been developed. We used Cox regression and three data mining-based classifiers (Naïve Bayes Classifier [NBC], Random Forests [RND-F] and logistic regression [Log-Reg]) to develop a robust and reproducible 5-year prognostic model. All the models were built and internally validated by means of 5-fold cross-validation on a population of 558 Italian SSc patients. Their predictive ability and capability of generalisation was then tested on an independent population of 356 patients recruited from 5 external centres and finally compared to the predictions made by two SSc domain experts on the same population. The NBC outperformed the Cox-based classifier and the other data mining algorithms after internal cross-validation (area under receiving operator characteristic curve, AUROC: NBC=0.759; RND-F=0.736; Log-Reg=0.754 and Cox= 0.724). The NBC had also a remarkable and better trade-off between sensitivity and specificity (e.g. Balanced accuracy, BA) than the Cox-based classifier, when tested on an independent population of SSc patients (BA: NBC=0.769, Cox=0.622). The NBC was also superior to domain experts in predicting 5-year survival in this population (AUROC=0.829 vs. AUROC=0.788 and BA=0.769 vs. BA=0.67). We provide a model to make consistent 5-year prognostic predictions in SSc patients. Its internal validity, as well as capability of generalisation and reduced uncertainty compared to human experts support its use at bedside. Available at: http://www.nd.edu/~nchawla/survival.xls.


Treatment approach and outcomes of vaginal melanoma.
To describe the characteristics of primary vaginal melanoma patients in the Surveillance, Epidemiology, and End Result database and to determine the outcome from the treatment approaches. From the Surveillance, Epidemiology, and End Result registry, 201 patients with vaginal melanoma were identified. Patients' characteristics and prognostic factors including age, race, extent of surgery, and use of radiation therapy were obtained. The median age was 68 years (range, 28-100 years). The population was 73% white, 11% black, and 16% Asian/American Indian. International Federation of Gynecology and Obstetrics staging results were stage I (46%), stage II (18%), stage III (3%), stage IVA (3%), stage IVB (12%), and unknown (18%). Treatment approach included surgical resection of the primary site in 70%, whereas 35% of the patients underwent lymph node resection. Approximately 40% of the patients received radiotherapy, which was primarily used in the adjuvant setting. Overall survival at 2 and 5 years was 24% and 15%, respectively. Presence of lymph nodes at diagnosis was associated with worse overall survival (hazard ratio, 1.98; P = 0.02). Adjuvant radiation did not offer a statistically significant overall survival advantage compared to surgery alone. Vaginal melanoma is a rare diagnosis primarily affecting the elderly. Overall survival is low even for patients presenting with disease limited to the vagina. Lymph node involvement at diagnosis is strongly predictive of worse overall survival. Most patients are treated with surgical resection with varying use of adjuvant radiotherapy. Further research is needed to identify the etiology and improve the outcome of this aggressive disease.


Display of a maize cDNA library on baculovirus infected insect cells.
Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.


Clinical phenotype is related to HLA genotype in the peripheral arthropathies of inflammatory bowel disease.
The detection of phenotype-determining genes as opposed to disease susceptibility genes requires precise phenotypic characterization of patients. Peripheral arthropathies in inflammatory bowel disease (IBD) are well recognized and are classified with the HLA-B*27-related spondyloarthropathies by the European Spondyloarthropathy Study Group. However, previous HLA studies in IBD have only shown this association with axial disease rather than peripheral arthropathy. We recently reported a clinical classification that describes 2 types of peripheral arthropathy, distinguished by their natural history and articular distribution. We now report the results of immunogenetic studies in these patients and compare them with other spondyloarthropathies. IBD patients with type 1 (n = 57) and type 2 (n = 45) peripheral arthropathy were identified by case note review and questionnaire. Patients and 603 controls from Oxfordshire were assigned HLA-A, -B, -C, -DR, and -DQ genotypes by sequence-specific primer polymerase chain reaction. Patient results were compared with controls (corrected for multiple comparisons), then with each other in light of existing hypotheses. The results were compared with those of a cohort of 30 patients with postenteric reactive arthritis (ReA) and 16 patients with IBD-associated ankylosing spondylitis (IBD-AS). Type 1 arthropathy was associated with HLA-DRB1*0103 (DR103; a rare subtype of DR1) in 33% (P < 0.0001; relative risk [RR], 12.1), B*35 in 30% (P = 0.01; RR, 2.2), and B*27 in 26% (P = 0. 001; RR, 4.0). In contrast, type 2 was associated with HLA-B*44 in 62% (P = 0.01; RR, 2.1). Similar significant associations to type 1 arthropathy were found in ReA, except that the HLA-B*27 association was significantly stronger and an association was found with DRB1*0101 (DR1) in 43% (P = 0.001; RR, 2.2). IBD-AS was associated only with HLA-B*27 and DRB1*0101. These data suggest that the clinical classification into type 1 and type 2 arthropathies describes immunogenetically distinct entities and establish that in polygenic disorders, genes may determine clinical phenotype without conferring overall disease susceptibility (in this case, HLA genes). Type 1 arthropathy is clinically and immunogenetically similar to the spondyloarthropathies, but different HLA associations may define phenotypically distinct groups. Type 2 arthropathy has different HLA associations and may have a different etiology. Further studies are now required to confirm these associations and to elucidate the different pathogenetic mechanisms.


Measuring micro-interactions between coagulating red blood cells using optical tweezers.
Agents that alter the dynamics of hemostasis form an important part in management of conditions such as atherosclerosis, cerebrovascular disease, and bleeding diatheses. In this study, we explored the effects of heparin and tranexamic acid on the efficiency of blood coagulation. Using optical tweezers, we evaluated the pN-range micro-interaction between coagulating red blood cells (RBCs) by measuring the minimum power required to trap them. By observing the mobility of RBCs and the intensity of cellular interactions, we found that the coagulation process can be separated into three phases. The effects of heparin and tranexamic acid were examined by observing variations in cellular interaction during the coagulation phases. Heparin attenuated the interaction between RBCs and prolonged the first phase whereas the samples containing tranexamic acid bypassed the first two phases and immediately proceeded to the final one.


Effect of propranolol on platelet signal transduction.
Propranolol inhibits platelet secondary aggregation and secretion by mechanisms unrelated to its beta-adrenergic-blocking activity. We previously reported that a major effect of the drug is perturbation of the physical microenvironment of the human platelet membrane. To explore further the molecular mechanisms underlying propranolol-mediated platelet inhibition, we studied protein kinase C activity, estimated from the phosphorylation of the substrate protein pleckstrin, in propranolol-treated human platelets. The drug inhibited activation of the enzyme in thrombin-stimulated platelets but not in platelets stimulated with phorbol esters, indicating that its site of action might be upstream of protein kinase C. It also inhibited the activity of phospholipase C, determined from the extent of generation of inositol phosphates and phosphatidic acid, in platelets stimulated with thrombin as well as the non-hydrolysable GTP analogue guanosine 5'-[beta, gamma-imido]triphosphate in a dose-dependent manner. These data suggest that propranolol inhibits signal transduction in thrombin-stimulated platelets by interacting at the level of phospholipase C and exclude interaction of the drug with the downstream effector enzyme protein kinase C.


Postnatal corneal transparency, keratocyte cell cycle exit and expression of ALDH1A1.
Recent studies have shown that rabbit corneal keratocytes abundantly express two water-soluble proteins, transketolase (TKT) and aldehyde dehydrogenase class 1A1 (ALDH1A1), in vivo and that these proteins may contribute to corneal transparency at the cellular level. The purpose of this study was to determine the relationship between the expression of these proteins and the development of postnatal corneal transparency. Rabbits 1 day to 42 days of postnatal age were evaluated by in vivo confocal microscopy (CM) to measure corneal epithelial thickness, stromal thickness, and corneal haze. Selected corneas were then processed for immunocytochemistry and Western and Northern blot analyses, to determine stromal cell density, cell cycle entry, and expression of ALDH1A1 and TKT. Quantitative measurement of corneal haze showed that the postnatal cornea was hazy after birth and became transparent during the first weeks after eyelid opening. Development of transparency was associated with decreased cytoplasmic light-scattering from postnatal corneal stromal cells, with the appearance of nuclear light-scattering after eyelid opening. Four days after birth, stromal cell density decreased rapidly, and the cells became quiescent, showing decreased staining by Ki67, a cell cycle marker. Whereas expression of TKT showed a gradual increase after birth, ALDH1A1 showed a marked increase after eyelid opening, and the combined expression significantly correlated with the reduction in light-scattering by postnatal stromal cells. The data suggest that development of postnatal corneal transparency is associated with decreased keratocyte density and quiescence and the expression of TKT/ALDH1A1.


Intensity-modulated radiation therapy for head and neck cancer: emphasis on the selection and delineation of the targets.
The head and neck contain many critical, noninvolved structures in close vicinity to the targets. The tightly conformal doses produced by intensity-modulated radiation therapy (IMRT), and the lack of internal organ motion in the head and neck, provide the potential for organ sparing and improved tumor irradiation. Many studies of treatment planning for head and neck cancer have demonstrated the dosimetric superiority of IMRT over conventional techniques in these respects. The initial results of clinical studies demonstrate reduced xerostomia. They suggest an improvement in tumor control, which needs to be verified in larger studies and longer follow-up. Critical issues for successful outcome of head and neck IMRT are accurate selection of the neck lymph nodes that require adjuvant treatment, and accurate delineation on the planning computed tomography (CT) of the lymph-node bearing areas and subclinical disease adjoining the gross tumor. This review emphasizes these topics and provides some guidelines.


Pathologic complete response to preoperative sequential doxorubicin/cyclophosphamide and single-agent taxane with or without trastuzumab in stage II/III HER2-positive breast cancer.
Four major clinical trials have established that trastuzumab added to adjuvant systemic chemotherapy for women with HER2+ breast cancer significantly improves disease-free and overall survival compared with chemotherapy alone. We evaluated pathologic complete response (pCR) rate and cardiac safety of preoperative doxorubicin and cyclophosphamide followed by a taxane with or without trastuzumab. We reviewed pCR rate and change in left ventricular ejection fraction in women with operable HER2+ breast cancer (defined as immunohistochemical 3+ or fluorescence in situ hybridization ratio > or = 2.2) who were treated between 2002 and 2008 with doxorubicin and cyclophosphamide followed by a taxane with or without trastuzumab before definitive breast surgery. We identified 33 patients, of whom 42.4% received preoperative chemotherapy without trastuzumab and 57.6% of whom received trastuzumab with chemotherapy. The pCR rates were 28.6% and 52.6% in the group that received chemotherapy alone or with trastuzumab, respectively (odds ratio, 2.78; 95% CI, 0.64-12.1; P = .173). Severe cardiac events or treatment delays as a result of cardiac toxicity were not observed. With a median follow-up time of 14 months, 21.4% of patients in the non-trastuzumab group and 10.5% in the trastuzumab group had disease recurrence. Sequential administration of preoperative doxorubicin and cyclophosphamide followed by a taxane and trastuzumab combination is safe in women with primary operable HER2+ breast cancer and is associated with a high pCR rate. Large randomized phase III clinical trials are evaluating the role of preoperative trastuzumab when added to anthracycline- and/or taxane-based regimens.


6,7-Dihydroxy-2-(4'-hydroxyphenyl)naphthalene induces HCT116 cell apoptosis through activation of endoplasmic reticulum stress and the extrinsic apoptotic pathway.
Colorectal cancer is the third leading cause of cancer-related deaths worldwide, and therefore, the development of novel drugs for its prevention and therapy are urgently required. This study aimed to determine the molecular mechanism of 6,7-dihydroxy-2-(4'-hydroxyphenyl) naphthalene (PNAP-6)-induced cytotoxicity in human colorectal cancer (HCT116) cells. The effects of 2-phenylnaphthalene derivatives on HCT116 cell growth and viability were assessed by MTT assays. The mechanisms involved in the regulation of the extrinsic apoptosis and endoplasmic reticulum (ER) stress pathways by PNAP-6 were analyzed by annexin-V/propidium iodide flow cytometric analysis, Hoechst 33342 fluorescent staining, and Western blotting. PNAP-6 was shown to have an IC50 value 15.20 μM. It induced G2/M phase arrest in HCT116 cells, associated with a marked decrease in cyclin B and CDK1 protein expression and increased caspase activation, PARP cleavage, chromatin condensation, and sub-G1 apoptosis. Moreover, we found that the apoptotic effects of PNAP-6 proceeded through extrinsic apoptosis and ER stress pathways, by increasing the expression of Fas protein and ER stress markers, including PERK, ATF4, CHOP, p-IRE1α, and XBP-1s. These results suggest that 2-phenylnaphthalene derivatives, such as PNAP-6, have potential as new treatments for colorectal cancer.


Serum Oxidative/anti-oxidative Stress Balance Is Dysregulated in Potentially Pulmonary Hypertensive Patients with Liver Cirrhosis: A Case Control Study.
Hepatopulmonary syndrome (HPS) is characterized by vascular dilatation and hyperdynamic circulation, while portopulmonary hypertension (POPH) is characterized by vasoconstriction with fibrous obliteration of the vascular bed. Vasoactive molecules such as nitric oxide (NO) are candidate factors for cirrhotic complications associated with these diseases. However, oxidative stress balance is not well characterized in HPS and POPH. The present objective is to investigate the oxidative stress and anti-oxidative stress balance and NO pathway balance in patients with potential HPS and POPH. We recruited patients with decompensated cirrhosis (n=69) admitted to our hospital as liver transplantation candidates. Patients exhibiting partial pressure of oxygen lower than 80 mmHg and alveolar-arterial oxygen gradient (AaDO2) ≥15 mmHg were categorized as potentially having HPS (23 of 69 patients). Patients exhibiting a tricuspid regurgitation pressure gradient ≥25 mmHg were categorized as potentially having POPH (29 of 61 patients). Serum reactive oxygen metabolites were measured and anti-oxidative OXY-adsorbent test (OXY) were performed, and the balance of these tests was defined as the oxidative index. The correlation between these values and the clinical characteristics of the patients were assessed in a cross-sectional study. Potential HPS patients exhibited no correlation with oxidative stress markers. Potential POPH patients exhibited lower OXY (p=0.037) and higher oxidative index values (p=0.001). Additionally, the vascular NO synthase enzyme inhibiting protein, asymmetric dimethylarginine, was higher in potential POPH patients (p=0.049). The potential POPH patients exhibited elevated AaDO2, suggesting the presence of pulmonary shunting. Potential POPH patients exhibited elevated oxidative stress with decreased anti-oxidative function accompanied by inhibited NO production. Anti-oxidants represent a candidate treatment for potential POPH patients.


The non-transplant treatment of myelodysplastic syndromes-what's on the horizon?
The therapeutic advance in the field of myelodysplastic syndromes (MDS) has evolved over the last decade with efficacy of hypomethylating agents and immunomodulatory drugs. Currently, several promising agents with diverse mechanisms of actions are in various stages of clinical development and will expand the therapeutic armamentarium for MDS. The advent of next generation sequencing technologies has improved the understanding of disease biology and this will drive further activity in the near future with new targeted therapies, like exploitation of newly identified molecular aberrations, such as SF3BI and STAG2, as a therapeutic target and also further refinement of existing treatments.


Differentiation and proliferation of hematopoietic stem cells.
Available evidence indicates that qualitative changes in hematopoietic stem cells and progenitors, such as the decision of stem cells to self-renew or differentiate, or selection of lineage potentials by the multipotential progenitors during differentiation (commitment), are intrinsic properties of the progenitors and are stochastic in nature. In-contrast, proliferative kinetics of the progenitors, namely survival and expansion of the progenitors, appear to be controlled by a number of interacting cytokines. While proliferation and maturation of committed progenitors is controlled by late-acting lineage-specific factors such as Ep, M-CSF, G-CSF, and IL-5, progenitors at earlier stages of development are controlled by a group of several overlapping cytokines. IL-3, GM-CSF, and IL-4 regulate proliferation of multipotential progenitors only after they exit from G0 and begin active cell proliferation. Triggering of cycling by dormant primitive progenitors and maintenance of B-cell potential of the primitive progenitors appears to require interactions of early acting cytokines including IL-6, G-CSF, IL-11, IL-12, LIF, and SF. Currently, this simple model fits our understanding of the interactions of growth factors with hematopoietic progenitors. Naturally the model risks oversimplification of a very complex process. However, because the model is testable, it will hopefully challenge investigators to design new experiments to examine its validity.


Nanoparticle-Patterned Multicompartmental Chitosan Capsules for Oral Delivery of Oligonucleotides.
Orally administered antisense therapy has been introduced as an effective approach for treating cancer in the gastrointestinal tract. However, its practical application has been limited by the instability of oligonucleotides and their inefficient delivery. To overcome these problems, we synthesized size-dependent, oligonucleotide nanoparticle-patterned chitosan/phytic acid (ODN/CS/PA) capsules with protective shields via a three-step process of self-assembly, nanoparticle encapsulation, and shell formation. The multicompartmental capsule size and oligonucleotide nanoparticle-loading pattern were controlled by applying different potentials during the electrostatic extrusion process used for nanoparticle encapsulation. Over 95% of encapsulated oligonucleotides were protected from nuclease digestion (DNase I) and, depending on their size, showed 40-75% protection against simulated gastric fluid. Their controlled release from capsules correlated with the cellular delivery of released nanoparticles and the inhibition of protein expression in cancer cells. Specifically, large capsules showed approximately 32-fold greater delivery to cancer cells than nonencapsulated nanoparticles. We also confirmed delivery of oligonucleotide nanoparticles to the small intestine and colon of rats following oral administration. These findings demonstrate that the multicompartmental ODN/CS/PA capsules can facilitate efficient oral delivery of oligonucleotides for cancer treatment.


Serological investigation of bovine brucellosis in three cattle production systems in Yewa Division, south-western Nigeria.
Limited data are available on the risk factors responsible for the occurrence of brucellosis amongst different cattle production systems in Nigeria despite its significant impact on livestock production. Consequently, a cross-sectional study was conducted to determine the prevalence of bovine brucellosis in three cattle production systems in Yewa Division of Ogun State, south-western Nigeria. A total of 279 blood samples (sedentary = 88; transhumance = 64; trade = 127) were examined for antibodies to Brucella sp. using the Rose Bengal test (RBT) and competitive enzyme-linked immunosorbent assay (cELISA). Overall, 24 (8.6%) and 16 (5.7%) of the animals tested seropositive for Brucella using RBT and cELISA, respectively. The herd seroprevalences based on RBT and cELISA were 31.6% and 15.8%, respectively. The results using cELISA reveal higher seroprevalence in the trade cattle (7.9%; confidence intervals [CI] = 3.2% - 12.6%) and those in a sedentary system (5.7%; CI = 0.9% - 10.5%) than in cattle kept under a transhumant management system (1.6%; CI = 1.5% - 4.7%). Age (> 3 years; p = 0.043) and breed (Djali; p = 0.038) were statistically significant for seropositivity to brucellosis based on cELISA, but sex (female, p = 0.234), production system (trade and sedentary; p = 0.208) or herd size (> 120; p = 0.359) was not. Since breeding stock is mostly sourced from trade and sedentary cattle, it is important that routine serological screening should be conducted before introducing any animal into an existing herd.


Effect of experimentally reduced distal sensation on postural response to hip abductor/ankle evertor muscle vibration.
This study assessed whether postural responses induced by vibratory perturbations of the hip abductors and ankle evertors, were modified when distal tactile sensation was experimentally reduced through cooling. Sixteen healthy subjects were investigated pre and post cooling. Subjects stood with their eyes closed with a stance width of 4 cm. A 2s vibratory stimulus was applied to the left or right hip abductor or ankle evertor muscle. The order of the site and side of the stimulation was randomised. The postural response to hip abductor and ankle evertor vibration was recorded using 3D motion analysis (Codamotion, Leicestershire). Medio-lateral centre of pressure motion was simultaneously recorded during quiet standing via a force plate (Kistler, UK). Pre-cooling people responded to unilateral ankle vibration with an ipsilateral translation and tilt of the pelvis, and an ipsilateral tilt of the trunk. People responded to unilateral hip vibration with a contralateral translation and tilt of the pelvis, and an ipsilateral tilt of the trunk. Following an experimental reduction in distal tactile sensation there was a significant reduction in the amplitude of pelvic tilt in response to ankle vibration (F(6.2)=P<0.05) and a significant increase in amplitude of pelvic tilt in response to hip vibration (F(5.2)=P<0.05). This suggests that the sensitivity to artificial stimulation of hip proprioception increases with distal cooling, possibly indicating a change in the gain/weighting placed upon sensory information from the hips.


Electrophysiological correlates of the flexible allocation of visual working memory resources.
Visual working memory is a brief, capacity-limited store of visual information that is involved in a large number of cognitive functions. To guide one's behavior effectively, one must efficiently allocate these limited memory resources across memory items. Previous research has suggested that items are either stored in memory or completely blocked from memory access. However, recent behavioral work proposes that memory resources can be flexibly split across items based on their level of task importance. Here, we investigated the electrophysiological correlates of flexible resource allocation by manipulating the distribution of resources amongst systematically lateralized memory items. We examined the contralateral delay activity (CDA), a waveform typically associated with the number of items held in memory. Across three experiments, we found that, in addition to memory load, the CDA flexibly tracks memory resource allocation. This allocation occurred as early as attentional selection, as indicated by the N2pc. Additionally, CDA amplitude was better-described when fit with a continuous model predicted by load and resources together than when fit with either alone. Our findings show that electrophysiological markers of attentional selection and memory maintenance not only track memory load, but also the proportion of memory resources those items receive.


Spontaneous cortical activity transiently organises into frequency specific phase-coupling networks.
Frequency-specific oscillations and phase-coupling of neuronal populations are essential mechanisms for the coordination of activity between brain areas during cognitive tasks. Therefore, the ongoing activity ascribed to the different functional brain networks should also be able to reorganise and coordinate via similar mechanisms. We develop a novel method for identifying large-scale phase-coupled network dynamics and show that resting networks in magnetoencephalography are well characterised by visits to short-lived transient brain states, with spatially distinct patterns of oscillatory power and coherence in specific frequency bands. Brain states are identified for sensory, motor networks and higher-order cognitive networks. The cognitive networks include a posterior alpha (8-12 Hz) and an anterior delta/theta range (1-7 Hz) network, both exhibiting high power and coherence in areas that correspond to posterior and anterior subdivisions of the default mode network. Our results show that large-scale cortical phase-coupling networks have characteristic signatures in very specific frequency bands, possibly reflecting functional specialisation at different intrinsic timescales.


Idiopathic intracranial hypertension without papilledema.
We describe 10 patients with idiopathic intracranial hypertension who did not have papilledema. Idiopathic intracranial hypertension without papilledema, although rarely reported, may well be a clinically important headache syndrome. Historical and demographic features of patients with idiopathic intracranial hypertension without papilledema are similar to those of patients with papilledema. Obese women with chronic daily headache and symptoms of increased intracranial pressure, pulsatile tinnitus, history of head trauma or meningitis, an empty sella on imaging studies, or a headache that is unrelieved by standard therapy should have a diagnostic lumbar puncture. Findings from laboratory and neurologic investigations are normal in most patients with idiopathic intracranial hypertension without papilledema. Initial management should include removal of possible inciting agents, weight loss if applicable, and standard headache therapy. Lumbar puncture and diuretic therapy should precede a trial of corticosteroids. Surgery (lumboperitoneal or ventriculoperitoneal shunt or perhaps optic nerve sheath fenestration) may be indicated for prolonged incapacitating headache that is not responsive to medical management or lumbar puncture.


Fracture resistance of teeth restored with two different post-and-core designs cemented with two different cements: an in vitro study. Part I.
The purpose of this in vitro study was to investigate fracture resistance in teeth restored with cast post and cores with and without ferrule and using two different luting cements. Forty intact maxillary premolars were endodontically treated after their crowns were removed at 2.00 mm from the cementoenamel junction. Specimens were embedded in acrylic resin blocks in aluminum cylinders, 4.00 mm apical to the cementoenamel junction. Twenty specimens were ferruled, and half of the posts and cores were cemented with zinc-phosphate cement, while the other half was cemented with resin cement. The same procedures were followed for the nonferruled group. Loads were applied at an angle of 45 degrees and measured with a universal testing machine. ANOVA and Tukey test were used for statistical analyses; a significance level was established at 5%. Ferruled specimens showed greater resistance than nonferruled ones, regardless of the cement used. There was no statistical difference between the group of specimens cemented with resin cement and without ferrule and the ferruled groups. The non-ferruled group with zinc-phosphate cement showed the poorest results. A 2.00-mm cervical ferrule is important for fracture resistance of restored teeth, and resin cement has a better performance.


A woman's place: unpaid work in the home.
Household work has only recently become a valid topic of study. Feminist scholars were among the first social scientists to draw attention to women's unpaid work in the home. Although household work occupations are frequently used for assessment and treatment within practice, occupational therapy literature demonstrates a paucity in the area of these occupations. This review of the feminist literature summarizes theory and research that explore the historical, political, social, and personal meanings of household work. Feminist analysis of household work may sensitize occupational therapists to the complex interactions of these meanings and lead them to the realization that women's responsibility for unpaid work in the home may have repercussions in the daily lives of both women and men.


Expression and detrimental role of hematopoietic prostaglandin D synthase in spinal cord contusion injury.
Prostaglandin D(2) (PGD(2) ) is a potent inflammatory mediator, which is implicated in both the initiation and resolution of inflammation in peripheral non-neural tissues. Its role in the central nervous system has not been fully elucidated. Spinal cord injury (SCI) is associated with an acute inflammatory response, which contributes to secondary tissue damage that worsens functional loss. We show here, with the use of hematopoietic prostaglandin D synthase (HPGDS) deficient mice and a HPGDS selective inhibitor (HQL-79), that PGD(2) plays a detrimental role after SCI. We also show that HPGDS is expressed in macrophages in the injured mouse spinal cord and contributes to the increase in PGD(2) in the contused spinal cord. HPGDS(-/-) mice also show reduced secondary tissue damage and reduced expression of the proinflammatory chemokine CXCL10 as well as an increase in IL-6 and TGFβ-1 expression in the injured spinal cord. This was accompanied by a reduction in the expression of the microglia/macrophage activation marker Mac-2 and an increase in the antioxidant metallothionein III. Importantly, HPGDS deficient mice exhibit significantly better locomotor recovery after spinal cord contusion injury than wild-type (Wt) mice. In addition, systemically administered HPGDS inhibitor (HQL-79) also enhanced locomotor recovery after SCI in Wt mice. These data suggest that PGD(2) generated via HPGDS has detrimental effects after SCI and that blocking the activity of this enzyme can be beneficial.


Identification of targeted analyte clusters for studies of schizophrenia.
The search for biomarkers to diagnose psychiatric disorders such as schizophrenia has been underway for decades. Many molecular profiling studies in this field have focused on identifying individual marker signals that show significant differences in expression between patients and the normal population. However, signals for multiple analyte combinations that exhibit patterned behaviors have been less exploited. Here, we present a novel approach for identifying biomarkers of schizophrenia using expression of serum analytes from first onset, drug-naïve patients and normal controls. The strength of patterned signals was amplified by analyzing data in reproducing kernel spaces. This resulted in the identification of small sets of analytes referred to as targeted clusters that have discriminative power specifically for schizophrenia in both human and rat models. These clusters were associated with specific molecular signaling pathways and less strongly related to other neuropsychiatric disorders such as major depressive disorder and bipolar disorder. These results shed new light concerning how complex neuropsychiatric diseases behave at the pathway level and demonstrate the power of this approach in identification of disease-specific biomarkers and potential novel therapeutic strategies.


Honesty Speaks a Second Language.
Theories of dishonest behavior implicitly assume language independence. Here, we investigated this assumption by comparing lying by people using a foreign language versus their native tongue. Participants rolled a die and were paid according to the outcome they reported. Because the outcome was private, they could lie to inflate their profit without risk of repercussions. Participants performed the task either in their native language or in a foreign language. With native speakers of Hebrew, Korean, Spanish, and English, we discovered that, on average, people inflate their earnings less when they use a foreign language. The outcome is explained by a dual system account that suggests that self-serving dishonesty is an automatic tendency, which is supported by a fast and intuitive system. Because using a foreign language is less intuitive and automatic, it might engage more deliberation and reduce the temptation to lie. These findings challenge theories of ethical behavior to account for the role of the language in shaping ethical behavior.


Characterisation of the pathophysiology of neuropathy and sensory dysfunction in a mouse model of recessive dystrophic epidermolysis bullosa.
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic condition in which mutations in the type VII collagen gene ( COL7A1 ) lead to decreased expression of this anchoring protein of the skin, causing the loss of stability at the dermo-epidermal junction. Most patients with RDEB experience neuropathic pain and itch due to the development of a small fibre neuropathy, characterised by decreased intraepidermal innervation and thermal hypoaesthesia. To understand the physiopathology of this neuropathy, we used a mouse model of RDEB (Col7a1 flNeo/flNeo ) and performed a detailed characterisation of the somatosensory system. Col7a1 flNeo/flNeo mice showed a decrease in heat sensitivity, an increase in spontaneous scratching, and a significant decrease in intraepidermal nerve fibre density in the hindpaw; these changes were distal because there was no significant loss of unmyelinated or myelinated fibres in the nerve trunk. Of interest, we observed a decrease in axon diameter in both myelinated and unmyelinated fibres. This axonal damage was not associated with inflammation of the dorsal root ganglion or central projection targets at the time of assessment. These results suggest that in RDEB, there is a distal degeneration of axons produced by exclusive damage of small fibres in the epidermis, and in contrast with traumatic and acute neuropathies, it does not induce sustained neuroinflammation. Thus, this animal model emphasizes the importance of a healthy cutaneous environment for maintenance of epidermal innervation and faithfully replicates the pathology in humans, offering the opportunity to use this model in the development of treatments for pain for patients with RDEB.


The strength of the protein-material interaction determines cell fate.
Extracellular matrix (ECM) proteins are key mediators of cell/material interactions. The surface density and conformation of these proteins adsorbed on the material surface influence cell adhesion and the cellular response. We have previously shown that subtle variations in surface chemistry lead to drastic changes in the conformation of adsorbed fibronectin (FN). On poly(ethyl acrylate) (PEA), FN unfolds and displays domains for cell adhesion and FN-FN interaction, whereas on poly(methyl acrylate) (PMA) - with only one methyl group less - FN remains globular as it is in solution. The effect of the strength of the protein/material interaction in cell response, and its relation to protein density and conformation, has received limited attention so far. In this work, we used FN-functionalized AFM cantilevers to evaluate, via force spectroscopy, the strength of interaction between fibronectin and the underlying polymer which controls FN conformation (PEA and PMA). We found that the strength of FN/PEA interaction is significantly higher than FN/PMA, which limits the mobility of FN layer on PEA, reduces the ability of cells to mechanically reorganize FN and then leads to enhanced proteolysis and degradation of the surrounding matrix with compromised cell viability. By contrast, both PEA and PMA support cell adhesion when FN density is increased and also in the presence of serum or other serum proteins, including vitronectin (VN) and bovine serum albumin (BSA), which provide a higher degree of mobility to the matrix. The identification of parameters influencing cell response is of paramount importance for the design of biomaterials that will act as synthetic scaffolds for cells to anchor, grow and, eventually, become specialised tissues. Cells interact with materials through an intermediate layer of proteins adsorbed on the material surface. It is known that the density and conformation of these proteins determine cell behaviour. Here we show that the strength of protein/material interactions, which has received very limited attention so far, is key to understand the cellular response to biomaterials. Very strong protein/material interactions reduce the ability of cells to mechanically reorganize proteins at the material interface which results in enhanced matrix degradation, leading ultimately to compromised cell viability.


Surface-Bound Cucurbit[8]uril Catenanes on Magnetic Nanoparticles Exhibiting Molecular Recognition.
We demonstrate the preparation of surface-bound cucurbit[8]uril (CB[8]) catenanes on silica nanoparticles (NPs), where CB[8] was employed as a tethered supramolecular "handcuff" to selectively capture target guest molecules. In this catenane, CB[8] was threaded onto a methyl viologen (MV(2+) ) axle and immobilized onto silica NPs. The formation of CB[8] catenanes on NPs were confirmed by UV/Vis titration experiments and lithographic characterization, demonstrating a high density of CB[8] on the silica NPs surface, 0.56 nm(-2) . This CB[8] catenane system exhibits specific molecular recognition towards certain aromatic molecules such as perylene bis(diimide), naphthol and aromatic amino acids, and thus it can act as a nanoscale molecular receptor for target guests. Furthermore, we also demonstrate its use as an efficient and recyclable nano-platform for peptide separation. By embedding magnetic NPs inside silica NPs, separation could be achieved by simply applying an external magnetic field. Moreover, the peptides captured by the catenanes could be released by reversible single-electron reduction of MV(2+) . The entire process demonstrated high recoverability.


Ludwig Boltzmann Cluster Oncology (LBC ONC): first 10 years and future perspectives.
In 2008 the Ludwig Boltzmann Cluster Oncology (LBC ONC) was established on the basis of two previous Ludwig Boltzmann Institutes working in the field of hematology and cancer research. The general aim of the LBC ONC is to improve treatment of hematopoietic neoplasms by eradicating cancer-initiating and disease-propagating cells, also known as leukemic stem cells (LSC) in the context of leukemia. In a first phase, the LBC ONC characterized the phenotype and molecular aberration profiles of LSC in various malignancies. The LSC phenotypes were established in acute and chronic myeloid leukemia, in acute lymphoblastic leukemia and in chronic lymphocytic leukemia. In addition, the concept of preleukemic (premalignant) neoplastic stem cells (pre-L-NSC) was coined by the LBC ONC and was tested in myelodysplastic syndromes and myeloproliferative neoplasms. Phenotypic characterization of LSC provided a solid basis for their purification and for the characterization of specific target expression profiles. In a second phase, molecular markers and targets were validated. This second phase is ongoing and should result in the development of new diagnostics parameters and novel, more effective, LSC-eradicating, treatment strategies; however, many issues still remain to be solved, such as sub-clonal evolution, LSC niche interactions, immunologic control of LSC, and LSC resistance. In the forthcoming years, the LBC ONC will concentrate on developing LSC-eradicating strategies, with special focus on LSC resistance, precision medicine and translation of LSC-eradicating concepts into clinical application.


Pharmacokinetics of a CCR5 inhibitor in rhesus macaques following vaginal, rectal and oral application.
This study measured and compared the pharmacokinetics of CMPD167, a small molecule antiretroviral CCR5 inhibitor with potential as an HIV microbicide, following vaginal, rectal and oral administration in rhesus macaques. A vaginal hydroxyethylcellulose (HEC) gel, a rectal HEC gel, a silicone elastomer matrix-type vaginal ring and an oral solution, each containing CMPD167, were prepared and administered to rhesus macaques pretreated with Depo-Provera. CMPD167 concentrations in vaginal fluid, vaginal tissue (ring only), rectal fluid and blood plasma were quantified by HPLC-mass spectrometry. CMPD167 concentrations measured in rectal fluid, vaginal fluid and blood plasma were highly dependent on both the route of administration and the formulation type. Although rectal and vaginal fluid concentrations were highest when CMPD167 was administered locally (via either gel or ring), lower concentrations of the drug were also measured in these compartments following administration at the remote mucosal site or orally. CMPD167 levels in the vaginal and rectal fluid following oral administration were relatively low compared with local administration. The study provides clear evidence for vaginal-rectal and rectal-vaginal drug transfer pathways and suggests that oral pre-exposure prophylaxis with CMPD167 may be less efficacious at preventing sexual transmission of HIV-1 than topically applied products.


Type 1 insulin-like growth factor receptor (IGF-IR) signaling inhibits apoptosis signal-regulating kinase 1 (ASK1).
The type 1 insulin-like growth factor receptor (IGF-IR) is a receptor-tyrosine kinase that plays a critical role in signaling cell survival and proliferation. IGF-IR binding to its ligand, insulin-like growth factor (IGF-I) activates phosphoinositide 3-kinase (PI3K), promotes cell proliferation by activating the mitogen-activated protein kinase (MAPK) cascade, and blocks apoptosis by inducing the phosphorylation and inhibition of proapoptotic proteins such as BAD. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that is required for c-Jun N-terminal kinase (JNK) and p38 activation in response to Fas and tumor necrosis factor (TNF) receptor stimulation, and for oxidative stress- and TNFalpha-induced apoptosis. The results presented here indicate that ASK1 forms a complex with the IGF-IR and becomes phosphorylated on tyrosine residue(s) in a manner dependent on IGF-IR activity. IGF-IR signaling inhibited ASK1 irrespective of TNFalpha-induced ASK1 activation and resulted in decreased ASK1-dependent JNK1 stimulation. Signaling through IGF-IR rescued cells from ASK1-induced apoptotic cell death in a manner independent of PI3K activity. These results indicate that IGF-IR signaling suppresses the ASK-1-mediated stimulation of JNK/p38 and the induction of programmed cell death. The simultaneous activation of MAP kinases and the inhibition of the stress-activated arm of the cascade by IGF-IR may constitute a potent proliferative signaling system and is possibly a mechanism by which IGF-I can stimulate growth and inhibit cell death in a wide variety of cell types and biological settings.


Microbodies and the problem of mitochondrial regeneration in liver cells.
The cytoplasm of the hepatic cell contains, besides the well known organelles, microbodies, characterized by a single membrane, a finely granular matrix, and average dimensions below those of mitochondria. Microbodies, rare in normal cells, become more numerous in regenerative livers and in various pathological conditions. In these cases, they either evolve towards degenerative forms or else show structural modifications corresponding to transitional forms between them and mitochondria. This suggests to us that the microbodies can be precursors of mitochondria.


Comparison of bacterial genome assembly software for MinION data and their applicability to medical microbiology.
Translating the Oxford Nanopore MinION sequencing technology into medical microbiology requires on-going analysis that keeps pace with technological improvements to the instrument and release of associated analysis software. Here, we use a multidrug-resistant Enterobacter kobei isolate as a model organism to compare open source software for the assembly of genome data, and relate this to the time taken to generate actionable information. Three software tools (PBcR, Canu and miniasm) were used to assemble MinION data and a fourth (SPAdes) was used to combine MinION and Illumina data to produce a hybrid assembly. All four had a similar number of contigs and were more contiguous than the assembly using Illumina data alone, with SPAdes producing a single chromosomal contig. Evaluation of the four assemblies to represent the genome structure revealed a single large inversion in the SPAdes assembly, which also incorrectly integrated a plasmid into the chromosomal contig. Almost 50 %, 80 % and 90 % of MinION pass reads were generated in the first 6, 9 and 12 h, respectively. Using data from the first 6 h alone led to a less accurate, fragmented assembly, but data from the first 9 or 12 h generated similar assemblies to that from 48 h sequencing. Assemblies were generated in 2 h using Canu, indicating that going from isolate to assembled data is possible in less than 48 h. MinION data identified that genes responsible for resistance were carried by two plasmids encoding resistance to carbapenem and to sulphonamides, rifampicin and aminoglycosides, respectively.


Targeted alpha-radionuclide therapy of functionally critically located gliomas with 213Bi-DOTA-[Thi8,Met(O2)11]-substance P: a pilot trial.
Functionally critically located gliomas represent a challenging subgroup of intrinsic brain neoplasms. Standard therapeutic recommendations often cannot be applied, because radical treatment and preservation of neurological function are contrary goals. The successful targeting of gliomas with locally injected beta radiation-emitting (90)Y-DOTAGA-substance P has been shown previously. However, in critically located tumours, the mean tissue range of 5 mm of (90)Y may seriously damage adjacent brain areas. In contrast, the alpha radiation-emitting radionuclide (213)Bi with a mean tissue range of 81 microm may have a more favourable toxicity profile. Therefore, we evaluated locally injected (213)Bi-DOTA-substance P in patients with critically located gliomas as the primary therapeutic modality. In a pilot study, we included five patients with critically located gliomas (WHO grades II-IV). After diagnosis by biopsy, (213)Bi-DOTA-substance P was locally injected, followed by serial SPECT/CT and MR imaging and blood sampling. Besides feasibility and toxicity, the functional outcome was evaluated. Targeted radiopeptide therapy using (213)Bi-DOTA-substance P was feasible and tolerated without additional neurological deficit. No local or systemic toxicity was observed. (213)Bi-DOTA-substance P showed high retention at the target site. MR imaging was suggestive of radiation-induced necrosis and demarcation of the tumours, which was validated by subsequent resection. This study provides proof of concept that targeted local radiotherapy using (213)Bi-DOTA-substance P is feasible and may represent an innovative and effective treatment for critically located gliomas. Primarily non-operable gliomas may become resectable with this treatment, thereby possibly improving the prognosis.


Functional characterization of Kv channel beta-subunits from rat brain.
1. The potassium channel beta-subunit from rat brain, Kv beta 1.1, is known to induce inactivation of the delayed rectifier channel Kv1.1 and Kv1.4 delta 1-110. 2. Kv beta 1.1 was co-expressed in Xenopus oocytes with various other potassium channel alpha-subunits. Kv beta 1.1 induced inactivation in members of the Kv1 subfamily with the exception of Kv 1.6; no inactivation of Kv 2.1, Kv 3.4 delta 2-28 and Kv4.1 channels could be observed. 3. The second member of the beta-subunit subfamily, Kv beta 2, had a shorter N-terminal end, accelerated inactivation of the A-type channel Kv 1.4, but did not induce inactivation when co-expressed with delayed rectifiers of the Kv1 channel family. 4. To test whether this subunit co-assembles with Kv alpha-subunits, the N-terminal inactivating domains of Kv beta 1.1 and Kv beta 3 were spliced to the N-terminus of Kv beta 2. The chimaeric beta-subunits (beta 1/ beta 2 and beta 3/ beta 2) induced fast inactivation of several Kv1 channels, indicating that Kv beta 2 associates with these alpha-subunits. No inactivation was induced in Kv 1.3, Kv 1.6, Kv2.1 and Kv3.4 delta 2-28 channels. 5. Kv beta 2 caused a voltage shift in the activation threshold of Kv1.5 of about -10 mV, indicating a putative physiological role. Kv beta 2 had a smaller effect on Kv 1.1 channels. 6. Kv beta 2 accelerated the activation time course of Kv1.5 but had no marked effect on channel deactivation.


NCYM, a Cis-antisense gene of MYCN, encodes a de novo evolved protein that inhibits GSK3β resulting in the stabilization of MYCN in human neuroblastomas.
The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.


Oral naltrexone to enhance analgesia in patients receiving continuous intrathecal morphine for chronic pain: a randomized, double-blind, prospective pilot study.
Years' worth of observations suggest that morphine has both inhibitory and excitatory actions, and that selective blockade of excitatory effects by low doses of opioid antagonists (e.g., naltrexone) may paradoxically enhance morphine analgesia. The purpose of this pilot study was to evaluate and compare the analgesic efficacy and safety of two different low doses of oral naltrexone given in addition to chronic intrathecal morphine infusions in patients with chronic nonmalignant pain (CNMP). After institutional review board approval, 15 patients with CNMP receiving continuous intrathecal morphine were admitted into a prospective, randomized, double-blind, placebo-controlled, seven-day pilot study. Patients were randomized into three treatment groups based on oral naltrexone dose: 100 microg (Group A, n = 3), 10 microg (Group B, n = 7), or placebo (Group C, n = 5). All patients continued with their constant intrathecal morphine infusion, and in addition they received one capsule of study medication every 12 hours for seven days. Other analgesics or coanalgesics were kept at a constant dose level throughout the study. Patients rated pain scores (visual analogue score [VAS]; 0 = no pain, 10 = worst pain imaginable) and side effects three times daily throughout the study period. Efficacy measures included pain intensity difference (PID) scores, constructed so that positive scores indicate a reduction in pain intensity and negative scores indicate a worsening of pain. Fifteen patients (six male, nine female) with a mean (SD) age of 55 (10) years and weight of 81 (21) kg completed the study. The mean (SD) baseline VAS pain intensity rating was similar in all three groups (6.8 [1.5]). Baseline pain VAS score minus the lowest daily pain VAS score yielded the peak PID score. The peak PID score from Day 1 was statistically (p < 0.05) highest (median PID score: 5.9) in Group A compared with Group C. There was a trend in PID scores across Days 2 through 7, with median PID scores higher (i.e., greater pain relief p = 0.07) in Group A. In the daily global pain assessments, the pain scores across Days 2 through 7 approached significance (least pain) in Group A compared to Group C (p = 0.07) or B (p = 0.08). Side effects were common (93 percent of patients), minor (headache, nausea, sedation, dry mouth), and similar across treatment groups. No serious adverse events were observed, and no evidence of opioid withdrawal was seen. 1) Patients with chronic pain who received oral naltrexone 100 microg BID in addition to their chronic intrathecal morphine infusions demonstrated the greatest improvement (p = 0.07) in their daily pain scores. Because of the small sample size, the results did not reach traditional levels of significance. 2) Side effects were common, minor, and similar across treatment groups. 3) No serious adverse events were recorded. 4) No evidence of opioid antagonist toxicity or opioid withdrawal was observed.


Analysis of vkorc1 polymorphisms in Norway rats using the roof rat as outgroup.
Certain mutations in the vitamin K epoxide reductase subcomponent 1 gene (vkorc1) mediate rodent resistance to warfarin and other anticoagulants. Testing for resistance often involves analysis of the vkorc1. However, a genetic test for the roof rat (Rattus rattus) has yet to be developed. Moreover, an available roof rat vkorc1 sequence would enable species identification based on vkorc1 sequence and the evaluation of natural selection on particular vkorc1 polymorphisms in the Norway rat (R. norvegicus). We report the coding sequence, introns and 5' and 3' termini for the vkorc1 gene of roof rats (R. r. alexandrinus and R. r. frugivorus) from Uganda, Africa. Newly designed PCR primers now enable genetic testing of the roof rat and Norway rat. Only synonymous and noncoding polymorphisms were found in roof rats from Uganda. Both nominal subspecies of roof rats were indistinguishable from each other but were distinct from R. losea and R. flavipectus; however, the roof rat also shares at least three coding sequence polymorphisms with R. losea and R. flavipectus. Many of recently published vkorc1 synonymous and non-synonymous single nucleotide polymorphisms (SNPs) in Norway rats are likely SNPs from roof rats and/or other Rattus species. Tests applied to presumably genuine Norway rat vkorc1 SNPs are consistent with a role for selection in two populations carrying the derived Phe63Cys and Tyr139Cys mutations. Geographic mapping of vkorc1 SNPs in roof rats should be facilitated by our report. Our assay should be applicable to most species of Rattus, which are intermediate in genetic distance from roof and Norway rats. Vkorc1-mediated resistance due to non-synonymous coding SNPs is not segregating in roof rats from Uganda. By using the roof rat sequence as a reference vkorc1, SNPs now can be assigned to the correct rat species with more confidence. Sampling designs and genotyping strategies employed so far have helped detect candidate mutations underlying vkorc1-mediated resistance, but generally provided unsuitable data to test for selection. We propose that our understanding of vkorc1-mediated evolution of resistance in rodents would benefit from the adoption of sampling and genotyping designs that enable tests for selection on vkorc1.


Early interaction of rhinoviruses with host cells.
The rate of attachment of type 2 virions to suspensions of HeLa cells is much greater than that of type 14, but the number of receptor sites per cell is similar for each type. The receptor sites may be partly saturated with excess virions; attachment is greatly reduced after about 10(4) particles have been taken up per cell. A lack of saturation of type 14 receptors by excess type 2 indicates that their receptor sites are separate on the cell surface. Excess of type 2 blocks attachment of type 1A, however, and excess of type 14 blocks type 51. Attachment of the human rhinoviruses is temperature-dependent with a Q(10) of 2.7. The eclipse reaction is also temperature-dependent. At 34.5 C, the irreversible eclipse of cell-associated rhinovirus type 2 requires only a few minutes, whereas the rate of eclipse of cell-associated type 14 is considerably slower. The eclipse product of type 2 rhinovirus has been recovered from infected cells. It sediments at about 90% of the rate of the infective virions and is missing virus polypeptide 4 (the smallest of the capsid polypeptides). Upon being subjected to CsCl gradient centrifugation, virus polypeptide 2 is also lost but the product still contains ribonucleic acid and bands at about 1.45 g/cc.


Solubilization of Acid-swollen cellulose by an enzyme system from a species of alternaria.
An unknown species of Alternaria, when grown on a medium containing carboxymethylcellulose as a carbon source produced a mixture of extracellular enzymes which solubilized acid-swollen cellulose. The product of the hydrolysis was a 1:2 molar mixture of cellobiose and glucose. The organism apparently produced no cellobiase. It is suggested that the mixture of cellulolytic enzymes contains at least two different enzymes which degrade cellulose in an endwise manner.


Incidence, Characteristics, and Outcomes of Myocardial Infarction in Patients With Peripheral Artery Disease: Insights From the EUCLID Trial.
Patients with peripheral artery disease (PAD) are at high risk for myocardial infarction (MI). To characterize the incidence and types of MI in a PAD population, identify factors associated with MI, and determine the association of MI with cardiovascular mortality and acute limb ischemia. The Study Comparing Cardiovascular Effects of Ticagrelor and Clopidogrel in Patients With Peripheral Artery Disease (EUCLID) was a double-blind randomized clinical trial conducted at 811 sites in 28 countries that randomized 13 885 patients with symptomatic PAD to monotherapy with ticagrelor or clopidogrel. Participants had an ankle-brachial index (ABI) of 0.80 or less or previous lower extremity revascularization. Median follow-up was 30 months. For these analyses, patients were evaluated for MI occurrence during follow-up irrespective of treatment. Data were analyzed from June 2017 to September 2018. An adjudication clinical events committee classified MI as type 1 (spontaneous), type 2 (secondary), type 3 (sudden cardiac death), type 4a (less than 48 hours after percutaneous coronary intervention), type 4b (definite stent thrombosis), or type 5 (less than 72 hours after coronary artery bypass graft). A multivariate regression model was developed by stepwise selection to identify factors associated with MI, and a time-dependent multivariate Cox regression analysis was performed to determine the association of MI with cardiovascular death and acute limb ischemia requiring hospitalization. Of the 13 885 patients included in this analysis, 9997 (72.0%) were male, and the median (interquartile range) age was 66 (60-73) years. Myocardial infarction occurred in 683 patients (4.9%; 2.4 events per 100 patient-years) during a median follow-up of 30 months. Patients experiencing MI were older (median [interquartile range] age, 69 [62-75] vs 66 [60-72] years), more likely to have diabetes (349 of 683 [51.1%] vs 4996 of 13 202 [37.8%]) or a previous lower extremity revascularization (466 of 683 [68.2%] vs 7409 of 13 202 [56.1%]), and had a lower ABI (if included by ABI) compared with censored patients. Of the 683 patients with MI during follow-up, the most common MI type was type 1 (405 [59.3%]), followed by type 2 (236 [34.6%]), type 4a (14 [2.0%]), type 3 (12 [1.8%]), type 4b (11 [1.6%]), and type 5 (5 [0.7%]). Postrandomization MI was independently associated with cardiovascular death (adjusted hazard ratio, 9.0; 95% CI, 7.3-11.2; P < .001) and acute limb ischemia requiring hospitalization (adjusted hazard ratio, 2.5; 95% CI, 1.3-5.0; P = .008). Approximately 5% of patients with symptomatic PAD had an MI during a median follow-up of 30 months. Type 1 MI (spontaneous) was the most common MI type; however, one-third of MIs were type 2 MI (secondary). More research is needed to identify therapies to reduce the risk of MI in patients with PAD and to improve management of type 2 MI. ClinicalTrials.gov Identifier: NCT01732822.


Phase II study of rituximab in combination with chop chemotherapy in patients with previously untreated, aggressive non-Hodgkin's lymphoma.
To determine the safety and efficacy of the combination of the chimeric anti-CD20 antibody Rituxan (rituximab, IDEC-C2B8; Genentech Inc, South San Francisco, CA) and cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy in patients with aggressive non-Hodgkin's lymphoma (NHL). Thirty-three patients with previously untreated advanced aggressive B-cell NHL received six infusions of Rituxan (375 mg/m2 per dose) on day 1 of each cycle in combination with six doses of CHOP chemotherapy given on day 3 of each cycle. The ORR by investigator assessment confirmed by the sponsor was 94% (31 of 33 patients). Twenty patients experienced a complete response (CR) (61%), 11 patients had a partial response (PR) (33%), and two patients were classified as having progressive disease. In the 18 patients with an International Prognostic Index (IPI) score > or = 2, the combination of Rituxan plus CHOP achieved an ORR of 89% and CR of 56%. The median duration of response and time to progression had not been reached after a median observation time of 26 months. Twenty-nine of 31 responding patients remained in remission during this follow-up period, including 15 of 16 patients with an IPI score > or = 2. The most frequent adverse events attributed to Rituxan were fever and chills, primarily during the first infusion. Rituxan did not seem to compromise the ability of patients to tolerate CHOP; all patients completed the entire six courses of the combination. The bcl-2 translocation of blood or bone marrow was positive at baseline in 13 patients; 11 patients had follow-up specimens obtained (eight CR, three PR), and all had a negative bcl-2 status after therapy. Only one patient has reconverted to bcl-2 positivity, and all patients remain in clinical remission. This is the first report to demonstrate the safety and efficacy of the Rituxan chimeric anti-CD20 antibody in combination with standard-dose CHOP in the treatment of aggressive B-cell lymphoma. The clinical responses are at least comparable to those achieved with CHOP alone with no significant added toxicity. The presence or absence of the bcl-2 translocation did not affect the ability of patients to achieve a CR with this regimen. The ability to achieve sustained remissions in patients with an IPI score > or = 2 warrants further investigation with a randomized study.


CA1 and CA3 differentially support spontaneous retrieval of episodic contexts within human hippocampal subfields.
The hippocampus plays a critical role in spatial and episodic memory. Mechanistic models predict that hippocampal subfields have computational specializations that differentially support memory. However, there is little empirical evidence suggesting differences between the subfields, particularly in humans. To clarify how hippocampal subfields support human spatial and episodic memory, we developed a virtual reality paradigm where participants passively navigated through houses (spatial contexts) across a series of videos (episodic contexts). We then used multivariate analyses of high-resolution fMRI data to identify neural representations of contextual information during recollection. Multi-voxel pattern similarity analyses revealed that CA1 represented objects that shared an episodic context as more similar than those from different episodic contexts. CA23DG showed the opposite pattern, differentiating between objects encountered in the same episodic context. The complementary characteristics of these subfields explain how we can parse our experiences into cohesive episodes while retaining the specific details that support vivid recollection.


A molecular survey of Eimeria in chickens across Australia.
Coccidiosis is a costly enteric disease of chickens caused by protozoan parasites of the genus Eimeria. Disease diagnosis and management is complicated since there are multiple Eimeria species infecting chickens and mixed species infections are common. Current control measures are only partially effective and this, combined with concerns over vaccine efficacy and increasing drug resistance, demonstrates a need for improved coccidiosis diagnosis and control. Before improvements can be made, it is important to understand the species commonly infecting poultry flocks in both backyard and commercial enterprises. The aim of this project was to conduct a survey and assessment of poultry Eimeria across Australia using genetic markers, and create a collection of isolates for each Eimeria species. A total of 260 samples (faecal or caecal) was obtained, and survey results showed that Eimeria taxa were present in 98% of commercial and 81% of backyard flocks. The distribution of each Eimeria species was widespread across Australia, with representatives of all species being found in every state and territory, and the Eimeria species predominating in commercial flocks differed from those in backyard flocks. Three operational taxonomic units also occurred frequently in commercial flocks highlighting the need to understand the impact of these uncharacterised species on poultry production. As Eimeria infections were also frequent in backyard flocks, there is a potential for backyard flocks to act as reservoirs for disease, especially as the industry moves towards free range production systems. This Eimeria collection will be an important genetic resource which is the crucial first step in the development of more sophisticated diagnostic tools and the development of new live vaccines which ultimately will provide savings to the industry in terms of more efficient coccidiosis management.


Structure and population dynamics of the major satellite DNA in the red flour beetle Tribolium castaneum.
In the beetle genus Tribolium, satellite DNAs comprise a significant amount of pericentromeric heterochromatin and are characterized by rapid turnover resulting in species specific profiles. In the present work we characterize the major pericentromeric satellite DNA TCAST of the beetle T. castaneum and analyse its population dynamics. Using direct sequencing of genomic PCR products we show that the TCAST satellite exists in the form of two related subfamilies: Tcast1a and Tcast1b that make up 20 and 15% of the genome, respectively. Tcast1a and Tcast1b have consensus sequences of 377 and 362 bp respectively, share an average similarity of 79% and are characterized by a divergent, subfamily specific region of approximately 100 bp. The two subfamilies are prevalently organized in the interspersed form, although a portion exists in the form of homogenous tandem arrays composed of only Tcast1a or Tcast1b. The pattern of restriction enzyme digestion indicates that Tcast1a and Tcast1b are organized in composite higher order repeats. Comparison of sequence variability of Tcast1a and Tcast1b among ten strains reveals a difference in the frequency of particular mutations present at some positions. However, no difference in the organization and in the amount of subfamilies was detected among strains. The results show that direct genomic sequencing can be a useful method for the detection of population specific features of satellite DNA. In the case of TCAST satellite DNA, changes in the mutational profiles seem to represent the first step in the genesis of a population specific satellite profile.


Mitogenome sequencing reveals shallow evolutionary histories and recent divergence time between morphologically and ecologically distinct European whitefish (Coregonus spp.).
The advent of second-generation sequencing has made it possible to quickly and economically generate whole mitochondrial genome (mitogenome) sequences. To date, mitogenome studies of nonmodel organisms have demonstrated increased power for resolving interspecies relationships. We explored an alternate use of such data to recover relationships and population history of closely related lineages with a shallow evolutionary history. Using a GS-FLX platform, we sequenced 106 mitogenomes from the Coregonus lavaretus (Europe) and Coregonus clupeaformis (North America) species complexes to investigate the evolutionary history of the endangered Danish North Sea houting (NSH) and other closely related Danish and Baltic European lake whitefish (ELW). Two well-supported clades were found within both ELW and NSH, probably reflecting historical introgression via Baltic migrants. Although ELW and NSH are not reciprocally monophyletic, they share no haplotypes, suggesting recent, but strong, reproductive isolation. The divergence time between NSH and the geographically closest ELW population was estimated using IMa, assuming isolation with migration and a new mutation rate estimate chosen to avoid time-dependency effects. The estimate of c. 2700 bp was remarkably similar to results obtained using microsatellite markers. Within North American C. clupeaformis, the divergence time between the two lineages (Atlantic and Acadian) was estimated as between 20,000 and 60,000 bp. Under the assumption that NSH and ELW colonized Denmark following the last glacial maximum, Bayesian Serial SimCoal analysis showed consistency with a scenario of long-term stability, resulting from a rapid initial sixfold population expansion. The findings illustrate the utility of mitogenome data for resolving recent intraspecific divergence events and provide evidence for recent reproductive isolation of the phenotypically divergent NSH.


Effects of hypoxia on heparan sulfate in bovine aortic and pulmonary artery endothelial cells.
Newly synthesized heparan sulfates purified from the cell layer of bovine aortic endothelial cells (BAECs) and main pulmonary artery endothelial cells (BPAECs) cultured under either normoxic (21% oxygen) or hypoxic (3% oxygen) conditions were characterized by size, charge, and capacity to bind to antithrombin III. Incorporation of radiolabeled sulfate into cell layer-associated heparan sulfate was reduced by 70% in BAECs and by 45% in BPAECs during exposure to 3% oxygen; degradation of radiolabeled heparan sulfate was not affected by hypoxia. However, the percentage of total radiolabeled heparan sulfate that bound to antithrombin III was increased by 33% for BAECs and by 120% for BPAECs when compared with radiolabeled heparan sulfate synthesized during the 21% oxygen exposure. Both the high- and low-antithrombin III affinity radiolabeled heparan sulfate consisted of two components of different sizes; the low-affinity components (mean sizes, 60 and 40 kd) generated under normoxic conditions were smaller than their respective high-affinity components (mean sizes, 70 and 55 kd) by molecular sieve chromatography. The components of low-antithrombin III affinity heparan sulfate generated during exposure to 3% oxygen were increased in size compared with the corresponding low-affinity components generated during the 21% oxygen exposure for both BPAECs and BAECs. In addition, the amount of the larger high-antithrombin III affinity component was reduced in both cell types exposed to hypoxia. There was no difference in functional heparin-like activity per dish between cells cultured at 3% and 21% oxygen; BAECs had twofold to threefold greater activity per dish than did BPAECs at both levels of oxygen.(ABSTRACT TRUNCATED AT 250 WORDS)


The heart microbiome of insectivorous bats from Central and South Eastern Europe.
Host associated microbiome not only may affect the individual health-status or provide insights into the species- or group specific bacterial communities but may act as early warning signs in the assessment of zoonotic reservoirs, offering clues to predict, prevent and control possible episodes of emerging zoonoses. Bats may be carriers and reservoirs of multiple pathogens such as viruses, bacteria and parasites, showing in the same time robust immunity against many of them. The microbiota plays a fundamental role on the induction, training and function of the host immune system and the immune system has largely evolved in order to maintain the symbiotic relationship of the host with these diverse microbes. Thus, expanding our knowledge on bat-associated microbiome it can be usefully in understanding bats' outstanding immune capacities. The aim of this study was to investigate the presence of different bacterial communities in heart tissue of insectivorous bats, Nyctalus noctula, Pipistrellus pipistrellus and Rhinoplophus hipposideros, from Central and Eastern Europe using high-throughput sequencing of variable regions of the 16S rRNA. In addition, species-specific PCRs were used to validate the presence of the vector-borne pathogens Bartonella spp. and Rickettsia spp. In this study we identified a wide variety of bacterial groups, with the most abundant phyla being Proteobacteria and Firmicutes. The results showed that at individual level, the year or location had no effect on the diversity and composition of the microbiome, however host species determined both structure and abundance of the bacterial community. We report the presence of vector-borne bacteria Bartonella spp. in samples of N. noctula and indications of Rickettsia spp. in R. hipposideros. Our results provide a first insight into the bacterial community found in heart tissue of bats from Central and South Eastern Europe.


Human genomic regions with exceptionally high levels of population differentiation identified from 911 whole-genome sequences.
Population differentiation has proved to be effective for identifying loci under geographically localized positive selection, and has the potential to identify loci subject to balancing selection. We have previously investigated the pattern of genetic differentiation among human populations at 36.8 million genomic variants to identify sites in the genome showing high frequency differences. Here, we extend this dataset to include additional variants, survey sites with low levels of differentiation, and evaluate the extent to which highly differentiated sites are likely to result from selective or other processes. We demonstrate that while sites with low differentiation represent sampling effects rather than balancing selection, sites showing extremely high population differentiation are enriched for positive selection events and that one half may be the result of classic selective sweeps. Among these, we rediscover known examples, where we actually identify the established functional SNP, and discover novel examples including the genes ABCA12, CALD1 and ZNF804, which we speculate may be linked to adaptations in skin, calcium metabolism and defense, respectively. We identify known and many novel candidate regions for geographically restricted positive selection, and suggest several directions for further research.