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Introduction: The COVID-19 pandemic has had an impact on ischaemic stroke management, with a reported decrease in hospital admissions, and even disruptions in healthcare and increased in-hospital mortality. However, there is a lack of evidence on the impact of the pandemic on functional prognosis. The aim of this study is to analyse the effect of the COVID-19 pandemic on the 3-month functional outcomes of patients hospitalised due to acute ischaemic stroke in Aragon (Spain). Methods: We reviewed the data of all patients admitted due to ischaemic stroke to any hospital in our regional healthcare system between 30 December 2019 and 3 May 2020. We compared modified Rankin Scale scores and mortality at 3 months in patients hospitalised before and after the declaration of a state of emergency due to the COVID-19 pandemic. Results: In total, 318 patients with acute ischaemic stroke met our inclusion criteria. No differences were observed between periods in global or specific characteristics, with the exception of a higher proportion of patients older than 80 years during the first period (42.2% vs 29.0%, P = .028). In the comparative analysis, we found no significant differences in mortality (12.3 vs 7.9, P = .465) or in the proportion of patients with modified Rankin Scale scores ≤ 2 (57.7% vs 57.1%, P = .425) at 3 months. Conclusion: To our knowledge, this is the first study to analyse the impact of COVID-19 pandemic on the 3-month functional outcomes of patients with ischaemic stroke. In our region, there has been no increase in rates of mortality or disability at 3 months in patients admitted due to ischaemic stroke during the pandemic. Resumen: Introducción: La pandemia COVID-19 ha tenido un impacto en el manejo del ictus isquémico, se ha descrito una disminución de los ingresos hospitalarios e incluso una interrupción en la cadena de atención y aumento de la mortalidad intrahospitalaria. Sin embargo, falta evidencia sobre su impacto en el pronóstico funcional. El objetivo de este estudio es analizar el efecto de la pandemia COVID-19 en el pronóstico funcional a tres meses de los pacientes con ictus isquémico agudo hospitalizados en Aragón. Material y métodos: Revisamos los datos de todos los pacientes ingresados por ictus isquémico ​​en todos los hospitales de nuestro sistema sanitario entre el 30 de diciembre de 2019 y el 3 de mayo de 2020. Comparamos su mRS y mortalidad a tres meses de los hospitalizados antes y después de haberse establecido el estado de emergencia secundario a la pandemia COVID-19. Resultados: En total, 318 pacientes con ictus isquémico agudo cumplieron nuestros criterios de inclusión. No hubo diferencias en las características globales y específicas de cada período, excepto por una mayor proporción de pacientes mayores de 80 años de edad durante el período pre-CoV (42,2% vs 29,0%, p = 0,028). En el análisis comparativo, no encontramos una diferencia significativa en la mortalidad (12,3 vs.7,9, p = 0,465) o la proporción de pacientes con mRS ≤2 (57,7% vs 57,1%, p = 0,425) a los tres meses. Conclusión: Hasta donde sabemos, este es el primer estudio que analiza el impacto de la pandemia de COVID-19 en el pronóstico funcional a tres meses de pacientes con ictus isquémico. En nuestra Comunidad Autónoma, no ha habido un incremento en la mortalidad o discapacidad a tres meses de pacientes hospitalizados por ictus isquémico durante el período COVID-19.

Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer. We had previously identified a set of RBPs that were highly dysregulated in B-cell acute lymphoblastic leukemia (B-ALL) with MLL translocations, which carry a poor prognosis. Here, we sought to functionally characterize these dysregulated RBP genes by performing a focused CRISPR dropout screen in B-ALL cell lines, finding dependencies on several genes including EIF3E, EPRS and USO1. Validating our findings, CRISPR/Cas9-mediated disruption of USO1 in MLL-translocated B-ALL cells reduced cell growth, promoted cell death, and altered the cell cycle. Transcriptomic analysis of USO1-deficient cells revealed alterations in pathways related to mTOR signaling, RNA metabolism, and targets of MYC. In addition, USO1-regulated genes from these experimental samples were significantly and concordantly correlated with USO1 expression in primary samples collected from B-ALL patients. Lastly, we found that loss of Uso1 inhibited colony formation of MLL-transformed in primary bone marrow cells from Cas9-EGFP mice. Together, our findings demonstrate an approach to performing focused sub-genomic CRISPR screens and highlight a putative RBP vulnerability in MLL-translocated B-ALL, thus identifying potential therapeutic targets in this disease.

Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes. U2AF1 mutations that code for the most common amino acid substitutions are always heterozygous, and the retained wild-type allele is expressed, suggesting that mutant hematopoietic cells may require the residual wild-type allele to be viable. We show that hematopoiesis and RNA splicing in U2af1 heterozygous knock-out mice was similar to control mice, but that deletion of the wild-type allele in U2AF1(S34F) heterozygous mutant expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of wild-type U2AF1 for survival in vivo and that U2AF1 is a haplo-essential cancer gene. Mutant U2AF1 (S34F) expressing cells were also more sensitive to reduced expression of wild-type U2AF1 than non-mutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the wild-type U2af1 allele was deleted compared to when it was not deleted. These results suggest that selectively targeting the wild-type U2AF1 allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboring U2AF1 mutations.

Background: Missense mutations of the TP53 gene frequently occur in patients with myelodysplastic syndrome and acute myeloid leukemia. The functional consequences of mutant p53 expression on hematopoiesis remain incompletely studied. Whether TP53 mutations confer a loss-of-function, gain-of-function, or dominant-negative effect in response to chemotherapies has not been fully explored. One of these hotspot mutations is a G-to-A substitution at nucleotide 525 of TP53, which codes for an Arg175His (R175H) substitution. Thus, we characterized hematopoiesis in Trp53R172H constitutive knock-in mice, corresponding to R175H TP53 mutation in patients, and compared the results to Trp53 knock-out and control mice. Methods: Five genotypes of mice were analyzed (Trp53+/+ [WT], Trp53R172H/+, Trp53R172H/R172H, Trp53+/-, and Trp53-/-). Frequencies of peripheral blood (PB) and bone marrow (BM) cells were measured by flow cytometry and fluorouracil (5-FU) and N-ethyl-N-nitrosourea (ENU) were used as hematopoietic stressors. Results: There was no change in the PB counts or lineage distribution of mature PB and BM cells in Trp53R172H/+ and Trp53R172H/R172H mice compared to WT or Trp53 knock-out mice at 8-15 weeks of age. We found that expression of Trp53R172H mutation in mice does not cause hematopoietic stem cell (HSC) expansion under basal conditions, in contrast to heterozygous and homozygous deletion of Trp53, but does confer a functional advantage to HSCs in a competitive repopulation assay. Trp53R172H HSCs expand further following ENU exposure, similar to Trp53-/- cells and consistent with a dominant-negative effect of Trp53R172H. Mechanistically, mutant p53R172H has a dominant-negative effect on p21-induced expression following ENU treatment. Repeated 5-FU injections drive proliferation of progenitors. To test the hematopoietic cell-intrinsic effects of mutant p53R172H on white blood cell count recovery, we exposed mice reconstituted with Trp53+/+, Trp53+/-, Trp53-/- or Trp53R172H/+ BM cells to a single dose of 5-FU and followed mice weekly for a month. Trp53R172H/+ mice were least affected by the 5-FU challenge and showed the fastest and largest recovery of WBC count compared to Trp53+/+, Trp53+/- and Trp53-/- mice. These results demonstrate that mutant p53R172H confers a gain-of-function property following 5-FU exposure that was not seen with deletion of Trp53. Conclusion: Our data suggest that in vivo expression of mutant Trp53R172H has minimal effects on basal hematopoiesis. However, mutant Trp53R172H induces either a gain-of-function or a dominant-negative effect depending on the type of hematopoietic stress and hematopoietic phenotype being observed. Overall, our results indicate that the functional consequences induced by various Trp53 mutations (e.g., deletion and point mutation) are not identical, but are influenced by the type of hematopoietic stress. Citation Format: Tuoen Liu, Tanzir Ahmed, Michael O. Alberti, Brian A. Wadugu, Jin Shao, Sarah Grieb, Grace Przybyl, Matthew Ndonwi, Matthew J. Walter. Mutant Trp53 R172H has gain-of-function or dominant-negative effects in response to hematopoietic stressors in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 841.

Somatic mutations in the spliceosome geneU2AF1are common in patients with myelodysplastic syndromes.U2AF1mutations that code for the most common amino acid substitutions are always heterozygous, and the retained wild-type allele is expressed, suggesting that mutant hematopoietic cells may require the residual wild-type allele to be viable and cause disease. We show that hematopoiesis and RNA splicing inU2af1heterozygous knock-out mice was similar to control mice, but that deletion of the wild-type allele in U2AF1(S34F) heterozygous mutant expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of wild-type U2AF1 for survivalin vivoand thatU2AF1is a haplo-essential cancer gene. Mutant U2AF1 (S34F) expressing cells were also more sensitive to reduced, but not absent, expression of wild-type U2AF1 than non-mutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the wild-typeU2af1allele was deleted compared to when it was not deleted. These results suggest that selectively targeting the wild-typeU2AF1allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboringU2AF1mutations.

SUMMARY Somatic mutations in spliceosome genes are found in ~50% of patients with myelodysplastic syndromes (MDS), a myeloid malignancy associated with low blood counts. Expression of the mutant splicing factor U2AF1(S34F) alters hematopoiesis and mRNA splicing in mice. Our understanding of the functionally relevant alternatively spliced target genes that cause hematopoietic phenotypes in vivo remains incomplete. Here, we demonstrate that reduced expression of H2afy1.1, an alternatively spliced isoform of the histone H2A variant gene H2afy, is responsible for reduced B cells in U2AF1(S34F) mice. Deletion of H2afy or expression of U2AF1(S34F) reduces expression of Ebf1 (early B cell factor 1), a key transcription factor for B cell development, and mechanistically, H2AFY is enriched at the EBF1 promoter. Induced expression of H2AFY1.1 in U2AF1(S34F) cells rescues reduced EBF1 expression and B cells numbers in vivo. Collectively, our data implicate alternative splicing of H2AFY as a contributor to lymphopenia induced by U2AF1(S34F) in mice and MDS., Graphical Abstract, In brief Kim et al. demonstrate that H2AFY is a functional target of U2AF1(S34F)-induced alternative splicing, a common spliceosome gene mutation in myelodysplastic syndromes. H2afy−/− and U2AF1(S34F) mice have similar defective B cell development. H2AFY occupies the Ebf1 promoter, a key B cell differentiation transcription factor, providing a potential mechanism to regulate its expression.

Background: Measurement of tacrolimus using the ARCHITECT immunoassay analyzer requires a manual extraction step that puts clinical laboratory workers at risk for ergonomic injury. Therefore, we developed 2 batched extraction systems for tacrolimus measurement on the ARCHITECT analyzer and describe their features herein. Methods: Two batched extraction methods were developed at 2 different laboratories. The batched extraction methods allow processing of at least 20 specimens at a time. We evaluated the analytical performance of those methods and compared them with the United States Food and Drug Administration (FDA)–cleared process for manually extracting individual specimens. Results: Comparing the performance of batched- and individual-extraction methods revealed that both methods had comparable between-day imprecision, high patient-results correlation ( R 2 values ≥0.9869), equivalent functional sensitivity (0.48 ng/mL), and good linearity between 1 ng per mL and 25 ng per mL. Further, we observed decreased delta check–identified errors using the batched method. Conclusion: The 2 developed batched extraction methods for tacrolimus measurement that we describe herein demonstrate excellent performance and can replace individual specimen extraction. * Abbreviations: : TDM : therapeutic drug monitoring ELITE : Efficacy Limiting Toxicity Elimination LC/MS-MS : liquid chromatography–tandem mass spectrometry UCLA : University of California, Los Angeles EDTA : ethylenediaminetetraacetic acid RCF : relative centrifugal force QC : quality control FDA : United States Food and Drug Administration ANOVA : analysis of variance TAT : turnaround time

Introduction. Mutations in TP53 are common (~18%) in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia and often undergo loss of heterozygosity. Our understanding of the hematopoietic consequences of expressing mutant TP53-R175H, one of the most common mutations in MDS, is incomplete. In addition, whether TP53-R175H confers a loss-of-function, gain-of-function, or dominant-negative effect in response to chemotherapy has not been fully explored. Methods. We used a constitutive knock-in mouse model expressing TRP53-R172H (G-to-A substitution at nucleotide 515), corresponding to human mutant TP53-R175H. We generated Trp53R172H/+ and Trp53R172H/R172H mice and compared them to wild type (WT), Trp53+/-, and Trp53-/- mice. Peripheral blood (PB) and bone marrow (BM) was analyzed in non-BM transplant conditions, following a non-competitive BM transplant, and following a competitive BM transplant with or without exposure to N-ethyl-N-nitrosourea (ENU) and 5-fluorouracil (5FU). Results. BM hematopoietic stem and progenitor cells (HSPC), including LSK-SLAM cells, were increased in Trp53+/- and Trp53-/-mice (n=4-8, 8-15 weeks old, P TP53 mutant cells can clonally expand in patients following cytotoxic chemotherapy. Therefore, we investigated the response of TRP53R172H/+ mutant cells to alkylator (ENU) exposure. We created mixed BM chimeric mice by transplanting test (WT, Trp53+/-, Trp53R172H/+ and Trp53-/-) and WT competitor BM in a 1:3 ratio, respectively. Following engraftment, chimeric mice received vehicle or ENU (2 doses of 100 mg/kg, 9 days apart). ENU-exposed Trp53R172H/+ cells have a robust PB multilineage competitive advantage relative to placebo (Fig. 1A, n = 4-5, 2-fold increase at 10 weeks post-ENU, P Next, we asked whether mutant TRP53-R172H has similar or different effects as Trp53 deletion following exposure to an alternative chemotherapy (5FU). We first treated mice with a single dose of 5FU (200 mg/kg) to deplete cells and monitored WBC count recovery for 4 weeks. Trp53R172H/+ mice had significantly higher recovery WBC counts compared to WT, Trp53+/-, andTrp53-/- mice (n = 5-15, P Collectively, the results indicate that mutant TRP53-R172H may induce a gain-of-function or a dominant-negative effect depending on the exposure to specific hematopoietic stresses. Disclosures No relevant conflicts of interest to declare.

Introducción Alrededor de 15 millones de personas sufren un ictus cada año, de los que un 10-15% ocurre en menores de 50 años (ictus en el adulto joven). La prevalencia de los distintos factores de riesgo vascular y las estrategias sanitarias para el manejo del ictus varían a nivel mundial, siendo interesante conocer la epidemiología y las características específicas de cada región. El objetivo de este estudio fue determinar la prevalencia de los diferentes factores de riesgo vascular, la etiología y las características de los ictus isquémicos en el adulto joven en la comunidad autónoma de Aragón. Métodos Estudio multicéntrico, de corte transversal, realizado por los Servicios de Neurología de todos los hospitales del Servicio Aragonés de Salud (SALUD). Se identificó a todos los pacientes entre 18 y 50 años que ingresaron en cualquiera de estos hospitales con el diagnóstico de ictus isquémico o AIT entre enero del 2005 y diciembre del 2015. Se recogieron variables demográficas, factores de riesgo vascular y tipo de ictus isquémico entre otras. Resultados En el periodo de estudio, 786 pacientes entre 18 y 50 años ingresaron con el diagnóstico de ictus isquémico o AIT en algún hospital del SALUD, con una tasa anual promedio de 12, 3 por 100.000 habitantes. La mediana de su edad fue de 45 años (RIQ: 40-48 años). El factor de riesgo vascular más prevalente fue el tabaquismo, 404 (51, 4%). La mayoría fue de causa indeterminada (36, 2%), seguida por «otras causas» (26, 5%). La mediana de puntuación en la escala NIHSS fue de 3, 5 (RIQ: 2, 07, 0). En total, 211 (26, 8%) de los ingresos fueron por AIT. De los pacientes que ingresaron con el diagnóstico de ictus isquémico, 59 (10, 3%) se fibrinolizaron. Conclusiones El ictus isquémico en el adulto joven no es infrecuente en Aragón y en un importante número de casos es de etiología indeterminada, por lo que es necesario implementar medidas que nos permitan mejorar su estudio, disminuir su incidencia y prevenir su recurrencia. Introduction: Stroke affects around 15 million people per year, with 10%-15% occurring in individuals under 50 years old (stroke in young adults). The prevalence of different vascular risk factors and healthcare strategies for stroke management vary worldwide, making the epidemiology and specific characteristics of stroke in each region an important area of research. This study aimed to determine the prevalence of different vascular risk factors and the aetiology and characteristics of ischaemic stroke in young adults in the autonomous community of Aragon, Spain. Methods: A cross-sectional, multi-centre study was conducted by the neurology departments of all hospitals in the Aragonese Health Service. We identified all patients aged between 18 and 50 years who were admitted to any of these hospitals with a diagnosis of ischaemic stroke or TIA between January 2005 and December 2015. Data were collected on demographic variables, vascular risk factors, and type of stroke, among other variables. Results: During the study period, 786 patients between 18 and 50 years old were admitted with a diagnosis of ischaemic stroke or TIA to any hospital of Aragon, at a mean annual rate of 12.3 per 100 000 population. The median age was 45 years (IQR: 40-48 years). The most prevalent vascular risk factor was tobacco use, in 404 patients (51.4%). The majority of strokes were of undetermined cause (36.2%), followed by other causes (26.5%). The median NIHSS score was 3.5 (IQR: 2.0-7.0). In total, 211 patients (26.8%) presented TIA. Fifty-nine per cent of the patients admitted with a diagnosis of ischaemic stroke (10.3%) were treated with fibrinolysis. Conclusions: Ischaemic stroke in young adults is not uncommon in Aragon, and is of undetermined aetiology in a considerable number of cases; it is therefore necessary to implement measures to improve study of the condition, to reduce its incidence, and to prevent its recurrence.

Nucleic acid isolation is often the starting point for all downstream experiments in biomedical research. It is therefore the most crucial step in any molecular technique. DNA and RNA extraction follow protocols with standardized reagents, many of which are available in quality-controlled commercial kits. Irrespective of the protocol, successful extraction of high-quality nucleic acid from biological tissues requires sufficient disruption of the tissue and cellular structures, denaturation of nucleoprotein complexes, inactivation of nucleases, and nucleic acid purification. These steps can be modified based on nucleic acid of interest and biological sample source. This chapter addresses DNA and RNA extraction from a variety of sample and tissue types, including saliva, and formalin-fixed, paraffin-embedded tissues, which are often archived in clinical pathology laboratories. Special considerations and common pitfalls of each protocol will also be discussed, as will nucleic acid quantitation techniques.

Introduction Stroke affects around 15 million people per year, with 10%-–15% occurring in individuals under 50 years old (stroke in young adults). The prevalence of different vascular risk factors and healthcare strategies for stroke management vary worldwide, making the epidemiology and specific characteristics of stroke in each region an important area of research. This study aimed to determine the prevalence of different vascular risk factors and the aetiology and characteristics of ischaemic stroke in young adults in the autonomous community of Aragon, Spain. Methods A cross-sectional, multi-centre study was conducted by the neurology departments of all hospitals in the Aragonese Health Service. We identified all patients aged between 18 and 50 years who were admitted to any of these hospitals with a diagnosis of ischaemic stroke or TIA between January 2005 and December 2015. Data were collected on demographic variables, vascular risk factors, and type of stroke, among other variables. Results During the study period, 786 patients between 18 and 50 years old were admitted with a diagnosis of ischaemic stroke or TIA to any hospital of Aragon, at a mean annual rate of 12.3 per 100 000 population. The median age was 45 years (IQR: 40-−48 years). The most prevalent vascular risk factor was tobacco use, in 404 patients (51.4%). The majority of strokes were of undetermined cause (36.2%), followed by other causes (26.5%). The median NIHSS score was 3.5 (IQR: 2.0-–7.0). In total, 211 patients (26.8%) presented TIA. Fifty-nine per cent of the patients admitted with a diagnosis of ischaemic stroke (10.3%) were treated with fibrinolysis. Conclusions Ischaemic stroke in young adults is not uncommon in Aragon, and is of undetermined aetiology in a considerable number of cases; it is therefore necessary to implement measures to improve study of the condition, to reduce its incidence, and to prevent its recurrence.

Objectives Immunoglobulin paraproteins can interfere with multiple chemistry assays. We want to investigate the mechanisms of immunoglobulin interference. Design and methods Serum samples containing paraproteins from the index patient and eight additional patients were used to investigate the interference with the creatinine and total protein assays on the Beckman Coulter AU5400/2700 analyzer, and to determine the effects of pH and ionic strength on the precipitation of different immunoglobulins in these patient samples. Results The paraprotein interference with the creatinine and total protein assays was caused by the precipitation of IgM paraprotein in the index patient's samples under alkaline assay conditions. At extremely high pH (12–13) and extremely low pH (1–2) and low ionic strength, paraprotein formed large aggregates in samples from the index patient but not from other patients. Conclusions The pH and ionic strength are the key factors that contribute to protein aggregation and precipitation which interfere with the creatinine and total protein measurements on AU5400/2700. The different amino acid sequence of each monoclonal paraprotein will determine the pH and ionic strength at which the paraprotein will precipitate., Highlights • Investigation of the cause of IgM paraprotein interference with creatinine assay. • Paraprotein precipitation under assay conditions caused the interference. • Identified The pH of chemistry assays is a key factor causing paraprotein precipitation. • Results showed that not all paraproteins precipitate under the same assay conditions. • Different paraproteins react differently to changes in pH and ionic strength.

The Etest on chocolate Mueller-Hinton agar was compared to broth microdilution (BMD) for 125 isolates of nutritionally variant streptococci. Vancomycin Etests yielded 31.1% essential agreement (EA) and 20.0% categorical agreement (CA). Penicillin Etests yielded 86.0% EA and 85.6% CA, whereas ceftriaxone Etests yielded 73.6% EA and 68.0% CA.

The DNA repair gene O 6 -methylguanine-DNA methyltransferase ( MGMT ) allows efficient in vivo enrichment of transduced hematopoietic stem cells (HSC). Thus, linking this selection strategy to therapeutic gene expression offers the potential to reconstitute diseased hematopoietic tissue with gene-corrected cells. However, different dual-gene expression vector strategies are limited by poor expression of one or both transgenes. To evaluate different co-expression strategies in the context of MGMT-mediated HSC enrichment, we compared selection and expression efficacies in cells cotransduced with separate single-gene MGMT and GFP lentivectors to those obtained with dual-gene vectors employing either encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or foot and mouth disease virus (FMDV) 2A elements for co-expression strategies. Each strategy was evaluated in vitro and in vivo using equivalent multiplicities of infection (MOI) to transduce 5-fluorouracil (5-FU) or Lin − Sca-1 + c-kit + (LSK)-enriched murine bone marrow cells (BMCs). The highest dual-gene expression (MGMT + GFP + ) percentages were obtained with the FMDV-2A dual-gene vector, but half of the resulting gene products existed as fusion proteins. Following selection, dual-gene expression percentages in single-gene vector cotransduced and dual-gene vector transduced populations were similar. Equivalent MGMT expression levels were obtained with each strategy, but GFP expression levels derived from the IRES dual-gene vector were significantly lower. In mice, vector-insertion averages were similar among cells enriched after dual-gene vectors and those cotransduced with single-gene vectors. These data demonstrate the limitations and advantages of each strategy in the context of MGMT-mediated selection, and may provide insights into vector design with respect to a particular therapeutic gene or hematologic defect.

Background Long non-coding RNAs (lncRNAs) play a variety of cellular roles, including regulation of transcription and translation, leading to alterations in gene expression. Some lncRNAs modulate the expression of chromosomally adjacent genes. Here, we assess the roles of the lncRNA CASC15 in regulation of a chromosomally nearby gene, SOX4, and its function in RUNX1/AML translocated leukemia. Results CASC15 is a conserved lncRNA that was upregulated in pediatric B-acute lymphoblastic leukemia (B-ALL) with t (12; 21) as well as pediatric acute myeloid leukemia (AML) with t (8; 21), both of which are associated with relatively better prognosis. Enforced expression of CASC15 led to a myeloid bias in development, and overall, decreased engraftment and colony formation. At the cellular level, CASC15 regulated cellular survival, proliferation, and the expression of its chromosomally adjacent gene, SOX4. Differentially regulated genes following CASC15 knockdown were enriched for predicted transcriptional targets of the Yin and Yang-1 (YY1) transcription factor. Interestingly, we found that CASC15 enhances YY1-mediated regulation of the SOX4 promoter. Conclusions Our findings represent the first characterization of this CASC15 in RUNX1-translocated leukemia, and point towards a mechanistic basis for its action. Electronic supplementary material The online version of this article (doi:10.1186/s12943-017-0692-x) contains supplementary material, which is available to authorized users.

Myelodysplastic syndromes (MDS) are the most common myeloid malignancies among the elderly. Patients present with bone marrow (BM) failure manifested by low peripheral blood (PB) counts and are at increased risk of developing acute myeloid leukemia. Mutations of U2AF1, a gene that encodes a spliceosome protein, are identified in 11% of MDS patients. The two most common U2AF1 mutants, S34F and Q157P, alter the splicing of two distinct sets of pre-mRNA targets in vitro and each co-occur with unique gene mutations in MDS patients, suggesting these mutants may affect MDS pathogenesis differently. In mice, U2AF1S34F expression leads to altered splicing, reduced B-cell counts, and features of MDS. Similar studies have not been performed for U2AF1Q157P. To study the impact of U2AF1Q157P expression on splicing and hematopoiesis in vivo, we created a doxycycline (DOX)-inducible ("Tet-On") transgenic mouse that expresses mutant U2AF1Q157P and is isogenic to our previously reported U2AF1S34F and U2AF1WT transgenic mice. First, we confirmed DOX-inducible expression of the U2AF1Q157P transgene in BM by RT-PCR-seq. To study the hematopoietic cell-intrinsic effects of U2AF1Q157P, we performed non-competitive BM transplants into lethally irradiated congenic recipient mice. Donor BM from U2AF1WT or U2AF1S34F mice was also transplanted for comparison. Six weeks after transplant, mice were maintained on DOX chow to induce U2AF1 transgene (U2AF1WT, U2AF1S34F, or U2AF1Q157P) expression (n = 10 mice per genotype). After six weeks on DOX, there were no significant changes in PB counts for U2AF1Q157P mice compared to U2AF1WT controls. In contrast, white blood cell (WBC) and B-cell counts were significantly reduced in U2AF1S34F mice, as reported previously. Assessment of the BM revealed increased numbers (per five leg bones) of hematopoietic stem and progenitor cells (LSK [Lin− Sca-1+ c-kit+] and LK [Lin− Sca-1− c-kit+]) in U2AF1S34F mice (1.33×105 LSK and 7.13×105 LK cells) compared to U2AF1WT (1.04×105 LSK and 5.69×105 LK cells; p < 0.05 for LSK and LK), as reported previously. In contrast, there was no change in LSK cells (1.03×105, p = 0.9668) and a non-significant increase in LK cells (6.84×105, p = 0.0547) in U2AF1Q157P mice compared to U2AF1WT. Both U2AF1S34F and U2AF1Q157P mice shared a significant increase in the number of common myeloid progenitors (CMP) compared to U2AF1WT (2.43×105 and 2.39×105 vs. 1.66×105 cells; p < 0.001 and p < 0.01, respectively), although CFU-C interrogated by methylcellulose assay were significantly increased only for U2AF1S34F mice. To study the hematopoietic cell-intrinsic effects of U2AF1Q157P on stem cell function, we mixed equal numbers of whole BM test cells (CD45.2+; U2AF1Q157P or U2AF1WT) with congenic control wild-type BM competitor cells (CD45.1+/CD45.2+) and transplanted them into lethally irradiated congenic recipient mice (CD45.1+/CD45.2+ ; n = 6 per genotype). As in non-competitive transplants, DOX chow was administered six weeks after transplant. After six weeks on DOX, we observed a relative multi-lineage competitive disadvantage by analysis of peripheral blood chimerism (%CD45.2+ WBC) for U2AF1Q157P test compared to U2AF1WT test cells (49.5% vs. 71.7%, respectively, p < 0.001). In addition, stem and progenitor cells were all significantly reduced in the BM of U2AF1Q157P competitive transplant mice compared to U2AF1WT after 18 weeks of DOX (LSK, 36.1% vs. 92.2%, respectively, p < 0.001; LK, 53.1% vs. 92.0%, p < 0.001). Lastly, using a Nanostring array, we identified consensus 3' splice sites of cassette exons that were increased or decreased in RNA from c-kit enriched mutant (U2AF1S34F or U2AF1Q157P) BM cells relative to U2AF1WT (FDR < 0.1). As expected, we observed altered consensus 3' splice sites at the −3 position (for U2AF1S34F) and +1 position (for U2AF1Q157P) of differentially spliced exons, indicating altered but different pre-mRNA splicing induced by either U2AF1 mutant. In aggregate, hematopoietic expression of U2AF1Q157P causes a multi-lineage competitive disadvantage of BM stem cells and expanded myeloid progenitors in the non-competitive transplant setting, like U2AF1S34F. However, PB counts and lineage distribution are not affected, indicating that the two common U2AF1 mutants, Q157P and S34F, are associated with different hematopoietic phenotypes and alterations to splicing, and may have different roles in MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.

B-cell development in the bone marrow is followed by specification into functional subsets in the spleen, including marginal zone (MZ) B-cells. MZ B-cells are classically characterized by T-independent antigenic responses and require the elaboration of distinct gene expression programs for development. Given their role in gene regulation, it is not surprising that microRNAs are important factors in B-cell development. Recent work demonstrated that deficiency of the NFκB feedback regulator, miR-146a, led to a range of hematopoietic phenotypes, but B-cell phenotypes have not been extensively characterized. Here, we found that miR-146a-deficient mice demonstrate a reduction in MZ B-cells, likely from a developmental block. Utilizing high-throughput sequencing and comparative analysis of developmental stage-specific transcriptomes, we determined that MZ cell differentiation was impaired due to decreases in Notch2 signaling. Our studies reveal miR-146a-dependent B-cell phenotypes and highlight the complex role of miR-146a in the hematopoietic system.

B cell development in bone marrow is followed by specification into spleen subsets, including marginal zone (MZ) cells. MZ require elaboration of distinct gene expression programs for development. Given their role in gene regulation, its not surprising that microRNAs (miRNAs) influence cell development. Recent work demonstrated that deficiency of NF-κB feedback regulator, Mir146 (miR-146a), led to a range of hematopoietic phenotypes, but B cells have not been extensively characterized. Here, we found miR-146a deficient mice demonstrate a reduction in MZ B cells, likely from a T cell independent developmental block. Utilizing comparative analysis of developmental stage-specific transcriptomes, we show MZ cell differentiation was impaired due to decreases in Notch2 signaling. Further, we discovered that the cell-fate regulatory protein, Numb, is a direct target of miR-146a, and its derepression in miR-146a deficient B cells underlies the decreases in Notch2. Our studies reveal miR-146a-dependent B cell phenotypes regulated by the Numb-Notch2 pathway. Disclosures No relevant conflicts of interest to declare.

Background A new class of non-coding RNAs, known as long non-coding RNAs (lncRNAs), has been recently described. These lncRNAs are implicated to play pivotal roles in various molecular processes, including development and oncogenesis. Gene expression profiling of human B-ALL samples showed differential lncRNA expression in samples with particular cytogenetic abnormalities. One of the most promising lncRNAs identified, designated B-ALL associated long RNA-6 (BALR-6), had the highest expression in patient samples carrying the MLL rearrangement, and is the focus of this study. Results Here, we performed a series of experiments to define the function of BALR-6, including several novel splice forms that we identified. Functionally, siRNA-mediated knockdown of BALR-6 in human B-ALL cell lines caused reduced cell proliferation and increased cell death. Conversely, overexpression of BALR-6 isoforms in both human and mouse cell lines caused increased proliferation and decreased apoptosis. Overexpression of BALR-6 in murine bone marrow transplantation experiments caused a significant increase in early hematopoietic progenitor populations, suggesting that its dysregulation may cause developmental changes. Notably, the knockdown of BALR-6 resulted in global dysregulation of gene expression. The gene set was enriched for leukemia-associated genes, as well as for the transcriptome regulated by Specificity Protein 1 (SP1). We confirmed changes in the expression of SP1, as well as its known interactor and downstream target CREB1. Luciferase reporter assays demonstrated an enhancement of SP1-mediated transcription in the presence of BALR-6. These data provide a putative mechanism for regulation by BALR-6 in B-ALL. Conclusions Our findings support a role for the novel lncRNA BALR-6 in promoting cell survival in B-ALL. Furthermore, this lncRNA influences gene expression in B-ALL in a manner consistent with a function in transcriptional regulation. Specifically, our findings suggest that BALR-6 expression regulates the transcriptome downstream of SP1, and that this may underlie the function of BALR-6 in B-ALL. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0485-z) contains supplementary material, which is available to authorized users.

A comparison study is presented, which examines the outcome, complications and cost of stereotactic brain biopsy performed with a frameless versus a frame-based method. The technique of frameless stereotactic biopsy has been shown previously, in both laboratory and in vivo studies, to achieve a level of accuracy at least equal to frame-based biopsy. The investigators have validated the technique in a large clinical series. The frameless and frame-based series were concurrent, comprising 76 and 79 cases, respectively. The frameless stereotactic technique involved standard needle biopsy, targeted by an image-guidance system and directed by a novel rigid adjustable instrument-holder. Frame-based biopsies were performed with the CRW and Leksell systems. There were no significant differences in the demographics, lesion site, size and pathologies between the groups. Operating theatre occupancy and anaesthetic time were both significantly shorter for the frameless series than the frame-based series (p < 0.0001). In addition, the complication rate in the frameless biopsy series was significantly lower than in the frame-based series (p = 0.018). This resulted in lower ITU bed occupancy (p = 0.02), shorter mean hospital stay (p = 0.0013) and significant cost savings (p = 0.0022) for the frameless stereotactic biopsy group, despite the greater use of more expensive MRI in these cases. This comparison study demonstrates that the superior imaging, target visualization and flexibility of the technique of frameless stereotactic biopsy translates into tangible advantages for safety, time and cost when compared with the current gold-standard of frame-based biopsy. The principles are discussed and the authors propose a definition for the term 'frameless stereotaxy'.

Fortification of salt with both iodine and iron is an attractive approach to simultaneous reduction of anemia and iodine deficiency disorders (IDD), which affect some 2 billion people in the developing world. Unfortunately iodine is unstable under storage conditions found in most developing countries. Its disappearance is accelerated by iron compounds as well as impurities found in most salts in these markets. Potassium iodide and potassium iodate were encapsulated in modified starches, gelatin, sodium hexametaphosphate and purified sodium chloride by spray drying and fluidized bed drying to produce microcapsules containing 0.3 to 2% iodine. Salt mixed with ferrous fumarate or ferrous sulphate, and encapsulated iodine was then stored at high temperature and high humidity. The most stable combination, containing 50 mg iodine and 1000 mg iron per kg salt, retained more than 75% of the added iodine for a year at 40 °C, 100% RH. Double-fortified salt prepared using this approach has been successfully tested in large-scale field trials. The results will be published after completion of data analysis.

In the pediatric population, B-acute lymphoblastic leukemia (B-ALL) is the most prevalent childhood hematological malignancy, as well as the leading cause of childhood cancer-related mortality. Advances in cytogenetics utilizing array-based technologies and next-generation sequencing (NGS) techniques have revealed exciting insights into the genetic basis of this disease, with the hopes of developing individualized treatment plans for affected children. In this comprehensive review, we discuss our current understanding of childhood (pediatric) B-ALL and highlight the most recent genetic advances and their therapeutic implications.

Advances in computer technology have permitted virtual reality images of the ventricular system. To determine the relevance of these images we have compared virtual reality simulations of the ventricular system with endoscopic findings in three patients. The virtual fly-through can be simulated after definition of waypoints. Flight objects of interest can be viewed from all sides. Important drawbacks are that filigree structures may be missed and blood vessels cannot be distinguished clearly. However, virtual endoscopy can presently be used as a planning tool or for training and has future potential for neurosurgery.

Rapid field tests for the determination of iodide in salt were developed, based on the oxidation of iodide to iodine and reaction of the released iodine with starch to produce a coloured complex that could be visually related to the iodide content of the salt. Potassium iodate and ferric chloride were used as oxidizing agents in the two versions of the test that were examined in detail. the reaction with ferric chloride gave a test with a wider useful concentration range. It had the added advantage of not producing false readings for iodine by reacting with reducing agents that were present in the salt as natural impurities or as a result of fraud. A simple test kit suitable for field use was developed and tested with iodized salts produced using salts from Canada and Ecuador. the kit results were compared with results obtained by neutron activation analysis using the SLOWPOKE nuclear reactor facility of the University of Toronto and by standard iodometric titration. the test kits readily analysed for iodine levels between 0 and 50 ppm, with an accuracy of ± 10 ppm and a reproducibility of ± 5 ppm. Inexpensive rapid tests using these kits may be useful in monitoring iodine levels in areas where salt iodization is based on potassium iodide, because of the presence of impurities in the salt supply that reduce the stability of the usual iodizing agent, potassium iodate.

Commercially available field test kits for the determination of iodate in salt were tested to evaluate their performance and to devise improvements in their range and accuracy. Dropper tests based on the blue colour of the starch-iodine complex formed with iodine released by excess potassium iodide gave rapid qualitative indication of the presence of iodate. Because of the dark colour of the complex, the tests were found to be quantitative over only a limited range, typically 0 to 20 μg of iodine per gram of salt. We have developed methods for improving the reliability and colour reproducibility of these types of kits. Instructions for producing reliable kits of this type with improved colour reproducibility are presented. Improved qualitative tests in the range of 0 to 50 μg/g were obtained with a simple liquid-phase colourimetric system, based on the same chemistry. A field test kit was developed and tested in laboratories in India and Zimbabwe, as well as in our laboratory in Toronto. the test gave reproducible results, comparable to those of titration, with a typical standard deviation of ± 5 μg/g. the range of measurements can be extended to 100 μg/g by dilution. Instructions for preparing these kits are presented.

The purpose of this study was to assess the effect of humidity and packaging materials on the stability of iodine in typical salt samples from countries with tropical and subtropical climates, under controlled climatic conditions. Initially we examined eight samples. In the second phase we expanded the study to salts from 18 sources and attempted to correlate the observed stability with salt impurities naturally present in these samples. High humidity resulted in rapid loss of iodine from salt iodized with potassium iodate, ranging from 30% to 98% of the original iodine content. Solid low-density polyethylene packaging protected the iodine to a great extent. High losses were observed from woven high-density polyethylene bags, which are often the packaging material of choice in tropical countries. Impurities that provided moisture at the salt surface had the most deleterious effect. Although clear correlations were not obtained, the presence of reducing agents, hygroscopic compounds of magnesium, and so forth seemed to have the most adverse effects on the stability of iodine. Surprisingly, carbonates had little effect on stability over the range present in the samples. Packaging salt in low-density polyethylene bags, which provided a good moisture barrier, significantly reduced iodine losses, and in most cases the iodine content remained relatively stable for six months to a year. The findings from this study indicate that iodine can be highly unstable, and in order to ensure the effectiveness of local salt-iodization programmes, countries should determine iodine losses from local iodized salt under local conditions of production, climate, packaging, and storage.

Severe spinal spasticity has been shown to be a good indication for continuous intrathecal baclofen infusion (CIBI), but there is only limited experience with this treatment in patients with supraspinal spasticity. Eighteen patients with severe spasticity from traumatic or hypoxic brain injury were treated with CIBI. In all patients spasticity could be reduced significantly. The mean Ashworth score was reduced from 4.5 to 2.33 and the mean Spasm frequency score from 2.16 to 0.94. This reduction of spasticity led to a marked pain reduction. Nursing, perineal care and mobilization became much easier. The complication rate was low. In this series we saw one infection in the pump pocket, one epileptic seizure after a bolus application of baclofen and one spinal catheter displacement. The results are similar to those reported from series of patients with spinal spasticity and correspond to the limited experience we have so far with supraspinal spasticity patients. To prevent limb contractures CIBI should be performed as soon as the patient is in a stable clinical condition after brain injury. Further prospective clinical trials will be necessary to obtain more experience with patients suffering from supraspinal spasticity.

Nutritionally variant streptococci (NVS) are fastidious Gram-positive cocci comprised of the species Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans. NVS are an important cause of bacteremia and infective endocarditis (IE) associated with significant morbidity and mortality. Antimicrobial susceptibility testing (AST) was performed for 14 antimicrobials using the broth microdilution MIC method described in the Clinical and Laboratory Standards Institute (CLSI) M45 guideline. A total of 132 clinical NVS blood isolates collected from 2008 to 2014 were tested. Species level identification of NVS isolates was achieved by 16S rRNA gene sequencing and/or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Ninety isolates were identified as G. adiacens, 37 as A. defectiva, and 5 as G. elegans. All isolates were susceptible to vancomycin (MIC90 = 1 μg/ml), and none displayed high-level resistance to aminoglycosides. G. adiacens was considerably more susceptible to penicillin than A. defectiva (38.9% versus 10.8% of isolates susceptible) but was less susceptible to cephalosporins than was A. defectiva (43.3% versus 100% of isolates susceptible to ceftriaxone). Several isolates were resistant to levofloxacin (6%), erythromycin (51%), and clindamycin (10%). The MIC90 for daptomycin was ≥ 4 μg/ml for G. adiacens and A. defectiva. G. elegans isolates were 100% susceptible to all antimicrobials tested, with the exception of erythromycin, to which only 20% were susceptible. This study provides antimicrobial susceptibility data for a recent collection of NVS and demonstrates important NVS species-related differences with respect to susceptibility to penicillin, cephalosporins, carbapenems, and daptomycin. Species-level identification of NVS organisms when susceptibility testing is not readily available may aid in treatment decisions.

Specific and efficient gene delivery to the lung has been hampered by liver sequestration of adenovirus serotype 5 (Ad5) vectors. The complexity of Ad5 liver tropism has largely been unraveled, permitting improved efficacy of Ad5 gene delivery. However, Kupffer cell (KC) scavenging and elimination of Ad5 still represent major obstacles to lung gene delivery strategies. KC uptake substantially reduces bioavailability of Ad5 for target tissues and compensatory dose escalation leads to acute hepatotoxicity and a potent innate immune response. Here, we report a novel lung-targeting strategy through redirection of Ad5 binding to the concentrated leukocyte pool within the pulmonary microvasculature. We demonstrate that this leukocyte-binding approach retargets Ad5 specifically to lung endothelial cells and prevents KC uptake and hepatocyte transduction, resulting in 165,000-fold enhanced lung targeting, compared with Ad5. In addition, myeloid cell-specific binding is preserved in single-cell lung suspensions and only Ad.MBP-coated myeloid cells achieved efficient endothelial cell transduction ex vivo. These findings demonstrate that KC sequestration of Ad5 can be prevented through more efficient uptake of virions in target tissues and suggest that endothelial transduction is achieved by leukocyte-mediated 'hand-off' of Ad.

The gene therapy field is currently limited by the lack of vehicles that permit efficient gene delivery to specific cell or tissue subsets. Native viral vector tropisms offer a powerful platform for transgene delivery but remain nonspecific, requiring elevated viral doses to achieve efficacy. In order to improve upon these strategies, our group has focused on genetically engineering targeting domains into viral capsid proteins, particularly those based on adenovirus serotype 5 (Ad5). Our primary strategy is based on deletion of the fiber knob domain, to eliminate broad tissue specificity through the human coxsackie-and-adenovirus receptor (hCAR), with seamless incorporation of ligands to re-direct Ad tropism to cell types that express the cognate receptors. Previously, our group and others have demonstrated successful implementation of this strategy in order to specifically target Ad to a number of surface molecules expressed on immortalized cell lines. Here, we utilized phage biopanning to identify a myeloid cell-binding peptide (MBP), with the sequence WTLDRGY, and demonstrated that MBP can be successfully incorporated into a knob-deleted Ad5. The resulting virus, Ad.MBP, results in specific binding to primary myeloid cell types, as well as significantly higher transduction of these target populations ex vivo, compared to unmodified Ad5. These data are the first step in demonstrating Ad targeting to cell types associated with inflammatory disease.

Background Previously, we have reported the isolation and molecular characterization of human Hepatitis C virus genotype 1 (HCV-1) from infected patients. We are now reporting an analysis of HCV obtained from patients infected with HCV genotype 3 (HCV-3) as diagnosed by clinical laboratories. Results HCV was cultured in vitro using our system. HCV RNA was isolated from patients' blood and from HCV cultured in various cell types for up to three months. The 5'UTR of these isolates were used for comparisons. Results revealed a number of sequence changes as compared to the serum RNA. The HCV RNA produced efficiently by infected macrophages, B-cells, and T-cells had sequences similar to HCV-1, which suggests that selection of the variants was performed at the level of macrophages. Virus with sequences similar to HCV-1 replicated better in macrophages than HCV having a 5'UTR similar to HCV-3. Conclusions Although HCV-3 replicates in cell types such as B-cells, T-cells, and macrophages, it may require a different primary cell type for the same purpose. Therefore, in our opinion, HCV-3 does not replicate efficiently in macrophages, and patients infected with HCV-3 may contain a population of HCV-1 in their blood.

Differential diagnosis of migraine-like headache with and without aura needs to proceed with neuroimaging evaluation in order to rule out any secondary conditions. We report a patient with a 2 year history of migraine like headache with a good response to ergotics and due to pituitary macroadenoma.A 44 year old man with no familiar history of migraine presented to our hospital because of campimetric failure and a change of his migrainous features. The patient started with migraine without aura 2 years ago and was well treated with ergotics from the beginning. Field testing showed a bitemporal hemianopsia. Magnetic resonance imaging revealed an unknown pituitary macroadenoma.Pituitary macroadenoma must be included in the differential diagnosis of migraine headache although the presence of aura or the good response to ergotics.

Abiotrophia defectiva (AD), Granulicatella adiacens (GA), and Granulicatella elegans (GE), collectively known as nutritionally variant streptococci (NVS), are fastidious Gram-positive cocci that can cause bacteremia and infective endocarditis (IE). Treatment guidelines for NVS IE recommend combination penicillin (PCN) or vancomycin (VA), plus an aminoglycoside. Limited studies from 10 or more years ago documented PCN resistance in these organisms, which poses an important challenge to treatment of serious NVS infections. Given the importance of choosing appropriate therapy for NVS infections, quick and reliable antimicrobial susceptibility testing (AST) methods are needed to help guide antimicrobial selection. However, …

We recently reported the isolation and in vitro replication of hepatitis C virus. These isolates were termed CIMM-HCV and analyzed to establish genotypes and subtypes, which are reported elsewhere. During this analysis, an HCV isolated from a patient was discovered that had large deletions in the 5'UTR. 57% of the HCV RNA found in this patient's sera had 113 or 116 bp deletions. Sequence data showed that domains IIIa to IIIc were missing. Previous studies have suggested that these domains may be important for translation. In vitro replicated HCV from this patient did not contain these deletions, however, it contained a 148 bp deletion in the 5'UTR. Whereas the patient HCV lacked domains IIIa through IIIc, the isolate lacked domains IIIa through IIId. HCV from this patient continues to produce large deletions in vitro, suggesting that the deletion may not be important for the assembly or replication of the virus. This is the first report describing these large deletions.

Isolation and self-replication of infectious HCV has been a difficult task. However, this is needed for the purposes of developing rational drugs and for the analysis of the natural virus. Our recent report of an in vitro system for the isolation of human HCV from infected patients and their replication in tissue culture addresses this challenge. At California Institute of Molecular Medicine several isolates of HCV, called CIMM-HCV, were grown for over three years in cell culture. This is a report of the analysis of CIMM-HCV isolates for subtypes and quasispecies using a 269 bp segment of the 5'UTR. HCV RNA from three patients and eleven CIMM-HCV were analyzed for this purpose. All isolates were essentially identical. Isolates of HCV from one patient were serially transmitted into fresh cells up to eight times and the progeny viruses from each transmission were compared to each other and also to the primary isolates from the patient's serum. Some isolates were also transmitted to different cell types, while others were cultured continuously without retransmission for over three years. We noted minor sequence changes when HCV was cultured for extended periods of time. HCV in T-cells and non-committed lymphoid cells showed a few differences when compared to isolates obtained from immortalized B-cells. These viruses maintained close similarity despite repeated transmissions and passage of time. There were no subtypes or quasispecies noted in CIMM-HCV.

Transient global amnesia (TGA) is a clinical condition that has been described in many studies, but its pathophysiology is not fully understood. In recent years the theory of valvular insufficiency in the jugular vein has been added to the classical hypotheses that link it to migraine, to epilepsy and -the most widely accepted- to transient ischemic attacks (TIA), although the real origin of the condition has still not been determined.In a retrospective study we compared 131 patients diagnosed with TGA between 1993 and 2004 with 262 patients who were diagnosed as having TIA over the same period.Mean age was 65.94 years in TGA versus 71.11 years in the case of TIA. There was a higher rate of arterial hypertension among the patients with TGA and diabetes mellitus was more frequent among those with TIA (p0.05 in both cases). Emboligenic heart disease was scarce among patients with TGA. The number of patients with a history of ischaemic heart disease and a history and the development of cerebrovascular diseases was greater among those with TIA than in cases of TGA (p0.05). The TGA recurrence rate was 12%. The percentage of pathological findings in the CAT brain scan was higher in patients with TIA (p0.05). There are no significant differences between patients with TGA and TIA as far as treatment on hospital discharge is concerned.TGA does not seem to be a symptom of an arteriosclerotic pathology nor does it appear to offer a higher risk of heart or cerebrovascular disease and, therefore, antiaggregating therapy would not be indicated in such cases.

Deficiencies in small quantities of micronutrients, especially iodine and iron, severely affect more than a third of the world's population, resulting in serious public health consequences, especially for women and young children. Salt is an ideal carrier of micronutrients. the double fortification of salt with both iodine and iron is an attractive approach to the reduction of both anemia and iodine-deficiency disorders. Because iodine is unstable under the storage conditions found during the manufacturing, distribution, and sale of salt in most developing countries, the effects of packaging materials and environmental conditions on the stability of salt double-fortified with iron and iodine were investigated. Salt was double-fortified with potassium iodide or potassium iodate and with ferrous sulfate or ferrous fumarate. the effects of stabilizers on the stability of iodine and iron were followed by storing the salt under three conditions that represent the extremes of normal distribution and sale for salt in developing countries: room temperature (25°C) w ith 50%–70% relative humidity, 40° C with 60% relative humidity, and 40° C with 100% relative humidity. the effects of stabilizers, such as sodium hexametaphosphate (SHMP), calcium carbonate, calcium silicate, and dextrose were investigated. None of the combinations of iron and iodine compounds was stable at elevated temperatures. Essentially all of the iodine was lost over a period of six months. SHMP effectively slowed down the iodine loss, whereas magnesium chloride, a typical hygroscopic impurity, greatly accelerated this process. Calcium carbonate did not have a sparing effect on iodine, despite contrary indications in the literature. Ferrous sulfate–fortified salts generally turned yellow and developed an unpleasant rusty flavor. Salt fortified with ferrous fumarate and potassium iodide was reasonably stable and maintained its organoleptic properties, making it more likely to be acceptable to consumers. We confirmed that application of the iodine compounds as solutions resulted in a more even distribution of the iodine throughout the sample. the effect of the packaging materials was overshadowed by the other variables. None of the packaging materials was clearly better than any other. This may have been due to the fact that the polymer bags were not heat sealed, and thus some moisture penetration was possible. the results indicate that with careful control of processing, packaging, and storage conditions, a double-fortified salt could be stabilized for the six-month period required for distribution and consumption. Unfortunately, the processing and storage required are difficult to attain under typical conditions in developing countries.

Today, endoscopic third ventriculostomy is an established operative modality in occlusive hydrocephalus. The elemental step in third ventriculostomy is the perforation of the floor of the third ventricle. Especially with a thickened third ventricular floor, anatomical orientation can be disturbed and perforation of third ventricular floor technically difficult. The combination of a neuronavigation system with an endoscope provides interactive image-guided neuroendoscopy. Exact planning of the approach is thus possible and the ideal trajectory to the target area can be determined. We have combined interactive neuronavigation and intraoperative fluoroscopy for incorporating real-time feedback to optimize endoscopy in patients with a thickened third ventricular floor selected for third ventriculostomy.

The initial clinical experience with a prototype of the EasyGuide Neuro surgical navigation system is presented together with some measurements of intraoperative brain shift taken with the neuro-navigator. The EasyGuide Neuro is an infrared-based system employing hand-held localisers with light-emitting diodes tracked by a table-mounted camera array. Standard orthogonal reformats which intersect at the pointer tip position are displayed by the workstation in near real-time. We have prospectively studied the 31 consecutive patients undergoing surgery with the neuronavigator during the first 3 months of clinical use. General patient data, system accuracy checks and surgical impact are described. In a subgroup of 16 patients measurements of brain shift were taken at the brain surface, at the deepest point of surgery and at the bone surface offset. The latter was used to correct for registration errors. We found a significantly greater shift of the cortical surface than of deep structures (p < 0.02) with this method. These measurements are presented as evidence supporting the validity of navigational guidance in clinical practice.

Interactive image guidance is now in routine use for open neurosurgical procedures and has demonstrated patient benefits. However, freehand interactive guidance is not an appropriate replacement for the traditional frame-based stereotactic procedures of biopsy, electrode placement, and functional lesioning. These point-based procedures require precise target localization and direct instrument guidance to avoid collateral brain injury. To perform true frameless stereotactic procedures requires a guide that is also adjustable for positioning, lockable, and adaptable to multiple instruments. We describe such a device, which is employed for the guidance of biopsy needles, shunts, electrodes, and endoscopes during neuronavigation. The method of frameless stereotactic biopsy retrieval with an infrared-based neuronavigation system is described, clinical results are given, and further areas of application discussed.

It is uncertain whether the use of image-guided surgery has an influence on operating time. We prospectively studied the time requirements which have to be invested for using image-guided surgery and performed a comparison of operating room times from 125 matched pairs of image-guided and conventional operations. Our study revealed that neuronavigation has to be regarded as time neutral in general neurosurgery, whereas in stereotactic surgery, namely biopsy retrival, a significant reduction of anaesthetic time can be achieved through the complete separation of imaging and operation in the frameless approach.

Introduccion. La aplicacion de tratamientos en fase aguda del ictus obliga a reducir al maximo el tiempo de llegada de los pacientes al hospital. Uno de los factores de demora en la atencion es el desconocimiento de la enfermedad por la poblacion. Sujetos y metodos. Nuestro objetivo es analizar el conocimiento que tiene la poblacion del ictus y de los factores de riesgo vascular, asi como la actitud ante este. Para ello hemos realizado entrevistas a pacientes que acuden a consultas de neurologia y a sus familiares. Resultados. Han sido 386 entrevistas, 158 mujeres y 228 hombres, con una edad media de 52 anos. 230 sujetos (59%) no conocen el termino ictus, aunque si reconocen otros, como infarto o embolia. 175 (45%) no conocen ningun sintoma de infarto cerebral o responden incorrectamente. El sintoma mas reconocido (86%) es la perdida de fuerza en un hemicuerpo. Un 32% de los entrevistados no toma una actitud correcta ante los sintomas del ictus y, si se tratase de un ataque isquemico transitorio, un 25% no lo consideraria urgente. 198 (51%) no conocen ningun factor de riesgo vascular. De los que responden correctamente, el mas nombrado es la hipertension arterial. Los factores relacionados de forma independiente con un mejor conocimiento de la enfermedad son el nivel cultural alto y la edad joven. Conclusion. Existe un escaso conocimiento en la poblacion del ictus y los factores de riesgo vascular. Son necesarias campanas informativas para mejorar la actitud de la poblacion ante la enfermedad.