MLL fusion-driven activation of CDK6 potentiates proliferation in MLL-rearranged infant ALL

Acute lymphoblastic leukemia in infants (     

Confidence Intervals for Population Allele Frequencies: The General Case of Sampling from a Finite Diploid Population of Any Size

The estimation of population allele frequencies using sample data forms a central component of studies in population genetics. These estimates can be used to test hypotheses on the evolutionary processes governing changes in genetic variation among populations. However, existing studies frequently do not account for sampling uncertainty in these estimates, thus compromising their utility. Incorporation of this uncertainty has been hindered by the lack of a method for constructing confidence intervals containing the population allele frequencies, for the general case of sampling from a finite diploid population of any size. In this study, we address this important knowledge gap by presenting a rigorous mathematical method to construct such confidence intervals. For a range of scenarios, the method is used to demonstrate that for a particular allele, in order to obtain accurate estimates within 0.05 of the population allele frequency with high probability (%), a sample size of is often required. This analysis is augmented by an application of the method to empirical sample allele frequency data for two populations of the checkerspot butterfly (Melitaea cinxia L.), occupying meadows in Finland. For each population, the method is used to derive % confidence intervals for the population frequencies of three alleles. These intervals are then used to construct two joint % confidence regions, one for the set of three frequencies for each population. These regions are then used to derive a % confidence interval for Jost''s D, a measure of genetic differentiation between the two populations. Overall, the results demonstrate the practical utility of the method with respect to informing sampling design and accounting for sampling uncertainty in studies of population genetics, important for scientific hypothesis-testing and also for risk-based natural resource management.     

A Suitable Streptomycin-Resistant Mutant for Constructing Unmarked In-Frame Gene Deletions Using rpsL as a Counter-Selection Marker

The streptomycin counter-selection system is a useful tool for constructing unmarked in-frame gene deletions, which is a fundamental approach to study bacteria and their pathogenicity at the molecular level. A prerequisite for this system is acquiring a streptomycin-resistant strain due to rpsL mutations, which encodes the ribosomal protein S12. However, in this study no streptomycin resistance was found to be caused by rpsL mutations in all 127 clinical strains of Klebsiella pneumoniae isolated from liver abscess patients. By screening 107 spontaneous mutants of streptomycin resistance from a clinical strain of K. pneumoniae, nucleotide substitution or insertion located within the rpsL was detected in each of these strains. Thirteen different mutants with varied S12 proteins were obtained, including nine streptomycin-dependent mutants. The virulence of all four streptomycin-resistant mutants was further evaluated. Compared with the parental strain, the K42N, K42T and K87R mutants showed a reduction in growth rate, and the K42N and K42T mutants became susceptible to normal human serum. In the mice LD₅₀ (the bacterial dose that caused 50% death) assay, the K42N and K42T mutants were ∼1,000-fold less lethal (∼2×10⁵ CFU) and the K87R mutant was ∼50-fold less lethal (∼1×10⁴ CFU) than the parental strain (∼2×10² CFU). A K42R mutant showed non-observable effects on the above assays, while this mutant exhibited a small cost (P<0.01) in an in vitro growth competition experiment. In summary, most of the K. pneumoniae strains with streptomycin resistance caused by rpsL mutations are less virulent than their parental strain in the absence of streptomycin. The K42R mutant showed similar pathogenicity to its parental strain and should be one of the best choices when using rpsL as a counter-selection marker.     

Post Diagnosis Diet Quality and Colorectal Cancer Survival in Women

Background

Dietary factors are known to influence colorectal cancer (CRC) risk, however, their association with CRC survival is unclear. Therefore, we prospectively examined the association between diet quality scores, dietary patterns and colorectal cancer (CRC) survival.

Methods

1201 women diagnosed with stage I–III CRC between 1986 and 2008, were followed through 2010. Diet was assessed via a food frequency questionnaire administered at least 6 months after diagnosis. We computed the Alternate Healthy Eating Index-2010 (AHEI-2010), alternate Mediterranean Diet score (aMED) and Dietary Approaches to Stop Hypertension score (DASH) and derived two dietary patterns, Western (unhealthy) and prudent (healthy), by principal component analysis for each woman.

Results

During follow-up, we documented 435 deaths, including 162 from CRC. After adjusting for potential confounders, only a higher AHEI-2010 score was significantly associated with lower overall mortality (HR comparing extreme quintiles = 0.71, 95% CI 0.52–0.98, p trend = 0.01) as well as borderline significantly with lower risk of CRC mortality by the trend test (HR Q5 vs Q1 = 0.72, 95% CI = 0.43–1.21, p trend = 0.07). When AHEI-2010 components were examined separately, inverse associations for overall mortality were primarily accounted for by moderate alcohol intake (HR comparing abstainers vs 5–15 g/d = 1.30, 95%CI = 1.05–1.61) and lower intake of sugar sweetened beverages and fruit juices combined (HR for each additional serving = 1.11, 95% CI = 1.01–1.23). No other diet quality score or dietary pattern was associated with overall or CRC-specific mortality.

Conclusion

Higher AHEI-2010 score may be associated with lower overall mortality, moderate alcohol consumption and lower consumption of sugar sweetened beverages and juices combined appeared to account for most of the observed associations.     

Whole Pichia pastoris Yeast Expressing Measles Virus Nucleoprotein as a Production and Delivery System to Multimerize Plasmodium Antigens

Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the circumsporozoite protein (CS) of Plasmodium, the etiologic agent of malaria. The CS protein from Plasmodium berghei (Pb) was selected given the availability of the stringent C57Bl/6 mouse model of infection by Pb sporozoites, allowing the evaluation of vaccine efficacy in vivo. PbCS was multimerized by fusion to the measles virus (MV) nucleoprotein (N) known to auto-assemble in yeast in large-size ribonucleoprotein rods (RNPs). Expressed in P. pastoris, the N-PbCS protein generated highly multimeric and heterogenic RNPs bearing PbCS on their surface. Electron microscopy and immunofluorescence analyses revealed the shape of these RNPs and their localization in peripheral cytoplasmic inclusions. Subcutaneous immunization of C57Bl/6 mice with heat-inactivated whole P. pastoris expressing N-PbCS RNPs provided significant reduction of parasitemia after intradermal challenge with a high dose of parasites. Thus, in the absence of accessory adjuvants, a very low amount of PbCS expressed in whole yeast significantly decreased clinical damages associated with Pb infection in a highly stringent challenge model, providing a proof of concept of the intrinsic adjuvancy of this vaccine strategy. In addition to PbCS multimerization, the N protein contributed by itself to parasitemia delay and long-term mice survival. In the future, mixtures of whole recombinant yeasts expressing relevant Plasmodium antigens would provide a multivalent formulation applicable for antigen combination screening and possibly for large-scale production, distribution and delivery of a malaria vaccine in developing countries.     

Visual Sensorial Impairments in Neurodevelopmental Disorders: Evidence for a Retinal Phenotype in Fragile X Syndrome

Visual sensory impairments are common in Mental Deficiency (MD) and Autism Spectrum Disorder (ASD). These defects are linked to cerebral dysfunction in the visual cortical area characterized by the deregulation of axon growth/guidance and dendrite spine immaturity of neurons. However, visual perception had not been addressed, although the retina is part of the central nervous system with a common embryonic origin. Therefore, we investigated retinal perception, the first event of vision, in a murine model of MD with autistic features. We document that retinal function is altered in Fmr1 KO mice, a model of human Fragile X Syndrome. Indeed, In Fmr1 KO mice had a lower retinal function characterized by a decreased photoreceptors neuron response, due to a 40% decrease in Rhodopsin content and to Rod Outer Segment destabilization. In addition, we observed an alteration of the visual signal transmission between photoreceptors and the inner retina which could be attributed to deregulations of pre- and post- synaptic proteins resulting in retinal neurons synaptic destabilization and to retinal neurons immaturity. Thus, for the first time, we demonstrated that retinal perception is altered in a murine model of MD with autistic features and that there are strong similarities between cerebral and retinal cellular and molecular defects. Our results suggest that both visual perception and integration must be taken into account in assessing visual sensory impairments in MD and ASD.     

Physiological and Biochemical Responses of Yarrowia lipolytica to Dehydration Induced by Air-Drying and Freezing

Organisms that can withstand anhydrobiosis possess the unique ability to temporarily and reversibly suspend their metabolism for the periods when they live in a dehydrated state. However, the mechanisms underlying the cell’s ability to tolerate dehydration are far from being fully understood. The objective of this study was to highlight, for the first time, the cellular damage to Yarrowia lipolytica as a result of dehydration induced by drying/rehydration and freezing/thawing. Cellular response was evaluated through cell cultivability determined by plate counts, esterase activity and membrane integrity assessed by flow cytometry, and the biochemical composition of cells as determined by FT-IR spectroscopy. The effects of the harvesting time (in the log or stationary phase) and of the addition of a protective molecule, trehalose, were investigated. All freshly harvested cells exhibited esterase activity and no alteration of membrane integrity. Cells freshly harvested in the stationary phase presented spectral contributions suggesting lower nucleic acid content and thicker cell walls, as well as longer lipid chains than cells harvested in the log phase. Moreover, it was found that drying/rehydration induced cell plasma membrane permeabilization, loss of esterase activity with concomitant protein denaturation, wall damage and oxidation of nucleic acids. Plasma membrane permeabilization and loss of esterase activity could be reduced by harvesting in the stationary phase and/or with trehalose addition. Protein denaturation and wall damage could be reduced by harvesting in the stationary phase. In addition, it was shown that measurements of loss of membrane integrity and preservation of esterase activity were suitable indicators of loss and preservation of cultivability, respectively. Conversely, no clear effect of freezing/thawing could be observed, probably because of the favorable operating conditions applied. These results give insights into Y. lipolytica mechanisms of cellular response to dehydration and provide a basis to better understand its ability to tolerate anhydrobiosis.     

National Surveillance on Vancomycin-Resistant Enterococcus faecium in Taiwan: Emergence and Widespread of ST414 and a Tn1546-Like Element with Simultaneous Insertion of IS1251-like and IS1678

Cases of bacteremia caused by vancomycin-resistant E. faecium (VRE-fm) increased significantly in Taiwan. The present multicenter surveillance study was performed to reveal the associated epidemiological characteristics. In 2012, 134 non-repetitive VRE-fm isolates were prospectively collected from 12 hospitals in Taiwan. Antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and analysis of van genes and Tn1546 structures were investigated. Two isolates carried vanB genes, while all the remaining isolates carried vanA genes. Three isolates demonstrated a specific vanA genotype - vanB phenotype. Nine (6.7%) isolates demonstrated tigecycline resistance, and all were susceptible to daptomycin and linezolid. Molecular typing revealed 58 pulsotypes and 13 sequence types (STs), all belonged to three major lineages 17, 18, and 78. The most frequent STs were ST17 (n = 48, 35.8%), ST414 (n = 22, 16.4%), and ST78 (n = 16, 11.9%). Among the vanA harboring isolates, eight structure types of the Tn1546-like element were demonstrated. Type I (a partial deletion in the orf1 and insertion of IS1251-like between the vanS - vanH genes) and Type II (Type I with an additional insertion of IS1678 between orf2 - vanS genes) were the most predominant, consisted of 60 (45.5%) and 62 (47.0%) isolates, respectively. The increase of VRE-fm bacteremia in Taiwan may be associated with the inter- and intra-hospital spread of some major STs and horizontal transfer of vanA genes mostly carried on two efficient Tn1546-like elements. The prevailing ST414 and widespread of the Type II Tn1546-like elements are an emerging problem that requires continuous monitoring.     

Pharmacokinetics of Naja sumatrana (Equatorial Spitting Cobra) Venom and Its Major Toxins in Experimentally Envenomed Rabbits

Background

The optimization of snakebite management and the use of antivenom depend greatly on the knowledge of the venom''s composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on cobra venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting cobra) venom and its major toxins (phospholipase A₂, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits.

Principal findings

The serum antigen concentration-time profile of the N. sumatrana venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana venom injected intramuscularly were similar to those of N. sputatrix venom determined in an earlier study. The venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A₂ and whole venom (T_max = 2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of envenoming. The more prominent role of the neurotoxin in N. sumatrana systemic envenoming is further supported by its significantly higher intramuscular bioavailability (F_i.m. = 81.5%) compared to that of the phospholipase A₂ (F_i.m. = 68.6%) or cardiotoxin (F_i.m. = 45.6%). The incomplete absorption of the phospholipase A₂ and cardiotoxin may infer the toxins'' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions.

Conclusion/Significance

Our results suggest that the venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic envenoming.     

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial world. Animal models are of pivotal importance to study such diseases. In this regard the photoreceptor-specific toxin N-methyl-N-nitrosourea (MNU) has been widely used in rodents to pharmacologically induce retinal degeneration. Previously, we have established a MNU-induced retinal degeneration model in the zebrafish, another popular model system in visual research.A fascinating difference to mammals is the persistent neurogenesis in the adult zebrafish retina and its regeneration after damage. To quantify this observation we have employed visual acuity measurements in the adult zebrafish. Thereby, the optokinetic reflex was used to follow functional changes in non-anesthetized fish. This was supplemented with histology as well as immunohistochemical staining for apoptosis (TUNEL) and proliferation (PCNA) to correlate the developing morphological changes.In summary, apoptosis of photoreceptors occurs three days after MNU treatment, which is followed by a marked reduction of cells in the outer nuclear layer (ONL). Thereafter, proliferation of cells in the inner nuclear layer (INL) and ONL is observed. Herein, we reveal that not only a complete histological but also a functional regeneration occurs over a time course of 30 days. Now we illustrate the methods to quantify and follow up zebrafish retinal de- and regeneration using MNU in a video-format.