Thr199 phosphorylation targets nucleophosmin to nuclear speckles and represses pre-mRNA processing

Nucleophosmin (NPM) is a multifunctional phosphoprotein, being involved in ribosome assembly, pre-ribosomal RNA processing, DNA duplication, nucleocytoplasmic protein trafficking, and centrosome duplication. NPM is phosphorylated by several kinases, including nuclear kinase II, casein kinase 2, Polo-like kinase 1 and cyclin-dependent kinases (CDK1 and 2), and these phosphorylations modulate the activity and function of NPM. We have previously identified Thr(199) as the major phosphorylation site of NPM mediated by CDK2/cyclin E (and A), and this phosphorylation is involved in the regulation of centrosome duplication. In this study, we further examined the effect of CDK2-mediated phosphorylation of NPM by using the antibody that specifically recognizes NPM phosphorylated on Thr(199). We found that the phospho-Thr(199) NPM localized to dynamic sub-nuclear structures known as nuclear speckles, which are believed to be the sites of storage and/or assembly of pre-mRNA splicing factors. Phosphorylation on Thr(199) by CDK2/cyclin E (and A) targets NPM to nuclear speckles, and enhances the RNA-binding activity of NPM. Moreover, phospho-Thr(199) NPM, but not unphosphorylated NPM, effectively represses pre-mRNA splicing. These findings indicate the involvement of NPM in the regulation of pre-mRNA processing, and its activity is controlled by CDK2-mediated phosphorylation on Thr(199).     

Pre-exposure to a novel nutritional mixture containing a series of phytochemicals prevents acetaminophen-induced programmed and unprogrammed cell deaths by enhancing BCL-XL expression and minimizing oxidative stress in the liver

From a disease-prevention perspective, recent progress in phytochemical and nutraceutical research clearly suggests (benefits outweigh the risk pattern). Although powerful antioxidant properties have been the most acclaimed mechanism of action for these entities, the individual antioxidants studied in clinical trials do not appear to have consistent preventative effects. The actions of the antioxidant nutrients alone do not explain the observed health benefits of diets rich in fruits and vegetables for chronic diseases. Therefore, we proposed that the additive and synergistic effects of phytochemicals in fruits and vegetables are responsible for these potent antioxidant and anticancer activities, and that the benefit of a diet rich in fruits and vegetables is attributed to the complex mixture of phytochemicals present in plants [1]. Surprisingly, however, no studies have attempted to evaluate the combined antitoxic potential of a phytochemical-nutraceutical mixture (PNM) in in vivo models. Therefore, this study, for the first time, was designed to investigate whether pre-exposure to a unique PNM has the ability to impede mechanistic events involved in acetaminophen (APAP)-induced hepatotoxicity. Besides several vitamins and minerals in balanced proportions (~US RDA), the PNM used in this investigation contained several well-known phytochemicals such as citrus flavonoids, red wine polyphenols, Garcinia, Gymnema, Ginkgo, Ephedra sinica, Camelia sinensis, Silybum, Guarana, Eluthero, Allium sativum and Ocimum basilicum extracts. To evaluate PNM's antitoxic potential, groups of animals ICR mice, 3 months old) received either a control diet or PNM containing diets (1X and 10X) for 4 weeks. On day-28, animals were divided into two subgroups. Half the animals were administered normal saline and the other half received 400mg/kg ip injections of APAP. All the animals were sacrificed 24h after APAP exposure. Serum and tissue (liver and kidneys) samples were analyzed. APAP alone caused massive liver injury (nearly 495-fold increase in ALT) and oxidative stress (Lipid peroxidation: 268% increase in MDA) coupled with genomic DNA fragmentation (288% increase). Exposure to 1X-PNM for 28 days significantly reduced animal mortality and all the APAP-induced biochemical events (In 1X-PNM + AP: ALT leakage decreased to 54 fold; MDA accumulation decreased to 125%, and DNA fragmentation decreased to 122%), whereas 10X-PNM + APAP slightly escalated both oxidative stress and genomic DNA fragmentation preceding liver injury. Liver homogenates subjected to western blot analysis disclosed the ability of 1X-PNM to counteract APAP-induced decrease in Bcl-xL expression. Histopathological evaluation of stained liver tissue sections indicated anti-apoptogenic and anti-necrogenic reponses coupled with near complete prevention of glycogen depletion by 1X-PNM. Collectively, our investigation suggests that a mixture containing an assortment of phytochemicals/nutraceuticals may serve as a much more powerful blend in preventing drug or chemical-induced organ injuries than a single phytochemical or nutraceutical entity. In addition, ephedra and caffeine containing PNM-exposure in a controlled manner may potentially shield vital target organs from toxicities caused by intentional, unintentional or accidental exposures to structurally and functionally diverse drug and chemical entities.     

A novel rat orthologue and homologue for the Drosophila crooked neck gene in neural stem cells and their immediate descendants

The crooked neck (crn) gene of Drosophila melanogaster encodes a scaffold protein carrying multiple tetratricopeptide repeat (TPR) motifs, and its mutation results in a reduction in the number of neuroblasts and lethality during larval stages. Here, we isolated two structurally related genes from a rat embryonic brain cDNA library. One gene is the rat orthologue of crn, which encodes 690 amino acids including 16 copies of TPR. The other gene, ATH55, encodes an 855 amino acid protein including 21 TPR motifs, which presumably represents a rat crn homologue and an orthologue of human XAB2. Both genes are highly expressed in embryonic brain but their expressions decrease during development. ATH55-like immunoreactivity is present in the ventricular zone and newly formed cortical plate, while CRN-like immunoreactivity is more abundant in a younger ventricular zone. In agreement, both proteins were found to be enriched in cultured neural stem cells and to decrease in response to cell differentiation signals. As indicated for the yeast CRN-like protein, ATH55 and CRN immunoreactivities were both recovered in the nuclear fraction and detected in the splicing complex carrying pre-mRNA. These findings suggest that both TPR-motif-containing proteins are involved in RNA processing of mammalian neural stem cells and their immediate descendants.     

Genetic analysis of Tn7 transposition

Summary The purpose of this work was to localize the DNA regions necessary for the transposition of Tn7. Several deletions of Tn7 were constructed by the excision of DNA fragments between restriction sites. The ability of these deleted Tn7s to transpose onto the recipient plasmid RP4 was examined. All the deleted Tn7s isolated in this work had lost their transposing capability. The possibility of complementing them was studied using plasmids containing all or part of Tn7. Two deleted Tn7s could not be complemented by an entire Tn7 indicating that a DNA sequence greater than the 42 bp terminal sequence is needed for recognition of the transposon by a transposition function. Four other deleted Tn7s could be complemented by Tn7. One of these was studied intensively in complementation experiments using different parts of Tn7 to obtain transposition. The results obtained allow us to propose that all genes needed for transposition of Tn7 onto plasmids are contained in a DNA segment of between 6.0 and 7.4 kb. Furthermore, one essential function must be contained in a DNA fragment longer than 2.5 kb on the right-hand end of Tn7. The classification of Tn7 with regard to the other transposable elements is discussed.     

High throughput single-cell genome sequencing gives insights into the generation and evolution of mosaic aneuploidy in Leishmania donovani

Leishmania, a unicellular eukaryotic parasite, is a unique model for aneuploidy and cellular heterogeneity, along with their potential role in adaptation to environmental stresses. Somy variation within clonal populations was previously explored in a small subset of chromosomes using fluorescence hybridization methods. This phenomenon, termed mosaic aneuploidy (MA), might have important evolutionary and functional implications but remains under-explored due to technological limitations. Here, we applied and validated a high throughput single-cell genome sequencing method to study for the first time the extent and dynamics of whole karyotype heterogeneity in two clonal populations of Leishmania promastigotes representing different stages of MA evolution in vitro. We found that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid founder cell. This possibly involves polyploidization/hybridization at an early stage of population expansion, followed by assorted ploidy reduction. During further stages of expansion, MA increases by moderate and gradual karyotypic alterations, affecting a defined subset of chromosomes. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies.     

Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells

Cancer cells are often associated with secondary chromosomal rearrangements, such as deletions, inversions, and translocations, which could be the consequence of unrepaired/misrepaired DNA double strand breaks (DSBs). Nonhomologous DNA end joining is one of the most common pathways to repair DSBs in higher eukaryotes. By using oligomeric DNA substrates mimicking various endogenous DSBs in a cell-free system, we studied end joining (EJ) in different cancer cell lines. We found that the efficiency of EJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of EJ proteins. Interestingly, cancer cells with lower levels of EJ possessed elevated expression of BCL2 and vice versa. Removal of BCL2 by immunoprecipitation or protein fractionation led to elevated EJ. More importantly, we show that overexpression of BCL2 or the addition of purified BCL2 led to the down-regulation of EJ. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo. Hence, our results suggest that EJ in cancer cells could be negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute toward increased chromosomal abnormalities in cancer.     

Effect of D222G mutation in the hemagglutinin protein on receptor binding, pathogenesis and transmissibility of the 2009 pandemic H1N1 influenza virus

Influenza viruses isolated during the 2009 H1N1 pandemic generally lack known molecular determinants of virulence associated with previous pandemic and highly pathogenic avian influenza viruses. The frequency of the amino acid substitution D222G in the hemagglutinin (HA) of 2009 H1N1 viruses isolated from severe but not mild human cases represents the first molecular marker associated with enhanced disease. To assess the relative contribution of this substitution in virus pathogenesis, transmission, and tropism, we introduced D222G by reverse genetics in the wild-type HA of the 2009 H1N1 virus, A/California/04/09 (CA/04). A dose-dependent glycan array analysis with the D222G virus showed a modest reduction in the binding avidity to human-like (α2-6 sialylated glycan) receptors and an increase in the binding to avian-like (α2-3 sialylated glycan) receptors in comparison with wild-type virus. In the ferret pathogenesis model, the D222G mutant virus was found to be similar to wild-type CA/04 virus with respect to lethargy, weight loss and replication efficiency in the upper and lower respiratory tract. Moreover, based on viral detection, the respiratory droplet transmission properties of these two viruses were found to be similar. The D222G virus failed to productively infect mice inoculated by the ocular route, but exhibited greater viral replication and weight loss than wild-type CA/04 virus in mice inoculated by the intranasal route. In a more relevant human cell model, D222G virus replicated with delayed kinetics compared with wild-type virus but to higher titer in human bronchial epithelial cells. These findings suggest that although the D222G mutation does not influence virus transmission, it may be considered a molecular marker for enhanced replication in certain cell types.     

Universal highly efficient conditional knockout system in Leishmania,with a focus on untranscribed region preservation

Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR‐Cas9 has been applied successfully to these parasites providing a robust tool to study non‐essential gene functions. Here, we have developed a versatile inducible system combining Di‐Cre recombinase and CRISPR‐Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre‐recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di‐Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed “universal” template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR‐based (molecular cloning‐free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.     

Comparative Assessment of Cytokines and Other Inflammatory Markers for the Early Diagnosis of Neonatal Sepsis–A Case Control Study

Objective

Cytokines (IL-6, IL-8 and TNF-α), sCD163, and C-reactive protein were serially measured in an attempt to identify a set of tests which can reliably confirm or refute the diagnosis of neonatal sepsis at an early stage.

Methods

One hundred neonates suspected to have sepsis on clinical grounds and who met the inclusion criteria were enrolled for the study. Based on the positive or negative blood culture reports they were classified as infected (n = 50) and non-infected (n = 50) neonates respectively. Fifty healthy neonates without any signs of sepsis were also included in the study as control group. The initial blood sample was taken on day 0 (at the time of sepsis evaluation) and two further samples were taken on days 1 and 2 for monitoring the clinical progress and response to treatment. In the control group the cord blood and 48 hours venous sample was collected. Plasma CRP (ng/ml), IL-6 (pg/ml), IL-8 (pg/ml), TNF-α (ng/ml) and sCD163 (ng/ml) were determined by double antibody method Enzyme Linked Immunosorbent Assay in all the three blood samples.

Results

The cut of levels for CRP at >19,689 ng/ml had a sensitivity of 68%, specificity of 92%, for IL-6 at >95.32 pg/ml had a sensitivity of 54%, specificity of 96%, for IL-8 at >70.86 pg/ml had a sensitivity of 78%, specificity of 70%, for sCD163 at >896.78 ng/ml had a sensitivity of 100%, specificity of 88% for the diagnosis of infection before antibiotics. TNF-α levels of >12.6 ng/ml showed 100% sensitivity and 72% specificity for the diagnosis of inflammation.

Conclusion

The most powerful predictor to differentiate between the non-infected and infected neonates before antibiotics was sCD163. The most powerful indicator for evaluation of prognosis is IL-6. sCD163 can be used alone to screen for sepsis in neonates before the results of blood culture are received.