Chromosome catastrophes involve replication mechanisms generating complex genomic rearrangements

Complex genomic rearrangements (CGRs) consisting of two or more breakpoint junctions have been observed in genomic disorders. Recently, a chromosome catastrophe phenomenon termed chromothripsis, in which numerous genomic rearrangements are apparently acquired in one single catastrophic event, was described in multiple cancers. Here, we show that constitutionally acquired CGRs share similarities with cancer chromothripsis. In the 17 CGR cases investigated, we observed localization and multiple copy number changes including deletions, duplications, and/or triplications, as well as extensive translocations and inversions. Genomic rearrangements involved varied in size and complexities; in one case, array comparative genomic hybridization revealed 18 copy number changes. Breakpoint sequencing identified characteristic features, including small templated insertions at breakpoints and microhomology at breakpoint junctions, which have been attributed to replicative processes. The resemblance between CGR and chromothripsis suggests similar mechanistic underpinnings. Such chromosome catastrophic events appear to reflect basic DNA metabolism operative throughout an organism's life cycle.     

Contrasting effects of a cladoceran (Daphnia galeata) and a calanoid copepod (Eodiaptomus japonicus) on algal and microbial plankton in a Japanese lake, Lake Biwa

Macrozooplankton may affect algal and microbial plankton directly through grazing or predation and indirectly through nutrient regeneration. They may also affect potential prey positively by removing alternative predators. Here, we examined the effects of a cladoceran (Daphnia) and a calanoid copepod (Eodiaptomus) on algal and microbial plankton in a Japanese lake using in situ experiments in which we manipulated the nutrient supply and biomass of these macrozooplankton. The response of algal and microbial plankton to macrozooplankton was diverse and varied depending on the level of nutrient supply. Eodiaptomus seemed to feed mainly on large algae (>20 µm) and microzooplankton, while direct grazing by Daphnia on algae, bacteria, heterotrophic nanoflagellates (HNF), and microzooplankton (ciliates, heliozoa, and rotifers) was pronounced. Trophic linkages within these microbial plankton was also suggested; bacteria were grazed by HNF and these in turn were grazed by microzooplankton. When the nutrient supply was high, both HNF and microzooplankton were exposed to higher amounts of algae and lower bacterial abundance. Moreover, nutrient regeneration by daphnids and Eodiaptomus copepods seemed to differentially stimulate the growth of algae and bacteria. The results suggest that the relationship between macrozooplankton and microbial plankton cannot be fully understood without taking into consideration not only the feeding characteristics of the macrozooplankton, but also the food web structure, the subsidized algal resource, and nutrient regeneration from the macrozooplankton.     

Characterization of two isoforms of the skeletal muscle LIM protein 1, SLIM1. Localization of SLIM1 at focal adhesions and the isoform slimmer in the nucleus of myoblasts and cytoplasm of myotubes suggests distinct roles in the cytoskeleton and in nuclear-cytoplasmic communication.

We have cloned and characterized a novel isoform of the skeletal muscle LIM protein 1 (SLIM1), designated SLIMMER. SLIM1 contains an N-terminal single zinc finger followed by four LIM domains. SLIMMER is identical to SLIM1 over the first three LIM domains but contains a novel C-terminal 96 amino acids with three potential bipartite nuclear localization signals, a putative nuclear export sequence, and 27 amino acids identical to the RBP-J binding region of KyoT2, a murine isoform of SLIM1. SLIM1 localized to the cytosol of Sol8 myoblasts and myotubes. SLIMMER was detected in the nucleus of myoblasts and, following differentiation into myotubes, was exclusively cytosolic. Recombinant green fluorescent protein-SLIM1 localized to the cytoplasm and associated with focal adhesions and actin filaments in COS-7 cells, while green fluorescent protein-SLIMMER was predominantly nuclear. SLIMMER truncation mutants revealed that the first nuclear localization signal mediates nuclear localization. The addition of the proposed nuclear export sequence decreased the level of exclusively nuclear expression and increased cytosolic SLIMMER expression in COS-7 cells. The leucine-rich nuclear export signal was required for the export of SLIMMER from the nucleus of myoblasts to the cytoplasm of myotubes. Collectively, these results suggest distinct roles for SLIM1 and SLIMMER in focal adhesions and nuclear-cytoplasmic communication.     

Genome-wide analyses of Liberibacter species provides insights into evolution,phylogenetic relationships,and virulence factors

‘Candidatus Liberibacter’ species are insect-transmitted, phloem-limited α-Proteobacteria in the order of Rhizobiales. The citrus industry is facing significant challenges due to huanglongbing, associated with infection from ‘Candidatus Liberibacter asiaticus’ (Las). In order to gain greater insight into ‘Ca. Liberibacter’ biology and genetic diversity, we have performed genome sequencing and comparative analyses of diverse ‘Ca. Liberibacter’ species, including those that can infect citrus. Our phylogenetic analysis differentiates ‘Ca. Liberibacter’ species and Rhizobiales in separate clades and suggests stepwise evolution from a common ancestor splitting first into nonpathogenic Liberibacter crescens followed by diversification of pathogenic ‘Ca. Liberibacter’ species. Further analysis of Las genomes from different geographical locations revealed diversity among isolates from the United States. Our phylogenetic study also indicates multiple Las introduction events in California and spread of the pathogen from Florida to Texas. Texan Las isolates were closely related, while Florida and Asian isolates exhibited the most genetic variation. We have identified conserved Sec translocon (SEC)-dependent effectors likely involved in bacterial survival and virulence of Las and analysed their expression in their plant host (citrus) and insect vector (Diaphorina citri). Individual SEC-dependent effectors exhibited differential expression patterns between host and vector, indicating that Las uses its effector repertoire to differentially modulate diverse organisms. Collectively, this work provides insights into the evolution of ‘Ca. Liberibacter’ species, the introduction of Las in the United States and identifies promising Las targets for disease management.     

First results on bathymetry and limnology of high-altitude lakes in the Gokyo Valley, Sagarmatha (Everest) National Park, Nepal

The aim of this study was to investigate for the first time the morphology, physical and chemical characteristics of three high-altitude lakes in the Gokyo Valley, Everest National Park, Nepal. The moraine-dammed glacial lakes were studied for three seasons to create baseline data. The second, third, and fourth lakes in the Gokyo Valley are deeper than previously assumed. The vertical profiles of temperature and dissolved oxygen indicated that the thermocline zone varied between 10 and 20 m below the surface during the post-monsoon season. Although most of the analyzed metal concentrations were below the level recommended by the WHO for safe drinking water, lead (Pb) and cadmium (Cd) levels exceeded the limits in some of the samples. This has created great concern for human health. The probable route of these two pollutants is monsoon precipitation that carries industrial pollutants along its route. The sedimentation rates of Gokyo Lakes range from 0.069 to 0.089 cm per annum, which is within the limits of other high-altitude lakes. The present findings created a baseline database for some of the remote high-altitude lakes in Nepal that can be used for lake management and for assessing future changes in lake characteristics in the Himalayan region.     

Targeted Delivery of Small Interfering RNA to Human Dendritic Cells To Suppress Dengue Virus Infection and Associated Proinflammatory Cytokine Production

Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), in which both viral and host factors have been implicated. RNA interference (RNAi) is a potent antiviral strategy and a potential therapeutic option for dengue if a feasible strategy can be developed for delivery of small interfering RNA (siRNA) to dendritic cells (DCs) and macrophages, the major in vivo targets of the virus and also the source of proinflammatory cytokines. Here we show that a dendritic cell-targeting 12-mer peptide (DC3) fused to nona-d-arginine (9dR) residues (DC3-9dR) delivers siRNA and knocks down endogenous gene expression in heterogenous DC subsets, (monocyte-derived DCs [MDDCs], CD34⁺ hematopoietic stem cell [HSC])-derived Langerhans DCs, and peripheral blood DCs). Moreover, DC3-9dR-mediated delivery of siRNA targeting a highly conserved sequence in the dengue virus envelope gene (siFvE^D) effectively suppressed dengue virus replication in MDDCs and macrophages. In addition, DC-specific delivery of siRNA targeting the acute-phase cytokine tumor necrosis factor alpha (TNF-α), which plays a major role in dengue pathogenesis, either alone or in combination with an antiviral siRNA, significantly reduced virus-induced production of the cytokine in MDDCs. Finally to validate the strategy in vivo, we tested the ability of the peptide to target human DCs in the NOD/SCID/IL-2Rγ^−/− mouse model engrafted with human CD34⁺ hematopoietic stem cells (HuHSC mice). Treatment of mice by intravenous (i.v.) injection of DC3-9dR-complexed siRNA targeting TNF-α effectively suppressed poly(I:C)-induced TNF-α production by DCs. Thus, DC3-9dR can deliver siRNA to DCs both in vitro and in vivo, and this delivery approach holds promise as a therapeutic strategy to simultaneously suppress virus replication and curb virus-induced detrimental host immune responses in dengue infection.Dengue is a mosquito-borne flavivirus infection that has emerged as a serious public health problem worldwide. Four serotypes of dengue virus (DEN-1 to DEN-4) are capable of causing human disease varying in severity from acute self-limiting febrile illness to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The plasma leakage, hemorrhagic manifestations, and shock that characterize DHF/DSS are considered to have an immunological basis, as they are more common during secondary infection with a heterologous dengue virus strain (15, 28, 33). However, severe clinical manifestations can also occur during primary dengue infection, pointing to a contributory role of viral virulence factors. The WHO estimates that more than 20,000 people worldwide, mainly children, die each year from serious complications of dengue. No specific antiviral therapies are currently available for treating the infection, and efforts to develop a safe prophylactic vaccine have been hindered by the complex role of the immune system in disease pathogenesis (39, 52, 57). Thus, novel treatment strategies that block viral replication and/or to attenuate the exaggerated cytokine response associated with DHF/DSS complications are urgently needed.Potent and specific gene silencing mediated by RNA interference (RNAi) has generated a great deal of interest in development of RNAi as a therapeutic strategy against viral infections (50, 54). Many studies have demonstrated the effectiveness of the RNAi approach to suppress flavivirus infection, including dengue virus replication in experimental cell lines (3, 23, 26, 42, 60). In addition, the versatility of RNAi could also be exploited to block important host mediators that contribute to dengue pathogenesis. However, the existence of four distinct dengue virus serotypes and the ability of viruses to develop resistance to RNAi by mutating their sequences will have to be taken into account before clinical use can be contemplated. A more serious hurdle for RNAi therapeutics is the specific delivery of small interfering RNA (siRNA) to relevant cell types.Even though dengue virus antigens have been detected in many tissues, including liver, spleen, lymph node, and skin of patients with DHF/DSS, macrophages and dendritic cells (DCs) are considered the predominant infected cell types (9, 36, 59). Following the bite of an infected Aedes mosquito, the initial local viral replication is believed to take place in the skin DCs, including myeloid DCs and Langerhans cells (31, 53, 59). Dengue-infected DCs play a key role in the immunopathogenesis of DHF/DSS, as, along with macrophages, they release proinflammatory cytokines and soluble factors that mediate plasma leakage, thrombocytopenia, and hypovolemic shock associated with severe dengue infection (14, 15, 29, 38). Therefore, development of a method to introduce siRNA into DCs would be an important step toward using RNAi therapeutically to suppress viral replication and/or to attenuate the vigorous host cytokine responses in dengue infection (7, 19).To target DCs, we used a previously characterized 12-amino-acid peptide identified from a phage display peptide library that specifically binds to a ligand expressed on DCs (10). In an earlier study, we demonstrated that fusing nucleic acid-binding nine d-arginine residues to a neuronal cell-targeting peptide enabled siRNA delivery to neuronal cells (27). Here, in a similar approach, we synthesized a chimeric peptide consisting of the DC-targeting peptide fused to nona-D-arginines (9dR) to target siRNA selectively to DCs. We investigated whether the DC3-9dR peptide could deliver siRNA targeting a dengue virus envelope sequence to reduce the viral load in DCs. As tumor necrosis factor alpha (TNF-α) is one of the acute-phase cytokines with a major role in inducing plasma leakage in dengue infection (8, 12, 17, 20), we also explored the possibility of reducing TNF-α expression in DC in vitro and in vivo. Our findings demonstrate the potential of a targeted RNAi-based approach for simultaneously decreasing viral load and reducing aberrant cytokine responses in DCs.     

Molecular Characterization of CTX-M β-Lactamase and Associated Addiction Systems in Escherichia coli Circulating among Cattle,Farm Workers,and the Farm Environment

A total of 84 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from cattle, farm workers, and the farm environment isolated from February to September 2008 in the Republic of Korea were investigated. All 84 ESBL-producing isolates carried bla_CTX-M genes that belonged to the CTX-M-1 (n = 35) or CTX-M-9 (n = 49) family. The most predominant CTX-M type identified was CTX-M-14 (n = 49), followed by CTX-M-32 (n = 26). The bla_CTX-M genes were identified most commonly in E. coli isolates from feces (n = 29), teats (n = 25), and milk (n = 14). A bla_CTX-M-14 gene was also detected in an E. coli isolate from a farmer''s hand. Transfer of the bla_CTX-M gene from 60 bla_CTX-M-positive E. coli isolates to the recipient E. coli J53 strain by conjugation was demonstrated. Plasmid isolation from bla_CTX-M-positive transconjugants revealed a large (95- to 140-kb) conjugative plasmid. Almost all (82/84) bla_CTX-M genes possessed an insertion sequence, ISEcp1, upstream of the bla_CTX-M gene. Only in the case of the CTX-M-14 genes was IS903 downstream of the gene. The bla_CTX-M genes were associated with seven kinds of addiction systems. Among them, pndAC, hok-sok, and srnBC were the most frequently identified addiction systems in both wild strains and transconjugants. The spread of bla_CTX-M genes was attributed to both clonal expansion and horizontal dissemination. Our data suggest that a combination of multiple addiction systems in plasmids carrying bla_CTX-M genes could contribute to their maintenance in the host cells. To our knowledge, the bla_CTX-M-32 gene has not previously been reported in animal isolates from the Republic of Korea.     

Aspirin in Primary Prevention of Cardiovascular Disease and Cancer: A Systematic Review of the Balance of Evidence from Reviews of Randomized Trials

Background

Aspirin has been recommended for primary prevention of cardiovascular disease (CVD) and cancer, but overall benefits are unclear. We aimed to use novel methods to re-evaluate the balance of benefits and harms of aspirin using evidence from randomised controlled trials, systematic reviews and meta-analyses.

Methods and Findings

Data sources included ten electronic bibliographic databases, contact with experts, and scrutiny of reference lists of included studies. Searches were undertaken in September 2012 and restricted to publications since 2008. Of 2,572 potentially relevant papers 27 met the inclusion criteria. Meta-analysis of control arms to estimate event rates, modelling of all-cause mortality and L''Abbé plots to estimate heterogeneity were undertaken. Absolute benefits and harms were low: 60-84 major CVD events and 34-36 colorectal cancer deaths per 100,000 person-years were averted, whereas 46-49 major bleeds and 68-117 gastrointestinal bleeds were incurred. Reductions in all-cause mortality were minor and uncertain (Hazard Ratio 0.96; 95% CI: 0.90-1.02 at 20 years, Relative Risk [RR] 0.94, 95% CI: 0.88-1.00 at 8 years); there was a non-significant change in total CVD (RR 0.85, 95% CI: 0.69-1.06) and change in total cancer mortality ranged from 0.76 (95% CI: 0.66-0.88) to 0.93 (95% CI: 0.84-1.03) depending on follow-up time and studies included. Risks were increased by 37% for gastrointestinal bleeds (RR 1.37, 95% CI: 1.15-1.62), 54%-66% for major bleeds (Rate Ratio from IPD analysis 1.54, 95% CI: 1.30-1.82, and RR 1.62, 95% CI: 1.31-2.00), and 32%-38% for haemorrhagic stroke (Rate Ratio from IPD analysis 1.32; 95% CI: 1.00-1.74; RR 1.38; 95% CI: 1.01-1.82).

Conclusions

Findings indicate small absolute effects of aspirin relative to the burden of these diseases. When aspirin is used for primary prevention of CVD the absolute harms exceed the benefits. Estimates of cancer benefit rely on selective retrospective re-analysis of RCTs and more information is needed.     

Efficient enzymatic systems for synthesis of novel α-mangostin glycosides exhibiting antibacterial activity against Gram-positive bacteria

Two enzymatic systems were developed for the efficient synthesis of glycoside products of α-mangostin, a natural xanthonoid exhibiting anti-oxidant, antibacterial, anti-inflammatory, and anticancer activities. In these systems, one-pot reactions for the synthesis of UDP-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined with a glycosyltransferase (GT) from Bacillus licheniformis DSM-13 to afford C-3 and C-6 position modified glucose and 2-deoxyglucose conjugated novel α-mangostin derivatives. α-Mangostin 3-O-β-D-glucopyranoside, α-mangostin 6-O-β-D-glucopyranoside, α-mangostin 3,6-di-O-β-D-glucopyranoside, α-mangostin 3-O-β-D-2-deoxyglucopyranoside, α-mangostin 6-O-β-D-2-deoxyglucopyranoside, and α-mangostin 3,6-di-O-β-D-2-deoxyglucopyranoside were successfully produced in practical quantities and characterized by high-resolution quadruple time-of-flight electrospray ionization-mass spectrometry (HR-QTOF ESI/MS), ¹H and ¹³C NMR analyses. In excess of the substrate, the maximum productions of three α-mangostin glucopyranosides (4.8 mg/mL, 86.5 % overall conversion of α-mangostin) and three α-mangostin 2-deoxyglucopyronosides (4.0 mg/mL, 79 % overall conversion of α-mangostin) were achieved at 4-h incubation period. All the α-mangostin glycosides exhibited improved water solubility, and their antibacterial activity against three Gram-positive bacteria Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus was drastically enhanced by the glucosylation at C-3 position. In this study, diverse glycosylated α-mangostin were produced in significant quantities by using inexpensive starting materials and recycling co-factors within a reaction vessel without use of expensive NDP-sugars in the glycosylation reactions.     

Azide-dependent nitric oxide emission from the water fern Azolla pinnata

Nitric oxide (NO) is involved in versatile functions in plant growth and development as a signaling molecule. To date, plants have been reported to produce NO following exposure to nitrite (N O ₂ ^? ) the amino acid L-arginine, hydroxylamine, or polyamines. Here we demonstrate azide-dependent NO production in plants. The water fern Azolla pinnata emitted NO into air upon exposure to sodium azide (NaN₃). The NO production was dependent on azide concentration and was strongly inhibited by potassium cyanide (KCN). Incubation of A. pinnata with the catalase inhibitor 3-aminotriazole (3-AT) abolished the azide-dependent NO production. Although nitrite-dependent NO production was inhibited by sodium azide, azide-dependent NO production was not affected by nitrite. These results indicate that A. pinnata enzymatically produces NO using azide as a substrate. We suggest that plants are also capable of producing NO from azide by the action of catalase as previously reported in animals.