Application of Ion Torrent Sequencing to the Assessment of the Effect of Alkali Ballast Water Treatment on Microbial Community Diversity

The impact of NaOH as a ballast water treatment (BWT) on microbial community diversity was assessed using the 16S rRNA gene based Ion Torrent sequencing with its new 400 base chemistry. Ballast water samples from a Great Lakes ship were collected from the intake and discharge of both control and NaOH (pH 12) treated tanks and were analyzed in duplicates. One set of duplicates was treated with the membrane-impermeable DNA cross-linking reagent propidium mono-azide (PMA) prior to PCR amplification to differentiate between live and dead microorganisms. Ion Torrent sequencing generated nearly 580,000 reads for 31 bar-coded samples and revealed alterations of the microbial community structure in ballast water that had been treated with NaOH. Rarefaction analysis of the Ion Torrent sequencing data showed that BWT using NaOH significantly decreased microbial community diversity relative to control discharge (p<0.001). UniFrac distance based principal coordinate analysis (PCoA) plots and UPGMA tree analysis revealed that NaOH-treated ballast water microbial communities differed from both intake communities and control discharge communities. After NaOH treatment, bacteria from the genus Alishewanella became dominant in the NaOH-treated samples, accounting for <0.5% of the total reads in intake samples but more than 50% of the reads in the treated discharge samples. The only apparent difference in microbial community structure between PMA-processed and non-PMA samples occurred in intake water samples, which exhibited a significantly higher amount of PMA-sensitive cyanobacteria/chloroplast 16S rRNA than their corresponding non-PMA total DNA samples. The community assembly obtained using Ion Torrent sequencing was comparable to that obtained from a subset of samples that were also subjected to 454 pyrosequencing. This study showed the efficacy of alkali ballast water treatment in reducing ballast water microbial diversity and demonstrated the application of new Ion Torrent sequencing techniques to microbial community studies.     

Caffeine reduces 11β-hydroxysteroid dehydrogenase type 2 expression in human trophoblast cells through the adenosine A(2B) receptor

Maternal caffeine consumption is associated with reduced fetal growth, but the underlying molecular mechanisms are unknown. Since there is evidence that decreased placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) is linked to fetal growth restriction, we hypothesized that caffeine may inhibit fetal growth partly through down regulating placental 11β-HSD2. As a first step in examining this hypothesis, we studied the effects of caffeine on placental 11β-HSD2 activity and expression using our established primary human trophoblast cells as an in vitro model system. Given that maternal serum concentrations of paraxanthine (the primary metabolite of caffeine) were greater in women who gave birth to small-for-gestational age infants than to appropriately grown infants, we also studied the effects of paraxanthine. Our main findings were: (1) both caffeine and paraxanthine decreased placental 11β-HSD2 activity, protein and mRNA in a concentration-dependent manner; (2) this inhibitory effect was mediated by the adenosine A(2B) receptor, since siRNA-mediated knockdown of this receptor prevented caffeine- and paraxanthine-induced inhibition of placental 11β-HSD2; and (3) forskolin (an activator of adenyl cyclase and a known stimulator of 11β-HSD2) abrogated the inhibitory effects of both caffeine and paraxanthine, which provides evidence for a functional link between exposure to caffeine and paraxanthine, decreased intracellular levels of cAMP and reduced placental 11β-HSD2. Taken together, these findings reveal that placental 11β-HSD2 is a novel molecular target through which caffeine may adversely affect fetal growth. They also uncover a previously unappreciated role for the adenosine A(2B) receptor signaling in regulating placental 11β-HSD2, and consequently fetal development.     

Synthesis, structure, photophysical and electrochemical behavior of 2-amino-anthracene triosmium clusters

The reactions of 2-amino-anthracene with [Os₃(CO)₁₀(CH₃CN)₂] have been studied and the products structurally characterized by spectroscopic, X-ray diffraction, photophysical and electrochemical techniques. At room temperature in CH₂Cl₂ two major, isomeric products are obtained [Os₃(CO)₁₀(μ-η²-(N-C(1))-NH₂C₁₄H₈)(μ-H)] (1, 14%) and [Os₃(CO)₁₀(μ-η²-(N-C(3))-NHC₁₄H₉)(μ-H)] (2, 35%) along with a trace amount of the dihydrido complex [Os₃(CO)₉(μ-η²-(N-C(3))-NHC₁₄H₈)(μ-H)₂] (3). In refluxing tetrahydrofuran only complexes 2 and 3 are obtained in 24% and 28%, respectively. A separate experiment shows that complex 1 slowly converts to 2 and that the rearrangement is catalyzed by adventitious water and involves proton transfer to the anthracene ring. Complex 1 is stereochemically non-rigid; exhibiting edge to edge hydride migration while 2 is stereochemically rigid. Complex 3 is also stereochemically non-rigid showing a site exchange process of the magnetically nonequivalent hydrides typical for trinuclear dihydrides. Interestingly, 2 decarbonylates cleanly to the electronically unsaturated 46e⁻ cluster [Os₃(CO)₉(μ₃-η²-(N-C(3))-NHC₁₀H₉)(μ-H)] (4, 68%) in refluxing cyclohexane, while photolysis of 2 in CH₂Cl₂ yields only a small amount of 3 along with considerable decomposition. The mechanism of the conversion of 1 to 2 and the dependence of the product distribution on solvent are discussed. All four compounds are luminescent with compounds 1-3 showing emissions that can be assigned to radiative decay associated with the anthracene ligand. Complexes 1-3 all show irreversible 1e⁻ reductions in the range of −1.85-2.14 V while 4 shows a nicely reversible 1e⁻ wave at −1.16 V and a quasi-reversible second 1e⁻ wave at −1.62 V. Irreversible oxidations are observed in the range from +0.35 to +0.49 V. The relationship between the cluster ligand configurations and the observed electrochemical and photochemical behavior is discussed and compared with that of the free ligand.     

In vitro plantlet regeneration of “dwarf” Indian olive (Elaeocarpus robustus Roxb.): a fruit plant of Bangladesh

A plantlet regeneration protocol was developed on pot-grown mature plants of Elaeocarpus robustus Roxb. cv. Dwarf from nodal and leaf explants. The best yield of adventitious shoots was achieved from the leaf-derived calli in a modified MS (MMS₁, half strength of major salts, full strength of minor salts, and vitamins) medium containing 4.0 μM BA + 4.0 μM Kn + 0.5 μM NAA + 15% coconut water (CW). The shoot multiplication rate was amplified about twofold per culture after the addition of 15% CW to the medium. The rate of shoot multiplication reached maximum at the 5th subculture, and it maintained this rate throughout the 3 subsequent subcultures. The best rooting in vitro was investigated by subculturing the microcuttings in an MMS₂ (half strength of both major salts and minor salts and full strength of vitamins) medium containing 1.0 μM IBA in the dark for one initial week at 30°C, followed by subculturing them in a plant-growth regulator (PGR)-free medium in the light. The plantlets raised in vitro were successfully established under ex vitro conditions.     

Identification of ChChd3 as a novel substrate of the cAMP-dependent protein kinase (PKA) using an analog-sensitive catalytic subunit

Due to the numerous kinases in the cell, many with overlapping substrates, it is difficult to find novel substrates for a specific kinase. To identify novel substrates of cAMP-dependent protein kinase (PKA), the PKA catalytic subunit was engineered to accept bulky N(6)-substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src (1). Methionine 120 was mutated to glycine in the ATP-binding pocket of the catalytic subunit. To express the stable mutant C-subunit in Escherichia coli required co-expression with PDK1. This mutant protein was active and fully phosphorylated on Thr(197) and Ser(338). Based on its kinetic properties, the engineered C-subunit preferred N(6)(benzyl)-ATP and N(6)(phenethyl)-ATP over other ATP analogs, but still retained a 30 microm K(m) for ATP. This mutant recombinant C-subunit was used to identify three novel PKA substrates. One protein, a novel mitochondrial ChChd protein, ChChd3, was identified, suggesting that PKA may regulate mitochondria proteins.     

Validity of Antibodies in Lymphocyte Supernatant in Diagnosing Tuberculosis in Severely Malnourished Children Presenting with Pneumonia

BackgroundThe diagnosis of tuberculosis (TB) in young children can be challenging, especially in severely malnourished children. There is a critical need for improved diagnostics for children. Thus, we sought to evaluate the performance of a technique that measures antibodies in lymphocyte supernatant (ALS) for the diagnosis of TB in severely malnourished children presenting with suspected pneumonia.MethodsChildren less than 5 years with severe acute malnutrition and radiological features of pneumonia admitted to the Dhaka Hospital of International Centre for Diarrhoeal Disease Research, Bangladesh, were enrolled consecutively following informed written consent. In addition to clinical and radiological assessment, samples taken for TB diagnosis included gastric lavage fluid and induced sputum for microbiological confirmation. ALS was measured from venous blood, and results were evaluated in children classified as “confirmed”, “non-confirmed TB” or “not TB”.ResultsAmong 224 children who had ALS analysis, 12 (5.4%) children had microbiologically “confirmed TB”, a further 41 (18%) had clinically diagnosed “non-confirmed TB” and the remaining 168 (75%) were considered not to have TB. ALS was positive in 89 (40%) and negative in 85 (39%) of children, with a large number (47 or 21%) reported as “borderline”. These proportions were similar between the three diagnostic groups. The sensitivity and specificity of ALS when comparing “Confirmed TB” to “Not TB” was only 67% (95% CI: 31–91%) and 51% (95% CI: 42–60%), respectively.

Conclusions and Significance

Our data suggest that ALS is not sufficiently accurate to improve the diagnosis of TB in children with severe malnutrition.     

Cigarette Smoking Causes Hearing Impairment among Bangladeshi Population

Lifestyle including smoking, noise exposure with MP3 player and drinking alcohol are considered as risk factors for affecting hearing synergistically. However, little is known about the association of cigarette smoking with hearing impairment among subjects who carry a lifestyle without using MP3 player and drinking alcohol. We showed here the influence of smoking on hearing among Bangladeshi subjects who maintain a lifestyle devoid of using MP3 player and drinking alcohol. A total of 184 subjects (smokers: 90; non-smokers: 94) were included considering their duration and frequency of smoking for conducting this study. The mean hearing thresholds of non-smoker subjects at 1, 4, 8 and 12 kHz frequencies were 5.63±2.10, 8.56±5.75, 21.06±11.06, 40.79±20.36 decibel (dB), respectively and that of the smokers were 7±3.8, 13.27±8.4, 30.66±12.50 and 56.88±21.58 dB, respectively. The hearing thresholds of the smokers at 4, 8 and 12 kHz frequencies were significantly (p<0.05) higher than those of the non-smokers, while no significant differences were observed at 1 kHz frequency. We also observed no significant difference in auditory thresholds among smoker subgroups based on smoking frequency. In contrast, subjects smoked for longer duration (>5 years) showed higher level of auditory threshold (62.16±19.87 dB) at 12 kHz frequency compared with that (41.52±19.21 dB) of the subjects smoked for 1-5 years and the difference in auditory thresholds was statistically significant (p<0.0002). In this study, the Brinkman Index (BI) of smokers was from 6 to 440 and the adjusted odds ratio showed a positive correlation between hearing loss and smoking when adjusted for age and body mass index (BMI). In addition, age, but not BMI, also played positive role on hearing impairment at all frequencies. Thus, these findings suggested that cigarette smoking affects hearing level at all the frequencies tested but most significantly at extra higher frequencies.     

Dynamic morphometrics reveals contributions of dendritic growth cones and filopodia to dendritogenesis in the intact and awake embryonic brain

Using in vivo rapid and long-interval two-photon time-lapse imaging of brain neuronal growth within the intact and unanesthetized Xenopus laevis tadpole, we characterize dynamic dendritic growth behaviors of filopodia, branches, and dendritic growth cones (DGCs), and analyze their contribution to persistent arbor morphology. The maturational progression of dynamic dendritogenesis was captured by short-term, 5 min interval, imaging for 1 h every day for 5 days, and the contribution of short-term growth to persistent structure was captured by imaging at 5 min intervals for 5 h, and at 2 h intervals for 10 h during the height of arbor growth. We find that filopodia and branch stability increases with neuronal maturation, and while the majority of dendritic filopodia rapidly retract, 3% to 7% of interstitial filopodia transition into persistent branches with lifetimes greater than 90 min. Here, we provide the first characterization of DGC dynamics, including morphology and behavior, in the intact and awake developing vertebrate brain. We find that DGCs occur on all growing branches indicating an essential role in branch elongation, and that DGC morphology correlates with dendritic branch growth behavior and varies with maturation. These results demonstrate that dendritogenesis involves a remarkable amount of continuous remodeling, with distinct roles for filopodia and DGCs across neuronal maturation. ? 2011 Wiley Periodicals, Inc. Develop Neurobiol 72: 615-627, 2012.     

Aspergillus nidulans galactofuranose biosynthesis affects antifungal drug sensitivity

The cell wall is essential for fungal survival in natural environments. Many fungal wall carbohydrates are absent from humans, so they are a promising source of antifungal drug targets. Galactofuranose (Galf) is a sugar that decorates certain carbohydrates and lipids. It comprises about 5% of the Aspergillus fumigatus cell wall, and may play a role in systemic aspergillosis. We are studying Aspergillus wall formation in the tractable model system, A. nidulans. Previously we showed single-gene deletions of three sequential A. nidulans Galf biosynthesis proteins each caused similar hyphal morphogenesis defects and 500-fold reduced colony growth and sporulation. Here, we generated ugeA, ugmA and ugtA strains controlled by the alcA(p) or niiA(p) regulatable promoters. For repression and expression, alcA(p)-regulated strains were grown on complete medium with glucose or threonine, whereas niiA(p)-regulated strains were grown on minimal medium with ammonium or nitrate. Expression was assessed by qPCR and colony phenotype. The alcA(p) and niiA(p) strains produced similar effects: colonies resembling wild type for gene expression, and resembling deletion strains for gene repression. Galf immunolocalization using the L10 monoclonal antibody showed that ugmA deletion and repression phenotypes correlated with loss of hyphal wall Galf. None of the gene manipulations affected itraconazole sensitivity, as expected. Deletion of any of ugmA, ugeA, ugtA, their repression by alcA(p) or niiA(p), OR, ugmA overexpression by alcA(p), increased sensitivity to Caspofungin. Strains with alcA(p)-mediated overexpression of ugeA and ugtA had lower caspofungin sensitivity. Galf appears to play an important role in A. nidulans growth and vigor.     

Proteome analysis of soybean roots under waterlogging stress at an early vegetative stage

To gain better insight into how soybean roots respond to waterlogging stress, we carried out proteomic profiling combined with physiological analysis at two time points for soybean seedlings in their early vegetative stage. Seedlings at the V2 stage were subjected to 3 and 7 days of waterlogging treatments. Waterlogging stress resulted in a gradual increase of lipid peroxidation and in vivo H₂O₂ level in roots. Total proteins were extracted from root samples and separated by two-dimensional gel electrophoresis (2-DE). A total of 24 reproducibly resolved, differentially expressed protein spots visualized by Coomassie brilliant blue (CBB) staining were identified by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis. Of these, 14 proteins were upregulated; 5 proteins were decreased; and 5 were newly induced in waterlogged roots. The identified proteins include well-known classical anaerobically induced proteins as well as novel waterlogging-responsive proteins that were not known previously as being waterlogging responsive. The novel proteins are involved in several processes, i.e. signal transduction, programmed cell death, RNA processing, redox homeostasis and metabolisms of energy. An increase in abundance of several typical anaerobically induced proteins, such as glycolysis and fermentation pathway enzymes, suggests that plants meet energy requirement via the fermentation pathway due to lack of oxygen. Additionally, the impact of waterlogging on the several programmed cell death- and signal transduction-related proteins suggest that they have a role to play during stress. RNA gel blot analysis for three programmed cell death-related genes also revealed a differential mRNA level but did not correlate well with the protein level. These results demonstrate that the soybean plant can cope with waterlogging through the management of carbohydrate consumption and by regulating programmed cell death. The identification of novel proteins such as a translation initiation factor, apyrase, auxin-amidohydrolase and coproporphyrinogen oxidase in response to waterlogging stress may provide new insight into the molecular basis of the waterlogging-stress response of soybean.