Life-cycle modification in open oceans accounts for genome variability in a cosmopolitan phytoplankton

Emiliania huxleyi is the most abundant calcifying plankton in modern oceans with substantial intraspecific genome variability and a biphasic life cycle involving sexual alternation between calcified 2N and flagellated 1N cells. We show that high genome content variability in Emiliania relates to erosion of 1N-specific genes and loss of the ability to form flagellated cells. Analysis of 185 E. huxleyi strains isolated from world oceans suggests that loss of flagella occurred independently in lineages inhabiting oligotrophic open oceans over short evolutionary timescales. This environmentally linked physiogenomic change suggests life cycling is not advantageous in very large/diluted populations experiencing low biotic pressure and low ecological variability. Gene loss did not appear to reflect pressure for genome streamlining in oligotrophic oceans as previously observed in picoplankton. Life-cycle modifications might be common in plankton and cause major functional variability to be hidden from traditional taxonomic or molecular markers.

A Combined Molecular Cloning and Mass Spectrometric Method to Identify, Characterize, and Design Frenatin Peptides from the Skin Secretion of Litoria infrafrenata

Amphibian skin secretions are unique sources of bioactive molecules, particularly bioactive peptides. In this study, the skin secretion of the white-lipped tree frog (Litoria infrafrenata) was obtained to identify peptides with putative therapeutic potential. By utilizing skin secretion-derived mRNA, a cDNA library was constructed, a frenatin gene was cloned and its encoded peptides were deduced and confirmed using RP-HPLC, MALDI-TOF and MS/MS. The deduced peptides were identified as frenatin 4.1 (GFLEKLKTGAKDFASAFVNSIKGT) and a post-translationally modified peptide, frenatin 4.2 (GFLEKLKTGAKDFASAFVNSIK.NH2). Antimicrobial activity of the peptides was assessed by determining their minimal inhibitory concentrations (MICs) using standard model microorganisms. Through studying structure–activity relationships, analogues of the two peptides were designed, resulting in synthesis of frenatin 4.1a (GFLEKLKKGAKDFASALVNSIKGT) and frenatin 4.2a (GFLLKLKLGAKLFASAFVNSIK.NH2). Both analogues exhibited improved antimicrobial activities, especially frenatin 4.2a, which displayed significant enhancement of broad spectrum antimicrobial efficiency. The peptide modifications applied in this study, may provide new ideas for the generation of leads for the design of antimicrobial peptides with therapeutic applications.

Bayesian alignment of continuous molecular shapes using random fields

Statistical methodology is proposed for comparing molecular shapes. In order to account for the continuous nature of molecules, classical shape analysis methods are combined with techniques used for predicting random fields in spatial statistics. Applying a modification of Procrustes analysis, Bayesian inference is carried out using Markov chain Monte Carlo methods for the pairwise alignment of the resulting molecular fields. Superimposing entire fields rather than the configuration matrices of nuclear positions thereby solves the problem that there is usually no clear one--to--one correspondence between the atoms of the two molecules under consideration. Using a similar concept, we also propose an adaptation of the generalised Procrustes analysis algorithm for the simultaneous alignment of multiple molecular fields. The methodology is applied to a dataset of 31 steroid molecules.

Targeted gene modification in Fragaria vesca mediated by CRISPR/Cas9 system

Genome editing is becoming an important biotechnological tool for gene function analysis and crop improvement, being the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system the most widely used. The natural CRISPR/Cas9 system has been reduced to two components: a single-guide RNA (sgRNA) for target recognition via RNA-DNA base pairing, which is commonly expressed using a promoter for small-RNAs (U6 promoter), and the Cas9 endonuclease for DNA cleavage (1). To validate the CRISPR/Cas9 system in strawberry plants, we designed two sgRNAs directed against the floral homeotic gene APETALA3 (sgRNA-AP3#1 and sgRNA-AP3#2). This gene was selected because ap3 mutations induce clear developmental phenotypes in which petals and stamens are missing or partially converted to sepals and carpels respectively (2). In this work, we used two different U6 promoters to drive the sgRNA-AP3s expression: AtU6-26 from Arabidopsis (4), and a U6 promoter from Fragaria vesca (FvU6) (this work). We also tested two different coding sequences of Cas9: a human- (hSpCas9) (3) and a plant-codon optimized (pSpCas9) (this work). Transient expression experiments using both CRISPR/Cas9 systems (AtU6-26:sgRNA-AP3#1_35S:hSpCas9_AtU6-26:sgRNA-AP3#2 and FvU6:sgRNA-AP3#1_35S:pSpCas9_FvU6:sgRNA-AP3#2) were performed infiltrating Agrobacterium tumefaciens into F. vesca fruits. PCR amplification and sequencing analyses across the target sites showed a deletion of 188-189 bp corresponding to the region comprised between the two cutting sites of Cas9, confirming that the CRISPR/Cas9 system is functional in F. vesca. Remarkably, the two systems showed different mutagenic efficiency that could be related to differences in expression of the U6 promoters as well as differences in the Cas9 transcripts stability and translation. Stable transformants for both F. vesca (2n) and Fragaria X anannassa (8n) are currently being established to test whether is possible to obtain heritable homozygous mutants derived from CRISPR/Cas9 strategies in strawberry. Thus, our work offers a promising tool for genome editing and gene functional analysis in strawberry. This tool might represent a more efficient alternative to the sometimes inefficient RNAi silencing methods commonly used in this species.

THE PERFORMANCE AND MODIFICATION OF RECYCLED ELECTRONIC WASTE PLASTICS FOR THE IMPROVEMENT OF ASPHALT PAVEMENT MATERIALS

Bulk electric waste plastics were recycled and reduced in size into plastic chips before pulverization or cryogenic grinding into powders. Two major types of electronic waste plastics were used in this investigation: acrylonitrile butadiene styrene (ABS) and high impact polystyrene (HIPS). This research investigation utilized two approaches for incorporating electronic waste plastics into asphalt pavement materials. The first approach was blending and integrating recycled and processed electronic waste powders directly into asphalt mixtures and binders; and the second approach was to chemically treat recycled and processed electronic waste powders with hydro-peroxide before blending into asphalt mixtures and binders. The chemical treatment of electronic waste (e-waste) powders was intended to strengthen molecular bonding between e-waste plastics and asphalt binders for improved low and high temperature performance. Superpave asphalt binder and mixture testing techniques were conducted to determine the rheological and mechanical performance of the e-waste modified asphalt binders and mixtures. This investigation included a limited emissions-performance assessment to compare electronic waste modified asphalt pavement mixture emissions using SimaPro and performance using MEPDG software. Carbon dioxide emissions for e-waste modified pavement mixtures were compared with conventional asphalt pavement mixtures using SimaPro. MEPDG analysis was used to determine rutting potential between the various e-waste modified pavement mixtures and the control asphalt mixture. The results from this investigation showed the following: treating the electronic waste plastics delayed the onset of tertiary flow for electronic waste mixtures, electronic waste mixtures showed some improvement in dynamic modulus results at low temperatures versus the control mixture, and tensile strength ratio values for treated e-waste asphalt mixtures were improved versus the control mixture.

Vibrational spectroscopy of phthalocyanine and naphthalocyanine in sandwich-type (na)phthalocyaninato and porphyrinato rare earth complexes : (Part 10) The infrared and Raman characteristics of phthalocyanine in heteroleptic bis(phthalocyaninato) rare earth complexes with decreased molecular symmetry

The infrared (IR) spectroscopic data for a series of eleven heteroleptic bis(phthalocyaninato) rare earth complexes MIII(Pc)[Pc(α-OC5H11)4] (M = Sm–Lu, Y) [H2Pc = unsubstituted phthalocyanine, H2Pc(α-OC5H11)4 = 1,8,15,22-tetrakis(3-pentyloxy)phthalocyanine] have been collected with 2 cm−1 resolution. Raman spectroscopic properties in the range of 500–1800 cm−1 for these double-decker molecules have also been comparatively studied using laser excitation sources emitting at 632.8 and 785 nm. Both the IR and Raman spectra for M(Pc)[Pc(α-OC5H11)4] are more complicated than those of homoleptic bis(phthalocyaninato) rare earth analogues due to the decreased molecular symmetry of these double-decker compounds, namely C4. For this series, the IR Pc√− marker band appears as an intense absorption at 1309–1317 cm−1, attributed to the pyrrole stretching. With laser excitation at 632.8 nm, Raman vibrations derived from isoindole ring and aza stretchings in the range of 1300–1600 cm−1 are selectively intensified. In contrast, when excited with laser radiation of 785 nm, the ring radial vibrations of isoindole moieties and dihedral plane deformations between 500 and 1000 cm−1 for M(Pc)[Pc(α-OC5H11)4] intensify to become the strongest scatterings. Both techniques reveal that the frequencies of pyrrole stretching, isoindole breathing, isoindole stretchings, aza stretchings and coupling of pyrrole and aza stretchings depend on the rare earth ionic size, shifting to higher energy along with the lanthanide contraction due to the increased ring-ring interaction across the series. The assignments of the vibrational bands for these compounds have been made and discussed in relation to other unsubstituted and substituted bis(phthalocyaninato) rare earth analogues, such as M(Pc)2 and M(OOPc)2 [H2OOPc = 2,3,9,10,16,17,23,24-octakis(octyloxy)phthalocyanine].

Time-Dependent Density Functional Molecular Orbital and Excited State Calculations on Bis(porphyrinyl)butadiynes in the Monocationic, Neutral, Monoanionic, and Dianionic Oxidation States

We report a theoretical study of the multiple oxidation states (1+, 0, 1−, and 2−) of a meso,meso-linked diporphyrin, namely bis[10,15,20-triphenylporphyrinatozinc(II)-5-yl]butadiyne (4), using Time-Dependent Density Functional Theory (TDDFT). The origin of electronic transitions of singlet excited states is discussed in comparison to experimental spectra for the corresponding oxidation states of the close analogue bis{10,15,20-tris[3‘,5‘-di-tert-butylphenyl]porphyrinatozinc(II)-5-yl}butadiyne (3). The latter were measured in previous work under in situ spectroelectrochemical conditions. Excitation energies and orbital compositions of the excited states were obtained for these large delocalized aromatic radicals, which are unique examples of organic mixed-valence systems. The radical cations and anions of butadiyne-bridged diporphyrins such as 3 display characteristic electronic absorption bands in the near-IR region, which have been successfully predicted with use of these computational methods. The radicals are clearly of the “fully delocalized” or Class III type. The key spectral features of the neutral and dianionic states were also reproduced, although due to the large size of these molecules, quantitative agreement of energies with observations is not as good in the blue end of the visible region. The TDDFT calculations are largely in accord with a previous empirical model for the spectra, which was based simplistically on one-electron transitions among the eight key frontier orbitals of the C4 (1,4-butadiyne) linked diporphyrins.

Enhanced human bone marrow stromal cell affinity for modified poly(L-lactide) surfaces by the upregulation of adhesion molecular genes

To enhance and regulate cell affinity for poly (l-lactic acid) (PLLA) based materials, two hydrophilic ligands, poly (ethylene glycol) (PEG) and poly (l-lysine) (PLL), were used to develop triblock copolymers: methoxy-terminated poly (ethylene glycol)-block-poly (l-lactide)-block-poly (l-lysine) (MPEG-b-PLLA-b-PLL) in order to regulate protein absorption and cell adhesion. Bone marrow stromal cells (BMSCs) were cultured on different composition of MPEG-b-PLLA-b-PLL copolymer films to determine the effect of modified polymer surfaces on BMSC attachment. To understand the molecular mechanism governing the initial cell adhesion on difference polymer surfaces, the mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analysed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was found that down regulation of adhesion molecules was responsible for the impaired BMSC attachment on PLLA surface. MPEG-b-PLLA-b-PLL copolymer films improved significantly the cell adhesion and cytoskeleton expression by upregulation of relevant molecule genes significantly. Six adhesion genes (CDH1, ITGL, NCAM1, SGCE, COL16A1, and LAMA3) were most significantly influenced by the modified PLLA surfaces. In summary, polymer surfaces altered adhesion molecule gene expression of BMSCs, which consequently regulated cell initial attachment on modified PLLA surfaces.

Comparison of molecular markers to detect fresh sewage in environmental waters

Human-specific Bacteroides HF183 (HS-HF183), human-specific Enterococci faecium esp (HS-esp), human-specific adenoviruses (HS-AVs) and human-specific polyomaviruses (HS-PVs) assays were evaluated in freshwater, seawater and distilled water to detect fresh sewage. The sewage spiked water samples were also tested for the concentrations of traditional fecal indicators (i.e., Escherichia coli, enterococci and Clostridium perfringens) and enteric viruses such as enteroviruses (EVs), sapoviruses (SVs), and torquetenoviruses (TVs). The overall host-specificity of the HS-HF183 marker to differentiate between humans and other animals was 98%. However, the HS-esp, HS-AVs and HS-PVs showed 100% hostspecificity. All the human-specific markers showed >97% sensitivity to detect human fecal pollution. E. coli, enterococci and, C. perfringens were detected up to dilutions of sewage 10_5, 10_4 and 10_3 respectively.HS-esp, HS-AVs, HS-PVs, SVs and TVs were detected up to dilution of sewage 10_4 whilst EVs were detected up to dilution 10_5. The ability of the HS-HF183 marker to detect freshsewagewas3–4 orders ofmagnitude higher than that of the HS-esp and viral markers. The ability to detect fresh sewage in freshwater, seawater and distilled water matrices was similar for human-specific bacterial and viral marker. Based on our data, it appears that human-specific molecular markers are sensitive measures of fresh sewage pollution, and the HS-HF183 marker appears to be the most sensitive among these markers in terms of detecting fresh sewage. However, the presence of the HS-HF183 marker in environmental waters may not necessarily indicate the presence of enteric viruses due to their high abundance in sewage compared to enteric viruses. More research is required on the persistency of these markers in environmental water samples in relation to traditional fecal indicators and enteric pathogens.

Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker.

BACKGROUND: Although we know much about the molecular makeup of the sinus node (SN) in small mammals, little is known about it in humans. The aims of the present study were to investigate the expression of ion channels in the human SN and to use the data to predict electrical activity. METHODS AND RESULTS: Quantitative polymerase chain reaction, in situ hybridization, and immunofluorescence were used to analyze 6 human tissue samples. Messenger RNA (mRNA) for 120 ion channels (and some related proteins) was measured in the SN, a novel paranodal area, and the right atrium (RA). The results showed, for example, that in the SN compared with the RA, there was a lower expression of Na(v)1.5, K(v)4.3, K(v)1.5, ERG, K(ir)2.1, K(ir)6.2, RyR2, SERCA2a, Cx40, and Cx43 mRNAs but a higher expression of Ca(v)1.3, Ca(v)3.1, HCN1, and HCN4 mRNAs. The expression pattern of many ion channels in the paranodal area was intermediate between that of the SN and RA; however, compared with the SN and RA, the paranodal area showed greater expression of K(v)4.2, K(ir)6.1, TASK1, SK2, and MiRP2. Expression of ion channel proteins was in agreement with expression of the corresponding mRNAs. The levels of mRNA in the SN, as a percentage of those in the RA, were used to estimate conductances of key ionic currents as a percentage of those in a mathematical model of human atrial action potential. The resulting SN model successfully produced pacemaking. CONCLUSIONS: Ion channels show a complex and heterogeneous pattern of expression in the SN, paranodal area, and RA in humans, and the expression pattern is appropriate to explain pacemaking.