PcoB is a defense outer membrane protein that facilitates cellular uptake of copper

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid‐encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical β‐barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion‐conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco‐harboring pathogens.     

Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM

Over the last three decades, Cryo-TEM has developed into a powerful technique for high-resolution imaging of biological macromolecules in their native vitrified state. However, the method for vitrifying specimens onto EM grids is essentially unchanged - application of ～3 μL sample to a grid, followed by blotting and rapid plunge freezing into liquid ethane. Several trials are often required to obtain suitable thin (few hundred nanometers or less) vitrified layers amenable for cryo-TEM imaging, which results in waste of precious sample and resources. While commercially available instruments provide some level of automation to control the vitrification process in an effort to increase quality and reproducibility, obtaining satisfactory vitrified specimens remains a bottleneck in the Cryo-TEM pipeline. We describe here a completely novel method for EM specimen preparation based on small volume (picoliter to nanoliter) dispensing using inkjet technology. A first prototype system (Spotiton v0.5) demonstrates feasibility of this new approach for specimen vitrification. A piezo-electric inkjet dispenser is integrated with optical real-time cameras (100 Hz frame rate) to analyze picoliter to nanoliter droplet profiles in-flight and spreading dynamics on the grid, and thus provides a method to optimize timing of the process. Using TEM imaging and biochemical assays we demonstrate that the piezo-electric inkjet mechanism does not disrupt the structural or functional integrity of macromolecules. These preliminary studies provide insight into the factors and components that will need further development to enable a robust and repeatable technique for specimen vitrification using this novel approach.     

Chemoselective Deprotection of Triethylsilyl Ethers

An efficient and selective method was developed for the deprotection of triethylsilyl (TES) ethers using formic acid in methanol (5–10%) or in methylene chloride 2–5%) with excellent yields. TES ethers are selectively deprotected to the corresponding alcohols in high yields using formic acid in methanol under mild reaction conditions. Other hydroxyl protecting groups like t-butyldimethylsilyl (TBDMS) remain unaffected.     

Improved Chiral SFC Screening for Analytical Method Development

In this study we describe the evaluation of a recently developed supercritical fluid chromatography (SFC) instrument for automated chiral SFC method development. The greatly improved gradient dwell volume and liquid flow control of the new instrument in combination with the use of shorter columns containing smaller stationary phase particles affords chiral SFC method development that is faster and more universal than previous systems. Chirality 25:799–804, 2013. © 2013 Wiley Periodicals, Inc.     

Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery

A novel water-soluble lipopolymer was synthesized by linking cholesteryl chloroformate to the secondary amino groups of branched poly(ethylenimine) (PEI) of 1,800 and 10,000 Da. Conjugation through PEI secondary amines gives this newly synthesized lipopolymer (abbreviated as PEI-Chol) special advantage over our previously synthesized lipopolymers, which utilized the primary amino groups for conjugation, as the primary amino groups have a significant role in DNA condensation. Also, significantly, only one cholesterol molecule was grafted onto each PEI molecule (confirmed by (1)H NMR and MALDI-TOF mass spectrometry), leaving enough space for the steric interactions of the PEI's primary amines with the DNA. The PEI-Chol lipopolymer was characterized for the critical micellar concentration (cmc), buffer capacity, DNA condensation (by band retardation and circular dichroism), in vitro transfection efficiency, and cell viability. The cmcs of PEI-Chol 1,800 and PEI-Chol 10,000 were 496.6 and 1,330.5 microg/mL, respectively. The acid-base titration indicated high buffering capacity of the polymers around the pH range of 5-7, which indicated their potential for buffering in the acidic pH environment of the endosomes. The band retardation studies indicated that efficient condensation of the plasmid DNA could be achieved using these lipopolymers. The circular dichroism spectra indicated a change in DNA conformation and adoption of lower energy state upon condensation with these lipopolymers when an N/P ratio of 2.5/1 or above was formulated. The mean particle size of these complexes was in the range 110-205 nm, except for the complexes prepared using PEI of 1,800 Da, which had a mean particle size of 384 +/- 300 nm. The zeta potential of DNA complexes prepared using PEI-Chol 1,800, PEI-Chol 10,000 and PEI of 1,800, 10,000, and 25,000 Da at an N/P ratio of 15/1 was in the range 23-30 mV and was dependent on the N/P ratios. The in vitro transfection of PEI-Chol/pCMS-EGFP complexes in Jurkat cells showed high levels of expressed Green Fluorescent Protein (GFP) with little toxicity as determined by flow cytometry. These novel water-soluble lipopolymers provided good transfection efficiency with other desirable characteristics such as water solubility, free primary amino groups for efficient DNA condensation and high buffer capacity that indicated the possibility of efficient endosomal release.     

First fossil evidence of Palaeocarya (Engelhardioideae: Juglandaceae) from India and its biogeographical implications

Even though presently indigenous to eastern Himalaya in India, no Engelhardioideae have been reported from the Cenozoic sediments of India till date. Here, we report the first Indian occurrence of a characteristic engelhardioid winged samaroid fruit having a tri-lobed wing (oblong-ovate median lobe and two lateral lobes) and a globose nut from the latest Neogene (Pliocene: Rajdanda Formation) sediments of Chotanagpur Plateau, eastern India. This is the first fossil evidence of relict family Juglandaceae from the Indian Cenozoic. We determine its taxonomic position on the basis of detailed macromorphological comparison with similar extant and fossil specimens and discuss its palaeoclimatic significance in terms of the present-day distribution of modern analogous species. We assign this Pliocene winged fruit specimen to the morphogenus Palaeocarya sect. Monocosta Manchester and describe it as a new species, namely Palaeocarya indica Hazra, Hazra M & Khan sp. nov. Palaeocarya sect. Monocosta has rich fossil records from the Cenozoic sediments of Europe, North America, and eastern Asia (China, Korea), but the modern analog, Engelhardia, is presently native only to India and neighboring Southeast Asia. We discuss the possible causes of disappearance of Engelhardia from the present-day vegetation of Chotanagpur Plateau. Its disappearance may be related to the gradual intensification of monsoonal rainfall seasonality since the Pliocene. Here, we also review in detail the biogeographic history of Palaeocarya sect. Monocosta and suggest its possible migration routes.     

Biotransformation of lantadene A (22β-angeloyloxy-3-oxoolean-12-en-28-oic acid), the pentacyclic triterpenoid, by Alcaligenes faecalis

A bacterial strain capable of biotransformation oflantadene A(22-angeloyloxy-3-oxo-olean-12-en-28-oic acid),the pentacyclic hepatotoxin of lantana (Lantanacamara var. aculeata) has been isolated fromsoil using lantadene A as the sole carbon source. Theorganism is Gram negative, rod shaped, motile,catalase positive and has been identified as Alcaligenes faecalis. The isolate has been found tobe specific for lantadene A and did not utilizelantadene B. In studies using sucrose as an additionalcarbon source, A. faecalis elicitedbiotransformation of lantadene A to its trans isomer22-tigloyloxy-3-oxoolean-12-en-28-oic acid,designated as lantadene X and two other minormetabolites which could not be isolated in pure state.     

New DNA data from a transthyretin nuclear intron suggest an Oligocene to Miocene diversification of living South America opossums (Marsupialia: Didelphidae)

Phylogenetic relationships of 19 species of didelphid marsupials were studied using two nuclear markers, the non-coding transthyretin intron 1 (TTR) and the coding interphotoreceptor retinoid binding protein exon 1 (IRBP), and two mitochondrial genes, the protein-coding cytochrome b (cyt-b) and the structural 12S ribosomal DNA (12S rDNA). Evolutionary dynamics of these four markers were compared to each other, revealing the appropriate properties presented by TTR intron 1 together with its well supported and resolved phylogenetic signal. Nuclear markers supported the monophyly of medium and large-sized opossums Metachirus+(Chironectes, Lutreolina, Didelphis, Philander), and the paraphyly of mouse-sized opossums, with the genera Gracilinanus, Thylamys, and Marmosops as a sister group to medium and large-sized didelphids. Conflicting branching patterns between mitochondrial and nuclear data involved the phylogenetic position of Marmosa-Micoureus-Monodelphis relative to other mouse-sized opossums. Nuclear phylogenetic inferences among genera were confirmed by the presence of synapomorphic indels observed in TTR intron 1. A Bayesian relaxed molecular clock dating of didelphid evolution using nuclear markers estimated their origin in the Middle Eocene (39.8 million years ago), with subsequent diversification during the Oligocene (Deseadan) and Miocene.     

Genome-wide distribution and organization of microsatellites in plants: an insight into marker development in Brachypodium

Plant genomes are complex and contain large amounts of repetitive DNA including microsatellites that are distributed across entire genomes. Whole genome sequences of several monocot and dicot plants that are available in the public domain provide an opportunity to study the origin, distribution and evolution of microsatellites, and also facilitate the development of new molecular markers. In the present investigation, a genome-wide analysis of microsatellite distribution in monocots (Brachypodium, sorghum and rice) and dicots (Arabidopsis, Medicago and Populus) was performed. A total of 797,863 simple sequence repeats (SSRs) were identified in the whole genome sequences of six plant species. Characterization of these SSRs revealed that mono-nucleotide repeats were the most abundant repeats, and that the frequency of repeats decreased with increase in motif length both in monocots and dicots. However, the frequency of SSRs was higher in dicots than in monocots both for nuclear and chloroplast genomes. Interestingly, GC-rich repeats were the dominant repeats only in monocots, with the majority of them being present in the coding region. These coding GC-rich repeats were found to be involved in different biological processes, predominantly binding activities. In addition, a set of 22,879 SSR markers that were validated by e-PCR were developed and mapped on different chromosomes in Brachypodium for the first time, with a frequency of 101 SSR markers per Mb. Experimental validation of 55 markers showed successful amplification of 80% SSR markers in 16 Brachypodium accessions. An online database 'BraMi' (Brachypodium microsatellite markers) of these genome-wide SSR markers was developed and made available in the public domain. The observed differential patterns of SSR marker distribution would be useful for studying microsatellite evolution in a monocot-dicot system. SSR markers developed in this study would be helpful for genomic studies in Brachypodium and related grass species, especially for the map based cloning of the candidate gene(s).