Electron Tomography of Cryo-Immobilized Plant Tissue: A Novel Approach to Studying 3D Macromolecular Architecture of Mature Plant Cell Walls In Situ

Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the primary cell walls of a mutant (cob-6) and wild type Arabidopsis hypocotyl parenchyma cells by RT-tomography of HPF-FS-resin sections, and detected a small but significant difference in spatial organization of cellulose microfibrils in the mutant walls.     

Genotyping of Plasmodium vivax reveals both short and long latency relapse patterns in Kolkata

Background

The Plasmodium vivax that was once prevalent in temperate climatic zones typically had an interval between primary infection and first relapse of 7–10 months, whereas in tropical areas P.vivax infections relapse frequently at intervals of 3–6 weeks. Defining the epidemiology of these two phenotypes from temporal patterns of illness in endemic areas is difficult or impossible, particularly if they overlap.

Methods

A prospective open label comparison of chloroquine (CQ) alone versus CQ plus unobserved primaquine for either 5 days or 14 days was conducted in patients presenting with acute vivax malaria in Kolkata. Patients were followed for 15 months and primary and recurrent infections were genotyped using three polymorphic antigen and up to 8 microsatellite markers.

Results

151 patients were enrolled of whom 47 (31%) had subsequent recurrent infections. Recurrence proportions were similar in the three treatment groups. Parasite genotyping revealed discrete temporal patterns of recurrence allowing differentiation of probable relapse from newly acquired infections. This suggested that 32 of the 47 recurrences were probable relapses of which 22 (69%) were genetically homologous. The majority (81%) of probable relapses occurred within three months (16 homologous, 10 heterologous) and six genetically homologous relapses (19%) were of the long latency (8–10 month interval) phenotype.

Conclusions

With long follow-up to assess temporal patterns of vivax malaria recurrence, genotyping of P.vivax can be used to assess relapse rates. A 14 day unobserved course of primaquine did not prevent relapse. Genotyping indicates that long latency P.vivax is prevalent in West Bengal, and that the first relapses after long latent periods are genetically homologous.

Trial Registration

Controlled-Trials.com ISRCTN14027467     

The Accidental Ally: Nucleosome Barriers Can Accelerate Cohesin-Mediated Loop Formation in Chromatin

An important question in the context of the three-dimensional organization of chromosomes is the mechanism of formation of large loops between distant basepairs. Recent experiments suggest that the formation of loops might be mediated by loop extrusion factor proteins such as cohesin. Experiments on cohesin have shown that cohesins walk diffusively on the DNA and that nucleosomes act as obstacles to the diffusion, lowering the permeability and hence reducing the effective diffusion constant. An estimation of the times required to form the loops of typical sizes seen in Hi-C experiments using these low-effective-diffusion constants leads to times that are unphysically large. The puzzle then is the following: how does a cohesin molecule diffusing on the DNA backbone achieve speeds necessary to form the large loops seen in experiments? We propose a simple answer to this puzzle and show that although at low densities, nucleosomes act as barriers to cohesin diffusion, beyond a certain concentration they can reduce loop formation times because of a subtle interplay between the nucleosome size and the mean linker length. This effect is further enhanced on considering stochastic binding kinetics of nucleosomes on the DNA backbone and leads to predictions of lower loop formation times than might be expected from a naive obstacle picture of nucleosomes.     

Mistic: Cellular localization,solution behavior,polymerization, and fibril formation

Mistic represents a family of unique membrane‐associating proteins originally found in Bacillus subtilis (M110). As a fusion partner, it has been shown to assist overexpression of foreign integral membrane proteins in E. coli. We have expressed shorter Mistic homologs from other Bacillus species and surprisingly, unlike M110, found them abundant in the cytoplasm. These Mistic homologs including the corresponding shorter sequence (amino acids 27 through 110 of M110) exist as multimeric assemblies in solution in the absence of detergent. Crystals of Mistic from B. leicheniformis (M2) diffracted to 3.2 Å resolution, indicating that it exists as a multimer in the crystalline state as well. Moreover, we show that although M2 is mostly α‐helical, it tends to polymerize and form fibrils. Such oligomerization could potentially mask the charged surface of the monomeric Mistic to assist membrane integration.     

Amyloid as a depot for the formulation of long-acting drugs

Amyloids are highly organized protein aggregates that are associated with both neurodegenerative diseases such as Alzheimer disease and benign functions like skin pigmentation. Amyloids self-polymerize in a nucleation-dependent manner by recruiting their soluble protein/peptide counterpart and are stable against harsh physical, chemical, and biochemical conditions. These extraordinary properties make amyloids attractive for applications in nanotechnology. Here, we suggest the use of amyloids in the formulation of long-acting drugs. It is our rationale that amyloids have the properties required of a long-acting drug because they are stable depots that guarantee a controlled release of the active peptide drug from the amyloid termini. This concept is tested with a family of short- and long-acting analogs of gonadotropin-releasing hormone (GnRH), and it is shown that amyloids thereof can act as a source for the sustained release of biologically active peptides.     

Elucidating the interaction of Spathodea campanulata leaf extracts mediated potential bactericidal gold nanoparticles with human serum albumin: spectroscopic analysis

Nanomaterials in different form have been thoroughly used in the area of pharmaceutics and medicine for drug delivery. The large scale of nanoparticles (NPs) synthesis from plant extract is much safe, cheap and eco-friendly. Here, we demonstrated a new, one-step, ultra-fast biosynthesis of gold nanoparticles (sc-AuNPs, 19.54?nm) by using aqueous Spathodea campanulata leaf extracts as a reducing and capping agent. And also, we presented the synthesis of citrate capped gold nanoparticles (cit-AuNPs) of approximately same size (19.66?nm). These two NPs were characterized by UV-Visible, dynamic light scattering, transmission electron microscope and energy dispersive X-ray spectroscopy. Fourier transform infrared spectroscopy confirmed that the functional groups like OH, NH, OH of COOH and CO were contributed in the sc-AuNPs formation. The negative zeta potential (?20.5, ?22.8?mV) established the stability and dispersion of the sc- and cit-AuNPs. The anti-bacterial activity of the sc- and cit-AuNPs were checked against Escherichia coli (DH5-Alpha). Minimum inhibitory concentration was 2.4 and 3.0?nM, respectively for sc- and cit-AuNPs. The interaction study of the sc-AuNPs/cit-AuNPs-human serum albumin (HSA) system was done by UV-Visible absorbance, fluorescence, circular dichroism, time resolved fluorescence spectroscopy and the measurement of zeta potential. Absorbance, three dimensional fluorescence, synchronous fluorescence and circular dichroism spectroscopy showed a minor conformational change of HSA upon interaction with the sc-AuNPs compared to cit-AuNPs. The present comparative study will advance our knowledge about the binding mode, mechanism and conformational change of the protein upon interaction with green synthesized sc-AuNPs and cit-AuNPs.

Communicated by Ramaswamy H. Sarma     

Home ranges, movements, and activity of wolves (Canis lupus) in the Dalmatian part of Dinarids, Croatia

Home-range sizes, movements, and daily activity of wolves (Canis lupus L. 1758) were studied in Dalmatia, Croatia in 1998–2001. The total home ranges (100% MCP) of two packs were 160 km² and 141 km², mean=150.5 km². Core areas (50% kernel) were 26.2 km² and 3.3 km², respectively. Differences in core area sizes were influenced by human activity—hunting and sheep grazing. Compared with random locations, wolf locations were closer to the nearest water source (mean=937 m) and farther from houses (mean=653 m). Wolves were significantly more active during the night than during the day (activity indexes were 0.53 vs. 0.35), and night activity was higher during summer (0.58), and lower during winter (0.48). A correlation was found between distances traveled and activity index (r=0.58, p=0.003). Home range, seasonal variations in home-range size, habitat use, and activity of wolves in Dalmatia were oriented to make the compromise from danger of proximity to humans and also to benefit from human-related food sources.     

Uranyl tris nitrato as a luminescent probe for trace water detection in acetonitrile

Uranyl tris nitrato i.e. [UO₂(NO₃)₃]^– was formed by adding tetramethylammonium nitrate to uranyl nitrate in acetonitrile medium. The luminescence features of this complex in acetonitrile are very sensitive to water content, which could lead to the use of it as a luminescent probe for water present in acetonitrile. The luminescence intensity ratio of 507 to 467 nm peak of uranyl tris nitrato showed a linear response in the range 0–5% (v/v) water content in acetonitrile. The present method was applied for three synthetic samples of acetonitrile for water detection and the results obtained were compared using Karl Fischer titration. There was a good agreement in the values obtained by both the methods.     

Diversity oriented synthesis of chromene-xanthene hybrids as anti-breast cancer agents

A diverse library of chromene-xanthene hybrids were synthesized through intramolecular Friedel-Crafts reaction of the arenoxy carbinols. Examples include first incorporation of amino acid tyrosine into xanthene skeletons with polar functionalities. A careful structural evaluation revealed that tyrosine crafted chromene-xanthene hybrids exhibited good activities against breast cancer cell lines MCF-7, MDA-MB-231. The lead compound 16 displays significant cell cycle arrest at G1 phase and induces apoptosis in MDA-MB-231 cells.     

Cell Adhesion on Amyloid Fibrils Lacking Integrin Recognition Motif

Amyloids are highly ordered, cross-β-sheet-rich protein/peptide aggregates associated with both human diseases and native functions. Given the well established ability of amyloids in interacting with cell membranes, we hypothesize that amyloids can serve as universal cell-adhesive substrates. Here, we show that, similar to the extracellular matrix protein collagen, amyloids of various proteins/peptides support attachment and spreading of cells via robust stimulation of integrin expression and formation of integrin-based focal adhesions. Additionally, amyloid fibrils are also capable of immobilizing non-adherent red blood cells through charge-based interactions. Together, our results indicate that both active and passive mechanisms contribute to adhesion on amyloid fibrils. The present data may delineate the functional aspect of cell adhesion on amyloids by various organisms and its involvement in human diseases. Our results also raise the exciting possibility that cell adhesivity might be a generic property of amyloids.