DNA Repair (Amst). 2013 Mar 1;12(3):162-76

Alonso JC, Cardenas PP, Sanchez H, Hejna J, Suzuki Y, Takeyasu K.

All organisms rely on integrated networks to repair DNA double-strand breaks (DSBs) in order to preserve the integrity of the genetic information, to re-establish replication, and to ensure proper chromosomal segregation. Genetic, cytological, biochemical and structural approaches have been used to analyze how Bacillus subtilis senses DNA damage and responds to DSBs.

RecN, which is among the first responders to DNA DSBs, promotes the ordered recruitment of repair proteins to the site of a lesion. Cells have evolved different mechanisms for efficient end processing to create a 3′-tailed duplex DNA, the substrate for RecA binding, in the repair of one- and two-ended DSBs. Strand continuity is re-established via homologous recombination (HR), utilizing an intact homologous DNA molecule as a template. In the absence of transient diploidy or of HR, however, two-ended DSBs can be directly re-ligated via error-prone non-homologous end-joining. Here we review recent findings that shed light on the early stages of DSB repair in Firmicutes.

Plant Cell. 2013 Aug 6

Castrillo G, Sánchez-Bermejo E, de Lorenzo L, Crevillén P, Fraile-Escanciano A, Tc M, Mouriz A, Catarecha P, Sobrino-Plata J, Olsson S, Leo Del Puerto Y, Mateos I, Rojo E, Hernández LE, Jarillo JA, Piñeiro M, Paz-Ares J, Leyva A.

Stress constantly challenges plant adaptation to the environment. Of all stress types, arsenic was a major threat during the early evolution of plants. The most prevalent chemical form of arsenic is arsenate, whose similarity to phosphate renders it easily incorporated into cells via the phosphate transporters.

Here, we found that arsenate stress provokes a notable transposon burst in plants, in coordination with arsenate/phosphate transporter repression, which immediately restricts arsenate uptake. This repression was accompanied by delocalization of the phosphate transporter from the plasma membrane. When arsenate was removed, the system rapidly restored transcriptional expression and membrane localization of the transporter.

We identify WRKY6 as an arsenate-responsive transcription factor that mediates arsenate/phosphate transporter gene expression and restricts arsenate-induced transposon activation. Plants therefore have a dual WRKY-dependent signaling mechanism that modulates arsenate uptake and transposon expression, providing a coordinated strategy for arsenate tolerance and transposon gene silencing.

J Biol Chem. 2013 Jul 19;288(29):20830-6

Rico AI, Krupka M, Vicente M.

Cell division in Escherichia coli begins by assembling three proteins, FtsZ, FtsA, and ZipA, to form a proto-ring at midcell. These proteins nucleate an assembly of at least 35 components, the divisome. The structuring of FtsZ to form a ring and the processes that effect constriction have been explained by alternative but not mutually exclusive mechanisms.

We discuss how FtsA and ZipA provide anchoring of the cytoplasmic FtsZ to the membrane and how a temporal sequence of alternative protein interactions may operate in the maturation and stability of the proto-ring. How the force needed for constriction is generated and how the proto-ring proteins relate to peptidoglycan synthesis remain as the main challenges for future research.

Mol Cell Proteomics. 2013 Aug;12(8):2332-40

Walzer M, Qi D, Mayer G, Uszkoreit J, Eisenacher M, Sachsenberg T, Gonzalez-Galarza FF, Fan J, Bessant C, Deutsch EW, Reisinger F, Vizcaíno JA, Medina-Aunon JA, Albar JP, Kohlbacher O, Jones AR.

The range of heterogeneous approaches available for quantifying protein abundance via mass spectrometry (MS) leads to considerable challenges in modeling, archiving, exchanging, or submitting experimental data sets as supplemental material to journals. To date, there has been no widely accepted format for capturing the evidence trail of how quantitative analysis has been performed by software, for transferring data between software packages, or for submitting to public databases.

In the context of the Proteomics Standards Initiative, we have developed the mzQuantML data standard. The standard can represent quantitative data about regions in two-dimensional retention time versus mass/charge space (called features), peptides, and proteins and protein groups (where there is ambiguity regarding peptide-to-protein inference), and it offers limited support for small molecule (metabolomic) data. The format has structures for representing replicate MS runs, grouping of replicates (for example, as study variables), and capturing the parameters used by software packages to arrive at these values. The format has the capability to reference other standards such as mzML and mzIdentML, and thus the evidence trail for the MS workflow as a whole can now be described. Several software implementations are available, and we encourage other bioinformatics groups to use mzQuantML as an input, internal, or output format for quantitative software and for structuring local repositories. All project resources are available in the public domain from the HUPO Proteomics Standards Initiative.

J Immunol. 2013 191:2742-2751

Saez de Guinoa J, Barrio L, Carrasco YR.

Lymphocytes use integrin-based platforms to move and adhere firmly to the surface of other cells. The molecular mechanisms governing lymphocyte adhesion dynamics are however poorly understood. In this study, we show that in mouse B lymphocytes, the actin binding protein vinculin localizes to the ring-shaped integrin-rich domain of the immune synapse (IS); the assembly of this platform, triggered by cognate immune interactions, is needed for chemokine-mediated B cell motility arrest and leads to firm, long-lasting B cell adhesion to the APC.

Vinculin is recruited early in IS formation, in parallel to a local phosphatidylinositol (4,5)-bisphosphate wave, and requires spleen tyrosine kinase activity. Lack of vinculin at the IS impairs firm adhesion, promoting, in turn, cell migration with Ag clustered at the uropod. Vinculin localization to the B cell contact area depends on actomyosin. These results identify vinculin as a major controller of integrin-mediated adhesion dynamics in B cells.

Fungal Genet Biol. 2013 Jul 18. pii: S1087-1845(13)00126-6

Valencia EY, Chambergo FS.

Brazil houses over 10% of the total number of known species on Earth, with a great diversity of plants and fungi. The collection, isolation, identification and conservation of filamentous fungi with relevance to agriculture, pharmaceutical, food and biotechnological industries in Biological Resource Centers (CRBs) is very important to the development of a nation's scientific and technological infrastructure. In Brazil, 36 fungal collections are registered in the database of International Collections. Several federal and state programs have encouraged the formation of a researcher's network in order to study natural resources and the nation's biodiversity.

In this context, Brazilian researchers have been on the frontiers of knowledge, investigating the enzymatic systems from native filamentous fungi with potential for biomass degradation and biotechnological application. In this review, we address recent progress in Brazilian fungal research, focusing on the identification and study of fungi and enzymes with potential for biomass degradation and application in bioenergy.

J Struct Biol. 2013 Feb;181(2):136-48

Vargas J, Otón J, Marabini R, Jonic S, de la Rosa-Trevín JM, Carazo JM, Sorzano CO.

In this work we present a fast and automated algorithm for estimating the contrast transfer function (CTF) of a transmission electron microscope. The approach is very suitable for High Throughput work because:

• (a) it does not require any initial defocus estimation,
• (b) it is almost an order of magnitude faster than existing approaches
• (c) it opens the way to well-defined extensions to the estimation of higher order aberrations, at the same time that provides defocus and astigmatism estimations comparable in accuracy to well established methods, such as Xmipp and CTFFIND3 approaches

The new algorithm is based on obtaining the wrapped modulating phase of the power spectra density pattern by the use of a quadrature filter. This phase is further unwrapped in order to obtain the continuous and smooth absolute phase map; then a Zernike polynomial fitting is performed and the defocus and astigmatism parameters are determined. While the method does not require an initial estimation of the defocus parameters or any non-linear optimization procedure, these approaches can be used if further refinement is desired. Results of the CTF estimation method are presented for standard negative stained images, cryo-electron microscopy images in the absence of carbon support, as well as micrographs with only ice. Additionally, we have also tested the proposed method with micrographs acquired from tilted and untilted samples, obtaining good results. The algorithm is freely available as a part of the Xmipp package [http://xmipp.cnb.csic.es].

Protein Expr Purif. 2013 Jul 12. pii: S1046-5928(13)00120-4

Salema V, Fernández LA.

Nanobodies (Nbs) are single domain antibodies based on the variable domains of heavy chain only antibodies (HCAbs) found in camelids, also referred to as VHHs. Their small size (ca. 12-15 kDa), superior biophysical and antigen binding properties have made Nbs very attractive molecules for multiple biotechnological applications, including human therapy. The most widely used system for the purification of Nbs is their expression in the periplasm of E. coli with a C-terminal hexa-histidine (His₆) tag followed by immobilized metal affinity chromatography (IMAC). However, significant variability in the expression levels of different Nbs are routinely observed and a single affinity chromatography step is often not sufficient to obtain Nbs of high purity.

Here, we report an alternative method for expression and purification of Nbs from the periplasm of E. coli based on their fusion to maltose binding protein (MBP) in the N-terminus and His₆ tag in the C-terminus (MBP-Nb_His6). Soluble MBP-Nb_His6 fusions were consistently expressed at high levels (⩾12 mg/L of induced culture in shake flasks) in the periplasm of E. coli HM140, a strain deficient in several periplasmic proteases. Highly pure MBP-Nb_His6 fusions and free Nb_His6 (after site specific proteolysis of the fusions), were recovered by amylose and metal affinity chromatography steps. The monomeric nature of the purified Nb_His6 was determined by gel filtration chromatography. Lastly, we demonstrated by ELISA that both monomeric Nb_His6 and MBP-Nb_His6 fusions retained antigen binding activity and specificity, thus facilitating their direct use in antigen recognition assays.

Mol Plant Microbe Interact. 2013 Jun 7

Carbonell A, Maliogka VI, Pérez JD, Salvador Esteban B, San León D, Garcia JA, Simón-Mateo C.

PPV-D and PPV-R are two isolates from strain D of Plum pox virus (PPV) that differ in host specificity. Previous analyses of chimeras originating from PPV-R and PPV-D suggested that the N-terminus of the coat protein (CP) includes host specific pathogenicity determinants.

Here, these determinants were mapped precisely by analyzing the infectivity in herbaceous and woody species of chimeras containing a fragment of the 3’ region of PPV-D –including the region coding for the CP– in a PPV-R backbone. These chimeras were not infectious in Prunus persica, but systemically infected Nicotiana clevelandii and Nicotiana benthamiana when specific amino acids (aa) were modified or deleted in a short 30-aa region of the N-terminus of the CP. Most of these mutations did not reduce PPV fitness in Prunus although others impaired systemic infection in this host.

We propose a model in which the N-terminus of the CP, highly relevant for virus systemic movement, is targeted by a host defense mechanism in Nicotiana. Mutations in this short region allow PPV to overcome the defense response in this host but can compromise the efficiency of PPV systemic movement in other hosts such as Prunus.

Environ Microbiol Rep. 2012 Jun;4(3):335-41

Tamames J, de la Peña S, de Lorenzo V.

In any metagenomic project, the coverage obtained for each particular species depends on its abundance. This makes it difficult to determine a priori the amount of DNA sequencing necessary to obtain a high coverage for the dominant genomes in an environment.

To aid the design of metagenomic sequencing projects, we have developed COVER, a web-based tool that allows the estimation of the coverage achieved for each species in an environmental sample. COVER uses a set of 16S rRNA sequences to produce an estimate of the number of operational taxonomic units (OTUs) in the sample, provides a taxonomic assignment for them, estimates their genome sizes and, most critically, corrects for the number of unobserved OTUs. COVER then calculates the amount of sequencing needed to achieve a given goal. Our tests and simulations indicate that the results obtained through COVER are in very good agreement with the experimental results.