In and out of refugia: historical patterns of diversity and demography in the North American Caesar's mushroom species complex

Some of the effects of past climate dynamics on plant and animal diversity make‐up have been relatively well studied, but to less extent in fungi. Pleistocene refugia are thought to harbour high biological diversity (i.e. phylogenetic lineages and genetic diversity), mainly as a product of increased reproductive isolation and allele conservation. In addition, high extinction rates and genetic erosion are expected in previously glaciated regions. Some of the consequences of past climate dynamics might involve changes in range and population size that can result in divergence and incipient or cryptic speciation. Many of these dynamic processes and patterns can be inferred through phylogenetic and coalescent methods. In this study, we first delimit species within a group of closely related edible ectomycorrhizal Amanita from North America (the American Caesar's mushrooms species complex) using multilocus coalescent‐based approaches; and then address questions related to effects of Pleistocene climate change on the diversity and genetics of the group. Our study includes extensive geographical sampling throughout the distribution range, and DNA sequences from three nuclear protein‐coding genes. Results reveal cryptic diversity and high speciation rates in refugia. Population sizes and expansions seem to be larger at midrange latitudes (Mexican highlands and SE USA). Range shifts are proportional to population size expansions, which were overall more common during the Pleistocene. This study documents responses to past climate change in fungi and also highlights the applicability of the multispecies coalescent in comparative phylogeographical analyses and diversity assessments that include ancestral species.     

Nfu facilitates the maturation of iron‐sulfur proteins and participates in virulence in Staphylococcus aureus

The acquisition and metabolism of iron (Fe) by the human pathogen Staphylococcus aureus is critical for disease progression. S. aureus requires Fe to synthesize inorganic cofactors called iron‐sulfur (Fe‐S) clusters, which are required for functional Fe‐S proteins. In this study we investigated the mechanisms utilized by S. aureus to metabolize Fe‐S clusters. We identified that S. aureus utilizes the Suf biosynthetic system to synthesize Fe‐S clusters and we provide genetic evidence suggesting that the sufU and sufB gene products are essential. Additional biochemical and genetic analyses identified Nfu as an Fe‐S cluster carrier, which aids in the maturation of Fe‐S proteins. We find that deletion of the nfu gene negatively impacts staphylococcal physiology and pathogenicity. A nfu mutant accumulates both increased intracellular non‐incorporated Fe and endogenous reactive oxygen species (ROS) resulting in DNA damage. In addition, a strain lacking Nfu is sensitive to exogenously supplied ROS and reactive nitrogen species. Congruous with ex vivo findings, a nfu mutant strain is more susceptible to oxidative killing by human polymorphonuclear leukocytes and displays decreased tissue colonization in a murine model of infection. We conclude that Nfu is necessary for staphylococcal pathogenesis and establish Fe‐S cluster metabolism as an attractive antimicrobial target.     

Zinc-, cadmium-, and mercury-containing one-dimensional tetraphenylporphyrin arrays: a DFT study

This work describes theoretical and experimental studies on glycerol esterification to obtain acetins focusing on the obtained isomers. The reaction of glycerol with acetic acid was carried out on Amberlyst 36 wet. Density functional theory calculations on the level of M06-2X functional and 6-311+G(d,p) basis set are carried out and the most stable structures of the reactants and products are located by considering a large number of conformers. The thermodynamics is discussed in terms of the calculated reaction Gibbs free energy. The AIM theory was used to characterize reactants and products. The glycerol esterification with acetic acid is found to be thermodynamically favored, with exothermal property. These agree well with experiments and allow us to explain the relative selectivity of products.

Quantitative Analysis of the Processes and Signaling Events Involved in Early HIV-1 Infection of T Cells

Lymphocyte invasion by HIV-1 is a complex, highly regulated process involving many different types of molecules that is prompted by the virus''s association with viral receptors located at the cell-surface membrane that culminates in the formation of a fusion pore through which the virus enters the cell. A great deal of work has been done to identify the key actors in the process and determine the regulatory interactions; however, there have been no reports to date of attempts being made to fully understand the system dynamics through a systemic, quantitative modeling approach. In this paper, we introduce a dynamic mathematical model that integrates the available information on the molecular events involved in lymphocyte invasion. Our model shows that moesin activation is induced by virus signaling, while filamin-A is mobilized by the receptor capping. Actin disaggregation from the cap is facilitated by cofilin. Cofilin is inactivated by HIV-1 signaling in activated lymphocytes, while in resting lymphocytes another signal is required to activate cofilin in the later stages in order to accelerate the decay of the aggregated actin as a restriction factor for the viral entry. Furthermore, stopping the activation signaling of moesin is sufficient to liberate the actin filaments from the cap. The model also shows the positive effect of gelsolin on actin capping by means of the nucleation effect. These findings allow us to propose novel approaches in the search for new therapeutic strategies. In particular, gelsolin inhibition is seen as a promising target for preventing HIV-1 entry into lymphocytes, due to its role in facilitating the capping needed for the invasion. Also it is shown that HIV-1 should overcome the cortical actin barrier during early infection and predicts the different susceptibility of CD4+ T cells to be infected in terms of actin cytoskeleton dynamics driven by associated cellular factors.     

In-depth Characterization of the Secretome of Colorectal Cancer Metastatic Cells Identifies Key Proteins in Cell Adhesion,Migration, and Invasion

Liver metastasis in colorectal cancer is the major cause of cancer-related deaths. To identify and characterize proteins associated with colon cancer metastasis, we have compared the conditioned serum-free medium of highly metastatic KM12SM colorectal cancer cells with the parental, poorly metastatic KM12C cells using quantitative stable isotope labeling by amino acids in cell culture (SILAC) analyses on a linear ion trap-Orbitrap Velos mass spectrometer. In total, 1337 proteins were simultaneously identified in SILAC forward and reverse experiments. For quantification, 1098 proteins were selected in both experiments, with 155 proteins showing >1.5-fold change. About 52% of these proteins were secreted directly or using alternative secretion pathways. GDF15, S100A8/A9, and SERPINI1 showed capacity to discriminate cancer serum samples from healthy controls using ELISAs. In silico analyses of deregulated proteins in the secretome of metastatic cells showed a major abundance of proteins involved in cell adhesion, migration, and invasion. To characterize the tumorigenic and metastatic properties of some top up- and down-regulated proteins, we used siRNA silencing and antibody blocking. Knockdown expression of NEO1, SERPINI1, and PODXL showed a significant effect on cellular adhesion. Silencing or blocking experiments with SOSTDC1, CTSS, EFNA3, CD137L/TNFSF9, ZG16B, and Midkine caused a significant decrease in migration and invasion of highly metastatic cells. In addition, silencing of SOSTDC1, EFNA3, and CD137L/TNFSF9 reduced liver colonization capacity of KM12SM cells. Finally, the panel of six proteins involved in invasion showed association with poor prognosis and overall survival after dataset analysis of gene alterations. In summary, we have defined a collection of proteins that are relevant for understanding the mechanisms underlying adhesion, migration, invasion, and metastasis in colorectal cancer.Despite the efforts for colorectal cancer (CRC)¹ prevention using different strategies (1–6), 30–40% of patients have regionally advanced disease or suffer from metastasis when diagnosed (7). Moreover, half of the CRC patients will develop recurrence and liver metastasis within 5 years (8). Although genetic changes leading to the development of sporadic colorectal cancer primary tumors in intestinal cells have been relatively well characterized (9), further efforts are necessary to better understand the biology of CRC metastasis and to identify associated markers that can be used as diagnostic/prognostic biomarkers or potential drug targets. Metastasis is a complex process involving different steps from extravasation to liver colonization and requires the concerted action of a large number of proteins to modulate different effects on adhesion, migration, invasion, and survival at the target organ (10).Cancer cells secrete proteins or protein fragments to body fluids, such as blood, that can be used as biomarkers (11, 12) and/or potential therapeutic targets (13). In the case of CRC, there are only three proteins currently used as biomarkers: the carcinoembryonic antigen (CEA) for recurrence and metastasis (1), deleted in colorectal carcinoma (DCC), and vascular endothelial growth factor (VEGF). The secretome constitutes a rich source of information not only for the identification of biomarkers but for the characterization of altered molecules like growth factors, cytokines, proteases, etc., which are vital for cancer progression and metastasis.We are using the well known human KM12 cell system (14) to study the biology of CRC metastasis. KM12SM cells, which possess high metastatic capacity to liver, were isolated from liver metastases in nude mice after five cycles of intrasplenic injection of the poorly metastatic cell line KM12C (14, 15). Multiple studies support a good correlation between the findings observed in the KM12 cell model and patient samples, indicating that KM12 isogenic cell lines recapitulate quite effectively some of the critical issues in CRC metastasis (16–21). In a previous study, we carried out a characterization of plasma membrane proteins of metastatic KM12 cells using a SILAC assay but with a low accuracy and resolution linear ion trap (17). About 60 proteins that showed ≥1.5-fold-change between both types of cells were identified. Recent studies applied iTRAQ or label-free quantification to other pairs of isogenic, nonmetastatic-metastatic colorectal cancer cell lines, SW480 and SW620, for the characterization of protein differences in the whole cell proteome (22) and secretome (23), respectively. The SW620 cell line was isolated from a metastatic lymph node of the same patient as SW480 (24). In contrast, KM12SM cells were chosen based on their capacity for liver metastasis, which makes them most appropriate for the study of liver homing and late stages of metastasis.We are analyzing different fractions of KM12 cells, including the secretome, for a deeper analysis of functionally relevant proteins in metastasis. In a previous report, we analyzed the cytokine/chemokine profiles released in the conditioned media by colorectal metastatic cancer KM12SM cells compared with KM12C using antibody microarrays (20). We found an important role for T_H2 cytokine IL-13 and its receptor IL13Rα2 in cell adhesion, migration, invasion, and liver colonization. Here, we continued this in-depth characterization of the secretome compartment using SILAC analysis with a high accuracy and resolution mass spectrometer, the linear ion trap Orbitrap Velos. The proteomic characterization resulted in the identification and quantification of 1337 and 1098 proteins, respectively, in the conditioned medium. In silico studies demonstrated a predominant association of deregulated proteins in metastatic cells to adhesion, migration, and invasion processes. Three candidates (GDF15, S100A8/A9, and SERPINI1) showed promise as CRC diagnostic markers in serum samples from CRC patients using ELISA. Functional studies using siRNA silencing and antibody blocking experiments demonstrated important tumorigenic and invasive properties in some previously uncharacterized proteins in CRC. In addition, three proteins, EFNA3, CD137L/TNFSF9, and SOSTDC1, demonstrated a critical role in liver homing for metastasis. Finally, meta-analysis of mRNA alterations data indicated that CD137L/TNFSF9, CTSS, SOSTDC1, ZG16B, EFNA3, and MDK were associated with poor prognosis in colorectal cancer.     

Development of amplified fragment length polymorphism (AFLP)‐derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot

Aims

The objective of this work was to design an amplified fragment length polymorphism (AFLP)‐derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot (PBRR) in plant material and soil.

Methods and Results

Specific primers for the detection of the pathogen were designed based on an amplified region using AFLPs. The banding patterns by AFLPs showed that isolates from diseased roots were clearly distinguishable from others members of the F. solani species complex. Many bands were specific to F. solani PBRR, one of these fragments was selected and sequenced. Sequence obtained was used to develop specific PCR primers for the identification of pathogen in pure culture and in plant material and soil. Primer pair FS1/FS2 amplified a single DNA product of 175 bp. Other fungal isolates occurring in soil, included F. solani non‐PBRR, were not detected by these specific primers. The assay was effective for the detection of pathogen from diseased root and infected soils.

Conclusions

The designed primers for F. solani causing PBRR can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen.

Significance and Impact of the Study

These diagnostic PCR primers will aid the detection of F. solani causing PBRR in diseased root and natural infected soils. The method developed could be a helpful tool for epidemiological studies and to avoid the spread of this serious disease in new areas.     

Index for estimation of muscle force from mechanomyography based on the Lempel–Ziv algorithm

The study of the amplitude of respiratory muscle mechanomyographic (MMG) signals could be useful in clinical practice as an alternative non-invasive technique to assess respiratory muscle strength. The MMG signal is stochastic in nature, and its amplitude is usually estimated by means of the average rectified value (ARV) or the root mean square (RMS) of the signal. Both parameters can be used to estimate MMG activity, as they correlate well with muscle force. These estimations are, however, greatly affected by the presence of structured impulsive noise that overlaps in frequency with the MMG signal. In this paper, we present a method for assessing muscle activity based on the Lempel–Ziv algorithm: the Multistate Lempel–Ziv (MLZ) index. The behaviour of the MLZ index was tested with synthesised signals, with various amplitude distributions and degrees of complexity, and with recorded diaphragm MMG signals. We found that this index, like the ARV and RMS parameters, is positively correlated with changes in amplitude of the diaphragm MMG components, but is less affected by components that have non-random behaviour (like structured impulsive noise). Therefore, the MLZ index could provide more information to assess the MMG–force relationship.