Inference from clustering with application to gene-expression microarrays.

There are many algorithms to cluster sample data points based on nearness or a similarity measure. Often the implication is that points in different clusters come from different underlying classes, whereas those in the same cluster come from the same class. Stochastically, the underlying classes represent different random processes. The inference is that clusters represent a partition of the sample points according to which process they belong. This paper discusses a model-based clustering toolbox that evaluates cluster accuracy. Each random process is modeled as its mean plus independent noise, sample points are generated, the points are clustered, and the clustering error is the number of points clustered incorrectly according to the generating random processes. Various clustering algorithms are evaluated based on process variance and the key issue of the rate at which algorithmic performance improves with increasing numbers of experimental replications. The model means can be selected by hand to test the separability of expected types of biological expression patterns. Alternatively, the model can be seeded by real data to test the expected precision of that output or the extent of improvement in precision that replication could provide. In the latter case, a clustering algorithm is used to form clusters, and the model is seeded with the means and variances of these clusters. Other algorithms are then tested relative to the seeding algorithm. Results are averaged over various seeds. Output includes error tables and graphs, confusion matrices, principal-component plots, and validation measures. Five algorithms are studied in detail: K-means, fuzzy C-means, self-organizing maps, hierarchical Euclidean-distance-based and correlation-based clustering. The toolbox is applied to gene-expression clustering based on cDNA microarrays using real data. Expression profile graphics are generated and error analysis is displayed within the context of these profile graphics. A large amount of generated output is available over the web.     

Cloning and functional expression of human short TRP7, a candidate protein for store-operated Ca2+ influx.

The regulation and control of plasma membrane Ca(2+) fluxes is critical for the initiation and maintenance of a variety of signal transduction cascades. Recently, the study of transient receptor potential channels (TRPs) has suggested that these proteins have an important role to play in mediating capacitative calcium entry. In this study, we have isolated a cDNA from human brain that encodes a novel transient receptor potential channel termed human TRP7 (hTRP7). hTRP7 is a member of the short TRP channel family and is 98% homologous to mouse TRP7 (mTRP7). At the mRNA level hTRP7 was widely expressed in tissues of the central nervous system, as well as some peripheral tissues such as pituitary gland and kidney. However, in contrast to mTRP7, which is highly expressed in heart and lung, hTRP7 was undetectable in these tissues. For functional analysis, we heterologously expressed hTRP7 cDNA in an human embryonic kidney cell line. In comparison with untransfected cells depletion of intracellular calcium stores in hTRP7-expressing cells, using either carbachol or thapsigargin, produced a marked increase in the subsequent level of Ca(2+) influx. This increased Ca(2+) entry was blocked by inhibitors of capacitative calcium entry such as La(3+) and Gd(3+). Furthermore, transient transfection of an hTRP7 antisense expression construct into cells expressing hTRP7 eliminated the augmented store-operated Ca(2+) entry. Our findings suggest that hTRP7 is a store-operated calcium channel, a finding in stark contrast to the mouse orthologue, mTRP7, which is reported to enhance Ca(2+) influx independently of store depletion, and suggests that human and mouse TRP7 channels may fulfil different physiological roles.     

Arf6 anchors Cdr2 nodes at the cell cortex to control cell size at division

Fission yeast cells prevent mitotic entry until a threshold cell surface area is reached. The protein kinase Cdr2 contributes to this size control system by forming multiprotein nodes that inhibit Wee1 at the medial cell cortex. Cdr2 node anchoring at the cell cortex is not fully understood. Through a genomic screen, we identified the conserved GTPase Arf6 as a component of Cdr2 signaling. Cells lacking Arf6 failed to divide at a threshold surface area and instead shifted to volume-based divisions at increased overall size. Arf6 stably localized to Cdr2 nodes in its GTP-bound but not GDP-bound state, and its guanine nucleotide exchange factor (GEF), Syt22, was required for both Arf6 node localization and proper size at division. In arf6Δ mutants, Cdr2 nodes detached from the membrane and exhibited increased dynamics. These defects were enhanced when arf6Δ was combined with other node mutants. Our work identifies a regulated anchor for Cdr2 nodes that is required for cells to sense surface area.     

Morphological and molecular characterization of a new autochthonous Trichoderma sp. isolate and its biocontrol efficacy against Alternaria sp.

The development of agriculture requires the use of microorganisms in the management of phytopathogens as a way to compensate for the use of chemical pesticides, in order to produce healthy crops. The objective of this study was to characterize a new isolate of Trichoderma sp. based on morphological and molecular features, and its potential ability to control the pathogen Alternaria sp. The antagonistic isolate was isolated from soil samples of potato fields in Guasave Sinaloa, Mexico, whereas the pathogen was collected from infected apple leaves in the orchard “La Escondida” in Guerrero County, Chihuahua, Mexico. For morphological characterization both fungi were grown on solid PDA medium. DNA of Trichoderma sp. was isolated using the CTAB method and PCR analyses were done using ITS1, ITS4 primers resulting in amplified products of 600 bp. These were sequenced, submitted to Genbank (acc. no. MN950427) and used for further phylogenetic analysis through Bayesian inference approach. Five clades were identified and the polytome topography recovered from clade 4 indicates a high genetic similarity with T. asperellum. A BLAST examination of the resulting sequence in GenBank showed 98.11% similarity with T. asperellum. This result together with the morphological and the phylogenetic analyses indicates that the isolate belongs to Trichoderma asperellum Samuels, Lieckfeldt & Nirenberg. Biocontrol tests of this isolate showed inhibition of Alternaria sp. between 50% and 93%. These results are essential for biodiversity research and give some new possibilities for pest management.     

Effects of apolipoprotein B-100 on the metabolism of a lipid microemulsion model in rats

In previous studies, it was shown that lipid microemulsions resembling LDL (LDE) but not containing protein, acquire apolipoprotein E when injected into the bloodstream and bind to LDL receptors (LDLR) using this protein as ligand. Aiming to evaluate the effects of apolipoprotein (apo) B-100 on the catabolism of these microemulsions, LDE with incorporated apo B-100 (LDE-apoB) and native LDL, all labeled with radioactive lipids were studied after intraarterial injection into Wistar rats. Plasma decay curves of the labels were determined in samples collected over 10 h and tissue uptake was assayed from organs excised from the animals sacrificed 24 h after injection. LDE-apo B had a fractional clearance rate (FCR) similar to native LDL (0.40 and 0.33, respectively) but both had FCR pronouncedly smaller than LDE (0.56, P<0.01). Liver was the main uptake site for LDE, LDE-apoB, and native LDL, but LDE-apoB and native LDL had lower hepatic uptake rates than LDE. Pre-treatment of the rats with 17α-ethinylestradiol, known to upregulate LDLR, accelerated the removal from plasma of both LDE and LDE-apoB, but the effect was greater upon LDE than LDE-apoB. These differences in metabolic behavior documented in vivo can be interpreted by the lower affinity of LDLR for apo B-100 than for apo E, demonstrated in in vitro studies. Therefore, our study shows in vivo that, in comparison with apo E, apo B is a less efficient ligand to remove lipid particles such as microemulsions or lipoproteins from the intravascular compartment.     

Exploring the Limiting Open‐Circuit Voltage and the Voltage Loss Mechanism in Planar CH3NH3PbBr3 Perovskite Solar Cells

Perovskite solar cells based on CH₃NH₃PbBr₃ with a band gap of 2.3 eV are attracting intense research interests due to their high open‐circuit voltage (V_oc) potential, which is specifically relevant for the use in tandem configuration or spectral splitting. Many efforts have been performed to optimize the V_oc of CH₃NH₃PbBr₃ solar cells; however, the limiting V_oc (namely, radiative V_oc:V_oc,rad) and the corresponding ΔV_oc (the difference between V_oc,rad and V_oc) mechanism are still unknown. Here, the average V_oc of 1.50 V with the maximum value of 1.53 V at room temperature is achieved for a CH₃NH₃PbBr₃ solar cell. External quantum efficiency measurements with electroluminescence spectroscopy determine the V_oc,rad of CH₃NH₃PbBr₃ cells with 1.95 V and a ΔV_oc of 0.45 V at 295 K. When the temperature declines from 295 to 200 K, the obtained V_oc remains comparably stable in the vicinity of 1.5 V while the corresponding ΔV_oc values show a more significant increase. Our findings suggest that the V_oc of CH₃NH₃PbBr₃ cells is primarily limited by the interface losses induced by the charge extraction layer rather than by bulk dominated recombination losses. These findings are important for developing strategies how to further enhance the V_oc of CH₃NH₃PbBr₃‐based solar cells.     

Preparation of inert magnetic nano-particles for the directed immobilization of antibodies

Various activated supports (cyanogen bromide, glutaraldehyde, epoxy-chelates, primary amino) were evaluated for the immobilization of IgG anti-horseradish peroxidase. Cyanogen bromide and glutaraldehyde supports greatly reduced the recognition capacity of the antigen, probably due to the incorrect orientation of the antibody on the support. Hetero-functional epoxy-chelate and immobilization by the sugar chain on primary amino groups had little effect on high recognition of the antigen (near to the theoretically expected value). However, the immobilization by the sugar chain resulted in a higher adsorption rate of horseradish peroxidase, possibly due to a favourable orientation on a flexible spacer arm). Antibodies immobilized on aminated surfaces showed two major drawbacks. Firstly, the biological activity of the immobilized antibody sharply decreased over several days when stored at low ionic strength, although this effect could be partially reversed by incubation at high ionic strength. Secondly, a high level of non-specific proteins adsorption on the support surface was observed. Both problems could be successfully resolved by controlling the coating of the support with aldehyde-aspartic-dextran. We propose that the loss of biological activity was related to the ionic adsorption of the immobilized antibody on the support surface, leading to a blocking of the recognition areas. This optimized protocol was applied to the immobilization of IgG anti-horseradish peroxidase from rabbit on magnetic nano-particles. A 10 microg preparation of nano-particles was able to capture more than 75% of the 0.1 microgram of recombinant horseradish peroxidase present in 10 L of crude protein extract (1g/L) from Escherichia coli.     

Uterine infusion of conceptus fragments changes the protein profile from cyclic mares

This experiment aimed to compare at day seven after ovulation, the protein profile of uterine fluid in cyclic mares with mares infused two days before with Day 13 conceptus fragments. Experimental animals were ten healthy cyclic mares, examined daily to detect ovulation (Day 0) as soon as estrus was confirmed. On day seven, after ovulation, uterine fluid was collected, constituting the Cyclic group (n = 10). The same mares were examined in the second cycle until ovulation was detected. On day five, after ovulation, fragments from a previously collected concepti were infused into each mare''s uterus. Two days after infusion, uterine fluid was collected, constituting the Fragment group (n = 10). Two-dimensional electrophoresis technique processed uterine fluid samples. A total of 373 spots were detected. MALDI-TOF/TOF and NanoUHPLC-QTOF mass spectrometry identified twenty spots with differences in abundance between the Cyclic and Fragment group. Thirteen proteins were identified, with different abundance between groups. Identified proteins may be related to embryo-maternal communication, which involves adhesion, nutrition, endothelial cell proliferation, transport, and immunological tolerance. In conclusion, conceptus fragments signalized changes in the protein profile of uterine fluid seven days after ovulation in comparison to the observed at Day 7 in the same cyclic mares.