Hyperproliferative response of a monoclonal memory CD8 T cell population is characterized by an increased frequency of clonogenic precursors

Strong memory T cell responses result partly from the selection of Ag-specific clones during immunization. In this study, we show that a monoclonal CD8 T cell population expressing a unique TCR is heterogeneous in terms of clonogenic potential following activation under optimal conditions. More importantly, the frequency of clonogenic cells is strongly increased among Ag-experienced cells, indicating that these cells were either generated or selected during the in vivo primary response. Moreover, strong proliferative responses of primed cells result from this enhanced frequency, as proliferating naive and primed cells display the same cycling parameters, i.e., lag time and intermitotic interval. Hence, these results suggest that the clonogenic potential of individual cells is imprinted before Ag encounter and that clonogenic precursors are selected or generated following in vivo activation.     

Human activity recognition based on Evolving Fuzzy Systems

Environments equipped with intelligent sensors can be of much help if they can recognize the actions or activities of their users. If this activity recognition is done automatically, it can be very useful for different tasks such as future action prediction, remote health monitoring, or interventions. Although there are several approaches for recognizing activities, most of them do not consider the changes in how a human performs a specific activity. We present an automated approach to recognize daily activities from the sensor readings of an intelligent home environment. However, as the way to perform an activity is usually not fixed but it changes and evolves, we propose an activity recognition method based on Evolving Fuzzy Systems.     

Toxicity of indoxyl derivative accumulation in bacteria and its use as a new counterselection principle

In this work we describe the conditional toxic effect of the expression of enzymes that cleave 5-bromo-4-chloro-3-indolyl (BCI) substrates and its use as a new counterselection principle useful for the generation of clean and unmarked mutations in the genomes of bacteria. The application of this principle was demonstrated in the thermophile Thermus thermophilus HB27 and in a mesophile for which currently no counterselection markers are available, Micrococcus luteus ATCC 27141. For T. thermophilus, the indigogenic substrate BCI-β-glucoside was used in combination with the T. thermophilus β-glucosidase gene (bgl). For M. luteus, a combination of BCI-β-galactoside and the E. coli lacZ gene was implemented. We observed a strong growth-inhibiting effect when the strains were grown on agar plates containing the appropriate BCI substrates, the inhibition being proportional to the substrate concentration and the level of bgl/lacZ expression. The growth inhibition apparently depends on intracellular BCI substrate cleavage and accumulation of toxic indoxyl precipitates. The bgl and lacZ genes were used as counterselection markers for the rapid generation of scar-less chromosomal deletions in T. thermophilus HB27 (both in a Δbgl and in a wild type background) and in M. luteus ATCC 27141.     

Bacteriocin production by strain <Emphasis Type="Italic">Lactobacillus delbruecki</Emphasis>i ssp. <Emphasis Type="Italic">bulgaricus</Emphasis> BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk

By screening for bacteriocin-producing lactic acid bacteria of 1,428 strains isolated from authentic Bulgarian dairy products, Lb. bulgaricus BB18 strain obtained from kefir grain was selected. Out of 11 yogurt starters containing Lb. bulgaricus BB18 and S. thermophilus strains resistant to bacteriocin secreted by Lb. bulgaricus BB18 a yogurt culture (S. thermophilus 11A+Lb. bulgaricus BB18) with high growth and bacteriocinogenic activity in milk was selected. Continuous (pH-stat 5.7) prefermentation processes were carried out in milk at 37 degrees C in a 2l MBR bioreactor (MBR AG, Zurich, Switzerland) with an IMCS controller for agitation speed, temperature, dissolved oxygen, CO2 and pH. Prefermented milk with pH 5.7 coagulated in a thermostat at 37 degrees C until pH 4.8-4.9. S. thermophilus 11A and Lb. bulgaricus BB18 grew independently in a continuous mode at similar and sufficiently high-dilution rates (D=1.83 h(-1)-S. thermophilus 11A; D=1.80 h(-1)-Lb. bulgaricus BB18). The yogurt cultures developed in a stream at a high-dilution rate (D=2.03-2.28 h(-1)). The progress of both processes (growth and bacteriocin production) depended on the initial ratio between the two microorganisms. The continuous prefermentation process promoted conditions for efficient fermentation and bacteriocinogenesis of the starter culture during the batch process: strong reduction of the times for bacteriocin production and coagulation of milk (to 4.5-5.0 h); high cell productivity (lactobacilli-4x10(12) CFU ml(-1), streptococci-6x10(12) CFU ml(-1)); high productivity of bacteriocins (4,500 BU ml(-1))-1.7 times higher than the bacteriocinogenic activity of the batch starter culture.     

Alternative hosts for functional (meta)genome analysis

Microorganisms are ubiquitous on earth, often forming complex microbial communities in numerous different habitats. Most of these organisms cannot be readily cultivated in the laboratory using standard media and growth conditions. However, it is possible to gain access to the vast genetic, enzymatic, and metabolic diversity present in these microbial communities using cultivation-independent approaches such as sequence- or function-based metagenomics. Function-based analysis is dependent on heterologous expression of metagenomic libraries in a genetically amenable cloning and expression host. To date, Escherichia coli is used in most cases; however, this has the drawback that many genes from heterologous genomes and complex metagenomes are expressed in E. coli either at very low levels or not at all. This review emphasizes the importance of establishing alternative microbial expression systems consisting of different genera and species as well as customized strains and vectors optimized for heterologous expression of membrane proteins, multigene clusters encoding protein complexes or entire metabolic pathways. The use of alternative host-vector systems will complement current metagenomic screening efforts and expand the yield of novel biocatalysts, metabolic pathways, and useful metabolites to be identified from environmental samples.     

Base excision repair of 8-oxoG in dinucleosomes

In this work we have studied the effect of chromatin structure on the base excision repair (BER) efficiency of 8-oxoG. As a model system we have used precisely positioned dinucleosomes assembled with linker histone H1. A single 8-oxoG was inserted either in the linker or the core particle DNA within the dinucleosomal template. We found that in the absence of histone H1 the glycosylase OGG1 removed 8-oxoG from the linker DNA and cleaved DNA with identical efficiency as in the naked DNA. In contrast, the presence of histone H1 resulted in close to 10-fold decrease in the efficiency of 8-oxoG initiation of repair in linker DNA independently of linker DNA length. The repair of 8-oxoG in nucleosomal DNA was very highly impeded in both absence and presence of histone H1. Chaperone-induced uptake of H1 restored the efficiency of the glycosylase induced removal of 8-oxoG from linker DNA, but not from the nucleosomal DNA. We show, however, that removal of histone H1 and nucleosome remodelling are both necessary and sufficient for an efficient removal of 8-oxoG in nucleosomal DNA. Finally, a model for BER of 8-oxoG in chromatin templates is suggested.     

Satellite DNA repeat sequence variation is low in three species of burying beetles in the genus Nicrophorus (Coleoptera: Silphidae)

Three satellite DNA families were identified in three species of burying beetles, Nicrophorus orbicollis, N. marginatus, and N. americanus. Southern hybridization and nucleotide sequence analysis of individual randomly cloned repeats shows that these satellite DNA families are highly abundant in the genome, are composed of unique repeats, and are species-specific. The repeats do not have identifiable core elements or substructures that are similar in all three families, and most interspecific sequence similarity is confined to homopolymeric runs of A and T. Satellite DNA from N. marginatus and N. americanus show single-base-pair indels among repeats, but single-nucleotide substitutions characterize most of the repeat variability. Although the repeat units are of similar lengths (342, 350, and 354 bp) and A + T composition (65%, 71%, and 71%, respectively), the average nucleotide divergence among sequenced repeats is very low (0.18%, 1.22%, and 0.71%, respectively). Transition/transversion ratios from the consensus sequence are 0.20, 0.69, and 0.70, respectively.      

Insights into extreme thermoacidophily based on genome analysis of Picrophilus torridus and other thermoacidophilic archaea

Thermoacidophiles are prokaryotic microorganisms with the stunning capability to survive and multiply at extremely low pH and simultaneously at high temperatures. The mechanisms by which these organisms, exclusively members of the Archaea, cope with their harsh surroundings are poorly understood. The genome sequences of several representatives of the thermoacidophilic genera Picrophilus, Thermoplasma and Sulfolobus have recently become available. Genome-wide comparison has revealed a number of features as possible facets of the overall acidophilic survival strategy of the most thermoacidophilic organisms known, such as a high ratio of secondary over primary transport systems, the composition of the respiratory chain, and the frequent genetic input via lateral gene transfer (LGT) during evolution.     

Novel characteristics of cassava,Manihot esculenta Crantz,a reputed C3-C4 intermediate photosynthesis species

The cassava plant, Manihot esculenta, grows exceptionally well in low fertility and drought prone environments, but the mechanisms that allow this growth are unknown. Earlier, and sometimes contradictory, work speculated about the presence of a C₄-type photosynthesis in cassava leaves. In the present work we found no evidence for a C₄ metabolism in mature attached cassava leaves as indicated i) by the low, 2 to 8%, incorporation of ¹⁴CO₂ into C₄ organic acids in short time periods, 10 s, and the lack of ¹⁴C transfer from C₄ acids to other compounds in ¹²CO₂, ii) by the lack of C₄ enzyme activity changes during leaf development and the inability to detect C₄ acid decarboxylases, and iii) by leaf CO₂ compensation values between 49 and 65 l of CO₂ 1^–1 and by other infrared gas exchange photosynthetic measurements. It is concluded that the leaf biochemistry of cassava follows the C₃ pathway of photosynthesis with no indication of a C₃-C₄ mechanism.However, cassava leaves exhibit several novel characteristics. Attached leaves have the ability to effectively partition carbon into sucrose with nearly 45% of the label in sucrose in about one min of ¹⁴CO₂ photosynthesis, contrasting with 34% in soybean (C₃) and 25% in pigweed (C₄). Cassava leaves displayed a strong preference for the synthesis of sucrose versus starch. Field grown cassava leaves exhibited high rates of photosynthesis and curvilinear responses to increasing sunlight irradiances with a tendency to saturate only at high irradiances, above 1500 mol m^–2 s^–1. Morphologically, the cassava leaf has papillose epidermal cells on its lower mesophyll surface that form fence-like arrangements encircling guard cells. It is proposed that the active synthesis of sugars has osmotic functions in the cassava plant and that the papillose epidermal cells function to maintain a healthy leaf water status in various environments.Abbreviations ADP adenosine diphosphate - Asp aspartate - BSA bovine serum albumin - CoA coenzyme A - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - FBP fructose-1,6-biphosphate - Gly glycine - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - Mal malate - NAD nicotinamide adenine dinucleotide (oxidized form) - NADH nicotinamide adenine dinucleotide (reduced form) - NADP nicotinamide adenine dinucleotide phosphate (oxidized form) - PAR photosynthetic active radiation (400–700 nm) - PEP phosphenolpyruvate carboxylase - p-FBPase plastid fructose-1,6-biphosphatase - PGA 3-phosphoglyceric acid - PMSF phenylmethylsulfonyl fluoride - PVP polyvinylpyrrolidone - Rubisco ribulose-1,5-biphosphate carboxylase/oxygenase - RuBP ribulose-1,5-biphosphate - Ser serine - sugar-P sugar-phosphates