Real-time detection of viable microorganisms by intracellular phototautomerism

Background  

To date, the detection of live microorganisms present in the environment or involved in infections is carried out by enumeration of colony forming units on agar plates, which is time consuming, laborious and limited to readily cultivable microorganisms. Although cultivation-independent methods are available, they involve multiple incubation steps and do mostly not discriminate between dead or live microorganisms. We present a novel generic method that is able to specifically monitor living microorganisms in a real-time manner.     

The R-spondin protein family

The four vertebrate R-spondin proteins are secreted agonists of the canonical Wnt/β-catenin signaling pathway. These proteins are approximately 35 kDa, and are characterized by two amino-terminal furin-like repeats, which are necessary and sufficient for Wnt signal potentiation, and a thrombospondin domain situated more towards the carboxyl terminus that can bind matrix glycosaminoglycans and/or proteoglycans. Although R-spondins are unable to initiate Wnt signaling, they can potently enhance responses to low-dose Wnt proteins. In humans, rare disruptions of the gene encoding R-spondin1 cause a syndrome of XX sex reversal (phenotypic male), palmoplantar keratosis (a thickening of the palms and soles caused by excess keratin formation) and predisposition to squamous cell carcinoma of the skin. Mutations in the gene encoding R-spondin4 cause anonychia (absence or hypoplasia of nails on fingers and toes). Recently, leucine-rich repeat-containing G-protein-coupled receptor (Lgr)4, Lgr5 and Lgr6, three closely related orphans of the leucine-rich repeat family of G-protein-coupled receptors, have been identified as receptors for R-spondins. Lgr5 and Lgr6 are markers for adult stem cells. Because R-spondins are potent stimulators of adult stem cell proliferation in vivo and in vitro, these findings might guide the therapeutic use of R-spondins in regenerative medicine.     

Assessment of changes in the brca2 and p53 genes in breast invasive ductal carcinoma in northeast Brazil

Background

BRCA protein interacts with at least 13 different proteins that have been implicated with cancer susceptibility and loss of BRCA function is correlated to sensitivity to DNA crosslinking agents in preclinical models.

Results

BRCA2 methylation frequency was 44%, p53 Pro22 allele frequency was 32% and heterozygous frequency of Arg/Pro72 genotype was 60% which could be associated as risk factor for metastasis (p = 0.046 OR = 4.190). Regarding to polymorphism of codon 249 the frequency of Arg249 allele presented 82% which was considered not statistically significant.

Conclusions

There was not statistical significance to BRCA2 promoter methylation with any parameters chosen. However, our findings suggest that patients who present heterozygous genotype at codon 72 of p53 gene may have a major susceptibility to any type of metastasis and this could serve as potential auxiliary biomarker for poor prognosis.     

Effects of Isoflurane Anesthesia on Ensemble Patterns of Ca2+ Activity in Mouse V1: Reduced Direction Selectivity Independent of Increased Correlations in Cellular Activity

Anesthesia affects brain activity at the molecular, neuronal and network level, but it is not well-understood how tuning properties of sensory neurons and network connectivity change under its influence. Using in vivo two-photon calcium imaging we matched neuron identity across episodes of wakefulness and anesthesia in the same mouse and recorded spontaneous and visually evoked activity patterns of neuronal ensembles in these two states. Correlations in spontaneous patterns of calcium activity between pairs of neurons were increased under anesthesia. While orientation selectivity remained unaffected by anesthesia, this treatment reduced direction selectivity, which was attributable to an increased response to the null-direction. As compared to anesthesia, populations of V1 neurons coded more mutual information on opposite stimulus directions during wakefulness, whereas information on stimulus orientation differences was lower. Increases in correlations of calcium activity during visual stimulation were correlated with poorer population coding, which raised the hypothesis that the anesthesia-induced increase in correlations may be causal to degrading directional coding. Visual stimulation under anesthesia, however, decorrelated ongoing activity patterns to a level comparable to wakefulness. Because visual stimulation thus appears to ‘break’ the strength of pairwise correlations normally found in spontaneous activity under anesthesia, the changes in correlational structure cannot explain the awake-anesthesia difference in direction coding. The population-wide decrease in coding for stimulus direction thus occurs independently of anesthesia-induced increments in correlations of spontaneous activity.     

Abiotic and Microbiotic Factors Controlling Biofilm Formation by Thermophilic Sporeformers

One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) spore-forming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus, whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans, which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.     

The lactosylceramide binding specificity of Helicobacter pylori

The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.      

Molecular evolution of a Y-chromosomal repetitive sequence family in the genus Mus

A 522-base-long Y-chromosomal sequence was isolated from a BALB/c genomic library and was designated "BF046." It is repeated about 200 times in the male genome, and a difference was detected between the Mus musculus musculus and the M. m. domesticus type Y chromosomes. BF046- related sequences were present over the entire length of the Y chromosome as visualized by in situ hybridization. Southern blot analysis against DNAs isolated from eight species in the genus Mus showed that BF046-related sequences were amplified in the Y chromosomes of three closely related species: M. musculus, M. spicilegus, and M. spretus. To gain insight into the stability of the BF046 sequence family, we isolated 18 additional clones from these three mouse species and compared their sequences. The M. musculus sequences differed from the M. spicilegus and M. spretus sequences by two indels. The remaining parts of the sequences were very similar, but both parsimony and distance-based analytical methods divided the sequences into the same four subgroups, with each species having its own subgroup(s). Thus, the Y chromosomes of M. musculus, M. spicilegus, and M. spretus can be distinguished from one another.      

Production of tissue plasminogen activator (t-PA) in Aspergillus niger

A protease-deficient strain of Aspergillus niger has been used as a host for the production of human tissue plasminogen activator (t-PA). In defined medium, up to 0.07 mg t-PA (g biomass)(-1) was produced in batch and fed-batch cultures and production was increased two- to threefold in two-phase batch cultures in which additional glucose was provided as a single pulse at the end of the first batch growth phase. Production was increased [up to 1.9 mg t-PA (g biomass)(-1)] by the addition of soy peptone to the defined medium. The rate of t-PA production in batch cultures supplemented with soy peptone (0.2 to 0.6 mg t-PA L(-1) h(-1)) was comparable to rates observed previously in high-producing mammalian or insect cell cultures. In glucose-limited chemostat culture supplemented with soy peptone, t-PA was produced at a rate of 0.7 mg t-PA L(-1) h(-1). Expression of t-PA in A. niger resulted in increased expression of genes (bipA, pdiA, and cypB) involved in the unfolded protein response (UPR). However, when cypB was overexpressed in a t-PA-producing strain, t-PA production was not increased. The t-PA produced in A. niger was cleaved into two chains of similar molecular weight to two-chain human melanoma t-PA. The two chains appeared to be stable for at least 16 h in culture supernatant of the host strain. However, in general, <1% of the t-PA produced in A. niger was active, and active t-PA disappeared from the culture supernatant during the stationary phase of batch cultures, suggesting that the two-chain t-PA may have been incorrectly processed or that initial proteolytic cleavage occurred within the proteolytic domain of the protein. Total t-PA (detected by enzyme-linked immunoassay) also eventually disappeared from culture supernatants, confirming significant extracellular proteolytic activity, even though the host strain was protease-deficient.