Co-expression and prognostic value of gross cystic disease fluid protein 15 and mammaglobin in primary breast cancer

Gross cystic disease fluid protein (GCDFP-15) and mammaglobin are both widely used and accepted markers for epithelia of breast origin. We aimed to evaluate their relation of expression on parallel whole tissue sections in primary breast cancer by immunohistochemistry and also to correlate it with clinico-pathological parameters including patient survival. Primary breast carcinomas from 165 patients with a mean clinical follow-up of 73 months were immunostained using commercially available antibodies against GCDFP-15 and mammaglobin. An immunoreactive score (IRS) was calculated based on the cytoplasmic staining intensity and the number of cells stained. Cytoplasmic expression of GCDFP-15 and mammaglobin was observed in 73.3% and 72.1% of invasive breast carcinomas respectively. 91.8% of breast cancer cases expressed at least one of both markers. Both markers strongly correlated with each other and were significantly associated with lower tumour grading. Additionally, GCDFP-15 negativity was significantly associated with shortened disease-free survival times in univariate and multivariate analyses. We demonstrated the strong correlation of GCDFP-15 and mammaglobin with each other and showed that only very few primary breast cancers are completely negative for both markers. The significantly longer disease free survival times for patients with GCDFP-15 positive tumours clearly warrants further study.     

An agr quorum sensing system that regulates granulose formation and sporulation in Clostridium acetobutylicum

The Gram-positive, anaerobic, endospore-forming bacterium Clostridium acetobutylicum has considerable biotechnological potential due to its ability to produce solvents as fermentation products, in particular the biofuel butanol. Its genome contains a putative agr locus, agrBDCA, known in staphylococci to constitute a cyclic peptide-based quorum sensing system. In staphylococci, agrBD is required for the generation of a peptide signal that, upon extracellular accumulation, is sensed by an agrCA-encoded two-component system. Using ClosTron technology, agrB, agrC, and agrA mutants of C. acetobutylicum ATCC 824 were generated and phenotypically characterized. Mutants and wild type displayed similar growth kinetics and no apparent differences in solvent formation under the conditions tested. However, the number of heat-resistant endospores formed by the mutants in liquid culture was reduced by about one order of magnitude. On agar-solidified medium, spore formation was more strongly affected, particularly in agrA and agrC mutants. Similarly, accumulation of the starch-like storage compound granulose was almost undetectable in colonies of agrB, agrA, and agrC mutants. Importantly, these defects could be genetically complemented, demonstrating that they were directly linked to agr inactivation. A diffusible factor produced by agrBD-expressing strains was found to restore granulose and spore formation in the agrB mutant. Furthermore, a synthetic cyclic peptide, designed on the basis of the C. acetobutylicum AgrD sequence, was also capable of complementing the defects of the agrB mutant when added exogenously to the culture. Together, these findings support the hypothesis that agr-dependent quorum sensing is involved in the regulation of sporulation and granulose formation in C. acetobutylicum.     

Helicobacter pylori FlhB function: the FlhB C-terminal homologue HP1575 acts as a "spare part" to permit flagellar export when the HP0770 FlhBCC domain is deleted

In Helicobacter pylori 26695, a gene annotated HP1575 encodes a putative protein of unknown function which shows significant similarity to part of the C-terminal domain of the flagellar export protein FlhB. In Salmonella enterica, this part (FlhB(CC)) is proteolytically cleaved from the full-length FlhB, a processing event that is required for flagellar protein export and, thus, motility. The role of FlhB (HP0770) and its C-terminal homologue HP1575 was studied in H. pylori using a range of nonpolar deletion mutants defective in HP1575, HP0770, and the CC domain of HP0770 (HP0770(CC)). Deletion of HP0770 abolished swimming motility, whereas mutants carrying a deletion of either HP1575 or HP0770(CC) retained their ability to swim. An H. pylori strain containing deletions in both HP1575 and HP0770(CC) was nonmotile and did not produce flagella, suggesting that at least one of the two proteins had to be present for flagellar assembly to occur. Indeed, motility was restored when HP1575 was reintroduced into this strain immediately downstream of, but not fused to, the truncated HP0770 gene. Thus, HP1575 can functionally replace HP0770(CC) in this background. Like FlhB in S. enterica, HP0770 appeared to be proteolytically processed at a conserved NPTH processing site. However, mutation of the proline contained within the NPTH site of HP0770 did not affect motility and flagellar assembly, although it clearly interfered with processing when the protein was heterologously produced in Escherichia coli.     

Liquid chromatographic method for the determination of uridine in human serum

A validated gas chromatography (GC)-mass spectrometric (MS) method for the analysis of hydroxyproline in rat femur is reported. Hydroxyproline in bone hydrolysates was extracted with an anion exchange resin and the N(O)-tert-butyldimethylsilyl derivatives analyzed by GC-MS. The hydroxyproline concentration was estimated relative to pipecolic acid, 3,4-dehydroproline and n-tetracosane as internal standards. The mass-to-charge ratios (m/z) for the ions used for quantitation by single ion monitoring were 314 m/z for hydroxyproline, 198 m/z for pipecolic acid, 256 m/z for dehydroproline and 57 m/z for n-tetracosane. A coefficient of variation of 5.8% was achieved and the limit of detection was calculated to be 0.233 micromol/l bone hydrolysate.     

AgNOR analysis of atypical ductal hyperplasia and intraductal carcinoma of the breast

OBJECTIVE: To determine the diagnostic and prognostic value of argyrophilic nucleolar organizer regions (AgNORs) in atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and microinvasive ductal carcinoma (MDCA) of the breast. STUDY DESIGN: Image analysis of histologic sections from biopsies of 46 breast ADH and DCIS and 18 cases of MDCA. Determination of morphometric features of cell nuclei and nucleolar organizer regions by using AMBA software system. Data were compared with the estrogen receptor/progesterone receptor (ER/PR) content as well as with the growth fraction, determined immunohistochemically. RESULTS: AgNOR number and total AgNOR area increased from ADH to DCIS. The highest values were recorded in cases of DCIS with microinvasion. Differences between ADH and intraductal or microinvasive ductal carcinoma were statistically significant. Within the group of intraductal carcinomas, the lowest values were measured in the solid type and highest values in the comedo type. A correlation was found between AgNOR features and growth fraction but not between these features and ER/PR status. CONCLUSION: Selected AgNOR features are relevant for differentiation between ADH and DCIS as well as between low and high grade DCIS and microinvasive ductal carcinoma. Therefore, objective and reproducible data obtained by AgNOR analysis may allow better evaluation of the prognostic significance of these lesions.     

Bioprocess development for endospore production by Bacillus coagulans using an optimized chemically defined medium

Bacillus coagulans is a promising probiotic, because it combines probiotic properties of Lactobacillus and the ability of Bacillus to form endospores. Due to this hybrid relationship, cultivation of this organism is challenging. As the probiotics market continues to grow, there is a new focus on the production of these microorganisms. In this work, a strain-specific bioprocess for B. coagulans was developed to support growth on one hand and ensure sporulation on the other hand. This circumstance is not trivial, since these two metabolic states are contrary. The developed bioprocess uses a modified chemically defined medium which was further investigated in a one-factor-at-a-time assay after adaptation. A transfer from the shake flask to the bioreactor was successfully demonstrated in the scope of this work. The investigated process parameters included temperature, agitation and pH-control. Especially the pH-control improved the sporulation in the bioreactor when compared to shake flasks. The bioprocess resulted in a sporulation efficiency of 80%–90%. This corresponds to a sevenfold increase in sporulation efficiency due to a transfer to the bioreactor with pH-control. Additionally, a design of experiment (DoE) was conducted to test the robustness of the bioprocess. This experiment validated the beforementioned sporulation efficiency for the developed bioprocess. Afterwards the bioprocess was then scaled up from a 1 L scale to a 10 L bioreactor scale. A comparable sporulation efficiency of 80% as in the small scale was achieved. The developed bioprocess facilitates the upscaling and application to an industrial scale, and can thus help meet the increasing market for probiotics.     

Look who's talking: communication and quorum sensing in the bacterial world

For many years bacteria were considered primarily as autonomous unicellular organisms with little capacity for collective behaviour. However, we now appreciate that bacterial cells are in fact, highly communicative. The generic term 'quorum sensing' has been adopted to describe the bacterial cell-to-cell communication mechanisms which co-ordinate gene expression usually, but not always, when the population has reached a high cell density. Quorum sensing depends on the synthesis of small molecules (often referred to as pheromones or autoinducers) that diffuse in and out of bacterial cells. As the bacterial population density increases, so does the synthesis of quorum sensing signal molecules, and consequently, their concentration in the external environment rises. Once a critical threshold concentration has been reached, a target sensor kinase or response regulator is activated (or repressed) so facilitating the expression of quorum sensing-dependent genes. Quorum sensing enables a bacterial population to mount a co-operative response that improves access to nutrients or specific environmental niches, promotes collective defence against other competitor prokaryotes or eukaryotic defence mechanisms and facilitates survival through differentiation into morphological forms better able to combat environmental threats. Quorum sensing also crosses the prokaryotic-eukaryotic boundary since quorum sensing-dependent signalling can be exploited or inactivated by both plants and mammals.