Integrating macrophages into organotypic co-cultures: a 3D in vitro model to study tumor-associated macrophages

Tumor progression is controlled by signals from cellular and extra-cellular microenvironment including stromal cells and the extracellular matrix. Consequently, three-dimensional in vitro tumor models are essential to study the interaction of tumor cells with their microenvironment appropriately in a biologically relevant manner. We have previously used organotypic co-cultures to analyze the malignant growth of human squamous cell carcinoma (SCC) cell lines on a stromal equivalent in vitro. In this model, SCC cell lines are grown on a collagen-I gel containing fibroblasts. Since macrophages play a critical role in the progression of many tumor types, we now have expanded this model by integrating macrophages into the collagen gel of these organotypic tumor co-cultures. This model was established as a murine and a human system of skin SCCs. The effect of macrophages on tumor progression depends on their polarization. We demonstrate that macrophage polarization in organotypic co-cultures can be modulated towards and M1 or an M2 phenotype by adding recombinant IFN-γ and LPS or IL-4 respectively to the growth medium. IL-4 stimulation of macrophage-containing cultures resulted in enhanced tumor cell invasion evidenced by degradation of the basement membrane, enhanced collagenolytic activity and increased MMP-2 and MMP-9. Interestingly, extended co-culture with tumor cells for three weeks resulted in spontaneous M2 polarization of macrophages without IL-4 treatment. Thus, we demonstrate that macrophages can be successfully integrated into organotypic co-cultures of murine or human skin SCCs and that this model can be exploited to analyze macrophage activation towards a tumor supporting phenotype.     

The crystal structure of the ttCsaA protein: an export-related chaperone from Thermus thermophilus

The CsaA protein was first characterized in Bacillus subtilis as a molecular chaperone with export-related activities. Here we report the 2.0 Angstrom-resolution crystal structure of the Thermus thermophilus CsaA protein, designated ttCsaA. Atomic structure and experiments in solution revealed a homodimer as the functional unit. The structure of the ttCsaA monomer is reminiscent of the well known oligonucleotide-binding fold, with the addition of extensions at the N- and C-termini that form an extensive dimer interface. The two identical, large, hydrophobic cavities on the protein surface are likely to constitute the substrate binding sites. The CsaA proteins share essential sequence similarity with the tRNA-binding protein Trbp111. Structure-based sequence analysis suggests a close structural resemblance between these proteins, which may extend to the architecture of the binding sites at the atomic level. These results raise the intriguing possibility that CsaA proteins possess a second, tRNA-binding activity in addition to their export-related function.     

Variation for neutral markers is correlated with variation for quantitative traits in the plant pathogenic fungus Mycosphaerella graminicola

We compared genetic variation and population differentiation at RFLP marker loci with seven quantitative characters including fungicide resistance, temperature sensitivity, pycnidial size, pycnidial density, colony size, percentage of leaves covered by pycnidia (PLACP) and percentage of leaves covered by lesions (PLACL) in Mycosphaerella graminicola populations sampled from four regions. Wide variation in population differentiation was found across the quantitative traits assayed. Fungicide resistance, temperature sensitivity, and PLACP displayed a significantly higher Q(ST) than G(ST), consistent with selection for local adaptation, while pycnidial size, pycnidial density and colony size displayed a lower or significantly lower Q(ST) than G(ST), consistent with constraining selection. There was not a statistical difference between Q(ST) and G(ST) in PLACL. We also found a positive and significant correlation between genetic variation in molecular marker loci and quantitative traits at the multitrait scale, suggesting that estimates of overall genetic variation for quantitative traits in M. graminicola could be derived from analysis of the molecular genetic markers.     

Non-collagenous proteins of predentine from dentinogenically active bovine teeth.

Predentin(e) was dissected out from unerupted permanent bovine teeth. The non-collagenous proteins were extracted at -13 degrees C by 4 M-guanidinium chloride containing proteinase inhibitors and separated by DEAE-Sepharose and Sephadex G-100 chromatography. In addition to a few minor constituents, the only major non-collagenous components that could be demonstrated were albumin and proteoglycan. The localization of the former, demonstrated by optical-microscopical immunochemistry, was such that it was concluded that albumin is not a constituent of predentin matrix. Very low amounts of phosphoprotein were found in predentin matrix. This was of two types, high- and low-phosphorylated. Larger amounts of phosphoprotein were not present until the dissection was carried deeper into newly formed dentin(e). On the basis of the present results and previously obtained morphological data the conclusion was drawn that predentin matrix, containing virtually only collagen type I and proteoglycan, is similar in composition to that of loose connective tissue and primarily aimed at the production and maturation of collagen fibres. Only immediately before the mineralization front are the non-collagenous protein components secreted that initiate and govern calcium-phosphate mineral formation.     

Testing the cranial evolutionary allometric ‘rule’ in Galliformes

Recent comparative studies have indicated the existence of a common cranial evolutionary allometric (CREA) pattern in mammals and birds, in which smaller species have relatively smaller faces and bigger braincases than larger species. In these studies, cranial allometry was tested using a multivariate regression between shape (described using landmarks coordinates) and size (i.e. centroid size), after accounting for phylogenetic relatedness. Alternatively, cranial allometry can be determined by comparing the sizes of two anatomical parts using a bivariate regression analysis. In this analysis, a slope higher or lower than one indicates the existence of positive or negative allometry, respectively. Thus, in those species that support the CREA ‘rule’, positive allometry is expected for the association between face size and braincase size, which would indicate that larger species have disproportionally larger faces. In this study, I applied these two approaches to explore cranial allometry in 83 Galliformes (Aves, Galloanserae), ranging in mean body weight from 30 g to 2.5 kg. The multivariate regression between shape and centroid size revealed the existence of a significant allometric pattern resembling CREA, whereas the second analysis revealed a negative allometry for beak size and braincase size (i.e. contrary to the CREA ‘rule’, larger galliform species have disproportionally shorter beaks than smaller galliform species). This study suggests that the presence of CREA may be overestimated when using cranium size as the standard measurement.     

Cryopreservation at −80°C of Agaricus blazei on rice grains

The preservation of Agaricus blazei is generally done by mycelial subculturing, but this technique may cause genetic degenerations. Despite this, there is not an efficient protocol established to preserve this fungus and cryopreservation could be an alternative. This study aimed to evaluate two freezing protocols for cryopreservation at −80°C of A. blazei strains. Five fungus strains grown on rice grains with husk and were transferred to glycerol (10%) in cryovials. Next, the cryovials were submitted to two freezing temperature protocols: (1) cryopreservation starting at 25°C, then at 8°C for 30 min and kept at −80°C; (2) cryopreservation starting at 25°C, then 8°C for 30 min, −196°C for 15 min and kept at −80°C. After 1 year of cryopreservation, the cryovials were thawed in a water bath at 30°C for 15 min and transferred to malt extract agar medium. It was concluded that the one-year cryopreservation process of A. blazei, grown on rice grains and cryopreserved at −80°C in glycerol 10%, is viable. The slow freezing, from 8 to −80°C, is effective whereas the fast freezing, from 8 to −196°C and then to −80°C, is ineffective. The different genetic characteristics among the strains of this fungus do not interfere in the cryopreservation process.     

Characterisation of preYvaY export reveals differences in the substrate specificities of Bacillus subtilis and Escherichia coli leader peptidases

Translocation, processing and secretion of YvaY, a Bacillus subtilis protein of unknown function, were characterised both in B. subtilis and in Escherichia coli. In its natural host B. subtilis, YvaY was transiently synthesised at the end of the exponential growth phase. It was efficiently secreted into the culture supernatant in spite of a calculated membrane spanning domain in the mature part of the protein. In E. coli, despite the high conservation of Sec-dependent transport components, processing of preYvaY was strongly impaired. To uncover which elements of E. coli and B. subtilis translocation systems are responsible for the observed substrate specificity, components of the B. subtilis Sec-system were co-expressed besides yvaY in E. coli. Expression of B. subtilis secA or secYEG genes did not affect processing, but expression of B. subtilis signal peptidase genes significantly enhanced processing of preYvaY in E. coli. While the major signal peptidases SipS or SipT had a strong stimulatory effect on preYvaY processing, the minor signal peptidases SipU, SipV or SipW had a far less stimulatory effect in E. coli. These results reveal that targeting and translocation of preYvaY is mediated by the E. coli Sec proteins but processing of preYvaY is not performed by E. coli signal peptidase LepB. Thus, differences in substrate specificities of E. coli LepB and the B. subtilis Sip proteins provide the bottleneck for export of YvaY in E. coli. Significant slower processing of preYvaY in absence of SecB indicated that SecB mediates targeting of the B. subtilis precursor.     

St John's wort for depression--an overview and meta-analysis of randomised clinical trials.

OBJECTIVE--To investigate if extracts of Hypericum perforatum (St John''s wort) are more effective than placebo in the treatment of depression, are as effective as standard antidepressive treatment, and have fewer side effects than standard antidepressant drugs. DESIGN--Systematic review and meta-analysis of trials revealed by searches. TRIALS--23 randomised trials including a total of 1757 outpatients with mainly mild or moderately severe depressive disorders: 15 (14 testing single preparations and one a combination with other plant extracts) were placebo controlled, and eight (six testing single preparations and two combinations) compared hypericum with another drug treatment. MAIN OUTCOME MEASURES--A pooled estimate of the responder rate ratio (responder rate in treatment group/responder rate in control group), and numbers of patients reporting and dropping out for side effects. RESULTS--Hypericum extracts were significantly superior to placebo (ratio = 2.67; 95% confidence interval 1.78 to 4.01) and similarly effective as standard antidepressants (single preparations 1.10; 0.93 to 1.31, combinations 1.52; 0.78 to 2.94). There were two (0.8%) drop outs for side effects with hypericum and seven (3.0%) with standard antidepressant drugs. Side effects occurred in 50 (19.8%) patients on hypericum and 84 (52.8%) patients on standard antidepressants. CONCLUSION--There is evidence that extracts of hypericum are more effective than placebo for the treatment of mild to moderately severe depressive disorders. Further studies comparing extracts with standard antidepressants in well defined groups of patients and comparing different extracts and doses are needed.     

Differential Use of Trails by Forest Mammals and the Implications for Camera-Trap Studies: A Case Study from Belize

Relative abundance indices are often used to compare species abundance between sites. The indices assume that species have similar detection probabilities, or that differences between detection probabilities are known and can be corrected for. Indices often consist of encounter frequencies of footprints, burrows, markings or photo captures along trails or transect lines, but the assumption of equal detection probabilities is rarely validated. This study analyzes detection probabilities of a range of Neotropical mammals on trails in dense secondary forests, using camera-trap and track data. Photo captures of the two large cats, jaguars ( Panthera onca ) and pumas ( Puma concolor ), were correlated solely with trail variables, while photo captures of their potential prey species had no correlation or negative correlation with trail variables. The Neotropical mammals varied greatly in their tendency to follow or cross trails based on footprints surveys. This indicates that camera locations on trails will have varying detection probability for these Neotropical mammals. Even the two similar-sized jaguars and pumas, occupying relatively similar niches, differed subtly in their use of trails. Pumas followed trails more completely while jaguars were more likely to deviate from trails. The ecological significance of these findings is that jaguars seem to be more willing to use the forest matrix away from trails than do pumas. We conclude that trail-based indices, such as photographic captures or tracks along trails, are not appropriate for comparison between Neotropical species, and not even between relatively similar species like jaguars and pumas.