Crystal Structure of Two Anti-Porphyrin Antibodies with Peroxidase Activity

We report the crystal structures at 2.05 and 2.45 Å resolution of two antibodies, 13G10 and 14H7, directed against an iron(III)-αααβ-carboxyphenylporphyrin, which display some peroxidase activity. Although these two antibodies differ by only one amino acid in their variable λ-light chain and display 86% sequence identity in their variable heavy chain, their complementary determining regions (CDR) CDRH1 and CDRH3 adopt very different conformations. The presence of Met or Leu residues at positions preceding residue H101 in CDRH3 in 13G10 and 14H7, respectively, yields to shallow combining sites pockets with different shapes that are mainly hydrophobic. The hapten and other carboxyphenyl-derivatized iron(III)-porphyrins have been modeled in the active sites of both antibodies using protein ligand docking with the program GOLD. The hapten is maintained in the antibody pockets of 13G10 and 14H7 by a strong network of hydrogen bonds with two or three carboxylates of the carboxyphenyl substituents of the porphyrin, respectively, as well as numerous stacking and van der Waals interactions with the very hydrophobic CDRH3. However, no amino acid residue was found to chelate the iron. Modeling also allows us to rationalize the recognition of alternative porphyrinic cofactors by the 13G10 and 14H7 antibodies and the effect of imidazole binding on the peroxidase activity of the 13G10/porphyrin complexes.     

Do MCF7 cells cope with metformin treatment under energetic stress in low glucose conditions?

There is a growing body of evidence about metformin being effective in cancer therapy. Despite controversies about the ways of its effectiveness, several ongoing clinical trials are evaluating the drug when used as an adjuvant or a neo-adjuvant agent. We aimed to investigate metformin’s effects on proliferation, metastasis, and hormone receptor expressions in breast cancer cell line MCF-7 incubated in two different glucose conditions. MCF-7 cells were incubated in high or low glucose media and treated with various doses of metformin. The cell viability was studied using MTT test. The Ki-67, estrogen and progesterone receptor expression were evaluated by ICC and galectin-3 expression was evaluated by ELISA or spectrophotometrically. The cell viability following consecutive metformin doses in either glucose condition for 24 and 48 h represented a significant decrease when compared to control. The proliferation detected in low glucose medium following metformin at doses < 20 mM was found significantly decreased when compared to high glucose medium at 48 h. In terms of galectin-3 levels, the increase in high glucose medium treated with metformin and the decrease in low glucose medium were found statistically significant when compared to control. Progesterone receptor staining demonstrated a significant increase in low glucose medium. Our findings represent better outcomes for cancer lines incubated in low glucose medium treated with metformin in terms of viability, receptor expression and metastatic activity, and highlight the potential benefit of metformin especially in restraining the cancer cell’s ability to cope energetic stress in low glucose conditions.     

Separating Bedtime Rest from Activity Using Waist or Wrist-Worn Accelerometers in Youth

Recent interest in sedentary behavior and technological advances expanded use of watch-size accelerometers for continuous monitoring of physical activity (PA) over extended periods (e.g., 24 h/day for 1 week) in studies conducted in natural living environment. This approach necessitates the development of new methods separating bedtime rest and activity periods from the accelerometer recordings. The goal of this study was to develop a decision tree with acceptable accuracy for separating bedtime rest from activity in youth using accelerometer placed on waist or wrist. Minute-by-minute accelerometry data were collected from 81 youth (10–18 years old, 47 females) during a monitored 24-h stay in a whole-room indirect calorimeter equipped with a force platform covering the floor to detect movement. Receiver Operating Characteristic (ROC) curve analysis was used to determine the accelerometer cut points for rest and activity. To examine the classification differences, the accelerometer bedtime rest and activity classified by the algorithm in the development group (n = 41) were compared with actual bedtime rest and activity classification obtained from the room calorimeter-measured metabolic rate and movement data. The selected optimal bedtime rest cut points were 20 and 250 counts/min for the waist- and the wrist-worn accelerometer, respectively. The selected optimal activity cut points were 500 and 3,000 counts/min for waist and wrist-worn accelerometers, respectively. Bedtime rest and activity were correctly classified by the algorithm in the validation group (n = 40) by both waist- (sensitivity: 0.983, specificity: 0.946, area under ROC curve: 0. 872) and wrist-worn (0.999, 0.980 and 0.943) accelerometers. The decision tree classified bedtime rest correctly with higher accuracy than commonly used automated algorithm for both waist- and wrist-warn accelerometer (all p<0.001). We concluded that cut points developed and validated for waist- and wrist-worn uniaxial accelerometer have a good power for accurate separation of time spent in bedtime rest from activity in youth.     

An Integrated Understanding of the Rapid Metabolic Benefits of a Carbohydrate-Restricted Diet on Hepatic Steatosis in Humans

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Multiple Feedbacks Contribute to a Centennial Legacy of Reindeer on Tundra Vegetation

Historical contingency is the impact of past events, like the timing and order of species arrival, on community assembly, and can sometimes result in alternative stable states of ecological communities. Large herbivores, wild and domestic, can cause profound changes in the structure and functioning of plant communities and therefore probably influence historical contingency; however, little empirical data on the stability of such shifts or subsequent drivers of stability are available. We studied the centennial legacy of reindeer (Rangifer tarandus) pressure on arctic tundra vegetation by considering historical milking grounds (HMGs): graminoid- and forb-dominated patches amid shrub-dominated tundra, formed by historical Sami reindeer herding practices that ended approximately 100 years ago. Our results show that the core areas of all studied HMGs remained strikingly stable, being hardly invaded by surrounding shrubs. Soil nitrogen concentrations were comparable to heavily grazed areas. However, the HMGs are slowly being reinvaded by vegetative growth of shrubs at the edges, and the rate of ingrowth increased with higher mineral N availability. Furthermore, our data indicate that several biotic feedbacks contribute to the stability of the HMGs: increased nutrient turnover supporting herbaceous vegetation, strong interspecific competition preventing invasion and herbivore damage to invading shrubs. In particular, voles and lemmings appear to be important, selectively damaging shrubs in the HMGs. We concluded that HMGs provide clear evidence for historical contingency of herbivore effects in arctic ecosystems. We showed that several biotic feedbacks can contribute to subsequent vegetation stability, but their relative importance will vary in time and space.     

Evapotranspiration and water yield of a pine‐broadleaf forest are not altered by long‐term atmospheric [CO2] enrichment under native or enhanced soil fertility

Changes in evapotranspiration (ET) from terrestrial ecosystems affect their water yield (WY), with considerable ecological and economic consequences. Increases in surface runoff observed over the past century have been attributed to increasing atmospheric CO₂ concentrations resulting in reduced ET by terrestrial ecosystems. Here, we evaluate the water balance of a Pinus taeda (L.) forest with a broadleaf component that was exposed to atmospheric [CO₂] enrichment (ECO₂; +200 ppm) for over 17 years and fertilization for 6 years, monitored with hundreds of environmental and sap flux sensors on a half‐hourly basis. These measurements were synthesized using a one‐dimensional Richard's equation model to evaluate treatment differences in transpiration (T), evaporation (E), ET, and WY. We found that ECO₂ did not create significant differences in stand T, ET, or WY under either native or enhanced soil fertility, despite a 20% and 13% increase in leaf area index, respectively. While T, ET, and WY responded to fertilization, this response was weak (<3% of mean annual precipitation). Likewise, while E responded to ECO₂ in the first 7 years of the study, this effect was of negligible magnitude (<1% mean annual precipitation). Given the global range of conifers similar to P. taeda, our results imply that recent observations of increased global streamflow cannot be attributed to decreases in ET across all ecosystems, demonstrating a great need for model–data synthesis activities to incorporate our current understanding of terrestrial vegetation in global water cycle models.     

Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells

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Correlation between stand structure and ground vegetation: an analytical approach

The effect of stand structure on the diversity of the forest's ground vegetation was examined based on data on permanent sample plots collected in the northern parts of North-Carelia, eastern Finland. Different ordination methods (DCA, GNMDS, LNMDS, and HMDS) were used together with the TWINSPAN classification method. The aim was to construct a basis for classifying forests with respect to the biodiversity of the forest vegetation. Fertility and stand age showed the strongest correlation with the variation in ground vegetation. Other important factors were basal area, tree species composition, and crown cover. These variables were important in the division of the sample plots into different classes. According to the diversity indices, species diversity was at its highest in young stands on fertile forest sites. As a result, twenty-one different classes were formed based on the relative abundance of understorey species. The variables with the highest correlation were used to describe the stand structure in these classes.     

Structural basis for Myf and Psa fimbriae‐mediated tropism of pathogenic strains of Yersinia for host tissues

Three pathogenic species of the genus Yersinia assemble adhesive fimbriae via the FGL‐chaperone/usher pathway. Closely related Y. pestis and Y. pseudotuberculosis elaborate the pH6 antigen (Psa), which mediates bacterial attachment to alveolar cells of the lung. Y. enterocolitica, instead, assembles the homologous fimbriae Myf of unknown function. Here, we discovered that Myf, like Psa, specifically recognizes β1‐3– or β1‐4–linked galactose in glycosphingolipids, but completely lacks affinity for phosphatidylcholine, the main receptor for Psa in alveolar cells. The crystal structure of a subunit of Psa (PsaA) complexed with choline together with mutagenesis experiments revealed that PsaA has four phosphatidylcholine binding pockets that enable super‐high‐avidity binding of Psa‐fibres to cell membranes. The pockets are arranged as six tyrosine residues, which are all missing in the MyfA subunit of Myf. Conversely, the crystal structure of the MyfA‐galactose complex revealed that the galactose‐binding site is more extended in MyfA, enabling tighter binding to lactosyl moieties. Our results suggest that during evolution, Psa has acquired a tyrosine‐rich surface that enables it to bind to phosphatidylcholine and mediate adhesion of Y. pestis/pseudotuberculosis to alveolar cells, whereas Myf has specialized as a carbohydrate‐binding adhesin, facilitating the attachment of Y. enterocolitica to intestinal cells.