Structure of beta-antithrombin and the effect of glycosylation on antithrombin's heparin affinity and activity

Antithrombin is a member of the serpin family of protease inhibitors and the major inhibitor of the blood coagulation cascade. It is unique amongst the serpins in that it circulates in a conformation that is inactive against its target proteases. Activation of antithrombin is brought about by a conformational change initiated upon binding heparin or heparan sulphate. Two isoforms exist in the circulation, alpha-antithrombin and beta-antithrombin, which differ in the amount of glycosylation present on the polypeptide chain; beta-antithrombin lacks the carbohydrate present at Asn135 in alpha-antithrombin. Of the two forms, beta-antithrombin has the higher affinity for heparin and thus functions as the major inhibitor in vivo even though it is the less abundant form. The reason for the differences in heparin affinity between the alpha and beta-forms have been shown to be due to the additional carbohydrate changing the rate of the conformational change. Here, we describe the most accurate structures of alpha-antithrombin and alpha-antithrombin+heparin pentasaccharide reported to date (2.6A and 2.9A resolution, respectively, both re-refinements using old data), and the structure of beta-antithrombin (2.6A resolution). The new structures have a remarkable degree of ordered carbohydrate and include parts of the antithrombin chain not modeled before. The structures have allowed a detailed comparison of the conformational differences between the three. They show that the structural basis of the lower affinity for heparin of alpha-antithrombin over beta-antithrombin is due to the conformational change that occurs upon heparin binding being sterically hindered by the presence of the additional bulky carbohydrate at Asn135.     

Keratinocyte growth factor induces expansion of murine peripheral CD4+Foxp3+ regulatory T cells and increases their thymic output

Keratinocyte growth factor (KGF) has been shown to reduce the incidence and severity of graft-versus-host disease by prevention of epithelial damage and by modulating alloreactivity. Since regulatory T cells (Treg) play a crucial role in immune modulation, we evaluated the effects of exogenous KGF on peripheral CD4(+)Foxp3(+) Treg and the generation of Treg in the thymus of normal mice. A 3-day course of KGF induced a rapid selective increase in the number of highly suppressive CD4(+)Foxp3(+) Treg. Blood Treg numbers remained elevated for >2 mo, but the frequency normalized after 2 wk due to a concomitant increase in CD4(+)Foxp3(-) T cells. Analysis of single joint TCR excision circles frequency and Ki-67 expression in peripheral blood Treg showed that the early selective increase of Treg was predominantly accounted for by peripheral expansion. Thymectomy before KGF administration did not affect the early selective increase of Treg but abrogated the late increase in CD4(+) T cell numbers, thereby showing its dependence on thymic output. Collectively, these results show that KGF induces an increase in blood CD4(+)Foxp3(+) Treg numbers via two independent mechanisms. First by selective peripheral expansion of Treg and thereafter by enhanced thymic output of newly developed Treg.     

Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction

Regeneration of the myocardium by transplantation of cardiomyocytes is an emerging therapeutic strategy. Human embryonic stem cells (HESC) form cardiomyocytes readily but until recently at low efficiency, so that preclinical studies on transplantation in animals are only just beginning. Here, we show the results of the first long-term (12 weeks) analysis of the fate of HESC-derived cardiomyocytes transplanted intramyocardially into healthy, immunocompromised (NOD-SCID) mice and in NOD-SCID mice that had undergone myocardial infarction (MI). Transplantation of mixed populations of differentiated HESC containing 20–25% cardiomyocytes in control mice resulted in rapid formation of grafts in which the cardiomyocytes became organized and matured over time and the noncardiomyocyte population was lost. Grafts also formed in mice that had undergone MI. Four weeks after transplantation and MI, this resulted in significant improvement in cardiac function measured by magnetic resonance imaging. However, at 12 weeks, this was not sustained despite graft survival. This suggested that graft size was still limiting despite maturation and organization of the transplanted cells. More generally, the results argued for requiring a minimum of 3 months follow-up in studies claiming to observe improved cardiac function, independent of whether HESC or other (adult) cell types are used for transplantation.     

A sensitive Tg assay or rhTSH stimulated Tg: what's the best in the long-term follow-up of patients with differentiated thyroid carcinoma?

Sensitivity of thyroglobulin (Tg) measurement in the follow-up of differentiated thyroid carcinoma (DTC) can be optimized by using a sensitive Tg assay and rhTSH stimulation. We evaluated the diagnostic yield of a sensitive Tg assay and rhTSH stimulated Tg in the detection of recurrences in the follow-up of DTC. Additionally the value of imaging techniques for the localization of recurrences was evaluated. We included 121 disease free patients in long-term follow-up for DTC (median 10 years, range 1-34). Tg during thyroid hormone suppression therapy (Tg-on) and rhTSH stimulated Tg were measured with a sensitive Tg assay. Patients with rhTSH stimulated Tg > or =1.0 ng/ml underwent imaging with neck ultrasound, FDG-PET and post therapy 131I WBS. Sensitive Tg measurement resulted in 3 patients with Tg-on > or =1.0 ng/ml, recurrence could be localized in 2 of them. RhTSH stimulation resulted in Tg > or =1.0 ng/ml in another 17 of 118 patients. Recurrence could be localized in only 1 additional patient (1 out of 118 patients). Recurrence was localized by neck ultrasound in 1 of 3, by FDG-PET in 2 of 3 and by post therapy 131I WBS in 2 of 3 patients. In the detection of recurrences in DTC, rhTSH stimulation had very limited additional value in comparison to Tg-on measurement with a sensitive Tg assay. We consider this too low to justify rhTSH stimulation in all patients during long-term follow up. Neck ultrasound, FDG-PET and post therapy 131I WBS showed complementary value in localization of disease, but were only positive in a small fracture of all procedures.     

More Malpighiales: Woods of Achariaceae and/or Salicaceae from the Deccan Intertrappean beds,India

The families Achariaceae and Salicaceae (Malpighiales) are characterized by wood anatomical ranges that partly overlap. Formerly these families were treated together in the polyphyletic Flacourtiaceae and a much more narrowly circumscribed Salicaceae. Here we attribute two recently collected fossil woods from the Deccan Intertrappean Beds to the clade that contain these two families, i.e., the Parietal Clade of the Malpighiales. The new genus Elioxylon shares features with several extant genera of Achariaceae and Salicaceae, but does not completely match with any of them. A new record of Hydnocarpoxylon indicum Bande & Khatri is a good match for extant Hydnocarpus Gaertn. (Achariaceae). Elioxylon and Hydnocarpoxylon share an absence of parenchyma, the presence of septate fibres and 1–3 seriate heterocellular rays with long uniseriate margins consistent with Achariaceae and Salicaceae. Elioxylon has mixed simple and scalariform perforations, whereas Hydnocarpxylon has exclusively scalariform perforations. Other Deccan fossils formerly attributed to “Flacourtiaceae” in the literature are critically discussed and mostly excluded from Achariaceae and Salicaceae. Elioxylon and Hydnocarpoxylon from the Maastrichtian ‐ Danian of India are the oldest fossil records of the Parietal Clade of the Malpighiales. With their occurrence on the Indian plate during its northward journey from Gondwana to Laurasia, these fossils provide further support for an ‘out‐of‐India’ hypothesis for Achariaceae and/or Salicaceae. “Baileyan trends” in vessel perforation plate and vessel grouping evolution are apparent in the phylogeny of the Parietal Clade.     

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta)

Diatoms are one of the most abundant and arguably the most species‐rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon‐rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single‐locus automated species delimitation methods. We compared the DNA‐based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.     

Comprehensive mass spectrometry‐guided phenotyping of plant specialized metabolites reveals metabolic diversity in the cosmopolitan plant family Rhamnaceae

Plants produce a myriad of specialized metabolites to overcome their sessile habit and combat biotic as well as abiotic stresses. Evolution has shaped the diversity of specialized metabolites, which then drives many other aspects of plant biodiversity. However, until recently, large‐scale studies investigating the diversity of specialized metabolites in an evolutionary context have been limited by the impossibility of identifying chemical structures of hundreds to thousands of compounds in a time‐feasible manner. Here we introduce a workflow for large‐scale, semi‐automated annotation of specialized metabolites and apply it to over 1000 metabolites of the cosmopolitan plant family Rhamnaceae. We enhance the putative annotation coverage dramatically, from 2.5% based on spectral library matches alone to 42.6% of total MS/MS molecular features, extending annotations from well‐known plant compound classes into dark plant metabolomics. To gain insights into substructural diversity within this plant family, we also extract patterns of co‐occurring fragments and neutral losses, so‐called Mass2Motifs, from the dataset; for example, only the Ziziphoid clade developed the triterpenoid biosynthetic pathway, whereas the Rhamnoid clade predominantly developed diversity in flavonoid glycosides, including 7‐O‐methyltransferase activity. Our workflow provides the foundations for the automated, high‐throughput chemical identification of massive metabolite spaces, and we expect it to revolutionize our understanding of plant chemoevolutionary mechanisms.     

Can diatom-based pollution indices be used for biomonitoring in South Africa? A case study of the Crocodile West and Marico water management area

The inclusion of diatoms into the current suite of biomonitoring tools in use in South Africa, as well as the use of European and other diatom indices in South Africa, and in particular the Crocodile and West Marico water management area, is discussed. The indices, when compared to chemical analyses, proved useful in providing an indication of the quality of the investigated waters. Several widely distributed diatom species were shown to have similar ecological tolerances in South Africa when compared to Europe. Although most of the diatoms encountered in the study were cosmopolitan, several possibly endemic species were recorded. The occurrence of endemic species, not included in existing diatom indices may lead to miscalculations of diatom indices. It is concluded that although diatom indices developed in Europe and elsewhere are useful at the present to indicate water quality, a diatom index unique to South Africa including endemic species will have to be formulated. Handling editor: D. Hamilton