Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in pathogenic fungi Magnaporthe grisea and Botryotinia fuckeliana

There is increasing evidence that in several fungi, rhamnose-containing glycans are involved in processes that affect host-pathogen interactions, including adhesion, recognition, virulence, and biofilm formation. Nevertheless, little is known about the pathways for the synthesis of these glycans. We show that rhamnose is present in glycans isolated from the rice pathogen Magnaporthe grisea and from the plant pathogen Botryotinia fuckeliana. We also provide evidence that these fungi produce UDP-rhamnose. This is in contrast to bacteria where dTDP-rhamnose is the activated form of this sugar. In bacteria, formation of dTDP-rhamnose requires three enzymes. Here, we demonstrate that in fungi only two genes are required for UDP-Rha synthesis. The first gene encodes a UDP-glucose-4,6-dehydratase that converts UDP-glucose to UDP-4-keto-6-deoxyglucose. The product was shown by time-resolved (1)H NMR spectroscopy to exist in solution predominantly as a hydrated form along with minor amounts of a keto form. The second gene encodes a bifunctional UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase that converts UDP-4-keto-6-deoxyglucose to UDP-rhamnose. Sugar composition analysis and gene expression studies at different stages of growth indicate that the synthesis of rhamnose-containing glycans is under tissue-specific regulation. Together, our results provide new insight into the formation of rhamnose-containing glycans during the fungal life cycle. The role of these glycans in the interactions between fungal pathogens and their hosts is discussed. Knowledge of the metabolic pathways involved in the formation of rhamnose-containing glycans may facilitate the development of drugs to combat fungal diseases in humans, as to the best of our knowledge mammals do not make these types of glycans.     

Primary xenografts of human prostate tissue as a model to study angiogenesis induced by reactive stroma

Characterization of the mechanism(s) of androgen-driven human angiogenesis could have significant implications for modeling new forms of anti-angiogenic therapies for CaP and for developing targeted adjuvant therapies to improve efficacy of androgen-deprivation therapy. However, models of angiogenesis by human endothelial cells localized within an intact human prostate tissue architecture are until now extremely limited. This report characterizes the burst of angiogenesis by endogenous human blood vessels in primary xenografts of fresh surgical specimens of benign prostate or prostate cancer (CaP) tissue that occurs between Days 6-14 after transplantation into SCID mice pre-implanted with testosterone pellets. The wave of human angiogenesis was preceded by androgen-mediated up-regulation of VEGF-A expression in the stromal compartment. The neo-vessel network anastomosed to the host mouse vascular system between Days 6-10 post-transplantation, the angiogenic response ceased by Day 15, and by Day 30 the vasculature had matured and stabilized, as indicated by a lack of leakage of serum components into the interstitial tissue space and by association of nascent endothelial cells with mural cells/pericytes. The angiogenic wave was concurrent with the appearance of a reactive stroma phenotype, as determined by staining for α-SMA, Vimentin, Tenascin, Calponin, Desmin and Masson's trichrome, but the reactive stroma phenotype appeared to be largely independent of androgen availability. Transplantation-induced angiogenesis by endogenous human endothelial cells present in primary xenografts of benign and malignant human prostate tissue was preceded by induction of androgen-driven expression of VEGF by the prostate stroma, and was concurrent with and the appearance of a reactive stroma phenotype. Androgen-modulated expression of VEGF-A appeared to be a causal regulator of angiogenesis, and possibly of stromal activation, in human prostate xenografts.     

Conformational states of a bacterial α2-macroglobulin resemble those of human complement C3

α(2) macroglobulins (α(2)Ms) are broad-spectrum protease inhibitors that play essential roles in the innate immune system of eukaryotic species. These large, multi-domain proteins are characterized by a broad-spectrum bait region and an internal thioester, which, upon cleavage, becomes covalently associated to the target protease, allowing its entrapment by a large conformational modification. Notably, α(2)Ms are part of a larger protein superfamily that includes proteins of the complement system, such as C3, a multi-domain macromolecule which is also characterized by an internal thioester-carrying domain and whose activation represents the pivotal step in the complement cascade. Recently, α(2)M/C3-like genes were identified in a large number of bacterial genomes, and the Escherichia coli α(2)M homolog (ECAM) was shown to be activated by proteases. In this work, we have structurally characterized ECAM by electron microscopy and small angle scattering (SAXS) techniques. ECAM is an elongated, flexible molecule with overall similarities to C3 in its inactive form; activation by methylamine, chymotrypsin, or elastase induces a conformational modification reminiscent of the one undergone by the transformation of C3 into its active form, C3b. In addition, the proposed C-terminus of ECAM displays high flexibility and different conformations, and could be the recognition site for partner macromolecules. This work sheds light on a potential bacterial defense mechanism that mimics structural rearrangements essential for activation of the complement cascade in eukaryotes, and represents a possible novel target for the development of antibacterials.     

Biomarkers of Good EULAR Response to the B Cell Depletion Therapy in All Seropositive Rheumatoid Arthritis Patients: Clues for the Pathogenesis

Objective

To find out whether a high number of auto-antibodies can increase the probability of a “good-EULAR response” and to identify the possible biomarkers of response in seropositive rheumatoid arthritis (RA) patients undergoing the B cell depletion therapy (BCDT).

Patients and Methods

One hundred and thirty-eight patients with long standing RA (LSRA), 75% non or poorly responsive to one or more TNFα blockers, all seropositive for at least one autoantibody (AAB) (RF-IgM, RF-IgA, RF-IgG, anti-MCV, ACPA-IgG, ACPA-IgA, ACPA-IgM) received one full course of BCDT. The major outcomes (moderate or good-EULAR response) were assessed after 6 months of therapy. The IL6 and BAFF levels were also determined.

Results

At a 6-month follow-up, 33 (23.9%) of the RA patients achieved a good EULAR response. Having up to 5-AABs positivity increased the chances for treatment response. After a logistic regression analysis, however, only 4 baseline factors arose as associated with a good-EULAR response: no steroid therapy (OR = 6.25), a lymphocyte count <1875/uL (OR = 10.74), a RF-IgG level >52.1 IU/ml (OR = 8.37) and BAFF levels <1011 pg/ml (OR = 7.38). When all the AABs, except for RF-IgM and ACPA-IgG, were left in the analysis, the two final predictors were no-steroid therapy and low lymphocyte count.

Discussion

The number of AABs increased the chances of being a “good-EULAR” responder. The only predictors, however, at the baseline of a good response in this seropositive cohort of RA patients were 2 simple variables – no steroids and lymphocyte count – and two laboratory assays – IgG-RF and BAFF.     

Human chorionic villus mesenchymal stromal cells reveal strong endothelial conversion properties

Chorion, amnion and villi are reservoirs of mesenchymal stromal cells (StC) and the hypothesis that StC from fetal tissues retain higher plasticity compared to adult StC has been suggested. Aimed at investigating this aspect, a series of in vitro experiments were performed with StC isolated from first trimester human chorionic villi (CVStC). CVStC were cultured in: (i) standard mesenchymal medium (MM) and (ii) AmniomaxII? (AM), specifically designed to grow amnion-derived cells in prenatal diagnostic procedures. Cells were then exposed to distinct differentiation treatments and distinguished according to morphology, immunophenotype and molecular markers. Human StC obtained from adult bone marrow (BMStC) were used as control. CVStC cultured either in MM or AM presented stromal morphology and immunophenotype, were negative for pluripotency factors (Nanog, Oct-4 and Sox-2), lacked detectable telomerase activity and retained high genomic stability. In AM, however, CVStC exhibited a faster proliferation rate compared to BMStC or CVStC kept in MM. During differentiation, CVStC were less efficient than BMStC in acquiring adipocytes and osteocytes features; the cardiomyogenic conversion occurred at low efficiency in both cell types. Remarkably, in the presence of pro-angiogenic factors, CVStC reprogrammed toward an endothelial-like phenotype at significantly higher efficiency than BMStC. This effect was particularly evident in CVStC expanded in AM. Mechanistically, the reduced CVStC expression of anti-angiogenic microRNA could support this process. The present study demonstrates that, despite of fetal origin, CVStC exhibit restricted plasticity, distinct from that of BMStC and predominantly directed toward the endothelial lineage.     

Interannual fluctuations in copepod abundance and contribution of small forms in the Drake Passage during austral summer

The relative importance of small forms of copepods has been historically underestimated by the traditional use of 200?C300-??m mesh nets. This work quantified the distribution and abundance of copepods, considering two size fractions (<300???m and >300???m), in superficial waters (9?m deep) of the Drake Passage and contributed to the knowledge of their interannual fluctuations among three summers. Four types of nauplii and eleven species of copepods at copepodite and adult stages were identified, with abundance values of up to 13 ind L^?1 and 28,300???g C m^?3. The <300-??m fraction, composed of Oithona similis, small cyclopoids and nauplii, dominated the copepod communities in the 3?years; it accounted for more than 77% of the total number and for between 40 and 63% of the total biomass. Changes in density and biomass values among the three cruises differed according to copepod size fraction and water mass; the >300-??m fraction showed no changes among the 3?years, both in Antarctic (density and biomass) and in Subantarctic waters (density), whereas the <300-??m fraction showed higher (density and biomass) values in 2001 both in Subantarctic and in Antarctic waters. Sea surface temperature and its anomaly accounted for the largest proportion of variability in copepod density and biomass, particularly for the <300-??m fraction.     

Screening of Amazonian plants from the Adolpho Ducke forest reserve, Manaus, state of Amazonas, Brazil, for antimicrobial activity

Tropical forests are species-rich reserves for the discovery and development of antimicrobial drugs. The aim of this work is to investigate the in vitro antimicrobial potential of Amazon plants found within the National Institute on Amazon Research's Adolpho Ducke forest reserve, located in Manaus, state of Amazonas, Brazil. 75 methanol, chloroform and water extracts representing 12 plant species were tested for antimicrobial activity towards strains of Mycobacterium smegmatis, Escherichia coli, Streptococcus sanguis, Streptococcus oralis, Staphylococcus aureus and Candida albicans using the gel-diffusion method. Active extracts were further evaluated to establish minimum inhibitory concentrations (MIC) and antimicrobial profiles using bioautography on normal-phase thin-layer chromatography plates. Diclinanona calycina presented extracts with good antimicrobial activity and S. oralis and M. smegmatis were the most sensitive bacteria. D. calycina and Lacmellea gracilis presented extracts with the lowest MIC (48.8 microg/ml). D. calycina methanol and chloroform leaf extracts presented the best overall antimicrobial activity. All test organisms were sensitive to D. calycina branch chloroform extract in the bioautography assay. This is the first evaluation of the biological activity of these plant species and significant in vitro antimicrobial activity was detected in extracts and components from two species, D. calycina and L. gracilis.