Reproductive success of wild Lesser Rheas (Pterocnemia -Rhea- pennata pennata) in north-western Patagonia, Argentina

We studied the reproductive success of a wild Lesser Rhea population (Pterocnemia -Rhea- pennata pennata) during two reproductive seasons (2004/2005 and 2005/2006) in north-western Patagonia, Argentina. The parameters recorded included population and nest density, clutch size, hatching success, chick survival (up to 3 months of age) and percentage of chicks that reached the juvenile stage after the winter. We also estimated the percentage of males that attempted to nest and of those that were successful (those producing at least one chick), daily nest mortality rates (DNMR) at different stages of the nesting cycle and the probability that an egg that has been recently laid will produce a chick. On average, both years pooled, the density of this population of Lesser Rheas was 1.55 ± 0.2 individuals/km² (SE), nest density was 0.17 ± 0.04 per km ² , clutch size was 20.8 ± 6.4 eggs, hatching success was 74.4% ± 11.3, Mayfield’s probability of an egg that will produce a chick was 0.64, chick survival was 65.4% ± 14.5 and percentage of chicks that reached the juvenile stage was 26.3%. Nearly a quarter of Lesser Rhea males in the population attempted to nest during a breeding season, and the DNMR was significantly higher during the laying stage (most nest failures were due to anthropogenic disturbances related to livestock raising activities). Nesting success, hatching success, and chick survival of Lesser Rheas were higher than those of their most closely related species, the Greater Rhea (Rhea americana), whereas the percentage of chicks that reached the juvenile stage was similar due to high winter mortalities of chicks. We suggest that the increase in reproductive effort is a strategy of this species to overcome environmental constraints.     

Effect of Dissemination of 2,4-Dichlorophenoxyacetic Acid (2,4-D) Degradation Plasmids on 2,4-D Degradation and on Bacterial Community Structure in Two Different Soil Horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10⁵ CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10⁵ CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.     

Conformation of pseudoazurin in the 152 kDa electron transfer complex with nitrite reductase determined by paramagnetic NMR

Copper-containing nitrite reductase is able to catalyze the reduction of nitrite with a turnover rate of several hundreds per second. Electrons for the reaction are donated by the electron transfer protein pseudoazurin. The process of protein complex formation, electron transfer and dissociation must occur on the millisecond timescale to enable the fast turnover of the enzyme. The structure of this transient protein complex has been studied using paramagnetic NMR spectroscopy. Gadolinium complexes were attached specifically through two engineered Cys residues on three sites on the surface of nitrite reductase, causing strong distance-dependent relaxation effects on the residues of pseudoazurin. Docking of the two proteins based on these NMR-derived distance restraints and the chemical shift perturbation data shows convergence to a cluster of structures with an average root-mean-square deviation of 1.5 Å. The binding interface consists of polar and non-polar residues surrounded by charges. The interprotein distance between the two type-1 copper sites is 15.5(± 0.5) Å, enabling fast interprotein electron transfer. The NMR-based lower limit estimate of 600 s⁻¹ for the dissociation rate constant and the fast electron transfer are consistent with the transient nature of the complex.     

Synthesis and structure-activity relationship of 7-azaindole piperidine derivatives as CCR2 antagonists

The synthesis and structure-activity relationship of a series of 7-azaindole piperidine derivatives are described. SAR studies led to the discovery of the potent CCR2 antagonists displaying IC(50) values in the nanomolar range. The representative compound 15 showed reasonable P450 and pharmacokinetics profile.     

Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells

We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 μM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (Δ⁴Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 μM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to Δ⁴Ad. In either cell line, T transformation to Δ⁴Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.     

A new species of Taeniastrotos Cressey (Copepoda: Poecilostomatoida) from southern Brazil

A new species of Taeniastrotos is described from an ariid host, Cathrops spixii, caught in southern Brazil. It is the first member of the genus to be recorded from the Atlantic Ocean and can be distinguished from its four known congeners by the setation of the terminal endopodal segment of the fourth leg.     

First Administration of the Fc-Attenuated Anti-β Amyloid Antibody GSK933776 to Patients with Mild Alzheimer’s Disease: A Randomized,Placebo-Controlled Study

Objective

To assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of the Fc-inactivated anti-β amyloid (Aβ) monoclonal antibody (mAb) GSK933776 in patients with mild Alzheimer’s disease (AD) or mild cognitive impairment (MCI).

Methods

This was a two-part, single blind, placebo-controlled, first-time-in-human (FTIH) study of single (n = 18) and repeat dose (n = 32) intravenous GSK933776 0.001–6 mg/kg (ClinicalTrials.gov: NCT00459550). Additional safety data from an open-label, uncontrolled, single dose study of intravenous GSK933776 1–6 mg/kg (n = 18) are included (ClinicalTrials.gov: NCT01424436).

Results

There were no cases of amyloid-related imaging abnormalities-edema (ARIA-E) or –hemorrhage (ARIA-H) after GSK933776 administration in both studies. Three patients across the two studies developed anti-GSK933776 antibodies. Plasma GSK933776 half-life (t_1/2) was 10–15 days after repeat dosing. After each of three administrations of GSK933776, plasma levels of total Aβ42 and Aβ increased whereas plasma levels of free Aβ decreased dose dependently; no changes were observed for placebo. For total Aβ42 the peak:trough ratio was ≤2 at doses ≥3 mg/kg; for total Aβ the ratio was ≤2 at 6 mg/kg. CSF concentrations of Aβ showed increases from baseline to week 12 for Aβ X–38 (week 12:baseline ratio: 1.65; 95%CI: 1.38, 1.93) and Aβ X–42 (week 12:baseline ratio: 1.18; 95%CI: 1.06, 1.30) for values pooled across doses.

Conclusion

In this FTIH study the Fc-inactivated anti-Aβ mAb GSK933776 engaged its target in plasma and CSF without causing brain ARIA-E/H in patients with mild AD or MCI.

Trial Registration

ClinicalTrials.gov NCT00459550     

IL-21 counteracts the regulatory T cell-mediated suppression of human CD4+ T lymphocytes

High expression of IL-21 and/or IL-21R has been described in T cell-mediated inflammatory diseases characterized by defects of counterregulatory mechanisms. CD4(+)CD25(+) regulatory T cells (Treg) are a T cell subset involved in the control of the immune responses. A diminished ability of these cells to inhibit T cell activation has been documented in immune-inflammatory diseases, raising the possibility that inflammatory stimuli can block the regulatory properties of Treg. We therefore examined whether IL-21 controls CD4(+)CD25(+) T cell function. We demonstrate in this study that IL-21 markedly enhances the proliferation of human CD4(+)CD25(-) T cells and counteracts the suppressive activities of CD4(+)CD25(+) T cells on CD4(+)CD25(-) T cells without affecting the percentage of Foxp3(+) cells or survival of Treg. Additionally, CD4(+)CD25(+) T cells induced in the presence of IL-21 maintain the ability to suppress alloresponses. Notably, IL-21 enhances the growth of CD8(+)CD25(-) T cells but does not revert the CD4(+)CD25(+) T cell-mediated suppression of this cell type, indicating that IL-21 makes CD4(+) T cells resistant to suppression rather than inhibiting CD4(+)CD25(+) T cell activity. Finally, we show that IL-2, IL-7, and IL-15, but not IL-21, reverse the anergic phenotype of CD4(+)CD25(+) T cells. Data indicate that IL-21 renders human CD4(+)CD25(-) T cells resistant to Treg-mediated suppression and suggest a novel mechanism by which IL-21 could augment T cell-activated responses in human immune-inflammatory diseases.     

Increased kynurenine-to-tryptophan ratio in the serum of patients infected with SARS-CoV2: An observational cohort study.

Immune dysregulation is a hallmark of patients infected by SARS-CoV2 and the balance between immune reactivity and tolerance is a key determinant of all stages of infection, including the excessive inflammatory state causing the acute respiratory distress syndrome. The kynurenine pathway (KP) of tryptophan (Trp) metabolism is activated by pro-inflammatory cytokines and drives mechanisms of immune tolerance. We examined the state of activation of the KP by measuring the Kyn:Trp ratio in the serum of healthy subjects (n = 239), and SARS-CoV2-negative (n = 305) and -positive patients (n = 89). Patients were recruited at the Emergency Room of St. Andrea Hospital (Rome, Italy). Kyn and Trp serum levels were assessed by HPLC/MS-MS. Compared to healthy controls, both SARS-CoV2-negative and -positive patients showed an increase in the Kyn:Trp ratio. The increase was larger in SARS-CoV2-positive patients, with a significant difference between SARS-CoV2-positive and -negative patients. In addition, the increase was more prominent in males, and positively correlated with age and severity of SARS-CoV2 infection, categorized as follows: 1 = no need for intensive care unit (ICU); 2 ≤ 3 weeks spent in ICU; 3 ≥ 3 weeks spent in ICU; and 4 = death. The highest Kyn:Trp values were found in SARS-CoV2-positive patients with severe lymphopenia. These findings suggest that the Kyn:Trp ratio reflects the level of inflammation associated with SARS-CoV2 infection, and, therefore, might represent a valuable biomarker for therapeutic intervention.     

A Novel Spore Protein,ExsM, Regulates Formation of the Exosporium in Bacillus cereus and Bacillus anthracis and Affects Spore Size and Shape

Bacillus cereus spores are assembled with a series of concentric layers that protect them from a wide range of environmental stresses. The outermost layer, or exosporium, is a bag-like structure that interacts with the environment and is composed of more than 20 proteins and glycoproteins. Here, we identified a new spore protein, ExsM, from a β-mercaptoethanol extract of B. cereus ATCC 4342 spores. Subcellular localization of an ExsM-green fluorescent protein (GFP) protein revealed a dynamic pattern of fluorescence that follows the site of formation of the exosporium around the forespore. Under scanning electron microscopy, exsM null mutant spores were smaller and rounder than wild-type spores, which had an extended exosporium (spore length for the wt, 2.40 ± 0.56 μm, versus that for the exsM mutant, 1.66 ± 0.38 μm [P < 0.001]). Thin-section electron microscopy revealed that exsM mutant spores were encased by a double-layer exosporium, both layers of which were composed of a basal layer and a hair-like nap. Mutant exsM spores were more resistant to lysozyme treatment and germinated with higher efficiency than wild-type spores, and they had a delay in outgrowth. Insertional mutagenesis of exsM in Bacillus anthracis ΔSterne resulted in a partial second exosporium and in smaller spores. In all, these findings suggest that ExsM plays a critical role in the formation of the exosporium.Bacillus cereus and Bacillus anthracis are closely related members of the Bacillus cereus group (47). Although B. cereus is mainly an apathogenic organism, certain isolates can cause two different types of food poisoning, emetic syndrome and diarrheal disease (18). The emetic syndrome is caused by ingestion of cereulide, a heat-resistant toxin produced by vegetative cells contaminating the food (30), while the diarrheal disease occurs when spores germinate in the intestinal tract. Spores are also the infective agent in anthrax, a disease caused by B. anthracis (64).B. cereus and B. anthracis differentiate into spores when faced with nutrient deprivation. The spore is a dormant cell type that can remain viable for decades until favorable conditions induce germination and the resumption of vegetative growth. The remarkable resistance properties of the spore result from its unique architecture, consisting of a series of concentric protective layers (51). The spore core contains the genetic material and is surrounded by the cortex, a thick layer of modified peptidoglycan that promotes a highly dehydrated state. Encasing the core and the cortex, the coat is a multilayer protein shell that provides mechanical and chemical resistance. In addition, both the cortex and coat contribute to spore germination (17). Separated from the coat by an interspace, the exosporium encloses the rest of the spore, and it is composed of an inner basal layer and an outer hair-like nap (25).Being the most external layer of the spore, the exosporium interacts directly with the environment and as such provides a semipermeable barrier that may exclude large molecules, like antibodies and hydrolytic enzymes (3, 23, 24, 54). However, the exosporium does not appear to contribute to the typical resistance properties of the spore (6, 35, 60). Also, the exosporium is not necessary in anthrax pathogenesis when tested under laboratory conditions (7, 27, 59), although it is able to down-modulate the innate immune response to spores and mediate adhesion to host tissues (4, 8, 43, 44). The exosporium may also help the spore avoid premature germination in unsustainable environments, since it contains two enzymes, alanine racemase (Alr) and inosine hydrolase (Iunh), that can inactivate low quantities of the germinants l-alanine and inosine, respectively (6, 48, 55, 61). However, regulation of germination by the exosporium is poorly understood. Mutation of exosporial proteins has resulted in only negligible and inconsistent germination phenotypes (2, 5, 27, 28, 52, 54).The exosporium is composed of at least 20 proteins and glycoproteins in tight or loose association (48, 53, 57, 61, 65). These proteins are synthesized in the mother cell and always start self-assembly at the forespore pole near the middle of the mother cell, concurrently with the cortex and coat formation (42). Exosporium assembly is discontinuous and starts with a synthesis of a substructure known as the cap, which likely contains only a subset of the proteins present in the exosporium (55). After cap formation, construction of the rest of the exosporium requires the expression of ExsY (6). BclA is the main component of the hair-like nap on the external side of the exosporium, and it is linked to the basal layer through interaction with ExsFA/BxpB (54, 58). In addition, CotE participates in the correct attachment of the exosporium to the spore (27).Despite these findings, exosporium assembly continues to be a poorly understood process, and many questions remain regarding its composition and the regulation of its synthesis. In this study, we characterized a new spore protein, ExsM, which plays a key role in assembly of the exosporium. In B. cereus, inactivation of exsM resulted in spores with an unusual double-layer exosporium, and a similar phenotype was also observed in B. anthracis exsM null mutant spores. Finally, double-layer exosporium spores allowed us to study the role of the exosporium in germination and outgrowth.