Influence of muskmelon cell wall polysaccharides on roridin E production by a pathogenic strain of Myrothecium roridum

Addition of fruit cell wall extracts from two muskmelon cultivars into liquid media affected mycotoxin production by a strain of Myrothecium roridum pathogenic to muskmelon. Cell wall extracts from a susceptible cultivar (Iroquois) significantly increased toxin production while cell wall extracts from a resistant cultivar (Hales Best) significantly inhibited toxin production. Media containing 0.1 or 1.0 mg ml^–1 stimulated toxin production more than media containing 10 or 100 mg ml^–1 of cell wall extracts. Previous studies in our laboratory suggest that roridin E may be involved in virulence or pathogenicity of M. roridum; the present study indicates that cell wall polysaccharides as well as other materials present in cell wall preparations from susceptible host tissue provide a better substrate for toxin production than cell wall preparation from resistant host tissue.     

Social Behavioral Deficits Coincide with the Onset of Seizure Susceptibility in Mice Lacking Serotonin Receptor 2c

The development of social behavior is strongly influenced by the serotonin system. Serotonin 2c receptor (5-HT_2cR) is particularly interesting in this context considering that pharmacological modulation of 5-HT_2cR activity alters social interaction in adult rodents. However, the role of 5-HT_2cR in the development of social behavior is unexplored. Here we address this using Htr2c knockout mice, which lack 5-HT_2cR. We found that these animals exhibit social behavior deficits as adults but not as juveniles. Moreover, we found that the age of onset of these deficits displays similar timing as the onset of susceptibility to spontaneous death and audiogenic-seizures, consistent with the hypothesis that imbalanced excitation and inhibition (E/I) may contribute to social behavioral deficits. Given that autism spectrum disorder (ASD) features social behavioral deficits and is often co-morbid with epilepsy, and given that 5-HT_2cR physically interacts with Pten, we tested whether a second site mutation in the ASD risk gene Pten can modify these phenotypes. The age of spontaneous death is accelerated in mice double mutant for Pten and Htr2c relative to single mutants. We hypothesized that pharmacological antagonism of 5-HT_2cR activity in adult animals, which does not cause seizures, might modify social behavioral deficits in Pten haploinsufficient mice. SB 242084, a 5-HT_2cR selective antagonist, can reverse the social behavior deficits observed in Pten haploinsufficient mice. Together, these results elucidate a role of 5-HT_2cR in the modulation of social behavior and seizure susceptibility in the context of normal development and Pten haploinsufficiency.     

Cerebral nitric oxide represses choroid plexus NFκB‐dependent gateway activity for leukocyte trafficking

Chronic neuroinflammation is evident in brain aging and neurodegenerative disorders and is often associated with excessive nitric oxide (NO) production within the central nervous system (CNS). Under such conditions, increased NO levels are observed at the choroid plexus (CP), an epithelial layer that forms the blood–cerebrospinal fluid barrier (BCSFB) and serves as a selective gateway for leukocyte entry to the CNS in homeostasis and following injury. Here, we hypothesized that elevated cerebral NO levels interfere with CP gateway activity. We found that induction of leukocyte trafficking determinants by the CP and sequential leukocyte entry to the CSF are dependent on the CP epithelial NFκB/p65 signaling pathway, which was inhibited upon exposure to NO. Examining the CP in 5XFAD transgenic mouse model of Alzheimer''s disease (AD-Tg) revealed impaired ability to mount an NFκB/p65-dependent response. Systemic administration of an NO scavenger in AD-Tg mice alleviated NFκB/p65 suppression at the CP and augmented its gateway activity. Together, our findings identify cerebral NO as a negative regulator of CP gateway activity for immune cell trafficking to the CNS.     

Structural insights into FRS2α PTB domain recognition by neurotrophin receptor TrkB

The fibroblast growth factor receptor (FGFR) substrate 2 (FRS2) family proteins function as scaffolding adapters for receptor tyrosine kinases (RTKs). The FRS2α proteins interact with RTKs through the phosphotyrosine‐binding (PTB) domain and transfer signals from the activated receptors to downstream effector proteins. Here, we report the nuclear magnetic resonance structure of the FRS2α PTB domain bound to phosphorylated TrkB. The structure reveals that the FRS2α‐PTB domain is comprised of two distinct but adjacent pockets for its mutually exclusive interaction with either nonphosphorylated juxtamembrane region of the FGFR, or tyrosine phosphorylated peptides TrkA and TrkB. The new structural insights suggest rational design of selective small molecules through targeting of the two conjunct pockets in the FRS2α PTB domain. Proteins 2014; 82:1534–1541. © 2014 Wiley Periodicals, Inc.     

Cell Wall Monosaccharide Composition and Muskmelon Resistance to Myrothecium roridum1

In vitro growth of Myrothecium roridum, a pathogen of muskmelon (Cucumis melo L.), on media supplemented with eight cell wall-related monosaccharides revealed that germination and germ tube elongation were enhanced in the presence of arabinose, galactose and glucose. Colony expansion of established mycelia of M. roridum was also enhanced by arabinose and glucose but inhibited by galactose, Non-cellulosic neutral sugar analysis of fruit cell walls from muskmelon cultivars resistant or susceptible to M. roridum revealed that susceptible cultivars had consistently higher arabinosyl, galactosyl and glucosyl residue content than resistant cultivars, while a net loss of galaciosyl and arabinosyl residues occurred in cell walls of fruits between 20- and 27-days post-anthesis. M. roridum germinated more rapidly on isolated fruit cell walls from susceptible than resistant cultivars, but no correlation was found between cultivar resistance to M. roridum and inhibitin of fungal colony expansion on cell walls. Although factors affecting spore germination and mycelial growth of M. roridum, in vitro and in vivo, may differ, any factor that increases cell wall polysaccharide hydrolysis may contribute to ability of M. roridum to become established in immature fruit of muskmelon.     

The solution structure of a gallium-substituted putidaredoxin mutant: GaPdx C85S

The Fe₂S₂ cluster of the ferredoxin putidaredoxin (Pdx) can be replaced by a single gallium ion, giving rise to a colorless, diamagnetic protein in which, apart from the metal binding site, the major structural features of the native ferredoxin are conserved. The solution structure of the C85S variant of gallium putidaredoxin (C85S GaPdx), in which a non-ligand cysteine is replaced by a serine, has been determined via multidimensional NMR methods using uniformly ¹⁵N,¹³C labeled samples of C85S GaPdx. Stereospecific assignments of leucine and valine methyl resonances were made using ¹³C,¹H HSQC spectra obtained with fractionally ¹³C-labeled samples, and backbone dihedral angle restraints were obtained using a combination of two-dimensional J-modulated ¹⁵N,¹H HSQC and three-dimensional (HN)CO(CO)NH experiments. A total of 1117 NOE-derived distance restraints were used in the calculations, including 454 short range ($i - j 3$), 456 long range (i - j 4) interresidue restraints and 207 non-trivial intraresidue restraints. 97 and 55 ₁ angular restraints were also included in the calculation of a family of 20 structures using a combined distance geometry-simulated annealing protocol. Most regions of the protein are well defined in the calculations, with an RMSD of 0.525 Å for backbone atoms excluding the metal binding loop (residues 34–48) and the last three C-terminal residues (residues 103–106). Where comparison is possible, these regions show an increase in dynamic behavior over the native protein, as does the loop containing residues 74–76. Structural and dynamic differences between native Pdx and GaPdx are discussed in relation to charge and packing of the metal binding site.     

The Pattern of Attrition from an Antiretroviral Treatment Program in Nigeria

Objective

To evaluate the rate and factors associated with attrition of patients receiving ART in tertiary and secondary hospitals in Nigeria.

Methods and Findings

We reviewed patient level data collected between 2007 and 2010 from 11 hospitals across Nigeria. Kaplan-Meier product-limit and Cox regression were used to determine probability of retention in care and risk factors for attrition respectively. Of 6,408 patients in the cohort, 3,839 (59.9%) were females, median age of study population was 33years (IQR: 27–40) and 4,415 (69%) were from secondary health facilities. The NRTI backbone was Stavudine (D4T) in 3708 (57.9%) and Zidovudine (ZDV) in 2613 (40.8%) of patients. Patients lost to follow up accounted for 62.7% of all attrition followed by treatment stops (25.3%) and deaths (12.0%). Attrition was 14.1 (N = 624) and 15.1% (N = 300) in secondary and tertiary hospitals respectively (p = 0.169) in the first 12 months on follow up. During the 13 to 24 months follow up period, attrition was 10.7% (N = 407) and 19.6% (N = 332) in secondary and tertiary facilities respectively (p<0.001). Median time to lost to follow up was 11.1 (IQR: 6.1 to 18.5) months in secondary compared with 13.6 (IQR: 9.9 to 17.0) months in tertiary sites (p = 0.002). At 24 months follow up, male gender [AHR 1.18, 95% CI: 1.01–1.37, P = 0.038]; WHO clinical stage III [AHR 1.30, 95%CI: 1.03–1.66, P = 0.03] and clinical stage IV [AHR 1.90, 95%CI: 1.20–3.02, p = 0.007] and care in a tertiary hospital [AHR 2.21, 95% CI: 1.83–2.67, p<0.001], were associated with attrition.

Conclusion

Attrition could potentially be reduced by decentralizing patients on ART after the first 12 months on therapy to lower level facilities, earlier initiation on treatment and strengthening adherence counseling amongst males.     

Growth hormone-like activities of macrocyclic trichothecenes in in vitro callus induction and growth of four Baccharis species

The ability of two plant-produced macrocyclic trichothecenes (baccharinoid B4 and roridin E) to induce callus growth of two trichothecene-producing Baccharis species (B. coridifolia and B. megapotamica) and two nontrichothecene-producing species (B. halimifolia and B. neglecta) was investigated. Roridin E had no effect in the induction of callus of B. coridifolia, a roridin-producing plant, but induced callus of nonroridin-producing plants (B. megapotamica, B. halimifolia, and B. neglecta). Baccharinoid B4 stimulated callus growth of B. megapotamica, a baccharinoid-producing plant, and inhibited growth of B. coridifolia, B. halimifolia, and B. neglecta callus tissues. The ability of roridin E to induce callus was most effective at concentrations of 10^–8 and 10^–6 M and when synergistically coupled with auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). The ability of baccharinoid B4 to stimulate callus growth appeared to increase with increased concentration in the culture medium. Analysis of callus cultures grown in medium amended with roridin E showed that B4, roridin E, and 8-hydroxyroridin E and verrucarols were formed in the tissues but not in the medium. The results of this study indicated that while the callus-inducing ability of roridin E seemed to be nonspecies-specific in nature, the ability of B4 to stimulate callus was a highly species-specific phenomena. Callus-inducing activity of roridin E may depend on the capacity of plant species to transform exogenous roridin E into baccharinoids or other macrocyclic trichothecene derivatives.     

Role of the armadillo Dasypus novemcinctus in the epidemiology of paracoccidioidomycosis

A study conducted in a rural area of Ibiá, State of Minas Gerais, Brazil, where Paracoccidioides brasiliensis was recently isolated from soil, sought to determine if the armadillo Dasypus novemcinctus developed paracoccidioidomycosis. Out of 21 armadillos captured in the area, one had a lung granuloma containing fungal cells attributable to those of P. brasiliensis. The present report presents the first histopathological evidence for the presence of this pathogen in the lungs of an armadillo. This finding permitted us to establish a linkage between the two species and to suggest that fungal infections of humans and of armadillos occur in the same agricultural and forest microenvironment that they share. This revised version was published online in August 2006 with corrections to the Cover Date.     

A refined model for the solution structure of oxidized putidaredoxin

A refined model for the solution structure of oxidized putidaredoxin (Pdxo), a Cys4Fe2S2 ferredoxin, has been determined. A previous structure (Pochapsky et al. (1994) Biochemistry 33, 6424-6432; PDB entry ) was calculated using the results of homonuclear two-dimensional NMR experiments. New data has made it possible to calculate a refinement of the original Pdxo solution structure. First, essentially complete assignments for diamagnetic 15N and 13C resonances of Pdxo have been made using multidimensional NMR methods, and 15N- and 13C-resolved NOESY experiments have permitted the identification of many new NOE restraints for structural calculations. Stereospecific assignments for leucine and valine CH3 resonances were made using biosynthetically directed fractional 13C labeling, improving the precision of NOE restraints involving these residues. Backbone dihedral angle restraints have been obtained using a combination of two-dimensional J-modulated 15N,1H HSQC and 3D (HN)CO(CO)NH experiments. Second, the solution structure of a diamagnetic form of Pdx, that of the C85S variant of gallium putidaredoxin, in which a nonligand Cys is replaced by Ser, has been determined (Pochapsky et al. (1998) J. Biomol. NMR 12, 407-415), providing information concerning structural features not observable in the native ferredoxin due to paramagnetism. Third, a crystal structure of a closely related ferredoxin, bovine adrenodoxin, has been published (Müller et al. (1998) Structure 6, 269-280). This structure has been used to model the metal binding site structure in Pdx. A family of fourteen structures is presented that exhibits an rmsd of 0.51 A for backbone heavy atoms and 0.83 A for all heavy atoms. Exclusion of the modeled metal binding loop region reduces overall the rmsd to 0.30 A for backbone atoms and 0.71 A for all heavy atoms.