Association Mapping Provides Insights into the Origin and the Fine Structure of the Sorghum Aluminum Tolerance Locus,AltSB

Root damage caused by aluminum (Al) toxicity is a major cause of grain yield reduction on acid soils, which are prevalent in tropical and subtropical regions of the world where food security is most tenuous. In sorghum, Al tolerance is conferred by SbMATE, an Al-activated root citrate efflux transporter that underlies the major Al tolerance locus, Alt_SB, on sorghum chromosome 3. We used association mapping to gain insights into the origin and evolution of Al tolerance in sorghum and to detect functional variants amenable to allele mining applications. Linkage disequilibrium across the Alt_SB locus decreased much faster than in previous reports in sorghum, and reached basal levels at approximately 1000 bp. Accordingly, intra-locus recombination events were found to be extensive. SNPs and indels highly associated with Al tolerance showed a narrow frequency range, between 0.06 and 0.1, suggesting a rather recent origin of Al tolerance mutations within Alt_SB. A haplotype network analysis suggested a single geographic and racial origin of causative mutations in primordial guinea domesticates in West Africa. Al tolerance assessment in accessions harboring recombinant haplotypes suggests that causative polymorphisms are localized to a ∼6 kb region including intronic polymorphisms and a transposon (MITE) insertion, whose size variation has been shown to be positively correlated with Al tolerance. The SNP with the strongest association signal, located in the second SbMATE intron, recovers 9 of the 14 highly Al tolerant accessions and 80% of all the Al tolerant and intermediately tolerant accessions in the association panel. Our results also demonstrate the pivotal importance of knowledge on the origin and evolution of Al tolerance mutations in molecular breeding applications. Allele mining strategies based on associated loci are expected to lead to the efficient identification, in diverse sorghum germplasm, of Al tolerant accessions able maintain grain yields under Al toxicity.     

Compositional and immunobiological analyses of extracellular vesicles released by Candida albicans

The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transport mechanism to trans‐cell wall passage of macromolecules. Previous studies have revealed the presence of EV in culture supernatants from fungal pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Malassezia sympodialis and Candida albicans. Here we investigated the size, composition, kinetics of internalization by bone marrow‐derived murine macrophages (MO) and dendritic cells (DC), and the immunomodulatory activity of C. albicans EV. We also evaluated the impact of EV on fungal virulence using the Galleria mellonella larvae model. By transmission electron microscopy and dynamic light scattering, we identified two populations ranging from 50 to 100 nm and 350 to 850 nm. Two predominant seroreactive proteins (27 kDa and 37 kDa) and a group of polydispersed mannoproteins were observed in EV by immunoblotting analysis. Proteomic analysis of C. albicans EV revealed proteins related to pathogenesis, cell organization, carbohydrate and lipid metabolism, response to stress, and several other functions. The major lipids detected by thin‐layer chromatography were ergosterol, lanosterol and glucosylceramide. Short exposure of MO to EV resulted in internalization of these vesicles and production of nitric oxide, interleukin (IL)‐12, transforming growth factor‐beta (TGF‐β) and IL‐10. Similarly, EV‐treated DC produced IL‐12p40, IL‐10 and tumour necrosis factor‐alpha. In addition, EV treatment induced the up‐regulation of CD86 and major histocompatibility complex class‐II (MHC‐II). Inoculation of G. mellonella larvae with EV followed by challenge with C. albicans reduced the number of recovered viable yeasts in comparison with infected larvae control. Taken together, our results demonstrate that C. albicans EV were immunologically active and could potentially interfere with the host responses in the setting of invasive candidiasis.     

Impact of an Educational Intervention Implanted in a Neurological Intensive Care Unit on Rates of Infection Related to External Ventricular Drains

Background

Studies on the implantation of care routines showed reduction on EVD catheter-related infections rates; however zero tolerance is difficult to be achieved. The objective of this study was to assess the impact of an educational intervention on the maximal reduction on rates of EVD-related infections.

Methodology/Principal Findings

The quasi-experimental (before-after intervention) study occurred in two phases: pre-intervention, from April 2007 to July 2008, and intervention, from August 2008 to July 2010. Patients were followed for 30 days after the removal of the EVD, and EVD-related infections were considered as only those with laboratorial confirmation in the CSF. Observations were made of the care of the EVD and compliance with Hygiene of the Hands (HH), a routine of care was drawn up, training was given, and intervention was made to reduce the time the EVD catheter remained in place.

Results

during the study, 178 patients were submitted to 194 procedures, corresponding to 1217 EVD catheters-day. Gram-negative agents were identified in 71.4% of the infections during the pre-intervention period and in 60% during the intervention period. During the study, EVD-related infection rates were reduced from 9.5% to 4.8% per patient, from 8.8% to 4.4% per procedure, and the incidence density dropped from 14.0 to 6.9 infections per 1000 catheters-day (p = 0.027). The mortality reduced 12% (from 42% to 30%).

Conclusions/Significance

During one year after the fourth intervention, no microbiologically identified infection was documented. In light of these results, educational intervention proved to be a useful tool in reducing these rates and showed also impact on mortality.     

Complete genome sequences of two hemotropic mycoplasmas, Mycoplasma haemofelis strain Ohio2 and Mycoplasma suis Strain Illinois

We report the complete and fully assembled genomes of Mycoplasma haemofelis strain Ohio2 and Mycoplasma suis strain Illinois, which are the first available genomes of these uncultivatable hemoplasma species. The single circular chromosomes of 1,152,484 bp and 742,431 bp for M. haemofelis and M. suis, respectively, are typical of mycoplasma species, having reduced size and low G+C content (38.8% for M. haemofelis and 31.1% for M. suis). Their metabolic pathways are reduced, with evidence of adaption to the blood environment.     

Site-Specific Chemoenzymatic Labeling of Aerolysin Enables the Identification of New Aerolysin Receptors

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.     

A Paracoccidioides brasiliensis glycan shares serologic and functional properties with cryptococcal glucuronoxylomannan

The cell wall of the yeast form of the dimorphic fungus Paracoccidioides brasiliensis is enriched with α1,3-glucans. In Cryptococcus neoformans, α1,3-glucans interact with glucuronoxylomannan (GXM), a heteropolysaccharide that is essential for fungal virulence. In this study, we investigated the occurrence of P. brasiliensis glycans sharing properties with cryptococcal GXM. Protein database searches in P. brasiliensis revealed the presence of sequences homologous to those coding for enzymes involved in the synthesis of GXM and capsular architecture in C. neoformans. In addition, monoclonal antibodies (mAbs) raised to cryptococcal GXM bound to P. brasiliensis cells. Using protocols that were previously established for extraction and analysis of C. neoformans GXM, we recovered a P. brasiliensis glycan fraction composed of mannose and galactose, in addition to small amounts of glucose, xylose and rhamnose. In comparison with the C. neoformans GXM, the P. brasiliensis glycan fraction components had smaller molecular dimensions. The P. brasiliensis components, nevertheless, reacted with different GXM-binding mAbs. Extracellular vesicle fractions of P. brasiliensis also reacted with a GXM-binding mAb, suggesting that the polysaccharide-like molecule is exported to the extracellular space in secretory vesicles. An acapsular mutant of C. neoformans incorporated molecules from the P. brasiliensis extract onto the cell wall, resulting in the formation of surface networks that resembled the cryptococcal capsule. Coating the C. neoformans acapsular mutant with the P. brasiliensis glycan fraction resulted in protection against phagocytosis by murine macrophages. These results suggest that P. brasiliensis and C. neoformans share metabolic pathways required for the synthesis of similar polysaccharides and that P. brasiliensis yeast cell walls have molecules that mimic certain aspects of C. neoformans GXM. These findings are important because they provide additional evidence for the sharing of antigenically similar components across phylogenetically distant fungal species. Since GXM has been shown to be important for the pathogenesis of C. neoformans and to elicit protective antibodies, the finding of similar molecules in P. brasiliensis raises the possibility that these glycans play similar functions in paracoccidiomycosis.     

Toward Developing a Universal Treatment for Fungal Disease Using Radioimmunotherapy Targeting Common Fungal Antigens

Background

Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall.

Methods

MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth (²¹³Bi) using the chelator CHXA”. B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium (¹⁸⁸Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls.

Results

²¹³Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80–100%. The ²¹³Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing.

Conclusion

Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

     

Clinical, epidemiological and molecular features of the HIV-1 subtype C and recombinant forms that are circulating in the city of São Paulo, Brazil

ABSTRACT: BACKGROUND: The city of Sao Paulo has the highest AIDS case rate, with nearly 60% in Brazil. Despite,several studies involving molecular epidemiology, lack of data regarding a large cohort studyhas not been published from this city.ObjectivesThis study aimed to describe the HIV-1 subtypes, recombinant forms and drug resistancemutations, according to subtype, with emphasis on subtype C and BC recombinants in thecity of S?o Paulo, Brazil.Study designRNA was extracted from the plasma samples of 302 HIV-1-seropositive subjects, of which211 were drug-naive and 82 were exposed to ART. HIV-1 partial pol region sequences wereused in phylogenetic analyses for subtyping and identification of drug resistance mutations.The envelope gene of subtype C and BC samples was also sequenced. RESULTS: From partial pol gene analyses, 239 samples (79.1%) were assigned as subtype B, 23 (7.6%)were F1, 16 (5.3%) were subtype C and 24 (8%) were mosaics (3 CRF28/CRF29-like). Thesubtype C and BC recombinants were mainly identified in drug-na?ve patients (72.7%) andthe heterosexual risk exposure category (86.3%), whereas for subtype B, these values were69.9% and 57.3%, respectively (p = 0.97 and p = 0.015, respectively). An increasing trend ofsubtype C and BC recombinants was observed (p < 0.01). CONCLUSION: The HIV-1 subtype C and CRFs seem to have emerged over the last few years in the city ofS?o Paulo, principally among the heterosexual population. These findings may have animpact on preventive measures and vaccine development in Brazil.