New developments of RNAi in Paracoccidioides brasiliensis: prospects for high-throughput, genome-wide, functional genomics.

BACKGROUND: The Fungal Genome Initiative of the Broad Institute, in partnership with the Paracoccidioides research community, has recently sequenced the genome of representative isolates of this human-pathogen dimorphic fungus: Pb18 (S1), Pb03 (PS2) and Pb01. The accomplishment of future high-throughput, genome-wide, functional genomics will rely upon appropriate molecular tools and straightforward techniques to streamline the generation of stable loss-of-function phenotypes. In the past decades, RNAi has emerged as the most robust genetic technique to modulate or to suppress gene expression in diverse eukaryotes, including fungi. These molecular tools and techniques, adapted for RNAi, were up until now unavailable for P. brasiliensis. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we report Agrobacterium tumefaciens mediated transformation of yeast cells for high-throughput applications with which higher transformation frequencies of 150±24 yeast cell transformants per 1×106 viable yeast cells were obtained. Our approach is based on a bifunctional selective marker fusion protein consisted of the Streptoalloteichus hindustanus bleomycin-resistance gene (Shble) and the intrinsically fluorescent monomeric protein mCherry which was codon-optimized for heterologous expression in P. brasiliensis. We also report successful GP43 gene knock-down through the expression of intron-containing hairpin RNA (ihpRNA) from a Gateway-adapted cassette (cALf) which was purpose-built for gene silencing in a high-throughput manner. Gp43 transcript levels were reduced by 73.1±22.9% with this approach. CONCLUSIONS/SIGNIFICANCE: We have a firm conviction that the genetic transformation technique and the molecular tools herein described will have a relevant contribution in future Paracoccidioides spp. functional genomics research.

Osteogenic differentiation of bone marrow mesenchymal stem cells of ovariectomized and non-ovariectomized female rats with thyroid dysfunction.

The objective of this study was to verify the osteogenic potential of the bone marrow mesenchymal stem cells (MSCs) of ovariectomized and non-ovariectomized female rats with hypo- and hyperthyroidism. Sixty two-month-old female rats were assigned to the following groups: (1) control (sham-operated), (2) ovariectomized (OVX'd), (3) hypothyroid sham-operated (Hypo-), (4) hypothyroid OVX'd, (5) hyperthyroid sham-operated (Hyper-) and (6) hyperthyroid OVX'd. After 135 days of treatment, the female rats were euthanized. We collected plasma to measure the levels of free T4, and the femur for extraction of MSCs. At 7 and 21 days of osteogenic differentiation of MSCs, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion, alkaline phosphatase activity, mineralized nodule number and gene expression for collagen I, osteocalcin, bone sialoprotein and osteopontin were analyzed. The hypothyroid group presented a significant reduction in the osteogenic differentiation of MSCs. The hyperthyroid group did not present changes in the synthesis of mineralized nodules for MSCs at day 21 of differentiation. However, in ovariectomized rats, hyperthyroidism increased the osteogenic differentiation of MSCs characterized by the increase of the alkaline phosphatase activity, the number of mineralized nodules and the expression of osteocalcin, sialoprotein and osteopontin. Our results demonstrated that the hypothyroidism reduces the osteogenic differentiation of MSCs only in non-ovariectomized rats and that the hyperthyroidism increases the osteogenic differentiation of MSCs only in ovariectomized rats.

Identification and characterization of immune complexes in the cerebrospinal fluid of patients with spinal cord schistosomiasis.

The pathogenesis of neuroschistosomiasis is largely unknown. Available evidence suggests that it depends on the presence of parasite eggs in the nervous tissue and on the host's immune response. We investigated the presence of immune complexes (ICs) in the cerebrospinal fluid (CSF) of four patients with spinal cord schistosomiasis (SCS), and performed their characterization. ICs containing soluble egg antigen of Schistosoma mansoni (SEA) were found in the CSF of all the SCS patients. To our knowledge, this is the first evidence of ICs containing schistosomal antigens in the CSF of patients with SCS. Further studies are necessary to confirm our findings and investigate the possible roles of ICs in the pathogenesis of this disease.