Somatic homozygous loss of SH2B3, and a non-Robertsonian translocation t(15;21)(q25.3;q22.1) with NTRK3 rearrangement, in an adolescent with progenitor B-cell acute lymphoblastic leukemia with the iAMP21.

Intrachromosomal amplification of chromosome 21 (iAMP21) occurs in ∼2% of B-cell acute lymphoblastic leukemia (ALL) and is considered to confer a poor prognosis. The relapse risk is associated with therapy intensity, suggesting that other somatic mutations may influence iAMP21-ALL prognosis. This abnormality is characterized by multiple copies of the RUNX1 gene in chromosome 21 and appears to arise through multiple breakage-fusion bridge cycles and chromothripsis. Rob(15;21) or a ring chromosome 21 have been associated with an increased risk for iAMP21-ALL, suggesting that constitutional genetic abnormalities may also drive leukemogenesis. Here we describe homozygous deletion of the SH2B3 gene, chromothripsis of chromosome 21, and a non-Robertsonian somatic t(15;21)(q25.3;q22.1) with NTRK3 gene rearrangement in an adolescent with iAMP21-B-ALL. Molecular cytogenetic studies detected iAMP21 with aCGH analysis revealing further genomic imbalances. The RT-qPCR analysis detected elevated expression levels of RUNX1 (68-fold) and reduced expression of CDK6 (0.057-fold). Studies with constitutive cells collected from mouth swabs showed that SH2B3 biallelic deletion was a somatic alteration occurring during clonal evolution. The identification of novel secondary genetic changes was valuable to discuss sporadic iAMP21 leukemogenic mechanisms. For the first time, we show a t(15;21)(q25.3;q22.1) with NTRK3 rearrangement in an adolescent with iAMP21-ALL.

Biocompatibility, induction of mineralization and antimicrobial activity of experimental intracanal pastes based on glass and glass-ceramic materials.

AIM: To evaluate the biocompatibility, induction of mineralization and antimicrobial activity of experimental intracanal pastes based on two glass and glass-ceramic materials. Calcium hydroxide (Ca(OH)2 ) paste was used as the positive control. METHODOLOGY: The glass-ceramic powder [two-phased Biosilicate (BS-2P)] and F18 bioactive glass were mixed with distilled water (ratio 2 : 1), inserted in polyethylene tubes and implanted in the subcutaneous tissues of 16 rats. Empty tubes were used as negative control. After 7 and 30 days (n = 8), the rats were euthanized for haematoxylin-eosin, von Kossa, polarized light and osteopontin (OPN) immunolabeling analysis. Direct contact tests using a suspension of each paste were performed with Enterococcus faecalis planktonic cells to evaluate antimicrobial activity (24 h of contact), in a pilot study. The number of CFU mL-1 was calculated for each group. The antimicrobial analysis data were submitted to one-way anova and Tukey tests, whilst biocompatibility and immunohistochemical data were submitted to the Kruskal-Wallis and Dunn tests (P < 0.05). RESULTS: Most specimens of the control, BS-2P and Ca(OH)2 groups were associated with moderate inflammation seven days following implantation, whilst F18 was associated with moderate to severe inflammation, without differences amongst the groups (P > 0.05). At 30 days, most specimens of control, F18 and BS-2P groups had mild inflammation, whilst Ca(OH)2 had mild to moderate inflammation; however, no differences were determined amongst the groups (P > 0.05). The fibrous capsule was thick at 7 days, becoming thin at 30 days. All pastes induced von Kossa-positive structures and were birefringent to polarized light. At seven days, the BS-2P group had significantly more OPN immunolabeling compared to the control and Ca(OH)2 groups (P < 0.05). At 30 days, the F18 group had significantly more OPN immunolabeling compared to the control and Ca(OH)2 groups (P < 0.05). All pastes reduced the total number of E. faecalis; however, the reduction was only significant when comparing BS-2P and Ca(OH)2 groups to the control (P < 0.05). Only calcium hydroxide eliminated E. faecalis. CONCLUSIONS: Experimental BS-2P and F18 pastes were biocompatible, stimulated biomineralization and induced significant OPN immunolabeling compared to Ca(OH)2 . Only the BS-2P paste demonstrated antimicrobial activity comparable to Ca(OH)2 .

Phylogenetic diversity of aerobic spore-forming Bacillalles isolated from Brazilian soils.

The phylum Firmicutes comprises seven classes where most species are either aerobic or anaerobic endospore former. Inside Firmicutes, species allocated in the genus Bacillus and related genera are collectively named aerobic endospore-forming bacteria (AEFB), and the soil is their major reservoir. AEFB have great importance in health, agriculture, and biotechnology although the more studied species are Bacillus subtilis and the human pathogens Bacillus cereus and Bacillus anthracis. AEFB have great importance in health, agriculture, and biotechnology; although the knowledge about these organisms is based on few species, notably, Bacillus subtilis, Bacillus cereus, and Bacillus anthracis. In this work, we generated partial 16S rRNA gene sequences of both strands of 192 AEFB strains isolated from soils of Distrito Federal, Brazil (SDF strains). The resulting consensus sequences were used to obtain taxonomic assignment and establish the phylogenetic relationships among these strains. Through this approach, we could observe that classified SDF strains were distributed among genera Bacillus (169 strains; 88.02%), Paenibacillus (11; 5.73%), Lysinibacillus (6; 3.13%), Brevibacillus (4; 2.08%), Terribacillus (1; 0.52%), and Rummeliibacillus (1; 0.52%). Phylogenetic trees revealed these 192 SDF strains can be segregated into eight groups spanning families Bacillaceae and Paenibacillaceae belonging to the order Bacillales. To expand the knowledge about the diversity of these SDF strains, further studies regarding characterization with different methodologies are underway.

Broad-spectrum bactericidal activity of a new bioactive grafting material (F18) against clinically important bacterial strains.

Infection is the most relevant surgical complication in implant or grafting procedures. Osteomyelitis and other chronic conditions pose a constant challenge in current medical practice. In this context, a grafting biomaterial that possesses antibacterial properties combined with bioactivity could have great clinical impact. Researchers at the Vitreous Materials Laboratory (LaMaV-UFSCar) recently developed a glass composition, named F18, that presents an improved workability range combined with high bioactivity. With F18, one can easily manufacture complex shapes, such as scaffolds, continuous fibres and coat implants. This biomaterial has proven to be a viable alternative for bone and skin regeneration in in vivo tests, however its antimicrobial properties have not been explored. Hence, the purpose of this study was to systematically investigate the antibacterial activity of F18 in powder and fibre forms according to the JIS Z 2801:2010 standard. Whether incorporation of silver into F18 glass could impact its antimicrobial activity was also evaluated. Four clinically relevant Gram-positive and Gram-negative pathogenic bacterial strains (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa) were used in this study. In both powder and fibre forms, F18 presented extremely efficient bactericidal activity against all strains tested, eliminating virtually 100% of the bacterial cells after 24 h. Kinetic tests showed that silver doping further increased the bactericidal activity, leading to S. aureus eradication in only 30 min after incubation. Both doped and non-doped glasses demonstrated very high bactericidal activity, making F18 a promising infection-preventing alternative for bone and wound regeneration in clinical practice.

Development of a bioactive glass-polymer composite for wound healing applications.

This study reports the production and characterization of a composite material for wound healing applications. A bioactive glass obtained by sol-gel process and doped with two different metal ions was investigated. Silver (Ag) and cobalt (Co) were chosen due to their antibacterial and angiogenic properties, respectively, very beneficial in the wound healing process. Poly(ε-caprolactone) (PCL) fibers were produced by electrospinning (ES) from a polymeric solution using acetone as a solvent. After optimization of the ES parameters, two main suspensions were prepared, namely: PCL containing bioactive glass nanoparticles (BG-NP) and PCL with Ag2O and CoO doped BG-NP (DP BG-NP), which were processed with different concentrations of BG-NP (0.25%, 0.5% and 0.75wt%). The composite membranes were characterized in terms of morphology, fiber diameter, weight loss, mineralization potential and mechanical performance.

Characterization and biocompatibility of a fibrous glassy scaffold.

Bioactive glasses (BGs) are known for their ability to bond to living bone and cartilage. In general, they are readily available in powder and monolithic forms, which are not ideal for the optimal filling of bone defects with irregular shapes. In this context, the development of BG-based scaffolds containing flexible fibres is a relevant approach to improve the performance of BGs. This study is aimed at characterizing a new, highly porous, fibrous glassy scaffold and evaluating its in vitro and in vivo biocompatibility. The developed scaffolds were characterized in terms of porosity, mineralization and morphological features. Additionally, fibroblast and osteoblast cells were seeded in contact with extracts of the scaffolds to assess cell proliferation and genotoxicity after 24, 72 and 144 h. Finally, scaffolds were placed subcutaneously in rats for 15, 30 and 60 days. The scaffolds presented interconnected porous structures, and the precursor bioglass could mineralize a hydroxyapatite (HCA) layer in simulated body fluid (SBF) after only 12 h. The biomaterial elicited increased fibroblast and osteoblast cell proliferation, and no DNA damage was observed. The in vivo experiment showed degradation of the biomaterial over time, with soft tissue ingrowth into the degraded area and the presence of multinucleated giant cells around the implant. At day 60, the scaffolds were almost completely degraded and an organized granulation tissue filled the area. The results highlight the potential of this fibrous, glassy material for bone regeneration, due to its bioactive properties, non-cytotoxicity and biocompatibility. Future investigations should focus on translating these findings to orthotopic applications. Copyright © 2015 John Wiley & Sons, Ltd.

Quality improvement and geographical indication of cachaça (Brazilian spirit) by using locally selected yeast strains.

AIMS: In order to improve the quality and to create a biological basis for obtainment of the protected denomination of origin (PDO), indigenous yeast were isolated and characterized for use in Salinas city (the Brazilian region of quality cachaça production). MATERIAL AND METHODS: Seven thousand and two hundred yeast colonies from 15 Salinas city distilleries were screened based on their fermentative behaviour and the physicochemical composition of cachaça. Molecular polymorphic analyses were performed to characterize these isolates. RESULTS: Two Saccharomyces cerevisiae strains (nos. 678 and 680) showed appropriate characteristics to use in the cachaça production: low levels of acetaldehyde and methanol, and high ethyl lactate/ethyl acetate ratio respectively. They also presented polymorphic characteristics more closely related between themselves even when compared to other strains from Salinas. CONCLUSIONS: The application of selected yeast to cachaça production can contribute for the improvement of the quality product as well as be used as a natural marker for PDO. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the use of selected yeast strains could contribute to obtain a cachaça similar to those produced traditionally, while getting wide acceptation in the market, yet presenting more homogeneous organoleptic characteristics, and thus contributing to the PDO implementation.

Larvicidal activity of Bacillus circulans against the gastrointestinal nematode Haemonchus contortus in sheep.

Efficient control of gastrointestinal parasites is necessary in sheep breeding. However, the available chemically based anthelmintics are becoming less effective due to the development of parasite resistance. An alternative to this problem is biological control. In the present study, we tested the larvicidal effect of Bacillus circulans by administering a spore suspension (2 × 109 colony forming units/ml) orally to lambs naturally infected with Haemonchus contortus. The number of faecal larvae was quantified daily and a significant reduction (~87%, P< 0.05) of larval development was observed after administration of B. circulans. Using a transformed B. circulans with green fluorescent protein, we were able to detect B. circulans in the faeces at 4 h post-administration and 72 h after cessation of its administration. These results suggest the use of B. circulans as a promising biological alternative for parasite control.

Effect of a new bioactive fibrous glassy scaffold on bone repair.

Researchers have investigated several therapeutic approaches to treat non-union fractures. Among these, bioactive glasses and glass ceramics have been widely used as grafts. This class of biomaterial has the ability to integrate with living bone. Nevertheless, bioglass and bioactive materials have been used mainly as powder and blocks, compromising the filling of irregular bone defects. Considering this matter, our research group has developed a new bioactive glass composition that can originate malleable fibers, which can offer a more suitable material to be used as bone graft substitutes. Thus, the aim of this study was to assess the morphological structure (via scanning electron microscope) of these fibers upon incubation in phosphate buffered saline (PBS) after 1, 7 and 14 days and, also, evaluate the in vivo tissue response to the new biomaterial using implantation in rat tibial defects. The histopathological, immunohistochemistry and biomechanical analyzes after 15, 30 and 60 days of implantation were performed to investigate the effects of the material on bone repair. The PBS incubation indicated that the fibers of the glassy scaffold degraded over time. The histological analysis revealed a progressive degradation of the material with increasing implantation time and also its substitution by granulation tissue and woven bone. Histomorphometry showed a higher amount of newly formed bone area in the control group (CG) compared to the biomaterial group (BG) 15 days post-surgery. After 30 and 60 days, CG and BG showed a similar amount of newly formed bone. The novel biomaterial enhanced the expression of RUNX-2 and RANK-L, and also improved the mechanical properties of the tibial callus at day 15 after surgery. These results indicated a promising use of the new biomaterial for bone engineering. However, further long-term studies should be carried out to provide additional information concerning the material degradation in the later stages and the bone regeneration induced by the fibrous material.

Molecular identification of Amazonian stingless bees using polymerase chain reaction single-strand conformation polymorphism.

In countries containing a mega diversity of wildlife, such as Brazil, identifying and characterizing biological diversity is a continuous process for the scientific community, even in face of technological and scientific advances. This activity demands initiatives for the taxonomic identification of highly diverse groups, such as stingless bees, including molecular analysis strategies. This type of bee is distributed in all of the Brazilian states, with the highest species diversity being found in the State of Amazônia. However, the estimated number of species diverges among taxonomists. These bees are considered the main pollinators in the Amazon rainforest, in which they obtain food and shelter; however, their persistence is constantly threatened by deforestation pressure. Hence, it is important to classify the number and abundance of bee specie, to measure their decline and implement meaningful, priority conservation strategies. This study aims to maximize the implementation of more direct, economic and successful techniques for the taxonomic identification of stingless bees. Specifically, the genes 16S rRNA and COI from mitochondrial DNA were used as molecular markers to differentiate 9 species of Amazonian stingless bees based on DNA polymorphism, using the polymerase chain reaction-single-strand conformation polymorphism technique. We registered different, exclusive SSCP haplotypes for both genes in all species analyzed. These results demonstrate that SSCP is a simple and cost-effective technique that is applicable to the molecular identification of stingless bee species.

Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants.

Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation.

Antioxidant and orofacial anti-nociceptive activities of the stem bark aqueous extract of Anadenanthera colubrina (Velloso) Brenan (Fabaceae).

The anti-nociceptive and antioxidant activities of the Anadenantheracolubrina stem bark aqueous extract (AEAC) were investigated. AEAC (30 µg/mL) reduced 94.8% of 2,2-diphenyl-1-picrylhydrazyl radical and prevented 64% (200 µg/mL) of lipid peroxidation caused by 2,2'-azobis(2-methylpropionamidine) dihydrochloride-induced peroxyl radicals. AEAC treatment (200 and 400 mg/kg) significantly (p < 0.001) reduced mice orofacial nociception in the first (61.4% and 62.6%, respectively) and second (48.9% and 61.9%, respectively) phases of the formalin test. Nociception caused by glutamate was significantly (p < 0.001) reduced by up to 79% at 400 mg/kg, while 56-60% of the nociceptive behaviour induced by capsaicin was significantly inhibited by AEAC (100-400 mg/kg). Mice treated with AEAC did not show changes in motor performance in the Rota-rod apparatus. It appears that AEAC is of pharmacological importance in treating pain due to its anti-nociceptive effects, which were shown to be mediated by central and peripheral mechanisms.

An unusual cytogenetic rearrangement originating from two different abnormalities in chromosome 6 in a child with acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is usually associated with a favorable outcome, but about 10% of patients tend to relapse. The genetic hallmark of APL is a balanced translocation involving chromosomes 15 and 17, and the PML-RARa gene fusion is found in more than 90% of these cases. Other chromosomal abnormalities are commonly found in APL, but their clinical significance has yet to be determined. Here we report a case of childhood APL that was studied by conventional cytogenetics along with molecular cytogenetic techniques. The patient showed a complex karyotype with an unusual cytogenetic rearrangement originating from two different abnormalities in a single chromosome 6. Our case is an exceptional example of a cryptic cytogenetic anomaly in APL and underscores the importance of detailed genetic characterization.

Variable number of tandem repeat markers in the genome sequence of Mycosphaerella fijiensis, the causal agent of black leaf streak disease of banana (Musa spp).

We searched the genome of Mycosphaerella fijiensis for molecular markers that would allow population genetics analysis of this plant pathogen. M. fijiensis, the causal agent of banana leaf streak disease, also known as black Sigatoka, is the most devastating pathogen attacking bananas (Musa spp). Recently, the entire genome sequence of M. fijiensis became available. We screened this database for VNTR markers. Forty-two primer pairs were selected for validation, based on repeat type and length and the number of repeat units. Five VNTR markers showing multiple alleles were validated with a reference set of isolates from different parts of the world and a population from a banana plantation in Costa Rica. Polymorphism information content values varied from 0.6414 to 0.7544 for the reference set and from 0.0400 and 0.7373 for the population set. Eighty percent of the polymorphism information content values were above 0.60, indicating that the markers are highly informative. These markers allowed robust scoring of agarose gels and proved to be useful for variability and population genetics studies. In conclusion, the strategy we developed to identify and validate VNTR markers is an efficient means to incorporate markers that can be used for fungicide resistance management and to develop breeding strategies to control banana black leaf streak disease. This is the first report of VNTR-minisatellites from the M. fijiensis genome sequence.

DNA sequencing of 13 cytokine gene fragments of Aotus infulatus and Saimiri sciureus, two non-human primate models for malaria.

Aotus and Saimiri are non-human primate models recommended by the World Health Organization for experimental studies in malaria, especially for vaccine pre-clinical trials. However, research using these primates is hindered by the lack of specific reagents to evaluate immune responses to infection or vaccination. As a step toward developing molecular tools for cytokine expression studies in these species, primer pairs for 18 cytokine gene fragments were designed based on human DNA sequences and used to amplify the corresponding genes in Aotus infulatus and Saimiri sciureus genomic DNA samples. IFNγ, TNFα, LTA, IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, CSF2 and TGFß2 gene fragments were amplified and sequenced. Primer pairs for IL8, IL17, IL18, IL27 and MIF failed to generate amplification products. When compared to the available corresponding human and non-human primate sequences, most--except IL3 and IL4--showed identity degrees above 90%. Small variations in sequence can help to explain the failure to amplify certain genes or the amplification only at lower annealing temperatures as compared to human DNA samples for several primer pairs. The sequences made available provide the basis for designing molecular tools such as primers for real time PCR specific for A. infulatus and/or S. sciureus. The nucleotide sequences reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers DQ985386 to DQ985389, DQ989356 to DQ989369, FJ89020 to FJ89024, and FJ89029.

FISH, PCR and cytogenetic characterization in a girl with ambiguous genitalia and karyotype mos46,X,iso(Y)(qter-->p11.3::p11.3-->qter)[80]/45,X[17]/46,X,+mar[3].

A cytogenetic study was carried out in a girl with virilized external genitalia, who showed a karyotype containing a Y isochromosome in mosaic form: mos46,X,iso(Y)(qter-->p11.3::p11.3-->qter)[80]/45,X[17]/46,X,+mar[3]. The chromosome aberrations were confirmed by fluorescence in situ hybridization analysis, with both whole chromosome paint Y probe and centromeric X chromosome probe. The molecular analyses by PCR detected the presence of the SRY, DAZ and AMGY genes, confirming the presence of the whole long arm and almost whole short arm of the Y chromosome. We suggest that the structural alteration of the Y chromosome was a new mutation, which occurred in the initial mitotic divisions of the embryo, originally 46,XY. The breakpoints occurred on the distal extremity of the short arm with later fusion of its extremities producing a Y isochromosome. The later numerical alteration occurred as a consequence of chromosomal instability. Although almost all cells (80%) in peripheral blood belonged to the iso(Y) line with a duplicated SRY gene, this did not determine male sexual differentiation in the patient. The result of accurate evaluation provides correct sex assignment and the prevention of the neoplastic degeneration of a dysgenetic gonad.

Mononeuropathy of the suprascapular nerve: study of 3 South American cases.

In this work 3 new cases of suprascapular nerve mononeuropathy are described. ENMG diagnosis criteria were: a) normal sensory conduction studies of the ipsolateral ulnar, median and radial nerves; b) bilateral suprascapular nerve latencies with bilateral compound muscle action potential, obtained from the infraspinatus muscle with symmetrical techniques; and c) abnormal neurogenic infraspinatus muscle electromyographic findings, coexisting with normal electromyographical data of the ipsolateral deltoideus and supraspinatus muscles. These 3 cases of suprascapular mononeurpathy were found in 6,080 ENMG exams from our University Hospital. For us this mononeuropathy is rare with a 0.05% occurrence.

Cell cycle, DNA replication, repair, and recombination in the dimorphic human pathogenic fungus Paracoccidioides brasiliensis

DNA replication, together with repair mechanisms and cell cycle control, are the most important cellular processes necessary to maintain correct transfer of genetic information to the progeny. These processes are well conserved throughout the Eukarya, and the genes that are involved provide essential information for understanding the life cycle of an organism. We used computational tools for data mining of genes involved in these processes in the pathogenic fungus Paracoccidiodes brasiliensis. Data derived from transcriptome analysis revealed that the cell cycle of this fungus, as well as DNA replication and repair, and the recombination machineries, are highly similar to those of the yeast Saccharomyces cerevisiae. Among orthologs detected in both species, there are genes related to cytoskeleton structure and assembly, chromosome segregation, and cell cycle control genes. We identified at least one representative gene from each step of the initiation of DNA replication. Major players in the process of DNA damage and repair were also identified.

Analysis of expressed sequence tags from Musa acuminata ssp. burmannicoides, var. Calcutta 4 (AA) leaves submitted to temperature stresses.

In order to discover genes expressed in leaves of Musa acuminata ssp. burmannicoides var. Calcutta 4 (AA), from plants submitted to temperature stress, we produced and characterized two full-length enriched cDNA libraries. Total RNA from plants subjected to temperatures ranging from 5 degrees C to 25 degrees C and from 25 degrees C to 45 degrees C was used to produce a COLD and a HOT cDNA library, respectively. We sequenced 1,440 clones from each library. Following quality analysis and vector trimming, we assembled 2,286 sequences from both libraries into 1,019 putative transcripts, consisting of 217 clusters and 802 singletons, which we denoted Musa acuminata assembled expressed sequence tagged (EST) sequences (MaAES). Of these MaAES, 22.87% showed no matches with existing sequences in public databases. A global analysis of the MaAES data set indicated that 10% of the sequenced cDNAs are present in both cDNA libraries, while 42% and 48% are present only in the COLD or in the HOT libraries, respectively. Annotation of the MaAES data set categorized them into 22 functional classes. Of the 2,286 high-quality sequences, 715 (31.28%) originated from full-length cDNA clones and resulted in a set of 149 genes.