Analysis of anti-Müllerian hormone (AMH) and its receptor (AMHR2) genes in patients with persistent Müllerian duct syndrome

OBJECTIVE: To screen for mutations in AMH and AMHR2 genes in patients with persistent Müllerian duct syndrome (PMDS). PATIENTS AND METHOD: Genomic DNA of eight patients with PMDS was obtained from peripheral blood leukocytes. Directed sequencing of the coding regions and the exon-intron boundaries of AMH and AMHR2 were performed. RESULTS: The AMH mutations p.Arg95*, p.Arg123Trp, c.556-2A>G, and p.Arg502Leu were identified in five patients; and p.Gly323Ser and p.Arg407* in AMHR2 of two individuals. In silico analyses of the novel c.556-2A>G, p.Arg502Leu and p.Arg407* mutations predicted that they were harmful and were possible causes of the disease. CONCLUSION: A likely molecular etiology was found in the eight evaluated patients with PMDS. Four mutations in AMH and two in AMHR2 were identified. Three of them are novel mutations, c.556-2A>G, and p.Arg502Leu in AMH; and p.Gly323Ser in AMHR2. Arq Bras Endocrinol Metab. 2012;56(8):473-8

Deep sequencing of New World screw-worm transcripts to discover genes involved in insecticide resistance

Background: The New World screw-worm (NWS), Cochliomyia hominivorax, is one of the most important myiasis-causing flies, causing severe losses to the livestock industry. In its current geographical distribution, this species has been controlled by the application of insecticides, mainly organophosphate (OP) compounds, but a number of lineages have been identified that are resistant to such chemicals. Despite its economic importance, only limited genetic information is available for the NWS. Here, as a part of an effort to characterize the C. hominivorax genome and identify putative genes involved in insecticide resistance, we sampled its transcriptome by deep sequencing of polyadenylated transcripts using the 454 sequencing technology. Results: Deep sequencing on the 454 platform of three normalized libraries (larval, adult male and adult female) generated a total of 548,940 reads. Eighteen candidate genes coding for three metabolic detoxification enzyme families, cytochrome P450 monooxygenases, glutathione S transferases and carboxyl/cholinesterases were selected and gene expression levels were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Of the investigated candidates, only one gene was expressed differently between control and resistant larvae with, at least, a 10-fold down-regulation in the resistant larvae. The presence of mutations in the acetylcholinesterase (target site) and carboxylesterase E3 genes was investigated and all of the resistant flies presented E3 mutations previously associated with insecticide resistance. Conclusions: Here, we provided the largest database of NWS expressed sequence tags that is an important resource, not only for further studies on the molecular basis of the OP resistance in NWS fly, but also for functional and comparative studies among Calliphoridae flies. Among our candidates, only one gene was found differentially expressed in resistant individuals, and its role on insecticide resistance should be further investigated. Furthermore, the absence of mutations in the OP target site and the high frequency of mutant carboxylesterase E3 indicate that metabolic resistance mechanisms have evolved predominantly in this species.

Complete Genome Sequence of Mycoplasma suis and Insights into Its Biology and Adaption to an Erythrocyte Niche

Mycoplasma suis, the causative agent of porcine infectious anemia, has never been cultured in vitro and mechanisms by which it causes disease are poorly understood. Thus, the objective herein was to use whole genome sequencing and analysis of M. suis to define pathogenicity mechanisms and biochemical pathways. M. suis was harvested from the blood of an experimentally infected pig. Following DNA extraction and construction of a paired end library, whole-genome sequencing was performed using GS-FLX (454) and Titanium chemistry. Reads on paired-end constructs were assembled using GS De Novo Assembler and gaps closed by primer walking; assembly was validated by PFGE. Glimmer and Manatee Annotation Engine were used to predict and annotate protein-coding sequences (CDS). The M. suis genome consists of a single, 742,431 bp chromosome with low G+C content of 31.1%. A total of 844 CDS, 3 single copies, unlinked rRNA genes and 32 tRNAs were identified. Gene homologies and GC skew graph show that M. suis has a typical Mollicutes oriC. The predicted metabolic pathway is concise, showing evidence of adaptation to blood environment. M. suis is a glycolytic species, obtaining energy through sugars fermentation and ATP-synthase. The pentose-phosphate pathway, metabolism of cofactors and vitamins, pyruvate dehydrogenase and NAD(+) kinase are missing. Thus, ribose, NADH, NADPH and coenzyme A are possibly essential for its growth. M. suis can generate purines from hypoxanthine, which is secreted by RBCs, and cytidine nucleotides from uracil. Toxins orthologs were not identified. We suggest that M. suis may cause disease by scavenging and competing for host nutrients, leading to decreased life-span of RBCs. In summary, genome analysis shows that M. suis is dependent on host cell metabolism and this characteristic is likely to be linked to its pathogenicity. The prediction of essential nutrients will aid the development of in vitro cultivation systems.

Effect of specific feed volume on the performance of an anaerobic sequencing batch biofilm reactor (AnSBBR) with circulation treating different wastewaters under different organic loads

The objective of this research was to study the behavior of two anaerobic sequencing batch reactors, containing immobilized biomass (AnSBBR), as a function of the ratio of the volume of treated medium in each cycle to the total volume of reaction medium. The reactors, in which mixing was accomplished by recirculation of the liquid phase, were maintained at 30 +/- 1 degrees C and treated different wastewaters in 8-h cycles. The operational conditions imposed had the objective to investigate whether maintenance of a residual volume in the reactor would affect, at the end of each cycle, process efficiency and stability, as well as to verify the intensity of the effect for different types of wastewaters and organic loading rates. The first reactor, with work volume of 2.5 L, treated reconstituted cheese whey at an organic loading rate of 12 g COD.L(-1).d(-1) and presented similar effluent quality for the four conditions under which it was operated: renewal of 100, 70, 50 and 25 % of its work volume at each cycle. Despite the fact that reduction in the renewed volume did not significantly affect effluent quality, in quantitative terms, this reduction resulted in an increase in the amount of organic matter removed by the first reactor. The second reactor, with work volume of 1.8 L, treated synthetic wastewater at organic loading rates of 3 and 5 g COD.L(-1).d(-1) and operated under two conditions for each loading: renewal of 100 and 50 % of its work volume. At the organic loading rate of 3 g COD.L(-1).d(-1), the results showed that both effluent quality and amount of organic matter removed by the second reactor were independent of the treated volume per cycle. At the organic loading rate of 5 g COD.L(-1).d(-1), although the reduction in the renewed volume did not affect the amount of organic matter removed by the reactor, effluent quality improved during reactor operation with total discharge of its volume. In general, results showed process stability under all conditions, evidencing reactor flexibility and the potential to apply this technology in the treatment of different types of wastewater.

Effects of bed materials on the performance of an anaerobic sequencing batch biofilm reactor treating domestic sewage

The objective of this study was to determine the best performance of an anaerobic sequencing batch biofilm reactor (AnSBBR) based on the use of four different bed materials as support for biomass immobilization. The bed materials utilized were Polyurethane foam (PU), vegetal carbon (VC), synthetic pumice (SP), and recycled low-density polyethylene (PE). The AnSBBR. with I total volume Of 7.2 L, was operated in 8-h batch cycles over 10 months, and fed with domestic sewage with an average influent chemical oxygen demand (COD) of 358 +/- 110 mg/L. The average effluent COD values were 121 +/- 31, 208 +/- 54, 233 +/- 52, and 227 +/- 51 mg/L. for PU, VC, SP, and PE, respectively. A modified first-order kinetic model was adjusted to temporal profiles of COD during a batch cycle, and the apparent kinetic constants were 0.52 +/- 0.05, 0.37 +/- 0.05, 0.80 +/- 0.04, and 0.30 +/- 0.021h(-1) for PU, VC, SP, and PE, respectively. Specific substrate utilization rates of 1.08, 0.11, and 0.86 mg COD/mg VS day were obtained for PU, VC, and PE, respectively. Although SP yielded the highest kinetic coefficient, PU was considered the best support, since SP presented loss of chemical constituents during the reactor`s operational phase. In addition, findings oil the microbial community were associated with the reactor`s performance data. Although PE did not show a satisfactory performance, an interesting microbial diversity was found oil its surface. Based oil the morphology and denaturing gradient gel electrophoresis (DGGE) results, PE showed the best capacity for promoting the attachment of methanogenic organisms, and is therefore a material that merits further analysis. PU was considered the Most suitable material showing the best performance in terms of efficiency of solids and COD removal. (C) 2007 Elsevier Ltd. All rights reserved.

HFE gene mutations in patients with primary iron overload: Is there a significant improvement in molecular diagnosis yield with HFE sequencing?

Rare HFE variants have been shown to be associated with hereditary hemochromatosis (HH), an iron overload disease. The low frequency of the HFE p.C282Y mutation in HH-affected Brazilian patients may suggest that other HFE-related mutations may also be implicated in the pathogenesis of HH in this population. The main aim was to screen for new HFE mutations in Brazilian individuals with primary iron overload and to investigate their relationship with HH. Fifty Brazilian patients with primary iron overload (transferrin saturation >50% in females and 60% in males) were selected. Subsequent bidirectional sequencing for each HFE exon was performed. The effect of HFE mutations on protein structure were analyzed by molecular dynamics simulation and free binding energy calculations. p.C282Y in homozygosis or in heterozygosis with p.H63D were the most frequent genotypic combinations associated with HH in our sample population (present in 17 individuals, 34%). Thirty-six (72.0%) out of the 50 individuals presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 22.0%). One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior indicated that the p.V256I mutation does not reduce the binding affinity between HFE and beta 2-microglobulin ((beta 2M) in the same way the p.C282Y mutation does compared with the native HFE protein. In conclusion, screening of HFE through direct sequencing, as compared to p.C282Y/p.H63D genotyping, was not able to increase the molecular diagnosis yield of HH. The novel p.V256I mutation could not be implicated in the molecular basis of the HH phenotype, although its role cannot be completely excluded in HH-phenotype development. Our molecular modeling analysis can help in the analysis of novel, previously undescribed, HFE mutations. (C) 2010 Elsevier Inc. All rights reserved.

Sequence analysis of rabbit haemorrhagic disease virus (RHDV) in Australia: alterations after its release

Liver samples from rabbits killed by RHDV, collected from five States in Australia in 1996 and 1997 were analysed by RT-PCR. A 398 bp fragment of the capsid protein (VP60) gene was amplified by PCR and directly sequenced. The alignment of the nucleotide and amino acid sequences and their comparison with the original strain of the virus released in Australia indicated genetic changes after two years have been small with 98.2% to 100% identity. The constructed phylogenetic tree suggests slight differences in nucleotide substitutions in various States but there is no clear evidence of clustering of sequences according to their geographic origin. In practical terms, sequencing of viral RNA provides a means of testing the efficacy of further releases and subsequent spread of the virus if such a strategy is employed as a means of enhancing RHD as a biological control of the wild rabbit in Australia.

Reverse transcription of a naturally occurring nonretroviral RNA produces a precise deletion in the majority of its cDNA products

A precise, reproducible deletion made during in vitro reverse transcription of RNA2 from the icosahedral positive-stranded Helicoverpa armigera stunt virus (Tetraviridae) is described. The deletion, located between two hexamer repeats, is a 50-base sequence that includes one copy of the hexamer repeat. Only the Moloney murine leukemia virus reverse transcriptase and its derivative Superscript I, carrying a deletion of the carboxy-terminal RNase H region, showed this response, indicating a template-switching mechanism different from one proposed that involves a RNase H-dependent strand transfer, Superscript II, however, which carries point mutations to reduce RNase H activity, does not cause a deletion. A possible mechanism involves the enzyme pausing at the 3' side of a stem-loop structure and the 3' end of the nascent DNA strand separating from the template and reannealing to the upstream hexamer repeat.

Microsatellite allele sequencing in population analyses of the South American cactophilic species Drosophila antonietae (Diptera: Drosophilidae)

Drosophila antonietae belongs to the Drosophila buzzatii cluster, a cactophilic group of species naturally endemic to South America. Morphological and genetic analyses indicate that its populations are the most homogenous in the cluster and that the diversity observed is mainly a result of variation within populations. Seven polymorphic microsatellite loci were described for this species and used in the present study to investigate the genetic diversity of natural populations of D. antonietae by both length and sequence variation. The study aimed to understand how homoplasy and null alleles affect inferences about the population history of this species and to obtain an accurate interpretation of population inferences where these loci could be applied. The results provide useful information on the interpretation of genetic data derived from the microsatellite loci described for D. antonietae and on evolutionary aspects of cactophilic Drosophila. Importantly, the results indicate that size homoplasy and null alleles do not represent significant problems for the population genetics analyses because the large amount of variability at microsatellite loci compensate the low frequency of these problems in the populations. (C) 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 573-584.

Molecular cloning, sequencing, and expression analysis of presenilin cDNA from Schistosoma mansoni

Presenilins (PS) are integral membrane proteins involved, among other functions, in regulated intramembrane proteolysis. In this study, we report the identification and characterization of a complementary DNA from Schistosoma mansoni exhibiting a significant homology to human and nonvertebrate presinilins. S. mansoni contained a 1,485 bp open reading frame encoding a predicted protein of 494 amino acids. Alignment of predicted amino acid sequence of S. mansoni with PS (SmPS) from other species revealed up to 40% similarity shared among the investigated organisms. In addition, phylogenetic analyses demonstrated SmPS being closely related to its orthologues found in Schistosoma japonicum and Caenorhabditis elegans. Expression analysis of SmPS using quantitative real-time PCR revealed that the transcript is up-regulated in the egg stage. We hypothesize that the high level of SmPS in the S. mansoni embryo correlates to an important role during cellular signaling associated to larval development. To our knowledge, this study represents the first attempt to investigate the existence and abundance of PS from a helminth parasite.

Phylogeny of the Sporobolus indicus complex, based on internal transcribed spacer (ITS) sequences

The entire internal transcribed spacer ( ITS) region, including the 5.8S subunit of the nuclear ribosomal DNA ( rDNA), was sequenced by direct double-stranded sequencing of polymerase chain reaction (PCR) amplified fragments. The study included 40 Sporobolus ( Family Poaceae, subfamily Chloridoideae) seed collections from 14 putative species ( all 11 species from the S. indicus complex and three Australian native species). These sequences, along with those from two out-group species [ Pennisetum alopecuroides ( L.) Spreng. and Heteropogon contortus ( L.) P. Beauv. ex Roemer & Schultes, Poaceae, subfamily Panicoideae], were analysed by the parsimony method (PAUP; version 4.0b4a) to infer phylogenetic relationships among these species. The length of the ITS1, 5.8S subunit and ITS2 region were 222, 164 and 218 base pairs ( bp), respectively, in all species of the S. indicus complex, except for the ITS2 region of S. diandrus P. Beauv. individuals, which was 217 bp long. Of the 624 characters included in the analysis, 245 ( 39.3%) of the 330 variable sites contained potential phylogenetic information. Differences in sequences among the members of the S. pyramidalis P. Beauv., S. natalensis (Steud.) Dur & Schinz and S. jacquemontii Kunth. collections were 0%, while differences ranged from 0 to 2% between these and other species of the complex. Similarly, differences in sequences among collections of S. laxus B. K. Simon, S. sessilis B. K. Simon, S. elongatus R. Br. and S. creber De Nardi were 0%, compared with differences of 1-2% between these four species and the rest of the complex. When comparing S. fertilis ( Steud.) Clayton and S. africanus (Poir.) Robyns & Tourney, differences between collections ranged from 0 to 1%. Parsimony analysis grouped all 11 species of the S. indicus complex together, indicating a monophyletic origin. For the entire data set, pair-wise distances among members of the S. indicus complex varied from 0.00 to 1.58%, compared with a range of 20.08-21.44% among species in the complex and the Australian native species studied. A strict consensus phylogenetic tree separated 11 species of the S. indicus complex into five major clades. The phylogeny, based on ITS sequences, was found to be congruent with an earlier study on the taxonomic relationship of the weedy Sporobolus grasses revealed from random amplified polymorphic DNA ( RAPD). However, this cladistic analysis of the complex was not in agreement with that created on past morphological analyses and therefore gives a new insight into the phylogeny of the S. indicus complex.

Sequencing cutting patterns with colored interval graphs

The problem addressed here originates in the industry of flat glass cutting and wood panel sawing, where smaller items are cut from larger items accordingly to predefined cutting patterns. In this type of industry the smaller pieces that are cut from the patterns are piled around the machine in stacks according to the size of the pieces, which are moved to the warehouse only when all items of the same size have been cut. If the cutting machine can process only one pattern at a time, and the workspace is limited, it is desirable to set the sequence in which the cutting patterns are processed in a way to minimize the maximum number of open stacks around the machine. This problem is known in literature as the minimization of open stacks (MOSP). To find the best sequence of the cutting patterns, we propose an integer programming model, based on interval graphs, that searches for an appropriate edge completion of the given graph of the problem, while defining a suitable coloring of its vertices.

Inoculation with Metal-Mobilizing Plant-GrowthPromoting Rhizobacterium Bacillus sp. SC2b and Its Role in Rhizoremediation

A plant growth-promoting bacterial (PGPB) strain SC2b was isolated from the rhizosphere of Sedum plumbizincicola grown in lead (Pb)/zinc (Zn) mine soils and characterized as Bacillus sp. based on (1) morphological and biochemical characteristics and (2) partial 16S ribosomal DNA sequencing analysis. Strain SC2b exhibited high levels of resistance to cadmium (Cd) (300 mg/L), Zn (730 mg/L), and Pb (1400 mg/L). This strain also showed various plant growth-promoting (PGP) features such as utilization of 1-aminocyclopropane-1-carboxylate, solubilization of phosphate, and production of indole-3-acetic acid and siderophore. The strain mobilized high concentration of heavy metals from soils and exhibited different biosorption capacity toward the tested metal ions. Strain SC2b was further assessed for PGP activity by phytagar assay with a model plant Brassica napus. Inoculation of SC2b increased the biomass and vigor index of B. napus. Considering such potential, a pot experiment was conducted to assess the effects of inoculating the metal-resistant PGPB SC2b on growth and uptake of Cd, Zn and Pb by S. plumbizincicola in metal-contaminated agricultural soils. Inoculation with SC2b elevated the shoot and root biomass and leaf chlorophyll content of S. plumbizincicola. Similarly, plants inoculated with SC2b demonstrated markedly higher Cd and Zn accumulation in the root and shoot system, indicating that SC2b enhanced Cd and Zn uptake by S. plumbizincicola through metal mobilization or plant-microbial mediated changes in chemical or biological soil properties. Data demonstrated that the PGPB Bacillus sp. SC2b might serve as a future biofertilizer and an effective metal mobilizing bioinoculant for rhizoremediation of metal polluted soils.

Aspergilli and Penicillia related to the surface of ripening italian grana type cheese and its ripening environment

Information available on the mycoflora associated to ripening Italian “grana type” cheese is very poor. Recently, ochratoxin A (OTA) was detected in samples of packed grated cheese [1]; therefore, the need of information to perform a risk management was highlighted. Moreover, sterigmatocystin (STC) has been reported in cheese and it is considered an emerging problem. Despite the fact that both of them are mycotoxins included in group 2B by IARC [2,3], no European regulation exists. So, the main goal of this work is to give for the first time a general overview about Penicillia and Aspergilli growing on the surface of ripening “grana type” cheese, with particular attention on mycotoxigenic species. To perform this, in 2013 and 2014 crust samples were scratched from ripening grana cheese wheels and also Potato Dextrose Agar plates were exposed to monitor ripening house air. Then, 140 fungal isolates were randomly chosen, purified and monosporic colonies were obtained for their identification at specie level. A polyphasic approach is followed, based on morphological characterisation, toxic extrolites profiling and gene sequencing. The identification is still in progress, but the first results based on the morphological approach showed the presence of mycotoxigenic Aspergilli (Aspergillus flavus and A. versicolor) and various Penicillium species; among them Penicillium chrysogenum, P. implicatum and P. solitum were identified. Only P. chrysogenum was reported to produce the mycotoxins cyclopiazonic acid (CPA) and roquefortine-C (ROQ-C) [4]. These results will be presented and discussed. [1] A. Biancardi, R. Piro, G. Galaverna, C. Dall’Asta, "A simple and reliable liquid chromatography–tandem mass spectrometry method for determination of ochratoxin A in hard cheese" International Journal of Food Sciences and Nutrition 64 (5), 2013, 632 – 640. [2] International Agency for Research on Cancer (IARC) “IARC Monographs on the Evaluation of Carcinogenic Risks to Humans” 31, 1983, 191 – 199. [3] International Agency for Research on Cancer (IARC) “IARC Monographs on the Evaluation of carcinogenic Risks to Humans”, suppl. 7, 1987, 72. [4] J. I. Pitt, D. A. Hocking, “Fungi and Food Spoilage” 1997, 291.