Production of xylanase and beta-xylosidase from autohydrolysis liquor of corncob using two fungal strains

Agroindustrial residues are materials often rich in cellulose and hemicellulose. The use of these substrates for the microbial production of enzymes of industrial interest is mainly due to their high availability associated with their low cost. In this work, corncob (CCs) particles decomposed to soluble compounds (liquor) were incorporated in the microbial growth medium through autohydrolysis, as a strategy to increase and undervalue xylanase and beta-xylosidase production by Aspergillus terricola and Aspergillus ochraceus. The CCs autohydrolysis liquor produced at 200 A degrees C for 5, 15, 30 or 50 min was used as the sole carbon source or associated with untreated CC. The best condition for enzyme synthesis was observed with CCs submitted to 30 min of autohydrolysis. The enzymatic production with untreated CCs plus CC liquor was higher than with birchwood xylan for both microorganisms. A. terricola produced 750 total U of xylanase (144 h cultivation) and 30 total U of beta-xylosidase (96-168 h) with 0.75% untreated CCs and 6% CCs liquor, against 650 total U of xylanase and 2 total U of beta-xylosidase in xylan; A. ochraceus produced 605 total U of xylanase and 56 total U of beta-xylosidase (168 h cultivation) with 1% untreated CCs and 10% CCs liquor against 400 total U of xylanase and 38 total U of beta-xylosidase in xylan. These results indicate that the treatment of agroindustrial wastes through autohydrolysis can be a viable strategy in the production of high levels of xylanolytic enzymes.

Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -beta and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The Delta pmcA and Delta pmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the Delta calA and Delta crzA mutant strains. However, only the A. fumigatus Delta pmcA was avirulent in the murine model of invasive pulmonary aspergillosis.

An ancient founder mutation in PROKR2 impairs human reproduction

Congenital gonadotropin-releasing hormone (GnRH) deficiency manifests as absent or incomplete sexual maturation and infertility. Although the disease exhibits marked locus and allelic heterogeneity, with the causal mutations being both rare and private, one causal mutation in the prokineticin receptor, PROKR2 L173R, appears unusually prevalent among GnRH-deficient patients of diverse geographic and ethnic origins. To track the genetic ancestry of PROKR2 L173R, haplotype mapping was performed in 22 unrelated patients with GnRH deficiency carrying L173R and their 30 first-degree relatives. The mutations age was estimated using a haplotype-decay model. Thirteen subjects were informative and in all of them the mutation was present on the same approximate to 123 kb haplotype whose population frequency is 10. Thus, PROKR2 L173R represents a founder mutation whose age is estimated at approximately 9000 years. Inheritance of PROKR2 L173R-associated GnRH deficiency was complex with highly variable penetrance among carriers, influenced by additional mutations in the other PROKR2 allele (recessive inheritance) or another gene (digenicity). The paradoxical identification of an ancient founder mutation that impairs reproduction has intriguing implications for the inheritance mechanisms of PROKR2 L173R-associated GnRH deficiency and for the relevant processes of evolutionary selection, including potential selective advantages of mutation carriers in genes affecting reproduction.

Chiral HPLC analysis of donepezil, 5-O-desmethyl donepezil and 6-O-desmethyl donepezil in culture medium: application to fungal biotransformation studies

An high performance liquid chromatography (HPLC) method for the enantioselective determination of donepezil (DPZ), 5-O-desmethyl donepezil (5-ODD), and 6-O-desmethyl donepezil (6-ODD) in Czapek culture medium to be applied to biotransformation studies with fungi is described for the first time. The HPLC analysis was carried out using a Chiralpak AD-H column with hexane/ethanol/methanol (75:20:5, v/v/v) plus 0.3 % triethylamine as mobile phase and UV detection at 270 nm. Sample preparation was carried out by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 100-10,000 ng mL(-1) for each enantiomer of DPZ (r a parts per thousand yenaEuro parts per thousand 0.9985) and of 100-5,000 ng mL(-1) for each enantiomer of 5-ODD (r a parts per thousand yenaEuro parts per thousand 0.9977) and 6-ODD (r a parts per thousand yenaEuro parts per thousand 0.9951). Within-day and between-day precision and accuracy evaluated by relative standard deviations and relative errors, respectively, were lower than 15 % for all analytes. The validated method was used to assess DPZ biotransformation by the fungi Beauveria bassiana American Type Culture Collection (ATCC) 7159 and Cunninghamella elegans ATCC 10028B. Using the fungus B. bassiana ATCC 7159, a predominant formation of (R)-5-ODD was observed while for the fungus C. elegans ATCC 10028B, DPZ was biotransformed to (R)-6-ODD with an enantiomeric excess of 100 %.

Comparative EST analysis provides insights into the basal aquatic fungus Blastocladiella emersonii

Abstract Background Blastocladiella emersonii is an aquatic fungus of the Chytridiomycete class, which is at the base of the fungal phylogenetic tree. In this sense, some ancestral characteristics of fungi and animals or fungi and plants could have been retained in this aquatic fungus and lost in members of late-diverging fungal species. To identify in B. emersonii sequences associated with these ancestral characteristics two approaches were followed: (1) a large-scale comparative analysis between putative unigene sequences (uniseqs) from B. emersonii and three databases constructed ad hoc with fungal proteins, animal proteins and plant unigenes deposited in Genbank, and (2) a pairwise comparison between B. emersonii full-length cDNA sequences and their putative orthologues in the ascomycete Neurospora crassa and the basidiomycete Ustilago maydis. Results Comparative analyses of B. emersonii uniseqs with fungi, animal and plant databases through the two approaches mentioned above produced 166 B. emersonii sequences, which were identified as putatively absent from other fungi or not previously described. Through these approaches we found: (1) possible orthologues of genes previously identified as specific to animals and/or plants, and (2) genes conserved in fungi, but with a large difference in divergence rate in B. emersonii. Among these sequences, we observed cDNAs encoding enzymes from coenzyme B12-dependent propionyl-CoA pathway, a metabolic route not previously described in fungi, and validated their expression in Northern blots. Conclusion Using two different approaches involving comparative sequence analyses, we could identify sequences from the early-diverging fungus B. emersonii previously considered specific to animals or plants, and highly divergent sequences from the same fungus relative to other fungi.

Evaluating methods for the isolation of marine-derived fungal strains and production of bioactive secondary metabolites

In the present investigation we evaluate methods for the isolation and growth of marine-derived fungal strains in artificial media for the production of secondary metabolites. Inoculation of marine macroorganisms fragments in Petri dishes proved to be the most convenient procedure for the isolation of the largest number of strains. Among the growth media used, 3% malt extract showed the best result for strains isolation and growth, and yielded the largest number of strains from marine macroorganisms. The percentage of strains isolated using each of the growth media which yielded cytotoxic and/or antibiotic extracts was in the range of 23-35%, regardless of the growth media used. Further investigation of extracts obtained from different marine-derived fungal strains yielded several bioactive secondary metabolites, among which (E)-4-methoxy-5-(3-methoxybut-1-enyl)-6-methyl-2H-pyran-2-one is a new metabolite isolated from the Penicillium paxilli strain Ma(G)K.

Endophytic fungi from the Amazonian plant Paullinia cupana and from Olea europaea isolated using cassava as an alternative starch media source

Endophytic fungi live inside plants, apparently do not cause any harm to their hosts and may play important roles in defense and growth promotion. Fungal growth is a routine practice at microbiological laboratories, and the Potato Dextrose Agar (PDA) is the most frequently used medium because it is a rich source of starch. However, the production of potatoes in some regions of the world can be costly. Aiming the development of a new medium source to tropical countries, in the present study, we used leaves from the guarana (a tropical plant from the Amazon region) and the olive (which grows in subtropical and temperate regions) to isolate endophytic fungi using PDA and Manihot Dextrose Agar (MDA). Cassava (Manihot esculenta) was evaluated as a substitute starch source. For guarana, the endophytic incidence (EI) was 90% and 98% on PDA and MDA media, respectively, and 65% and 70% for olive, respectively. The fungal isolates were sequenced using the ITS- rDNA region. The fungal identification demonstrated that the isolates varied according to the host plant and media source. In the guarana plant, 13 fungal genera were found using MDA and six were found using PDA. In the olive plant, six genera were obtained using PDA and 4 were obtained using MDA. The multivariate analysis results demonstrated the highest fungal diversity from guarana when using MDA medium. Interestingly, some genera were isolated from one specific host or in one specific media, suggesting the importance of these two factors in fungal isolation specificity. Thus, this study indicated that cassava is a feasible starch source that could serve as a potential alternative medium to potato medium.

Effects of thyroid status on NEI concentration in specific brain areas related to reproduction during the estrous cycle

We previously showed that short-term hypo- and hyperthyroidism induce changes in neuropeptide glutamic-acid-isoleucine-amide (NEI) concentrations in discrete brain areas in male rats. To investigate the possible effects of hypo- and hyperthyroidism on NEI concentrations mainly in hypothalamic areas related to reproduction and behavior, female rats were sacrificed at different days of the estrous cycle. Circulating luteinizing hormone (LH), estradiol and progesterone concentrations were measured in control, hypothyroid (hypoT, treated with PTU during 7-9 days) and hyperthyroid (hyperT, l-T4 during 4-7 days) animals. Both treatments blunted the LH surge. Hypo- and hyperthyroidism increased estradiol concentrations during proestrus afternoon (P-PM), although hypoT rats showed lower values compared to control during proestrus morning (P-AM). Progesterone levels were higher in all groups at P-PM and in the hyperT during diestrus morning (D2). NEI concentrations were lower in hypoT rats during the estrous cycle except in estrus (E) in the peduncular part of the lateral hypothalamus (PLH). They were also reduced by both treatments in the perifornical part of the lateral hypothalamus (PeFLH) during P-PM. Hypothyroidism led to higher NEI concentrations during P-PM in the organum vasculosum of the lamina terminalis and anteroventral periventricular nucleus (OVLT+AVPV). The present results indicate that NEI concentration is regulated in a complex manner by hypo- and hyperthyroidism in the different areas studied, suggesting a correlation between NEI values and the variations of gonadal steroid levels during estrous cycle. These changes could be, in part, responsible for the alterations observed in the hypothalamic-pituitary-gonadal axis in these pathologies.

Binding of the wheat germ lectin to Cryptococcus neoformans chitooligomers affects multiple mechanisms required for fungal pathogenesis

The principal capsular component of Cryptococcus neoformans, glucuronoxylomannan (GXM), interacts with surface glycans, including chitin-like oligomers. Although the role of GXM in cryptococcal infection has been well explored, there is no information on how chitooligomers affect fungal pathogenesis. In this study, surface chitooligomers of C. neoformans were blocked through the use of the wheat germ lectin (WGA) and the effects on animal pathogenesis, interaction with host cells, fungal growth and capsule formation were analyzed. Treatment of C. neoformans cells with WGA followed by infection of mice delayed mortality relative to animals infected with untreated fungal cells. This observation was associated with reduced brain colonization by lectin-treated cryptococci. Blocking chitooligomers also rendered yeast cells less efficient in their ability to associate with phagocytes. WGA did not affect fungal viability, but inhibited GXM release to the extracellular space and capsule formation. In WGA-treated yeast cells, genes that are involved in capsule formation and GXM traffic had their transcription levels decreased in comparison with untreated cells. Our results suggest that cellular pathways required for capsule formation and pathogenic mechanisms are affected by blocking chitin-derived structures at the cell surface of C. neoformans. Targeting chitooligomers with specific ligands may reveal new therapeutic alternatives to control cryptococcosis.

On reproduction in red algae: futher research needed at the molecular level

[EN]Multicellular red algae (Rhodophyta) have some of the most complex life cycles known in living organisms. Economically valuable seaweeds, such as phycocolloid producers, have a triphasic (gametophyte, carposporophyte, and tetrasporophyte) life cycle, not to mention the intricate alternation of generations in the edible “sushi-alga” nori. It is a well-known fact that reproductive processes are controlled by one or more abiotic factor(s), including day length, light quality, temperature, and nutrients. Likewise, endogenous chemical factors such as plant growth regulators have been reported to affect reproductive events in some red seaweeds. Still, in the genomic era and given the high throughput techniques at our disposal, our knowledge about the endogenous molecular machinery lags far behind that of higher plants.

Transposable elements: structure and dynamic of the autonomous retrotransposon R2 in Arthropoda with non-canonical reproduction

Eukaryotic ribosomal DNA constitutes a multi gene family organized in a cluster called nucleolar organizer region (NOR); this region is composed usually by hundreds to thousands of tandemly repeated units. Ribosomal genes, being repeated sequences, evolve following the typical pattern of concerted evolution. The autonomous retroelement R2 inserts in the ribosomal gene 28S, leading to defective 28S rDNA genes. R2 element, being a retrotransposon, performs its activity in the genome multiplying its copy number through a “copy and paste” mechanism called target primed reverse transcription. It consists in the retrotranscription of the element’s mRNA into DNA, then the DNA is integrated in the target site. Since the retrotranscription can be interrupted, but the integration will be carried out anyway, truncated copies of the element will also be present in the genome. The study of these truncated variants is a tool to examine the activity of the element. R2 phylogeny appears, in general, not consistent with that of its hosts, except some cases (e.g. Drosophila spp. and Reticulitermes spp.); moreover R2 is absent in some species (Fugu rubripes, human, mouse, etc.), while other species have more R2 lineages in their genome (the turtle Mauremys reevesii, the Japanese beetle Popilia japonica, etc). R2 elements here presented are isolated in 4 species of notostracan branchiopods and in two species of stick insects, whose reproductive strategies range from strict gonochorism to unisexuality. From sequencing data emerges that in Triops cancriformis (Spanish gonochoric population), in Lepidurus arcticus (two putatively unisexual populations from Iceland) and in Bacillus rossius (gonochoric population from Capalbio) the R2 elements are complete and encode functional proteins, reflecting the general features of this family of transposable elements. On the other hand, R2 from Italian and Austrian populations of T. cancriformis (respectively unisexual and hermaphroditic), Lepidurus lubbocki (two elements within the same Italian population, gonochoric but with unfunctional males) and Bacillus grandii grandii (gonochoric population from Ponte Manghisi) have sequences that encode incomplete or non-functional proteins in which it is possible to recognize only part of the characteristic domains. In Lepidurus couesii (Italian gonochoric populations) different elements were found as in L. lubbocki, and the sequencing is still in progress. Two hypothesis are given to explain the inconsistency of R2/host phylogeny: vertical inheritance of the element followed by extinction/diversification or horizontal transmission. My data support previous study that state the vertical transmission as the most likely explanation; nevertheless horizontal transfer events can’t be excluded. I also studied the element’s activity in Spanish populations of T. cancriformis, in L. lubbocki, in L. arcticus and in gonochoric and parthenogenetic populations of B. rossius. In gonochoric populations of T. cancriformis and B. rossius I found that each individual has its own private set of truncated variants. The situation is the opposite for the remaining hermaphroditic/parthenogenetic species and populations, all individuals sharing – in the so far analyzed samples - the majority of variants. This situation is very interesting, because it isn’t concordant with the Muller’s ratchet theory that hypothesizes the parthenogenetic populations being either devoided of transposable elements or TEs overloaded. My data suggest a possible epigenetic mechanism that can block the retrotransposon activity, and in this way deleterious mutations don’t accumulate.

Reproduction and maternal health care among young women in Kenya: geographic and socio-economic determinants

Many factors influence the propensity of young women to seek appropriate maternal healthcare, and they need to be considered when analyzing these women’s reproductive behavior. This study aimed to contribute to the analysis concerning Kenyan young women’s determinants on maternal healthcare-seeking behavior for the 5 years preceding the 2008/9 Kenya Demographic and Health Survey. The specific objectives were to: investigate the individual and contextual variables that may explain maternal healthcare habits; measure the individual, household and community effect on maternal healthcare attitudes in young women; assess the link between young women’s characteristics and the use of facilities for maternal healthcare; find a relationship between young women’s behavior and the community where they live; examine how the role of the local presence of healthcare facilities influences reproductive behavior, and if the specificity of services offered by healthcare facilities affects their inclination to use healthcare facilities, and measure the geographic differences that influence the propensity to seek appropriate maternal healthcare. The analysis of factors associated with maternal healthcare-seeking behavior for young women in Kenya was investigated using multilevel models. We performed three major analyses, which concerned the individual and contextual determinants influencing antenatal care (discussed in Part 6), delivery care (Part 7), and postnatal care (Part 8). Our results show that there is a significant variation in antenatal, delivery and postnatal care between communities, even if the majority of variability is explained by individual characteristics. There are differences at the women’s level on the probability of receiving antenatal care and delivering in a healthcare facility instead of at home. Moreover, community factors and availability of healthcare facilities on the territory are also crucial in influencing young women’s behavior. Therefore, policies addressed to youth’s reproductive health should also consider geographic inequalities and different types of barriers in access to healthcare facilities.

Epidemiology and population genetics of Podosphaera fusca and Golovinomyces orontii, causal agents of cucurbit powdery mildew

In 2010, 2011 and 2012 growing seasons, the occurrence of the ascomycetes Podosphaera fusca and Golovinomyces orontii, causal agents of powdery mildew disease, was monitored on cultivated cucurbits located in Bologna and Mantua provinces to determine the epidemiology of the species. To identify the pathogens, both morphological and molecular identifications were performed on infected leaf samples and a Multiplex-PCR was performed to identify the mating type genes of P. fusca isolates. The investigations indicated a temporal succession of the two species with the earlier infections caused by G. orontii, that seems to be the predominant species till the middle of July when it progressively disappears and P. fusca becomes the main species infecting cucurbits till the end of October. The temporal variation is likely due to the different overwintering strategies of the two species instead of climatic conditions. Only chasmothecia of P. fusca were recorded and mating type alleles ratio tended to be 1:1. Considering that only chasmothecia of P. fusca were found, molecular-genetic analysis were carried out to find some evidence of recombination within this species by MLST and AFLP methods. Surprisingly, no variations were observed within isolates for the 8 MLST markers used. According to this result, AFLP analysis showed a high similarity within isolates, with SM similarity coefficient ranging between 0.91-1.00 and also, sequencing of 12 polymorphic bands revealed identity to some gene involved in mutation and selection. The results suggest that populations of P. fusca are likely to be a clonal population with some differences among isolates probably due to agricultural practices such as fungicides treatments and cultivated hosts. Therefore, asexual reproduction, producing a lot of fungal biomass that can be easily transported by wind, is the most common and useful way to the spread and colonization of the pathogen.