Studies toward the structural optimization of novel thiazolylhydrazone-based potent antitrypanosomal agents

In previous studies, we identified promising anti-Trypanosoma cruzi cruzain inhibitors based on thiazolylhydrazones. To optimize this series, a number of medicinal chemistry directions were explored and new thiazolylhydrazones and thiosemicarbazones were thus synthesized. Potent cruzain inhibitors were identified, such as thiazolylhydrazones 3b and 3j, which exhibited IC(50) of 200-400nM. Furthermore, molecular docking studies showed concordance with experimentally derived structure-activity relationships (SAR) data. In the course of this work, lead compounds exhibiting in vitro activity against both the epimastigote and trypomastigote forms of T. cruzi were identified and in vivo general toxicity analysis was subsequently performed. Novel SAR were documented, including the importance of the thiocarbonyl carbon attached to the thiazolyl ring and the direct comparison between thiosemicarbazones and thiazolylhydrazones.     

The Extremophile Endolithella mcmurdoensis gen. et sp. nov. (Trebouxiophyceae,Chlorellaceae), A New Chlorella-like Endolithic Alga From Antarctica

The McMurdo Dry Valleys constitute the largest ice-free region of Antarctica and one of the most extreme deserts on Earth. Despite the low temperatures, dry and poor soils and katabatic winds, some microbes are able to take advantage of endolithic microenvironments, inhabiting the pore spaces of soil and constituting photosynthesis-based communities. We isolated a green microalga, Endolithella mcmurdoensis gen. et sp. nov, from an endolithic sandstone sample collected in the McMurdo Dry Valleys (Victoria Land, East Antarctica) during the K020 expedition, in January 2013. The single non-axenic isolate (E. mcmurdoensis LEGE Z-009) exhibits cup-shaped chloroplasts, electron-dense bodies, and polyphosphate granules but our analysis did not reveal any diagnostic morphological characters. On the basis of phylogenetic analysis of the 18S rRNA (SSU) gene, the isolate was found to represent a new genus within the family Chlorellaceae.     

Decreased length of telomeric DNA sequences and increased numerical chromosome aberrations in human osteoarthritic chondrocytes

Length of telomeric DNA sequences and numerical chromosome aberrations from uncultured human osteoarthritic (OA) articular chondrocytes were compared with those from peripheral blood leukocytes (PBL) from the same individual and from chondrocytes and PBL from control subjects. Cells were both obtained from 39 OA patients (age range: 43-80 years) and from 20 control subjects (age range: 39-94 years). Mean length of telomeric DNA sequences was determined using a quantitative real-time polymerase chain reaction (qPCR) assay and numerical chromosome aberrations were identified in interphase nuclei by Fluorescence In Situ Hybridization (FISH) using cocktails of specific DNA probes for chromosomes 7, 8 and for 18, X and Y. Chondrocytes revealed higher telomere size than PBL, both in control subjects and in OA patients, being 2 and 1.6 times higher respectively, thus revealing cell type specific differences. However, chondrocytes from OA patients showed significantly shorter telomere size than chondrocytes from control subjects (T/S ratio 1.64±0.41 vs. 1.99±0.54; mean±sd; p=0.008). Regarding the percentage of numerical chromosome aberrations, OA chondrocytes showed 1.7 times higher than chondrocytes from control subjects (19.80±3.31 vs.11.48±4.11; p<0.01) and 1.5 times average higher than that from PBL from the own OA patient (13.06±1.45; p<0.001). Moreover, PBL from OA patients also showed 1.4 times more anomalies than PBL from controls (13.06±1.45 vs. 9.54±1.61; p<0.001). No significant differences were found between chondrocytes and PBL in control subjects. Chromosome loss was the more frequent aneuploidy, mainly monosomy 18. The decreased telomere size and increased chromosome instability in chondrocytes from OA affected joints may imply a local advanced senescence that could contribute to the pathogenesis or progression of the degenerative articular disease. Moreover, the increased chromosomal abnormalities in PBL from OA patients suggest a more general accelerated senescence phenotype that could promote the age-related degenerative joint pathology.     

Pyrene-p-tert-butylcalixarenes inclusion complexes formation: a surface photochemistry study.

Diffuse reflectance and luminescence techniques were used to study the photophysics and photochemistry of pyrene within p-tert-butylcalix[n]arenes with n = 4, 6, and 8, and to study their ability to form inclusion complexes in heterogeneous media. Evidences for inclusion complex formation were found for the three hosts under study. Ground state diffuse reflectance results have shown the formation of ground state dimers of pyrene inside the cavity of calix[6]arene and calix[8]arene, with this feature much more evident for calix[6]arene. For calix[4]arene, only a monomer fits inside the cavity and the presence of pyrene microcrystals outside the cavity was detected. A luminescence lifetime distribution analysis was performed, revealing the presence of prompt emissions from the pyrene microcrystals outside the cavity in the case of calix[4]arene and from the constrained dimers inside the cavities of calix[6]arene and calix[8]arene. Transient absorption results have shown the presence of pyrene radical cation and also of trapped electrons for the three hosts under study. The formation of the phenoxyl radical of the calixarene following the laser pulsed excitation of pyrene at 355 nm is increased for calix[6]arene and calix[8]arene. This feature is particularly relevant for calix[6]arene, suggesting a very favourable situation for the hydrogen atom abstraction to occur. The analysis of the degradation products revealed the presence of hydroxypyrene as a major photodegradation product for the three hosts. Dihydro-hydroxypyrene was also formed in the case of calix[6]arene and calix[8]arene. The formation of the calixarene's phenoxyl radical and subsequent hydrogen abstraction is consistent with the formation of dihydro-dihydroxypyrene.     

Cork oak vulnerability to fire: the role of bark harvesting, tree characteristics and abiotic factors

Forest ecosystems where periodical tree bark harvesting is a major economic activity may be particularly vulnerable to disturbances such as fire, since debarking usually reduces tree vigour and protection against external agents. In this paper we asked how cork oak Quercus suber trees respond after wildfires and, in particular, how bark harvesting affects post-fire tree survival and resprouting. We gathered data from 22 wildfires (4585 trees) that occurred in three southern European countries (Portugal, Spain and France), covering a wide range of conditions characteristic of Q. suber ecosystems. Post-fire tree responses (tree mortality, stem mortality and crown resprouting) were examined in relation to management and ecological factors using generalized linear mixed-effects models. Results showed that bark thickness and bark harvesting are major factors affecting resistance of Q. suber to fire. Fire vulnerability was higher for trees with thin bark (young or recently debarked individuals) and decreased with increasing bark thickness until cork was 3-4 cm thick. This bark thickness corresponds to the moment when exploited trees are debarked again, meaning that exploited trees are vulnerable to fire during a longer period. Exploited trees were also more likely to be top-killed than unexploited trees, even for the same bark thickness. Additionally, vulnerability to fire increased with burn severity and with tree diameter, and was higher in trees burned in early summer or located in drier south-facing aspects. We provided tree response models useful to help estimating the impact of fire and to support management decisions. The results suggested that an appropriate management of surface fuels and changes in the bark harvesting regime (e.g. debarking coexisting trees in different years or increasing the harvesting cycle) would decrease vulnerability to fire and contribute to the conservation of cork oak ecosystems.     

Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams

The present study is focused on the proteome of reproductive tract fluids from tropically-adapted Santa Ines rams. Seminal plasma, cauda epididymal (CEF) and vesicular gland fluid (VGF) proteins were analyzed by 2-D electrophoresis and mass spectrometry. Seminal plasma maps contained 302 ± 16 spots, within the 4-7 pH range. From these maps, 73 spots were identified, corresponding to 41 proteins. Ram Seminal Vesicle Proteins (RSVP) 14 and 22kDa and bodhesins 1 and 2 represented the most abundant seminal components. Other seminal proteins included clusterin, angiotensin-converting enzyme, matrix metalloproteinase-2, tissue-inhibitor of metalloproteinase-2, plasma glutamate carboxypeptidase, albumin, lactoferrin, alpha enolase, peroxiredoxin, leucine aminopeptidase, β-galactosidase, among others. Later, seminal plasma gels were run within narrow pH intervals (3.9-5.1; 4.7-5.9; 5.5-6.7), allowing the additional identification of 21 proteins not detected in 4-7 pH maps. Major proteins of CEF and VGF were albumin and transferrin, and RSVPs, respectively. Western blots confirmed that RSVPs were mainly present in VGF while bodhesins, in VGF and CEF. Based on RT-PCR, RSVP and bodhesin genes were primarily expressed in the vesicular glands. In summary, the reproductive tract fluids of Brazilian hairy rams contain several categories of proteins, with potential roles in sperm protection, capacitation, acrosome reaction and sperm-oocyte interaction.     

Multi-gene analysis and morphology reveal novel Ilyonectria species associated with black foot disease of grapevines

Black foot is an important disease of grapevines, which has in recent years been recorded with increased incidence and severity throughout the world, affecting grapevines both in nurseries and young vineyards. In the past the disease has been associated with infections by Ilyonectria macrodidyma, Ilyonectria liriodendri, Campylocarpon fasciculare, and Campylocarpon pseudofasciculare. Based on published data, a high level of genetic diversity was detected among isolates of I. macrodidyma. To resolve this issue, we employed a multigene analysis strategy (based on the β-tubulin, histone H3, translation elongation factor 1-α, and the internal transcribed spacers on both sides of the 5.8S nuclear ribosomal RNA gene) along with morphological characterisation to study a collection of 81 I. macrodidyma-like isolates from grapevine and other hosts. Morphological characters (particularly conidial size) and molecular data (highest resolution achieved with histone H3 nucleotide sequence) enabled the distinction of six monophyletic species within the I. macrodidyma complex, four of which (Ilyonectria alcacerensis, Ilyonectria estremocensis, Ilyonectria novozelandica, and Ilyonectria torresensis) are described here. This work forms part of an effort by the International Council on Grapevine Trunk Diseases to resolve the species associated with black foot disease, which we believe will clarify their taxonomy, and therefore help researchers to devise control strategies to reduce the devastating impact of this disease.     

Low prevalence of glucokinase gene mutations in gestational diabetic patients with good glycemic control

Glucokinase (GCK) plays a key role in glucose homeostasis. Gestational diabetes mellitus increases the risk of gestational complications in pregnant women and fetuses. We screened for mutations in coding and flanking regions of the GCK gene in pregnant women with or without gestational diabetes in a Brazilian population. A sample of 200 pregnant women classified as healthy (control, N = 100) or with gestational diabetes (N = 100) was analyzed for mutations in the GCK gene. All gestational diabetes mellitus patients had good glycemic control maintained by diet alone and no complications during pregnancy. Mutations were detected by single-strand conformation polymorphism and DNA sequencing. Thirteen of the 200 subjects had GCK gene mutations. The mutations detected were in intron 3 (c.43331A>G, new), intron 6 (c.47702T>C, rs2268574), intron 9 (c.48935C>T, rs2908274), and exon 10 (c.49620G>A, rs13306388). None of these GCK mutations were found to be significantly associated with gestational diabetes mellitus. In summary, we report a low frequency of GCK mutations in a pregnant Brazilian population and describe a new intronic variation (c.43331A>G, intron 3). We conclude that mutations in GCK introns and in non-translatable regions of the GCK gene do not affect glycemic control and are not correlated with gestational diabetes mellitus.     

Dominant-Negative Effects of Adult-Onset Huntingtin Mutations Alter the Division of Human Embryonic Stem Cells-Derived Neural Cells

Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington’s disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150^Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150^Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150^Glued complex interactions.