Interference of aging media on the assessment of yeast chronological life span by propidium iodide staining

An increasing number of researchers are using the Saccharomyces cerevisiae chronological aging model to gain insight into the post-mitotic cellular aging. Recently, an alternative approach to the traditional cellular viability assay by colony-forming unit (CFU) counts, based on the propidium iodide (PI) staining combined with flow cytometry (PI-FCM), was proposed for the assessment of yeast chronological aging. Since the chronological aging assessment shows variations particularly concerning the aging media, in this work, the influence of the most common aging media (exhausted media or water) on the assessment of chronological aging by PI staining was studied. Our results show that this methodology is highly affected by the aging media. Indeed, a correlation between CFU counts and the percentage of PI-stained cells is only achieved with the exhausted media. As such, the assessment of yeast chronological aging by PI-FCM water should not be used.     

Effects of an evaporative cooling system on plasma cortisol, IGF-I, and milk production in dairy cows in a tropical environment

Access to an evaporative cooling system can increase production in dairy cows because of improved thermal comfort. This study aimed to evaluate the impact of ambient temperature on thermoregulation, plasma cortisol, insulin-like growth factor 1 (IGF-I), and productive status, and to determine the efficiency of an evaporative cooling system on physiological responses under different weather patterns. A total of 28 Holstein cows were divided into two groups, one with and the other without access to a cooling system with fans and mist in the free stall. The parameters were analyzed during morning (0700 hours) and afternoon milking (1430 hours) under five different weather patterns throughout the year (fall, winter, spring, dry summer, and rainy summer). Rectal temperature (RT), body surface temperature (BS), base of tail temperature (TT), and respiratory frequency (RF) were lower in the morning (P?<?0.01). The cooling system did not affect RT, and both the groups had values below 38.56 over the year (P?=?0.11). Cortisol and IGF-I may have been influenced by the seasons, in opposite ways. Cortisol concentrations were higher in winter (P?<?0.05) and IGF-I was higher during spring-summer (P?<?0.05). The air temperature and the temperature humidity index showed positive moderate correlations to RT, BS, TT, and RF (P?<?0.001). The ambient temperature was found to have a positive correlation with the physiological variables, independent of the cooling system, but cooled animals exhibited higher milk production during spring and summer (P?<?0.01).     

Using common mycorrhizal networks for controlled inoculation of Quercus spp. with Tuber melanosporum: the nurse plant method

The high cost and restricted availability of black truffle spore inoculum for controlled mycorrhiza formation of host trees produced for truffle orchards worldwide encourage the search for more efficient and sustainable inoculation methods that can be applied globally. In this study, we evaluated the potential of the nurse plant method for the controlled inoculation of Quercus cerris and Quercus robur with Tuber melanosporum by mycorrhizal networks in pot cultures. Pine bark compost, adjusted to pH?7.8 by liming, was used as substrate for all assays. Initially, Q. robur seedlings were inoculated with truffle spores and cultured for 12 months. After this period, the plants presenting 74 % mycorrhizal fine roots were transferred to larger containers. Nurse plants were used for two treatments of two different nursling species: five sterilized acorns or five 45-day-old, axenically grown Q. robur or Q. cerris seedlings, planted in containers around the nurse plant. After 6 months, colonized nursling plant root tips showed that mycorrhiza formation by T. melanosporum was higher than 45 % in the seedlings tested, with the most successful nursling combination being Q. cerris seedlings, reaching 81 % colonization. Bulk identification of T. melanosporum mycorrhizae was based on morphological and anatomical features and confirmed by sequencing of the internal transcribed spacer region of the ribosomal DNA of selected root tips. Our results show that the nurse plant method yields attractive rates of mycorrhiza formation by the Périgord black truffle and suggest that establishing and maintaining common mycorrhizal networks in pot cultures enables sustained use of the initial spore inoculum.     

Disturbances, elevation, topography and spatial proximity drive vegetation patterns along an altitudinal gradient of a top biodiversity hotspot

The correlation between vegetation patterns (species distribution and richness) and altitudinal variation has been widely reported for tropical forests, thereby providing theoretical basis for biodiversity conservation. However, this relationship may have been oversimplified, as many other factors may influence vegetation patterns, such as disturbances, topography and geographic distance. Considering these other factors, our primary question was: is there a vegetation pattern associated with substantial altitudinal variation (10–1,093 m a.s.l.) in the Atlantic Rainforest—a top hotspot for biodiversity conservation—and, if so, what are the main factors driving this pattern? We addressed this question by sampling 11 1-ha plots, applying multivariate methods, correlations and variance partitioning. The Restinga (forest on sandbanks along the coastal plains of Brazil) and a lowland area that was selectively logged 40 years ago were floristically isolated from the other plots. The maximum species richness (>200 spp. per hectare) occurred at approximately 350 m a.s.l. (submontane forest). Gaps, multiple stemmed trees, average elevation and the standard deviation of the slope significantly affected the vegetation pattern. Spatial proximity also influenced the vegetation pattern as a structuring environmental variable or via dispersal constraints. Our results clarify, for the first time, the key variables that drive species distribution and richness across a large altitudinal range within the Atlantic Rainforest.     

Diversity and abundance of climbers from the Atlantic Forest, southeastern Brazil

Climbing plant community composition and abundance was compared between the Alto da Serra de Paranapiacaba Biological Reserve (ASPRB) and the Nascentes de Paranapiacaba Municipal Natural Park (NPMNP), two protected areas in the largest Atlantic Forest remnant, southeastern Brazil. Climbing plants ≥1 cm in diameter were sampled in 52 quadrats of 10 × 20 m (1.04 ha). From these data we calculated the Shannon–Wiener diversity index, calculated abundance and importance values for families, performed a principal coordinate analysis, and created a similarity dendrogram (TWINSPAN). The composition and abundance of species between ASPRB and NPMNP differed significantly due to the successional state of the ASPRB forest, which was affected by more recent disturbance. There we found 34 species, while in the NPMNP, species richness was 72, with only 24 species in common. The two areas formed two groups corresponding to two distinct floristic subsets. These montane forests were characterized by a predominance of species of the Asteraceae. The high species richness, floristic composition, and community composition reflect the greater degree of conservation in the area of NPMNP. The high density and community composition of climbers in the more conserved area of rainforest may be due to the low intensity of disturbance.     

Alarm scent-marking during predatory attempts in the Cabrera vole (Microtus cabrerae Thomas, 1906)

The alarm pheromones often released by animals under stressful situations seem to elicit behavioral changes in conspecifics, which in the appropriate context can be viewed as anti-predatory responses. However, the releasing of alarm pheromones associated with predatory events has not been demonstrated in mammals. In the current study with wild-caught Cabrera voles, we carried out experiments in the laboratory and in the field to assess the release of alarm pheromones in scent-marks during simulated predatory events and disclose their effects on conspecifics. We first conducted an assay wherein voles where let to scent-mark a clean substrate in the absence of disturbance (control) and under the simulation of predatory events. Contrarily to the control, no fecal boli were released and the area marked with urine was significantly larger during the predatory simulation. In a subsequent assay, we assessed the voles’ preference between urine-marks released under predatory simulation and in control conditions. Voles showed a significant preference by control substrates. Finally, a third assay was carried out in the vole’s habitat wherein the individual activity was monitored by radio-tracking before and after placement of urine-marks obtained during the conditions described above. The vole’s activity was only reduced near the urine-marks released during the simulated predatory events. The results suggest that: (1) during predatory attempts, Cabrera voles release an alarm pheromone in their urine-marks; (2) the putative alarm pheromone reduces the voles’ activity in the surroundings of the marked area; (3) the putative alarm pheromone persists in the field affecting conspecifics’ activity for several days.     

Nosocomial Bloodstream Infections in Brazilian Pediatric Patients: Microbiology,Epidemiology, and Clinical Features

Background

Nosocomial bloodstream infections (nBSIs) are an important cause of morbidity and mortality and are the most frequent type of nosocomial infection in pediatric patients.

Methods

We identified the predominant pathogens and antimicrobial susceptibilities of nosocomial bloodstream isolates in pediatric patients (≤16 years of age) in the Brazilian Prospective Surveillance for nBSIs at 16 hospitals from 12 June 2007 to 31 March 2010 (Br SCOPE project).

Results

In our study a total of 2,563 cases of nBSI were reported by hospitals participating in the Br SCOPE project. Among these, 342 clinically significant episodes of BSI were identified in pediatric patients (≤16 years of age). Ninety-six percent of BSIs were monomicrobial. Gram-negative organisms caused 49.0% of these BSIs, Gram-positive organisms caused 42.6%, and fungi caused 8.4%. The most common pathogens were Coagulase-negative staphylococci (CoNS) (21.3%), Klebsiella spp. (15.7%), Staphylococcus aureus (10.6%), and Acinetobacter spp. (9.2%). The crude mortality was 21.6% (74 of 342). Forty-five percent of nBSIs occurred in a pediatric or neonatal intensive-care unit (ICU). The most frequent underlying conditions were malignancy, in 95 patients (27.8%). Among the potential factors predisposing patients to BSI, central venous catheters were the most frequent (66.4%). Methicillin resistance was detected in 37 S. aureus isolates (27.1%). Of the Klebsiella spp. isolates, 43.2% were resistant to ceftriaxone. Of the Acinetobacter spp. and Pseudomonas aeruginosa isolates, 42.9% and 21.4%, respectively, were resistant to imipenem.

Conclusions

In our multicenter study, we found a high mortality and a large proportion of gram-negative bacilli with elevated levels of resistance in pediatric patients.     

Assessment of the Relationship between Self-Declared Ethnicity,Mitochondrial Haplogroups and Genomic Ancestry in Brazilian Individuals

In populations that have a high degree of admixture, such as in Brazil, the sole use of ethnicity self-declaration information is not a good method for classifying individuals regarding their ethnicity. Here, we evaluate the relationship of self-declared ethnicities with genomic ancestry and mitochondrial haplogroups in 492 individuals from southeastern Brazil. Mitochondrial haplogroups were obtained by analyzing the hypervariable regions of the mitochondrial DNA (mtDNA), and the genomic ancestry was obtained using 48 autosomal insertion-deletion ancestry informative markers (AIM). Of the 492 individuals, 74.6% self-declared as White, 13.8% as Brown and 10.4% as Black. Classification of the mtDNA haplogroups showed that 46.3% had African mtDNA, and the genomic ancestry analysis showed that the main contribution was European (57.4%). When we looked at the distribution of mtDNA and genomic ancestry according to the self-declared ethnicities from 367 individuals who self-declared as White, 37.6% showed African mtDNA, and they had a high contribution of European genomic ancestry (63.3%) but also a significant contribution of African ancestry (22.2%). Of the 68 individuals who self-declared as Brown, 25% showed Amerindian mtDNA and similar contribution of European and African genomic ancestries. Of the 51 subjects who self-declared as black, 80.4% had African mtDNA, and the main contribution of genomic ancestry was African (55.6%), but they also had a significant proportion of European ancestry (32.1%). The Brazilian population had a uniform degree of Amerindian genomic ancestry, and it was only with the use of genetic markers (autosomal or mitochondrial) that we were able to capture Amerindian ancestry information. Additionally, it was possible to observe a high degree of heterogeneity in the ancestry for both types of genetic markers, which shows the high genetic admixture that is present in the Brazilian population. We suggest that in epidemiological studies, the use of these methods could provide complementary information.     

Silencing Mutant Ataxin-3 Rescues Motor Deficits and Neuropathology in Machado-Joseph Disease Transgenic Mice

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein – ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.