Características e controle da podridão "olho de boi" nas maçãs do sul do Brasil.

bitstream/item/55164/1/cir066.pd

Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1) Mutant Zebrafish.

Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b) have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing "dark-flash" visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations

Standardized and reproducible measurement of decision-making in mice

Progress in science requires standardized assays whose results can be readily shared, compared, and reproduced across laboratories. Reproducibility, however, has been a concern in neuroscience, particularly for measurements of mouse behavior. Here we show that a standardized task to probe decision-making in mice produces reproducible results across multiple laboratories. We designed a task for head-fixed mice that combines established assays of perceptual and value-based decision making, and we standardized training protocol and experimental hardware, software, and procedures. We trained 140 mice across seven laboratories in three countries, and we collected 5 million mouse choices into a publicly available database. Learning speed was variable across mice and laboratories, but once training was complete there were no significant differences in behavior across laboratories. Mice in different laboratories adopted similar reliance on visual stimuli, on past successes and failures, and on estimates of stimulus prior probability to guide their choices. These results reveal that a complex mouse behavior can be successfully reproduced across multiple laboratories. They establish a standard for reproducible rodent behavior, and provide an unprecedented dataset and open-access tools to study decision-making in mice. More generally, they indicate a path towards achieving reproducibility in neuroscience through collaborative open-science approaches

Risk factors of one year increment of coronary calcifications and survival in hemodialysis patients

<p>Abstract</p> <p>Background</p> <p>Heart and coronary calcifications in hemodialysis patients are of very common occurrence and linked to cardiovascular events and mortality. Several studies have been published with similar results. Most of them were mainly cross-sectional and some of the prospective protocols were aimed to evaluate the results of the control of altered biochemical parameters of mineral disturbances with special regard to serum calcium, phosphate and CaxP with the use of calcium containing and calcium free phosphate chelating agents. The aim of the present study was to evaluate in hemodialysis patients classic and some non classic risk factors as predictors of calcification changes after one year and to evaluate the impact of progression on survival.</p> <p>Methods</p> <p>81 patients on hemodialysis were studied, with a wide age range and HD vintage. Several classic parameters and some less classic risk factors were studied like fetuin-A, CRP, 25-OHD and leptin. Calcifications, as Agatston scores, were evaluated with Multislice CT basally and after 12-18 months.</p> <p>Results</p> <p>Coronary artery calcifications were observed in 71 of 81 patients. Non parametric correlations between Agatston scores and Age, HD Age, PTH and CRP were significant. Delta increments of Agatston scores correlated also with serum calcium, CaxP, Fetuin-A, triglycerides and serum albumin. Logistic regression analysis showed Age, PTH and serum calcium as important predictors of Delta Agatston scores. LN transformation of the not normally distributed variables restricted the significant correlations to Age, BMI and CRP. Considering the Delta Agatston scores as dependent, significant predictors were Age, PTH and HDL. A strong association was found between basal calcification scores and Delta increment at one year. By logistic analysis, the one year increments in Agatston scores were found to be predictors of mortality. Diabetic and hypertensive patients have significantly higher Delta scores.</p> <p>Conclusions</p> <p>Progression of calcification is of common occurrence, with special regard to elevated basal scores, and is predictive of survival. Higher predictive value of survival is linked to the one year increment of calcification scores. Some classic and non classic risk factors play an important role in progression. Some of them could be controlled with appropriate management with possible improvement of mortality.</p

Morphology of <i>stxbp1a</i>^s3000 mutant zebrafish.

<p>(A) Heterozygous <i>stxbp1a</i>^{<i>s3000/+</i>} mutant larvae (5 dpf) are morphologically indistinguishable from wild-type siblings. Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae are immobile and fail to hatch out of the chorion. Scale bar = 500 μm. (B) Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae (n = 10) removed from their chorions are not significantly different in length from their siblings (n = 30; p = 0.0592, two-tailed t-test). (C-D) At 5 dpf, the dorsal surface of homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae that were removed from their chorions at 2 dpf (D) show dispersed melanin and foreshortened craniofacial structure compared to siblings (C). Scale bar = 300 μm.</p

Zebrafish <i>stxbp1a</i> CRISPR/Cas9 mutant allele.

<p>(A) Sites of human and zebrafish mutations in highly conserved Stxbp1 sequence. Human STXBP1A protein sequence was aligned to zebrafish Stxbp1a and Stxbp1b. Black background indicates amino acid residues that are similar in all three proteins (BLOSUM 62). Grey background indicates amino acid residues that are similar between two of the three proteins. The salmon-colored background indicates the position of the deletion mutation in zebrafish mutant alleles. (B) Alignment of the mutant <i>stxbp1a</i>^s3000 allele sequence (top) with wild-type zebrafish <i>stxbp1a</i> sequence (bottom). The CRISPR/Cas9 target site is shown as a wide purple arrow below the plot. The site of the 4bp deletion <i>stxbp1a</i>^s3000 allele is highlighted in salmon, and corresponds to amino acids 211–212 highlighted in (A). (C) Alignment of the mutant <i>stxbp1b</i>^<i>s3001</i> allele sequence with wild-type zebrafish <i>stxbp1b</i> sequence.</p

Dark-flash response deficit in <i>stxbp1b</i> mutant zebrafish larvae.

<p>(A) Normal mobility in homozygous <i>stxbp1b</i>^<i>-/-</i> mutant larval zebrafish. Cumulative plots of the position and velocity of 10 representative wild-type larvae, 10 representative <i>stxbp1b</i>^{<i>s3001/+</i>} heterozygous mutants, and 10 <i>stxbp1b</i>^{<i>s30010/s3001</i>} homozygous mutants during 10 minutes of behavioral recording. Larval zebrafish (5 dpf) were placed in individual wells of a flat-bottom 96-well plate and acclimated to the Daniovision recording chamber before tracking began. Yellow indicates low velocity movement; red indicates high velocity movement. Scale bar = 1 cm. (B) Larval zebrafish (5 dpf) were placed in individual wells of a flat-bottom 96-well plate and acclimated to the Daniovision recording chamber. 24 hours of movement data were collected beginning at 4:00 PM. Data shown are sums of 10-minute bins (mean ± SD; n = 13 WT, 23 Het, 12 Mut). (C) Homozygous <i>stxbp1b</i>^{<i>s3001/s3001</i>} and heterozygous <i>stxbp1b</i>^{<i>s3001/+</i>} mutants’ baseline movement did not differ statistically from wild-type baseline movement. There were no significant differences in movement (seconds spent moving) between heterozygous <i>stxbp1b</i>^{<i>s3001/+</i>} and wild-type siblings (two-tailed t-tests, n = 13 WT, 23 Het, 12 Mut), or for velocity or distance traveled (not shown; mean ± SD). (D) In response to transition from 100% light intensity to darkness (0% light intensity), homozygous <i>stxbp1b</i>^{<i>s3001/s3001</i>} larvae (6 dpf) responded with less movement than either their wild-type siblings (two-tailed t-test, p = 0.021) or their heterozygous siblings (two-tailed t-test, p = 0.00024) mean ± SE (n = 13 WT, 23 Het, 12 Mut).</p

Ontogeny of <i>stxbp1a</i> and <i>stxbp1b</i> expression in zebrafish larvae.

<p>Wild-type zebrafish were probed for expression of <i>stxbp1a</i> mRNA (A-E) or <i>stxbp1b</i> mRNA (F-J). Abbreviations: CeP: cerebellar plate, Ha: habenula, IR: inner retina, MO: medulla oblongata, OB: olfactory bulb, P: pallium, Ret: retina, SC: spinal cord, Tel: telencephalon, TeO: optic tectum, Scale bar = 100μm.</p

Normal metabolic function in <i>stxbp1b</i>^<i>s3001</i> mutants.

<p>Homozygous <i>stxbp1b</i>^{<i>s30010/s3001</i>} mutant larvae have unaffected metabolism compared to controls (siblings). (A) 5 dpf <i>stxbp1b</i>^{<i>s30010/s3001</i>} homozygous mutants (n = 11) did not differ from siblings (n = 52) in extracellular acidification (ECAR) (two-tailed t-test; mean ± SD). (B) 5 dpf <i>stxbp1b</i>^{<i>s30010/s3001</i>} homozygous mutants (n = 11) did not differ from siblings (n = 52) in oxygen consumption (OCR) (two-tailed t-test; mean ± SD). (C) Heart rates of <i>stxbp1b</i>^{<i>s30010/s3001</i>} mutant larvae (n = 16) at 3 dpf were not significantly different from heart rates of their siblings (n = 34). All measured values are plotted; mean values are indicated by horizontal lines. (D) All <i>stxbp1b</i>^{<i>s30010/s3001</i>} mutant larvae tested (n = 8) survived until 10 dpf; 12.5% of their control siblings (n = 48) died by 10dpf.</p

Metabolic and survival deficits caused by <i>stxbp1a</i>^s3000 mutation.

<p>Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae have lower metabolism than controls (siblings) and die prematurely. (A) 5 dpf <i>stxbp1a</i>^{<i>s3000/s3000</i>} homozygous mutants (n = 10) had significantly lower extracellular acidification (ECAR) than siblings (n = 10; mean ± SD). * = p < 0.0001 (two-tailed t-test). (B) 5 dpf <i>stxbp1a</i>^{<i>s3000/s3000</i>} homozygous mutants (n = 10) had significantly lower oxygen consumption (OCR) than siblings (n = 10; mean ± SD). * = p < 0.0001 (two-tailed t-test). (C) Heart rates of <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae (n = 15) at 3 dpf were significantly lower than heart rates of their siblings (n = 33). All measured values are plotted; mean values are indicated by horizontal lines. (D) Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutants die as larvae. Homozygous mutants (n = 50) and siblings (n = 50) were maintained in petri dishes without food and counted each day from 2 dpf until 10dpf. The <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutants began dying at 6 dpf, and 98% (49) died by 10dpf. Only 2% (1) of siblings died at 10dpf.</p