Ribo-seQC: comprehensive analysis of cytoplasmic and organellar ribosome profiling data

Summary: Ribosome profiling enables genome-wide analysis of translation with unprecedented resolution. We present Ribo-seQC, a versatile tool for the comprehensive analysis of Ribo-seq data, providing in-depth insights on data quality and translational profiles for cytoplasmic and organelle ribosomes. Ribo-seQC automatically generates platform-independent HTML reports, offering a detailed and easy-to-share basis for collaborative Ribo-seq projects. Availability: Ribo-seQC is available at https://github.com/ohlerlab/RiboseQC and submitted to Bioconductor. Contact: uwe.ohler{at}mdc-berlin.d

Reactivity of Nelore cows during reproductive management

The Nelore breed is recognized as rustic and well adapted to parasites and high temperatures, and these features composes a good part of the Zebu genetic group. However, they are considered more reactive than other breeds and for this reason they require more time to be managed, putting at risk the safety of employees, the animals themselves and also compromise installations. In order to solve problems such as short duration and high nocturnal occurrence of oestrus many properties choose to perform artificial insemination during the breeding season. At this time, there is an intensification of human-animal interaction in short periods and at different times. When animals are more reactive on handling and are exposed to an uncomfortable thermal environment the stress rises causing impacts on reproduction and production. Stress is comprehended by a set of behavioral and physiological changes when in threatening or uncomfortable situations caused by stressors (physical environment, social or management practices). Some farms have already made the selection for temperament, using different methods of evaluation in accordance with the conditions of creation and infrastructure available at the property. Thus, the aim of this research was to evaluate the reactivity of Nelore cows in different managements that occur in the breeding season. Was evaluated 20 multiparous Nelore cows from October 2013 to March 2014, during the spring and summer seasons with air temperature averages ranging from 22.7 ± 4.9°C to 25.2 ± 4.7°C, in the Campus’ beef cattle sector of the Universidade de São Paulo in Pirassununga. Reactivity was evaluated using scores of escape velocity or output speed using subjective scores: 1 - does not leave the chute, needs to be touched; 2 - walking out the chute; 3 - exits the chute at average speed; 4 – exists the chute quickly. So, as higher is the score as higher is the stress. The scores were given immediately after the animals exists the chute from the managements of sync (M1), artificial insemination (M2), pregnancy diagnosis at 30 days (M3), pregnancy diagnosis at 60 days (M4), pregnancy diagnosis at 90 days (M5). The data of the escape score was analyzed using analysis of variance, using the GLM procedure of SAS and means were compared using the Tukey test at 5%. It was noted that, there was no differences on the average of escape score (2.34) at the managements M1, M2, M3 and M4. However, in management M5 the average was 1.79 differing from the other managements (P<0.05). In this way, at the last management, there was a reduction of 23.50% in escape score. So, we conclude that with the increase in frequency of human-animal interactions during the reproductive managements, there is a decrease in the escape score and consequently the stress, contributing to the agility on each management, security of employees, lower physical trauma on animals and damages to facilities

Reactivity in ewes submitted to invasive and non-invasive techniques of samples collections

The first-time experience of a procedure or with a person should be as positive as possible. If a procedure is very aversive or painful at first contact, it can be difficult to persuade the animal to return to the local where this occurred. For this reason it is important to develop a strategy in experimental and routine activities, taking into account that sheep has often reactive temperament. This study aims to evaluate the possible decreased of the reactivity in sheep across the prior presentation of invasive and non-invasive techniques for collecting saliva and blood samples for cortisol analysis and transepidermal water loss through the use of Vapometer®. Saliva was collected using Sallivetes® and blood was taken from the jugular vein in heparinized tubes, placed on ice, and then centrifuged at 4 °C and 3,000 for 15 min. The tubes containing serum were stored at −20 °C until CORT and IGF-I determination using an enzyme immunoassay kit (Diagnostic Systems Laboratory, Webster, TX). The collection of transepidermal water loss (TEWL) was performed using a Vapometer ® (Dolphin, Finland) device. In the three techniques the ewes were always contained by the same person for the time necessary to collect the variable. To take saliva samples the animals were contained during 2 minutes, for blood samples during 1 minute and for TEWL during 30 seconds. The test consists of six data collections performed with increasing intervals between days, checking the possible adaptation of the animal management factors. The first interval was 2 days and after 3 days, then after 4 days, and the next was 7 days and again 7 days, totaling 24 days of experiment. Before sampling saliva, blood or TEWL the reactivity of the ewes was analyzed by a composite score scale when animals were inside the chute. The composite score (CS) is a combination of the scores of: entrance (1 to 4), respiratory rate (1 to 4), vocalization (0 or 1), movement (1 to 4) and flight speed (1 to 4). After the chute a score of containment (1 to 4) was attributed by the same person, which had contained the animal. The experimental unit was each animal and the average of the observations in each sheep was used for the statistical analysis. The observed data of transepidermal water loss, salivary cortisol and serum cortisol were subjected to analysis of variance, with the days and the groups as fixed effect. In case of significant results (P<0.05) it was adopted Tukey-Kramer Test as the procedure for multiple comparisons. Cortisol decreased for saliva and blood groups during the experiment days (P<0.05), showing habituation of the animals to the management. In the same way, TEWL had the highest mean value on day 1, and decreased afterwards (P<0.05). The score of containment was lower for the TEWL group compared to the other two groups (P<0.05), however the higher value was observed on day 5, seven days after a sampling (P<0.05). The score of containment of saliva and blood groups decrease in day 2, but return to increase in the next sampling day (P<0.05). The CS was higher for saliva group (P<0.05). On day 5 CS increased for blood group, but decreased for TEWL (P<0.05). The invasive techniques (saliva and blood) increased the reactivity of sheep before and after retention. Sheep are reactive animals therefore need a more gentle handling during the experiment with invasive samples

Anticancer properties of propofol-docosahexaenoate and propofol-eicosapentaenoate on breast cancer cells

INTRODUCTION: Epidemiological evidence strongly links fish oil, which is rich in docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), with low incidences of several types of cancer. The inhibitory effects of omega-3 polyunsaturated fatty acids on cancer development and progression are supported by studies with cultured cells and animal models. Propofol (2,6-diisopropylphenol) is the most extensively used general anesthetic–sedative agent employed today and is nontoxic to humans at high levels (50 μg/ml). Clinically relevant concentrations of propofol (3 to 8 μg/ml; 20 to 50 μM) have also been reported to have anticancer activities. The present study describes the synthesis, purification, characterization and evaluation of two novel anticancer conjugates, propofol-docosahexaenoate (propofol-DHA) and propofol-eicosapentaenoate (propofol-EPA). METHODS: The conjugates linking an omega-3 fatty acid, either DHA or EPA, with propofol were synthesized and tested for their effects on migration, adhesion and apoptosis on MDA-MB-231 breast cancer cells. RESULTS: At low concentrations (25 μM), DHA, EPA or propofol alone or in combination had minimal effect on cell adhesion to vitronectin, cell migration against serum and the induction of apoptosis (only 5 to 15% of the cells became apoptotic). In contrast, the propofol-DHA or propofol-EPA conjugates significantly inhibited cell adhesion (15 to 30%) and migration (about 50%) and induced apoptosis (about 40%) in breast cancer cells. CONCLUSION: These results suggest that the novel propofol-DHA and propofol-EPA conjugates reported here may be useful for the treatment of breast cancer

RNA localization is a key determinant of neurite-enriched proteome

Protein subcellular localization is fundamental to the establishment of the body axis, cell migration, synaptic plasticity, and a vast range of other biological processes. Protein localization occurs through three mechanisms: protein transport, mRNA localization, and local translation. However, the relative contribution of each process to neuronal polarity remains unknown. Using neurons differentiated from mouse embryonic stem cells, we analyze protein and RNA expression and translation rates in isolated cell bodies and neurites genome-wide. We quantify 7323 proteins and the entire transcriptome, and identify hundreds of neurite-localized proteins and locally translated mRNAs. Our results demonstrate that mRNA localization is the primary mechanism for protein localization in neurites that may account for half of the neurite-localized proteome. Moreover, we identify multiple neurite-targeted non-coding RNAs and RNA-binding proteins with potential regulatory roles. These results provide further insight into the mechanisms underlying the establishment of neuronal polarity. Subcellular localization of RNAs and proteins is important for polarized cells such as neurons. Here the authors differentiate mouse embryonic stem cells into neurons, and analyze the local transcriptome, proteome, and translated transcriptome in their cell bodies and neurites, providing a unique resource for future studies on neuronal polarity

DDX3 depletion represses translation of mRNAs with complex 5' UTRs

DDX3 is an RNA chaperone of the DEAD-box family that regulates translation. Ded1, the yeast ortholog of DDX3, is a global regulator of translation, whereas DDX3 is thought to preferentially affect a subset of mRNAs. However, the set of mRNAs that are regulated by DDX3 are unknown, along with the relationship between DDX3 binding and activity. Here, we use ribosome profiling, RNA-seq, and PAR-CLIP to define the set of mRNAs that are regulated by DDX3 in human cells. We find that while DDX3 binds highly expressed mRNAs, depletion of DDX3 particularly affects the translation of a small subset of the transcriptome. We further find that DDX3 binds a site on helix 16 of the human ribosomal rRNA, placing it immediately adjacent to the mRNA entry channel. Translation changes caused by depleting DDX3 levels or expressing an inactive point mutation are different, consistent with different association of these genetic variant types with disease. Taken together, this work defines the subset of the transcriptome that is responsive to DDX3 inhibition, with relevance for basic biology and disease states where DDX3 is altered

Consumption of an omega-3 fatty acids product, INCELL AAFA™, reduced side-effects of CPT-11 (irinotecan) in mice

INCELL AAFA™, an omega-3 polyunsaturated fatty acid product containing a high concentration of long chain fatty acids, was tested for its ability to ameliorate the harmful side effects of CPT-11 chemotherapy including: leukopenia, anaemia, asthenia, weight loss and liver involvement. Four groups of mice were fed an AIN-76 diet modified to contain: 10% w/w corn oil (CO), 0% AAFA™; 9% CO, 1% AAFA™; 8% CO, 2% AAFA™; or 7% CO, 3% AAFA™. After 2 weeks on the diets, half of the mice received CPT-11 chemotherapy (60 mg kg−1 q 4 days, i.v.) the rest of the mice received vehicle for 2 weeks. It was found that 2% AAFA™ in the diet of the CPT-11 treated mice: decreased apoptotic figures in the duodenal crypts; markedly suppressed the inflammatory eicosanoid, prostaglandin E2 in the liver; prevented liver hypertrophy; improved white blood cell counts; significantly increased red blood cell counts; decreased numbers of CPT-11 induced immature red blood cell and micronuclei in red blood cells of the peripheral blood; increased eicosapentaenoic acid and docosahexaenoic acid in liver cell membranes and maintained normal grooming behaviour. Thus 2% AAFA™ in the diet reduced the side effects of CPT-11 treatment in mice