Acute and chronic pain in calves after different methods of rubber-ring castration

The goal of the present study was to evaluate the effect of different methods of rubber-ring castration on acute and chronic pain in calves. Sixty-three 4-6 week-old calves were randomly and sequentially allocated to one of five groups: Group RR (traditional rubber ring castration); group BRR (combination of one rubber ring with Burdizzo); group Rcut (one rubber ring applied with the scrotal tissue and rubber ring removed on day 9); group 3RR (three rubber rings placed one above the other around the scrotal neck); and group CO (controls; sham-castrated). All calves received 0.2 mL/kg bodyweight lidocaine 2%, injected into the spermatic cords and around the scrotal neck 15 min before castration. The presence of acute and chronic pain was assessed using plasma cortisol concentrations, response to palpation of scrotal area, time from castration until complete wound healing, and behavioural signs. Calves of group 3RR showed severe swelling and inflammation, and licking of the scrotal area occurred significantly more often than in groups Rcut and CO. Technique 3RR was discontinued for welfare reasons before the end of the study. All castration groups had significantly more pain upon palpation than calves of group CO, but palpation elicited markedly less pain in group Rcut than in the other castration groups. The most rapid healing time and shortest duration of chronic pain after castration was achieved in group Rcut. For welfare reasons, the Rcut technique should be considered as a valuable alternative to traditional rubber ring castration of calves at 4-6 weeks of age.

An mRNA-derived ncRNA targets and regulates the ribosome

Small non-protein-coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily, not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. This is unexpected, given the central position the ribosome plays during gene expression. To investigate whether such a class of regulatory ncRNAs does exist we performed genomic screens for small ribosome-associated RNAs in various model organisms of all three domains [1,2]. Here we focus on the functional characterisation of an 18 nucleotide long ncRNA candidate derived from an open reading frame (ORF) of an annotated S. cerevisiae gene, which encodes a tRNA methyltransferase. Yeast cells lacking this tRNA methyltransferase showed clear growth defects in high salt containing media. Genetic analysis showed that the absence of the mRNA-derived ncRNA rather than the absence of the tRNA methyltransferase activity is responsible for the observed phenotype. Since we performed a screen for small ribosome-associated RNAs we examined the regulatory potential of the synthetic 18mer during translation in vitro and in vivo. Metabolic labeling experiments in the presence of the synthetic 18mer RNA revealed an inhibitory potential on the global protein biosynthesis rate. In vitro translation and northern blot analysis further strengthen the hypothesis, that this RNA is a ribosome-associated regulatory ncRNA. Our studies in pro- and eukaryotic model organisms reveal the ribosome as a novel target for small regulatory ncRNAs in all domains of life. Ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory ncRNAs.

An mRNA-derived ncRNA targets and regulates the ribosome

Small non-protein-coding RNA (ncRNA) molecules are key players in controlling gene expression at multiple steps in all domains of life. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily (such as micro RNAs and small-interfering RNAs), not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). This is unexpected, given the central position the ribosome plays during gene expression. To investigate whether such a class of regulatory ncRNAs does exist we performed genomic screens for small ribosome-associated RNAs in various model organisms of all three domains [1,2]. Here we show that an mRNA-derived 18 nucleotide long ncRNA is capable of down-regulating translation in Saccharomyces cerevisiae by directly targeting the ribosome [3]. This 18-mer ncRNA binds to polysomes upon salt stress and is crucial for efficient growth under hyperosmotic conditions. Although the 18-mer RNA originates from the TRM10 locus, which encodes a tRNA methyltransferase, genetic analyses revealed the 18-mer RNA nucleotide sequence, rather than the mRNA-encoded enzyme, as the translation regulator under these stress conditions. Our data reveal the ribosome as a target for small regulatory ncRNAs and unveil the existence of a novel mechanism of translation regulation. Analogous genomic screens in organisms spanning all three domains of life demonstrate the existence of thousands of ncRNA candidates putatively regulating the ribosome. We therefore anticipate that ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory ncRNAs.

Trypanosoma brucei RRM1 is a nuclear RNA-binding protein and modulator of chromatin structure

TbRRM1 of Trypanosoma brucei is a nucleoprotein that was previously identified in a search for splicing factors in T. brucei. We show that TbRRM1 associates with mRNAs and with the auxiliary splicing factor polypyrimidine tract-binding protein 2, but not with components of the core spliceosome. TbRRM1 also interacts with several retrotransposon hot spot (RHS) proteins and histones. RNA immunoprecipitation of a tagged form of TbRRM1 from procyclic (insect) form trypanosomes identified ca. 1,500 transcripts that were enriched and 3,000 transcripts that were underrepresented compared to cellular mRNA. Enriched transcripts encoded RNA-binding proteins, including TbRRM1 itself, several RHS transcripts, mRNAs with long coding regions, and a high proportion of stage-regulated mRNAs that are more highly expressed in bloodstream forms. Transcripts encoding ribosomal proteins, other factors involved in translation, and procyclic-specific transcripts were underrepresented. Knockdown of TbRRM1 by RNA interference caused widespread changes in mRNA abundance, but these changes did not correlate with the binding of the protein to transcripts, and most splice sites were unchanged, negating a general role for TbRRM1 in splice site selection. When changes in mRNA abundance were mapped across the genome, regions with many downregulated mRNAs were identified. Two regions were analyzed by chromatin immunoprecipitation, both of which exhibited increases in nucleosome occupancy upon TbRRM1 depletion. In addition, subjecting cells to heat shock resulted in translocation of TbRRM1 to the cytoplasm and compaction of chromatin, consistent with a second role for TbRRM1 in modulating chromatin structure. IMPORTANCE: Trypanosoma brucei, the parasite that causes human sleeping sickness, is transmitted by tsetse flies. The parasite progresses through different life cycle stages in its two hosts, altering its pattern of gene expression in the process. In trypanosomes, protein-coding genes are organized as polycistronic units that are processed into monocistronic mRNAs. Since genes in the same unit can be regulated independently of each other, it is believed that gene regulation is essentially posttranscriptional. In this study, we investigated the role of a nuclear RNA-binding protein, TbRRM1, in the insect stage of the parasite. We found that TbRRM1 binds nuclear mRNAs and also affects chromatin status. Reduction of nuclear TbRRM1 by RNA interference or heat shock resulted in chromatin compaction. We propose that TbRRM1 regulates RNA polymerase II-driven gene expression both cotranscriptionally, by facilitating transcription and efficient splicing, and posttranscriptionally, via its interaction with nuclear mRNAs.

The Caenorhabditis elegans histone hairpin-binding protein is required for core histone gene expression and is essential for embryonic and postembryonic cell division.

As in all metazoans, the replication-dependent histone genes of Caenorhabditis elegans lack introns and contain a short hairpin structure in the 3' untranslated region. This hairpin structure is a key element for post-transcriptional regulation of histone gene expression and determines mRNA 3' end formation, nuclear export, translation and mRNA decay. All these steps contribute to the S-phase-specific expression of the replication-dependent histone genes. The hairpin structure is the binding site for histone hairpin-binding protein that is required for hairpin-dependent regulation. Here, we demonstrate that the C. elegans histone hairpin-binding protein gene is transcribed in dividing cells during embryogenesis and postembryonic development. Depletion of histone hairpin-binding protein (HBP) function in early embryos using RNA-mediated interference leads to an embryonic-lethal phenotype brought about by defects in chromosome condensation. A similar phenotype was obtained by depleting histones H3 and H4 in early embryos, indicating that the defects in hairpin-binding protein-depleted embryos are caused by reduced histone biosynthesis. We have confirmed this by showing that HBP depletion reduces histone gene expression. Depletion of HBP during postembryonic development also results in defects in cell division during late larval development. In addition, we have observed defects in the specification of vulval cell fate in animals depleted for histone H3 and H4, which indicates that histone proteins are required for cell fate regulation during vulval development.

White lupin (Lupinus albus L.), the neglected multipurpose crop: Its production and utilization in the mixed crop-livestock farming system of Ethiopia

A survey was conducted to generate holistic information on the production and utilization of local white lupin in two lupin growing districts, namely, Mecha and Sekela, representing mid and high altitude areas, respectively in North-western Ethiopia. During the survey, two types of participatory rural appraisal (PRA) techniques, namely, individual farmer interview (61 farmers from Mecha and 51 from Sekela) and group discussion (with 20 farmers from each district) were employed. There are significant differences (P<0.05) between the two study districts for the variables like total land holding, frequency of ploughing during lupin planting, days to maturity, lupin productivity, and number of days of soaking lupin in running water. However, there are no significant differences (P>0.05) between the two study districts for the variables like land allocated for lupin cultivation, lupin seed rate, lupin soaking at home, lupin consumption per family per week and proportion of lupin used for household consumption. The use of the crop as livestock feed is negligible due to its high alkaloid content. It is concluded that the local white lupin in Ethiopia is a valuable multipurpose crop which is being cultivated in the midst of very serious shortage of cropland. Its ability to maintain soil fertility and serve as a source of food in seasons of food scarcity makes it an important crop. However, its bitter taste due to its high alkaloid content remains to be a big challenge and any lupin improvement strategy has to focus on minimizing the alkaloid content of the crop.

Long-term results of a prospective randomised trial assessing the impact of readaptation of the dorsolateral peritoneal layer following extended pelvic lymph node dissection and cystectomy.

OBJECTIVE To evaluate the long term oncological and functional outcomes after readaptation of the dorsolateral peritoneal layer following pelvic lymph node dissection (PLND) and cystectomy . PATIENTS AND METHODS A randomised, single-center, single-blinded, two-arm trial was conducted on 200 consecutive cystectomy patients who underwent PLND and cystectomy for bladder cancer (and September 2009. Patients were randomised into two groups: group A with readaptation of the dorsolateral peritoneal layer (n=100; 73 male, 27 female; median age 68 yrs, range 35-86 yrs) and group B without readapation (n=100; 66 male, 34 female; median age 65 yrs, range 30-86 yrs). Regular postoperative follow-up was performed at our outpatient clinic. Median follow-up was 59 months (range 3-100 months), five patients were lost to follow-up in group A, seven in group B. Bowel function was evaluated using the validated Gastrointestinal Quality of Life Index questionnaire and an institutional questionnaire regarding post-cystectomy outcome. Local recurrences and distal metastases were evaluated using computed tomography and bone scan at the regular follow-up visits. RESULTS There was no significant difference between the two groups in terms of the rate of local (pelvic) recurrence (5/95 [5.3%] in group A; 7/93 [7.5%] in group B; p = 0.53), the rate of distant metastases (21/95 [22.1%] in group A; 23/93 [24.7%] in group B; p = 0.67), cancer-specific survival (p = 0.37), and overall survival (p = 0.59). Group A had significantly better bowel function at 3 (p < 0.001), 6 (p < 0.006), 12 (p <0.006) and 24 months (p = 0.04), and significantly less postoperative abdominal pain and bloating at 3 (p = 0.002) and 6 months (p = 0.01). CONCLUSION Readaptation of the dorsolateral peritoneal layer following PLND and cystectomy has a beneficial long-term impact on bowel function and postoperative pain without compromising oncological radicality. This article is protected by copyright. All rights reserved.

RNAs and ribonucleoproteins in recognition and catalysis.

CONTENTS. 1. Did life begin with catalytic RNA?–2. Self-splicing and self-cleaving RNAs–2.1 Self-splicing of group I introns – 2.2 Self-splicing of group II introns – 2.3 Self-cleaving RNAs–3. Splicing mediated by trans-acting factors–3.1 Group III introns – 3.2 Splicing of nuclear pre-mRNAs – 3.3 Trans-splicing – 3.4 Is nuclear pre-mRNA splicing evolutionarily related to group I and group II self-splicing?– 3.5 Non-RNA mediated splicing of tRNAs–4. Processing of ribosomal precursor RNAs–5. Processing of pre-mRNA 3′ ends–5.1 Polyadenylation – 5.2 Histone pre-mRNA 3′ processing–6. Other RNPs involved in metabolic mechanisms–6.1 5′ end processing of pre-tRNAs by RNase P – 6.2 The signal recognition particle – 6.3 Telomerase – 6.4 RNA editing in trypanosomatid mitochondria–7. Why RNA?

Growth Cone Localization of the mRNA Encoding the Chromatin Regulator HMGN5 Modulates Neurite Outgrowth

Neurons exploit local mRNA translation and retrograde transport of transcription factors to regulate gene expression in response to signaling events at distal neuronal ends. Whether epigenetic factors could also be involved in such regulation is not known. We report that the mRNA encoding the high-mobility group N5 (HMGN5) chromatin binding protein localizes to growth cones of both neuron-like cells and of hippocampal neurons, where it has the potential to be translated, and that HMGN5 can be retrogradely transported into the nucleus along neurites. Loss of HMGN5 function induces transcriptional changes and impairs neurite outgrowth, while HMGN5 overexpression induces neurite outgrowth and chromatin decompaction; these effects are dependent on growth cone localization of Hmgn5 mRNA. We suggest that the localization and local translation of transcripts coding for epigenetic factors couple the dynamic neuronal outgrowth process with chromatin regulation in the nucleus.

Use of Extended Characteristics of Locomotion and Feeding Behavior for Automated Identification of Lame Dairy Cows.

This study was carried out to detect differences in locomotion and feeding behavior in lame (group L; n = 41; gait score ≥ 2.5) and non-lame (group C; n = 12; gait score ≤ 2) multiparous Holstein cows in a cross-sectional study design. A model for automatic lameness detection was created, using data from accelerometers attached to the hind limbs and noseband sensors attached to the head. Each cow's gait was videotaped and scored on a 5-point scale before and after a period of 3 consecutive days of behavioral data recording. The mean value of 3 independent experienced observers was taken as a definite gait score and considered to be the gold standard. For statistical analysis, data from the noseband sensor and one of two accelerometers per cow (randomly selected) of 2 out of 3 randomly selected days was used. For comparison between group L and group C, the T-test, the Aspin-Welch Test and the Wilcoxon Test were used. The sensitivity and specificity for lameness detection was determined with logistic regression and ROC-analysis. Group L compared to group C had significantly lower eating and ruminating time, fewer eating chews, ruminating chews and ruminating boluses, longer lying time and lying bout duration, lower standing time, fewer standing and walking bouts, fewer, slower and shorter strides and a lower walking speed. The model considering the number of standing bouts and walking speed was the best predictor of cows being lame with a sensitivity of 90.2% and specificity of 91.7%. Sensitivity and specificity of the lameness detection model were considered to be very high, even without the use of halter data. It was concluded that under the conditions of the study farm, accelerometer data were suitable for accurately distinguishing between lame and non-lame dairy cows, even in cases of slight lameness with a gait score of 2.5.

Effect of soy on faecal dry matter content and excretion of Brachyspira hyodysenteriae in pigs.

The aim of this study was to investigate the effect of a soy diet on the excretion of Brachyspira hyodysenteriae in five farms with subclinically infected pigs. The effects on general health, faecal consistency and dry matter were analysed. In total, 200 pigs of different ages (group 1 <100 days of age (n=120) and group 2 ≥100 days (n=80)) were randomly assigned to the control (C) and the treatment (T) groups. Group C received the farm's standard diet. In group T half of the daily feed ration was replaced by pure soy on two consecutive days. Faecal scores were used to determine faecal consistency and a microwave method to assess faecal dry matter content (FDMC). In age group 1, soy feeding resulted in a statistically significant decrease of the FDMC of 2.5 per cent compared with group C and in age group 2 in a significant increase of 2.2 per cent compared with group C at day 2. Overall seven (T: 5, C: 2) out of 597 faecal samples tested positive for B hyodysenteriae by PCR. In conclusion, a high soy diet applied over two days influenced the faecal consistency and the FDMC in growers, finishers and sows under field conditions. Further investigations with more sensitive diagnostic methods are needed to prove a potential influence of a high soy diet on the detection rate of B hyodysenteriae in subclinically infected herds.