Native RNA sequencing in fission yeast reveals frequent alternative splicing isoforms.

The unicellular yeast Schizosaccharomyces pombe (fission yeast) retains many of the splicing features observed in humans and is thus an excellent model to study the basic mechanisms of splicing. Nearly half the genes contain introns, but the impact of alternative splicing in gene regulation and proteome diversification remains largely unexplored. Here we leverage Oxford Nanopore Technologies native RNA sequencing (dRNA), as well as ribosome profiling data, to uncover the full range of polyadenylated transcripts and translated open reading frames. We identify 332 alternative isoforms affecting the coding sequences of 262 different genes, 97 of which occur at frequencies higher than 20%, indicating that functional alternative splicing in S. pombe is more prevalent than previously suspected. Intron retention events make about 80% of the cases; these events may be involved in the regulation of gene expression and, in some cases, generate novel protein isoforms, as supported by ribosome profiling data in 18 of the intron retention isoforms. One example is the rpl22 gene, in which intron retention is associated with the translation of a protein of only 13 amino acids. We also find that lowly expressed transcripts tend to have longer poly(A) tails than highly expressed transcripts, highlighting an interdependence between poly(A) tail length and transcript expression level. Finally, we discover 214 novel transcripts that are not annotated, including 158 antisense transcripts, some of which also show translation evidence. The methodologies described in this work open new opportunities to study the regulation of splicing in a simple eukaryotic model.

Identification of Taxonomically Restricted Transcripts from Illumina RNA Sequencing Data.

In order to perform a well-balanced comparative transcriptomic analysis, the reference genome and annotations for all species included in the comparison must be of a similar quality and completeness. Frequently, comparative transcriptomic analyses include non-model organisms whose annotations are not as well curated; this inequality can lead to biases.To avoid potential biases stemming from incomplete annotations, a comparative transcriptomic analysis can incorporate de novo transcriptome assemblies for each species, which reduces this disparity. This chapter covers all of the steps which are necessary to run a comparative transcriptomic analysis with de novo transcriptome assemblies, from the first step of the experimental design to the sequencing, and ultimately the bioinformatic analysis.

Uncovering de novo gene birth in yeast using deep transcriptomics.

De novo gene origination has been recently established as an important mechanism for the formation of new genes. In organisms with a large genome, intergenic and intronic regions provide plenty of raw material for new transcriptional events to occur, but little is know about how de novo transcripts originate in more densely-packed genomes. Here, we identify 213 de novo originated transcripts in Saccharomyces cerevisiae using deep transcriptomics and genomic synteny information from multiple yeast species grown in two different conditions. We find that about half of the de novo transcripts are expressed from regions which already harbor other genes in the opposite orientation; these transcripts show similar expression changes in response to stress as their overlapping counterparts, and some appear to translate small proteins. Thus, a large fraction of de novo genes in yeast are likely to co-evolve with already existing genes.

Extensive post-transcriptional buffering of gene expression in the response to severe oxidative stress in baker's yeast.

Cells responds to diverse stimuli by changing the levels of specific effector proteins. These changes are usually examined using high throughput RNA sequencing data (RNA-Seq); transcriptional regulation is generally assumed to directly influence protein abundances. However, the correlation between RNA-Seq and proteomics data is in general quite limited owing to differences in protein stability and translational regulation. Here we perform RNA-Seq, ribosome profiling and proteomics analyses in baker's yeast cells grown in rich media and oxidative stress conditions to examine gene expression regulation at various levels. With the exception of a small set of genes involved in the maintenance of the redox state, which are regulated at the transcriptional level, modulation of protein expression is largely driven by changes in the relative ribosome density across conditions. The majority of shifts in mRNA abundance are compensated by changes in the opposite direction in the number of translating ribosomes and are predicted to result in no net change at the protein level. We also identify a subset of mRNAs which is likely to undergo specific translational repression during stress and which includes cell cycle control genes. The study suggests that post-transcriptional buffering of gene expression may be more common than previously anticipated.

Transcriptomics data of 11 species of yeast identically grown in rich media and oxidative stress conditions.

OBJECTIVE: The objective of this experiment was to identify transcripts in baker's yeast (Saccharomyces cerevisiae) that could have originated from previously non-coding genomic regions, or de novo. We generated this data to be able to compare the transcriptomes of different species of Ascomycota. DATA DESCRIPTION: We generated high-depth RNA sequencing data for 11 species of yeast: Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces kudriavzevii, Saccharomyces bayanus, Naumovia castelii, Kluyveromyces lactis, Lachancea waltii, Lachancea thermotolerans, Lachancea kluyveri, and Schizosaccharomyces pombe. Using RNA-Seq from yeast grown in rich and oxidative conditions we created genome-guided de novo assemblies of the transcriptomes for each species. We included synthetic spike-in transcripts in each sample to determine the lower limit of detection of the sequencing platform as well as the reliability of our de novo transcriptome assembly pipeline. We subsequently compared the de novo transcripts assemblies to the reference gene annotations and generated assemblies that comprised both annotated and novel transcripts.

Chronic desmitis and enthesiophytosis of the radio-ulnar interosseous ligament in a dog.

A 10-year-old golden retriever dog was presented for chronic right forelimb lameness associated with a painful swelling at the lateral aspect of the proximal ulna. Proximal ulnar ostectomy and stabilization resulted in a good clinical outcome. The proposed diagnosis is chronic desmitis and enthesiophytosis of the radio-ulnar interosseous ligament.

Desmite chronique et enthésiophytose du ligament interosseux radio-ulnaire chez un chien. Un Golden retriever de 10 ans a été présenté pour boiterie chronique du membre thoracique droite associée à un gonflement douloureux à l'aspect latéral de l'ulna proximal. Une ostéotomie ulnaire proximale avec stabilisation ont permit un bon résultat clinique. Le diagnostic proposé est une desmite chronique et enthésiophytose du ligament interosseux radio-ulnaire.(Traduit par Isabelle Vallières).

Slow-growing cells within isogenic populations have increased RNA polymerase error rates and DNA damage.

Isogenic cells show a large degree of variability in growth rate, even when cultured in the same environment. Such cell-to-cell variability in growth can alter sensitivity to antibiotics, chemotherapy and environmental stress. To characterize transcriptional differences associated with this variability, we have developed a method--FitFlow--that enables the sorting of subpopulations by growth rate. The slow-growing subpopulation shows a transcriptional stress response, but, more surprisingly, these cells have reduced RNA polymerase fidelity and exhibit a DNA damage response. As DNA damage is often caused by oxidative stress, we test the addition of an antioxidant, and find that it reduces the size of the slow-growing population. More generally, we find a significantly altered transcriptome in the slow-growing subpopulation that only partially resembles that of cells growing slowly due to environmental and culture conditions. Slow-growing cells upregulate transposons and express more chromosomal, viral and plasmid-borne transcripts, and thus explore a larger genotypic--and so phenotypic--space.

Bilateral iliopsoas muscle contracture and spinous process impingement in a German Shepherd dog.

OBJECTIVE: To report diagnosis and treatment of bilateral iliopsoas muscle contracture in a dog with spinous process impingement. STUDY DESIGN: Case report. ANIMALS: German Shepherd dog. METHODS: A dog with chronic progressive lameness, flexion contracture of the coxofemoral joints, severe pain, and decreased femoral reflexes had severe spondylosis bridging the vertebral bodies from L1 to L4 and enlarged dorsal spinous processes from T8 to L6 with impingement and bony proliferation. Ultrasonographic and magnetic resonance imaging (MRI) findings were consistent with fibrosis, mineralization, and atrophy of the iliopsoas muscles bilaterally which was treated by staged tenectomy of the insertions of the iliopsoas muscles. RESULTS: Because of severe perivascular fibrosis, the femoral vessels required ligation. Bilateral iliopsoas muscle tenectomy improved gait and provided pain relief. Histologic findings were consistent with fibrotic myopathy. CONCLUSIONS: Slow progression of severe clinical signs observed bilaterally in this dog differs from previous reports of iliopsoas myopathy. Findings were similar to the fibrotic myopathy of the gracilis or semitendinosus muscles described in dogs. CLINICAL RELEVANCE: Iliopsoas muscle abnormalities should be considered in dogs with limited hip extension and pain. MRI is useful for diagnosing muscle fibrosis. Iliopsoas tenectomy may improve clinical function in dogs with fibrotic myopathy.

Osteocartilaginous metaplasia in the right atrial myocardium of healthy adult sheep.

Among 172 hearts from clinically normal adult sheep aged 1.5 to 7 years evaluated for the presence of cartilage and/or bone in the right atrial myocardium, 3.49% (6/172) had palpable evidence of osteocartilaginous foci. An additional 8% prevalence was estimated based on radiographs of hearts that contained < or =1 mm foci of nonpalpable, radiographically dense bone. Microscopically, the nodules in the atria were characterized by mature lamellar bone enclosing adipose tissue, with occasional new bone formation by endochondral ossification. No degenerative changes were evident in the affected atrial myocardium, suggesting that these lesions were clinically insignificant background changes.

Spontaneous retrograde movement of ureteroliths in two dogs and five cats.

CASE DESCRIPTION: 2 dogs and 5 cats were evaluated for treatment of ureteroliths. CLINICAL FINDINGS: Spontaneous retrograde movement of 1 or more ureteroliths was detected by radiography, ultrasonography, fluoroscopy, and a combination of fluoroscopy and ultrasonography. The ureteroliths moved retrograde up to 4 centimeters. Retrograde movement of ureteroliths into the renal pelvis resulted in improved renal function in some patients but made complete surgical removal of all uroliths more difficult. TREATMENT AND OUTCOME: Medical management was not successful, and ureteroliths were surgically removed. Surgical management of ureteroliths was complicated by retrograde movement of ureteroliths in the perioperative period. CLINICAL RELEVANCE: Ureteroliths can move retrograde within the ureter and even back into the renal pelvis. Retrograde movement of ureteroliths may make surgical planning more difficult.

Extraskeletal osteosarcoma associated with retained surgical sponge in a dog.

Retained surgical sponges are usually discovered in the abdominal cavity, sometimes years after the surgical procedure, and the typical reaction is formation of a foreign-body granuloma, often called gossypiboma or textiloma. In this instance, an extraskeletal osteosarcoma, associated with the granulomatous reaction to a retained surgical sponge adjacent to the stifle, was diagnosed in an 11-year-old Labrador Retriever 9 years after repair of a ruptured cranial cruciate ligament. Radiographic detection of linear foreign material in the soft tissue mass was the basis for a diagnosis of gossypiboma. The mass was surgically excised. Histologically, fibers consistent with those of cotton gauze were associated with the granulomatous inflammation and the osteosarcoma. Amputation or radiation therapy was declined; the dog was treated conservatively with doxycycline and deracoxib. Three months after surgical excision, the dog was euthanized because of local recurrence of the mass along with lameness and decreased appetite.

Songbirds tune their vocal tract to the fundamental frequency of their song.

In human speech, the sound generated by the larynx is modified by articulatory movements of the upper vocal tract, which acts as a variable resonant filter concentrating energy near particular frequencies, or formants, essential in speech recognition. Despite its potential importance in vocal communication, little is known about the presence of tunable vocal tract filters in other vertebrates. The tonal quality of much birdsong, in which upper harmonics have relatively little energy, depends on filtering of the vocal source, but the nature of this filter is controversial. Current hypotheses treat the songbird vocal tract as a rigid tube with a resonance that is modulated by the end-correction of a variable beak opening. Through x-ray cinematography of singing birds, we show that birdsong is accompanied by cyclical movements of the hyoid skeleton and changes in the diameter of the cranial end of the esophagus that maintain an inverse relationship between the volume of the oropharyngeal cavity and esophagus and the song's fundamental frequency. A computational acoustic model indicates that this song-related motor pattern tunes the major resonance of the oropharyngeal-esophageal cavity to actively track the song's fundamental frequency.

Secretion of sex hormones in dogs with adrenal dysfunction.

OBJECTIVE: To evaluate adrenal sex hormone concentrations in response to ACTH stimulation in healthy dogs, dogs with adrenal tumors, and dogs with pituitary-dependent hyperadrenocorticism (PDH). DESIGN: Prospective study. ANIMALS: 11 healthy control dogs, 9 dogs with adrenal-dependent hyperadrenocorticism (adenocarcinoma [ACA] or other tumor); 11 dogs with PDH, and 6 dogs with noncortisol-secreting adrenal tumors (ATs). PROCEDURE: Hyperadrenocorticism was diagnosed on the basis of clinical signs; physical examination findings; and results of ACTH stimulation test, low-dose dexamethasone suppression test, or both. Dogs with noncortisol-secreting ATs did not have hyperadrenocorticism but had ultrasonographic evidence of an AT. Concentrations of cortisol, androstenedione, estradiol, progesterone, testosterone, and 17-hydroxyprogesterone were measured before and 1 hour after i.m. administration of 0.25 mg of synthetic ACTH. RESULTS: All dogs with ACA, 10 dogs with PDH, and 4 dogs with ATs had 1 or more sex hormone concentrations greater than the reference range after ACTH stimulation. The absolute difference for progesterone, 17-hydroxyprogesterone, and testosterone concentrations (value obtained after ACTH administration minus value obtained before ACTH administration) was significantly greater for dogs with ACA, compared with the other 3 groups. The absolute difference for androstenedione was significantly greater for dogs with ACA, compared with dogs with AT and healthy control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs with ACA secrete increased concentrations of adrenal sex hormones, compared with dogs with PDH, noncortisol-secreting ATs, and healthy dogs. Dogs with noncortisol-secreting ATs also have increased concentrations of sex hormones. There is great interdog variability in sex hormone concentrations in dogs with ACA after stimulation with ACTH.

Surgical management of compound odontoma in two horses.

Two horses were admitted for evaluation of mandibular swelling (horse 1) or maxillary distortion (horse 2). Both horses had radiographic evidence of tumors of dental origin that had the appearance of a compound odontoma. Extensive surgical resection was performed for treatment. Horse 1 was treated with 1-stage surgical resection, but an iatrogenic fracture occurred during surgery, which was managed successfully with a type I external fixator and extraoral alimentation. Horse 2 was treated in multiple stages to remove all portions of the tumor. To manage an extensive orosinal fistula, a custom-designed dental bridge was constructed to occlude the fistula. For both horses, the histopathologic diagnosis was compound odontoma. Compound odontomas are benign, locally expansive tumors of dental origin. Compound odontomas can be treated successfully; however, multiple surgeries may be necessary.

Inflation of the esophagus and vocal tract filtering in ring doves.

Ring doves vocalize with their beaks and nostrils closed, exhaling into inflatable chambers in the head and neck region. The source sound produced at the syrinx contains a fundamental frequency with prominent second and third harmonic overtones, but these harmonics are filtered out of the emitted signal. We show by cineradiography that the upper esophagus, oral and nasal cavities collect the expired air during vocalization and that the inflated esophagus becomes part of the suprasyringeal vocal tract. The level of the second and third harmonics, relative to the fundamental frequency (f(0)), is reduced in the esophagus and emitted vocalization compared with in the trachea, although these harmonics are still considerably higher in the esophagus than in the emitted signal. When the esophagus is prevented from fully inflating, there is a pronounced increase in the level of higher harmonics in the emitted vocalization. Our data suggest that the trachea and esophagus act in series as acoustically separate compartments attenuating harmonics by different mechanisms. We hypothesize that the trachea behaves as a tube closed at the syringeal end and with a variable, restricted opening at the glottal end that lowers the tracheal first resonance to match the f(0) of the coo. The inflated esophagus may function as a Helmholtz resonator in which the elastic walls form the vibrating mass. Such a resonator could support the f(0) over a range of inflated volumes.

Treatment of tracheal collapse with an intraluminal stent in a miniature horse.

A 7-month-old miniature horse was referred for respiratory distress. Tracheal collapse at the level of the thoracic inlet was diagnosed. An intraluminal nitinol stent was placed with endoscopic guidance. Respiratory function was restored immediately after stent placement. The main complication observed during a 14-month follow-up period was growth of granulation tissue through the stent, which was controlled satisfactorily by electrocautery performed during endoscopy with the horse standing. Treatment of tracheal collapse with an intraluminal stent is an effective, practical, and minimally invasive procedure in miniature and young equids and ponies.