Expression and localization of the neuropeptide phoenixin-14 and its receptor GRP173 in the canine reproductive organs and periovarian adipose tissue.

Phoenixin-14 (PNX-14) is a regulatory neuropeptide encoded by the SMIM20 gene, which has been implicated in the reproductive cycle by modulating the hypothalamic-pituitary-gonadal (HPG) axis. Recently, we showed that PNX-14 is downregulated in bitches with cystic endometrial hyperplasia and pyometra. The objective of this study was to determine the expression of Smim20, PNX-14, and its putative receptor GRP173 in the canine ovary (both healthy and those with ovarian cysts), periovarian adipose tissue (PAT) and in the endometrium during the oestrous cycle. The expression was analysed by RT-qPCR and Western blot. In tissue sections, peptides were localised by immunofluorescent assays, and blood plasma concentrations of PNX-14 were detected by EIA. The results demonstrated increased levels of PNX in bitches in the anestrus groups compared to diestrus animals. The expression of GPR173 increased in PAT during the diestrus phase and endometrial tissue in late diestrus bitches. In the ovary, strong signals of PNX-14 and GPR173 were detected in the luteal and follicular cells. Furthermore, bitches with cystic ovaries were characterised by elevated circulating PNX levels and a significantly higher expression of PNX and GPR173 in gonadal tissues, when compared with healthy animals. Moreover, a positive correlation between PNX and progesterone in the blood of healthy bitches was noted, which changed to a negative correlation in females affected by cystic ovaries. These studies expand the knowledge regarding the expression and localization of the PNX/GRP173 system in canine reproductive organs during physiological and pathological conditions.

Novel functions of a retroposed gene.

Retroposed protein-coding genes are commonly considered to be nonfunctional duplicates. However, they often gain transcriptional capability and have important roles. Amici et al. recently identified novel functions of a retroposed gene. HAPSTR2, a retrocopy of HAPSTR1, encodes a protein that stabilizes the HAPSTR1 protein and functionally buffers its loss.

Prevalence and characterisation of antimicrobial resistance genes and class 1 and 2 integrons in multiresistant Escherichia coli isolated from poultry production.

A global increase in the populations of drug resistant bacteria exerts negative effects on animal production and human health. Our study has been focused on the assessment of resistance determinants in relation to phenotypic resistance of the 74 commensal E. coli isolates present in different ecological environments. The samples were collected from poultry litter, feces, and neck skin. Among the microorganisms isolated from the poultry litter (group A), the highest resistance was noted against AMP and DOX (100%). In the E. coli extracts from the cloacal swabs (group B), the highest resistance was observed against AMP (100%) and CIP (92%). The meat samples (group C) were characterized by resistance to AMP (100%) and STX (94.7%). Genes encoding resistance to ß-lactams (blaTEM, blaCTX-M), fluoroquinolones (qnrA, qnrB, qnrS), aminoglycosides (strA-strB, aphA1, aac(3)-II), sulfonamides (sul1, sul2, sul3), trimethoprim (dfr1, dfr5, dfr7/17) and tetracyclines (tetA, tetB) were detected in the studied bacterial isolates. The presence of class 1 and 2 integrons was confirmed in 75% of the MDR E. coli isolates (plasmid DNA), of which 60% contained class 1 integrons, 15% contained class 2 integrons, and 11.7% carried integrons of both classes. Thus, it may be concluded that integrons are the common mediators of antimicrobial resistance among commensal multidrug resistant Escherichia coli at important stages of poultry production.

Canine cystic endometrial hyperplasia and pyometra may downregulate neuropeptide phoenixin and GPR173 receptor expression.

The most common uterine diseases affecting bitches are cystic endometrial hyperplasia (CEH) and pyometra. The neuropeptide phoenixin (PNX) and its receptor (GPR173) are potential key factors involved in the proliferative and inflammatory regulation of the reproductive system in females. This study aimed to evaluate the expression of PNX and GPR173 by qPCR, western blot and immunofluorescence assays in the endometrium of bitches suffering from CEH or pyometra compared to clinically healthy females. Additionally, PNX and progesterone (P4) plasma concentrations were analysed. The results showed a significantly lower expression levels of PNX and GPR173 (mRNA and protein production) in bitches with the CEH or pyometra groups compared to healthy animals. Immunofluorescence staining examination also confirmed a lower concentration of PNX and GPR173 signals in bitches with pathological uteri. Moreover, a lower concentration of PNX blood levels in bitches suffering from pyometra was observed. The PNX concentration was negatively correlated with P4 but only in healthy bitches. These results illustrate that the development of canine uterine disorders may cause a lower expression of PNX and its receptor GPR173.

A cross-sectional retrospective study of SARS-CoV-2 seroprevalence in domestic cats, dogs and rabbits in Poland.

BACKGROUND: Coronaviruses (CoVs) have long been known to cause infection in domestic and free-living birds and mammals including humans. The zoonotic origin of SARS-CoV-2 and the biological properties of CoVs, including ability to cross interspecies barriers, enable its emergence in populations of various animals, including companion animals (cats, dogs, rabbits) an area requiring further study. To date, several cases of cats and dogs positive for SARS-CoV-2 and/or specific antibodies have been described. The aim of our cross-sectional retrospective study is to determine seroprevalence of SARS-CoV-2 in domestic dog, cat and rabbit population during recent COVID-19 pandemic in Poland. RESULTS: In total, serum samples from 279 cats and 343 dogs and 29 rabbits were used in the study. The seroprevalence of SARS-CoV-2 in cats and dogs reached 1.79% (95% CI: 0.77 - 4.13) and 1.17% (95% CI 0.45 - 2.96), respectively (p ≥ 0.05). Anti- SARS-CoV-2 antibodies were detected in 5 cats (mean S/P% 106 ± 48.23) and 4 dogs (mean S/P% 78.5 ± 16.58). All 29 samples from rabbits were negative for SARS-CoV-2 antibodies. No significant gender or age differences in seroprevalence in dogs and cats (p ≥ 0.05) were found. None of the animals with anti-SARS-CoV-2 antibodies displayed respiratory or gastrointestinal signs at the time of sampling. CONCLUSIONS: Our results confirmed previous findings that SARS-CoV-2 infections in companion animals occurs but are not frequent. Future serological testing of large pet population may provide a comprehensive picture of disease dynamics in companion animals.

Expression of Transforming Growth Factor Beta Isoforms in Canine Endometrium with Cystic Endometrial Hyperplasia-Pyometra Complex.

Cystic endometrial hyperplasia (CEH) and pyometra are the most frequently diagnosed uterine diseases affecting bitches of different ages. Transforming growth factor beta (TGF-ß) has been classified in females as a potential regulator of many endometrial changes during the estrous cycle or may be involved in pathological disorders. The aim of this study was to determine the expression of TGF-ß1, -ß2 and -ß3 in the endometrium of bitches suffering from CEH or a CEH-pyometra complex compared to clinically healthy females (control group; CG). A significantly increased level of TGF-ß1 mRNA expression was observed in the endometrium with CEH-pyometra compared to CEH and CG. Protein production of TGF-ß1 was identified only in the endometrium of bitches with CEH-pyometra. An increase in TGF-ß3 mRNA expression was observed in all the studied groups compared to CG. The expression of TGF-ß2 mRNA was significantly higher in CEH and lower in CEH-pyometra uteri. The results indicate the presence of TGF-ß cytokines in canine endometrial tissues affected by proliferative and degenerative changes. However, among all TGF-ß isoforms, TGF-ß1 could potentially be a key factor involved in the regulation of the endometrium in bitches with CEH-pyometra complex.

Promoter switching in response to changing environment and elevated expression of protein-coding genes overlapping at their 5' ends.

Despite the number of studies focused on sense-antisense transcription, the key question of whether such organization evolved as a regulator of gene expression or if this is only a byproduct of other regulatory processes has not been elucidated to date. In this study, protein-coding sense-antisense gene pairs were analyzed with a particular focus on pairs overlapping at their 5' ends. Analyses were performed in 73 human transcription start site libraries. The results of our studies showed that the overlap between genes is not a stable feature and depends on which TSSs are utilized in a given cell type. An analysis of gene expression did not confirm that overlap between genes causes downregulation of their expression. This observation contradicts earlier findings. In addition, we showed that the switch from one promoter to another, leading to genes overlap, may occur in response to changing environment of a cell or tissue. We also demonstrated that in transfected and cancerous cells genes overlap is observed more often in comparison with normal tissues. Moreover, utilization of overlapping promoters depends on particular state of a cell and, at least in some groups of genes, is not merely coincidental.

Not So Dead Genes-Retrocopies as Regulators of Their Disease-Related Progenitors and Hosts.

Retroposition is RNA-based gene duplication leading to the creation of single exon nonfunctional copies. Nevertheless, over time, many of these duplicates acquire transcriptional capabilities. In human in most cases, these so-called retrogenes do not code for proteins but function as regulatory long noncoding RNAs (lncRNAs). The mechanisms by which they can regulate other genes include microRNA sponging, modulation of alternative splicing, epigenetic regulation and competition for stabilizing factors, among others. Here, we summarize recent findings related to lncRNAs originating from retrocopies that are involved in human diseases such as cancer and neurodegenerative, mental or cardiovascular disorders. Special attention is given to retrocopies that regulate their progenitors or host genes. Presented evidence from the literature and our bioinformatics analyses demonstrates that these retrocopies, often described as unimportant pseudogenes, are significant players in the cell's molecular machinery.

Comparative genomics in the search for conserved long noncoding RNAs.

Long noncoding RNAs (lncRNAs) have emerged as prominent regulators of gene expression in eukaryotes. The identification of lncRNA orthologs is essential in efforts to decipher their roles across model organisms, as homologous genes tend to have similar molecular and biological functions. The relatively high sequence plasticity of lncRNA genes compared with protein-coding genes, makes the identification of their orthologs a challenging task. This is why comparative genomics of lncRNAs requires the development of specific and, sometimes, complex approaches. Here, we briefly review current advancements and challenges associated with four levels of lncRNA conservation: genomic sequences, splicing signals, secondary structures and syntenic transcription.

A chromatin-associated splicing isoform of OIP5-AS1 acts in cis to regulate the OIP5 oncogene.

A large portion of the human genome is transcribed into long noncoding RNAs that can range from 200 nucleotides to several kilobases in length. The number of identified lncRNAs is still growing, but only a handful of them have been functionally characterized. However, it is known that the functions of lncRNAs are closely related to their subcellular localization. Cytoplasmic lncRNAs can regulate mRNA stability, affect translation and act as miRNA sponges, while nuclear-retained long noncoding RNAs have been reported to be involved in transcriptional control, chromosome scaffolding, modulation of alternative splicing and chromatin remodelling. Through these processes, lncRNAs have diverse regulatory roles in cell biology and diseases. OIP5-AS1 (also known as Cyrano), a poorly characterized lncRNA expressed antisense to the OIP5 oncogene, is deregulated in multiple cancers. We showed that one of the OIP5-AS1 splicing forms (ENST00000501665.2) is retained in the cell nucleus where it associates with chromatin, thus narrowing down the spectrum of its possible mechanisms of action. Its knockdown with antisense LNA gapmeRs led to inhibited expression of a sense partner, OIP5, strongly suggesting a functional coupling between OIP5 and ENST00000501665.2. A subsequent bioinformatics analysis followed by RAP-MS and RNA Immunoprecipitation experiments suggested its possible mode of action; in particular, we found that ENST00000501665.2 directly binds to a number of nuclear proteins, including SMARCA4, a component of the SWI/SNF chromatin remodelling complex, whose binding motif is located in the promoter of the OIP5 oncogene.

Complex Analysis of Retroposed Genes' Contribution to Human Genome, Proteome and Transcriptome.

Gene duplication is a major driver of organismal evolution. One of the main mechanisms of gene duplications is retroposition, a process in which mRNA is first transcribed into DNA and then reintegrated into the genome. Most gene retrocopies are depleted of the regulatory regions. Nevertheless, examples of functional retrogenes are rapidly increasing. These functions come from the gain of new spatio-temporal expression patterns, imposed by the content of the genomic sequence surrounding inserted cDNA and/or by selectively advantageous mutations, which may lead to the switch from protein coding to regulatory RNA. As recent studies have shown, these genes may lead to new protein domain formation through fusion with other genes, new regulatory RNAs or other regulatory elements. We utilized existing data from high-throughput technologies to create a complex description of retrogenes functionality. Our analysis led to the identification of human retroposed genes that substantially contributed to transcriptome and proteome. These retrocopies demonstrated the potential to encode proteins or short peptides, act as cis- and trans- Natural Antisense Transcripts (NATs), regulate their progenitors' expression by competing for the same microRNAs, and provide a sequence to lncRNA and novel exons to existing protein-coding genes. Our study also revealed that retrocopies, similarly to retrotransposons, may act as recombination hot spots. To our best knowledge this is the first complex analysis of these functions of retrocopies.

Proportion of acute ischaemic strokes attributable to a cardiac aetiology in an unselected young patient population: A single centre experience.

The incidence of acute ischaemic stroke in young patients is increasing and identifying the underlying cause is critically important with regards to their optimal management. The true proportion of cardiac causes of stroke in young patients is poorly defined. We aimed to determine the proportion of strokes attributable to cardiac causes in an unselected, consecutive cohort of young patients. We used the database of a large stroke service to identify patients aged ≤55 years presenting with stroke between 01 January 2015 and 31 December 2017. We reviewed their clinical notes and investigations and then categorised patients by the cause of their stroke.We screened 202 cases, and excluded 35, resulting in a study population of 167 patients; 24.0% (40/167) had a cardiac cause of stroke including 9.6% (16/167) had patent foramen ovale, 9.0% (15/167) had intracardiac source of embolus and 5.4% (9/167) had atrial fibrillation; 50.8% (85/167) had other more likely causes; and 25.1% (42/167) had no clear underlying cause.A high proportion (24%) of strokes in young patients are secondary to a cardiac cause. Thorough investigation in these patients is warranted and requires close interdisciplinary links between cardiologists and stroke physicians to ensure optimal management.

Essen Risk Score in Prediction of Myocardial Infarction After Transient Ischemic Attack or Ischemic Stroke Without Prior Coronary Artery Disease.

Background and Purpose- More intensive secondary prevention with newer drugs may be cost-effective in patients with coronary artery disease (CAD). Whether some subgroups of patients who had a transient ischemic attack (TIA) or ischemic stroke, but no prior CAD are at similar high risk of myocardial infarction as those with prior CAD remains unclear. We determined whether the Essen score identified a subset of TIA/stroke patients without known prior CAD who, nevertheless, had a high risk of myocardial infarction on current secondary prevention management. Methods- In a population-based cohort (Oxford Vascular Study) of consecutive TIA or ischemic stroke patients recruited from 2002 to 2014, 10-year actuarial risks of myocardial infarction and of recurrent ischemic stroke were determined by face-to-face follow-up in patients with and without prior CAD using Kaplan-Meier analyses. Predictive value of the Essen score was assessed with C statistic. Results- Of 2555 patients with TIA/stroke (13 070 patient-years of follow-up), 10-year risk of myocardial infarction in those without prior CAD (n=2017, 78.9%) ranged from 0.9% (95% CI, 0-1.9) at Essen score ≤1 to 29.8% (95% CI, 7.7-46.6) in those with a score ≥5 (C statistic =0.64 [95% CI, 0.57-0.71]; P<0.001). The score tended to be less predictive (difference: P=0.0460) for the risk of recurrent ischemic stroke (C statistic =0.57 [95% CI, 0.54-0.60]). Compared with patients with prior CAD (n=538, 21.1%), an Essen risk score of ≥4 (n=294, 11.5%) in those without prior CAD identified a subgroup at similar high 10-year risks of myocardial infarction (17.2% [95% CI, 6.9-26.3] versus 16.9% [95% CI, 11.5-22.0]) and of recurrent stroke (40.4% [95% CI, 26.7-51.6] versus 32.4% [95% CI, 25.2-38.8]). Conclusions- The Essen score is a simple clinical score to risk-stratify patients with TIA/stroke without prior CAD and to identify subsets who may be at sufficiently high risk of myocardial infarction and recurrent stroke to justify more intensive treatment or inclusion in trials.

Effect of coexisting vascular disease on long-term risk of recurrent events after TIA or stroke.

OBJECTIVE: To determine whether patients with TIA or ischemic stroke with coexisting cardiovascular disease (i.e., history of coronary or peripheral artery disease) are still at high risk of recurrent ischemic events despite current secondary prevention guidelines. METHODS: In a population-based study in Oxfordshire, UK (Oxford Vascular Study), we studied consecutive patients with TIA or ischemic stroke for 2002-2014. Patients were treated according to current secondary prevention guidelines and we determined risks of coronary events, recurrent ischemic stroke, and major bleeding stratified by the presence of coexisting cardiovascular disease. RESULTS: Among 2,555 patients (9,148 patient-years of follow-up), those (n = 640; 25.0%) with coexisting cardiovascular disease (449 coronary only; 103 peripheral only; 88 both) were at higher 10-year risk of coronary events than those without (22.8%, 95% confidence interval 17.4-27.9; vs 7.1%, 5.3-8.8; p < 0.001; age- and sex-adjusted hazard ratio [HR] 3.07, 2.24-4.21) and of recurrent ischemic stroke (31.5%, 25.1-37.4; vs 23.4%, 20.5-26.2; p = 0.0049; age- and sex-adjusted HR 1.23, 0.99-1.53), despite similar rates of use of antithrombotic and lipid-lowering medication. However, in patients with noncardioembolic TIA/stroke, risk of extracranial bleeds was also higher in those with coexisting cardiovascular disease, particularly in patients aged <75 years (8.1%, 2.8-13.0; vs 3.4%, 1.6-5.3; p = 0.0050; age- and sex-adjusted HR 2.71, 1.16-6.30), although risk of intracerebral hemorrhage was not increased (age- and sex-adjusted HR 0.36, 0.04-2.99). CONCLUSIONS: As in older studies, patients with TIA/stroke with coexisting cardiovascular disease remain at high risk of recurrent ischemic events despite current management. More intensive lipid-lowering might therefore be justified, but benefit from increased antithrombotic treatment might be offset by the higher risk of extracranial bleeding.

Prognostic value of "tissue-based" definitions of TIA and minor stroke: Population-based study.

OBJECTIVE: Since use of diffusion-weighted imaging (DWI) positivity in the "tissue-based" definition of stroke in patients with a clinical TIA is supported by the high associated 90-day risk of recurrent stroke, we aimed to determine long-term prognostic significance, stratified by etiologic subtype, and whether the same tissue-based distinction is predictive in minor strokes. METHODS: Consecutive eligible patients with TIA or minor stroke (NIH Stroke Scale [NIHSS] ≤3) in the population-based Oxford Vascular Study underwent brain MRI at baseline. Stroke risk on 10-year follow-up was stratified by NIHSS (0/1 vs 2/3) and Trial of Org 10172 in Acute Stroke Treatment classification of the initial event. RESULTS: Among 1,033 patients (633 TIA; 400 minor stroke), 248 (24.0%) had acute lesions on DWI (13.9% of TIAs; 40.0% of minor strokes). A positive DWI was associated with an increased 10-year risk of recurrent ischemic stroke after an index TIA (hazard ratio [HR] 2.66, 95% confidence interval [CI] 1.28-5.54, p = 0.009) or a stroke with NIHSS 0-1 (3.03, 1.29-7.08, p = 0.011), but not after a stroke with NIHSS 2-3 (0.70, 0.24-2.10, p = 0.53). Ischemic stroke risk after DWI-positive TIA was at least equivalent to that after DWI-negative stroke (1.81, 0.82-4.00, p = 0.14). Among all patients, DWI positivity was most predictive of 10-year risk after cryptogenic events (4.68, 1.70-12.92, p = 0.003). CONCLUSION: DWI positivity is associated with an increased long-term risk of recurrent stroke after TIA and minor stroke, supporting a tissue-based definition of minor stroke as well as TIA. Prognostic value is greatest after cryptogenic events.

Electroretinographic findings in day-blind dogs.

Cone degeneration (cd; day blindness) is one of the inherited retinal diseases of dogs. Its diagnosis is based on vision testing, fundoscopy, electroretinography (ERG) and, for some breeds, on genetic tests. Typical signs of the disease are day blindness and cone dysfunction during ERG while fundoscopy does not show any abnormalities. The aim of this study was to compare behavioural findings, fundoscopic lesions and electroretinographic alterations in 12 cd-affected dogs (Alaskan Malamute, Labrador Retriever, German Shepherd, Dachshund, Yorkshire Terrier, Shih Tzu, Siberian Husky and crossbreeds) examined at our clinic. None of the examined dogs had any fundoscopic lesions, and all of them had normal scotopic vision with strongly impaired or absent photopic vision. Light-adapted transient, cone-mediated and steady-state, 31-Hz cone flicker ERGs were much below the 5th percentile limits of normality or even unrecordable, while the rod-driven ERGs were within normal values. Vision test and ERG results corresponded to each other and, combined with the results of fundoscopy, were typical of cd. To date, our research is one of the few studies in the world presenting ERG alterations compared with vision test findings and fundoscopic results in the course of cd.

Long-Term Prognosis of Patients With Transient Ischemic Attack or Stroke and Symptomatic Vascular Disease in Multiple Arterial Beds.

BACKGROUND AND PURPOSE: Cerebrovascular, coronary, and peripheral vascular disease have common underlying arterial pathology and risk factors, but the clinical significance of multiple-territory disease in patients with transient ischemic attack (TIA)/ischemic stroke is unclear, particularly whether the number of clinically affected territories still predicts long-term outcome on current standard secondary prevention therapies. METHODS: In a population-based study of 92 728 individuals in Oxfordshire, United Kingdom (Oxford Vascular Study), we studied patients presenting with TIA/ischemic stroke (2002-2014) in relation to the number of other vascular beds (coronary, peripheral) affected by symptomatic (current or previous) disease. We compared the risk factor profile and long-term prognosis in patients with single- versus multiple-territory disease. RESULTS: Among 2554 patients with 10 679 patient-years of follow-up, 1842 (72.1%) had single- (TIA/stroke only), 608 (23.8%) double-, and 104 (4.1%) triple-territory symptomatic vascular disease. The number of affected vascular beds increased with the number of atherosclerotic risk factors (Ptrend<0.0001). Compared with patients with TIA/stroke only, those with multiple-territory disease had more hypertension (age/sex-adjusted odds ratio [OR], 3.43; 95% confidence interval [CI], 2.76-4.27; P<0.0001), diabetes mellitus (OR, 2.89; 95% CI, 2.27-3.66; P<0.0001), hypercholesterolemia (OR, 4.67; 95% CI, 3.85-5.66; P<0.0001), and current or previous smoking (OR, 1.52; 95% CI, 1.26-1.84; P<0.0001). Triple-territory disease was particularly strongly associated with hypercholesterolemia (OR, 6.80; 95% CI, 4.39-10.53; P<0.0001). Despite more intensive secondary prevention in patients with multiple-territory disease, the 5-year risk of vascular death increased steeply with the number of territories affected (17.2% versus 30.0% versus 42.9%; P<0.0001). Compared with patients with single-territory, patients with multiple-territory disease also had higher postacute long-term risks (90 days to 10 years) of recurrent ischemic stroke (age/sex-adjusted hazard ratio, 1.38; 95% CI, 1.04-1.81; P=0.02) and nonstroke acute vascular events (hazard ratio, 3.06; 95% CI, 2.23-4.20; P<0.0001). CONCLUSIONS: Number of affected vascular beds appeared to be a simple clinical rule in identifying TIA/ischemic stroke patients who are at high long-term risk of nonstroke vascular events and vascular death.

Natural antisense transcripts in diseases: From modes of action to targeted therapies.

Antisense transcription is a widespread phenomenon in mammalian genomes, leading to production of RNAs molecules referred to as natural antisense transcripts (NATs). NATs apply diverse transcriptional and post-transcriptional regulatory mechanisms to carry out a wide variety of biological roles that are important for the normal functioning of living cells, but their dysfunctions can be associated with human diseases. In this review, we attempt to provide a molecular basis for the involvement of NATs in the etiology of human disorders such as cancers and neurodegenerative and cardiovascular diseases. We also discuss the pros and cons of oligonucleotide-based therapies targeted against NATs, and we comment on state-of-the-art progress in this promising area of clinical research. WIREs RNA 2018, 9:e1461. doi: 10.1002/wrna.1461 This article is categorized under: RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions.

RetrogeneDB-a database of plant and animal retrocopies.

Database URL: http://yeti.amu.edu.pl/retrogenedb. Secondary database URL: http://rhesus.amu.edu.pl/retrogenedb.