COVID-19 Vaccine and Fertility: The Male Perspective.

Aims The expedited development of multiple COVID-19 vaccines has raised concerns for some, with vaccine hesitancy described in many populations. A U.S. study assessing fertility patients attitudes towards the COVID -19 vaccine revealed that over half were unsure, or would not accept the vaccine if offered. Only 7.4% of participants in this study were male. We therefore sought to assess the perspective of male fertility patients towards COVID-19 vaccination. Methods Men with a fertility appointment were invited to complete an anonymous 21-item questionnaire. Results Willingness to accept the COVID-19 vaccination was influenced by stage of fertility journey. Overall, 76% (n=102) of participants were willing to receive the COVID-19 vaccine. Men with a pregnant partner were most likely to accept or have already accepted the vaccine (97%, 30/31). Conclusion Although concerns around COVID-19 vaccines persist, this study demonstrates the growing rate of acceptance and engagement among the male fertility population.

Dysregulation of the interleukin-17A pathway in endometrial tissue from women with unexplained infertility affects pregnancy outcome following assisted reproductive treatment.

STUDY QUESTION: Which transcriptomic alterations in mid-luteal endometrial scratch biopsies, taken prior to the assisted reproductive treatment (ART) treatment cycle are associated with unsuccessful pregnancy? SUMMARY ANSWER: Dysregulated interleukin-17 (IL-17) pathway components are demonstrated in women who fail to become pregnant after ART. WHAT IS KNOWN ALREADY: Implantation failure is now recognised as a critical factor in unexplained infertility and may be an important component of failed ART. STUDY DESIGN, SIZE, DURATION: Using a prospective longitudinal study design, 29 nulliparous women with unexplained infertility undergoing ART were recruited between October 2016 and February 2018. Mid-luteal stage endometrium and matched serum samples were collected, and patients underwent a single embryo transfer in the subsequent cycle. RNA-seq analysis of endometrial biopsies was performed on the discovery cohort (n = 20). PARTICIPANTS/MATERIALS, SETTING, METHODS: Gene set enrichment analysis of the differentially expressed genes (DEGs) was performed. Endometrium and serum were then prepared for IL-17A analysis by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: There were 204 differentially expressed protein-coding genes identified in tissue from women who became pregnant (n = 9) compared with tissue from women who failed to become pregnant (n = 11) (false discovery rate; P < 0.05). Of the 204 DEGs, 166 were decreased while 38 were increased in the pregnant compared to the non-pregnant groups. Gene set enrichment analysis of the DEGs identified an over-representation of IL-17 and Pl3K-Akt signalling pathways. All the DEGs within the IL-17 signalling pathway (MMP3, MMP1, IL1ß, LCN2, S100A9 and FOSL1) demonstrated decreased expression in the pregnant group. Serum IL-17 protein levels were increased in the non-pregnant discovery cohort (n = 11) and these findings were confirmed a validation cohort (n = 9). LIMITATIONS, REASONS FOR CAUTION: Limitations of our study include the cohort size and the lack of aneuploidy data for the embryos; however, all embryos transferred were single good or top-quality blastocysts. WIDER IMPLICATIONS OF THE FINDINGS: These findings demonstrate dysregulated IL-17 pathway components in women who fail to become pregnant after ART. Elevated serum levels of the pro-inflammatory cytokine IL-17 may predict failure of ART in women with unexplained infertility. Future trials of anti-IL-17 therapies in this cohort warrant further investigation. STUDY FUNDING/COMPETING INTEREST(S): Funding from the UCD Wellcome Institutional Strategic Support Fund, which was financed jointly by University College Dublin and the SFI-HRB-Wellcome Biomedical Research Partnership (ref 204844/Z/16/Z), is acknowledged. The authors have no competing interests. TRIAL REGISTRATION NUMBER: NA.

Fertility Preservation in Adolescent Males.

Aim Survival rates of childhood cancer are now above 80%, so there is increasing emphasis on survivorship. A major late effect of cancer treatment is fertility loss. International best practice indicates that post-pubertal boys with cancer should be offered sperm cryopreservation prior to treatment. The aim of this study was to demonstrate the feasibility of a national sperm cryopreservation program for adolescent males and to examine outcomes for a pilot. Methods Patient demographics and semen parameters of adolescent male oncology patients referred to our service were analysed. Sperm analyses were performed in accordance with WHO guidelines. Results Fifteen patients were referred, 12 of whom (80%) attempted sperm production. Of these 12 samples, 25% (3/12) were unsuitable for freezing. One patient was too unwell to produce a sample. Eight patients (age range 12-17 years) had sperm successfully cryopreserved. Of these 8 samples, 25% were within WHO 'normal' limits, 50% had reduced sperm concentration. The number of cryopreserved samples (straws) ranged from 4-8 per patient. Conclusion We have established a successful, structured fertility preservation service for adolescent males in Ireland. Sperm cryopreservation is an accessible method of safeguarding fertility in male patients facing cancer treatment and should be offered to all.

CA125 measured during menstruation can be misleading.

The aim of these case reports and literature review is to report the importance of cyclical variation of serum CA-125 levels in two patients with endometriosis. Two case reports and a literature review of cyclical variation in serum CA-125 levels are discussed. There was significant variation in serum CA-125 levels taken during menses and mid-cycle in these two cases. Serum CA-125 levels increase dramatically during menstruation in women with endometriosis. This is important when assessing disease status.

Exploring the psychological processes underlying touch: lessons from the Alexander Technique.

UNLABELLED: The experience of touch is significant; both in its positive implications and in how it attracts caution and controversy. Accordingly, physical contact within psychological therapy has been shown to improve well-being and the therapeutic relationship, yet the majority of therapists never or rarely use touch. This research aimed to explore psychological processes underlying touch through the Alexander Technique, a psycho-physical technique taught one to one using touch. Six individuals who had received the Alexander Technique were interviewed, and 111 completed surveys. Interview data suggested an incompatibility between touch and the spoken word, which was understood through the way touch lacks verbal discourses in our society. The largely simplistic and dichotomous verbal understanding we have (either only very positive or very negative) could help understand some of the societal-level caution surrounding touch. Touch was seen also as a nurturing experience by interviewees, which influenced inter-personal and intra-personal relational processes. Developmental models were used to frame the way touch strengthened the pupil-teacher relationship and the way pupils' intra-personal psychological change seemed linked to this relational experience. The surveys largely supported these findings, and discussion is made around the notable way pupils negatively interpreted the intention of the survey. Implications for the use of touch in psychological therapies are discussed, as are limitations and ideas for future research. KEY PRACTITIONER MESSAGE: Touch is a powerful experience, and physical contact within psychological therapy has been shown to improve well-being and the therapeutic relationship, yet the majority of therapists never or rarely use touch. The AT is an alternative therapeutic approach to psycho-physical well-being that offers an interesting model to study the impact of touch. Findings from those that have used the technique reaffirmed that touch can improve well-being and can be a powerful force in the 'therapeutic relationship'. Accounts drew strong parallels with developmental experiences, which may be of particular interest to those working psychodynamically. Findings also highlighted the lack of discourses our culture has for touch and how the ones we share can be super-imposed onto experiences. This should be kept in mind when discussing all types of physical contact with clients. Outcomes from AT pupils cannot be generalized to those seeking psychological support; however, the findings accentuated the power of holistic working. This is important as we begin to understand more around how emotions are held in the body.

Assisted human reproduction legislation: Acknowledging the voice of health care professionals.

OBJECTIVE: Ireland is one of 5 European countries which currently lacks specific legislation on Assisted Human Reproduction (AHR). Draft legislation was introduced in 2017 and revised in 2022 with a view to enacting legislation this year (2022). This study sought to ascertain the views of healthcare professionals to proposed AHR legislation, prior to the implementation of that legislation. STUDY DESIGN: A survey questionnaire based on all clinically relevant aspects of the Irish draft AHR Bill 2017 was distributed to relevant healthcare professionals using an online platform. RESULTS: Over 200 healthcare personnel indicated strong support for the availability of AHR techniques, access to treatment for all patient populations regardless of relationship or gender status, and appropriate legislation and regulation in the field. Views of respondents are at variance with several proposals surrounding surrogacy, with 84 % favouring a pre-birth order to assign parentage from birth, rather than the proposed birth order 6 weeks after birth. The majority also support legislation around international surrogacy. Contrary to the draft Bill, respondents believe that men, as well as women, should be able to use posthumously any stored gametes or embryos belonging to the deceased partner or the couple. While the majority favour altruistic gamete donation, respondents support more generous compensation for donors, such as compensation for time lost at work. CONCLUSION: This study has uniquely ascertained the views of healthcare professionals to imminent AHR legislation. It is hoped that the results will help inform the national legislation as it nears completion. Similar studies could help other countries, and policy bodies such as ESHRE to frame good legislation in this extremely specialised and complex field.

Assisted human reproduction legislation: Listening to the voice of patients.

OBJECTIVE: Legislation and policies regarding assisted human reproduction (AHR) vary widely across nations and societies. As one of only 5 European countries which currently lacks legislation, Ireland now has a unique opportunity to learn from other jurisdictions and introduce AHR law that is reflective of the ongoing myriad developments in this complex field. Draft legislation, initially published in 2017, was revised in 2022 with strong political commitment to enacting in the same year. This study sought to ascertain the views of fertility patients (service users) to the proposed AHR legislation in its current format, prior to its implementation. STUDY DESIGN: A survey questionnaire, previously designed to investigate the attitudes and perceptions of healthcare professionals (HCPs) towards a broad range of issues contained within the draft AHR Bill, was adapted for a patient/service user population. The survey link was distributed via secure email to all patients that had a doctor consult at our fertility clinic in 2020-2021. RESULTS: The survey link was sent to 4420 patients/service users, of whom 1044 (23.6%) responded. A majority had experienced AHR treatment. Service users indicated strong support for AHR regulation and for access to all AHR techniques for all patients, irrespective of relationship or gender status. A majority of respondents disagreed with aspects of the draft bill regarding mandatory counselling, the timing of assignment of parentage in surrogacy, the exclusion of international surrogacy and the exclusion of men from posthumous AHR. Interestingly, the fertility patient cohort were more liberal in their views and opinions regarding AHR than the Irish HCPs previously surveyed. CONCLUSION: This study demonstrates the views of a large group of AHR patients/service users towards proposed AHR legislation. Many of their views concur with but others differ from those of the drafters of the legislation and from those of healthcare professionals. Consideration of the views of all these groups and a collaborative approach would help ensure that Ireland has AHR legislation that is inclusive and fit for purpose in the 21st century.

Self-Care for Family Carers: Can the Alexander Technique help?

BACKGROUND AND PURPOSE: Caring for a family member who is living with dementia can be incredibly challenging. Interventions to support family carers are vital and so carers should be supported to care for themselves and to maintain their own sense of self. The aim of this exploratory study was to explore the views of carers on the potential value of developing an Alexander Technique intervention for family carers of people with dementia. MATERIALS AND METHODS: We delivered a one-off taster session of the Alexander Technique to family carers of people with dementia. Eight carers of people with dementia attended the group session led by two registered Alexander teachers. Post-session questionnaires examined carers' thoughts on the content, context, and process of learning the Alexander technique. A focus group at the end of the session asked participants to provide feedback on their experience and the perceived benefits for carers. RESULTS: Carers' satisfaction with the session was high and they reported benefitting from it. Participants appreciated having time for themselves in which they were able to stop to enjoy a moment of calm. They felt they could use the ideas they gained from the session in everyday life. The use of touch in the sessions was also valued by carers. CONCLUSION: This study provides preliminary evidence that the Alexander Technique has the potential to increase carers' ability to self-care and to support them in their caring. In so doing it has the potential to indirectly help those they care for.

Bridging the phenotypic gap: real-time assessment of mitochondrial function and metabolism of the nematode Caenorhabditis elegans.

BACKGROUND: The ATP levels of an organism are an important physiological parameter that is affected by genetic make up, ageing, stress and disease. RESULTS: We have generated luminescent C. elegans through ubiquitous, constitutive expression of firefly luciferase, widely used for in vitro ATP determination. We hypothesise that whole animal luminescence reflects its intracellular ATP levels in vivo. To test this, we characterised the bioluminescence response of C. elegans during sublethal exposure to, and recovery from azide, a treatment that inhibits mitochondrial respiration reversibly, and causes ATP depletion. Consistent with our expectations, in vivo luminescence decreased with increasing sublethal azide levels, and recovered fully when worms were removed from azide. Firefly luciferase expression levels, stability and activity did not influence the final luminescence. Bioluminescence also reflected the lowered activity of the electron transport chain achieved with RNA interference (RNAi) of genes encoding respiratory chain components. CONCLUSION: Results indicated that C. elegans luminescence reports on ATP levels in real-time. For the first time, we are able to directly assess the metabolism of a whole, living, multicellular organism by determination of the relative ATP levels. This will enable genetic analysis based on a readily quantifiable metabolic phenotype and will provide novel insights into mechanisms of fitness and disease that are likely to be of relevance for other organisms, as well as the worm.

TGF-ß1 alters esophageal epithelial barrier function by attenuation of claudin-7 in eosinophilic esophagitis.

Barrier dysfunction has been implicated in the pathophysiology of eosinophilic esophagitis (EoE). Transforming growth factor-ß1 (TGF-ß1), a potent pleiotropic molecule, is increased in EoE; however, no study has evaluated its influence on esophageal epithelial barrier. We hypothesized that TGF-ß1 regulates barrier dysfunction in EoE. We aimed to determine the role of TGF-ß1 in the epithelial barrier in models of EoE. To examine the impact of TGF-ß1 on esophageal barrier, immortalized human esophageal epithelial (EPC2-hTERT) cells were exposed to TGF-ß1 during the three-dimensional air-liquid interface (3D-ALI) model in vitro. TGF-ß1 exposure diminished EPC2-hTERT barrier function as measured by transepithelial electrical resistance (TEER) and 3 kDa Fluorescein isothiocyanate dextran paracellular flux (FITC Flux), and hematoxylin and eosin (H&E) assessment revealed prominent cellular separation. In analysis of epithelial barrier molecules, TGF-ß1 led to the specific reduction in expression of the tight-junction molecule, claudin-7 (CLDN7), and this was prevented by TGF-ß-receptor I inhibitor. Short hairpin ribonucleic acid (shRNA)-mediated CLDN7 knockdown diminished epithelial barrier function, whereas CLDN7 overexpression resulted in protection from TGF-ß1-mediated barrier dysfunction. In pediatric EoE biopsies CLDN7 expression was decreased and altered localization was observed with immunofluorescence analysis, and the TGF-ß1 downstream transcription factor, phosphorylated SMAD2/3 (pSMAD2/3), was increased. Our data suggest that TGF-ß1 participates in esophageal epithelial barrier dysfunction through CLDN7 dysregulation.

Control of gag-pol gene expression in the Candida albicans retrotransposon Tca2.

BACKGROUND: In the C. albicans retrotransposon Tca2, the gag and pol ORFs are separated by a UGA stop codon, 3' of which is a potential RNA pseudoknot. It is unclear how the Tca2 gag UGA codon is bypassed to allow pol expression. However, in other retroelements, translational readthrough of the gag stop codon can be directed by its flanking sequence, including a 3' pseudoknot. RESULTS: The hypothesis was tested that in Tca2, gag stop codon flanking sequences direct translational readthrough and synthesis of a gag-pol fusion protein. Sequence from the Tca2 gag-UGA-pol junction (300 nt) was inserted between fused lacZ and luciferase (luc) genes in a Saccharomyces cerevisiae dual reporter construct. Although downstream of UGA, luc was expressed, but its expression was unaffected by inserting additional stop codons at the 3' end of lacZ. Luc expression was instead being driven by a previously unknown minor promoter activity within the gag-pol junction region. Evidence together indicated that junction sequence alone cannot direct UGA readthrough. Using reporter genes in C. albicans, the activities of this gag-pol junction promoter and the Tca2 long terminal repeat (LTR) promoter were compared. Of the two promoters, only the LTR promoter was induced by heat-shock, which also triggers retrotransposition. Tca2 pol protein, epitope-tagged in C. albicans to allow detection, was also heat-shock induced, indicating that pol proteins were expressed from a gag-UGA-pol RNA. CONCLUSION: This is the first demonstration that the LTR promoter directs Tca2 pol protein expression, and that pol proteins are translated from a gag-pol RNA, which thus requires a mechanism for stop codon bypass. However, in contrast to most other retroelement and viral readthrough signals, immediate gag UGA-flanking sequences were insufficient to direct stop readthrough in S. cerevisiae, indicating non-canonical mechanisms direct gag UGA bypass in Tca2.

Tryptophan metabolite activation of the aryl hydrocarbon receptor regulates IL-10 receptor expression on intestinal epithelia.

IL-10 is a potent anti-inflammatory cytokine that inhibits the production of proinflammatory mediators. Signaling by IL-10 occurs through the IL-10 receptor (IL-10R), which is expressed in numerous cell types, including intestinal epithelial cells (IECs), where it is associated with development and maintenance of barrier function. Guided by an unbiased metabolomics screen, we identified tryptophan (Trp) metabolism as a major modifying pathway in interferon-γ (IFNγ)-dominant murine colitis. In parallel, we demonstrated that IFNγ induction of indoleamine 2,3-dioxygenase 1, an enzyme that catalyzes the conversion of Trp to kynurenine (Kyn), induces IL-10R1 expression. Based on these findings, we hypothesized that IL-10R1 expression on IEC is regulated by Trp metabolites. Analysis of the promoter region of IL-10R1 revealed a functional aryl hydrocarbon response element, which is induced by Kyn in luciferase-based IL-10R1 promoter assays. Additionally, this analysis confirmed that IL-10R1 protein levels were increased in response to Kyn in IEC in vitro. Studies using in vitro wounding assays revealed that Kyn accelerates IL-10-dependent wound closure. Finally, reduction of murine dextran sodium sulfate colitis through Kyn administration correlates with colonic IL-10R1 expression. Taken together, these results provide evidence on the importance of IL-10 signaling in intestinal epithelia and implicate AHR in the regulation of IL-10R1 expression in the colon.

Calsequestrin binds to monomeric and complexed forms of key calcium-handling proteins in native sarcoplasmic reticulum membranes from rabbit skeletal muscle.

Ca(2+)-handling proteins are important regulators of the excitation-contraction-relaxation cycle in skeletal muscle fibres. Although domain binding studies suggest protein coupling between various Ca(2+)-regulatory elements of triad junctions, no direct biochemical evidence exists demonstrating high-molecular-mass complex formation in native microsomal membranes. Calsequestrin represents the protein backbone of the luminal Ca(2+) reservoir and thereby occupies a central position in Ca(2+) homeostasis; we therefore used calsequestrin blot overlay assays in order to determine complex formation between sarcoplasmic reticulum components. Peroxidase-conjugated calsequestrin clearly labelled four major protein bands in one-dimensional (1D) and 2D electrophoretically separated membrane preparations from adult skeletal muscle. Immunoblotting identified the calsequestrin-binding proteins of approximately 26, 63, 94 and 560 kDa as junctin, calsequestrin itself, triadin and the ryanodine receptor, respectively. Protein-protein coupling could be modified by ionic detergents, non-ionic detergents, changes in Ca(2+) concentration, as well as antibody and purified calsequestrin binding. Importantly, complex formation as determined by blot overlay assays was confirmed by differential co-immunoprecipitation experiments and chemical crosslinking analysis. Hence, the key Ca(2+)-regulatory membrane components of skeletal muscle form a supramolecular membrane assembly. The formation of this tightly associated junctional sarcoplasmic reticulum complex seems to underlie the physiological regulation of skeletal muscle contraction and relaxation, which supports the biochemical concept that Ca(2+) homeostasis is regulated by direct protein-protein interactions.

The 3' untranslated region plays a role in the targeting of metallothionein-I mRNA to the perinuclear cytoplasm and cytoskeletal-bound polysomes.

The mechanism of localisation of metallothionein-I (MT-I) mRNA was studied in transfected cells by in situ hybridisation and cell fractionation. Hepatoma cells were transfected with the 5'-untranslated region and coding region of the beta-globin gene alone or linked to either the beta-globin 3'-untranslated region (3'-UTR) or the MT-I 3'-UTR. The wild-type beta-globin mRNA and the beta-globin mRNA lacking its native 3'-UTR were present in free and cytoskeletal-bound polysomes to a similar extent and showed no localisation. Chimaeric globin-metallothionein transcripts were significantly enriched in cytoskeletal-bound polysomes and were localised in the perinuclear cytoplasm. Chimaeric globin-metallothionein and wild-type globin transcripts were of similar stability. Chinese Hamster Ovary cells were transfected with constructs in which the MT-I 5'-untranslated region and coding sequences were linked to either the endogenous 3'-UTR or the glutathione peroxidase 3'-UTR. Wild-type MT-I transcripts were localised in the perinuclear cytoplasm but the chimaeric MT-I-glutathione peroxidase transcripts showed no distinct localisation. The results indicate that the 3'-UTR of MT-I mRNA contains a localisation signal which promotes both the association of the mRNA with the cytoskeleton and its perinuclear localisation.

Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning.

We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening approximately 38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans.

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode's ATP levels.(1-3) Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.(4,5) In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

SERT and uncertainty: serotonin transporter expression influences information processing biases for ambiguous aversive cues in mice.

The long allele variant of the serotonin transporter (SERT, 5-HTT) gene-linked polymorphic region (5-HTTLPR) is associated with higher levels of 5-HTT expression and reduced risk of developing affective disorders. However, little is known about the mechanisms underlying this protective effect. One hypothesis is that 5-HTT expression influences aversive information processing, with reduced negative cognitive bias present in those with higher 5-HTT expression. Here we investigated this hypothesis using genetically-modified mice and a novel aversive learning paradigm. Mice with high levels of 5-HTT expression (5-HTT over-expressing, 5-HTTOE mice) and wild-type mice were trained to discriminate between three distinct auditory cues: one cue predicted footshock on all trials (CS+); a second cue predicted the absence of footshock (CS-); and a third cue predicted footshock on 20% of trials (CS20%), and was therefore ambiguous. Wild-type mice exhibited equivalently high levels of fear to the CS+ and CS20% and minimal fear to the CS-. In contrast, 5-HTTOE mice exhibited high levels of fear to the CS+ but minimal fear to the CS- and the CS20%. This selective reduction in fear to ambiguous aversive cues suggests that increased 5-HTT expression reduces negative cognitive bias for stimuli with uncertain outcomes.

Epithelial-specific A2B adenosine receptor signaling protects the colonic epithelial barrier during acute colitis.

Central to inflammatory bowel disease (IBD) pathogenesis is loss of mucosal barrier function. Emerging evidence implicates extracellular adenosine signaling in attenuating mucosal inflammation. We hypothesized that adenosine-mediated protection from intestinal barrier dysfunction involves tissue-specific signaling through the A2B adenosine receptor (Adora2b) at the intestinal mucosal surface. To address this hypothesis, we combined pharmacologic studies and studies in mice with global or tissue-specific deletion of the Adora2b receptor. Adora2b(-/-) mice experienced a significantly heightened severity of colitis, associated with a more acute onset of disease and loss of intestinal epithelial barrier function. Comparison of mice with Adora2b deletion on vascular endothelial cells (Adora2b(fl/fl)VeCadCre(+)) or intestinal epithelia (Adora2b(fl/fl)VillinCre(+)) revealed a selective role for epithelial Adora2b signaling in attenuating colonic inflammation. In vitro studies with Adora2b knockdown in intestinal epithelial cultures or pharmacologic studies highlighted Adora2b-driven phosphorylation of vasodilator-stimulated phosphoprotein (VASP) as a specific barrier repair response. Similarly, in vivo studies in genetic mouse models or treatment studies with an Adora2b agonist (BAY 60-6583) recapitulate these findings. Taken together, our results suggest that intestinal epithelial Adora2b signaling provides protection during intestinal inflammation via enhancing mucosal barrier responses.

Evidence for a localisation signal in the 3'-untranslated region from vimentin messenger RNA.

There is increasing evidence that some mRNAs are localised in eukaryotic somatic cells, but it is unclear what proportion of mRNAs are localised and whether this sorting involves 3'-untranslated sequences. The presence of a localisation signal within the 3'-untranslated region of vimentin mRNA was investigated by studying mRNA distribution in fibroblasts transfected with beta-globin and hybrid globin-vimentin gene constructs. In cells transfected with constructs containing either a fragment of the rabbit beta-globin gene containing both coding sequences and 3'untranslated region or the beta-globin coding sequences alone in situ hybridisation showed that beta-globin mRNA was distributed throughout the cytoplasm without any evident localisation. In contrast, in cells transfected with globin coding sequences linked to the vimentin 3'-untranslated region there was a strong perinuclear localisation of the hybrid mRNA. The results show that loss of its endogenous 3'-untranslated region does not affect distribution of beta-globin mRNA whereas the vimentin 3'-untranslated region causes an altered localisation of beta-globin mRNA. We conclude that the vimentin 3'-untranslated region contains a localisation signal which can direct reporter sequences to the perinuclear cytoplasm.