Metabolic imaging across scales reveals distinct prostate cancer phenotypes.

Hyperpolarised magnetic resonance imaging (HP-13C-MRI) has shown promise as a clinical tool for detecting and characterising prostate cancer. Here we use a range of spatially resolved histological techniques to identify the biological mechanisms underpinning differential [1-13C]lactate labelling between benign and malignant prostate, as well as in tumours containing cribriform and non-cribriform Gleason pattern 4 disease. Here we show that elevated hyperpolarised [1-13C]lactate signal in prostate cancer compared to the benign prostate is primarily driven by increased tumour epithelial cell density and vascularity, rather than differences in epithelial lactate concentration between tumour and normal. We also demonstrate that some tumours of the cribriform subtype may lack [1-13C]lactate labelling, which is explained by lower epithelial lactate dehydrogenase expression, higher mitochondrial pyruvate carrier density, and increased lipid abundance compared to lactate-rich non-cribriform lesions. These findings highlight the potential of combining spatial metabolic imaging tools across scales to identify clinically significant metabolic phenotypes in prostate cancer.

Uncertainty estimation using a 3D probabilistic U-Net for segmentation with small radiotherapy clinical trial datasets.

BACKGROUND AND OBJECTIVES: Bio-medical image segmentation models typically attempt to predict one segmentation that resembles a ground-truth structure as closely as possible. However, as medical images are not perfect representations of anatomy, obtaining this ground truth is not possible. A surrogate commonly used is to have multiple expert observers define the same structure for a dataset. When multiple observers define the same structure on the same image there can be significant differences depending on the structure, image quality/modality and the region being defined. It is often desirable to estimate this type of aleatoric uncertainty in a segmentation model to help understand the region in which the true structure is likely to be positioned. Furthermore, obtaining these datasets is resource intensive so training such models using limited data may be required. With a small dataset size, differing patient anatomy is likely not well represented causing epistemic uncertainty which should also be estimated so it can be determined for which cases the model is effective or not. METHODS: We use a 3D probabilistic U-Net to train a model from which several segmentations can be sampled to estimate the range of uncertainty seen between multiple observers. To ensure that regions where observers disagree most are emphasised in model training, we expand the Generalised Evidence Lower Bound (ELBO) with a Constrained Optimisation (GECO) loss function with an additional contour loss term to give attention to this region. Ensemble and Monte-Carlo dropout (MCDO) uncertainty quantification methods are used during inference to estimate model confidence on an unseen case. We apply our methodology to two radiotherapy clinical trial datasets, a gastric cancer trial (TOPGEAR, TROG 08.08) and a post-prostatectomy prostate cancer trial (RAVES, TROG 08.03). Each dataset contains only 10 cases each for model development to segment the clinical target volume (CTV) which was defined by multiple observers on each case. An additional 50 cases are available as a hold-out dataset for each trial which had only one observer define the CTV structure on each case. Up to 50 samples were generated using the probabilistic model for each case in the hold-out dataset. To assess performance, each manually defined structure was matched to the closest matching sampled segmentation based on commonly used metrics. RESULTS: The TOPGEAR CTV model achieved a Dice Similarity Coefficient (DSC) and Surface DSC (sDSC) of 0.7 and 0.43 respectively with the RAVES model achieving 0.75 and 0.71 respectively. Segmentation quality across cases in the hold-out datasets was variable however both the ensemble and MCDO uncertainty estimation approaches were able to accurately estimate model confidence with a p-value < 0.001 for both TOPGEAR and RAVES when comparing the DSC using the Pearson correlation coefficient. CONCLUSIONS: We demonstrated that training auto-segmentation models which can estimate aleatoric and epistemic uncertainty using limited datasets is possible. Having the model estimate prediction confidence is important to understand for which unseen cases a model is likely to be useful.

An RNA-dependent and phase-separated active subnuclear compartment safeguards repressive chromatin domains.

The nucleus is composed of functionally distinct membraneless compartments that undergo phase separation (PS). However, whether different subnuclear compartments are connected remains elusive. We identified a type of nuclear body with PS features composed of BAZ2A that associates with active chromatin. BAZ2A bodies depend on RNA transcription and BAZ2A non-disordered RNA-binding TAM domain. Although BAZ2A and H3K27me3 occupancies anticorrelate in the linear genome, in the nuclear space, BAZ2A bodies contact H3K27me3 bodies. BAZ2A-body disruption promotes BAZ2A invasion into H3K27me3 domains, causing H3K27me3-body loss and gene upregulation. Weak BAZ2A-RNA interactions, such as with nascent transcripts, promote BAZ2A bodies, whereas the strong binder long non-coding RNA (lncRNA) Malat1 impairs them while mediating BAZ2A association to chromatin at nuclear speckles. In addition to unraveling a direct connection between nuclear active and repressive compartments through PS mechanisms, the results also showed that the strength of RNA-protein interactions regulates this process, contributing to nuclear organization and the regulation of chromatin and gene expression.

Multi-omics and imaging mass cytometry characterization of human kidneys to identify pathways and phenotypes associated with impaired kidney function.

Despite the recent advances in our understanding of the role of lipids, metabolites, and related enzymes in mediating kidney injury, there is limited integrated multi-omics data identifying potential metabolic pathways driving impaired kidney function. The limited availability of kidney biopsies from living donors with acute kidney injury has remained a major constraint. Here, we validated the use of deceased transplant donor kidneys as a good model to study acute kidney injury in humans and characterized these kidneys using imaging and multi-omics approaches. We noted consistent changes in kidney injury and inflammatory markers in donors with reduced kidney function. Neighborhood and correlation analyses of imaging mass cytometry data showed that subsets of kidney cells (proximal tubular cells and fibroblasts) are associated with the expression profile of kidney immune cells, potentially linking these cells to kidney inflammation. Integrated transcriptomic and metabolomic analysis of human kidneys showed that kidney arachidonic acid metabolism and seven other metabolic pathways were upregulated following diminished kidney function. To validate the arachidonic acid pathway in impaired kidney function we demonstrated increased levels of cytosolic phospholipase A2 protein and related lipid mediators (prostaglandin E2) in the injured kidneys. Further, inhibition of cytosolic phospholipase A2 reduced injury and inflammation in human kidney proximal tubular epithelial cells in vitro. Thus, our study identified cell types and metabolic pathways that may be critical for controlling inflammation associated with impaired kidney function in humans.

High prevalence of hyperuricemia and the association with metabolic syndrome in the rural areas of Southwestern China: A structural equation modeling based on the Zhuang minority cohort.

BACKGROUND AND AIMS: The prevalence of hyperuricemia (HUA) and metabolic syndrome (MetS) in the Zhuang minority had not been examined. We aimed to determine the prevalence of HUA and MetS, and explore the interrelationship among the serum uric acid to creatinine (SUA/Cr) ratio, MetS, and its components. METHODS AND RESULTS: A cross-sectional study was conducted with structured questionnaire and physical examination based on the Zhuang minority cohort. A Structural Equation Model was performed to examine the hypothesis link between the SUA/Cr ratio, MetS, and its components. 10,902 aged 35-74 years Zhuang minority adults were included. The total prevalence of HUA and MetS was 17.5% and 23.7%, respectively. The SUA/Cr ratio had a positive effect on MetS (the standardized coefficient ßr was 0.311 in males and 0.401 in females). The SUA/Cr ratio was positively associated with obesity (ßr = 0.215), dyslipidemia (ßr = 0.177), and high blood pressure (ßr = 0.034) in males and was positively associated with obesity (ßr = 0.303), dyslipidemia (ßr = 0.162), and hyperglycemia (ßr = 0.036) in females. CONCLUSIONS: The prevalence of HUA in the aged 35-74 years Zhuang minority adults was high while the prevalence of MetS was relatively low. As HUA is an earlier-onset metabolic disorder and the SUA/Cr ratio had a positive effect on MetS and its components, the prevention measures of MetS should be strengthened. And the SUA/Cr ratio can be used as an early warning sign to implement the intervention measures of MetS.

EyeCee One preloaded intraocular lens: are patients with glaucoma more at risk?

OBJECTIVE: The Medicine and Healthcare products Regulatory Agency reported links of raised intraocular pressure (IOP) with recently implanted EyeCee One intraocular lens (IOL). This work investigates if glaucomatous eyes were more susceptible to these postoperative IOP rises and if they required more intensive management. METHODS: Retrospective observational study of all phacoemulsification surgery with implanted EyeCee One IOL, performed between 1 October 2022 and 26 January 2023 inclusive. ANALYSIS: A significant IOP elevation was defined as an IOP rise of 10 mm Hg or more from preoperative to maximal postoperative IOP reading. The management of all patients who had a significant IOP elevation was reviewed. Glaucoma/ocular hypertension cases were identified and analysed against non-glaucomatous eyes and statistical analysis performed. RESULTS: 112 glaucoma and 671 non-glaucoma cases identified; 19.6% of the glaucoma cohort had a significant postoperative IOP rise compared with 8.9% of patients without glaucoma (OR 2.49 (95% CI 1.45 to 4.20) p=0.0014). In the glaucoma cohort, 12.5% had an increase in the number of topical IOP-lowering agents (mean increase 1.65±1.58), 6.3% required systemic treatment and 2.7% surgical intervention. In the non-glaucoma group, 3.3% required topical treatment (mean number of agents 0.88±1.34), 0.8% required systemic treatment and 0.2% surgical intervention. CONCLUSION: This study shows that during the time frame in question, patients with glaucoma or ocular hypertension who had an EyeCee One IOL were almost two and a half times more likely to have a postoperative rise of 10 mm Hg or more in IOP following routine cataract surgery, requiring more aggressive management.

Intensification of Local Therapy With High Dose Rate, Intraoperative Radiation Therapy (HDR-IORT) and Extended Resection for Locally Advanced and Recurrent Colorectal Cancer.

BACKGROUND: We report our long-term experience with high dose rate intraoperative radiotherapy (HDR-IORT) in a single, quaternary institution. PATIENTS/METHODS: From 2004 to 2020, 60 HDR-IORT procedures for locally advanced colorectal cancer (LACC) and 81 for locally recurrent colorectal cancer (LRCC) were done in our institution. Preoperative radiotherapy was done prior to majority of the resections (89%, 125/141). Sixty-nine percent (58/84) of the resections involving pelvic exenterations had >3 en bloc organs resected. HDR-IORT was delivered using a Freiburg applicator. A single 10 Gy fraction was delivered. Margin status was R0 and R1 in 54% (76/141) and 46% (65/141) of the resections, respectively. RESULTS: With a median follow-up time of 4 years, 3-, 5-, and 7- year, overall survival (OS) rates were 84%, 58%, and 58% for LACC and 68%, 41%, and 37% for LRCC, respectively. Local progression-free survival (LPFS) rates were 97%, 93%, and 93% for LACC and 80%, 80%, 80% for LRCC, respectively. For the LRCC group, an R1 resection was associated with worse OS, LPFS, and progression-free survival (PFS), preoperative EBRT was associated with improved LPFS and PFS, and ≥2 years disease-free interval was associated with improved PFS. The most common severe adverse events were postoperative abscess (n = 25) and bowel obstruction (n = 11). There were 68 grade 3 to 4 and no grade 5 adverse events. CONCLUSIONS: Favorable OS and LPFS can be achieved for LACC and LRCC with intensive local therapy. In patients with risk factors for poorer outcomes, optimization of EBRT and IORT, surgical resection, and systemic therapy are required.

Dengue in Pregnancy: A Southeast Asian Perspective.

Dengue cases have been rising in recent years. In 2019 alone, over 658,301 of the 5.6 million reported cases originated from Southeast Asia (SEA). Research has also shown detrimental outcomes for pregnant infected women. Despite this, existing literature describing dengue's effects on pregnancy in SEA is insufficient. Through this narrative review, we sought to describe dengue's effects on pregnancy systemically and emphasize the existing gaps in the literature. We extensively searched various journals cited in PubMed and Ovid Medline, national clinical practice guidelines, and governmental reports. Dengue in pregnancy increases the risk of pre-eclampsia, Dengue Hemorrhagic Fever (DHF), fetal distress, preterm delivery, Caesarean delivery, and maternal mortality. Vertical transmission, intrauterine growth restriction, and stillbirth are possible sequelae of dengue in fetuses. We found that trimester-specific physiological impacts of dengue in pregnancy (to both mother and child) and investigations and management methods demanded further research, especially in the SEA region.

Practice of immediate sequential bilateral cataract surgery (ISBCS): A teaching hospital experience in United Kingdom.

BACKGROUND / OBJECTIVES: Utilisation of ISBCS has been encouraged since COVID-19 in line with the RCOphth recommendations. This study aims to share experience from a UK teaching hospital on ISBCS and to evaluate pre-, intra- and post-operative outcomes from the ISBCS cohort. METHODS: Of 3402 cataract surgeries performed between July 2020 and July 2021 (1 year since the reopening of the cataract service from COVID-19), 208 eyes of 104 patients (6.1%) undergoing ISBCS were retrospectively studied on their demographics, biometry, surgeon grades, and pre-, intra- and post-operative data. RESULTS: The mean age was 74.5 ± 9.4 years and 62% were female. Eighty-nine percent of the eyes were performed under local anaesthesia and 70% were 'routine' cases. Other risk factors included: short eyes requiring pre-operative mannitol infusion (10%), high myopia (8%), poor dilation (2%) and a 'glaucoma' cohort [angle closure (2%) and prior trabeculectomy (1%)]. Three eyes (1%) had complications intra-operatively in second eye (1 case each: posterior capsule rupture, corneal oedema and zonular dehiscence). Two patients (1%) had complications in the first eye (1 case each: suprachoroidal haemorrhage, conjunctival & iris haemorrhage), hence had their second eye postponed. Twelve months post-operatively, 20 eyes (10%) had recorded post-op complications with cystoid macular oedema being the commonest (4.5%) and no endophthalmitis. Eighty-six percent were discharged with satisfaction. No significant differences were found in pre-operative features, complication rates and post-operative outcomes between consultant and non-consultant surgeons (p > 0.05). CONCLUSIONS: Our experience which included a cohort of high-risk patients showed safe and successful practice of ISBCS without having a negative impact on training.

Ophthalmology service recovery with 'general anaesthesia to local anaesthesia conversion' or GALA initiative: a new surgical service pilot.

We report on the establishment of "general anaesthesia (GA) to local anaesthesia (LA) conversion" or GALA initiative at Sheffield Teaching Hospitals NHS Foundation Trust. Through GALA initiative, long waiters for cataract surgery under GA or LA with intravenous sedation were counselled for LA surgery. With careful surgical planning and personalised solutions to LA barriers, LA-converted patients reported good visual and surgical experience outcomes. The GALA initiative reduced cataract surgery waiting times of participants by 11 weeks and increased GA list capacity by three to four theatre lists in its four months of pilot run. This initiative did not limit training opportunities for ophthalmology trainees whose training was affected significantly during the pandemic.

Lacrimal sac ciliated HPV16 positive, adenosquamous carcinoma-A case report of a unique histological variant at this site and a review of the literature.

A 47-year-old female developed a reddish swelling of the right medial canthus over 3 months. On examination, a red, firm mass, involving the right medial canthal and extending into the inferior fornix was present and the globe was displaced upwards and inwards. A staging MRI scan confirmed a lacrimal sac lesion with anterior orbit extension. After an equivocal biopsy, the patient underwent debulking surgery. Histology showed a lacrimal sac invasive adenosquamous carcinoma, comprising poorly differentiated squamous carcinoma and invasive adenocarcinoma areas arranged in a tubulo-glandular pattern. The adenocarcinoma harboured numerous cilia. p16 showed block positivity of both components and micro-dissected tissue from both areas showed the presence of HPV16 DNA by PCR. This is the first description of ciliated adenosquamous carcinoma of the lacrimal sac and this finding is placed into the context of what is known about ciliated head and neck adenosquamous carcinomas and the role of high-risk HPV.

Holding back the tears: is there a role for marsupialisation?

OBJECTIVE: Medial eyelid tumours may result in the loss of the proximal lacrimal system during staged excision and delayed reconstruction, to achieve tumour margin clearance. The remnant canaliculus was marsupialised during reconstruction. The aim was to understand how many patients experienced symptomatic epiphora as a consequence of this. METHODS AND ANALYSIS: A retrospective study including patients over a 15-year period with medial eyelid tumours, where the proximal lacrimal system was sacrificed to achieve tumour margin clearance. Included were all who had marsupialisation of the remnant distal stump as part of their delayed reconstruction. All who had pre-existing epiphora were excluded. The primary objective was the rate of epiphora following the procedure. A systematic literature review of postoperative epiphora occurring in patients with lid tumours requiring lacrimal system injury/sacrifice during tumour excision. RESULTS: There were 22 eyes (22 patients). All were basal cell carcinomas except for 1 (4.5%) tarsal conjunctival squamous cell carcinoma. All cases involved the lower lid. There were two (9.1%) patients who developed epiphora. One patient underwent a superior three-snip punctoplasty, botulinum toxin to the lacrimal gland and conjunctivodacryocystorhinostomy with Lester Jones tube insertion. The other patient was not overly troubled and did not require further treatment. The literature review showed the median postoperative rate of epiphora in these patients was 12.5% (range 0%-100%). CONCLUSION: Marsupialisation of the remnant canaliculus during delayed reconstruction is a straightforward and effective surgical option, which may help prevent postreconstruction epiphora when the proximal lacrimal system is sacrificed for tumour margin clearance. TRIAL REGISTRATION NUMBER: 10391.

A systematic review of patient-reported outcome measures in paediatric endocrinology.

CONTEXT: Patient reported outcome measures (PROMs) are useful tools in paediatric endocrinology to gauge health status in children, especially since they are often unable to clearly communicate it themselves. We aimed to systematically search and appraise all available PROMs relevant to paediatric endocrinology and provide a curated resource for health professionals to utilise. EVIDENCE ACQUISITION: We identified PROMs in paediatric endocrinology by systematically searching the Cochrane Library, MEDLINE, World Health Organisation International Clinical Trials Registry Platform, and the Cumulative Index to Nursing and Allied Health Literature on May 20, 2022. Additional studies were located through hand searching and content area expert contributions. We assessed the quality of each PROM using the COSMIN risk of bias checklist. EVIDENCE SYNTHESIS: We identified 5003 papers in the initial search. After applying exclusion criteria we included seven PROMs in the review. Six PROMs were specific to Type I Diabetes and one to Hypothyroidism. We gave all studies an overall COSMIN score of 'inadequate' due to poorly detailed PROM development. CONCLUSION: The scope and quality of PROMs in paediatric endocrinology is limited. Further research and development of PROM tools are required in paediatric endocrinology to allow for improved patient care.

Combining Radiotherapy and Immunotherapy in Metastatic Breast Cancer: Current Status and Future Directions.

The role of radiotherapy and immunotherapy with immune checkpoint inhibitors (ICI) is of emerging interest in many solid tumours, including breast cancer. There is increasing evidence that the host's immune system plays an important role in influencing the response to treatment and prognosis in breast cancer. Several pre-clinical studies and clinical trials have reported on the 'abscopal effect-regression of distant untreated tumour sites, mediated by an immunological response following ionizing radiation to a targeted tumour site. Stereotactic Ablative Body Radiotherapy (SABR) is a non-invasive technique used to augment various immune responses with an ablative tumoricidal dose when compared to conventional radiotherapy. SABR is characterized by typically 1-5 precision radiotherapy treatments that simultaneously deliver a high dose, whilst sparing normal tissues. Following SABR, there is evidence of systemic immune activation in patients with increased PD1 expression on CD8+ and CD4+ T cells. Studies continue to focus on metastatic triple-negative disease, a highly immunogenic subtype of breast cancer with poor prognosis. In this review, we discuss the immunological effect of SABR, alone and in combination with immunotherapy, and the importance of dose and fractionation. We also propose future strategies for treating oligometastatic disease, where this approach may be most useful for producing durable responses.

Construction and Comprehensive Analysis of a circRNA-miRNA-mRNA Regulatory Network to Reveal the Pathogenesis of Hepatocellular Carcinoma.

Background: Circular RNAs (circRNAs) have been demonstrated to be closely related to the carcinogenesis of human cancer in recent years. However, the molecular mechanism of circRNAs in the pathogenesis of hepatocellular carcinoma (HCC) has not been fully elucidated. We aimed to identify critical circRNAs and explore their potential regulatory network in HCC. Methods: The robust rank aggregation (RRA) algorithm and weighted gene co-expression network analysis (WGCNA) were conducted to unearth the differentially expressed circRNAs (DEcircRNAs) in HCC. The expression levels of DEcircRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). A circRNA-miRNA-mRNA regulatory network was constructed by computational biology, and protein-protein interaction (PPI) network, functional enrichment analysis, survival analysis, and infiltrating immune cells analysis were performed to uncover the potential regulatory mechanisms of the network. Results: A total of 22 DEcircRNAs were screened out from four microarray datasets (GSE94508, GSE97332, GSE155949, and GSE164803) utilizing the RRA algorithm. Meanwhile, an HCC-related module containing 404 circRNAs was identified by WGCNA analysis. After intersection, only four circRNAs were recognized in both algorithms. Following qRT-PCR validation, three circRNAs (hsa_circRNA_091581, hsa_circRNA_066568, and hsa_circRNA_105031) were chosen for further analysis. As a result, a circRNA-miRNA-mRNA network containing three circRNAs, 17 miRNAs, and 222 mRNAs was established. Seven core genes (ESR1, BUB1, PRC1, LOX, CCT5, YWHAZ, and DDX39B) were determined from the PPI network of 222 mRNAs, and a circRNA-miRNA-hubgene network was also constructed. Functional enrichment analysis suggested that these seven hub genes were closely correlated with several cancer related pathways. Survival analysis revealed that the expression levels of the seven core genes were significantly associated with the prognosis of HCC patients. In addition, we also found that these seven hub genes were remarkably related to the infiltrating levels of immune cells. Conclusion: Our research identified three pivotal HCC-related circRNAs and provided novel insights into the underlying mechanisms of the circRNA-miRNA-mRNA regulatory network in HCC.

Liver-specific LINC01146, a promising prognostic indicator, inhibits the malignant phenotype of hepatocellular carcinoma cells both in vitro and in vivo.

BACKGROUND: Long non-coding RNAs (lncRNAs) are involved in the development of hepatocellular carcinoma (HCC). We aimed to investigate the function of LINC01146 in HCC. METHODS: The expression of LINC01146 in HCC tissues was explored via The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and was verified using quantitative real-time polymerase chain reaction (qRT-PCR) in our HCC cohort. Kaplan-Meier analysis was used to assess the relationship between LINC01146 and the prognosis of HCC patients. Cell Counting Kit 8, colony formation assays, Transwell assays, flow cytometric assays, and tumour formation models in nude mice were conducted to reveal the effects of LINC01146 on HCC cells both in vitro and in vivo. Bioinformatic methods were used to explore the possible potential pathways of LINC01146 in HCC. RESULTS: LINC01146 was significantly decreased in HCC tissues compared with adjacent normal tissues and was found to be related to the clinical presentations of malignancy and the poor prognosis of HCC patients. Overexpression of LINC01146 inhibited the proliferation, migration, and invasion of HCC cells in vitro, while promoting their apoptosis. In contrast, downregulation of LINC01146 exerted the opposite effects on HCC cells in vitro. In addition, overexpression of LINC01146 significantly inhibited tumour growth, while downregulation of LINC01146 promoted tumour growth in vivo. Furthermore, the coexpressed genes of LINC01146 were mainly involved in the "metabolic pathway" and "complement and coagulation cascade pathway". CONCLUSION: LINC01146 expression was found to be decreased in HCC tissues and associated with the prognosis of HCC patients. It may serve as a cancer suppressor and prognostic biomarker in HCC.

The activity of human enhancers is modulated by the splicing of their associated lncRNAs.

Pervasive enhancer transcription is at the origin of more than half of all long noncoding RNAs in humans. Transcription of enhancer-associated long noncoding RNAs (elncRNA) contribute to their cognate enhancer activity and gene expression regulation in cis. Recently, splicing of elncRNAs was shown to be associated with elevated enhancer activity. However, whether splicing of elncRNA transcripts is a mere consequence of accessibility at highly active enhancers or if elncRNA splicing directly impacts enhancer function, remains unanswered. We analysed genetically driven changes in elncRNA splicing, in humans, to address this outstanding question. We showed that splicing related motifs within multi-exonic elncRNAs evolved under selective constraints during human evolution, suggesting the processing of these transcripts is unlikely to have resulted from transcription across spurious splice sites. Using a genome-wide and unbiased approach, we used nucleotide variants as independent genetic factors to directly assess the causal relationship that underpin elncRNA splicing and their cognate enhancer activity. We found that the splicing of most elncRNAs is associated with changes in chromatin signatures at cognate enhancers and target mRNA expression. We provide evidence that efficient and conserved processing of enhancer-associated elncRNAs contributes to enhancer activity.