We have compared genome-wide patterns of RNA 2'-O-methylation (Nm) between two isogenic pairs of neurons. Each pair includes one line harboring a small deletion of orphan box C/D snoRNAs (SNORD116s) from the paternal chr15q11-q13 region. One isogenic pair also differs in expression of SNORD113/114 snoRNAs from chr14q32.2. Wild-type and modified cells were differentiated into cortical neurons, and genome-wide patterns of Nm identified. Neurons display a distinctive signature of rRNA modification compared to undifferentiated stem cells. We further identified thousands of shared Nm sites in mRNAs, lncRNAs and small RNAs. Most sites do not exhibit canonical complementarity to snoRNAs, but a number exhibit strong complementarity to U3 snoRNA, not previously shown to direct Nm. Evidence from cross-linking and sequencing of hybrids (CLASH) suggests that U3 is proximally associated with a subset of 2'-O-methylation events. Finally, we identify a number of apparent canonical targets of SNORD113, SNORD114 and SNORD116 snoRNAs. These data present a comprehensive characterization of the Nm landscape in neurons and, for the first time, allow the assignment of Nm sites targeted by specific orphan snoRNAs associated with neurodevelopmental and other disorders.

Previously considered "housekeeping" genes, small nucleolar RNAs (snoRNAs) are increasingly understood to have wide-ranging functions in cancer, yet their role in metastasis has been less well studied. Here, we identify the snoRNA Snord67 as a regulator of lymph node (LN) metastasis in breast cancer. Snord67 expression is enriched in LN metastases in an immune-competent mouse model of female breast cancer. In an orthotopic breast cancer model, loss of Snord67 decreases LN metastasis. In a model of lymphatic metastasis, Snord67 loss decreases LN tumor growth and distant metastases. In breast cancer cell lines, Snord67 knockout results in loss of targeted 2'-O-methylation on U6 small nuclear RNA, as well as widespread changes in splicing. Together, these results demonstrate that Snord67 regulates splicing and promotes the growth of LN metastases and subsequent spread to distant metastases. SnoRNA-guided modifications of the spliceosome and regulation of splicing may represent a potentially targetable pathway in cancer.

BACKGROUND:Reactive oxygen species (ROS) augment the activation of vascular smooth muscle cells (SMCs) and promote neointimal hyperplasia evoked by arterial injury or atherogenesis. We have previously shown that small nucleolar RNAs (snoRNAs) from the Rpl13a locus are key regulators of cellular ROS levels. METHODS:Using mice deficient in the Rpl13a snoRNAs, we tested whether these snoRNAs regulate SMC activation in vitro and in vivo. Carotid endothelial denudation was used to provoke neointimal hyperplasia in wild-type (WT) and snoRNA knockout (snoKO) mice, which lack all four intronically-encoded Rpl13a snoRNAs. Primary SMCs from WT and snoKO mice were used for in vitro functional and proteomic analyses. HEK293T cells with specific snoRNA deletions were used to test for snoRNA-guided 2'-O-methylation of mRNA. RESULTS:Arterial ROS levels, inflammation, and carotid artery neointimal hyperplasia were reduced in snoKO compared with WT mice. In vitro, snoKO SMCs demonstrated lower ROS levels and less migration, proliferation, and inflammatory signaling than WT SMCs. Reduced ROS levels in snoKO SMCs and aortas correlated with upregulation of the mitochondrial protein COX4I2, which is associated with reduced mitochondrial ROS under normoxic conditions. Deleting the snoRNA U32A in human HEK293T cells decreased 2'-O-methylation of COX4I2 mRNA and upregulated COX4I2 protein without changing COX4I2 mRNA levels. Silencing Cox4i2 in snoKO SMCs upregulated SMC ROS to WT levels. CONCLUSIONS:Rpl13a snoRNAs are important drivers of SMC activation and neointimal hyperplasia. Rpl13a snoRNAs augment SMC ROS levels, at least in part, by post-transcriptional downregulation of COX4I2 expression.

The N 6 -methyladenosine (m 6 A) modification is found in thousands of cellular mRNAs and is a critical regulator of gene expression and cellular physiology. m 6 A dysregulation contributes to several human diseases, and the m 6 A methyltransferase machinery has emerged as a promising therapeutic target. However, current methods for studying m 6 A require RNA isolation and do not provide a real-time readout of mRNA methylation in living cells. Here we present a g enetically encoded m 6 A sensor (GEMS) technology, which couples a fluorescent signal with cellular mRNA methylation. GEMS detects changes in m 6 A caused by pharmacological inhibition of the m 6 A methyltransferase, giving it potential utility for drug discovery efforts. Additionally, GEMS can be programmed to achieve m 6 A-dependent delivery of custom protein payloads in cells. Thus, GEMS is a versatile platform for m 6 A sensing that provides both a simple readout for m 6 A methylation and a system for m 6 A-coupled protein expression.

Abstract Small nucleolar RNAs (snoRNAs) a class of ncRNAs that canonically guide post-transcriptional modifications, including 2'-O-methylation, on ribosomal RNA (rRNA) and small nuclear RNA (snRNA). While traditionally considered housekeeping genes, snoRNAs have increasingly been found to function in diverse physiologic and pathologic processes, including cancer. In an immune-competent murine model of triple negative breast cancer (TNBC) lymphatic metastasis, we identified the snoRNA Snord67 as one of the most upregulated noncoding RNAs in axillary LN (AxLN) tumors relative to mammary fat pad (MFP) tumors and lung metastases. Loss of Snord67 resulted in decreased colony formation and spheroid size in murine and human TNBC cell lines, and led to decreased lymph node tumor growth and decreased distant metastases in two immune-competent murine models of TNBC lymphatic dissemination. To determine the mechanism by which Snord67 promotes tumor growth and metastasis, we examined the impact of Snord67 on 2'-O-methylation, gene expression, and alternative splicing. Loss of Snord67 in TNBC cell lines led to decreased 2'-O-methylation at the C60 nucleotide (Cm60) in the core spliceosome component U6 snRNA. Re-introduction of wild-type Snord67 rescued Cm60 in U6 snRNA and rescued the colony formation and spheroid phenotypes of the Snord67 knockout cell lines. However, a mutant Snord67 incapable of guiding U6 Cm60 only partially rescued these in vitro phenotypes, suggesting that Snord67 promotes in vitro tumor cell growth at least in part by guiding Cm60 in U6 snRNA. We then performed RNA sequencing of Snord67 knockout and wild-type murine and human TNBC cell lines. We found that loss of Snord67 led to widespread changes in alternative splicing, consistent with its role in guiding 2'-O-methylation of the core spliceosome component U6. We further demonstrated that the inclusion of alternatively spliced cassette exons in MYO18A and NFYA was not only downregulated upon Snord67 knockout in a human TNBC cell line, but also positively correlated with Snord67 expression levels in primary breast tumors and lymph node metastases from breast cancer patients. Based on these results, we propose a model in which Snord67 guides U6 Cm60, which leads to a pro-metastatic alternative splicing program and thereby promotes lymph node tumor growth and distant metastasis. Citation Format: Katherine I Zhou, Yvonne Chao, Kwame K Forbes, Alessandro Porrello, Gabrielle M Gentile, Aaron C Chack, Dixcy J.S. John Mary, Haizhou Liu, Yinzhou Zhu, Eric Cockman, Lincy Edatt, Grant A Goda, Justin Zhao, Hala Abou Assi, Hannah J Wiedner, Yi-Hsuan Tsai, Lily Wilkinson, Amanda E Van Swearingen, Lisa A Carey, Jimena Giudice, Daniel Dominguez, Christopher L Holley, Chad V Pecot. Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A003.

N6-methyladenosine (m6A) is an abundant mRNA modification in the brain that has important roles in neurodevelopment and brain function. However, because of technical limitations, global profiling of m6A sites within the individual cell types that make up the brain has not been possible. Here, we develop a mouse model that enables transcriptome-wide m6A detection in any tissue of interest at single-cell resolution. We use these mice to map m6A across different brain regions and within single cells of the mouse cortex and discover a high degree of shared methylation across brain regions and cell types. However, we also identify a small number of differentially methylated mRNAs in neurons that encode important regulators of neuronal signaling, and we discover that microglia have lower levels of m6A than other cell types. Finally, we perform single-cell m6A mapping in aged mice and identify many transcripts with age-dependent changes in m6A.

Small nucleolar RNAs (snoRNAs) are critical in guiding post-transcriptional modifications like 2'- O -methylation (Nm), which play crucial roles in downstream processes such as splicing and translation. This study tests a novel method for Nm validation, addressing a significant gap in modern Nm research, and offers insight into the intricacies of snoRNA-guided Nm. While mapping of Nm modifications has seen significant improvement within the past decade, no major techniques have been able to validate these potential sites. Additionally, many mapping techniques lack consensus among proposed Nm sites, especially on mRNAs. Without a proper validation technique, Nm research lags compared to its peer post-transcriptional modifications. The RNase H-based Nm-VAQ assay used here quantifies 2'- O -methylation at single nucleotide resolution across various RNA species including rRNA, snRNA, and mRNA. Its optimization for mRNA allows for an unprecedented way to study the effects of Nm modifications in low abundance transcripts. Utilizing this, the study also explores the potential of creating synthetic snoRNAs to guide Nm modifications. Exogenous snoRNAs are shown to rescue Nm in genetic knockout models and can be mutated to guide Nm at any location along the target RNA transcript. Preliminary work indicates that synthetic snoRNAs demonstrate the ability to modify luciferase, impacting translation efficiency. Targeting an exon increases mRNA abundance but decreases protein expression, consistent with previous findings on Pxdn mRNA. These findings set the scene for novel understanding of the relationship between snoRNA abundance, 2'- O -methylation efficiency, and Nm's impact on gene expression.

Small nucleolar RNAs (snoRNAs) are a class of ncRNAs that canonically guide post-transcriptional modifications, including 2'-O-methylation, on ribosomal RNA (rRNA) and small nuclear RNA (snRNA). While traditionally considered housekeeping genes, snoRNAs have increasingly been found to function in diverse physiologic and pathologic processes, including cancer. In an immune-competent murine model of triple negative breast cancer (TNBC) lymphatic metastasis, we identified the snoRNA Snord67 as one of the most upregulated noncoding RNAs in axillary LN (AxLN) tumors relative to mammary fat pad (MFP) tumors and lung metastases. Loss of Snord67 resulted in decreased colony formation and spheroid size in murine and human TNBC cell lines, and led to decreased lymph node tumor growth and decreased distant metastases in two immune-competent murine models of TNBC lymphatic dissemination. To determine the mechanism by which Snord67 promotes tumor growth and metastasis, we examined the impact of Snord67 on 2'-O-methylation, gene expression, and alternative splicing. Loss of Snord67 in TNBC cell lines led to decreased 2'-O-methylation at the C60 nucleotide (Cm60) in the core spliceosome component U6 snRNA. Re-introduction of wild-type Snord67 rescued Cm60 in U6 snRNA and rescued the colony formation and spheroid phenotypes of the Snord67 knockout cell lines. However, a mutant Snord67 incapable of guiding U6 Cm60 only partially rescued these in vitro phenotypes, suggesting that Snord67 promotes in vitro tumor cell growth at least in part by guiding Cm60 in U6 snRNA. We then performed RNA sequencing of Snord67 knockout and wild-type murine and human TNBC cell lines. We found that loss of Snord67 led to widespread changes in alternative splicing, consistent with its role in guiding 2'-O-methylation of the core spliceosome component U6. We further demonstrated that the inclusion of alternatively spliced cassette exons in MYO18A and NFYA was not only downregulated upon Snord67 knockout in a human TNBC cell line, but also positively correlated with Snord67 expression levels in primary breast tumors and lymph node metastases from breast cancer patients. Based on these results, we propose a model in which Snord67 guides U6 Cm60, which leads to a pro-metastatic alternative splicing program and thereby promotes lymph node tumor growth and distant metastasis. Katherine Ismei Zhou, Yvonne L. Chao, Kwame K. Forbes, Alessandro Porrello, Gabrielle M. Gentile, Yinzhou Zhu, Aaron C. Chack, Dixcy J. John Mary, Haizhou Liu, Eric Cockman, Lincy Edatt, Grant A. Goda, Justin Zhao, Hala A. Assi, Hannah J. Wiedner, Yi-Hsuan Tsai, Lily Wilkinson, Amanda E. Van Swearingen, Lisa A. Carey, Jimena Giudice, Daniel Dominguez, Christopher L. Holley, Chad V. Pecot. Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 78.

BACKGROUND: Heart transplant (HT) in recipients with left ventricular assist devices (LVADs) is associated with poor early post-HT outcomes, including primary graft dysfunction (PGD). As complicated heart explants in recipients with LVADs may produce longer ischemic times, innovations in donor heart preservation may yield improved post-HT outcomes. The SherpaPak Cardiac Transport System is an organ preservation technology that maintains donor heart temperatures between 4 °C and 8 °C, which may minimize ischemic and cold-induced graft injuries. This analysis sought to identify whether the use of SherpaPak versus traditional cold storage was associated with differential outcomes among patients with durable LVAD undergoing HT. METHODS: Global Utilization and Registry Database for Improved Heart Preservation-Heart (NCT04141605) is a multicenter registry assessing post-HT outcomes comparing 2 methods of donor heart preservation: SherpaPak versus traditional cold storage. A retrospective review of all patients with durable LVAD who underwent HT was performed. Outcomes assessed included rates of PGD, post-HT mechanical circulatory support use, and 30-day and 1-year survival. RESULTS: SherpaPak (n=149) and traditional cold storage (n=178) patients had similar baseline characteristics. SherpaPak use was associated with reduced PGD (adjusted odds ratio, 0.56 [95% CI, 0.32–0.99]; P =0.045) and severe PGD (adjusted odds ratio, 0.31 [95% CI, 0.13–0.75]; P =0.009), despite an increased total ischemic time in the SherpaPak group. Propensity matched analysis also noted a trend toward reduced intensive care unit (SherpaPak 7.5±6.4 days versus traditional cold storage 11.3±18.8 days; P =0.09) and hospital (SherpaPak 20.5±11.9 days versus traditional cold storage 28.7±37.0 days; P =0.06) lengths of stay. The 30-day and 1-year survival was similar between groups. CONCLUSIONS: SherpaPak use was associated with improved early post-HT outcomes among patients with LVAD undergoing HT. This innovation in preservation technology may be an option for HT candidates at increased risk for PGD. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT04141605.

RNA 2 '-O-methylation (Nm) is highly abundant in noncoding RNAs including ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA), and occurs in the 5 ' cap of virtually all messenger RNAs (mRNAs) in higher eukaryotes. More recently, Nm has also been reported to occur at internal sites in mRNA. High-throughput methods have been developed for the transcriptome-wide detection of Nm. However, these methods have mostly been applied to abundant RNAs such as rRNA, and the validity of the internal mRNA Nm sites detected with these approaches remains controversial. Nonetheless, Nm in both coding and noncoding RNAs has been demonstrated to impact cellular processes, including translation and splicing. In addition, Nm modifications at the 5 ' cap and possibly at internal sites in mRNA serve to prevent the binding of nucleic acid sensors, thus preventing the activation of the innate immune response by self-mRNAs. Finally, Nm has been implicated in a variety of diseases including cancer, cardiovascular diseases, and neurologic syndromes. In this review, we discuss current challenges in determining the distribution, regulation, function, and disease relevance of Nm, as well as potential future directions for the field.

Introduction: Changes in substrate utilization may be a biomarker of, or a contributor to, heart failure with reduced ejection fraction (HFrEF). Previous work has described circulating metabolites, proteins and transcripts that reflect myocardial bioenergetics, but direct assessment in relevant tissues is lacking. Here, we leverage integrative omics to identify tissue-level biomarkers and pathways of HFrEF that may play a role in pathogenesis. Methods: Myocardial tissue was obtained from 27 HFrEF donors and 21 non-failing hearts (noHF) from Duke University and the University of Colorado Medical Centers. Targeted (n=139 metabolites) and nontargeted (Metabolon, n=817) metabolic profiling was performed; in addition, 496 proteins were assayed (Olink) and ~31,000 transcripts were quantified via RNA sequencing. Individual metabolites were tested for association with HFrEF in models adjusted for age, sex and diabetes. Metabolomics, proteomics and RNA-seq data were integrated using block sparse partial least squares discriminant analysis (sPLS-DA) to identify correlated biomarkers that discriminate HFrEF from noHF hearts. Results: The targeted metabolomics platform primarily assayed metabolic fuel substrate pathways; 71 analytes (51%) were associated with HFrEF after controlling the false discovery rate (FDR) at 5%. Medium- and long-chain acylcarnitines (C10s-C18s) were significantly lower in tissue from failing hearts, while branched-chain amino acids (BCAA) and ketoacids were higher. In the nontargeted set, 293 metabolites (36%) differed between HFrEF and noHF. BCAA and acylcarnitine associations were recapitulated along with a decrease in malonylcarnitine. The sPLS-DA model selected 25 targeted and 50 nontargeted metabolites, 35 proteins and 35 transcripts, and successfully discriminated HFrEF from no HF (balanced error rate = 3.2%). 39/186 tested KEGG pathways were overrepresented in these analytes, including fatty acid metabolism (FDR q=0.002). Conclusions: Analysis of tissue-level metabolites suggests both decreased use of fatty acids as a fuel and disrupted enzymatic breakdown of BCAAs in the failing heart, while integrative omics pathway analysis also supports the association of disrupted fatty acid metabolism with HF.

While many factors have been implicated in breast cancer progression, effective treatments are still lacking. In recent years, it has become clear that posttranscriptional regulation plays a key role in the aberrant gene expression underlying malignancy and metastasis. For example, the mRNA modification N6-methyladenosine (m6A) is involved in numerous post-transcriptional regulation processes and has been implicated in many cancer types, including breast cancer. Despite intense study, even within a single type of cancer, there is little consensus, and often conflicting results, as to the role of m6A, suggesting other factors must influence the process. The goal of this study was to determine if the effects of m6A manipulation on proliferation and migration differed based on the stage of disease progression. Using the MCF10 model of breast cancer, we reduced m6A levels by targeting METTL3, the main cellular m6A RNA methyltransferase. Knocking down Mettl3 at different stages of breast cancer progression indeed shows unique effects at each stage. The early-stage breast cancer line showed a more proliferative phenotype with the knockdown of Mettl3 while the transformed breast cancer line showed a more migratory phenotype. Interestingly, the metastasized breast cancer cell line showed almost no effect on phenotype with the knockdown of Mettl3. Furthermore, transcriptome wide analysis revealed EMT as the probable pathway influencing the phenotypic changes. The results of this study may begin to address the controversy of m6A’s role in cancer and suggest that m6A may have a dynamic role in cancer that depends on the stage of progression.

Specific non-coding, small nucleolar (sno) RNAs encoded within introns of the Rpl13a gene elevate cellular levels of reactive oxygen species (ROS), and ROS are known to aggravate atherosclerosis (athero). We found that genetic deficiency of Rpl13a -snoRNAs reduces brachiocephalic artery athero in Apoe -/- mice by 50%. We therefore tested the hypothesis that athero could be reduced by acute pharmacologic intervention targeting Rpl13a -snoRNAs in Apoe -/- mice. To that end, we injected male and female Apoe -/- mice subcutaneously with 48 mg/kg antisense oligonucleotides (ASOs) targeting (a) the four Rpl13a -snoRNAs (ASO-snoRNA), or (b) green fluorescent protein (ASO-control). We tested two distinct strategies: ( i ) preventive, in which ASO injections and Western diet were started in 10-wk-old mice, with 4 weekly and 5 bi-weekly ASO treatments over 14 wk, then sacrifice; and ( ii ) “therapeutic,” in which ASO injections were started in 24-wk-old mice that had been fed Western diet for 14 wk and then were continued on Western diet with 6 weekly ASO treatments and then sacrificed. ASO injections were administered as indicated until 1 wk prior to sacrifice. Compared with saline-injected mice, ASO-control and ASO-snoRNA showed no hepatotoxicity (as assessed by serum ALT and AST). Compared with ASO-control, ASO-snoRNA administration for 6 wk reduced Rpl13a -snoRNA levels in the aorta and spleen by 54-80% for snoRNAs U32a , U33 , and U34 (RT-qPCR). In the “therapeutic” cohort, brachiocephalic artery cross-sectional athero was reduced by 38% in ASO-snoRNA-treated as compared with ASO-control-treated mice (n=7/group, p <0.03). Additionally, male ASO-snoRNA-treated mice exhibited a 98.1% decrease in circulating IL1-β transcripts (n=6-8/group, p<0.003). In the “preventive” cohort, aortic athero was reduced by 40% (13±5 vs 7±3 % aortic lesion area, n= 11-13/group, p <0.02) in ASO-snoRNA-treated as compared with ASO-control-treated mice. We conclude that therapeutic targeting of Rpl13a -snoRNAs with ASOs mitigates athero, both during the early and later stages of atherogenesis.

BACKGROUND:Cardiac metabolism is altered in heart failure and ischemia-reperfusion injury states. We hypothesized that metabolomic profiling during ex situ normothermic perfusion before heart transplantation (HT) would lend insight into myocardial substrate utilization and report on subclinical and clinical allograft dysfunction risk. METHODS:Metabolomic profiling was performed on serial samples of ex situ normothermic perfusate assaying biomarkers of myocardial injury in lactate and cardiac troponin I (TnI) as well as metabolites (66 acylcarnitines, 15 amino acids, nonesterified fatty acids [NEFA], ketones, and 3-hydroxybutyrate). We tested for change over time in injury biomarkers and metabolites, along with differential changes by recovery strategy (donation after circulatory death [DCD] vs donation after brain death [DBD]). We examined associations between metabolites, injury biomarkers, and primary graft dysfunction (PGD). Analyses were performed using linear mixed models adjusted for recovery strategy, assay batch, donor-predicted heart mass, and time. RESULTS:A total of 176 samples from 92 ex situ perfusion runs were taken from donors with a mean age of 35 (standard deviation 11.3) years and a median total ex situ perfusion time of 234 (interquartile range 84) minutes. Lactate trends over time differed significantly by recovery strategy, while TnI increased during ex situ perfusion regardless of DCD vs DBD status. We found fuel substrates were rapidly depleted during ex situ perfusion, most notably the branched-chain amino acids leucine/isoleucine, as well as ketones, 3-hydroxybutyrate, and NEFA (least squares [LS] mean difference from the first to last time point -1.7 to -4.5, false discovery rate q < 0.001). Several long-chain acylcarnitines (LCAC), including C16, C18, C18:1, C18:2, C18:3, C20:3, and C20:4, increased during the perfusion run (LS mean difference 0.42-0.67, q < 0.001). Many LCACs were strongly associated with lactate and TnI. The change over time of many LCACs was significantly different for DCD vs DBD, suggesting differential trends in fuel substrate utilization by ischemic injury pattern. Changes in leucine/isoleucine, arginine, C12:1-OH/C10:1-DC, and C16-OH/C14-DC were associated with increased odds of moderate-severe PGD. Neither end-of-run nor change in lactate or TnI was associated with PGD. CONCLUSIONS:Metabolomic profiling of ex situ normothermic perfusion solution reveals a pattern of fuel substrate utilization that correlates with subclinical and clinical allograft dysfunction. This study highlights a potential role for interventions focused on fuel substrate modification in allograft conditioning during ex situ perfusion to improve allograft outcomes.

Heart transplantation remains the gold-standard therapy for end-stage heart failure; the expected median survival range is 12-13 years. More than 30,000 heart transplants have been performed globally in the past decade alone. With advances in medical and surgical therapies for heart failure, including durable left ventricular assist devices, an increasing number of patients are living with end-stage disease. Last year alone, more than 2500 patients were added to the heart-transplant waitlist in the United States. Despite recent efforts to expand the donor pool, including an increase in transplantation of hepatitis C-positive and extended-criteria donors, supply continues to fall short of demand. Donation after circulatory death (DCD), defined by irreversible cardiopulmonary arrest rather than donor brain death, is widely used in other solid-organ transplants, including kidney and liver, but has not been widely adopted in heart transplantation. However, resurging interest in DCD donation and the introduction of ex vivo perfusion technology has catalyzed recent clinical trials and the development of DCD heart-transplantation programs. Herein, we review the history of DCD heart transplantation, describe the currently used procurement protocols for it and examine clinical challenges and outcomes of such a procedure.

A 52-year-old man with ischemic cardiomyopathy presented with progressive, severe orthostatic hypotension refractory to medical therapy. Standard abdominal and leg compression devices were used without success. A novel, inflatable abdominal compression device was created that alleviated the patient's symptoms and maintained his blood pressure.

Abstract Background Despite optimal therapy, heart failure (HF) remains a relentless and deadly disease. Given the relative inaccessibility of myocardial human tissues, identification of circulating biomarkers mirroring myocardial pathological signaling pathways, especially in peripheral blood mononuclear cells (PBMC) is expected to be extremely relevant. Small Nucleolar RNAs (snoRNAs) have been shown to play important roles in various cellular physiological processes. However, the connection between snoRNAs and pathological dysfunction in the heart or peripheral blood mononuclear cells (PBMC) is still poorly understood. Purpose To identify novel circulating PBMC biomarkers linked to myocardial dysfunction and HF. Methods : Myocardial left ventricle (LV) samples and PBMC were obtained from patients affected by ischemic HF (HF, n =13) undergoing heart transplantation and control donors (CD, n=7) and analyzed by RNA sequencing analysis (RNASeq). SNORD3A expression levels in the different groups were evaluated by quantitative real-time PCR. HF was induced in 8-week-old wild type C57BL/6 mice by transverse aortic constriction (TAC). Sham-operated mice (sham) were used as controls. After twelve-week-TAC (12w) or sham operation, mice were anesthetized, cardiac function was analyzed by echocardiography, and cardiac/PBMC samples were collected after sacrifice. In order to test the role of SNORD3A in cardiomyocyte hypoxia, H9C2 cardiomyoblasts were transfected with SNORD3A-targeted antisense oligonucleotides (ASO) and cell survival was analyzed. Results RnaSeq analysis identified a small set of genes differentially expressed in the heart and PBMC from HF patients. Among these, SNORD3A was up-regulated in cardiac and PBMC samples from HF patients compared to CD (Figure 1A). Similarly, in murine HF induced by 12w TAC, SNORD3A levels were increased by rtPCR, both in the heart and PBMC (Figure 1B). SNORD3A expression levels were also significantly increased in H9C2 cells exposed to in vitro hypoxia (Figure 1C). Interestingly, H9C2 transfection with SNORD3A-specific ASO significantly reduced hypoxia-induced SNORD3A upregulation and reduced hypoxia-induced cell death (Figure 1D). Conclusions In this study, we identify SNORD3A as a novel possible biomarker in human HF, similarly up-regulated in the heart and PBMC, induced by hypoxia in vitro and modulating cell survival.

The ability to detect 2'-O-methylation sites (Nm) in high-throughput fashion is important, as increasing evidence points to a more diverse landscape for this RNA modification as well as the possibility of yet unidentified functions. Here we describe an optimized version of RibOxi-seq, which is built upon the original published method, that not only accurately profiles ribosomal RNA (rRNA) Nm sites with minimal RNA input but is also robust enough to identify mRNA intronic and exonic sites.