Technological advancements in deciphering RNA-RNA interactions.

RNA-RNA interactions (RRIs) can dictate RNA molecules to form intricate higher-order structures and bind their RNA substrates in diverse biological processes. To elucidate the function, binding specificity, and regulatory mechanisms of various RNA molecules, especially the vast repertoire of non-coding RNAs, advanced technologies and methods that globally map RRIs are extremely valuable. In the past decades, many state-of-the-art technologies have been developed for this purpose. This review focuses on those high-throughput technologies for the global mapping of RRIs. We summarize the key concepts and the pros and cons of different technologies. In addition, we highlight the novel biological insights uncovered by these RRI mapping methods and discuss the future challenges for appreciating the crucial roles of RRIs in gene regulation across bacteria, viruses, archaea, and mammals.

SARS-CoV-2 RNA stabilizes host mRNAs to elicit immunopathogenesis.

SARS-CoV-2 RNA interacts with host factors to suppress interferon responses and simultaneously induces cytokine release to drive the development of severe coronavirus disease 2019 (COVID-19). However, how SARS-CoV-2 hijacks host RNAs to elicit such imbalanced immune responses remains elusive. Here, we analyzed SARS-CoV-2 RNA in situ structures and interactions in infected cells and patient lung samples using RIC-seq. We discovered that SARS-CoV-2 RNA forms 2,095 potential duplexes with the 3' UTRs of 205 host mRNAs to increase their stability by recruiting RNA-binding protein YBX3 in A549 cells. Disrupting the SARS-CoV-2-to-host RNA duplex or knocking down YBX3 decreased host mRNA stability and reduced viral replication. Among SARS-CoV-2-stabilized host targets, NFKBIZ was crucial for promoting cytokine production and reducing interferon responses, probably contributing to cytokine storm induction. Our study uncovers the crucial roles of RNA-RNA interactions in the immunopathogenesis of RNA viruses such as SARS-CoV-2 and provides valuable host targets for drug development.

Capture RIC-seq reveals positional rules of PTBP1-associated RNA loops in splicing regulation.

RNA-binding proteins (RBPs) bind at different positions of the pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working principle of these positional effects, we develop a capture RIC-seq (CRIC-seq) method to enrich specific RBP-associated in situ proximal RNA-RNA fragments for deep sequencing. We determine hnRNPA1-, SRSF1-, and PTBP1-associated proximal RNA-RNA contacts and regulatory mechanisms in HeLa cells. Unexpectedly, the 3D RNA map analysis shows that PTBP1-associated loops in individual introns preferentially promote cassette exon splicing by accelerating asymmetric intron removal, whereas the loops spanning across cassette exon primarily repress splicing. These "positional rules" can faithfully predict PTBP1-regulated splicing outcomes. We further demonstrate that cancer-related splicing quantitative trait loci can disrupt RNA loops by reducing PTBP1 binding on pre-mRNAs to cause aberrant splicing in tumors. Our study presents a powerful method for exploring the functions of RBP-associated RNA-RNA proximal contacts in gene regulation and disease.

Genome-wide analysis of copy-number variation in humans with cleft lip and/or cleft palate identifies COBLL1, RIC1, and ARHGEF38 as clefting genes.

Cleft lip with or without cleft palate (CL/P) is a common birth defect with a complex, heterogeneous etiology. It is well established that common and rare sequence variants contribute to the formation of CL/P, but the contribution of copy-number variants (CNVs) to cleft formation remains relatively understudied. To fill this knowledge gap, we conducted a large-scale comparative analysis of genome-wide CNV profiles of 869 individuals from the Philippines and 233 individuals of European ancestry with CL/P with three primary goals: first, to evaluate whether differences in CNV number, amount of genomic content, or amount of coding genomic content existed within clefting subtypes; second, to assess whether CNVs in our cohort overlapped with known Mendelian clefting loci; and third, to identify unestablished Mendelian clefting genes. Significant differences in CNVs across cleft types or in individuals with non-syndromic versus syndromic clefts were not observed; however, several CNVs in our cohort overlapped with known syndromic and non-syndromic Mendelian clefting loci. Moreover, employing a filtering strategy relying on population genetics data that rare variants are on the whole more deleterious than common variants, we identify several CNV-associated gene losses likely driving non-syndromic clefting phenotypes. By prioritizing genes deleted at a rare frequency across multiple individuals with clefts yet enriched in our cohort of individuals with clefts compared to control subjects, we identify COBLL1, RIC1, and ARHGEF38 as clefting genes. CRISPR-Cas9 mutagenesis of these genes in Xenopus laevis and Danio rerio yielded craniofacial dysmorphologies, including clefts analogous to those seen in human clefting disorders.

The ric-8b protein (resistance to inhibitors of cholinesterase 8b) is key to preserving contractile function in the adult heart.

Resistance to inhibitors of cholinesterases (ric-8 proteins) are involved in modulating G-protein function, but little is known of their potential physiological importance in the heart. In the present study, we assessed the role of resistance to inhibitors of cholinesterase 8b (Ric-8b) in determining cardiac contractile function. We developed a murine model in which it was possible to conditionally delete ric-8b in cardiac tissue in the adult animal after the addition of tamoxifen. Deletion of ric-8b led to severely reduced contractility as measured using echocardiography days after administration of tamoxifen. Histological analysis of the ventricular tissue showed highly variable myocyte size, prominent fibrosis, and an increase in cellular apoptosis. RNA sequencing revealed transcriptional remodeling in response to cardiac ric-8b deletion involving the extracellular matrix and inflammation. Phosphoproteomic analysis revealed substantial downregulation of phosphopeptides related to myosin light chain 2. At the cellular level, the deletion of ric-8b led to loss of activation of the L-type calcium channel through the ß-adrenergic pathways. Using fluorescence resonance energy transfer-based assays, we showed ric-8b protein selectively interacts with the stimulatory G-protein, Gαs. We explored if deletion of Gnas (the gene encoding Gαs) in cardiac tissue using a similar approach in the mouse led to an equivalent phenotype. The conditional deletion of the Gαs gene in the ventricle led to comparable effects on contractile function and cardiac histology. We conclude that ric-8b is essential to preserve cardiac contractile function likely through an interaction with the stimulatory G-protein and downstream phosphorylation of myosin light chain 2.

RNA in situ conformation sequencing reveals novel long-range RNA structures with impact on splicing.

Over recent years, long-range RNA structure has emerged as a factor that is fundamental to alternative splicing regulation. An increasing number of human disorders are now being associated with splicing defects; hence it is essential to develop methods that assess long-range RNA structure experimentally. RNA in situ conformation sequencing (RIC-seq) is a method that recapitulates RNA structure within physiological RNA-protein complexes. In this work, we juxtapose pairs of conserved complementary regions (PCCRs) that were predicted in silico with the results of RIC-seq experiments conducted in seven human cell lines. We show statistically that RIC-seq support of PCCRs correlates with their properties, such as equilibrium free energy, presence of compensatory substitutions, and occurrence of A-to-I RNA editing sites and forked eCLIP peaks. Exons enclosed in PCCRs that are supported by RIC-seq tend to have weaker splice sites and lower inclusion rates, which is indicative of post-transcriptional splicing regulation mediated by RNA structure. Based on these findings, we prioritize PCCRs according to their RIC-seq support and show, using antisense nucleotides and minigene mutagenesis, that PCCRs in two disease-associated human genes, PHF20L1 and CASK, and also PCCRs in their murine orthologs, impact alternative splicing. In sum, we demonstrate how RIC-seq experiments can be used to discover functional long-range RNA structures, and particularly those that regulate alternative splicing.

Unraveling the molecular interactions between α7 nicotinic receptor and a RIC3 variant associated with backward speech.

Recent work putatively linked a rare genetic variant of the chaperone Resistant to Inhibitors of acetylcholinesterase (RIC3) (NM_024557.4:c.262G > A, NP_078833.3:p.G88R) to a unique ability to speak backwards, a language skill that is associated with exceptional working memory capacity. RIC3 is important for the folding, maturation, and functional expression of α7 nicotinic acetylcholine receptors (nAChR). We compared and contrasted the effects of RIC3G88R on assembly, cell surface expression, and function of human α7 receptors using fluorescent protein tagged α7 nAChR and Förster resonance energy transfer (FRET) microscopy imaging in combination with functional assays and 125I-α-bungarotoxin binding. As expected, the wild-type RIC3 protein was found to increase both cell surface and functional expression of α7 receptors. In contrast, the variant form of RIC3 decreased both. FRET analysis showed that RICG88R increased the interactions between RIC3 and α7 protein in the endoplasmic reticulum. These results provide interesting and novel data to show that a RIC3 variant alters the interaction of RIC3 and α7, which translates to decreased cell surface and functional expression of α7 nAChR.

N-Glycosylation Deficiency in Transgene α7 nAChR and RIC3 Expressing CHO Cells Without NACHO.

The human neuronal nicotinic acetylcholine receptor α7 (nAChR) is an important target implicated in diseases like Alzheimer's or Parkinson's, as well as a validated target for drug discovery. For α7 nAChR model systems, correct folding and ion influx functions are essential. Two chaperones, resistance to inhibitors of cholinesterase 3 (RIC3) and novel nAChR regulator (NACHO), enhance the assembly and function of α7 nAChR. This study investigates the consequence of NACHO absence on α7 nAChR expression and function. Therefore, the sequences of human α7 nAChR and human RIC3 were transduced in Chinese hamster ovary (CHO) cells. Protein expression and function of α7 nAChR were confirmed by Western blot and voltage clamp, respectively. Cellular viability was assessed by cell proliferation and lactate dehydrogenase assays. Intracellular and extracellular expression were determined by in/on-cell Western, compared with another nAChR subtype by novel cluster fluorescence-linked immunosorbent assay, and N-glycosylation efficiency was assessed by glycosylation digest. The transgene CHO cell line showed expected protein expression and function for α7 nAChR and cell viability was barely influenced by overexpression. While intracellular levels of α7 nAChR were as anticipated, plasma membrane insertion was low. The glycosylation digest revealed no appreciable N-glycosylation product. This study demonstrates a stable and functional cell line expressing α7 nAChR, whose protein expression, function, and viability are not affected by the absence of NACHO. The reduced plasma membrane insertion of α7 nAChR, combined with incorrect matured N-glycosylation at the Golgi apparatus, suggests a loss of recognition signal for lectin sorting.

Viral RNA Is a Hub for Critical Host-Virus Interactions.

RNA is a central molecule in the life cycle of viruses, acting not only as messenger (m)RNA but also as a genome. Given these critical roles, it is not surprising that viral RNA is a hub for host-virus interactions. However, the interactome of viral RNAs remains largely unknown. This chapter discusses the importance of cellular RNA-binding proteins in virus infection and the emergent approaches developed to uncover and characterise them.

Synthesis and Evaluation of a Cathepsin B-Recognizing Trifunctional Chelating Agent to Improve Tumor Retention of Radioimmunoconjugates.

Cathepsin B (CTSB) is a lysosomal protease that is overexpressed in tumor cells. Radioimmunoconjugates (RICs) composed of CTSB-recognizing chelating agents are expected to increase the molecular weights of their radiometabolites by forming conjugates with CTSB in cells, resulting in their improved retention in tumor cells. We designed a novel CTSB-recognizing trifunctional chelating agent, azide-[111In]In-DOTA-CTSB-substrate ([111In]In-ADCS), to synthesize a RIC, trastuzumab-[111In]In-ADCS ([111In]In-TADCS), and evaluated its utility to improve tumor retention of the RIC. [111In]In-ADCS and [111In]In-TADCS were synthesized with satisfactory yield and purity. [111In]In-ADCS was markedly stable in murine plasma until 96 h postincubation. [111In]In-ADCS showed binding to CTSB in vitro, and the conjugation was blocked by the addition of CTSB inhibitor. In the internalization assay, [111In]In-TADCS exhibited high-level retention in SK-OV-3 cells, indicating the in vitro utility of the CTSB-recognizing unit. In the biodistribution assay, [111In]In-TADCS showed high-level tumor accumulation, but the retention was hardly improved. In the first attempt to combine a CTSB-recognizing unit and RIC, these findings show the fundamental properties of the CTSB-recognizing trifunctional chelating agent to improve tumor retention of RICs.

Deletion induced splicing in RIC3 drives nicotinic acetylcholine receptor regulation with implications for endoplasmic reticulum stress in human astrocytes.

Nicotinic acetylcholine receptor (nAChR) dysregulation in astrocytes is reported in neurodegenerative disorders. Modulation of nAChRs through agonists confers protection to astrocytes from stress but regulation of chaperones involved in proteostasis with pathological implications is unclear. Resistance to inhibitors of cholinesterase 3 (RIC3), a potential chaperone of nAChRs is poorly studied in humans. We characterized RIC3 in astrocytes derived from an isogenic wild-type and Cas9 edited "del" human iPSC line harboring a 25 bp homozygous deletion in exon2. Altered RIC3 transcript ratio due to deletion induced splicing and an unexpected gain of α7nAChR expression were observed in "del" astrocytes. Transcriptome analysis showed higher expression of neurotransmitter/G-protein coupled receptors mediated by cAMP and calcium/calmodulin-dependent kinase signaling with increased cytokines/glutamate secretion. Functional implications examined using tunicamycin induced ER stress in wild-type astrocyte stress model showed cell cycle arrest, RIC3 upregulation, reduction in α7nAChR membrane levels but increased α4nAChR membrane expression. Conversely, tunicamycin-treated "del" astrocytes showed a comparatively higher α4nAChR membrane expression and upsurged cAMP signaling. Furthermore, reduced expression of stress markers CHOP, phospho-PERK and lowered XBP1 splicing in western blot and qPCR, validated by proteome-based pathway analysis indicated lowered disease severity. Findings indicate (i) a complex RNA regulatory mechanism via exonic deletion induced splicing; (ii) RIC-3 as a disordered protein having contrasting effects on co-expressed nAChR subtypes under basal/stress conditions; and (iii) RIC3 as a potential drug target against ER stress in astrocytes for neurodegenerative/nicotine-related brain disorders. Cellular rescue mechanism through deletion induced exon skipping may encourage ASO-based therapies for tauopathies.

RBPome identification in egg-cell like callus of Arabidopsis.

RNA binding proteins (RBPs) have multiple and essential roles in transcriptional and posttranscriptional regulation of gene expression in all living organisms. Their biochemical identification in the proteome of a given cell or tissue requires significant protein amounts, which limits studies in rare and highly specialized cells. As a consequence, we know almost nothing about the role(s) of RBPs in reproductive processes such as egg cell development, fertilization and early embryogenesis in flowering plants. To systematically identify the RBPome of egg cells in the model plant Arabidopsis, we performed RNA interactome capture (RIC) experiments using the egg cell-like RKD2-callus and were able to identify 728 proteins associated with poly(A+)-RNA. Transcripts for 97 % of identified proteins could be verified in the egg cell transcriptome. 46 % of identified proteins can be associated with the RNA life cycle. Proteins involved in mRNA binding, RNA processing and metabolism are highly enriched. Compared with the few available RBPome datasets of vegetative plant tissues, we identified 475 egg cell-enriched RBPs, which will now serve as a resource to study RBP function(s) during egg cell development, fertilization and early embryogenesis. First candidates were already identified showing an egg cell-specific expression pattern in ovules.

Impact of extramedullary disease in AML patients undergoing sequential RIC for HLA-matched transplantation: occurrence, risk factors, relapse patterns, and outcome.

We sought to evaluate the role of extramedullary disease (EMD) in sequential RIC retrospectively analyzing data of 144 high-risk AML patients undergoing HLA-matched transplantation. Median long-term follow-up was 11.6 years. Eighteen percent of patients (n = 26/144) presented with extramedullary AML (EM AML) or a history of EMD at time of transplantation. Overall relapse rate was 25% (n = 36/144) with 15% (n = 21/144) of all patients developing isolated BM relapse and 10% (n = 15/144) developing EM AML relapse with or without concomitant BM relapse (EM ± BM). Manifestation of EM relapse after transplantation occurred frequently at multiple sites and presented mostly as solid tumor mass. Only 3/15 patients with EM ± BM relapse showed a prior EMD manifestation. EMD prior to allogeneic transplantation had no impact on post-transplant OS when compared to non-EMD (median post-transplant OS 3.8 years versus 4.8 years; ns). Risk factors (p = < 0.1) for EM ± BM relapse included younger age and a higher number of prior intensive chemotherapies, whereas the presence of chronic GVHD was a protective factor. Median post-transplant OS (15.5 months vs. 15.5 months), RFS (9.6 months vs 7.3 months), and post-relapse OS (6.7 months vs. 6.3 months) were not significantly different between patients with isolated BM vs. EM ± BM relapse. Taken together, occurrence of EMD prior to as well as of EM ± BM AML relapse after transplantation was moderate, presenting mostly as solid tumor mass after transplantation. However, diagnosis of those does not seem to influence outcomes after sequential RIC. A higher number of chemotherapy cycles prior to transplantation was identified as recent risk factor for EM ± BM relapse.

Influence of conditioning regimen intensity on outcomes post-allogeneic hematopoietic cell transplantation for acute myeloid leukemia in complete morphological remission.

INTRODUCTION: The literature comparing outcomes between myeloablative (MAC) and reduced intensity conditioning (RIC) for acute myeloid leukemia (AML) is conflicting. METHODS: We retrospectively analyzed 451 patients who underwent allogenic hematopoietic cell transplantation (alloHCT) for AML in complete remission (CR) with either RIC (n = 331) or MAC (n = 120) with the use of dual T-cell depletion as graft-versus-host disease (GVHD) prophylaxis. RESULTS: Univariate analysis demonstrated nonrelapse mortality (NRM) at 2 years was 19.1% for MAC and 22.5% for RIC (p = .44). Two-year cumulative incidence of relapse (CIR) was 19.8% for MAC and 24.5% for RIC (p = .15). Two-year overall survival (OS) was 61% and 53% for MAC and RIC, respectively (p = .02). Two-year graft-versus-host disease relapse-free survival (GRFS) was 40.8% for MAC and 33.7% for RIC (p = .30). A propensity score-matched analysis was done matching patients for age, HLA match, in vivo T-cell depletion, and Disease Risk Index (DRI). Two-year OS was 67% for MAC, 66% for RIC (p = .95). A subgroup analysis identified that matched related donor transplants benefit from MAC with OS at 2 years 82.6% versus 57.3% for RIC (p = .006). CONCLUSIONS: In the matched-related donor setting, MAC regimens may offer superior survival. Overall, for our cohort of predominantly in vivo T-cell depleted patients the outcomes of MAC and RIC were similar.

Is Treosulfan-Based Conditioning Attractive as a Reduced-Intensity Conditioning Regimen in Korea?

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) was not actively performed in elderly acute myeloid leukemia (AML) or myelodysplastic syndrome patients who are at a high-risk based on hematopoietic cell transplantation-specific comorbidity index (HCT-CI). The advent of reduced-intensity conditioning (RIC) regimens has made HSCT applicable in this population. However, the selection of appropriate conditioning is a major concern for the attending physician. The benefits of combination of treosulfan and fludarabine (Treo/Flu) have been confirmed through many clinical studies. Korean data on treosulfan-based conditioning regimen are scarce. METHODS: A retrospective study was conducted to compare the clinical outcomes of allogeneic HSCT using RIC between 13 patients receiving Treo/Flu and 39 receiving busulfan/fludarabine (Bu/Flu). RESULTS: In terms of conditioning-related complications, the frequency of ≥ grade 2 nausea or vomiting was significantly lower and the duration of symptoms was shorter in the Treo/Flu group than in the Bu/Flu group. The incidence of ≥ grade 2 mucositis tended to be lower in the Treo/Flu group. In the analysis of transplant outcomes, all events of acute graft versus host disease (GVHD) and ≥ grade 2 acute GVHD occurred more frequently in the Treo/Flu group. The frequency of Epstein-Barr virus reactivation was significantly higher in the Treo/Flu group (53.8% vs. 23.1%, P = 0.037). Non-relapse mortality (NRM) at 12 months was higher in the Treo/Flu group (30.8% vs. 7.7%, P = 0.035). Significant prognostic factors included disease type, especially secondary AML, disease status and high-risk based on HCT-CI, ≥ grade 2 acute GVHD, and cases requiring ≥ 2 immunosuppressive drugs for treating acute GVHD. In the comparison of survival outcomes according to conditioning regimen, the Bu/Flu group seemed to show better results than the Treo/Flu group (60% vs. 46.2%, P = 0.092 for overall survival; 56.4% vs. 38.5%, P = 0.193 for relapse-free survival). In additional analysis for only HCT-CI high-risk groups, there was no difference in transplant outcomes except that the Treo/Flu group tended to have a higher NRM within one year after transplantation. Survival outcomes of both groups were similar. CONCLUSION: This study suggests that Treo/Flu conditioning may be an alternative to Bu/Flu regimen in elderly patients with high-risk who are not suitable for standard conditioning.

Two cases of Clostridium ramosum bacteremia with intestinal perforation: The antimicrobial susceptibility of clinical strains.

Clostridium ramosum is one of the obligate anaerobes that constitute the intestinal microbiota, and one of the rare Clostridia. With Clostridium ramosum, very few data have been reported to investigate antimicrobial susceptibility for clinical isolates that have caused bacteremia. Here, we report two cases of Clostridium ramosum bacteremia. The first case was a 54-year-old Japanese man with taking 20mg hydrocortisone for hypopituitarism. He presented to the emergency department for an unknown cause cardiopulmonary arrest. At the hospital day 36, he had fever and a drop in blood pressure. Abdomen computed tomography (CT) revealed free air around the ascending colon, we diagnosed with intestinal perforation, and peritonitis. Blood culture revealed Clostridium ramosum. We administered conservative management by 6-week of antibiotic treatment. The second case was a 78-year-old Japanese man with no significant medical history. He was referred to our hospital with fever and abdominal pain. Abdomen CT revealed perforated appendicitis, and blood cultures revealed Clostridium ramosum. We performed emergency surgery, and administered one-week course of antibiotic treatment. This report demonstrates two cases of Clostridium ramosum bacteremia with intestinal perforation, and the antimicrobial susceptibility of each clinical strain. For the future, it is necessary to accumulate data on the susceptibility of clinical isolates in order to find an appropriate treatment.

A Comparison of Two Types of Acoustic Emission Sensors for the Characterization of Hydrogen-Induced Cracking.

Acoustic emission (AE) technology is a non-destructive testing (NDT) technique that is able to monitor the process of hydrogen-induced cracking (HIC). AE uses piezoelectric sensors to convert the elastic waves generated from the growth of HIC into electric signals. Most piezoelectric sensors have resonance and thus are effective for a certain frequency range, and they will fundamentally affect the monitoring results. In this study, two commonly used AE sensors (Nano30 and VS150-RIC) were used for monitoring HIC processes using the electrochemical hydrogen-charging method under laboratory conditions. Obtained signals were analyzed and compared on three aspects, i.e., in signal acquisition, signal discrimination, and source location to demonstrate the influences of the two types of AE sensors. A basic reference for the selection of sensors for HIC monitoring is provided according to different test purposes and monitoring environments. Results show that signal characteristics from different mechanisms can be identified more clearly by Nano30, which is conducive to signal classification. VS150-RIC can identify HIC signals better and provide source locations more accurately. It can also acquire low-energy signals better, which is more suitable for monitoring over a long distance.

Hypertensive status is associated with renoprotection by remote ischemic conditioning for acute myocardial infarction-a meta-regression and trial sequential analysis of randomized clinical trials.

The potential modifiable factors for remote ischemic conditioning (RIC) in reducing contrast-associated acute kidney injury (CA-AKI) in patients with acute myocardial infarction (AMI) have not been investigated. The aim of this meta-regression was to address these issues.We searched Pubmed, Embase and the Cochrane Library database for published randomized controlled trials (RCTs) with registration number CRD42020155532. Nine RCTs comprising of 1540 subjects were included in our meta-analysis. Compared with control group, RIC was associated with reduced incidence of CA-AKI [(9 studies, 1540 subjects, relative risk (RR) 0.51, 95% confidence intervals (CI) 0.35 to 0.76, p = 0.000, I2 = 52%, p for heterogeneity 0.04)] and major adverse cardiovascular events (MACE) (5 studies, 1078 subjects, RR 0.52, 95% CI 0.38 to 0.73, p = 0.000, I2 = 9%, p for heterogeneity 0.36) for AMI. In addition, both meta-regression and subgroup analyses have shown that RIC was more effective in the hypertensive patients in reducing CA-AKI for AMI (regression coefficient = -0.05, p = 0.021; for subgroup with more hypertensive patients: RR 0.36, 95% CI 0.25 to 0.52 vs the one with less hypertensive patients: RR 0.72, 95% CI (0.40 to 1.30, p for subgroup difference 0.008). Subsequent trial sequential analysis confirmed the effect of RIC in both CA-AKI and MACE. RIC is an effective strategy in reducing CA-AKI and MACE in patients with AMI, especially for patients with hypertension.

Conditioning with fludarabine and treosulfan compared to FLAMSA-RIC in allogeneic stem cell transplantation for myeloid malignancies: a retrospective single-center analysis.

Reduced intensity conditioning (RIC) and reduced toxicity conditioning (RTC) regimens enable allogeneic hematopoietic stem cell transplantation (alloSCT) to more patients due to reduction in transplant-related mortality (TRM). The conditioning regimens with fludarabine and treosulfan (Flu/Treo) or fludarabine, amsacrine, cytarabine (FLAMSA)-RIC have shown their efficacy and tolerability in various malignancies. So far, no prospective study comparing the two regimens is available. Two studies compared the regimens retrospectively, in which both provided similar outcome. In this retrospective, single-center analysis, these two regimens were compared with regard to outcome, rate of acute and chronic graft versus host disease (GvHD), and engraftment. 113 consecutive patients with myeloid malignancies who received Flu/Treo or FLAMSA-RIC conditioning prior to alloSCT between 2007 and 2019 were included. Except for age, previous therapies, and remission status before alloSCT, patient characteristics were well balanced. The median follow-up time within this analysis was 44 months. There was no significant difference in absolute neutrophil count (ANC) or platelet engraftment between the two conditioning regimens. Overall survival (OS), the relapse-free survival (RFS), and the TRM were not significantly different between the two cohorts. The rate of GvHD did not differ between the two groups. In summary, this retrospective analysis shows that there is no major difference regarding tolerability and survival between the Flu/Treo and FLAMSA-RIC regimens. Despite several limitations due to uneven distribution concerning age and remission status, we demonstrate that Flu/Treo and FLAMSA-RIC provide similar outcomes and are feasible in older and intensively pre-treated patients.

Repetitive application of remote ischemic conditioning (RIC) in patients with peripheral arterial occlusive disease (PAOD) as a non-invasive treatment option: study protocol for a randomised controlled clinical trial.

BACKGROUND: The best medical treatment (BMT) for most patients with early stage of peripheral arterial occlusive disease (PAOD) is often limited to gait training and pharmacological therapy besides endovascular surgery. The application of remote ischemic conditioning (RIC) has been described as a promising experimental strategy for the improvement of therapeutic outcome in cardiovascular disease but has not proven beneficial effects in clinical practice and treatment of PAOD yet. METHODS: Here we describe a prospective, randomized trial for the evaluation of possible effects of repeated application of RIC in patients with PAOD. This monocentric study will enrol 200 participants distributed to an intervention group receiving RIC + BMT and a control group only receiving BMT for four weeks. Patients are at least 18 years of age and have diagnosed PAOD Fontaine stage II b. Pain-free and total walking distance will be measured via treadmill test (primary endpoints). In addition, ankle-brachial index (ABI) and quality of life (QoL) will be assessed using the SF-36 and VascuQoL-6 questionnaire. Moreover, evaluation of markers for atherosclerosis, angiogenic profiling and mononuclear cell characterization will be performed using biochemical assays, proteome profiling arrays and flow cytometry (secondary endpoints). DISCUSSION: Our prospective, randomized monocentric trial is the first of its kind to analyse the effects of chronic and repetitive treatment with RIC in patients with PAOD and might provide important novel information on the molecular mechanisms associated with RIC in PAOD patients. TRIAL REGISTRATION: Prospectively registered in the German Clinical Trials Register (Deutsche Register Klinischer Studien) Registration number: DRKS00025735; Date of registration: 01.07.2021.