Mapping the functional impact of non-coding regulatory elements in primary T cells through single-cell CRISPR screens.

BACKGROUND: Drug targets with genetic evidence are expected to increase clinical success by at least twofold. Yet, translating disease-associated genetic variants into functional knowledge remains a fundamental challenge of drug discovery. A key issue is that the vast majority of complex disease associations cannot be cleanly mapped to a gene. Immune disease-associated variants are enriched within regulatory elements found in T-cell-specific open chromatin regions. RESULTS: To identify genes and molecular programs modulated by these regulatory elements, we develop a CRISPRi-based single-cell functional screening approach in primary human T cells. Our pipeline enables the interrogation of transcriptomic changes induced by the perturbation of regulatory elements at scale. We first optimize an efficient CRISPRi protocol in primary CD4+ T cells via CROPseq vectors. Subsequently, we perform a screen targeting 45 non-coding regulatory elements and 35 transcription start sites and profile approximately 250,000 T -cell single-cell transcriptomes. We develop a bespoke analytical pipeline for element-to-gene (E2G) mapping and demonstrate that our method can identify both previously annotated and novel E2G links. Lastly, we integrate genetic association data for immune-related traits and demonstrate how our platform can aid in the identification of effector genes for GWAS loci. CONCLUSIONS: We describe "primary T cell crisprQTL" - a scalable, single-cell functional genomics approach for mapping regulatory elements to genes in primary human T cells. We show how this framework can facilitate the interrogation of immune disease GWAS hits and propose that the combination of experimental and QTL-based techniques is likely to address the variant-to-function problem.

Increased Seroprevalence of Campylobacter jejuni, but not HEV, in healthcare workers in gastroenterological endoscopy.

BACKGROUND: The exposure of healthcare workers (HCW) to fecal-orally transmitted pathogens like hepatitis E Virus (HEV), Campylobacter jejuni or Helicobacter pylori is still not known. The potential risk for employees or patients to acquire these infections through asymptomatic infected healthcare personnel has not yet been studied. Physicians and nurses in gastroenterology working in endoscopic workspaces were recruited. Employees from cardiology, presumed to possess a lower exposure, served as controls. The cytomegalovirus (CMV) seroprevalence was analyzed as a control pathogen without fecal-oral route of transmission. This study provides an objective view onto the potential exposure risk for HCW and patients in endoscopic workspaces. We hypothesize that HCW in gastroenterological endoscopy show a higher seroprevalence for fecal-oral pathogens like HEV, C. jejuni and H. pylori compared to HCW in cardiology. OBJECTIVE: Primary objective was the assessment of antibody titers against HEV, C. jejuni and H. pylori in serum of HCW from gastroenterological endoscopy as well as cardiology. As a secondary objective we analyzed the seroprevalence against CMV. METHODS: 65 HCW were from gastroenterological endoscopy (n=42) and cardiology (n=23) in three medical centers in the German federal states of Brandenburg, Hamburg and Schleswig-Holstein and were prospectively studied. Antibody titers were determined via ELISA in serum. RESULTS: HCW in gastroenterological endoscopy showed a significantly higher C. jejuni seroprevalence for IgG (19.1 %) compared to HCW from the field of cardiology (8.7 %; p=0.04). IgA titers against C. jejuni were negligible. HEV seroprevalence for IgG did not differ significantly between HCW in gastroenterological endoscopy (7.1 %) and cardiology (8.7 %), respectively. IgA and IgM titers against HEV were also negligible. All other antibody titers against CMV and H. pylori showed no significant difference. CONCLUSIONS: Only the C. jejuni seroprevalence was significantly increased in HCW from the field of gastroenterological endoscopy. HEV seroprevalence showed no differences. The results for CMV and H. pylori were without pathological findings. However, there is no elevated risk for HEV exposure in medical staff working at an endoscopy unit, but for C. jejuni the protective measures might need to be improved.

Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages.

CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify protein-coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that targeting the same gene simultaneously with two guide RNAs (paired guide RNAs, pgRNAs) synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next design a library to target, in parallel, pc-genes and lncRNAs known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four (two pc-genes and two lncRNAs) for deeper characterization. Our results suggest that in the case of the candidate lncRNAs, their impact in transdifferentiation may be actually mediated by enhancer regions at the targeted loci, rather than by the lncRNA transcripts themselves. The CRISPR-Cas9 coupled to a pgRNAs system is, therefore, a suitable tool to simultaneously target pc-genes and lncRNAs for genomic perturbation assays.

Diagnostic benefit of high b-value computed diffusion-weighted imaging in acute brainstem infarction.

BACKGROUND AND PURPOSE: Diffusion-weighted imaging (DWI) is a cornerstone in diagnostic of ischemic stroke. The aim of this study was to investigate the usefulness of high-b-value computed DWI (c-DWI) in comparison to standard DWI in patients with acute brainstem infarction. MATERIALS AND METHODS: 56 patients with acute brainstem infarction were retrospectively analysed by two readers. DWI was obtained with the b-values 0, 500 and 1000 s/mm² on either a 1.5 or 3 T magnetic resonance imaging (MRI) scanner. c-DWI was calculated with a monoexponential model with high b-values 2000, 3000, 4000 and 5000 s/mm². All c-DWI series with high-b-values were compared to the standard DWI sequence at b-value of 1000 s/mm² in terms of image artifacts, lesion extent and contrast. RESULTS: There was no statistically significant difference between 1.5 and 3 T MRI regarding the measured ischemic lesion size. There were no statistically significant differences between the ischemic lesion sizes on DWI at b-values of 1000 s/mm² and on c-DWI at higher b-values. Overall, the contrast between the lesion and the surrounding normal areas improved with increasing b-value on the isotropic DWIs: maximum at b = 5000, followed by that at b 2000 and b 1000 s/mm², in order. The best relation between artifacts and lesion contrast was identified for b 2000 s/mm². CONCLUSION: High b-value DWI derived from c-DWI has a higher visibility for ischemic brainstem lesions compared to standard DWI without additional time cost. The b-2000 image is recommended to use in clinical routine, higher b-value images lead to more imaging artifacts, which might result in misdiagnosis.

Low incidence of colonic complications after severe Shiga toxin-producing E. coli O104:H4 infection.

BACKGROUND: In summer 2011, Shiga toxin producing Escherichia coli (EHEC) serotype O104:H4 caused the most severe EHEC outbreak in Germany to date. The case of a previously recovered patient with symptomatic postinflammatory colonic stenosis following EHEC- infection prompted us to conduct a prospective study to assess the macro- and microscopic intestinal long-term damage in a cohort of patients who had suffered from severe EHEC colitis. METHODS: Following EHEC infection in 2011, 182 patients were offered to participate in this study between January 2013 and October 2014 as part of the post-inpatient follow-up care at the University Medical Center Hamburg-Eppendorf and to undergo colonoscopy with stepwise biopsies. Prior to colonoscopy, medical history and persistent post-infectious complaints were assessed. RESULTS: Out of 182 patients, 22 (12%) participated in the study, 18 (82%) were female. All patients had been hospitalized due severe EHEC enterocolitis: 20 patients (90%) had subsequently developed hemolytic uremic syndrome (HUS), 16 patients (72%) had additionally required dialysis. On assessment prior to colonoscopy, all patients denied any abdominal complaints before EHEC-infection but 8 (36%) patients reported persistent post-infectious symptoms. According to the ROME IV criteria, 4 (18%) patients met the definition for post-infectious irritable bowel syndrome (PI-IBS). In all patients with persistent symptoms, colonoscopies and histological examination were unremarkable. Only in one symptom-free patient, biopsy revealed a locally limited cryptitis of the caecum, while all patients without complaints had inconspicuous histological and endoscopical findings. CONCLUSION: Following infection colonic stenosis is a serious but rare long-term complication in patients who had suffered from severe enterocolitis. However, a significant proportion of these patients develop PI-IBS.

Multi-hallmark long noncoding RNA maps reveal non-small cell lung cancer vulnerabilities.

Long noncoding RNAs (lncRNAs) are widely dysregulated in cancer, yet their functional roles in cancer hallmarks remain unclear. We employ pooled CRISPR deletion to perturb 831 lncRNAs detected in KRAS-mutant non-small cell lung cancer (NSCLC) and measure their contribution to proliferation, chemoresistance, and migration across two cell backgrounds. Integrative analysis of these data outperforms conventional "dropout" screens in identifying cancer genes while prioritizing disease-relevant lncRNAs with pleiotropic and background-independent roles. Altogether, 80 high-confidence oncogenic lncRNAs are active in NSCLC, which tend to be amplified and overexpressed in tumors. A follow-up antisense oligonucleotide (ASO) screen shortlisted two candidates, Cancer Hallmarks in Lung LncRNA 1 (CHiLL1) and GCAWKR, whose knockdown consistently suppressed cancer hallmarks in two- and three-dimension tumor models. Molecular phenotyping reveals that CHiLL1 and GCAWKR control cellular-level phenotypes via distinct transcriptional networks. This work reveals a multi-dimensional functional lncRNA landscape underlying NSCLC that contains potential therapeutic vulnerabilities.

Granulomatous Inflammation in ANCA-Associated Vasculitis.

ANCA-associated vasculitis (AAV) comprises granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). While systemic vasculitis is a hallmark of all AAV, GPA is characterized by extravascular granulomatous inflammation, preferentially affecting the respiratory tract. The mechanisms underlying the emergence of neutrophilic microabscesses; the appearance of multinucleated giant cells; and subsequent granuloma formation, finally leading to scarred or destroyed tissue in GPA, are still incompletely understood. This review summarizes findings describing the presence and function of molecules and cells contributing to granulomatous inflammation in the respiratory tract and to renal inflammation observed in GPA. In addition, factors affecting or promoting the development of granulomatous inflammation such as microbial infections, the nasal microbiome, and the release of damage-associated molecular patterns (DAMP) are discussed. Further, on the basis of numerous results, we argue that, in situ, various ways of exposure linked with a high number of infiltrating proteinase 3 (PR3)- and myeloperoxidase (MPO)-expressing leukocytes lower the threshold for the presentation of an altered PR3 and possibly also of MPO, provoking the local development of ANCA autoimmune responses, aided by the formation of ectopic lymphoid structures. Although extravascular granulomatous inflammation is unique to GPA, similar molecular and cellular patterns can be found in both the respiratory tract and kidney tissue of GPA and MPA patients; for example, the antimicrobial peptide LL37, CD163+ macrophages, or regulatory T cells. Therefore, we postulate that granulomatous inflammation in GPA or PR3-AAV is intertwined with autoimmune and destructive mechanisms also seen at other sites.

Endoscopic neo-anastomosis by Rendez-vous technique for the treatment of complete occlusion of bilioenteric anastomoses and distal bile ducts (case series).

BACKGROUND AND AIMS: The complete occlusion of bilioenteric anastomoses is a rare and challenging clinical condition. Repeated surgery is burdened with technical difficulties and significant morbidity. We report the first series of completely occluded bilioenteric anastomoses resp. distal bile duct successfully treated by simultaneous percutaneous and retrograde endoscopic interventions. PATIENTS AND METHODS: This case series includes 4 patients with obstructive jaundice and/or recurring cholangitis and pain due to complete fibrotic occlusion of a hepaticojejunostomy (3 patients) and the distal bile duct (1 patient). After performing PTCD and stepwise dilation of the biliocutaneous tract, we tried to approach the occluded anastomosis from 2 sides by simultaneous percutaneous cholangioscopy and peroral device-assisted enteroscopy/duodenoscopy. By cutting through the separating tissue layer with a needle knife under endoscopic and fluoroscopic control using diaphanoscopy, a new anastomosis should be established followed by dilation of the neoanastomosis with subsequent percutaneous transhepatic drainage for a minimum of 1 year to prevent re-occlusion. RESULTS: The Rendez-vous maneuver was successful in 3/4 cases. In one case, the retrograde access to the anastomosis failed, so the neoanastomosis was cut under cholangioscopic and fluoroscopic guidance only. The neoanastomosis could be established successfully in all 4 cases. Jaundice, cholangitis, and pain disappeared. Minor periinterventional adverse events were cholangitis (n = 1) and pneumonia (n = 1) due to aspiration, which could be managed conservatively. No serious adverse events were observed, and no re-occlusion of any neoanastomosis occurred during the follow-up before and after removal of the percutaneous drainage. CONCLUSION: Simultaneous percutaneous cholangioscopy and device-assisted enteroscopy/duodenoscopy with endoscopic creation of a neoanastomosis is a possible concept for the treatment of completely occluded bilioenteric anastomoses and distal bile ducts. This case series confirms the feasibility, safety, and long-term effectiveness of this treatment.

[Clinical practicability of the cognitive screening battery BICAMS in patients with multiple sclerosis: results of the feasibility study in Germany]. / Klinische Umsetzbarkeit der kognitiven Screeningbatterie BICAMS bei Patienten mit Multipler Sklerose: Ergebnisse der Machbarkeitsstudie in Deutschland.

BACKGROUND: Patients with multiple sclerosis (MS) suffer from cognitive impairment in 40-70% of the cases. There is evidence that the cognitive status is predictive for working ability and early retirement. Regular assessment of cognitive functionality is therefore urgently needed. PURPOSE: The German validation of the Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) battery was evaluated in a multicentric way with respect to its feasibility in private neurological centers across Germany. METHODS: Physician assistants were trained with respect to application and scoring of BICAMS. All scored test materials were evaluated by independent neuropsychological experts. RESULTS: A total of 1606 BICAMS datasets were collected from 65 neurological centers. Of these 1573 datasets were analyzed of which 49.7% were correctly applied and scored while mistakes in application, scoring and transformation were found in 50.3%. Interrater reliability for each subtest was found to be ICC [Formula: see text] 0.953 when datasets containing mistakes were excluded. DISCUSSION: In general, BICAMS is highly recommended to be applied in standard clinical care; however, it should be emphasized that although the interrater reliability in the final sample was high, serious mistakes were found in 50.3% of cases. From these findings we conclude that nonpsychological staff have to be even more intensively trained and supervised by experts in the application and scoring of BICAMS.

Mental symptoms in MS (MeSyMS): Development and validation of a new assessment.

BACKGROUND: Patients with Multiple Sclerosis (MS) have an increased risk of suffering from mental and neuropsychiatric symptoms. So far, a fundamental problem in the clinical care of MS patients is that these symptoms are underdiagnosed and, as a consequence, often remain untreated. Present assessment tools have not been developed to be applied in patients with MS. This study aims to develop and validate a new questionnaire to identify disease-related mental symptoms in MS patients. METHODS: A questionnaire has been developed by including the following subscales: social and emotional health problems, anxiety, and depression. To evaluate test quality and internal consistency, an item analysis has been conducted. After matching MS patients and control subjects on age and gender, we conducted group comparisons, a Receiver Operating Characteristic (ROC) Curve analysis and a binary logistic regression model. RESULTS: In total, 314 MS patients and 100 matched control subjects were analysed. After performed item analysis, the questionnaire revealed an excellent internal consistency (α=0.94). Compared to control subjects, MS patients showed significant mental health problems in all three dimensions. In comparison to the subscales, the dimension of social and emotional health problems revealed the highest accuracy (AUC = 0.75; d = 0.948) and turned out to be the only scale that reliably differentiated between the groups. CONCLUSIONS: MeSyMS constitutes a valid screening instrument to detect mental symptoms in MS. Social and emotional health problems turned out to be the most important aspect when identifying disease-related mental health symptoms in MS.

CT imaging features of skeletal muscle metastasis: A rare tumour group with different patterns.

INTRODUCTION: Skeletal muscle metastasis (SMM) are rare and can have different patterns on computed tomography (CT). Our aim was to estimate the frequency of the different patterns, primary tumours and localizations of SMM in a large single-centre sample. METHODS: A retrospective search in the database of our radiological institution was performed for patients with SMM. The primary tumour, affected muscles and CT patterns were estimated. RESULTS: Overall, 104 SMM were diagnosed in 82 patients. In 11 patients (13.4%), SMM were the isolated manifestation of metastatic disease in clinical work up. Regarding primary tumours, gastrointestinal tumours (24.4%), lung cancer (20.4%) and malignant melanoma (13.4%) were identified most frequently. Other malignancies were rare. Most commonly, the trunk musculature was affected (41.3% of the cases). SMM type 1 were identified in 37 cases (35.6%), type 2 in 64 cases (61.5%), type 3 in 2 cases (1.9%) and type 4 in one case (1%). CONCLUSIONS: The present study shows typical imaging appearances of SMM based on a large patient sample in a single centre. SMM has a rare occurrence with several different patterns, which can impose diagnostic difficulties and varies possible differential diagnoses.

Fibrinolysis and the Influence of Tranexamic Acid Dosing in Cardiac Surgery.

OBJECTIVE: The present study aimed to determine whether underlying disease, performed surgery, and dose of tranexamic acid influence fibrinolysis measured with D-dimer levels. DESIGN: Retrospective analysis. SETTING: Single institution (Department of Cardiac Surgery and Section of Clinical Hemostaseology at the Düsseldorf University Hospital). PARTICIPANTS: The study comprised 3,152 adult patients undergoing elective cardiac surgery between February 2013 and October 2016. INTERVENTIONS: Two doses of tranexamic acid during surgery were administered. MEASUREMENTS AND MAIN RESULTS: D-dimer levels were analyzed at the start of surgery and before protamine administration. D-dimer levels at the start of surgery were compared according to disease. Intraoperative D-dimer development was analyzed according to the type of surgery and within 2 cohorts with different tranexamic acid doses. Interindividual variability was pronounced for D-dimer levels at the start of surgery, with significant differences among patients with coronary artery disease, valve disease, and aortic disease and patients undergoing heart transplantation compared with patients receiving a left ventricular assist device (p < 0.01). Aortic dissection, endocarditis, and extracorporeal life support were associated with higher D-dimer levels (p ≤ 0.01). With tranexamic acid at a fixed dose, intraoperative D-dimer levels decreased in on-pump and off-pump coronary bypass surgery, valve surgery, and left ventricular assist device surgery (p ≤ 0.02), but levels increased in aortic surgery and heart transplantations (p < 0.01). A decrease or increase in D-dimer levels during surgery was influenced significantly by a higher or lower tranexamic acid dose (p ≤ 0.01). CONCLUSIONS: D-dimer testing allows for the assessment of individual fibrinolytic activity in cardiac surgery, which is influenced by disease type, surgery type, and dose of tranexamic acid. The assessment of the fibrinolytic status may have the potential to facilitate dose-adjusted antifibrinolytic therapy in the future.

A case report of an unrecognized osteoid osteoma of the proximal femur.

We present the case of a 59-year-old male patient with unrecognized osteoid osteoma in radiography as well as in magnetic resonance imaging. Computed tomography revealed osteoid osteoma that was successfully treated with percutaneous computed tomography guided radiofrequency ablation. The osseous pathology was underestimated on magnetic resonance imaging in the presented case and bone marrow edema led to incorrect diagnosis. The particular case emphasizes the value of computed tomography scans diagnosing an osteoid osteoma.

Health-related quality of life after heart surgery - Identification of high-risk patients: A cohort study.

BACKGROUND: This cohort study evaluated factors, which have been shown to be relevant for Health-Related Quality of Live (HRQL) after cardiac surgery and investigated the combinatory impact on HRQL. Additionally, the aim was to introduce a first attempt to developing a risk estimation model which could identify patients at risk for impaired HRQL. METHODS: For this single-centre cohort study, 6099 cardiac surgical patients (60% isolated coronary bypass surgery) filled in the Nottingham Health Profile (NHP) for the evaluation of HRQL six months after surgery and provided information regarding their medical and socio-demographic status. For the NHP scores the deviation to the matched normative data of a healthy sample was calculated. A robust linear regression examined factors that influence HRQL. As a next step, based on the regression model, a risk estimation model was developed which is a first attempt to classify patients into risk categories. RESULTS: Male gender, age below 60 or between 60 and 74 years, living alone, no occupation, bypass surgery, NYHA status II, III or IV and chest pain were identified as risk factors to determine impaired HRQL. The model explains 29.13% of the variance. Based on the risk estimation model 27.4% were classified as medium or high risk. CONCLUSIONS: For the first time a multilevel method was applied to evaluate HRQL after heart surgery showing that socio-demographic variables are important co-factors to dyspnea and chest pain. We take a first attempt in developing a new approach that should encourage further research in this field to frame a screening tool that may help identifying patients at risk in the future.

Characterizing cognitive deficits and potential predictors in multiple sclerosis: A large nationwide study applying Brief International Cognitive Assessment for Multiple Sclerosis in standard clinical care.

With the proposal of the Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) battery, the need to screen for cognitive deficits within standard clinical care of patients with multiple sclerosis (MS) has been acknowledged. Data regarding how patient characteristics might predict low cognitive performance and therefore require particularly close monitoring is, however, limited so far. We investigated a large, nationwide patient cohort from ambulatory settings, representing the typical distribution of different subtypes, levels of physical disability, and disease durations. Besides cognitive testing with BICAMS, additional sampling of multiple demographics and clinical variables allowed us to characterize general and domain-specific prevalence patterns of cognitive impairment (CI) as well as to delineate which factors are associated with cognitive performance. In a total of 1,094 patients, CI was present in 28% (using a conservative cut-off of the 5th percentile below normative values), with information-processing speed being most frequently affected. Impairment was overall higher in patients with primary progressive (PPMS) and secondary progressive MS than in patients with relapsing-remitting (RR)MS. Regression modelling revealed that disease subtype (i.e., PPMS), long disease duration, high physical disability, unemployment, low educational level, high age, male sex, and the absence of current disease-modifying treatment were important predictors for worse BICAMS' test performance. These results emphasize the importance of continuous cognitive assessment during regular neurological follow-up visits, with a particular focus on patients being identified as high-risk subjects for CI according to the reported factors.

On the validity of single tests, two-test combinations and the full Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) in detecting patients with cognitive impairment.

BACKGROUND: The international standard to screen for cognitive impairment in multiple sclerosis (MS) is BICAMS (Brief International Cognitive Assessment for MS). However, with an application time of approximately 20 minutes, the battery might be too time consuming from a pragmatic perspective of a routine examination. OBJECTIVES: To examine the relative sensitivity and specificity of a BICAMS short version and its validity compared to the total battery. METHODS: The German BICAMS version was applied comprising the Symbol Digit Modalities Test (SDMT), the Brief Visuospatial Memory Test-Revised (BVMT-R) and the Rey Auditory Verbal Learning Test (RAVLT; German VLMT). Single tests and two-test combinations were compared regarding conformity with the total battery. RESULTS: Examining 1320 MS patients, the two-test combination of SDMT-BVMT-R was the most sensitive (92.7%) to impairment and showed the strongest agreement with the total battery (κ = 0.95). Performing binary logistic regression analyses, this combination was also validated by its association with employment status. CONCLUSION: Application of the total BICAMS battery should be the goal to strive for. However, in time-restricted clinical settings, the combined application of SDMT and BVMT-R is a recommendable alternative with an application time of 10 minutes, while single tests alone are not sufficiently sensitive.

Dynamic changes in intron retention are tightly associated with regulation of splicing factors and proliferative activity during B-cell development.

Intron retention (IR) has been proposed to modulate the delay between transcription and translation. Here, we provide an exhaustive characterization of IR in differentiated white blood cells from both the myeloid and lymphoid lineage where we observed highest levels of IR in monocytes and B-cells, in addition to previously reported granulocytes. During B-cell differentiation, we found an increase in IR from the bone marrow precursors to cells residing in secondary lymphoid organs. B-cells that undergo affinity maturation to become antibody producing plasma cells steadily decrease retention. In general, we found an inverse relationship between global IR levels and both the proliferative state of cells, and the global levels of expression of splicing factors. IR dynamics during B-cell differentiation appear to be conserved between human and mouse, suggesting that IR plays an important biological role, evolutionary conserved, during blood cell differentiation. By correlating the expression of non-core splicing factors with global IR levels, and analyzing RNA binding protein knockdown and eCLIP data, we identify a few splicing factors likely playing an evolutionary conserved role in IR regulation. Our work provides new insights into the role of IR during hematopoiesis, and on the main factors involved in regulating IR.

Basicity Enhancement by Multiple Intramolecular Hydrogen Bonding in Organic Superbase N,N',N″,N‴-Tetrakis(3-(dimethylamino)propyl)triaminophosphazene.

With the synthesis of N,N',N″,N‴-tetrakis(3-(dimethylamino)propyl)triaminophosphazene (TDMPP, 1), we present the first phosphazene superbase with enhanced basicity through the effect of multiple intramolecular hydrogen bonding (IHB). Due to intramolecular solvation of four NH protons, the proton affinity is even higher than that of second-order phosphazene (dma)P2-tBu. X-ray structural proof, NMR titration experiments, and computational investigations provide a more detailed quantitative description of the IHB influence on the superbasicity of 1 in solid-state, solution, and the gas-phase.

Phosphazenyl Phosphines: The Most Electron-Rich Uncharged Phosphorus Brønsted and Lewis Bases.

It was discovered that phosphazenyl phosphines (PAPs) can be stronger P-superbases than the corresponding Schwesinger type phosphazene N-superbases. A simple synthetic access to this class of PR3 derivatives including their homologization is described. XRD structures, proton affinities (PA), and gas-phase basicities (GB) as well as calculated and experimental pK BH + values in THF are presented. In contrast to their N-basic counterparts, PAPs are also privileged ligands in transition metal chemistry. In fact, they are currently the strongest uncharged P-donors known, exceeding classical and more recently discovered ligands such as PtBu3 and imidazolin-2-ylidenaminophosphines (IAPs) with respect to their low Tolman electronic parameters (TEPs) and large cone angles.