Search

Introduction: Cryptosporidiosis has emerged as a major cause of diarrheal disease worldwide. It has especially serious health consequences for young, malnourished children living in endemic areas and for individuals with highly impaired T-cell function, such as HIV-positive individuals with low CD4 counts or immunosuppressed solid-organ transplant recipients.Areas covered: A selective literature search using PubMed was performed to review the available therapeutics to treat cryptosporidiosis, as well as related advances in drug development.Expert opinion: The only FDA-approved antiparasitic treatment in immunocompetent patients is nitazoxanide; however, it has failed to demonstrate convincing effectiveness among HIV-positive patients, immunosuppressed individuals and malnourished children. Thus, restoring HIV-positive patients' cellular immune response through effective antiretroviral therapy (ART), or reducing or changing immunosuppressive drugs, is important. Several new targets have been identified for chemotherapy, and the development of drugs for these targets has progressed, including parasite kinases, nucleic acid synthesis and processing, proteases and lipid metabolism. Candidate drugs that have been shown to be effective and safe in a neonatal calf model will most likely constitute the next advance for clinical trials in humans. However, developing an effective and inexpensive vaccination, as well as complementing structural preventive measures, would most decisively reduce the global cryptosporidiosis burden.

Purpose Dioxygenases are oxidoreductase enzymes with roles in metabolic pathways necessary for aerobic life. 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL), encoded by HPDL, is an orphan paralogue of 4-hydroxyphenylpyruvate dioxygenase (HPD), an iron-dependent dioxygenase involved in tyrosine catabolism. The function and association of HPDL with human diseases remain unknown. Methods We applied exome sequencing in a cohort of over 10,000 individuals with neurodevelopmental diseases. Effects of HPDL loss were investigated in vitro and in vivo, and through mass spectrometry analysis. Evolutionary analysis was performed to investigate the potential functional separation of HPDL from HPD. Results We identified biallelic variants in HPDL in eight families displaying recessive inheritance. Knockout mice closely phenocopied humans and showed evidence of apoptosis in multiple cellular lineages within the cerebral cortex. HPDL is a single-exonic gene that likely arose from a retrotransposition event at the base of the tetrapod lineage, and unlike HPD, HPDL is mitochondria-localized. Metabolic profiling of HPDL mutant cells and mice showed no evidence of altered tyrosine metabolites, but rather notable accumulations in other metabolic pathways. Conclusion The mitochondrial localization, along with its disrupted metabolic profile, suggests HPDL loss in humans links to a unique neurometabolic mitochondrial infantile neurodegenerative condition.

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3–1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3–1 and RIG-I mRNA was also observed during decitabine treatment in vivo in serially sorted peripheral blood AML blasts. In patient-derived monosomal karyotype AML murine xenografts, decitabine treatment resulted in superior survival rates compared with cytarabine. Collectively, these data demonstrate preferential gene derepression and ERV reactivation in AML with chromosomal deletions, providing a mechanistic explanation that supports the clinical observation of superiority of HMA over cytarabine in this difficult-to-treat patient group. Significance: These findings unravel the molecular mechanism underlying the intriguing clinical activity of HMAs in AML/MDS patients with chromosome 7 deletions and other monosomal karyotypes. See related commentary by O'Hagan et al., p. 813

Core-binding factor (CBF) acute myeloid leukemia (AML) encompasses AML with inv(16)(p13.1q22) and AML with t(8;21)(q22;q22.1). Despite sharing a common pathogenic mechanism involving rearrangements of the CBF transcriptional complex, there is growing evidence for considerable genotypic heterogeneity. We comprehensively characterized the mutational landscape of 350 adult CBF-AML [inv(16): n = 160, t(8;21): n = 190] performing targeted sequencing of 230 myeloid cancer-associated genes. Apart from common mutations in signaling genes, mainly NRAS, KIT, and FLT3, both CBF-AML entities demonstrated a remarkably diverse pattern with respect to the underlying cooperating molecular events, in particular in genes encoding for epigenetic modifiers and the cohesin complex. In addition, recurrent mutations in novel collaborating candidate genes such as SRCAP (5% overall) and DNM2 (6% of t(8;21) AML) were identified. Moreover, aberrations altering transcription and differentiation occurred at earlier leukemic stages and preceded mutations impairing proliferation. Lasso-penalized models revealed an inferior prognosis for t(8;21) AML, trisomy 8, as well as FLT3 and KIT exon 17 mutations, whereas NRAS and WT1 mutations conferred superior prognosis. Interestingly, clonal heterogeneity was associated with a favorable prognosis. When entering mutations by functional groups in the model, mutations in genes of the methylation group (ie, DNMT3A, TET2) had a strong negative prognostic impact.

Key points CAST/ELKS are positive regulators of presynaptic growth and are suppressors of active zone expansion at the developing mouse calyx of Held. CAST/ELKS regulate all three CaV 2 subtype channel levels in the presynaptic terminal and not just CaV 2.1. The half-life of ELKS is on the timescale of days and not weeks. Synaptic transmission was not impacted by the loss of CAST/ELKS. CAST/ELKS are involved in pathways regulating morphological properties of presynaptic terminals during an early stage of circuit maturation. Abstract Many presynaptic active zone (AZ) proteins have multiple regulatory roles that vary during distinct stages of neuronal circuit development. The CAST/ELKS protein family are evolutionarily conserved presynaptic AZ molecules that regulate presynaptic calcium channels, synaptic transmission and plasticity in the mammalian CNS. However, how these proteins regulate synapse development and presynaptic function in a developing neuronal circuit in its native environment is unclear. To unravel the roles of CAST/ELKS in glutamatergic synapse development and in presynaptic function, we used CAST knockout (KO) and ELKS conditional KO (CKO) mice to examine how their loss during the early stages of circuit maturation impacted the calyx of Held presynaptic terminal development and function. Morphological analysis from confocal z-stacks revealed that combined deletion of CAST/ELKS resulted in a reduction in the surface area and volume of the calyx. Analysis of AZ ultrastructure showed that AZ size was increased in the absence of CAST/ELKS. Patch clamp recordings demonstrated a reduction of all presynaptic CaV 2 channel subtype currents that correlated with a loss in presynaptic CaV 2 channel numbers. However, these changes did not impair synaptic transmission and plasticity and synaptic vesicle release kinetics. We conclude that CAST/ELKS proteins are positive regulators of presynaptic growth and are suppressors of AZ expansion and CaV 2 subtype currents and levels during calyx of Held development. We propose that CAST/ELKS are involved in pathways regulating presynaptic morphological properties and CaV 2 channel subtypes and suggest there is developmental compensation to preserve synaptic transmission during early stages of neuronal circuit maturation.

ZusammenfassungTyphus (besser: Typhus abdominalis) und Paratyphus sind global bedeutsame systemische Infektionskrankheiten, die durch Salmonella enterica Subspezies enterica Serovar Typhi (Kurzbezeichnung: Salmonella Typhi) bzw. Serovar Paratyphi (Kurzbezeichnung: Salmonella Paratyphi) hervorgerufen werden. Der Verlauf der fäkal-oral übertragenen Erkrankung ist vor allem durch hohes Fieber gekennzeichnet. Unbehandelt kann Typhus schwer verlaufen und zum Tode führen. Die Erkrankung wird zu nahezu 100 % im außereuropäischen Ausland (v. a. in Indien) erworben und ist in Deutschland, Österreich und in der Schweiz meldepflichtig. Als Paratyphus bezeichnet man ein abgeschwächtes Krankheitsbild des Typhus, dessen Erreger Salmonella Paratyphi ist. Verfügbare Impfstoffe schützen nur gegen Salmonella Typhi. Die Resistenz gegen Antibiotika spiegelt die Situation in endemischen Ländern wider und zeigt sich in einem besorgniserregenden Zuwachs von Isolaten mit Multiresistenz. Gegenwärtig werden Cephalosporine der 3. Generation wie Ceftriaxon als Erstlinientherapie empfohlen; sofern es sich um Isolate mit Chinolonempfindlichkeit handelt, können auch weiterhin Fluorchinolone wie Ciprofloxacin verabreicht werden. Entscheidende vorbeugende Maßnahmen für Reisende sind konsequente Wasser- und Nahrungsmittelhygiene sowie Impfprävention, wobei sich mit den bislang verfügbaren Impfstoffen nur Protektionsraten von 50–70 % erreichen lassen. Mit der Zunahme von multiresistenten Erregerstämmen wird ein besser wirksamer Konjugatimpfstoff gegen Salmonella Typhi mit Kreuzreaktivität gegen Salmonella Paratyphi dringender denn je benötigt.

We report the case of a 65-year-old female patient with hepatic alveolar echinococcosis (AE) caused by Echinococcus multilocularis. This infrequent zoonosis has a considerable morbidity and mortality. The malignant appearing hepatic mass was initially misdiagnosed as cholangiocarcinoma of the right hepatic lobe (segments VII, VIII, and IVa, sized 10.9 cm × 7.6 cm) involving the right and middle hepatic vein and extending close to the left hepatic vein. During exploratory laparotomy, the frozen-section biopsy was indicative of AE (World Health Organization [WHO] classification: stage P3N0M0). Due to the high operative risk, it was decided to pretreat the patient with albendazole as inductive therapy in order to remove the AE secondarily in accordance with the patient’s request. After year-long treatment with albendazole (under strict control of the maximum blood levels), a right hemihepatectomy was successfully performed. Postoperative treatment with albendazole had to be stopped prematurely after 11 months due to considerable subjective intolerance and a more-than-tenfold elevation of transaminases despite normal therapeutic albendazole blood levels. A 18F-FDG-PET/CT scan revealed no evidence of AE residues. Conducting follow-up examinations by 18F-FDG-PET/CT scans every 2 years is planned in order to recognize possible recurrence at an early stage.

The prevalence of alveolar echinococcosis (AE) and cystic echinococcosis (CE) in Germany is increasing. The number of cases in northern and eastern regions is low, so there is little experience with regard to diagnosis and therapy. The objective of this paper was to evaluate the management of suspected and/or diagnosed echinococcosis at a university center in a low-prevalence region. All the patients at the Leipzig University Hospital between 2004 and 2018 who had been serologically examined for echinococci were included in a retrospective cohort study. Clinical course, imaging, histology, therapeutic characteristics, relevant comorbidities and risk factors for AE and CE were evaluated. A time-staggered prevalence estimation, as well as sensitivity and specificity calculations for the serological tests, were performed. A total of 382 patients were enrolled, with 11 AE and 7 CE cases identified. The mean prevalence rate of AE in this cohort was 2.9 % and that of CE was 1.8 %. Among the patients, 56 % had known risk factors for AE and CE. The serological tests showed a sensitivity of 86 % and a specificity of 91 %. Two patients with false-negative serology were diagnosed by biopsy. All CE and 5 AE patients (45 %) were operated on. Six AE patients received long-term treatment with albendazole. AE and CE are rare diseases in the greater Leipzig region; however, case numbers are on the rise. Due to favorable factors such as the escalation of migration, a further increase is expected. Diagnosis and therapy are challenging and should be supported by specialists (experienced infectiologists, imaging experts and skilled hepatobiliary surgeons) who should be integrated in a German network.EINFüHRUNG : Die Prävalenz der alveolären (AE) und zystischen Echinokokkose (CE) in Deutschland nimmt zu, aber die Gesamtzahl der Fälle in den nördlichen und östlichen Bundesländern ist noch immer gering. Betroffene Patienten werden überwiegend in wenigen spezialisierten Zentren in Süddeutschland behandelt, sodass in anderen Regionen Deutschlands bislang nur relativ wenig klinische Erfahrung mit AE und CE vorhanden ist.Ziel unserer Studie war es, das derzeitige Management von Patienten mit Verdacht auf oder bestätigter Echinokokkose an einem Zentrum der tertiären Versorgungsstufe in einer Niedrigprävalenzregion zu bewerten, um eine Verbesserung des Vorgehens bei Verdacht auf AE/CE bzw. bereits gesicherter Diagnose zu erreichen.Alle Patienten, die am Universitätsklinikum Leipzig zwischen dem 01.01.2004 und dem 30.09.2018 infektionsserologisch auf Echinokokken untersucht wurden oder die Diagnose „AE“ bzw. „CE“ erhalten hatten, wurden in eine retrospektive Kohortenstudie eingeschlossen. Klinischer Verlauf, bildgebende sowie histopathologische Befunde, Therapiecharakteristika, relevante Begleiterkrankungen und mögliche Risikofaktoren für AE bzw. CE wurden bewertet. Die Datenanalyse umfasste weiter eine zeitlich gestaffelte Prävalenzabschätzung für unser Zentrum sowie Sensitivitäts- und Spezifitätsberechnungen für die durchgeführten serologischen Tests.Insgesamt wurden 382 Patienten in die Analyse einbezogen. 11 AE- und 7 CE-Fälle wurden identifiziert. Die mittlere Prävalenzrate der AE in dieser Kohorte lag bei 2,9 % und die der CE bei 1,8 %. Bei 4 Patienten war die Diagnose bereits auswärtig gesichert worden und eine gezielte Überweisung zur spezialisierten Behandlung erfolgt. 56 % der Patienten wiesen bekannte Risikofaktoren für CE bzw. AE auf. Mit Ausnahme eines Falls waren alle CE-Fälle Migranten mit ausländischer Herkunft. Die durchgeführten serologischen Tests zeigten für beide Erkrankungen eine Sensitivität von 86 % und eine Spezifität von 91 %. 2 Patienten mit zunächst falsch negativer Diagnose (einer mit AE und einer mit CE) wurden durch ultraschallgestützte Biopsie korrekt diagnostiziert. Alle CE-Fälle und 5 AE-Fälle (45 %) wurden unter Albendazolschutz chirurgisch versorgt. Die restlichen 6 AE-Fälle erhielten eine Langzeitbehandlung mit Albendazol. Es wurden keine Todesfälle infolge Echinokokkose beobachtet.Sowohl AE als auch CE sind im Großraum Leipzig seltene Erkrankungen, wenngleich ein Anstieg der Fallzahlen an unserem Zentrum zu verzeichnen ist. Aufgrund unterschiedlicher begünstigender Faktoren wie z. B. Zunahme der Migration ist in Zukunft mit einem weiteren Anstieg zu rechnen. Diagnosestellung und Therapiemanagement stellen sowohl für die AE als auch für die CE eine Herausforderung dar und sollten von einem Expertenteam begleitet werden, darunter erfahrene Infektiologen, Bildgebungsspezialisten und versierte hepatobiliäre Chirurgen. Der Aufbau eines deutschen Echinokokkosenetzwerks wäre sinnvoll.

Key Points mRNA decapping gene EDC4 is a novel fusion partner of MLL in AML. Genes functioning in mRNA decapping may compose a distinct group of MLL fusion partners that links MLL function with mRNA decapping in AML.

Background Repeat elements constitute a large proportion of the human genome and recent evidence indicates that repeat element expression has functional roles in both physiological and pathological states. Specifically for cancer, transcription of endogenous retrotransposons is often suppressed to attenuate an anti-tumor immune response, whereas aberrant expression of heterochromatin-derived satellite RNA has been identified as a tumor driver. These insights demonstrate separate functions for the dysregulation of distinct repeat subclasses in either the attenuation or progression of human solid tumors. For hematopoietic malignancies, such as Acute Myeloid Leukemia (AML), only very few studies on the expression/dysregulation of repeat elements were done. Methods To study the expression of repeat elements in AML, we performed total-RNA sequencing of healthy CD34 + cells and of leukemic blast cells from primary AML patient material. We also developed an integrative bioinformatic approach that can quantify the expression of repeat transcripts from all repeat subclasses (SINE/ALU, LINE, ERV and satellites) in relation to the expression of gene and other non-repeat transcripts (i.e. R/G ratio). This novel approach can be used as an instructive signature for repeat element expression and has been extended to the analysis of poly(A)-RNA sequencing datasets from Blueprint and TCGA consortia that together comprise 120 AML patient samples. Results We identified that repeat element expression is generally down-regulated during hematopoietic differentiation and that relative changes in repeat to gene expression can stratify risk prediction of AML patients and correlate with overall survival probabilities. A high R/G ratio identifies AML patient subgroups with a favorable prognosis, whereas a low R/G ratio is prevalent in AML patient subgroups with a poor prognosis. Conclusions We developed an integrative bioinformatic approach that defines a general model for the analysis of repeat element dysregulation in physiological and pathological development. We find that changes in repeat to gene expression (i.e. R/G ratios) correlate with hematopoietic differentiation and can sub-stratify AML patients into low-risk and high-risk subgroups. Thus, the definition of a R/G ratio can serve as a valuable biomarker for AML and could also provide insights into differential patient response to epigenetic drug treatment.

Genomic surveillance can inform effective public health responses to pathogen outbreaks. However, integration of non-local data is rarely done. We investigate two large hospital outbreaks of a carbapenemase-carrying Klebsiella pneumoniae strain in Germany and show the value of contextual data. By screening more than ten thousand genomes, 500 thousand metagenomes, and two culture collections using in silico and in vitro methods, we identify a total of 415 closely related genomes reported in 28 studies. We identify the relationship between the two outbreaks through time-dated phylogeny, including their respective origin. One of the outbreaks presents extensive hidden transmission, with descendant isolates only identified in other studies. We then leverage the genome collection from this meta-analysis to identify genes under positive selection. We thereby identify an inner membrane transporter (ynjC) with a putative role in colistin resistance. Contextual data from other sources can thus enhance local genomic surveillance at multiple levels and should be integrated by default when available.

Red blood cell transfusions (RBCT) remain the cornerstone of supportive care in lower-risk myelodysplastic syndrome (LRMDS) [1]. Transfusion dependency in LRMDS patients is associated with inferior outcomes, mainly attributed to severe bone marrow failure [2]. However, iron toxicity, due to frequent RBCT or ineffective erythropoiesis, may be an additional negative prognostic factor [3,4,5,6]. Recently, much progress has been made in unraveling the iron metabolism. The peptide hormone hepcidin is the key regulator by inhibiting iron uptake through degradation of ferroportin, a cellular iron exporter [7]. Erythroferrone and GDF15, produced by erythroblasts, inhibit hepcidin production, which leads to increased uptake and cellular release of iron for the purpose of erythropoiesis [8]. The pathophysiology of iron metabolism in MDS is still not completely understood. Exceedingly high reactive oxygen species (ROS) levels are associated with iron toxicity, disease development, and progression in MDS patients [9,10,11,12]. Malondialdehyde (MDA), resulting from lipid peroxidation of polyunsaturated fatty acids, is a biomarker of oxidative stress [10, 12]. Currently, little is known about the prognostic impact of ROS in MDS patients. The aim of this study is twofold: (1) describe iron and oxidative stress parameters over time in LRMDS patients and (2) to assess their effect on overall and progression-free survival. The EUMDS registry prospectively collects observational data on newly diagnosed LRMDS patients from 148 centers in 16 countries in Europe and Israel as of January 2008. All patients provided informed consent. Clinical data were collected at baseline and at each six-monthly follow-up visit. Serum samples were collected prospectively at each visit from 256 patients included in six participating countries. Conventional iron parameters were measured with routine assays. We additionally analyzed hepcidin, growth differentiation factor 15 (GDF15), soluble transferrin receptor (sTfR), non-transferrin bound iron (NTBI), labile plasma iron (LPI), and MDA. Subjects were prospectively followed until death, loss to follow-up, or withdrawal of consent. All iron parameters were measured centrally at the department of Laboratory Medicine of the Radboudumc, Nijmegen, The Netherlands. Serum samples were collected just prior to transfusion in transfusion-dependent patients and stored at −80 °C. Details on the assays and reference ranges of hepcidin, GDF15, sTfR, NTBI, LPI, and MDA are provided in the supplement. The Spearman rank test was used to evaluate correlations between iron parameters. We stratified the results by transfusion dependency per visit and the presence of ring sideroblasts. When evaluating temporal changes in iron parameters, with linear quantile mixed models, we excluded patients from the timepoint they received iron chelation therapy. Overall survival (OS) was defined as the time from MDS diagnosis to death or, in case of progression-free survival, to date of progression or death; patients still alive at the end of follow-up were censored. Time-dependent Kaplan–Meier curves and cox proportional hazards models were used. In total, 256 consecutive patients, were included in this study. Over five six-monthly visits, 1040 samples were collected. Table 1 describes the patient characteristics. Most patients without ring sideroblasts were transfusion-independent at diagnosis (nonRS-TI; 55.9%), 18.8% with ring sideroblasts were transfusion-independent (RS-TI), 18.4% without ring sideroblasts were transfusion-dependent (nonRS-TD), and 7% with ring sideroblasts were transfusion-dependent patients (RS-TD). The median follow-up time was 6.6 years (95% CI 5.9–7.0). LPI was positively correlated with transferrin saturation (TSAT) (r = 0.15, p < 0.001, Fig. S1). LPI values increased exponentially at TSAT values above 80%. This effect was most pronounced in the transfusion-dependent groups, but also observed in the RS-TI group. MDA was weakly correlated with NTBI (r = 0.09, p = 0.069) and negatively correlated with hemoglobin level (r = −0.1, p = 0.033). GDF15 and hepcidin were negatively correlated in the RS-TI and nonRS-TD group and significantly negatively correlated in the RS-TD group (r = −0.34, p = 0.007, Fig. S2). Serum ferritin levels were elevated in all subgroups with a mean value of 858 µg/L at visit 5. The highest serum ferritin levels were observed in the RS-TD group (mean value at visit 5: 2092 µg/L, Table S1). Serum ferritin increased significantly per visit in the RS-TD group (beta 454.46 µg/L; 95% CI 334.65–574.27), but not in the other groups (Table S2). All subgroups, except for the nonRS-TI, had elevated TSAT levels. TSAT levels were most markedly increased in the RS-TD group with a mean TSAT of 88% at visit 5 (Table S1). In both transfusion-dependent groups the median increase per visit was significant (Table S2). LPI was elevated in the RS-TD group exclusively with a mean value of 0.59 µmol/L at visit 5 (Table S1). NTBI was elevated in all subgroups, with the highest values in the RS-TD group (Table S1). The increase in median NTBI level was significant in both transfusion-dependent groups (Table S2). Hepcidin levels were markedly elevated in the nonRS-TD group. Interestingly, hepcidin levels were lower in the RS-TD group, probably reflecting ineffective erythropoiesis, likewise supported by lower hepcidin/ferritin ratios in RS groups (Table S1). Median hepcidin levels increased over time in the transfusion-dependent subgroups only (Table S2). GDF15 levels, analyzed in the light of its potential role in hepcidin suppression, were increased in all subgroups (Table S1). The RS subgroups had higher GDF15 levels compared to the nonRS groups, reflecting increased erythropoiesis. Mean sTfR levels were within the reference range in all subgroups except for the RS-TI group, which showed elevated levels, reflecting...

An amendment to this paper has been published and can be accessed via the original article.

Monitoring the humoral protective immune response and its durability after SARS-CoV-2 infections is essential for risk assessment of reinfections, the improvement of diagnostic methods and the evaluation of vaccine trials. We have analyzed neutralizing antibodies and IgG responses specific to different antigens, including the inactivated whole-virion of SARS-CoV-2, the spike subunit 1 protein and its receptor binding domain, as well as the nucleocapsid protein. We show the dynamic developments of the responses from the early convalescent stages up to 9 months post symptoms onset in follow-up samples from 57 COVID-19 patients with varying clinical severity. By correlating antibody signals to neutralizing titres, valid diagnostic markers for the estimation of neutralizing protection could be identified.

The chromosomal translocation t(8;21) and the resulting oncofusion gene AML1/ETO have long served as a prototypical genetic lesion to model and understand leukemogenesis. In this review, we describe the wide-ranging role of AML1/ETO in AML leukemogenesis, with a particular focus on the aberrant epigenetic regulation of gene transcription driven by this AML-defining mutation. We begin by analyzing how structural changes secondary to distinct genomic breakpoints and splice changes, as well as posttranscriptional modifications, influence AML1/ETO protein function. Next, we characterize how AML1/ETO recruits chromatin-modifying enzymes to target genes and how the oncofusion protein alters chromatin marks, transcription factor binding, and gene expression. We explore the specific impact of these global changes in the epigenetic network facilitated by the AML1/ETO oncofusion on cellular processes and leukemic growth. Furthermore, we define the genetic landscape of AML1/ETO-positive AML, presenting the current literature concerning the incidence of cooperating mutations in genes such as KIT, FLT3, and NRAS. Finally, we outline how alterations in transcriptional regulation patterns create potential vulnerabilities that may be exploited by epigenetically active agents and other therapeutics.

Food hydrocolloids 110, 106132 (2021). doi:10.1016/j.foodhyd.2020.106132, Published by Elsevier, Amsterdam

Although, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents one of the biggest challenges in the world today, the exact immunopathogenic mechanism that leads to severe or critical Coronavirus Disease 2019 (COVID-19) has remained incompletely understood. Several studies have indicated that high systemic plasma levels of inflammatory cytokines result in the so-called “cytokine storm”, with subsequent development of microthrombosis, disseminated intravascular coagulation, and multiorgan-failure. Therefore, we reasoned those elevated inflammatory molecules might act as prognostic factors. Here, we analyzed 245 serum samples of patients with COVID-19, collected at hospital admission. We assessed the levels of heat shock protein 27 (HSP27), soluble suppressor of tumorigenicity-2 (sST2) and 20S proteasome at hospital admission and explored their associations with overall-, 30-, 60-, 90-day- and in-hospital mortality. Moreover, we investigated their association with the risk of ventilation. We demonstrated that increased serum sST2 was uni- and multivariably associated with all endpoints. Furthermore, we also identified 20S proteasome as independent prognostic factor for in-hospital mortality (sST2, AUC = 0.73, HSP27, AUC = 0.59, 20S proteasome = 0.67). Elevated sST2, HSP27, and 20S proteasome levels at hospital admission were univariably associated with higher risk of invasive ventilation (OR = 1.8, p &lt, 0.001, OR = 1.1, p = 0.04, OR = 1.03, p = 0.03, respectively). These findings could help to identify high-risk patients early in the course of COVID-19.

Hypervirulent Klebsiella pneumoniae (hvKp) is a novel pathotype that has been rarely described in Europe. This study characterizes a hvKp isolate that caused a community-acquired infection. The hypermucoviscous Klebsiella pneumoniae (K. pneumoniae) strain 18-0005 was obtained from a German patient with tonsillopharyngitis in 2017. Antibiotic susceptibility testing was performed and the genome was sequenced by Illumina and Nanopore technology. Whole genome data were analyzed by conducting core genome multilocus sequence typing (cgMLST) and single nucleotide polymorphism (SNP) analysis. Virulence genes were predicted by applying Kleborate. Phenotypic and whole genome analyses revealed a high similarity of the study isolate 18-0005 to the recently reported antibiotic-susceptible hvKp isolate SB5881 from France and the “ancestral” strain Kp52.145; both were assigned to the ST66-K2 lineage. Comparative genomic analysis of the three plasmids showed that the 18-0005 plasmid II differs from SB5881 plasmid II by an additional 3 kb integrated fragment of plasmid I. Our findings demonstrate the genetic flexibility of hvKp and the occurrence of a strain of the clonal group CG66-K2 in Germany. Hence, it emphasizes the need to improve clinical awareness and infection monitoring of hvKp.

Highlights • Deep learning technology applied to diagnosis of Acute Promyelocytic Leukemia (APL). • Convolutional neural networks (Mask R-CNN) may diagnose APL in bone marrow smear images with a high precision. • Data augmentation and pre-trained approach improves diagnostic accuracy., Background Serological severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody assays differ in the target antigen specificity, e.g. of antibodies directed against the viral spike or the nucleocapsid protein, and in the spectrum of detected immunoglobulins. The aim of the study was to evaluate the performance of two different routinely used immunoassays in hospitalized and outpatient COVID-19 cases. Methods The test characteristics of commercially available spike1 protein-based serological assays (Euroimmun, EI-assays), determining IgA or IgG and nucleocapsid-based assays (Virotech, VT-assays) determining IgA, IgM or IgG were compared in 139 controls and 116 hospitalized and outpatient COVID-19 cases. Results Hospitalized COVID-19 patients (n = 51; 115 samples) showed significantly higher concentrations of antibodies against SARS-CoV-2 and differed from outpatient cases (n = 65) by higher age, higher disease severity scores and earlier follow up blood sampling. Sensitivity of the two IgG assays was comparable in hospitalized patients tested ≥ 14 days (EI-assay: 88%, CI95% 67.6–99.9; VT-assay: 96%, CI95% 77.7–99.8). In outpatient COVID-19 cases sensitivity was significantly lower in the VT-assay (86.2%, CI95% 74.8–93.1) compared with the EI-assay (98.5%, CI95% 90.6–99.9). Assays for IgA and IgM demonstrated a lack of specificity or sensitivity. Conclusions Our results indicate that SARS-CoV-2 serological assays may need to be optimized to produce reliable results in outpatient COVID-19 cases who are low or even asymptomatic. Assays for IgA and IgM have limited diagnostic performance and do not prove an additional value for population-based screening approaches.

DNA methyltransferase inhibitors (DNMTi) approved for older AML patients are clinically tested in combination with histone deacetylase inhibitors (HDACi). The mechanism of action of these drugs is still under debate. In colon cancer cells, 5-aza-2'-deoxycytidine (DAC) can downregulate oncogenes and metabolic genes by reversing gene body DNA methylation, thus implicating gene body methylation as a novel drug target. We asked whether DAC-induced gene body demethylation in AML cells is also associated with gene repression, and whether the latter is enhanced by HDACi.Transcriptome analyses revealed that a combined treatment with DAC and the HDACi panobinostat or valproic acid affected significantly more transcripts than the sum of the genes regulated by either treatment alone, demonstrating a quantitative synergistic effect on genome-wide expression in U937 cells. This effect was particularly striking for downregulated genes. Integrative methylome and transcriptome analyses showed that a massive downregulation of genes, including oncogenes (e.g., MYC) and epigenetic modifiers (e.g., KDM2B, SUV39H1) often overexpressed in cancer, was associated predominantly with gene body DNA demethylation and changes in acH3K9/27. These findings have implications for the mechanism of action of combined epigenetic treatments, and for a better understanding of responses in trials where this approach is clinically tested.

The study analyzes the clonal architecture and the abnormalities involved in a series of 191 patients with myelodysplastic syndromes (MDS) and 2-3 clonal abnormalities. All patients were extracted from an international database. The patients were classified into six clonal subtypes (2A-3C) based on the number of abnormalities and the presentation of unrelated clones (UC) and/or a clonal evolution. UC were detected in 23/191 patients (12%). The composition of UC showed great variability. The only recurrent combination of abnormalities was del(5q) and + 8 in 8 of 23 patients (35%). In patients with clonal evolution, the clone size of the primary and secondary clone varied: Patients with -7 and + 8 in the primary clone showed a larger primary and a smaller secondary clone (-7: median 74% vs 10%; +8 73% vs 18%) while patients with del(5q) in the primary clone showed a smaller primary and a larger secondary clone (33% vs 61%). Univariate and multivariate analyses showed no significant differences regarding overall or AML-free survival between the clonal subtypes. Only the subtype 3C (3 abnormalities and clonal evolution) was an independent risk factor for developing AML (Hazard Ratio 5.5 as compared to subtype 2A, P < .05). Finally, our study confirms that the number of abnormalities clearly defines a significant risk factor for overall- as well as AML-free survival. Importantly, in patients with more than one clone, the calculation of the number of abnormalities in the entire sample instead of the number of abnormalities per clone allows a higher prognostic accuracy.

FAD-dependent lysine-specific demethylase 1 (LSD1) is overexpressed or deregulated in many cancers such as AML and prostate cancer and hence is a promising anticancer target with first inhibitors in clinical trials. Clinical candidates are N-substituted derivatives of the dual LSD1-/monoamine oxidase-inhibitor tranylcypromine (2-PCPA) with a basic amine function in the N-substituent. These derivatives are selective over monoamine oxidases. So far, only very limited information on structure-activity studies about this important class of LSD1 inhibitors is published in peer reviewed journals. Here, we show that N-substituted 2-PCPA derivatives without a basic function or even a polar group are still potent inhibitors of LSD1 in vitro and effectively inhibit colony formation of leukemic cells in culture. Yet, these lipophilic inhibitors also block the structurally related monoamine oxidases (MAO-A and MAO-B), which may be of interest for the treatment of neurodegenerative disorders, but this property is undesired for applications in cancer treatment. The introduction of a polar, non-basic function led to optimized structures that retain potent LSD1 inhibitors but exhibit selectivity over MAOs and are highly potent in the suppression of colony formation of cultured leukemic cells. Cellular target engagement is shown via a Cellular Thermal Shift Assay (CETSA) for LSD1.

Summary Mitochondria perform central functions in cellular bioenergetics, metabolism, and signaling, and their dysfunction has been linked to numerous diseases. The available studies cover only part of the mitochondrial proteome, and a separation of core mitochondrial proteins from associated fractions has not been achieved. We developed an integrative experimental approach to define the proteome of east mitochondria. We classified > 3,300 proteins of mitochondria and mitochondria-associated fractions and defined 901 high-confidence mitochondrial proteins, expanding the set of mitochondrial proteins by 82. Our analysis includes protein abundance under fermentable and nonfermentable growth, submitochondrial localization, single-protein experiments, and subcellular classification of mitochondria-associated fractions. We identified mitochondrial interactors of respiratory chain supercomplexes, ATP synthase, AAA proteases, the mitochondrial contact site and cristae organizing system (MICOS), and the coenzyme Q biosynthesis cluster, as well as mitochondrial proteins with dual cellular localization. The integrative proteome provides a high-confidence source for the characterization of physiological and pathophysiological functions of mitochondria and their integration into the cellular environment., Graphical Abstract, Highlights • Classification of > 3,300 proteins of mitochondria and associated fractions • High-confidence mitochondrial proteome with absolute quantification and topology • Interactors of oxidative phosphorylation complexes and cristae organizing system • Identification of system linking respiratory chain and AAA quality control, Morgenstern et al. describe an integrative organelle proteomics analysis to define the mitochondrial proteome in baker’s yeast. The study provides a quantitative footprint of the proteome and its dynamics under different conditions. The results expand the set of proteins assigned to the mitochondria and provide a resource for future mitochondrial research.

Hypomorphic germline variants in TERT, the gene encoding the reverse transcriptase component of the human telomerase complex, occur with a frequency of 3-5% in acute myeloid leukemia. We analyzed the clinical and prognostic impact of the most common TERT A1062T variant in younger patients with acute myeloid leukemia intensively treated within two prospective multicenter trials. Four hundred and twenty patients (age 17-60 years) were analyzed for the TERT A1062T variant by direct sequencing. Fifteen patients (3.6%) carried the TERT A1062T variant. Patients with the TERT A1062T variant had a trend towards less favorable and more intermediate 2/adverse karyotypes/genotypes according to the European Leukemia Net classification. In univariate and multivariate analysis, patients with the TERT A1062T variant had a significantly inferior overall survival compared to wild-type patients (6-year overall survival 20 vs. 41%, p = 0.005). Patients with the TERT A1062T variant showed a high rate of treatment-related mortality: 5/15 (33%) died during induction therapy or in complete remission as compared to 62/405 (15%) of the wild-type patients. In patients with the TERT variant, 14/15 (93%) suffered from non-hematological/non-infectious grade 3/4 adverse events (mostly hepatic and/or mucosal) as compared to 216/405 (53%) wild-type patients (p = 0.006). In multivariate analysis, the TERT A1062T variant was an independent risk factor predicting for adverse events during induction chemotherapy. In conclusion, the TERT A1062T variant is an independent negative prognostic factor in younger patients with acute myeloid leukemia and seems to predispose those patients to treatment-related toxicity.

Studies within the last decade have localized the functional expression of olfactory receptors (ORs) to cells outside of the olfactory epithelium. In human hepatocarcinoma and prostate cancer cells, the activation of ORs by odors modulates elementary physiological processes and leads to an inhibitory effect on proliferation. Cells of the respiratory tract are in direct contact with the surrounding air, in which a myriad of volatile molecules, especially odors, are present. Non-small-cell lung cancer (NSCLC) has a high prevalence, a high mortality rate and is difficult to treat. NSCLC cells are nearly resistant to common chemotherapeutic approaches, and surgical resection provides the only possible chance of a cure for most patients. New approaches for the treatment of NSCLC are the focus of many current studies. Thus, it is of interest to characterize the functional expression of ORs in cancer cells of the lung and to investigate the impact of ORs on pathophysiological processes. In the present study, we demonstrate that the expression of OR2J3 and cytosolic Ca2+ increase via the activation of the agonist helional in the NSCLC cell line A549. We further investigated the underlying pathway. Helional triggers phoshoinositol-3 kinase (PI3K), signaling the release of intracellular Ca2+ and phosphorylation of ERK. We observed that OR2J3 activation induces apoptosis and inhibits cell proliferation and migration in long-term stimulus experiments with helional. Our study provides the first evidence of the functional expression of an OR in NSCLC cells and its putative therapeutic impact.

Lysine‐specific demethylase 1 (LSD1) has evolved as a promising therapeutic target for cancer treatment, especially in acute myeloid leukaemia (AML). To approach the challenge of site‐specific LSD1 inhibition, we developed an enzyme‐prodrug system with the bacterial nitroreductase NfsB (NTR) that was expressed in the virally transfected AML cell line THP1‐NTR+. The cellular activity of the NTR was proven with a new luminescent NTR probe. We synthesised a diverse set of nitroaromatic prodrugs that by design do not affect LSD1 and are reduced by the NTR to release an active LSD1 inhibitor. The emerging side products were differentially analysed using negative controls, thereby revealing cytotoxic effects. The 2‐nitroimidazolyl prodrug of a potent LSD1 inhibitor emerged as one of the best prodrug candidates with a pronounced selectivity window between wild‐type and transfected THP1 cells. Our prodrugs are selectively activated and release the LSD1 inhibitor locally, proving their suitability for future targeting approaches., Site‐specific targeting: Nitroaromatic prodrugs were activated by the bacterial nitroreductase NfsB (NTR) to selectively inhibit lysine‐specific demethylase 1 (LSD1) in transfected leukaemic cells. The effects of LSD1 inhibition and cytotoxicity of the side products were distinguished by negative controls. This system may be applied in targeting approaches to reach site‐specific LSD1 inhibition.

For many years, researchers have postulated that helminthic infections may increase susceptibility to HIV, and that immune activation may have contributed to the extensive spread of HIV in sub-Saharan Africa. In the meantime, immunological studies have provided some evidence in support of this hypothesis, while cross-sectional clinical studies were able to further support the assumed association between HIV infection and selected helminthic co-infections. However, as many of the helminthic infections relevant to HIV-infected patients belong to the group of “neglected tropical diseases”, as defined by the World Health Organization, a certain lack of attention has inhibited progress in fully scaling up treatment and prevention efforts. In addition, despite the fact that the challenges of co-infections have preoccupied clinicians for over two decades, relevant research questions remain unanswered. The following review aims to provide a concise overview of associations between HIV and selected helminthic co-infections concerning aspects of HIV acquisition and transmission, clinical and immunological findings in co-infected individuals, as well as treatment and prevention efforts.

The karyotype is a strong independent prognostic factor in myelodysplastic syndromes (MDS). Since the implementation of the new comprehensive cytogenetic scoring system for MDS, chromosome 7 anomalies are no longer generally assigned to poor risk features but are thoroughly separated. However, der(1;7)(q10;p10), hereinafter der(1;7), is merged into the group labeled "any other single" and belongs to the intermediate risk group, just by definition due to lack of adequate clinical data. The aim of our international collaborative was to clarify the "real" prognostic impact of der(1;7) on a homogenous and well-documented data base. We performed detailed analysis of 63 MDS patients with isolated der(1;7) constituting the largest cohort hitherto reported. Furthermore, clinical data are compared with those of patients with isolated del(7q) and isolated monosomy 7. Median overall survival (OS) of patients with der(1;7) is 26 months (hazard ratio (HR) 0.91 for del(7q) vs der(1;7) and 2.53 for monosomy 7 vs der(1;7)). The der(1;7) is associated with profound thrombocytopenia most probably causing the reduced OS which is in striking contrast to the low risk for AML transformation (HR 3.89 for del(7q) vs der(1;7) and 5.88 for monosomy 7 vs der(1;7)). Molecular karyotyping indicates that der(1;7) is generated in a single step during mitosis and that a chromosomal imbalance rather than a single disrupted gene accounts for malignancy. Thus, the current cytogenetic scoring system assigning isolated der(1;7) to the intermediate risk group is now confirmed by a sufficient data set.

Summary Mitochondria are key organelles for cellular energetics, metabolism, signaling, and quality control and have been linked to various diseases. Different views exist on the composition of the human mitochondrial proteome. We classified >8,000 proteins in mitochondrial preparations of human cells and defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP). We identified interactors of translocases, respiratory chain, and ATP synthase assembly factors. The abundance of MitoCoP proteins covers six orders of magnitude and amounts to 7% of the cellular proteome with the chaperones HSP60-HSP10 being the most abundant mitochondrial proteins. MitoCoP dynamics spans three orders of magnitudes, with half-lives from hours to months, and suggests a rapid regulation of biosynthesis and assembly processes. 460 MitoCoP genes are linked to human diseases with a strong prevalence for the central nervous system and metabolism. MitoCoP will provide a high-confidence resource for placing dynamics, functions, and dysfunctions of mitochondria into the cellular context., Graphical abstract, Highlights • Human mitochondrial high-confidence proteome with >1,100 proteins (MitoCoP) • Mitochondria-specific protein copy numbers and half-lives • Interactors of protein translocases and oxidative phosphorylation assembly factors • >40% of mitochondrial proteome linked to human diseases, Mitochondria are crucial for cellular energy metabolism and human health. Morgenstern et al. present a high-confidence protein compendium of human mitochondria including mitochondria-specific protein copy numbers and half-lives. They identify interactors of key mitochondrial protein machineries and link >40% of the mitochondrial proteome to human diseases.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood characterized by mutations activating RAS signaling. Established clinical and genetic markers fail to fully recapitulate the clinical and biological heterogeneity of this disease. Here we report DNA methylome analysis and mutation profiling of 167 JMML samples. We identify three JMML subgroups with unique molecular and clinical characteristics. The high methylation group (HM) is characterized by somatic PTPN11 mutations and poor clinical outcome. The low methylation group is enriched for somatic NRAS and CBL mutations, as well as for Noonan patients, and has a good prognosis. The intermediate methylation group (IM) shows enrichment for monosomy 7 and somatic KRAS mutations. Hypermethylation is associated with repressed chromatin, genes regulated by RAS signaling, frequent co-occurrence of RAS pathway mutations and upregulation of DNMT1 and DNMT3B, suggesting a link between activation of the DNA methylation machinery and mutational patterns in JMML., Juvenile myelomonocytic leukemia (JMML) is an aggressive disease with limited options for treatment. Here, the authors analyse the DNA methylome and mutational profile of JMML to define three subgroups with unique molecular and clinical characteristics.

In recent years, global and regional crises have led to extraordinary worldwide migration, accompanied by an increase in long-distance travel from Western countries. Both are linked to a rising incidence of rare parasitic and infectious diseases in first world countries, including in the biliary tract. Areas covered: A selective literature research in PubMed was performed to review the most important parasitic and infectious biliary diseases, which are caused by a wide variety of pathogens and may be latent over long periods, with chronic courses leading to cholangitis, hepatic failure or development of cholangiocarcinoma. Parasites such as Ascaris, Fasciola and Clonorchis/Opisthorchis are particularly important and may trigger biliary diseases or predisposition for bacterial superinfections. Viral or protozoal cholangitis is mainly a problem of impaired immunity. Expert commentary: Currently, these entities are still rare in migrants and long-distance travelers. However, a significant increase in Western countries has to be expected. Incidences are most likely underestimated because of protracted clinical latency. Diagnosis depends on the relevant pathogens, the host's immune status and the extent or distribution of biliary obstruction. Modern tomographic methods, ERCP and specific microbiological/parasitological/virological tests are of crucial diagnostic importance. Antimicrobial/antiparasitic/antiviral therapy along with ERCP and interventional sonography/radiology provide effective treatment options.

We recently described the development of an inv(16) acute myeloid leukemia (AML) in a CBL mutated clonal hematopoiesis. Here, we further characterized the clonal composition and evolution of the AML based on the genetic information from the bulk specimen and analyses of individual bone marrow cells for mutations in CAND1, PTPRT, and DOCK6. To control for allele dropout, heterozygous polymorphisms located close to the respective mutation loci were assessed in parallel. The clonal composition concluded from exome sequencing suggested a proliferation advantage associated with the acquisition of mutations in CAND1, PTPRT, and DOCK6. Out of 102 single cell sequencing reactions on these mutations and the respective polymorphisms, analyses yielded conclusive results for at least 2 mutation sites in 12 cells. The single cell genotyping not only confirmed the co-occurrence of the PTPRT, CAND1 and DOCK6 mutations in the same AML clone but also revealed a clonal hierarchy, as the PTPRT mutation was likely acquired after the CAND1 and DOCK6 mutations. This insight had not been possible based solely on the exome sequencing data and suggests that the mutation in PTPRT, which encodes a STAT3-inhibiting protein tyrosine phosphatase, contributed to the AML development at a later stage by enhancing proliferation.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.

Rodent models have contributed significantly to the understanding of haematological malignancies. One important model system in this context are patient-derived xenografts (PDX). In the current study, we examined 20 acute leukaemia PDX models for growth behaviour, infiltration in haemopoietic organs and sensitivity towards cytarabine. PDX were injected intratibially (i.t.), intrasplenicaly (i.s.) or subcutaneously (s.c.) into immune compromised mice. For 18/20 models the engraftment capacity was independent of the implantation site. Two models could exclusively be propagated in one or two specific settings. The implantation site did influence tumour growth kinetics as median overall survival differed within one model depending on the injection route. The infiltration pattern was similar in i.t. and i.s. models. In contrast to the s.c. implantation, only one model displayed circulating leukaemic cells outside of the locally growing tumour mass. Cytarabine was active in all four tested models. Nevertheless, the degree of sensitivity was specific for an individual model and implantation site. In summary, all three application routes turned out to be feasible for the propagation of PDX. Nevertheless, the distinct differences between the settings highlight the need for well characterized platforms to ensure the meaningful interpretation of data generated using those powerful tools.

LSD1 has emerged as a promising epigenetic target in the treatment of acute myeloid leukemia (AML). We used two murine AML models based on retroviral overexpression of Hoxa9/Meis1 (H9M) or MN1 to study LSD1 loss of function in AML. The conditional knockout of Lsd1 resulted in differentiation with both granulocytic and monocytic features and increased ATRA sensitivity and extended the survival of mice with H9M-driven AML. The conditional knockout led to an increased expression of multiple genes regulated by the important myeloid transcription factors GFI1 and PU.1. These include the transcription factors GFI1B and IRF8. We also compared the effect of different irreversible and reversible inhibitors of LSD1 in AML and could show that only tranylcypromine derivatives were capable of inducing a differentiation response. We employed a conditional knock-in model of inactive, mutant LSD1 to study the effect of only interfering with LSD1 enzymatic activity. While this was sufficient to initiate differentiation, it did not result in a survival benefit in mice. Hence, we believe that targeting both enzymatic and scaffolding functions of LSD1 is required to efficiently treat AML. This finding as well as the identified biomarkers may be relevant for the treatment of AML patients with LSD1 inhibitors.

Knowledge of the molecular and clonal characteristics in the myelodysplastic syndromes (MDS) and during progression to acute myeloid leukaemia (AML) is essential to understand the disease dynamics and optimize treatment. Sequencing serial bone marrow samples of eight patients, we observed that MDS featured a median of 3 mutations. Mutations in genes involved in RNA-splicing or epigenetic regulation were most frequent, and exclusively present in the major clone. Minor subclones were distinguishable in three patients. As the MDS progressed, a median of one mutation was gained, leading to clonal outgrowth. No AML developed genetically independent of a pre-existing clone. The gained mutation mostly affected genes encoding signalling proteins. Additional acquisition of genomic aberrations frequently occurred. Upon treatment, emergence of new clones could be observed. As confirmed by single-cell sequencing, multiple mutations in identical genes in different clones were present within individual patients. DNA-methylation profiling in patients without identification of novel mutations in AML revealed methylation changes in individual genes. In conclusion, our data complement previous observations on the mutational and clonal characteristics in MDS and at progression. Moreover, DNA-methylation changes may be associated with progression in single patients. Redundancy of mutated genes in different clones suggests fertile grounds promoting clonal selection or acquisition.

Infektionen durch Carbapenem-resistente Enterobakterien (CRE) stellen aufgrund ihrer hohen Morbiditat und Mortalitat weltweit eine wachsende Bedrohung fur Lebertransplantatempfanger (LTR) dar, v. a. bei Nachweis von Klebsiella pneumoniae Carbapenemase (KPC)-bildenden Erregern. Nach der aktuellen Literatur entwickeln 3 bis 13 % der Organtransplantierten in Endemiegebieten Infektionen durch CRE, wobei der primare Infektionsort eng mit dem transplantierten Organ im Zusammenhang steht. Die kumulative 30-Tage-Sterblichkeit von LTR mit Infektionen durch Carbapenem-resistente K. pneumoniae liegt bei 36 %, und die 180-Tage-Mortalitatsrate betragt 58 %. Die hohe Anfalligkeit von LTR fur potenziell todlich verlaufende bakterielle Infektionen mit multiresistenten Erregern (MRE) fuhrt in der Praxis zu einer haufigeren empirischen Anwendung von erweiterten Breitspektrum-Antibiotikatherapien, die wiederum die Selektion extremer Resistenzen fordert. Dennoch besteht eine relevante Gefahr des Scheiterns der herkommlichen empirischen Behandlung, da kulturbasierte Techniken zur mikrobiologischen Identifikation von CRE eine 48- bis 72-stundige Verzogerung von der Abnahme der Blutkulturen bis zur mikrobiologischen Befunderstellung mit Resistenztestung beinhalten, sodass zielgerichtete Therapien nur verzogert begonnen werden konnen. Dieser Teufelskreis ist schwer zu durchbrechen und fuhrt zu einer fortwahrenden Einengung der antibiotischen Behandlungsmoglichkeiten. Da die derzeit verfugbaren therapeutischen Optionen auserst begrenzt sind, kommt der Infektionspravention eine herausragende Bedeutung zu, v. a. in der Fruhphase nach Lebertransplantation mit intensiver Immunsuppression. Die Verbesserung klinischer Behandlungsergebnisse ist eine grose Herausforderung und erfordert einen mehrgliedrigen Ansatz, bei dem stringent praktizierte Hygienemasnahmen, Screening-Tests auf MRE, zeitsparende molekularbiologische Untersuchungstechniken und Antibiotic Stewardship (ABS) kombiniert werden. Dieser Artikel stellt die aktuelle Literatur in Bezug auf die Haufigkeit und das klinische Outcome von CRE-Infektionen bei LTR vor und fasst aktuelle praventive und therapeutische Empfehlungen zusammen, um die Bedrohung durch CRE im Real-Life-Setting der klinischen Transplantationsmedizin zu minimieren.

In myelodysplastic syndromes (MDS), deletion of the short arm of chromosome 12 (del(12p)) is usually a small abnormality, rarely detected as a single aberration by chromosome banding analysis (CBA) of bone marrow metaphases. Del(12p) has been described in 0.6 to 5% of MDS patients at initial diagnosis and is associated with a good to intermediate prognosis as a sole anomaly according to current scoring systems. Here, we present the results of a systematic del(12p) testing in a German prospective diagnostic study (clinicaltrials.gov: NCT01355913) on 367 MDS patients in whom CD34+ peripheral blood cells were analysed for the presence of del(12p) by sequential fluorescence in situ hybridization (FISH) analyses. A cohort of 2,902 previously published MDS patients diagnosed by CBA served as control. We demonstrate that, using a sensitive FISH technique, 12p deletion occurs significantly more frequently in MDS than previously described (7.6% by CD34+ PB-FISH vs. 1.6% by CBA, P < 0.001) and is often associated with other aberrations (93% by CD34+ PB-FISH vs. 60% by CBA). Additionally, the detection rate can be increased by repeated analyses in a patient over time which is important for the patient´s prognosis to distinguish a sole anomaly from double or complex aberrations. To our knowledge, this is the first study to screen for 12p deletions with a suitable probe for ETV6/TEL in 12p13. Our data suggest that the supplement of a probe for the detection of a 12p deletion to common FISH probe panels helps to avoid missing a del(12p), especially as part of more complex aberrations.

Therapy resistance is a major clinical problem in cancer medicine and crucial for disease relapse and progression. Therefore, the clinical need to overcome it, particularly for aggressive tumors such as pancreatic cancer, is very high. Aberrant activation of an epithelial–mesenchymal transition (EMT) and an associated cancer stem cell phenotype are considered a major cause of therapy resistance. Particularly, the EMT-activator ZEB1 was shown to confer stemness and resistance. We applied a systematic, stepwise strategy to interfere with ZEB1 function, aiming to overcome drug resistance. This led to the identification of both its target gene miR-203 as a major drug sensitizer and subsequently the class I HDAC inhibitor mocetinostat as epigenetic drug to interfere with ZEB1 function, restore miR-203 expression, repress stemness properties, and induce sensitivity against chemotherapy. Thereby, mocetinostat turned out to be more effective than other HDAC inhibitors, such as SAHA, indicating the relevance of the screening strategy. Our data encourage the application of mechanism-based combinations of selected epigenetic drugs with standard chemotherapy for the rational treatment of aggressive solid tumors, such as pancreatic cancer.

We recently described mitochondrial pathology in neurons of transgenic mice with genes associated with Parkinson's disease (PD). Now we describe severe mitochondrial damage in glial cells of the mesencephalon in mice carrying a targeted deletion of parkin (PaKO) or overexpressing doubly mutated human alpha-synuclein (asyn). The number of mitochondria with altered morphology in glial cells is cell type-dependent, but always higher than in neurons. Interestingly, mitochondrial damage also occurs in mesencephalic glia of mice carrying mutated asyn controlled by the tyrosine hydroxylase promoter. Such mice do not show glial expression of the transgene, but show expression in neighboring neurons. However, we found strong overexpression of endogenous asyn in mesencephalic astrocytes from these mice. Cortical astrocytes neither display enhanced asyn expression nor mitochondrial damage. Cultivated mesencephalic astrocytes from newborn transgenic mice display various functional defects along with the morphological damage of mitochondria. First, the mitochondrial Ca(2+)-storage capacity is reduced in asyn transgenic mesencephalic astrocytes, but not in astrocytes from PaKO. Second, the expression of the mitochondrial protein PTEN-induced putative kinase is constitutively increased in asyn transgenic mice, while in PaKO it reacts to oxidative stress by overexpressing this protein along with other mitochondria-related proteins. Third, the neurotrophic effects exerted by control astrocytes, stimulating cortical neurons from healthy mice to develop longer processes and larger neuronal areas, are lacking in co-cultures with transgenic mesencephalic astrocytes. In summary, glial mitochondria from transgenic mice display morphological and functional alterations. Such transgenic astrocytes fail to influence neuronal differentiation, reflecting an important role that glia may play in PD pathogenesis.

In central nervous system (CNS) synapses, action potential-evoked neurotransmitter release is principally mediated by CaV2.1 calcium channels (CaV2.1) and is highly dependent on the physical distance between CaV2.1 and synaptic vesicles (coupling). Although various active zone proteins are proposed to control coupling and abundance of CaV2.1 through direct interactions with the CaV2.1 α1 subunit C-terminus at the active zone, the role of these interaction partners is controversial. To define the intrinsic motifs that regulate coupling, we expressed mutant CaV2.1 α1 subunits on a CaV2.1 null background at the calyx of Held presynaptic terminal. Our results identified a region that directly controlled fast synaptic vesicle release and vesicle docking at the active zone independent of CaV2.1 abundance. In addition, proposed individual direct interactions with active zone proteins are insufficient for CaV2.1 abundance and coupling. Therefore, our work advances our molecular understanding of CaV2.1 regulation of neurotransmitter release in mammalian CNS synapses. DOI: http://dx.doi.org/10.7554/eLife.28412.001, eLife digest The points of contact between nerve cells are called synapses, and nerve cells communicate across synapses via chemicals known as neurotransmitters. These chemical messengers are initially stored within bubble-like packages called synaptic vesicles that are released after they fuse with the membrane of the nerve cell at a specialized site referred to as the “active zone”. Calcium ions are one of the major factors that lead to the release of synaptic vesicles. Ion channel proteins in the membrane of the nerve cell control the flow of calcium ions into the cell. There are often many different ion channels at a synapse, but one type called CaV2.1 most effectively triggers the release of synaptic vesicles when a nerve impulse reaches the synapse. Various proteins at the active zone can bind directly to parts of the CaV2.1 channel that are identified by a short sequence of amino acids – the building blocks of all proteins. Several researchers have proposed that the interactions with some of these short sequences, which are also known as motifs, control how much of this ion channel is in the synapse and how it interacts with synaptic vesicles to regulate the release of neurotransmitters. However, other researchers do not agree with this proposed explanation. Lübbert, Goral et al. set out to determine which parts in a specific part of the CaV2.1 channel (called the “α1 subunit C-terminus”) are critical for its interaction with synaptic vesicles. The experiments revealed a new motif that regulates how many synaptic vesicles could be released in response to electrical impulses travelling along nerve cells from mice. The same motif also regulates the total number of synaptic vesicles at the active zone. Lübbert, Goral et al. went on to show that binding to known active proteins at most played a minor role in controlling the abundance of the CaV2.1 channels and how close they were to the synaptic vesicles. As such, these findings counter prevailing views of the roles of certain motifs in the α1 subunit of the CaV2.1 channel. Thus, it may be necessary to re-think how the CaV2.1 channel regulates the release of synaptic vesicles. Ion channels are vital to the activity of all nerve cells, and working out how the numbers and organization of CaV2.1 and related ion channels are regulated will be fundamental to understanding how information is encoded in brain. In addition, problems with these kinds of ion channel may result in disorders such as migraines and epilepsy. Therefore, the new findings may help to guide further studies investigating possible ways to treat these disorders. DOI: http://dx.doi.org/10.7554/eLife.28412.002

DNA methylation differences between normal tissue and cancerous tissue resulting in differential expression of genes are a hallmark of acute myeloid leukemia (AML) and can provide malignant cells with a growth advantage via silencing of specific genes, for example, transcription factors. Oligodendrocyte lineage transcription factor 2 (OLIG2) was reported to be differentially methylated and associated with prognosis in AML and, as reported for acute lymphoblastic leukemia and malignant glioma, may play a role in malignant transformation. We report that DNA methylation of OLIG2 is associated with decreased expression of mRNA in AML cell lines and patients. Moreover, in cell lines, decreased mRNA expression also translated into decreased OLIG2 protein expression. Treatment of non-expressing cell lines PL-21 and U-937 with the demethylating agent decitabine resulted in robust re-expression of OLIG2 on mRNA and protein levels. Furthermore, stable overexpression of OLIG2 in non-expressing cell lines Kasumi-1 and U-937, using a lentiviral vector system, led to moderate growth inhibition after 4 days and resulted in signs of differentiation in U-937 cells. Interestingly, although CD34 + cells from healthy donors and 10 of 12 AML patients exhibited no protein expression, OLIG2 was expressed in two patients, both bearing the translocation t(15;17), corresponding to OLIG2 expression in NB-4 cells, also harboring t(15;17). In conclusion, we provide first evidence that OLIG2 is epigenetically regulated via DNA methylation and expressed in a subset of AML patients. OLIG2 may exert antiproliferative activity in leukemia cell lines, and its potential leukemia-suppressing role in AML warrants further investigation.

Several mechanisms of action have been proposed for DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi), primarily based on candidate-gene approaches. However, less is known about their genome-wide transcriptional and epigenomic consequences. By mapping global transcription start site (TSS) and chromatin dynamics, we observed the cryptic transcription of thousands of treatment-induced non-annotated TSSs (TINATs) following DNMTi and HDACi treatment. The resulting transcripts frequently splice into protein-coding exons and encode truncated or chimeric ORFs translated into products with predicted abnormal or immunogenic functions. TINAT transcription after DNMTi treatment coincided with DNA hypomethylation and gain of classical promoter histone marks, while HDACi specifically induced a subset of TINATs in association with H2AK9ac, H3K14ac, and H3K23ac. Despite this mechanistic difference, both inhibitors convergently induced transcription from identical sites, as we found TINATs to be encoded in solitary long terminal repeats of the ERV9/LTR12 family, which are epigenetically repressed in virtually all normal cells.

Background Cytogenetic aberrations such as deletion of chromosome 5q (del(5q)) represent key elements in routine clinical diagnostics of haematological malignancies. Currently established methods such as metaphase cytogenetics, FISH or array-based approaches have limitations due to their dependency on viable cells, high costs or semi-quantitative nature. Importantly, they cannot be used on low abundance DNA. We therefore aimed to establish a robust and quantitative technique that overcomes these shortcomings. Methods For precise determination of del(5q) cell fractions, we developed an inexpensive multiplex-PCR assay requiring only nanograms of DNA that simultaneously measures allelic imbalances of 12 independent short tandem repeat markers. Results Application of this method to n=1142 samples from n=260 individuals revealed strong intermarker concordance (R²=0.77–0.97) and reproducibility (mean SD: 1.7%). Notably, the assay showed accurate quantification via standard curve assessment (R²>0.99) and high concordance with paired FISH measurements (R²=0.92) even with subnanogram amounts of DNA. Moreover, cytogenetic response was reliably confirmed in del(5q) patients with myelodysplastic syndromes treated with lenalidomide. While the assay demonstrated good diagnostic accuracy in receiver operating characteristic analysis (area under the curve: 0.97), we further observed robust correlation between bone marrow and peripheral blood samples (R²=0.79), suggesting its potential suitability for less-invasive clonal monitoring. Conclusions In conclusion, we present an adaptable tool for quantification of chromosomal aberrations, particularly in problematic samples, which should be easily applicable to further tumour entities.

Background From July 2010-April 2013, Leipzig University Hospital experienced the largest outbreak of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae (KPC-2- Kp ) strain observed in Germany to date. After termination of the outbreak, we aimed to reconstruct transmission pathways by phylogenetics based on whole-genome sequencing (WGS). Methods One hundred seventeen KPC-2- Kp isolates from 89 outbreak patients, 5 environmental KPC-2- Kp isolates, and 24 K pneumoniae strains not linked to the outbreak underwent WGS. Phylogenetic analysis was performed blinded to clinical data and based on the genomic reads. Results A patient from Greece was confirmed as the source of the outbreak. Transmission pathways for 11 out of 89 patients (12.4%) were plausibly explained by descriptive epidemiology, applying strict definitions. Five of these and an additional 15 (ie, 20 out of 89 patients [22.5%]) were confirmed by phylogenetics. The rate of phylogenetically confirmed transmissions increased significantly from 8 out of 66 (12.1% for the time period before) to 12 out of 23 patients (52.2% for the time period after; P .001) after implementation of systematic screening for KPC-2- Kp (33,623 screening investigations within 11 months). Using descriptive epidemiology, systematic screening showed no significant effect (7 out of 66 [10.6%] vs 4 out of 23 [17.4%] patients; P = .465). The phylogenetic analysis supported the assumption that a contaminated positioning pillow served as a reservoir for the persistence of KPC-2- Kp . Conclusions Effective phylogenetic identification of transmissions requires systematic microbiologic screening. Extensive screening and phylogenetic analysis based on WGS should be started as soon as possible in a bacterial outbreak situation.

High antibiotic and antifungal concentrations in wastewater from anti-infective drug production may exert selection pressure for multidrug-resistant (MDR) pathogens. We investigated the environmental presence of active pharmaceutical ingredients and their association with MDR Gram-negative bacteria in Hyderabad, South India, a major production area for the global bulk drug market. From Nov 19 to 28, 2016, water samples were collected from the direct environment of bulk drug manufacturing facilities, the vicinity of two sewage treatment plants, the Musi River, and habitats in Hyderabad and nearby villages. Samples were analyzed for 25 anti-infective pharmaceuticals with liquid chromatography–tandem mass spectrometry and for MDR Gram-negative bacteria using chromogenic culture media. In addition, specimens were screened with PCR for bla VIM, bla KPC, bla NDM, bla IMP-1, and bla OXA-48 resistance genes. All environmental specimens from 28 different sampling sites were contaminated with antimicrobials. High concentrations of moxifloxacin, voriconazole, and fluconazole (up to 694.1, 2500, and 236,950 µg/L, respectively) as well as increased concentrations of eight other antibiotics were found in sewers in the Patancheru–Bollaram industrial area. Corresponding microbiological analyses revealed an extensive presence of extended-spectrum beta-lactamase and carbapenemase-producing Enterobacteriaceae and non-fermenters (carrying mainly bla OXA-48, bla NDM, and bla KPC) in more than 95% of the samples. Insufficient wastewater management by bulk drug manufacturing facilities leads to unprecedented contamination of water resources with antimicrobial pharmaceuticals, which seems to be associated with the selection and dissemination of carbapenemase-producing pathogens. The development and global spread of antimicrobial resistance present a major challenge for pharmaceutical producers and regulatory agencies.