Learning through trial-and-error interactions allows animals to adapt innate behavioural ‘rules of thumb’ to the local environment, improving their prospects for survival and reproduction. Naive Drosophila melanogaster males, for example, court both virgin and mated females, but learn through experience to selectively suppress futile courtship towards females that have already mated. Here we show that courtship learning reflects an enhanced response to the male pheromone cis-vaccenyl acetate (cVA), which is deposited on females during mating and thus distinguishes mated females from virgins. Dissociation experiments suggest a simple learning rule in which unsuccessful courtship enhances sensitivity to cVA. The learning experience can be mimicked by artificial activation of dopaminergic neurons, and we identify a specific class of dopaminergic neuron that is critical for courtship learning. These neurons provide input to the mushroom body (MB) γ lobe, and the DopR1 dopamine receptor is required in MBγ neurons for both natural and artificial courtship learning. Our work thus reveals critical behavioural, cellular and molecular components of the learning rule by which Drosophila adjusts its innate mating strategy according to experience.

Mutations of spliceosome components are common in myeloid neoplasms. One of the affected genes, PRPF8, encodes the most evolutionarily conserved spliceosomal protein. We identified either recurrent somatic PRPF8 mutations or hemizygous deletions in 15/447 and 24/450 cases, respectively. 50% of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis. PRPF8 defects correlated with increased myeloblasts and ring sideroblasts in cases without SF3B1 mutations. Knockdown of PRPF8 in K562 and CD34+ primary bone marrow cells increased proliferative capacity. Whole RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects. In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process suggesting that the mutants have defects in proof-reading functions. In sum, the exploration of clinical and functional consequences suggests that PRPF8 is a novel leukemogenic gene in myeloid neoplasms with a distinct phenotype likely manifested through aberrant splicing.

Milk remains the most convenient, cheapest, and readily available solution in most situations while also being capable of keeping PDL cells alive. Further studies are required to evaluate the efficacy of more commonly found storage media besides milk.

Acute erythroleukemia (AML-M6 or AEL) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing three genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (e.g. DNMT3A, TET2 or IDH2), and undefined cases with low mutational burden. We established an erythroid vs. myeloid transcriptomics-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, more than 25% of AEL patients, including in the genetically-undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1 binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicates that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

The COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.

Recent application of whole exome next generation sequencing has led to the discovery of a new class of somatic mutations affecting various components of the spliceosomal machinery in myeloid neoplasms. All affected spliceosomal genes, except for LUC7L2 (which might function in the U1snRNP-related pathway) encode U2snRNP associated proteins, but the molecular consequences of individual mutations remain unclear. Based on 3 index cases where somatic PRPF8 mutations (M1307I, A687P and G1750E) were observed, we have screened a much larger cohort of patients with MDS and related conditions for the presence of acquired lesions affecting this gene. Somatic PRPF8 mutations and heterozygous deletions affecting the corresponding locus were found in 9/352 and 24/450 cases, respectively. Del17p13 was associated with haploinsufficient PRPF8 expression, but down-modulation of PRPF8 was also found in about 10% of additional cases with diploid 17p. PRPF8 mutations were mutually exclusive with other spliceosomal mutations, in particular with SF3B1. Serial studies of clonal architecture revealed that somatic PRPF8 mutation is an early event. PRPF8 encodes the largest and evolutionarily most conserved spliceosomal protein. It is essential for pre-mRNA splicing and required in all tissues. Germline mutations in human PRPF8 are associated with an autosomal dominant form of retinitis pigmentosa type 13. In total, around 1/2 of the PRPF8 mutant and del17p deletion cases were found in pAML and sAML. Accordingly, MDS with PRPF8 lesions conveyed poor prognosis as shown by KM statistics. Phenotypically, PRPF8 defects correlated with the presence of ring sideroblasts (83% of mutants and 65% of deletion cases), likely overseen in the context of a high blast count. Additionally, most of the cases also displayed pseudo Pelger-Huet anomaly. In in vitro experiments, decreased expression levels of PRPF8 due to experimental knockdown by lentiviral shRNA were associated with increased cellular proliferation and clonogenicity, suggesting that PRPF8 has a tumor suppressor role in myeloid malignancies. When we studied the consequences of PRPF8 defects in yeast models, mutations in PRPF8 abrogated a block in the second step of splicing introduced by an experimental intron mutation. RNA-sequencing of primary mutant and deletion cases as well as engineered shRNA knockdown cell lines showed that loss of PRPF8 function or mutation resulted in a global missplicing defect. Some of the most misspliced genes included those involved in mitochondrial function such as NDUFAF6, SFXN2, RPS24, and SLC25A19 and, perhaps most significantly, GATA1 which was mispliced in samples with either low or mutant PRPF8 as well as in the knockdown K562 cells. Similarly, analysis in patients with RS and defective PRPF8 showed common expression profiles of genes involved in mitochondrial electron transport chain complexes. These changes may correspond to ring sideroblasts associated with PRPF8 defects while missplicing of RNA helicases (frequently mutated in MDS, see other abstract from this group), may explain the mechanisms of lost tumor suppressor function. In sum, we describe here another important somatic spliceosomal mutation associated with myeloid neoplastic transformation and unique phenotypic features. Disclosures: Maciejewski: NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

During a pandemic, mitigation as well as protection of system-critical or vulnerable institutions requires massive parallel, yet cost effective testing to monitor the spread of agents such as the current SARS-CoV2 virus. Here we present SARSeq, saliva analysis by RNA sequencing, as an approach to monitor presence of SARS-CoV2 and other respiratory viruses performed on tens of thousands of samples in parallel. SARSeq is based on next generation sequencing of multiple amplicons generated in parallel in a multiplexed RT-PCR reaction. It relies on a two-dimensional unique dual indexing strategy using four indices in total for unambiguous and scalable assignment of reads to individual samples. We calibrated this method using dilutions of synthetic RNA and virions to show sensitivity down to few molecules, and applied it to hundreds of patient samples validating robust performance across various sample types. Double blinded benchmarking to gold-standard quantitative RT-PCR performed in a clinical setting and a human diagnostics laboratory showed robust performance up to a Ct of 36. The false positive rate, likely due to cross contamination during sample pipetting, was estimated at 0.04-0.1%. In addition to SARS-CoV2, SARSeq detects Influenza A and B viruses as well as human rhinovirus and can be easily expanded to include detection of other pathogens. In sum, SARSeq is an ideal platform for differential diagnostic of respiratory diseases at a scale, as is required during a pandemic.

Cell-to-cell communication is a fundamental process in every multicellular organism. In addition to membrane-bound and released factors, the sharing of cytosolic components represents a new, poorly explored signaling route. An extraordinary example of this communication channel is the direct transport of mitochondria between cells. In this review, we discuss how intercellular mitochondrial transfer can be used by cancer cells to sustain their high metabolic requirements and promote drug resistance and describe relevant molecular players in the context of current and future cancer therapy.