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Immune responses triggered by pathogen-associated molecular patterns (PAMPs) are key to pathogen defense, but drivers and stabilizers of the growth-to-defense genetic reprogramming remain incompletely understood in plants. Here, we report a time-course study of the establishment of PAMP-triggered immunity (PTI) using cap analysis of gene expression. We show that around 15% of all transcription start sites (TSSs) rapidly induced during PTI define alternative transcription initiation events. From these, we identify clear examples of regulatory TSS change via alternative inclusion of target peptides or domains in encoded proteins, or of upstream open reading frames in mRNA leader sequences. We also find that 60% of PAMP response genes respond earlier than previously thought. In particular, a cluster of rapidly and transiently PAMP-induced genes is enriched in transcription factors (TFs) whose functions, previously associated with biological processes as diverse as abiotic stress adaptation and stem cell activity, appear to converge on growth restriction. Furthermore, examples of known potentiators of PTI, in one case under direct mitogen-activated protein kinase control, support the notion that the rapidly induced TFs could constitute direct links to PTI signaling pathways and drive gene expression changes underlying establishment of the immune state.

Amplification of short interfering RNA (siRNAs) via RNA-dependent RNA polymerases (RdRPs) is of fundamental importance in RNA silencing. Plant microRNA (miRNA) action generally does not involve engagement of RdRPs, in part thanks to a poorly understood activity of the cytoplasmic exosome adaptor SKI2. Here, we show that inactivation of the exosome subunit RRP45B and SKI2 results in similar patterns of miRNA-induced siRNA production. Furthermore, loss of the nuclear exosome adaptor HEN2 leads to secondary siRNA production from miRNA targets largely distinct from those producing siRNAs in ski2. Importantly, mutation of the Release Factor paralogue PELOTA1 required for subunit dissociation of stalled ribosomes causes siRNA production from miRNA targets overlapping with, but distinct from, those affected in ski2 and rrp45b mutants. We also show that in exosome mutants, miRNA targets can be sorted into producers and non-producers of illicit secondary siRNAs based on trigger miRNA levels and miRNA:target affinity rather than on presence of 5′-cleavage fragments. We propose that stalled RNA-Induced Silencing Complex (RISC) and ribosomes, but not mRNA cleavage fragments released from RISC, trigger siRNA production, and that the exosome limits siRNA amplification by reducing RISC dwell time on miRNA target mRNAs while PELOTA1 does so by reducing ribosome stalling.

Background: Given the limitations in access and license status of commercially developed automated insulin delivery (AID) systems, open-source AID systems are becoming increasingly popular amongst people with diabetes, including children and adolescents. This study focused on lived experiences, physical and emotional health implications of children and their caregivers following the initiation of open-source AID, their perceived challenges, and sources of support, which have not been explored by the existing literature. Methods: Qualitative data were collected through two sets of open-ended questions of a web-based multinational study survey from 60 caregivers of children with diabetes from 16 countries. The narratives were thematically analysed and a coding framework was identified through an iterative process after the alignment discussions among three coders. Results: A range of emotions, improvements of quality of life and physical health were reported as open-source AID enabled the families to shift their focus away from diabetes therapy. Caregivers were less worried about hypoglycaemia at night-time and outside of their family home, leading to increased autonomy for the child. Simultaneously, glycaemic outcomes and sleep quality of both child and caregiver improved. Nonetheless, the acquisition of suitable hardware and technical set-up could be challenging. The #WeAreNotWaiting community was the primary source of practical but also emotional support. Conclusion: Our findings show the benefits and transformative impact open-source AID and peer-support have on children with diabetes, their caregivers, and families, where commercial AID systems are not available or suitable. Further efforts are required to improve effectiveness, usability, and facilitate access for children with diabetes worldwide to benefit from this innovative treatment. Funding: European Commission (H2020-MSCA-RISE-2018), Berlin Institute of Health Digital Clinician Scientist and SPOKES Wellcome Trust Translational Partnership Program. Declaration of Interest: KB received fees for medical consulting and public speaking from Roche Diabetes Care, Dexcom, Medtronic Diabetes, Diabeloop, Novo Nordisk, Sanofi Diabetes and BCG Digital Ventures; outside the submitted work. KR received fees for medical consulting and public speaking from Dexcom, Abbott, Lilly Diabetes Care, Novo Nordisk (Germany) and Springer Healthcare UK; outside the submitted work. All other authors have nothing to disclose. Ethical Approval: Ethical approval was provided by Charite – Universitatsmedizin Berlin (EA2/140/18).

BACKGROUND Given the limitations in the access and license status of commercially developed automated insulin delivery (AID) systems, open-source AID systems are becoming increasingly popular among people with diabetes, including children and adolescents. OBJECTIVE This study aimed to investigate the lived experiences and physical and emotional health implications of children and their caregivers following the initiation of open-source AID, their perceived challenges, and sources of support, which have not been explored in the existing literature. METHODS Data were collected through 2 sets of open-ended questions from a web-based multinational survey of 60 families from 16 countries. The narratives were thematically analyzed, and a coding framework was identified through iterative alignment. RESULTS A range of emotions and improvements in quality of life and physical health were reported, as open-source AID enabled families to shift their focus away from diabetes therapy. Caregivers were less worried about hypoglycemia at night and outside their family homes, leading to increased autonomy for the child. Simultaneously, the glycemic outcomes and sleep quality of both the children and caregivers improved. Nonetheless, the acquisition of suitable hardware and technical setup could be challenging. The #WeAreNotWaiting community was the primary source of practical and emotional support. CONCLUSIONS Our findings show the benefits and transformative impact of open-source AID and peer support on children with diabetes and their caregivers and families, where commercial AID systems are not available or suitable. Further efforts are required to improve the effectiveness and usability and facilitate access for children with diabetes, worldwide, to benefit from this innovative treatment. INTERNATIONAL REGISTERED REPORT RR2-10.2196/15368

Background 5′-end sequencing assays, and Cap Analysis of Gene Expression (CAGE) in particular, have been instrumental in studying transcriptional regulation. 5′-end methods provide genome-wide maps of transcription start sites (TSSs) with base pair resolution. Because active enhancers often feature bidirectional TSSs, such data can also be used to predict enhancer candidates. The current availability of mature and comprehensive computational tools for the analysis of 5′-end data is limited, preventing efficient analysis of new and existing 5′-end data. Results We present CAGEfightR, a framework for analysis of CAGE and other 5′-end data implemented as an R/Bioconductor-package. CAGEfightR can import data from BigWig files and allows for fast and memory efficient prediction and analysis of TSSs and enhancers. Downstream analyses include quantification, normalization, annotation with transcript and gene models, TSS shape statistics, linking TSSs to enhancers via co-expression, identification of enhancer clusters, and genome-browser style visualization. While built to analyze CAGE data, we demonstrate the utility of CAGEfightR in analyzing nascent RNA 5′-data (PRO-Cap). CAGEfightR is implemented using standard Bioconductor classes, making it easy to learn, use and combine with other Bioconductor packages, for example popular differential expression tools such as limma, DESeq2 and edgeR. Conclusions CAGEfightR provides a single, scalable and easy-to-use framework for comprehensive downstream analysis of 5′-end data. CAGEfightR is designed to be interoperable with other Bioconductor packages, thereby unlocking hundreds of mature transcriptomic analysis tools for 5′-end data. CAGEfightR is freely available via Bioconductor: bioconductor.org/packages/CAGEfightR .

Amplification of short interfering RNA (siRNAs) via RNA dependent RNA Polymerases (RdRPs) is of fundamental importance in RNA silencing. In plants, silencing by microRNAs (miRNAs) generally does not lead to engagement of RdRPs, in part thanks to an as yet poorly understood activity of the cytoplasmic exosome adaptor SKI2. Here, we show that mutation of the cytoplasmic exosome subunit RRP45B results in siRNA production very similar to what is observed in ski2 mutants. Furthermore, loss of the nuclear exosome adaptor HEN2 leads to secondary siRNA production from miRNA targets largely distinct from those producing siRNAs in ski2. Importantly, mutation of the Release Factor paralogue PELOTA1 required for subunit dissociation of stalled ribosomes causes siRNA production from miRNA targets overlapping with, but distinct from, those affected in ski2 and rrp45b mutants. We also show that miRNA-induced illicit secondary siRNA production correlates with miRNA levels rather than accumulation of stable 5’-cleavage fragments. We propose that stalled RNA-induced Silencing Complex (RISC) and ribosomes, but not stable target mRNA cleavage fragments released from RISC, trigger secondary siRNA production, and that the exosome limits siRNA amplification by reducing RISC dwell time on miRNA target mRNAs while PELOTA1 does so by reducing ribosome stalling.

Immune responses triggered by pathogen-associated molecular patterns (PAMPs) are key to pathogen defense, but drivers of the genetic reprogramming required to reach the immune state remain incompletely understood in plants. Here, we report a time-course study of the establishment of PAMP-triggered immunity (PTI) using cap analysis of gene expression (CAGE). Our results show that as much as 15% of all PAMP response genes display alternative transcription initiation. In several cases, use of alternative TSSs may be regulatory as it determines inclusion of target peptides or protein domains, or occurrence of upstream open reading frames (uORFs) in mRNA leader sequences. We also find that 60% of PAMP-response genes respond much earlier than previously thought. In particular, a previously unnoticed cluster of rapidly and transiently PAMP-induced genes is enriched in transcription factors whose functions, previously associated with biological processes as diverse as abiotic stress adaptation and stem cell activity, appear to converge on growth restriction. Furthermore, some examples of known potentiators of PTI, in one case under direct MAP kinase control, support the notion that the rapidly induced transcription factors could constitute direct links to PTI signaling pathways and drive gene expression changes underlying establishment of the immune state.

BACKGROUND Automated insulin delivery (AID) systems have been shown to be safe and effective in reducing hyperglycemia and hypoglycemia but are not universally available, accessible, or affordable. Therefore, user-driven open-source AID systems are becoming increasingly popular. OBJECTIVE This study aims to investigate the motivations for which people with diabetes (types 1, 2, and other) or their caregivers decide to build and use a personalized open-source AID. METHODS A cross-sectional web-based survey was conducted to assess personal motivations and associated self-reported clinical outcomes. RESULTS Of 897 participants from 35 countries, 80.5% (722) were adults with diabetes and 19.5% (175) were caregivers of children with diabetes. Primary motivations to commence open-source AID included improving glycemic outcomes (476/509 adults, 93.5%, and 95/100 caregivers, 95%), reducing acute (443/508 adults, 87.2%, and 96/100 caregivers, 96%) and long-term (421/505 adults, 83.3%, and 91/100 caregivers, 91%) complication risk, interacting less frequently with diabetes technology (413/509 adults, 81.1%; 86/100 caregivers, 86%), improving their or child’s sleep quality (364/508 adults, 71.6%, and 80/100 caregivers, 80%), increasing their or child’s life expectancy (381/507 adults, 75.1%, and 84/100 caregivers, 84%), lack of commercially available AID systems (359/507 adults, 70.8%, and 79/99 caregivers, 80%), and unachieved therapy goals with available therapy options (348/509 adults, 68.4%, and 69/100 caregivers, 69%). Improving their own sleep quality was an almost universal motivator for caregivers (94/100, 94%). Significant improvements, independent of age and gender, were observed in self-reported glycated hemoglobin (HbA1c), 7.14% (SD 1.13%; 54.5 mmol/mol, SD 12.4) to 6.24% (SD 0.64%; 44.7 mmol/mol, SD 7.0; PP CONCLUSIONS These results highlight the unmet needs of people with diabetes, provide new insights into the evolving phenomenon of open-source AID technology, and indicate improved clinical outcomes. This study may inform health care professionals and policy makers about the opportunities provided by open-source AID systems. CLINICALTRIAL INTERNATIONAL REGISTERED REPORT RR2-10.2196/15368

BACKGROUND The COVID-19 pandemic poses new challenges to health care providers and the delivery of continuous care. Although many diabetes technologies, such as insulin pumps and continuous glucose monitors, have been established, the data from these devices are rarely assessed. Furthermore, telemedicine has not been sufficiently integrated into clinical workflows. OBJECTIVE We sought to remotely support children with type 1 diabetes and their caregivers, enhance the clinical outcomes and quality of life of children with diabetes, increase multiple stakeholders’ engagement with digital care via a participatory approach, evaluate the feasibility of using an interoperable open-source platform in a university hospital setting, and analyze the success factors and barriers of transitioning from conventional care to digital care. METHODS Service design methods were used to adapt clinical workflows. Remote consultations were performed on a monthly and on-demand basis. Diabetes device data were uploaded from patients’ homes to an open-source platform. Clinical and patient-reported outcomes were assessed before, during, and after the COVID-19 lockdown period in Germany. RESULTS A total of 28 children with type 1 diabetes and their caregivers enrolled in this study and completed 6 months of remote visits. Of these 28 participants, 16 (57%) also opted to attend at least one of their regular visits remotely. After 3 months of remote visits, participants’ time in range (P=.001) and time in hyperglycemia (P=.004) significantly improved, and their time in hypoglycemia did not increase. These improvements were maintained during the COVID-19 lockdown period (ie, between months 3 and 6 of this study). Participants’ psychosocial health improved after 6 months. CONCLUSIONS Remote consultations and commonly shared data access can improve the clinical outcomes and quality of life of children with type 1 diabetes, even during challenging circumstances. A service design approach helped with the delivery of comprehensive and holistic solutions that accounted for the needs of multiple stakeholders. Our findings can inform the future integration of digital tools into clinical care during and beyond the pandemic. CLINICALTRIAL German Clinical Trials Register DRKS00016170; https://tinyurl.com/skz4wdk5

RNA polymerase II (RNAPII) transcription is crucial for gene expression. RNAPII density peaks at gene boundaries, associating these key regions for gene expression control with limited RNAPII movement. The connections between RNAPII transcription speed and gene regulation in multicellular organisms are poorly understood. Here, we directly modulate RNAPII transcription speed by point mutations in the second largest subunit of RNAPII in Arabidopsis thaliana. A RNAPII mutation predicted to decelerate transcription is inviable, while accelerating RNAPII transcription confers phenotypes resembling auto‐immunity. Nascent transcription profiling revealed that RNAPII complexes with accelerated transcription clear stalling sites at both gene ends, resulting in read‐through transcription. The accelerated transcription mutant NRPB2‐Y732F exhibits increased association with 5′ splice site (5′SS) intermediates and enhanced splicing efficiency. Our findings highlight potential advantages of RNAPII stalling through local reduction in transcription speed to optimize gene expression for the development of multicellular organisms., RNAPII mutations that accelerate transcription cause auto‐immunity‐like phenotypes, read‐through transcription at RNAPII stalling sites and enhanced splicing in Arabidopsis, indicating that controlled transcription speed is required for optimal gene expression and plant development.

Background: Automated insulin delivery (AID) systems have been shown to be safe and effective in reducing hyper- and hypoglycaemia, but are not universally available, accessible, and affordable. Therefore, user-driven open-source AID systems are becoming increasingly popular. Methods: This study investigated motivations why people with diabetes or their caregivers decide to build and use a personalised open-source AID. A cross-sectional online survey was conducted to assess personal motivations and associated self-reported clinical outcomes. Findings: Of 897 participants from 35 countries, 80·4% were adults with diabetes and 19·6% were caregivers of children with diabetes. Primary motivations to commence open-source AID included: improving glycaemic outcomes (94% of adults, 95% of caregivers), reducing acute (87%/96%) and long-term (83%/91%) complication risk, interacting less frequently with diabetes technology (81%/76%), improving their/child’s sleep quality (72%/80%), increasing their/child’s life expectancy (72%/85%), lack of commercially available AID systems (71%/70%), and unachieved therapy goals with available therapy options (68%/69%). Improving their own sleep quality was an almost universal motivator for caregivers (94%). Significant improvements, independent of age and gender, were observed in self-reported HbA1C, 7·14±1·13% (54·5±12·4 mmol/mol) to 6·24±0·64% (44·7±7·0 mmol/mol; p

Targeting of ArabidopsisPHABULOSA (PHB) mRNA by miR166 has been implicated in gene body methylation at the PHB locus. We report that the PHB locus produces an array of stable nuclear RNA species that are neither polyadenylated nor capped. Their biogenesis requires neither RNA polymerases IV/V nor miR166-guided cleavage. The PHB RNAs are insensitive to mutation of nuclear RNA decay pathways and are conserved in several Brassicaceae species, suggesting functional relevance. Similar RNA species are also produced by another body-methylated locus encoding the miR414 target eIF2. Our data reveal the existence of a new class of genic nuclear RNA species.

Aims To investigate self-reported out-of-pocket expenses (OoPE) associated with insulin and diabetes supplies for people living with type 1 diabetes (T1D) worldwide. Methods A web-based, cross-sectional survey was conducted from August to December 2020. The analysis included comparisons between responses from countries with no, partial, and full healthcare coverage. Results 1,066 participants from 64 countries took part in the study. ~25% of respondents reported having underused insulin at least once within the last year due to perceived cost. A significant correlation was observed between OoPEs and reported household income for respondents with partial healthcare coverage. 63.2% of participants reported disruption of insulin supplies and 25.3% reported an increase of prices related to the COVID-19 pandemic. Conclusions This study confirms previous reports of ~25% of people in the United States with T1D using less insulin and/or fewer supplies at least once in the last year due to cost, a trend associated with the extent of healthcare coverage. Similar trends were observed in some middle/low income countries. Moreover, patients reported an increase in insulin prices and disruption of supplies during the COVID-19 pandemic. This study highlights the importance of self-reported OoPEs and its association with underuse/rationing of insulin.

In animals, transcription by RNA polymerase II initiates bidirectionally from gene promoters to produce pre-mRNAs on the forward strand and promoter upstream transcripts (PROMPTs) on the reverse strand. PROMPTs are rapidly degraded by the nuclear exosome. Similarly, active enhancer regions in animals initiate transcription of exosome-sensitive enhancer RNAs (eRNAs). Previous studies based on nascent RNA approaches concluded that Arabidopsis thaliana does not produce PROMPTs. Here, we used steady-state RNA sequencing methods in mutants defective in nuclear RNA decay, including by the exosome, to reassess the existence of PROMPTs and eRNAs in A. thaliana. While PROMPTs are overall rare in A. thaliana, about 100 clear cases of exosome-sensitive PROMPTs and 113 loci producing eRNA-like transcripts were identified. In addition, we found ∼200 transcription start sites within 3’-UTR-encoding regions that produce unspliced exosome-sensitive antisense RNAs covering much of the cognate pre-mRNA. A typical representative of this class of RNAs is the previously characterized non-coding RNA controlling the expression of the key seed dormancy regulator, DELAY OF GERMINATION1. Exosome-sensitive antisense RNAs are overrepresented in transcription factor genes, suggesting a potential for widespread control of gene expression. Lastly, we assess the use of alternative promoters in A. thaliana and compare the accuracy of existing TSS annotations.

In animals, RNA polymerase II initiates transcription bidirectionally from gene promoters to produce pre-mRNAs on the forward strand and promoter upstream transcripts (PROMPTs) on the reverse strand. PROMPTs are degraded by the nuclear exosome. Previous studies based on nascent RNA approaches concluded that Arabidopsis (Arabidopsis thaliana) does not produce PROMPTs. Here, we used steady-state RNA sequencing in mutants defective in nuclear RNA decay including the exosome to reassess the existence of Arabidopsis PROMPTs. While they are rare, we identified ∼100 cases of exosome-sensitive PROMPTs in Arabidopsis. Such PROMPTs are sources of small interfering RNAs in exosome-deficient mutants, perhaps explaining why plants have evolved mechanisms to suppress PROMPTs. In addition, we found ∼200 long, unspliced and exosome-sensitive antisense RNAs that arise from transcription start sites within parts of the genome encoding 3'-untranslated regions on the sense strand. The previously characterized noncoding RNA that regulates expression of the key seed dormancy regulator, DELAY OF GERMINATION1, is a typical representative of this class of RNAs. Transcription factor genes are overrepresented among loci with exosome-sensitive antisense RNAs, suggesting a potential for widespread control of gene expression via this class of noncoding RNAs. Lastly, we assess the use of alternative promoters in Arabidopsis and compare the accuracy of existing TSS annotations.

Fission yeast, Schizosaccharomyces pombe, is an attractive model organism for transcriptional and chromatin biology research. Such research is contingent on accurate annotation of transcription start sites (TSSs). However, comprehensive genome-wide maps of TSSs and their usage across commonly applied laboratory conditions and treatments for S. pombe are lacking. To this end, we profiled TSS activity genome-wide in S. pombe cultures exposed to heat shock, nitrogen starvation, hydrogen peroxide and two commonly applied media, YES and EMM2, using Cap Analysis of Gene Expression (CAGE). CAGE-based annotation of TSSs is substantially more accurate than existing PomBase annotation; on average, CAGE TSSs fall 50-75 bp downstream of PomBase TSSs and co-localize with nucleosome boundaries. In contrast to higher eukaryotes, S. pombe does not show sharp and dispersed TSS distributions. Our data recapitulate known S. pombe stress expression response patterns and identify stress- and mediaresponsive alternative TSSs. Notably, alteration of growth medium induces changes of similar magnitude as some stressors. We show a link between nucleosome occupancy and genetic variation, and that the proximal promoter region is genetically diverse between S. pombe strains. Our detailed TSS map constitute a central resource for S. pombe gene regulation research.

We developed the CAGEfightR R/Biconductor-package for analyzing CAGE data. CAGEfightR allows for fast and memory efficient identification of transcription start sites (TSSs) and predicted enhancers. Downstream analysis, including annotation, quantification, visualization and TSS shape statistics are implemented in easy-to-use functions. The package is freely available at https://bioconductor.org/packages/CAGEfightR

The “Advanced Big Data Training School for Life Sciences” took place during September 3-7, 2018, organized by the Data Management Group (DAMA-UPC) at the Technical University of Catalonia (UPC) in Barcelona, Spain. It is the follow-up training school of the first “Big Data Training School for Life Sciences”, held in Uppsala, Sweden, in September 2017, which was defined and structured at the “Think Tank Hackathon”, held in Ljubljana, Slovenia, in February 2018. The aim of this training school was to get participants acquainted with emerging Big Data processing techniques in the field of Computational Biology and Bioinformatics.This article explains in detail the development of the training school, the covered contents and the interaction of the participants within and out of the training event by the student, organizer and lecturer perspective.

This report provides a short summary of the work and achievements of the EMBnet Publicity and Public Relations Project Committee (PP ii) coordination and monthly drafting, release and advertisement of EMBnet.digest ; iii) assistance in the production and release of new EMBnet QuickGuides ; iv) support in the production and advertisement of EMBnet.journal ; v) management of relationships within EMBnet’s communities and related networks/societies; vi) support in the organisation of the 2015 EMBnet AGM and of a series of workshops and tutorial events, mostly relating to EMBnet members’ involvement in affiliated projects.