The ALMA Interferometric Pipeline Heuristics

We describe the calibration and imaging heuristics developed and deployed in the ALMA interferometric data processing pipeline, as of ALMA Cycle 9. The pipeline software framework is written in Python, with each data reduction stage layered on top of tasks and toolkit functions provided by the Common Astronomy Software Applications package. This framework supports a variety of tasks for observatory operations, including science data quality assurance, observing mode commissioning, and user reprocessing. It supports ALMA and VLA interferometric data along with ALMA and NRO45m single dish data, via different stages and heuristics. In addition to producing calibration tables, calibrated measurement sets, and cleaned images, the pipeline creates a WebLog which serves as the primary interface for verifying the data quality assurance by the observatory and for examining the contents of the data by the user. Following the adoption of the pipeline by ALMA Operations in 2014, the heuristics have been refined through annual development cycles, culminating in a new pipeline release aligned with the start of each ALMA Cycle of observations. Initial development focused on basic calibration and flagging heuristics (Cycles 2-3), followed by imaging heuristics (Cycles 4-5), refinement of the flagging and imaging heuristics with parallel processing (Cycles 6-7), addition of the moment difference analysis to improve continuum channel identification (2020 release), addition of a spectral renormalization stage (Cycle 8), and improvement in low SNR calibration heuristics (Cycle 9). In the two most recent Cycles, 97% of ALMA datasets were calibrated and imaged with the pipeline, ensuring long-term automated reproducibility. We conclude with a brief description of plans for future additions, including self-calibration, multi-configuration imaging, and calibration and imaging of full polarization data.Comment: accepted for publication by Publications of the Astronomical Society of the Pacific, 65 pages, 20 figures, 10 tables, 2 appendice

Prime-boost immunization of rabbits with HIV-1 gp120 elicits potent neutralization activity against a primary viral isolate

<div><p>Development of a vaccine for HIV-1 requires a detailed understanding of the neutralizing antibody responses that can be experimentally elicited to difficult-to-neutralize primary isolates. Rabbits were immunized with the gp120 subunit of HIV-1 JR-CSF envelope (Env) using a DNA-prime protein-boost regimen. We analyzed five sera that showed potent autologous neutralizing activity (IC50s at ∼10³ to 10⁴ serum dilution) against pseudoviruses containing Env from the primary isolate JR-CSF but not from the related isolate JR-FL. Pseudoviruses were created by exchanging each variable and constant domain of JR-CSF gp120 with that of JR-FL or with mutations in putative N-glycosylation sites. The sera contained different neutralizing activities dependent on C3 and V5, C3 and V4, or V4 regions located on the glycan-rich outer domain of gp120. All sera showed enhanced neutralizing activity toward an Env variant that lacked a glycosylation site in V4. The JR-CSF gp120 epitopes recognized by the sera are generally distinct from those of several well characterized mAbs (targeting conserved sites on Env) or other type-specific responses (targeting V1, V2, or V3 variable regions). The activity of one serum requires specific glycans that are also important for 2G12 neutralization and this serum blocked the binding of 2G12 to gp120. Our findings show that different fine specificities can achieve potent neutralization of HIV-1, yet this strong activity does not result in improved breadth.</p> </div

Strategies for controlling the innate immune responses to in vitro transcribed messenger RNAs

Synthetic messenger RNA (mRNA) produced via in vitro transcription (IVT mRNA) has emerged as an appealing tool for the transient introduction of genes, particularly for vaccination applications. The interaction that IVT mRNA has with the innate immune system is centrally important to its performance as a vaccine. These innate immune responses can both interfere with the expression of the encoded antigenic protein and direct development of adaptive immunity. The objective of this thesis is to investigate the innate immune responses to IVT mRNA and to identify strategies to modulate these immune responses. We first demonstrated that substitution of the modified bases 5-methylcytosine and pseudouridine in IVT mRNA consistently reduces antiviral cytokine responses but affects transgene expression in a gene-specific manner. To assess the pathogen recognition receptors involved in detection of IVT mRNA, we developed proximity ligation assays, which allowed histological identification of PRR signaling complexes. We used these assays to identify that nanoparticle-mediated delivery modified PRR-activation following intramuscular delivery compared to delivery of the naked IVT mRNA molecule. Lastly, we developed a strategy to program the immunostimulatory properties of IVT mRNA by tethering adjuvants directly to the molecule. We show that upon intramuscular injection, the combination delivery of a TLR7 adjuvant and IVT mRNA lead to heightened local antiviral responses when delivered tethered, rather than as a cocktail. This work provides a foundation for the modulation and systematic study of IVT mRNA’s interaction with the innate immune system. Insights gained from this work may help direct and advance the design of IVT mRNA sequences for vaccination applications.Ph.D

Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed mRNA

International audienceThe characterization of innate immune activation is crucial for vaccine and therapeutic development, including RNA-based vaccines, a promising approach. Current measurement methods quantify type I interferon and inflammatory cytokine production, but they do not allow for the isolation of individual pathways, do not provide kinetic activation or spatial information within tissues, and cannot be translated into clinical studies. Here we demonstrated the use of proximity ligation assays (PLAs) to detect pattern recognition receptor (PRR) activation in cells and in tissue samples. First, we validated PLA's sensitivity and specificity using well-characterized soluble agonists. Next, we characterized PRR activation from in vitro-transcribed (IVT) mRNAs, as well as the effect of sequence and base modifications in vitro. Finally, we established the measurement of PRR activation in tissue sections via PLA upon IVT mRNA intramuscular (i.m.) injection in mice. Overall, our results indicate that PLA is a valuable, versatile , and sensitive tool to monitor PRR activation for vaccine, adjuvant, and therapeutic screening

Additional file 2 of A mixed community of skin microbiome representatives influences cutaneous processes more than individual members

Additional file 1. Microorganism Isolate Information. Table containing information for the bacterial microorganisms used in this study. Table containing the identifying genus, the isolate identifier used by the collecting study, the 16S rRNA sequence, and the least common ancestors of the bacterial isolate determined by classification to the Greengenes, RDP, and SILVA databases [42]

A mixed community of skin microbiome representatives influences cutaneous processes more than individual members

Abstract Background Skin, the largest organ of the human body by weight, hosts a diversity of microorganisms that can influence health. The microbial residents of the skin are now appreciated for their roles in host immune interactions, wound healing, colonization resistance, and various skin disorders. Still, much remains to be discovered in terms of the host pathways influenced by skin microorganisms, as well as the higher-level skin properties impacted through these microbe-host interactions. Towards this direction, recent efforts using mouse models point to pronounced changes in the transcriptional profiles of the skin in response to the presence of a microbial community. However, there is a need to quantify the roles of microorganisms at both the individual and community-level in healthy human skin. In this study, we utilize human skin equivalents to study the effects of individual taxa and a microbial community in a precisely controlled context. Through transcriptomics analysis, we identify key genes and pathways influenced by skin microbes, and we also characterize higher-level impacts on skin processes and properties through histological analyses. Results The presence of a microbiome on a 3D skin tissue model led to significantly altered patterns of gene expression, influencing genes involved in the regulation of apoptosis, proliferation, and the extracellular matrix (among others). Moreover, microbiome treatment influenced the thickness of the epidermal layer, reduced the number of actively proliferating cells, and increased filaggrin expression. Many of these findings were evident upon treatment with the mixed community, but either not detected or less pronounced in treatments by single microorganisms, underscoring the impact that a diverse skin microbiome has on the host. Conclusions This work contributes to the understanding of how microbiome constituents individually and collectively influence human skin processes and properties. The results show that, while it is important to understand the effect of individual microbes on the host, a full community of microbes has unique and pronounced effects on the skin. Thus, in its impacts on the host, the skin microbiome is more than the sum of its parts. Video abstract

Additional file 7 of A mixed community of skin microbiome representatives influences cutaneous processes more than individual members

Additional file 6. Antimicrobial Peptide Gene Expression. This file contains differential gene expression data (Log2 fold change and the adjusted p-value) for genes that encode for antimicrobial proteins and peptides. Citations for the individual genes are also includes. (XLSX 250 kb

Additional file 6 of A mixed community of skin microbiome representatives influences cutaneous processes more than individual members

Additional file 5. Gene Set Overrepresentation Test Data. This file contains input and output information important to the Gene Set Overrepresentation Test. Genes used for the background gene list. The output data from the PANTHER Overrepresentation tests is also shown. (PDF 1358 kb

Additional file 4 of A mixed community of skin microbiome representatives influences cutaneous processes more than individual members

Additional file 3. Quality Assessment of RNA-Sequencing Data. This file shows quality control metrics for the sequences of each individual sample. This includes the number of reads in the sample, the number and percentage of those reads that were mapped to the transcriptome, the number and percentage of total transcripts covered, and the number and percentage of total genes hit. Additionally, we assessed Pearson correlations of each individual sample within a biological treatment group