Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression.

The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3' UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels

Loss of miR-29a/b1 promotes inflammation and fibrosis in acute pancreatitis

MicroRNA-29 (miR-29) is a critical regulator of fibroinflammatory processes in human diseases. In this study, we found a decrease in miR-29a in experimental and human chronic pancreatitis, leading us to investigate the regulatory role of the miR-29a/b1 cluster in acute pancreatitis (AP) utilizing a conditional miR-29a/b1–KO mouse model. miR-29a/b1-sufficient (WT) and -deficient (KO) mice were administered supramaximal caerulein to induce AP and characterized at different time points, utilizing an array of IHC and biochemical analyses for AP parameters. In caerulein-induced WT mice, miR-29a remained dramatically downregulated at injury. Despite high-inflammatory milieu, fibrosis, and parenchymal disarray in the WT mice during early AP, the pancreata fully restored during recovery. miR-29a/b1–KO mice showed significantly greater inflammation, lymphocyte infiltration, macrophage polarization, and ECM deposition, continuing until late recovery with persistent parenchymal disorganization. The increased pancreatic fibrosis was accompanied by enhanced TGFβ1 coupled with persistent αSMA+ PSC activation. Additionally, these mice exhibited higher circulating IL-6 and inflammation in lung parenchyma. Together, this collection of studies indicates that depletion of miR-29a/b1 cluster impacts the fibroinflammatory mechanisms of AP, resulting in (a) aggravated pathogenesis and (b) delayed recovery from the disease, suggesting a protective role of the molecule against AP

Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity.

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for correcting variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations

Direct measurements of meltwater runoff on the Greenland ice sheet surface

Meltwater runoff from the Greenland ice sheet surface influences surface mass balance (SMB), ice dynamics, and global sea level rise, but is estimated with climate models and thus difficult to validate. We present a way to measure ice surface runoff directly, from hourly in situ supraglacial river discharge measurements and simultaneous high-resolution satellite/drone remote sensing of upstream fluvial catchment area. A first 72-h trial for a 63.1-km2 moulin-terminating internally drained catchment (IDC) on Greenland?s midelevation (1,207?1,381 m above sea level) ablation zone is compared with melt and runoff simulations from HIRHAM5, MAR3.6, RACMO2.3, MERRA-2, and SEB climate/SMB models. Current models cannot reproduce peak discharges or timing of runoff entering moulins but are improved using synthetic unit hydrograph (SUH) theory. Retroactive SUH applications to two older field studies reproduce their findings, signifying that remotely sensed IDC area, shape, and supraglacial river length are useful for predicting delays in peak runoff delivery to moulins. Applying SUH to HIRHAM5, MAR3.6, and RACMO2.3 gridded melt products for 799 surrounding IDCs suggests their terminal moulins receive lower peak discharges, less diurnal variability, and asynchronous runoff timing relative to climate/SMB model output alone. Conversely, large IDCs produce high moulin discharges, even at high elevations where melt rates are low. During this particular field experiment, models overestimated runoff by +21 to +58%, linked to overestimated surface ablation and possible meltwater retention in bare, porous, low-density ice. Direct measurements of ice surface runoff will improve climate/SMB models, and incorporating remotely sensed IDCs will aid coupling of SMB with ice dynamics and subglacial systemspublishersversionPeer reviewe

Cell-Type Specific Expression of a Dominant Negative PKA Mutation in Mice

We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any cell-type that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIα regulatory subunit (RIαB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIαB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIαB demonstrated a reduction in PKA activity and inhibition of cAMP-responsive gene expression. Mice expressing RIαB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIαB allele in vivo provides a novel system for the analysis of PKA function in physiology

First Results of an ALMA Band 10 Spectral Line Survey of NGC 6334I: Detections of Glycolaldehyde (HC(O)CH_2OH) and a New Compact Bipolar Outflow in HDO and CS

We present the first results of a pilot program to conduct an Atacama Large Millimeter Array (ALMA) band 10 spectral line survey of the high-mass star-forming region NGC 6334I. The observations were taken in exceptional weather conditions (0.19 mm precipitable water) with typical system temperatures T_(sys) < 950 K at ~890 GHz. A bright, bipolar north–south outflow is seen in HDO and CS emission, driven by the embedded massive protostar MM1B. This has allowed, for the first time, a direct comparison of the thermal water in this outflow to the location of water maser emission from prior 22 GHz Very Large Array observations. The maser locations are shown to correspond to the sites along the outflow cavity walls, where high-velocity gas impacts the surrounding material. We also compare our new observations to prior Herschel Heterodyne Instrument for the Far-infrared (HIFI) spectral line survey data of this field, detecting an order of magnitude more spectral lines (695 versus 65) in the Atacama Large Millimeter/submillimeter Array (ALMA) data. We focus on the strong detections of the complex organic molecule glycolaldehyde (HC(O)CH2OH) in the ALMA data that is not detected in the heavily beam-diluted HIFI spectra. Finally, we stress the need for dedicated THz laboratory spectroscopy to support and exploit future high-frequency molecular line observations with ALMA

Opportunistic Detection of Type 2 Diabetes Using Deep Learning From Frontal Chest Radiographs

Deep learning (DL) models can harness electronic health records (EHRs) to predict diseases and extract radiologic findings for diagnosis. With ambulatory chest radiographs (CXRs) frequently ordered, we investigated detecting type 2 diabetes (T2D) by combining radiographic and EHR data using a DL model. Our model, developed from 271,065 CXRs and 160,244 patients, was tested on a prospective dataset of 9,943 CXRs. Here we show the model effectively detected T2D with a ROC AUC of 0.84 and a 16% prevalence. The algorithm flagged 1,381 cases (14%) as suspicious for T2D. External validation at a distinct institution yielded a ROC AUC of 0.77, with 5% of patients subsequently diagnosed with T2D. Explainable AI techniques revealed correlations between specific adiposity measures and high predictivity, suggesting CXRs\u27 potential for enhanced T2D screening

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists