Programmable in situ amplification for multiplexed imaging of mRNA expression

In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR). With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

A second update on mapping the human genetic architecture of COVID-19

Matters Arising From: COVID-19 Host Genetics Initiative. Nature https://doi.org/10.1038/s41586-021-03767-x (2021)Data availability: Summary statistics generated by the COVID-19 HGI are available online, including per-ancestry summary statistics for African, admixed American, East Asian, European and South Asian ancestries (https://www.covid19hg.org/results/r7/). The analyses described here used the data release 7. If available, individual-level data can be requested directly from contributing studies, listed in Supplementary Table 1. We used publicly available data from GTEx (https://gtexportal.org/home/), the Neale laboratory (http://www.nealelab.is/uk-biobank/), the Finucane laboratory (https://www.finucanelab.org), the FinnGen Freeze 4 cohort (https://www.finngen.fi/en/access_results) and the eQTL catalogue release 3 (http://www.ebi.ac.uk/eqtl/).Code availability: The code for summary statistics lift-over, the projection PCA pipeline including precomputed loadings and meta-analyses (https://github.com/covid19-hg/); for heritability estimation (https://github.com/AndrewsLabUCSF/COVID19_heritability); for Mendelian randomization and genetic correlation (https://github.com/marcoralab/MRcovid); and subtype analyses (https://github.com/mjpirinen/covid19-hgi_subtypes) are available at GitHub.Reporting summary: Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article online at: https://www.nature.com/articles/s41586-023-06355-3#MOESM2 .Supplementary information is available online at: https://www.nature.com/articles/s41586-023-06355-3#Sec4 .Copyright © The Author(s) 2023. Investigating the role of host genetic factors in COVID-19 severity and susceptibility can inform our understanding of the underlying biological mechanisms that influence adverse outcomes and drug development1,2. Here we present a second updated genome-wide association study (GWAS) on COVID-19 severity and infection susceptibility to SARS-CoV-2 from the COVID-19 Host Genetic Initiative (data release 7). We performed a meta-analysis of up to 219,692 cases and over 3 million controls, identifying 51 distinct genome-wide significant loci—adding 28 loci from the previous data release2. The increased number of candidate genes at the identified loci helped to map three major biological pathways that are involved in susceptibility and severity: viral entry, airway defence in mucus and type I interferon

A novel Alzheimer disease locus located near the gene encoding tau protein

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this recordAPOE ε4, the most significant genetic risk factor for Alzheimer disease (AD), may mask effects of other loci. We re-analyzed genome-wide association study (GWAS) data from the International Genomics of Alzheimer's Project (IGAP) Consortium in APOE ε4+ (10 352 cases and 9207 controls) and APOE ε4- (7184 cases and 26 968 controls) subgroups as well as in the total sample testing for interaction between a single-nucleotide polymorphism (SNP) and APOE ε4 status. Suggestive associations (P<1 × 10-4) in stage 1 were evaluated in an independent sample (stage 2) containing 4203 subjects (APOE ε4+: 1250 cases and 536 controls; APOE ε4-: 718 cases and 1699 controls). Among APOE ε4- subjects, novel genome-wide significant (GWS) association was observed with 17 SNPs (all between KANSL1 and LRRC37A on chromosome 17 near MAPT) in a meta-analysis of the stage 1 and stage 2 data sets (best SNP, rs2732703, P=5·8 × 10-9). Conditional analysis revealed that rs2732703 accounted for association signals in the entire 100-kilobase region that includes MAPT. Except for previously identified AD loci showing stronger association in APOE ε4+ subjects (CR1 and CLU) or APOE ε4- subjects (MS4A6A/MS4A4A/MS4A6E), no other SNPs were significantly associated with AD in a specific APOE genotype subgroup. In addition, the finding in the stage 1 sample that AD risk is significantly influenced by the interaction of APOE with rs1595014 in TMEM106B (P=1·6 × 10-7) is noteworthy, because TMEM106B variants have previously been associated with risk of frontotemporal dementia. Expression quantitative trait locus analysis revealed that rs113986870, one of the GWS SNPs near rs2732703, is significantly associated with four KANSL1 probes that target transcription of the first translated exon and an untranslated exon in hippocampus (P≤1.3 × 10-8), frontal cortex (P≤1.3 × 10-9) and temporal cortex (P≤1.2 × 10-11). Rs113986870 is also strongly associated with a MAPT probe that targets transcription of alternatively spliced exon 3 in frontal cortex (P=9.2 × 10-6) and temporal cortex (P=2.6 × 10-6). Our APOE-stratified GWAS is the first to show GWS association for AD with SNPs in the chromosome 17q21.31 region. Replication of this finding in independent samples is needed to verify that SNPs in this region have significantly stronger effects on AD risk in persons lacking APOE ε4 compared with persons carrying this allele, and if this is found to hold, further examination of this region and studies aimed at deciphering the mechanism(s) are warranted

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Determination of the Gelation Mechanism of Freeze–Thawed Hen Egg Yolk

A study of yolks stored up to 168 d at −20 °C was conducted to determine the gelation behavior and mechanism of freeze–thawed yolk. Methods used were rheology, native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (native- and SDS-PAGE), differential scanning calorimetry (DSC), transmission electron microscopy (TEM), particle size analysis, and proton nuclear magnetic resonance (¹H NMR) spectroscopy for matrix mobility. Results indicate that both constituents of plasma and granules contributed to gelation of yolk under freezing. PAGE analyses suggest that granular proteins participated in aggregation during freeze–thaw. Increasing gel strength and particle size and decreasing water and lipid–water mobility indicate that lipoproteins or apolipoproteins aggregated. At storage times ≥84 d, increased protein and lipid mobility, the detection of smaller particles, and secondarily increased gel strength suggest the liberation of protein or lipoprotein components from previously formed aggregates and further aggregation of these constituents. Disruption of the gelled yolk matrix observed with TEM supported that ice crystal formation was required for gelation to occur. A two-stage dynamic gelation model is thus proposed