Photo-isolation chemistry for high-resolution and deep spatial transcriptome with mouse tissue sections

Photo-isolation chemistry (PIC) enables isolation of transcriptome information from locally defined areas by photo-irradiation. Here, we present an optimized PIC protocol for formalin-fixed frozen and paraffin mouse sections and fresh-frozen mouse sections. We describe tissue section preparation and permeabilization, followed by in situ reverse transcription using photo-caged primers. We then detail immunostaining and UV-mediated uncaging to the target areas, followed by linear amplification of uncaged cDNAs, library preparation, and quantification. This protocol can be applied to various animal tissue types

High-depth spatial transcriptome analysis by photo-isolation chemistry

光照射を用いた超高解像度な遺伝子解析技術の開発に成功 --組織内に潜むがん細胞の病理診断などに応用可能--. 京都大学プレスリリース. 2021-07-27.In multicellular organisms, expression profiling in spatially defined regions is crucial to elucidate cell interactions and functions. Here, we establish a transcriptome profiling method coupled with photo-isolation chemistry (PIC) that allows the determination of expression profiles specifically from photo-irradiated regions of interest. PIC uses photo-caged oligodeoxynucleotides for in situ reverse transcription. PIC transcriptome analysis detects genes specifically expressed in small distinct areas of the mouse embryo. Photo-irradiation of single cells demonstrated that approximately 8, 000 genes were detected with 7 × 10⁴ unique read counts. Furthermore, PIC transcriptome analysis is applicable to the subcellular and subnuclear microstructures (stress granules and nuclear speckles, respectively), where hundreds of genes can be detected as being specifically localised. The spatial density of the read counts is higher than 100 per square micrometre. Thus, PIC enables high-depth transcriptome profiles to be determined from limited regions up to subcellular and subnuclear resolutions

Exact results for the thermal and magnetic properties of strong coupling ladder compounds

We investigate the thermal and magnetic properties of the integrable su(4) ladder model by means of the quantum transfer matrix method. The magnetic susceptibility, specific heat, magnetic entropy and high field magnetization are evaluated from the free energy derived via the recently proposed method of high temperature expansion for exactly solved models. We show that the integrable model can be used to describe the physics of the strong coupling ladder compounds. Excellent agreement is seen between the theoretical results and the experimental data for the known ladder compounds (5IAP)$_2$CuBr$_4$$\cdot$2H$_2$O, Cu$_{2}$(C$_5$H$_{12}$N$_2$)$_2$Cl$_4$ etc.Comment: 10 pages, 5 figure

An Ecological Study on Edwardsiella tarda in Flounder Farms

An ecological investigation on Edwardsiella tarda was carried out in two Japanese flounder (Paralichthys olivaceus) farms in Fukuyama City from April to December 1992. E. tarda was isolated from 86% of water, 44% of sediment and 14% of fish samples in one pond, and 22% of water, 0% of sediment and 2% of fish samples in the other pond, although epizootics caused by E. tarda were not observed in either pond. Serological studies proved that all the isolates of E. tarda from flounder and its environment shared a common heat stable O-antigen. Almost all the isolates tested exhibited pathogenicity to flounder by intramuscular injection. In survivability tests of selected isolates in sea water and freshwater, they perished within 7 days in sea water but survived longer period in freshwater. These results suggest that there is only one serotype of E. tarda which has pathogenicity to flounder in the environment of flounder farms

Infection Experiments with Edwardsiella tarda in the Japanese Flounder

Infection experiments in the Japanese flounder (Paralichthys olivaceus) with Edwardsiella tarda were conducted by intramuscular injection, intraperitoneal injection, immersion, and oral administration methods. Mortalities were produced by all the methods tested, LD50 being determined as 7.1 X 10^1 CFU/fish by intramuscular injection, 1.7 X 10^2 CFU/fish by intraperitoneal injection, 3.6 X 10^6 CFU/ml by immersion, and 1.3 X 10^6 CFU/fish by oral administration. These results show that the Japanese flounder has high susceptibility to E. tarda. Mortalities continuously occurred during the observation period of 15 or 20 days showing a chronic nature of the disease

Vaccination Trials in the Japanese Flounder against Edwardsiellosis

ヒラメに対し, Edwardsiella tardaのホルマリン死菌を用いて, 注射免疫, 浸漬免疫, および経口免疫を施し, 注射, 浸漬もしくは経口攻撃を行った。注射免疫および経口免疫区では血清抗体価の上昇とある程度の死亡の遅れが認められたが, いずれの場合も免疫の効果は低かった。浸漬免疫区では抗体価の上昇はなく, 防御能の強化もほとんど認められなかった。E.tardaの菌体外産物および菌体成分にヒラメに対する致死毒性が認められたため, これらの粗毒素を用いて注射免疫を行ったが, ホルマリン死菌と同様防御効果は認められなかった。The Japanese flounder (Paralichthys alivaceus) were vaccinated with formalin-killed cells (FKC) of Edwardsiella tarda by intramuscular injection, immersion and oral administration. Fish were also immunized by injection with diluted extracellular products (ECP) and intracellular components (ICC) of the bacterium, both of which were found lethal to flounder. As a result, mean serum agglutinating antibody titers against FKC rose in all the immunized fish groups except those vaccinated by immersion with FKC. When the immunized and control fish were challenged by injection and immersion with live cells, death was delayed in most of the immunized groups. However, clear protection was not observed in any group immunized either with FKC, ECP or ICC

Effect of Ingestion of Medium-Chain Triglycerides on Substrate Oxidation during Aerobic Exercise Could Depend on Sex Difference in Middle-Aged Sedentary Persons

Fat oxidation (FAO) during aerobic exercise and whole-body FAO via lipid intake are thought to be important for the maintenance of health, such as the prevention of type 2 diabetes and obesity in sedentary persons in their 40s and 50s. Medium-chain triglycerides (MCTs) ingestion has been attracting attention. However, the effects of difference of sex and the composition of medium-chain fatty acids (MCFAs) are unclear, so we examined the effects of these factors on FAO during aerobic exercise. We conducted a randomized, double-blind, placebo-controlled, 3-arm, within-participants crossover trial. FAO during low- to moderate-intensity exercise was compared when octanoate-rich MCTs (C8R), decanoate-rich MCTs (C10R), or carbohydrate (control) was ingested. Three 2-week interventions were separated by two 2-week washout periods. An increase of FAO during exercise after the C8R diet was found in males, but not in females. An increase of carbohydrate oxidation (CAO) and oxygen uptake during exercise after the C10R diet was found in females, but not in males. In a pooled estimate of the effect of MCTs (C8R and C10R) in women and men, FAO increased during exercise. In conclusion, short-term ingestion of MCTs by middle-aged sedentary persons could increase FAO during aerobic exercise compared to carbohydrate ingestion, but the enhancing effect of MCTs on substrate utilization and oxygen uptake might vary, depending on sex and the composition of MCFAs