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

Electrospray Ionization Mass Spectrometry for Mechanism Analysis of Metal Ion Complexation and Photoionization of Malachite Green Derivatives Incorporating a Crown Ether Moiety

Electrospray-ionization mass spectrometry using a photoirradiation interface has been successfully applied to the investigation of metal-ion complexation and photoionization of crowned Malachite Green leuconitrile derivatives in solution. The mass spectrometry clearly showed that the Malachite Green derivatives in their electrically neutral forms can complex metal ions with their crown ether moiety. Ionization of the Malachite Green derivatives to their corresponding cationic forms, accompanied by metal-ion repelling from their crown ether moiety, was detected upon UV-light irradiation. A remarkably different photoionization behavior in various solvents, which is difficult to detect by other analytical techniques, was also found by the mass spectrometry

A Case of Advanced Hepatocellular Carcinoma with Portal Vein Tumor Thrombus Refractory to Epirubicin That Showed Marked Decrease in Tumor Markers after Transcatheter Arterial Infusion with Miriplatin

Miriplatin, a cisplatin derivative with a high affinity for iodized ethyl esters of fatty acids from poppy seed oil, is a novel chemotherapeutic agent designed for use in the transarterial treatment of hepatocellular carcinoma (HCC). Here, we describe transcatheter arterial infusion (TAI) using miriplatin to treat a case of advanced HCC with portal vein tumor thrombus (PVTT) refractory to TAI with epirubicin. A 66-year-old man with advanced hepatitis C virus-related HCC with PVTT in the right lobe of the liver was treated with TAI with epirubicin suspended in iodized oil; however, tumor marker levels (alpha-fetoprotein and des-gamma-carboxy protein) did not decrease. Next, he was treated twice with TAI with miriplatin suspended in iodized oil. The tumor marker levels markedly decreased to a nearly normal range and the size of the main tumor was markedly reduced according to dynamic computed tomography. No serious adverse events occurred during the course of treatment with TAI and miriplatin. Therefore, we suggest that TAI with miriplatin is a safe and effective treatment option for advanced HCCs refractory to TAI with epirubicin

Evaluation of transporter-mediated hepatobiliary transport of newly developed ¹⁸F-labeled pitavastatin derivative, PTV-F1, in rats by PET imaging

Quantitative evaluations of the functions of uptake and efflux transporters directly in vivo is desired to understand an efficient hepatobiliary transport of substrate drugs. Pitavastatin is a substrate of organic anion transporting polypeptides (OATPs) and canalicular efflux transporters; thus, it can be a suitable probe for positron-emission tomography (PET) imaging of hepatic transporter functions. To characterize the performance of [¹⁸F]PTV-F1, an analogue of pitavastatin, we investigated the impact of rifampicin (a typical OATP inhibitor) coadministration or Bcrp (breast cancer resistance protein) knockout on [¹⁸F]PTV-F1 hepatic uptake and efflux in rats by PET imaging. After intravenous administration, [¹⁸F]PTV-F1 selectively accumulated in the liver, and the radioactivity detected in plasma, liver, and bile mainly derived from the parent PTV-F1 during the PET study (∼40 min). Coadministration of rifampicin largely decreased the hepatic uptake of [¹⁸F]PTV-F1 by 73%. Because of its lower clearance in rats, [¹⁸F]PTV-F1 is more sensitive for monitoring changes in hepatic OATP1B function that other previously reported OATP1B PET probes. Rifampicin coadministration also significantly decreased the biliary excretion of radioactivity by 65%. Bcrp knockout did not show a significant impact on its biliary excretion.[¹⁸F]PTV-F1 enables quantitative analysis of the hepatobiliary transport system for organic anions

Studies on metal-ion complex formation of crown ether derivatives incorporating a photoionizable spirobenzopyran moiety by electrospray ionization mass spectrometry

Metal-ion complexation of crown ether derivatives incorporating one and two spirobenzopyran units was investigated by electrospray ionization mass spectrometry.The crowned spirobenzopyran derivatives exhibited very different metal ion-complexing behavior from their corresponding parent crown ether rings,preferring multivalent to monovalent metal ions owing to the additional ionic interaction with the nitrophenolate anion of the merocyanine moiety.The on-line photochemical reaction for mass spectrometry indicated that the metal ion-complexing ability and ion selectivities of the crowned spirobenzopyrans can be switched photochemically,taking advantage of the difference between the spiropyran and merocyanine isomers

Performance measurement of the 8-input SQUIDs for TES frequency domain multiplexing

金沢大学大学院理工研究域数物科学系We report on performance of 8-input superconducting quantum interference devices (SQUIDs) for multiplexing transition-edge sensor signals by using frequency-domain multiplexing. We found the typical critical current and the flux noise to be 17-19 μA and 0.7-1.1 μΦ0/Hz, respectively. We also measured the crosstalk current between the input coils of the SQUIDs, and found that the mutual inductance was consistent with the design value, 800 pH. We confirmed that the cross talk current due to the mutual inductance was reduced by the flux-locked-loop (FLL) feedback, and its reduction rate was consistent with 1/(1+L), where L is the FLL feedback gain. We also show the result of 2-channel DC-driven TES signals readout using the 8-input SQUIDs. © Springer Science+Business Media, LLC 2008

Outburst of LS V+44 17 Observed by MAXI and RXTE, and Discovery of a Dip Structure in the Pulse Profile

We report on the first observation of an X-ray outburst of a Be/X-ray binary pulsar LS V +44 17/RX J0440.9+4431, and the discovery of an absorption dip structure in the pulse profile. An outburst of this source was discovered by MAXI GSC in 2010 April. It was the first detection of the transient activity of LS V +44 17 since the source was identified as a Be/X-ray binary in 1997. From the data of the follow-up RXTE observation near the peak of the outburst, we found a narrow dip structure in its pulse profile which was clearer in the lower energy bands. The pulse-phase-averaged energy spectra in the 3$-$100 keV band can be fitted with a continuum model containing a power-law function with an exponential cutoff and a blackbody component, which are modified at low energy by an absorption component. A weak iron K$\alpha$ emission line is also detected in the spectra. From the pulse-phase-resolved spectroscopy we found that the absorption column density at the dip phase was much higher than those in the other phases. The dip was not seen in the subsequent RXTE observations at lower flux levels. These results suggest that the dip in the pulse profile originates from the eclipse of the radiation from the neutron star by the accretion column.Comment: 18 pages, 7 figures, accepted for publication in PAS