Lung adenocarcinoma in a patient with Li-Fraumeni syndrome bearing a novel germ-line mutation, TP53R333Vfs*12.

Germline mutations of TP53 are responsible for Li-Fraumeni syndrome in its 60-80%. We found a novel germline mutation, TP53: c.997del:p.R333Vfs*12 (NM_000546.6, GRCh, 17:7670713..7670713). The proband is a 40-year-old female, who was suffered from osteosarcoma in her right forearm at her age of 11. She was also suffered from lung adenocarcinoma in her right upper lobe and bone metastasis in her right scapula at her age of 37. She was treated with gefitinib, an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) because of EGFR mutation (L747-S752 del). Her bone metastasis became resistant after 1-year treatment. Bone metastasis had an additional EGFR mutation (T790M). The secondary treatment with osimertinib, an another EGFR-TKI, can successfully control the tumors for over 2 years. This TP53 mutation (R333Vfs*12) was first found in lung adenocarcinomas. The therapeutic effect of osimertinib for this triple mutant lung adenocarcinoma is better than the previous report.

Development of a Series of Practical Fluorescent Chemical Tools To Measure pH Values in Living Samples.

In biological systems, the pH in intracellular organelles or tissues is strictly regulated, and differences of pH are deeply related to key biological events such as protein degradation, intracellular trafficking, renal failure, and cancer. Ratiometric fluorescence imaging is useful for determination of precise pH values, but existing fluorescence probes have substantial limitations, such as inappropriate p Ka for imaging in the physiological pH range, inadequate photobleaching resistance, and insufficiently long excitation and emission wavelengths. Here we report a versatile scaffold for ratiometric fluorescence pH probes, based on asymmetric rhodamine. To demonstrate its usefulness for biological applications, we employed it to develop two probes. (1) SiRpH5 has suitable p Ka and water solubility for imaging in acidic intracellular compartments; by using transferrin tagged with SiRpH5, we achieved time-lapse imaging of pH in endocytic compartments during protein trafficking for the first time. (2) Me-pEPPR is a near-infrared (NIR) probe; by using dextrin tagged with Me-pEPPR, we were able to image extracellular pH of renal tubules and tumors in situ. These chemical tools should be useful for studying the influence of intra- and extracellular pH on biological processes, as well as for in vivo imaging.

Development of azo-based fluorescent probes to detect different levels of hypoxia.

Let it shine: New hypoxia-sensitive fluorescent probes were developed; they consist of a rhodamine moiety with an azo group directly conjugated to the fluorophore. Because of an ultrafast conformational change around the NN bond, the compounds are nonfluorescent under normoxia. However, under hypoxia, the azo group is reduced, and a strongly fluorescent rhodamine derivative is released.

Hyperthyroidism With Non-chylous Ascites: A Case Report.

Ascites is the accumulation of fluid in the abdominal cavity and is commonly attributed to various etiologies, including portal hypertension and peritoneal diseases. Hyperthyroidism is rarely associated with ascites, which is typically chylous and accompanied by high central venous pressure. We present a unique case of a 57-year-old woman with untreated hyperthyroidism who manifested non-chylous ascites without evidence of high venous pressure. Initially presenting with left lower leg pain, the patient presented with leg edema, abdominal distention, and diarrhea. A range of diagnostic tests ruled out common etiologies of ascites, such as liver cirrhosis, renal impairment, heart failure, infection, and malignancy. Ascites was characterized by low triglyceride levels, while no evidence of high venous pressure was found. Notably, the patient showed decreased levels of rapid turnover proteins, suggesting hypercatabolism and insufficient protein synthesis due to hyperthyroidism. Upon the initiation of antithyroid therapy, the patient's symptoms markedly improved. In conclusion, this report highlights a rare manifestation of hyperthyroidism that resulted in non-chylous ascites without high venous pressure. This underscores the need to include hyperthyroidism in the differential diagnosis of unexplained ascites, particularly in cases in which classical hyperthyroid symptoms are absent.

Reversible off-on fluorescence probe for hypoxia and imaging of hypoxia-normoxia cycles in live cells.

We report a fully reversible off-on fluorescence probe for hypoxia. The design employs QSY-21 as a Förster resonance energy transfer (FRET) acceptor and cyanine dye Cy5 as a FRET donor, based on our finding that QSY-21 undergoes one-electron bioreduction to the radical under hypoxia, with an absorbance decrease at 660 nm. At that point, FRET can no longer occur, and the dye becomes strongly fluorescent. Upon recovery of normoxia, the radical is immediately reoxidized to QSY-21, with loss of fluorescence due to restoration of FRET. We show that this probe, RHyCy5, can monitor repeated hypoxia-normoxia cycles in live cells.

Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor.

Adenosine 5' triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase ε subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.

Biologists often refer to a small molecule called adenosine triphosphate ­ or ATP for short ­ as 'the currency of life'. This molecule carries energy all through the body, and most cells and proteins require ATP to perform their various roles. Nerve cells (also known as neurons) in the brain release ATP when activated, and use this molecule to send signals to other active neurons or other cells in the brain. But ATP can also signal danger in the brain. A molecule derived from ATP is involved in transmitting the pain signals of migraines and severe headaches; and ATP levels can become imbalanced after strokes, when parts of the brain are deprived of blood. Despite its importance, ATP remains difficult to visualize in the body, and monitoring the molecule in the active brain in real time is challenging. To address this issue, Kitajima et al. designed an optical sensor that could monitor ATP in the healthy brain, and was sensitive enough to detect when and where it was released. First, Kitajima et al. made several potential sensors by attaching various fluorescent tags to different locations on a protein that binds ATP. Next each sensor was tested to determine whether it could bind ATP tightly and get bright upon binding. This is important because previous sensors could not detect ATP release in the brains of living animals. To illustrate the new sensors' potential, Kitajima et al. used the sensor to image ATP in the brains of live mice. A 'wave' of ATP was seen spreading through the brain after neurons were stimulated with a small electric pulse, mimicking a sudden migraine or stroke. The results confirm that this new sensor is suitable for imaging how ATP signals in the brain, and it may help resolve the underlying mechanisms of migraines and strokes. This sensor could also be used to understand other cellular process which rely on ATP to carry out their role.

Rational Design of a Near-infrared Fluorescence Probe for Ca2+ Based on Phosphorus-substituted Rhodamines Utilizing Photoinduced Electron Transfer.

Fluorescence imaging in the near-infrared (NIR) region (650-900 nm) is useful for bioimaging because background autofluorescence is low and tissue penetration is high in this range. In addition, NIR fluorescence is useful as a complementary color window to green and red for multicolor imaging. Here, we compared the photoinduced electron transfer (PeT)-mediated fluorescence quenching of silicon- and phosphorus-substituted rhodamines (SiRs and PRs) in order to guide the development of improved far-red to NIR fluorescent dyes. The results of density functional theory calculations and photophysical evaluation of a series of newly synthesized PRs confirmed that the fluorescence of PRs was more susceptible than that of SiRs to quenching via PeT. Based on this, we designed and synthesized a NIR fluorescence probe for Ca2+ , CaPR-1, and its membrane-permeable acetoxymethyl derivative, CaPR-1 AM, which is distributed to the cytosol, in marked contrast to our previously reported Ca2+ far-red to NIR fluorescence probe based on the SiR scaffold, CaSiR-1 AM, which is mainly localized in lysosomes as well as cytosol in living cells. CaPR-1 showed longer-wavelength absorption and emission (up to 712 nm) than CaSiR-1. The new probe was able to image Ca2+ at dendrites and spines in brain slices, and should be a useful tool in neuroscience research.

Synthesis of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability.

In this data article, we described the detailed synthetic procedure and the experimental data for the synthesis of a red-fluorescent probe for calcium ions (Ca2+) with improved water solubility. This Ca2+ red-fluorescent probe CaTM-3 AM could be applied to fluorescence imaging of physiological Ca2+ concentration changes in not only live cells, but also brain slices, with high cell-membrane permeability leading to bright fluorescence in biosamples. The data provided herein are in association with the research article "The Development of Practical Red Fluorescent Probe for Cytoplasmic Calcium Ions with Greatly Improved Cell-membrane Permeability" in Cell Calcium (Hirabayashi et al., 2016) [1].

Development of an Azoreductase-based Reporter System with Synthetic Fluorogenic Substrates.

Enzyme/substrate pairs, such as ß-galactosidase with chromogenic x-gal substrate, are widely used as reporters to monitor biological events, but there is still a requirement for new reporter systems, which may be orthogonal to existing systems. Here, we focused on azoreductase (AzoR). We designed and synthesized a library of azo-rhodamine derivatives as candidate fluorogenic substrates. These derivatives were nonfluorescent, probably due to ultrafast conformational change around the N═N bond after photoexcitation. We found that AzoR-mediated reduction of the azo bond of derivatives bearing an electron-donating group on the azobenzene moiety was followed by nonenzymatic cleavage to afford highly fluorescent 2-methyl-rhodamine green (2-Me RG), which was well retained in cells. We show that the AzoR/compound 9 reporter system can detect azoreductase-expressing live cells at the single cell level.

Development of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability.

Fluorescence imaging of calcium ions (Ca(2+)) has become an essential technique for investigation of signaling pathways involving Ca(2+) as a second messenger. But, Ca(2+) signaling is involved in many biological phenomena, and therefore simultaneous visualization of Ca(2+) and other biomolecules (multicolor imaging) would be particularly informative. For this purpose, we set out to develop a fluorescent probe for Ca(2+) that would operate in a different color region (red) from that of probes for other molecules, many of which show green fluorescence, as exemplified by green fluorescent protein (GFP). We previously developed a red fluorescent probe for monitoring cytoplasmic Ca(2+) concentration, based on our established red fluorophore, TokyoMagenta (TM), but there remained room for improvement, especially as regards efficiency of introduction into cells. We considered that this issue was probably mainly due to limited water solubility of the probe. So, we designed and synthesized a red-fluorescent probe with improved water solubility. We confirmed that this Ca(2+) red-fluorescent probe showed high cell-membrane permeability with bright fluorescence. It was successfully applied to fluorescence imaging of not only live cells, but also brain slices, and should be practically useful for multicolor imaging studies of biological mechanisms.