Imaging vs Nonimaging Raman Spectroscopy for High-Throughput Single-Cell Phenotyping.

Raman spectroscopy can provide nonbiased single-cell analysis based on the endogenous ensemble of biomolecules, with alterations in cellular content indicative of cell state and disease. The measurements themselves can be performed in a variety of modes: generally, full imaging takes the most time but can provide the most information. By reducing the imaging resolution and generating the most characteristic single-cell Raman spectrum in the shortest time, we optimize the utility of the Raman measurement for cell phenotyping. Here, we establish methods to compare these different measurement approaches and assess what, if any, undesired effects occur in the cell. Assuming that laser-induced damage should be apparent as a change in molecular spectra across sequential measurements, and by defining the information content as the Raman-based separability of two cell lines, we thereby establish a parameter range for optimum measurement sensitivity and single-cell throughput in single-cell Raman spectroscopic analysis. While the work here uses 532 nm irradiation, the same approach can be generalized to Raman analysis at other wavelengths.

Detecting nitrile-containing small molecules by infrared photothermal microscopy.

The use of infrared (IR) photothermal microscopy (IR-PTM) is emerging for imaging chemical substances in various samples. In this research, we demonstrated the use of a nitrile group as a vibrational tag to image target molecules in the low water-background region. We performed IR photothermal imaging of trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) in cells and confirmed the high spatial resolution by photothermal detection using visible light as a probe beam. We imaged FCCP-treated HeLa cells and confirmed that the photothermal signal was indeed produced from the vibrational tag in lipid droplets. We also compared the results with nitrile imaging by stimulated Raman scattering (SRS) microscopy. From both the calculated and experimental results, IR-PTM demonstrated a signal-to-noise ratio (SNR) several tens of times better than that of SRS microscopy on the basis of the same power input.

[A Case of Intraductal Papillary Mucinous Carcinoma of the Pancreas Extending Widely within the Pancreas Resected by Total Pancreatectomy].

A 78-year-old woman visiting our hospital with the chief complaint of abdominal pain was diagnosed with ischemic colitis. CT pointed out pancreatic enlargement, so detailed pancreatic examination was conducted in parallel with treatment. In enhanced CT and MRI, the main pancreatic duct significantly extended over the entire length, and mural nodules scattered in the main pancreatic duct were observed. It was diagnosed with main duct intraductal papillary mucinous neoplasm (IPMN), which is likely to be intraductal papillary mucinous carcinoma(IPMC), and total pancreatectomy was performed. Pathological findings showed diffuse dilation of the main pancreatic duct, non-invasive IPMC progressed over approximately the entire length of the main pancreatic duct, mucinous carcinoma-tubular adenocarcinoma was widely infiltrated in pancreatic tissues around mural nodules. The cancer remained in the pancreas and was eventually diagnosed with IPMC, invasive, pT2, pN0, and pStage ⅠB. This case was an IPMN with high-risk stigmata, and it was an adaptive case of total pancreatectomy because mural nodules extended to the entire pancreas. In recent years, total pancreatectomy has come to be expected good results by the progress of diabetes treatment and the development of pancreatic enzyme agents, it is necessary to carefully judge each case for adaptation.

[A case of gastrointestinal stromal tumor of the duodenum with ruptured liver metastasis during administration of imatinib].

A fifty-year-old man with a liver metastasis of a duodenal gastrointestinal stromal tumor (GIST) previously treated with imatinib. Thirty-three months following initiation of the therapy, he visited the emergency room of our hospital presenting with an upper abdominal pain. Dynamic CT scan revealed a ruptured liver metastasis of duodenal GIST. We used transcatheter arterial embolization to stop the bleeding. Due to the rarity of this condition, we herein report this case with an article review.

[Short-Term and Long-Term Outcomes of Colonic Stenting as a Bridge to Surgery for Obstructive Colorectal Cancer].

We examined the short-term and long-term outcomes in 50 patients who underwent stenting as a bridge to surgery(BTS) for obstructive colorectal cancer. The patients comprised 30 men and 20 women, with a mean age of 74.0 years. Stenting and decompression were successful in all patients, and the mean time to oral intake after stenting was 2.4 days. No serious complications related to stenting occurred. Colonoscopy after stenting was important for the preoperative diagnosis of coexisting lesions and planning of the extent of resection. Elective and one-stage surgeries could be performed in all patients after stenting. Regarding long-term outcomes, the 5-year overall survival rate and disease-free survival rate in the BTS patients with Stage Ⅱ plus Ⅲ cancer were 73.1% and 55.7%, respectively. The results of this study suggest that BTS for obstructive colorectal cancer is an effective treatment strategy for not only short-term but also long-term outcomes.

[Results of Preoperative Colon Stent Placement for Obstructive Colorectal Cancer].

We examined short-term outcomes in 34 patients who had stenting as a bridge to surgery(BTS)for obstructive colorectal cancer during the 5-year period between April 2012 and March 2017.T he patients were 22 men and 12 women with a mean age of 72.6 years. Stenting and decompression were successful in all patients, and the mean time to oral intake after stenting was 2.5 days.No serious complications related to stenting occurred.Elective surgery could be performed in all patients after stenting.The mean number of days to surgery was 24.7 days.Laparoscopic surgery was performed in 14 patients.Postoperative complications included minor leakage in 1 patient, an abdominal wall abscess due to tumor invasion of the abdominal wall in 1 patient, and heart failure and pneumonia, as serious complications, in 1 patient each.Colorectal stenting in patients with obstructive colorectal cancer is a safe and relatively simple procedure.This is an effective treatment strategy in which preoperative colorectal decompression enables a one-stage resection.

[Findings from Total Colonoscopy in Obstructive Colorectal Cancer Patients Who Underwent Stent Placement as a Bridge to Surgery(BTS)].

We clinically investigated 34 patients with obstructive colorectal cancer who underwent placement of a colonic stent as a bridge to surgery(BTS), focusing on endoscopic findings after stent placement.Twenty -nine patients(85.3%)underwent colonoscopy after stent placement, and the entire large intestine could be observed in 28(96.6%).Coexisting lesions were observed in 22(78.6%)of these 28 patients.The lesions comprised adenomatous polyps in 17 patients(60.7%), synchronous colon cancers in 5 patients(17.9%), and obstructive colitis in 3 patients(10.7%), with some overlapping cases.All patients with multiple cancers underwent one-stage surgery, and all lesions were excised at the same time.Colonoscopy after colonic stent placement is important for preoperative diagnosis of coexisting lesions and planning the extent of resection. These considerations support the utility of colonic stenting for BTS.

Solution-phase synthesis and biological evaluation of triostin A and its analogues.

Triostin A is a biosynthetic precursor of echinomycin which is one of the most potent hypoxia inducible factor 1 (HIF-1) inhibitors. An improved solution-phase synthesis of triostin A on a preparative scale has been achieved in 17.5% total yield in 13 steps. New analogues of triostin A with various aromatic chromophores, oxidized intra-peptide disulfide bridges and diastereoisomeric cyclic depsipeptide cores were also successfully synthesized. All analogues had a significant inhibitory effect on HIF-1 transcriptional activation in hypoxia and cytotoxicity on MCF-7 cells, with the exception of the derivatives containing a naphthalene chromophore or a thiosulfonate bridge. For the first time, triostin A, echinomycin and the thiosulfinate analogue of triostin A have been revealed to inhibit not only DNA binding of HIF-1 but also HIF-1α protein accumulation in MCF-7 cells. Furthermore, the thiosulfinate analogue and triostin A exhibited a hypoxia-selective cytotoxicity on MCF-7 cells. The improved solution-phase synthetic procedure described herein will contribute to the development of diverse bicyclic depsipeptide drug candidates with the potential to act as novel anti-cancer agents targeting hypoxic tumor microenvironments.

[Cases of Obstructive Colon Cancer for Which Elective Surgery Was Performed after Colonic Stent Placement].

The present study investigated the short-term outcomes of 20 patients with obstructive colon cancer who underwent colonic stent placement as a bridge to surgery (BTS) during the 3-year period between April 2012 and March 2015. Subjects comprised 13 men and 7 women, with a mean age of 68.3 years. Placement and decompression were successfully achieved in all of the patients. Oral ingestion became possible from a mean of 2.7 days after placement. No serious complications associated with placement were encountered. Total colonoscopy was performed after placement in 17 patients (85%), and independent advanced cancer was seen in the proximal portion of the colon in 1 patient. Elective surgery was performed for all of the patients after placement. Excluding the 2 patients for whom preoperative chemotherapy or treatment of another disease was prioritized, the mean interval to surgery for the remaining 18 patients was 23.2 days. The operative procedure performed was laparoscopic surgery in 8 patients (40%). Although minor leakage (n=1) and abdominal wall abscess (n=1) were observed as postoperative complications, the patients generally had an uneventful course. Colonic stent placement for obstructive colon cancer is relatively easy and safe, and may be considered as an effective treatment method that enables favorable intestinal decompression preoperatively and one-stage resection.

Simple purification of small-molecule-labelled peptides via palladium enolate formation from ß-ketoamide tags.

Palladium enolates derived from ß-ketocarbonyl compounds serve as key intermediates in various catalytic asymmetric reactions. We found that the palladium enolate formed from ß-ketoamide is stable in air and moisture and we applied this property to develop a peptide purification system using ß-ketoamide as a small affinity tag in aqueous media. A solid-supported palladium complex successfully captured ß-ketoamide-tagged molecules as palladium enolates and released them in high yield upon acid treatment. Optimum conditions for the catch and release of tagged peptides from a mixture of untagged peptides were established. To demonstrate the value of this methodology in identifying the binding site of a ligand to its target protein, we purified and identified a peptide containing the ligand-binding site from the tryptic digest of cathepsin B labelled with a covalent cathepsin B inhibitor containing a ß-ketoamide tag.

Lysine long-chain fatty acylation regulates the TEAD transcription factor.

TEAD transcription factors are responsible for the transcriptional output of Hippo signaling. TEAD activity is primarily regulated by phosphorylation of its coactivators, YAP and TAZ. In addition, cysteine palmitoylation has recently been shown to regulate TEAD activity. Here, we report lysine long-chain fatty acylation as a posttranslational modification of TEADs. Lysine fatty acylation occurs spontaneously via intramolecular transfer of acyl groups from the proximal acylated cysteine residue. Lysine fatty acylation, like cysteine palmitoylation, contributes to the transcriptional activity of TEADs by enhancing the interaction with YAP and TAZ, but it is more stable than cysteine acylation, suggesting that the lysine fatty-acylated TEAD acts as a "stable active form." Significantly, lysine fatty acylation of TEAD increased upon Hippo signaling activation despite a decrease in cysteine acylation. Our results provide insight into the role of fatty-acyl modifications in the regulation of TEAD activity.

Spectral focusing in picosecond pulsed stimulated Raman scattering microscopy.

We introduce spectral focusing of picosecond laser pulses in stimulated Raman scattering (SRS) microscopy to improve spectral resolution, reduce nonlinear background signals, and decrease nonlinear photodamage. We produce a pair of 14 ps pump and Stokes laser pulses by spectral focusing of a 2 ps laser and achieve a spectral resolution of 2 cm-1. Due to instantaneous narrow-band excitation, we find that the chirped 14 ps laser pulses can be used to improve the signal-to-background ratio in SRS microscopy of various samples such as polymer particles and small molecules in HeLa cells. The lower peak powers produced by chirped picosecond laser pulses also reduce nonlinear photodamage, allowing long-term SRS imaging of living cells with higher SNR.

Design, Synthesis, and Conformation-Activity Study of Unnatural Bridged Bicyclic Depsipeptides as Highly Potent Hypoxia Inducible Factor-1 Inhibitors and Antitumor Agents.

By carrying out structural modifications based on the bicyclic peptide structure of echinomycin, we successfully synthesized various powerful antitumor derivatives. The ring conformation in the obtained compounds was restricted by cross-linking with an unnatural bond. The prepared derivatives were demonstrated to strongly suppress the hypoxia inducible factor (HIF)-1 transcriptional activation and hypoxia induction of HIF-1 protein expression. Particularly, alkene-bridged derivative 12 exhibited remarkably potent cytotoxicity (IC50 = 0.22 nM on the MCF-7 cell line) and HIF-1 inhibition (IC50 = 0.09 nM), which considerably exceeded those of echinomycin. Conformational analyses and molecular modeling studies revealed that the biological activities were enhanced following restriction of the conformation by cross-linking through a metabolically stable and rigid bridge bond. In addition, we proposed a new globular conformation stabilized by intramolecular π stacking that can contribute to the biological effects of bicyclic depsipeptides. The developments presented in the current study serve as a useful guide to expand the chemical space of peptides in drug discovery.

Quantitative Drug Dynamics Visualized by Alkyne-Tagged Plasmonic-Enhanced Raman Microscopy.

Visualizing live-cell uptake of small-molecule drugs is paramount for drug development and pharmaceutical sciences. Bioorthogonal imaging with click chemistry has made significant contributions to the field, visualizing small molecules in cells. Furthermore, recent developments in Raman microscopy, including stimulated Raman scattering (SRS) microscopy, have realized direct visualization of alkyne-tagged small-molecule drugs in live cells. However, Raman and SRS microscopy still suffer from limited detection sensitivity with low concentration molecules for observing temporal dynamics of drug uptake. Here, we demonstrate the combination of alkyne-tag and surface-enhanced Raman scattering (SERS) microscopy for the real-time monitoring of drug uptake in live cells. Gold nanoparticles are introduced into lysosomes of live cells by endocytosis and work as SERS probes. Raman signals of alkynes can be boosted by enhanced electric fields generated by plasmon resonance of gold nanoparticles when alkyne-tagged small molecules are colocalized with the nanoparticles. With time-lapse 3D SERS imaging, this technique allows us to investigate drug uptake by live cells with different chemical and physical conditions. We also perform quantitative evaluation of the uptake speed at the single-cell level using digital SERS counting under different quantities of drug molecules and temperature conditions. Our results illustrate that alkyne-tag SERS microscopy has a potential to be an alternative bioorthogonal imaging technique to investigate temporal dynamics of small-molecule uptake of live cells for pharmaceutical research.