Optical imaging with fluorescence microscopy is a vital tool in the study of living systems. The most common method for cell imaging, one-photon microscopy (OPM), uses a single photon of higher ...energy to excite the fluorophore. However, two-photon microscopy (TPM), which uses two photons of lower energy as the excitation source, is growing in popularity among biologists because of several distinct advantages. Using TPM, researchers can image intact tissue for a long period of time with minimum interference from tissue preparation artifacts, self-absorption, autofluorescence, photobleaching, and photodamage. However, to make TPM a more versatile tool in biology, researchers need a wider variety of two-photon probes for specific applications. In this Account, we describe a series of two-photon probes that we developed that can visualize the distribution of intracellular metal ions, acidic vesicles, and lipid rafts in living cells and tissues. The development of these probes requires a significant two-photon cross section for the bright image and receptors (sensing moieties) that triggers the emission of the two-photon excited fluorescence upon binding with the ions or membrane in the living system. These probes also must be sensitive to the polarity of the environment to allow selective detection of cytosolic and membrane-bound probes. In addition, they need to be cell-permeable, water-soluble for the staining of cells and tissues, and highly photostable for long-term imaging. The resulting probesAMg1 (Mg2+), ACa1−ACa3 (Ca2+), AZn1 and AZn2 (Zn2+), AH1, AH2, and AL1 (acidic vesicles), and CL2 (membrane)use 2-acetyl-6-aminonaphthalene as the fluorophore and receptors for the target ions or membrane. All of these two-photon turn-on probes can detect the intracellular free metal ions, acidic vesicles, and lipid rafts at 100−300 μm depth in live tissues. Moreover, with ACa1-AM, we could simultaneously visualize the spontaneous Ca2+ waves in the somas of neurons and astrocytes at ∼120 μm depth in fresh hypothalamic slices for more than 1000 s without appreciable decay. Furthermore, AL1 could visualize the transport of the acidic vesicles between cell body and axon terminal along the axon in fresh rat hippocampal slices at ∼120 μm depth.
Endoscopic ultrasonography-guided transmural gallbladder drainage (EUS-GBD) has been proposed for the management of acute cholecystitis in high risk patients; however, little is known about the ...long-term outcomes of this treatment. The aim of this study was to evaluate the procedural and long-term outcomes of EUS-GBD with self-expandable metallic stent (SEMS).
Data for this retrospective study were obtained from a prospectively collected EUS database. Patients with acute cholecystitis who were deemed unsuitable for cholecystectomy were included. Study outcomes were technical and clinical success, adverse events, and stent patency.
EUS-GBD was technically and clinically successful in 62/63 patients (98.4 %; 95 % confidence interval CI 94.9 % - 100 %). Procedural adverse events included duodenal perforation (n = 1, 1.6 %) and self-limiting pneumoperitoneum (n = 2, 3.2 %), all of which resolved with conservative treatment. Long-term outcomes of EUS-GBD were evaluated in 56 patients who were followed for a median of 275 days (range 40 - 1185 days). Late adverse events developed in four patients (7.1 %; 95 %CI 5.7 % - 8.4 %), including asymptomatic distal stent migration (n = 2), and acute cholecystitis due to stent occlusion (n = 2). Two patients with occluded stent were successfully treated endoscopically (reintervention rate of 3.6 %). A total of 54 patients (96.4 %) had no recurrence of acute cholecystitis during follow-up. Median stent patency time was 190 days overall (range 15 - 1185 days) and 458 days (range 151 - 1185 days) for the 28 patients who were alive at the study end. The cumulative stent patency rate was 86 % at 3 years.
EUS-GBD with an SEMS for acute cholecystitis showed excellent long-term outcomes and may be a definitive treatment in patients who are unsuitable for cholecystectomy because of advanced malignancy or high surgical risk.
Background and Aims Preoperative biliary drainage (PBD) with stent placement has been commonly used for patients with malignant biliary obstruction. In PBD, the placement of fully covered ...self-expandable metal stents (FCSEMSs) may provide better patency duration and a lower incidence of cholangitis compared with plastic stents. We aimed to evaluate which type of stent showed better outcomes in PBD. Methods In this multicenter, prospective randomized trial, we compared PBD with FCSEMSs versus plastic stents in 86 patients with malignant biliary obstruction between January 2012 and December 2014. Patients with obstructive jaundice were randomly assigned to undergo PBD either with plastic stents or FCSEMS placement. Results Baseline characteristics were not significantly different between the 2 groups. Endoscopic stent placement was technically successful in all patients. Procedure-related adverse events were not significantly different between the 2 groups (plastic vs FCSEMS group; 16.3% vs 16.3%, P = 1.0). Reintervention was required in 16.3% of the plastic stent group and 14.0% of the FCSEMS group ( P = .763). The interval to surgery after PBD (plastic vs FCSEMS group; 14.2 ± 8.3 vs 12.3 ± 6.9 days, P = .426) was not significantly different between groups. Surgery-related adverse events occurred in 43.6% of the plastic stent group and 40.0% of the FCSEMS group ( P = .755). Conclusions In patients with resectable malignant biliary obstruction, the outcomes of PBD with plastic stents and FCSEMSs were similar. Considering the cost-effectiveness, PBD with plastic stents may be preferable to FCSEMS placement. (Clinical trial registration number: NCT01789502 .)
Acute myeloid leukemia (AML) is an aggressive malignancy characterized by rapid growth and uncontrolled proliferation of undifferentiated myeloid cells. Metabolic reprogramming is commonly observed ...in the bone marrow of AML patients, as leukemia cells require increased ATP supply to support disease progression. In this study, we examined the potential role of mesothelin as a metabolic modulator in myeloid cells in AML. Mesothelin is a well-known marker of solid tumors that promotes cancer cell proliferation and survival. We initially analyzed alterations in mesothelin expression in the myeloblast subpopulations, defined as SSC-Alow/CD45dim, obtained from the bone marrow of AML patients using flow cytometry. Our results showed overexpression of mesothelin in 34.8% of AML patients. Subsequently, metabolic changes in leukemia cells were evaluated by comparing the oxygen consumption rates (OCR) of bone marrow samples derived from adult AML patients. Notably, a higher OCR was observed in the mesothelin-positive compared to the mesothelin-low and non-expressing groups. Treatment with recombinant human mesothelin protein enhanced OCR and increased the mRNA expression of glycolytic enzymes and mitochondrial complex II in KG1α AML cells. Notably, siRNA targeting mesothelin in KG1α cells led to the reduction of glycolysis-related gene expression but had no effect on the mitochondrial complex gene. The collective results demonstrate that mesothelin induces metabolic changes in leukemia cells, facilitating the acquisition of a rapid supply of ATP for proliferation in AML. Therefore, the targeting of mesothelin presents a potentially promising approach to mitigating the progression of AML through the inhibition of glycolysis and mitochondrial respiration in myeloid cells.
The genetic landscape of medullary thyroid cancer (MTC) is not yet fully understood, although some oncogenic mutations have been identified. To explore genetic profiles of MTCs, formalin-fixed, ...paraffin-embedded tumor tissues from MTC patients were assayed on the Ion AmpliSeq Cancer Panel v2. Eighty-four sporadic MTC samples and 36 paired normal thyroid tissues were successfully sequenced. We discovered 101 hotspot mutations in 18 genes in the 84 MTC tissue samples. The most common mutation was in the ret proto-oncogene, which occurred in 47 cases followed by mutations in genes encoding Harvey rat sarcoma viral oncogene homolog (N = 14), serine/threonine kinase 11 (N = 11), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (N = 6), mutL homolog 1 (N = 4), Kiesten rat sarcoma viral oncogene homolog (N = 3) and MET proto-oncogene (N = 3). We also evaluated anaplastic lymphoma kinase (ALK) rearrangement by immunohistochemistry and break-apart fluorescence in situ hybridization (FISH). Two of 98 screened cases were positive for ALK FISH. To identify the genomic breakpoint and 5' fusion partner of ALK, customized targeted cancer panel sequencing was performed using DNA from tumor samples of the two patients. Glutamine:fructose-6-phosphate transaminase 1 (GFPT1)-ALK and echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusions were identified. Additional PCR analysis, followed by Sanger sequencing, confirmed the GFPT1-ALK fusion, indicating that the fusion is a result of intra-chromosomal translocation or deletion. Notably, a metastatic MTC case harboring the EML4-ALK fusion showed a dramatic response to an ALK inhibitor, crizotinib. In conclusion, we found several genetic mutations in MTC and are the first to identify ALK fusions in MTC. Our results suggest that the EML4-ALK fusion in MTC may be a potential driver mutation and a valid target of ALK inhibitors. Furthermore, the GFPT1-ALK fusion may be a potential candidate for molecular target therapy.
Background and Aims
Immunoglobulin G4‐related sclerosing cholangitis (IgG4‐SC) is considered a biliary manifestation of IgG4‐related diseases. However, there has been a controversy on the clinical ...outcomes according to the location of the involved bile duct. We therefore compared the clinical outcomes and long‐term prognosis of IgG4‐SC with proximal bile duct involvement (proximal IgG4‐SC) and IgG4‐SC with distal bile duct involvement (distal IgG4‐SC).
Methods
We reviewed the data of patients with IgG4‐SC that were prospectively collected at 10 tertiary centers between March 2002 and October 2020. Clinical manifestations, outcomes, association with autoimmune pancreatitis (AIP), steroid‐responsiveness, and relapse of IgG4‐SC were evaluated.
Results
A total of 148 patients (proximal IgG4‐SC, n = 59; distal IgG4‐SC, n = 89) were analyzed. The median age was 65 years (IQR, 56.25–71), and 86% were male. The two groups were similar in terms of jaundice at initial presentation (51% vs 65%; P = 0.082) and presence of elevated serum IgG4 (66% vs 70%; P = 0.649). The two groups showed significant differences in terms of steroid‐responsiveness (91% vs 100%; P = 0.008), association with AIP (75% vs 99%; P = 0.001), and occurrence of liver cirrhosis (9% vs 1%; P = 0.034). During a median follow‐up of 64 months (IQR, 21.9–84.7), the cumulative relapse‐free survival was significantly different between the two groups (67% vs 79% at 5 years; P = 0.035).
Conclusions
Relapse of IgG4‐SC frequently occurred during follow‐up. Proximal IgG4‐SC and distal IgG4‐SC had different long‐term outcomes in terms of steroid‐responsiveness, occurrence of liver cirrhosis, and recurrence. It may be advantageous to determine the therapeutic and follow‐up strategies according to the location of bile duct involvement.
Background and Aim
Endoscopic ultrasound‐guided pancreatic duct drainage (EUS‐PD) has been proposed for pancreatic duct obstruction after failure of endoscopic retrograde pancreatography. We evaluate ...the long‐term outcomes of EUS‐PD using a fully covered self‐expandable metal stent (FCSEMS) for pancreaticojejunal anastomosis (PJA) strictures following Whipple procedures.
Methods
Twenty‐three patients with PJA strictures underwent EUS‐PD according to the findings of EUS‐guided pancreatogram and the passage of the guidewire through PJA stricture (complete vs partial stricture) after failure of endoscopic retrograde pancreatography. Technical and clinical success, adverse events (AEs), and long‐term outcomes were assessed.
Results
Technical and clinical success was achieved in all patients. The complete and partial strictures were 11 and 12, respectively. The direct transanastomotic and transmural plastic stenting in partial PJA stricture was successful in only three patients (13%). Therefore, 20 patients underwent EUS‐guided transmural FCSEMS placement during the initial attempt. Early AEs, including abdominal pain (n = 3) and peripancreatic fluid collection (n = 1), occurred in four patients (17.4%). During the follow‐up periods (median, 27.2 months; interquartile range IQR, 18.7–40.6), five patients (21.7%) developed late AEs, including asymptomatic stent fracture at the gastric end (n = 3), asymptomatic stent migration (n = 1), and stent occlusion (n = 1). The total duration of stent placement was 27.2 months (IQR, 18.7–40.6), and the median number of stent revision was 2 (IQR, 1–2).
Conclusions
In terms of safety and efficacy, EUS‐PD with an FCSEMS showed favorable success and acceptable AEs rate and durable long‐term outcomes.
Surface gravity waves, crucial for sediment transport in coastal regions, undergo wave modulation owing to local hydrodynamic conditions. However, the cause of the spatial and temporal distribution ...of the dominant factors in wave modulation remains vague. This study was aimed at identifying the causes of tidal modulation of waves in Gyeonggi Bay, which exhibits shallow water and macro-tidal environment, and estimating site-specific dominant factors using numerical modeling. The results indicated that tides significantly impact wave dynamics at semidiurnal tidal frequencies, with spatiotemporal variability. The most dominant factor in wave modulation in the nearshore region is the depth change caused by tides, and the most notable impact was found in tidal flats. Depth dependence is mainly evident in surf zones, with a clear distinction at locations where the wave height to water depth ratio reaches 0.2. While idealized studies highlight the significant role of wind in governing incoming waves, it is not the primary cause of modulation. Nevertheless, stronger tidal strength can lead to more pronounced modulation, which is further amplified when accompanied by strong wind conditions. This finding indicates that the spatiotemporal distribution of wave modulation in macro-tidal regions may have a significant impact on various estuarine dynamics including sediment transport.
•In the macro-tidal region, wave modulations were dominated at tidal frequencies.•The dominant factors in wave modulation were determined by the ratio between water depth and wave height.•The intensity of wave modulations is determined by the magnitude of incoming waves and tidal strength.•The spatiotemporal distribution of wave modulation in macro-tidal regions may impact estuarine dynamics.
We reported a ratiometric two-photon fluorescent probe (SG1) for β-galactosidase (β-gal) and its application to quantitative detection of β-gal activity during cellular senescence in live cells and ...in aged tissues. This probe is characterized by a significant two-photon excited fluorescence, a marked blue-to-yellow emission color change in response to β-gal, easy loading, insensitivity to pH and reactive oxygen species (ROS), high photostability, and low cytotoxicity. In addition, we show that SG1 labeling is an effective tool for quantitative detection of senescence-associated β-gal activity at the subcellular level in situ. This finding demonstrates that SG1 will find useful applications in biomedical research, including studies of cell aging.
TFEB, a key regulator of lysosomal biogenesis and autophagy, is induced not only by nutritional deficiency but also by organelle stress. Here, we find that Tfeb and its downstream genes are ...upregulated together with lipofuscin accumulation in adipose tissue macrophages (ATMs) of obese mice or humans, suggestive of obesity-associated lysosomal dysfunction/stress in ATMs. Macrophage-specific TFEB-overexpressing mice display complete abrogation of diet-induced obesity, adipose tissue inflammation and insulin resistance, which is independent of autophagy, but dependent on TFEB-induced GDF15 expression. Palmitic acid induces Gdf15 expression through lysosomal Ca
-mediated TFEB nuclear translocation in response to lysosomal stress. In contrast, mice fed a high-fat diet with macrophage-specific Tfeb deletion show aggravated adipose tissue inflammation and insulin resistance, accompanied by reduced GDF15 level. Finally, we observe activation of TFEB-GDF15 in ATMs of obese humans as a consequence of lysosomal stress. These findings highlight the importance of the TFEB-GDF15 axis as a lysosomal stress response in obesity or metabolic syndrome and as a promising therapeutic target for treatment of these conditions.
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