Optimizing immunofluorescence with high-dynamic-range imaging to enhance PD-L1 expression evaluation for 3D pathology assessment from NSCLC tumor tissue.

Assessing programmed death ligand 1 (PD-L1) expression through immunohistochemistry (IHC) is the golden standard in predicting immunotherapy response of non-small cell lung cancer (NSCLC). However, observation of heterogeneous PD-L1 distribution in tumor space is a challenge using IHC only. Meanwhile, immunofluorescence (IF) could support both planar and three-dimensional (3D) histological analyses by combining tissue optical clearing with confocal microscopy. We optimized clinical tissue preparation for the IF assay focusing on staining, imaging, and post-processing to achieve quality identical to traditional IHC assay. To overcome limited dynamic range of the fluorescence microscope's detection system, we incorporated a high dynamic range (HDR) algorithm to restore the post imaging IF expression pattern and further 3D IF images. Following HDR processing, a noticeable improvement in the accuracy of diagnosis (85.7%) was achieved using IF images by pathologists. Moreover, 3D IF images revealed a 25% change in tumor proportion score for PD-L1 expression at various depths within tumors. We have established an optimal and reproducible process for PD-L1 IF images in NSCLC, yielding high quality data comparable to traditional IHC assays. The ability to discern accurate spatial PD-L1 distribution through 3D pathology analysis could provide more precise evaluation and prediction for immunotherapy targeting advanced NSCLC.

Chiral anomaly and Weyl orbit in three-dimensional Dirac semimetal Cd3As2grown on Si.

Preparing Cd3As2, which is a three-dimensional (3D) Dirac semimetal in certain crystal orientation, on Si is highly desirable as such a sample may well be fully compatible with existing Si CMOS technology. However, there is a dearth of such a study regarding Cd3As2films grown on Si showing the chiral anomaly. Here,for the first time, we report the novel preparation and fabrication technique of a Cd3As2(112) film on a Si (111) substrate with a ZnTe (111) buffer layer which explicitly shows the chiral anomaly with a nontrivial Berry's phase ofπ. Despite the Hall carrier density (n3D≈9.42×1017cm-3) of our Cd3As2film, which is almost beyond the limit for the portents of a 3D Dirac semimetal to emerge, we observe large linear magnetoresistance in a perpendicular magnetic field and negative magnetoresistance in a parallel magnetic field. These results clearly demonstrate the chiral magnetic effect and 3D Dirac semimetallic behavior in our silicon-based Cd3As2film. Our tailoring growth of Cd3As2on a conventional substrate such as Si keeps the sample quality, while also achieving a low carrier concentration.

FOXM1 is required for small cell lung cancer tumorigenesis and associated with poor clinical prognosis.

Small cell lung cancer (SCLC) continues to cause poor clinical outcomes due to limited advances in sustained treatments for rapid cancer cell proliferation and progression. The transcriptional factor Forkhead Box M1 (FOXM1) regulates cell proliferation, tumor initiation, and progression in multiple cancer types. However, its biological function and clinical significance in SCLC remain unestablished. Analysis of the Cancer Cell Line Encyclopedia and SCLC datasets in the present study disclosed significant upregulation of FOXM1 mRNA in SCLC cell lines and tissues. Gene set enrichment analysis (GSEA) revealed that FOXM1 is positively correlated with pathways regulating cell proliferation and DNA damage repair, as evident from sensitization of FOXM1-depleted SCLC cells to chemotherapy. Furthermore, Foxm1 knockout inhibited SCLC formation in the Rb1fl/flTrp53fl/flMycLSL/LSL (RPM) mouse model associated with increased levels of neuroendocrine markers, Ascl1 and Cgrp, and decrease in Yap1. Consistently, FOXM1 depletion in NCI-H1688 SCLC cells reduced migration and enhanced apoptosis and sensitivity to cisplatin and etoposide. SCLC with high FOXM1 expression (N = 30, 57.7%) was significantly correlated with advanced clinical stage, extrathoracic metastases, and decrease in overall survival (OS), compared with the low-FOXM1 group (7.90 vs. 12.46 months). Moreover, the high-FOXM1 group showed shorter progression-free survival after standard chemotherapy, compared with the low-FOXM1 group (3.90 vs. 8.69 months). Our collective findings support the utility of FOXM1 as a prognostic biomarker and potential molecular target for SCLC.

Copper-catalysed synthesis of 3-hydroxyisoindolin-1-ones from benzylcyanide 2-iodobenzamides.

An efficient one-pot two-step sequential reaction for the synthesis of biologically active 3-hydroxyisoindolin-1-one derivatives from 2-iodobenzamide derivatives and various substituted benzyl cyanides in the presence of CuCl and cesium carbonate in DMSO is reported. Furthermore, 3-hydroxyisoindolinone derivatives possessing bromo substituents were obtained from 2-iodobenzamide and 2-bromobenzyl cyanide substrates in two steps. Benzyl cyanide has been successfully used for the first time as a benzoyl synthon for the synthesis of 3-hydroxyisoindolin-1-ones. Interestingly, the mechanism of formation of 3-hydroxyisoindolin-1-ones is a novel pathway that involves carbon degradation followed by ring contraction.

Disparity in Tumor Immune Microenvironment of Breast Cancer and Prognostic Impact: Asian Versus Western Populations.

BACKGROUND: The clinicopathological features and prognosis of breast cancer in Asia are different from those in the Western countries. Tumor-infiltrating immune cells can influence the outcome of patients with breast cancer, but they have not been systemically evaluated in Asian patients with breast cancer. METHODS: We compared the immune score, composition, and prognostic impact of infiltrating immune cells between Asian and Western patients with breast cancer by analyzing gene expression profiles from eight Gene Expression Omnibus data sets and The Cancer Genome Atlas data set. The Estimation of Stromal and Immune Cells in Malignant Tumours Using Expression Data (ESTIMATE) and Cell Type Identification by Estimating Relative Subsets of Known RNA Transcripts (CIBERSORT) algorithms were used to determine the immune score and composition of tumor-infiltrating immune cells, respectively. FINDINGS: This study included 462 Asian patients and 2,186 Western patients. Tumors of Asian patients had significantly higher immune score, particularly in the luminal B and HER2-enriched subtypes. High immune score was associated with favorable prognosis in both Asian and Western patients, and Asian race with a high ESTIMATE immune score provided additional power to predict longer disease-free survival. Activated CD4 T cells and M2 macrophages were the most strongly associated with survival in both Asian and Western patients. INTERPRETATION: Our study highlights the difference in tumor immune microenvironments between Asian and Western patients. The higher ESTIMATE immune score, which represents more abundant tumor-infiltrating immune cells, in tumors of Asian patients partly explains their favorable prognosis. IMPLICATIONS FOR PRACTICE: The tumor microenvironment serves as an interface that affects the human body's reaction to cancer cells. Evidence has revealed that tumor-infiltrating immune cells were associated with patient prognosis. This study demonstrated the disparity of tumor microenvironments and their prognostic impact between Asian and Western patients with breast cancer. The differences in immune score partially explained the racial survival differences noted in recent studies. Integrated analysis of tumor cells, tumor microenvironment, and racial effect may significantly improve recurrence risk prediction for patients with stage I-III breast cancer. Because the effect of tumor microenvironment varies across different populations, a model of interaction between immune score and race/ethnicity is recommended in accessing the risk of patients with cancer.

Frequent CTNNB1 or PIK3CA Mutations Occurred in Endometrial Endometrioid Adenocarcinoma With High Levels of Microsatellite Instability and Loss of MSH2/MSH6 Expression.

BACKGROUND: DNA mismatch repair (MMR) proteins form 2 heterodimers-MutSα formed by MSH2 and MSH6, and MutLα by MLH1 and PMS2. In endometrial endometrioid adenocarcinomas, cases with MMR protein defect also usually harbor other recurrent genetic mutations of the neoplasm. However, it remains unknown whether defects of the 2 functionally different heterodimers are linked to mutations in different genes. We aimed to study the MMR protein expression, microsatellite instability (MSI), and other common genetic mutations of endometrial endometrioid adenocarcinoma. MATERIALS AND METHODS: We investigated the MSI status of 107 endometrial endometrioid adenocarcinoma patients. MMR protein expression, and mutation of KRAS, CTNNB1, and PIK3CA were also evaluated by immunohistochemistry and sequencing. RESULTS: An overall 34.6% (37/107) of endometrial endometrioid adenocarcinomas were MSI-H. All MSI-H tumors exhibited loss of MMR protein expression (loss of MLH1, PMS2, MSH6, and MSH2 was noted in 22, 25, 12, and 7 cases, respectively). CTNNB1, PIK3CA, and KRAS mutation were present in 9, 7, and 7 MSI-H tumors. Compared with patients with loss of PMS2 and/or MLH1 expression, patients with loss of MSH6 and/or MSH2 expression were associated with higher frequencies of CTNNB1 mutation (P=0.036) and PIK3CA mutation (P=0.025). CONCLUSIONS: In MSI-H endometrial endometrioid adenocarcinomas, different types of MMR protein deficiency indicate different molecular genetic alterations.

Synthesis of Polysubstituted Cyclopentene and Cyclopenta[ b]carbazole Analogues from Unsymmetrical 4-Arylidene-3,6-diarylhex-2-en-5-ynal and Indole Derivatives via an Iodine Mediated Electrocyclization Reaction.

An efficient method for the synthesis of polysubstituted cyclopentene and cyclopenta[ b]carbazole derivatives through the iodine-promoted electrocyclization of substituted indoles and 4-arylidene-3,6-diarylhex-2-en-5-ynal derivatives is reported. Polysubstituted cyclopentene derivatives were produced through 4π electrocyclization reactions with indole, 7-methylindole, and 5-bromoindole as coupling partners, whereas cyclopenta[ b]carbazole derivatives were produced via 6π electrocyclization in the case of methoxy (-OMe)-substituted indoles. The methods reported herein diastereo- and regioselectively proceed under straightforward and mild conditions.

Copper-Catalyzed Cascade Synthesis of 2-Aryl-3-cyanobenzofuran and Dibenzo[ b, f]oxepine-10-carbonitrile Derivatives.

The copper-catalyzed reaction of aryl aldehydes with 2-iodobenzylcyanides afforded 2-aryl-3-cyanobenzofurans in isolated yields of up to 74% in a cascade manner, which involves Knoevenagel condensation, aryl hydroxylation, oxa-Michael addition, and aromatization reactions. Conversely, 2-halo benzaldehydes as reacting partners with 2-iodobenzylcyanide regioselectively furnished dibenzo[ b,f]oxepine-10-carbonitrile derivatives up to 85% isolated yields via tandem Knoevenagel condensation, aryl hydroxylation, and Ullmann coupling reactions.

Synthesis of Benzopyridoindolone Derivatives via a One-Pot Copper Catalyzed Tandem Reaction of 2-Iodobenzamide Derivatives and 2-Iodobenzylcyanides.

An efficient approach for the synthesis of benzo-fused pyridoindolone derivatives via a one-pot copper catalyzed tandem reaction of 2-iodobenzamides with 2-iodobenzylcyanides has been developed. Using this protocol, benzo-fused pyridoindolone derivatives are obtained in high yields in a relatively short period of time under mild reaction conditions. To the best of our knowledge, this is the first approach where one can synthesize free indole N-H benzo-fused pyridoindolones. Also, both indole and pyridone cores are constructed during the course of the reaction. The methodology shows good functional group tolerance and allows synthesis of thiophene-fused pyridoindolones and fused indolobenzonaphthyridone derivatives.

Synthesis of Biologically Active Indenoisoquinoline Derivatives via a One-Pot Copper(II)-Catalyzed Tandem Reaction.

A concise route for the synthesis of indenoisoquinoline derivatives from 2-iodobenzamide and 1,3-indanedione derivatives in the presence of copper(II) chloride and cesium carbonate in acetonitrile solvent is reported. A wide variety of 2-iodobenzamide derivatives and indandiones could be used to synthesize indenoisoquinoline derivatives and other fused indenoisoquinoline in moderate to good yields. This methodology was adapted for the one-step synthesis of a series of clinically active topoisomerase I inhibitors such as NSC 314622, LMP-400, LMP-776.

Regioselective switching approach for the synthesis of α and δ carboline derivatives.

A metal-free protocol for accessing both α and δ-carboline derivatives, starting from a common indolylchalcone oxime ester precursor is reported. The reaction involves mild conditions and uses a regiodivergent approach. DDQ is used as a switching agent in selectively generating α and δ-carboline derivatives in good to moderate yields.

Palladium-Catalyzed Tandem C-H Functionalization/Cyclization Strategy for the Synthesis of 5-Hydroxybenzofuran Derivatives.

A palladium-catalyzed benzoquinone C-H functionalization/cyclization strategy with terminal alkynes was employed for the synthesis of some biologically relevant 2,3-disubstituted 5-hydroxybenzofuran derivatives. The benzoquinone acts as a reactant as well as an oxidant. During the process, an additional alkyne functionality can be introduced at the C3 position of the benzofuran. Base, ligand, and external oxidant are not required in this protocol.

Synthesis of benzo[a]carbazole derivatives from 3-ethylindoles by exploiting the dual character of benzoquinone as an oxidizing agent and dienophile.

The dual character of benzoquinone is exploited for the synthesis of benzo[a]carbazole derivatives starting from 3-ethylindole derivatives. A diene is generated in situ from the 3-ethylindole derivative with the help of benzoquinone, where it acts as an oxidizing agent. Later it serves as a dienophile and participates in a Diels-Alder reaction with the diene to afford benzo[a]carbazole derivatives.

Seasonal distribution and prevalence of diarrheagenic Escherichia coli in different aquatic environments in Taiwan.

Diarrheagenic Escherichia coli (DEC) are the most common agents of diarrhea. Waterborne DEC could pose a potential health risk to human through agricultural, household, recreational, and industrial use. There are few published reports on the detection of DEC and its seasonal distribution in aquatic environments. The presence of DEC in different types of aquatic environments was investigated in this study. Water samples were collected from major rivers, water reservoirs, and recreational hot springs throughout Taiwan. Moreover, an intensive water sampling plan was carried out along Puzih River. The detection of DEC target genes was used to determine the presence of enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), and Shiga toxin-producing E. coli (STEC). Among the 383 water samples analyzed, DEC was found in 122 (31.8%) samples. The detection rate varied by genotype, raging from 3.6% for STEC to 17.2% for EPEC. The DEC detection rate was higher from river waters than reservoirs and hot springs. In addition, DEC was detected at a higher rate in spring and summer. The presence of EPEC was significantly associated with total coliform levels among hot spring samples. Moreover, the presence of ETEC in river water samples was associated with heterotrophic plate counts. Water with EPEC differed significantly in pH from Puzih River samples. These results suggest that seasonal characteristics may affect the presence of DEC in different aquatic environments, and water quality indicators may be indicative of the presence of DEC.

Syntheses of 4-Indolylquinoline Derivatives via Reductive Cyclization of Indolylnitrochalcone Derivatives by Fe/HCl.

An easy and efficient procedure for the synthesis of 4-indolylquinoline derivatives is described. This process involves two steps, the first of which is the Michael addition of indole to nitrochalcones promoted by sulfamic acid under solvent free conditions and the second step is a reductive cyclization of the indolylnitrochalcone intermediates to 4-indolylquinoline derivatives by Fe/HCl in ethanol. In both steps, the reactions are clean and the yields of products are high.

Synthesis of Bicyclic Isoxazoles and Isoxazolines via Intramolecular Nitrile Oxide Cycloaddition.

An efficient and straight forward procedure for the syntheses of bicyclic isoxazole/isoxazoline derivatives from the corresponding dimethyl-2-(2-nitro-1-aryl/alkyl)-2-(prop-2-yn-1yl)malonates or dimethyl 2-allyl-2-(2-nitro-1-aryl/alkyl ethyl)malonate is described. High yields and simple operations are important features of this methodology.

Perivascular Interstitial Cells of Cajal in Human Colon.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) closely associate with nerves and smooth muscles to modulate gut motility. In the ICC microenvironment, although the circulating hormones/factors have been shown to influence ICC activities, the association between ICC and microvessels in the gut wall has not been described. We applied three-dimensional (3D) vascular histology with c-kit staining to identify the perivascular ICC and characterize their morphologic and population features in the human colon wall. METHODS: Full-thickness colons were obtained from colectomies performed for colorectal cancer. We targeted the colon wall away from the tumor site. Confocal microscopy with optical clearing (use of immersion solution to reduce scattering in optical imaging) was performed to simultaneously reveal the ICC and vascular networks in space. 3D image rendering and projection were digitally conducted to illustrate the ICC-vessel contact patterns. RESULTS: Perivascular ICC were identified in the submucosal border, myenteric plexus, and circular and longitudinal muscles via high-definition 3D microscopy. Through in-depth image projection, we specified two contact patterns-the intimate cell body-to-vessel contact (type I, 18% of ICC in circular muscle) and the long-distance process-to-vessel contact (type II, 16%)-to classify perivascular ICC. Particularly, type I perivascular ICC were detected with elevated c-kit staining levels and were routinely found in clusters, making them readily distinguishable from other ICC in the network. CONCLUSIONS: We propose a new subclass of ICC that closely associates with microvessels in the human colon. Our finding suggests a functional relationship between these mural ICC and microvessels based on the morphologic proximity.

Molecular iodine-mediated reaction of 2-(2-phenylethynyl)-Morita-Baylis-Hillman adducts: an easy route to naphthyl ketones and iodo-substituted isochromenes.

The molecular iodine-promoted reaction of 2-(2-phenylethynyl)-Morita-Baylis-Hillman adducts is reported. In the presence of I2, naphthyl ketone derivatives are produced, whereas in the presence of I2/K3PO4, iodo-substituted isochromene derivatives are produced.