Development of a sensitive LC-MS/MS method for determination of N-nitrosopiperazine in levocetirizine.

Levocetirizine is one of the widely used antihistamines and has the potential to form N-nitrosopiperazine (NPZ) during drug synthesis, manufacturing, or storage. NPZ classified as a nitrosamine is a genotoxic impurity with carcinogenic properties. Controlling the presence of NPZ in the active pharmaceutical ingredient (API) and drug products is crucial with levels ideally maintained below 80 ppm. Herein, we developed a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique to analyze NPZ levels in levocetirizine API and various formulations. Chromatographic separation was carried out using an F5 column with mobile phases consisting of 2 mM ammonium formate in water and acetonitrile, employing gradient elution mode at a flow rate of 0.2 mL min-1. The column oven temperature was set at 30 °C, and the injection volume was 2 µL. NPZ quantification was achieved using positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The developed method underwent rigorous validation according to regulatory guidelines. The limit of quantification (LOQ) was established at 1 ng mL-1 within the range of 1-50 ng mL-1, covering 10-500% of the specified NPZ limit in drugs. The effectiveness of the method was shown by utilizing it to analyze the NPZ impurity in both levocetirizine API and various drug products, including tablets, capsules, chewables, and syrups. The proposed method and the resulting data would be valuable for determining potentially present impurities in drug substances or products for quality assessment.

Histopathologic image-based deep learning classifier for predicting platinum-based treatment responses in high-grade serous ovarian cancer.

Platinum-based chemotherapy is the cornerstone treatment for female high-grade serous ovarian carcinoma (HGSOC), but choosing an appropriate treatment for patients hinges on their responsiveness to it. Currently, no available biomarkers can promptly predict responses to platinum-based treatment. Therefore, we developed the Pathologic Risk Classifier for HGSOC (PathoRiCH), a histopathologic image-based classifier. PathoRiCH was trained on an in-house cohort (n = 394) and validated on two independent external cohorts (n = 284 and n = 136). The PathoRiCH-predicted favorable and poor response groups show significantly different platinum-free intervals in all three cohorts. Combining PathoRiCH with molecular biomarkers provides an even more powerful tool for the risk stratification of patients. The decisions of PathoRiCH are explained through visualization and a transcriptomic analysis, which bolster the reliability of our model's decisions. PathoRiCH exhibits better predictive performance than current molecular biomarkers. PathoRiCH will provide a solid foundation for developing an innovative tool to transform the current diagnostic pipeline for HGSOC.

Eco-friendly natural mineral biotite as a cesium adsorbent: Utilizing low-concentration acid and hydrogen peroxide.

Biotite, a phyllosilicate mineral, possesses significant potential for cesium (Cs) adsorption owing to its negative surface charge, specific surface area (SSA), and frayed edge sites (FES). Notably, FES are known to play an important role in the adsorption of Cs. The objectives of this study were to investigate the Cs adsorption capacity and behavior of artificially weathered biotite and identify mineralogical characteristics for the development of an eco-friendly geologically-based Cs adsorbent. Through various analyses, it was confirmed that the FES of biotite was mainly formed by mineral structural distortion during artificial weathering. The Cs adsorption capacity is improved by approximately 39% (from 20.53 to 28.63 mg g-1) when FES are formed in biotite through artificial weathering using a low-concentration acidic solution mixed with hydrogen peroxide (H2O2). Especially, the Cs selectivity in Cs-containing seawater, including high concentrations of cations and organic matter, was significantly enhanced from 203.2 to 1707.6 mL g-1, an increase in removal efficiency from 49.5 to 89.2%. These results indicate that FES of artificially weathered biotite play an essential role in Cs adsorption. Therefore, this simple and economical weathering method, which uses a low-concentration acidic solution mixed with H2O2, can be applied to natural minerals for use as Cs adsorbents.

Beyond the Microscope: A Technological Overture for Cervical Cancer Detection.

Cervical cancer is a common and preventable disease that poses a significant threat to women's health and well-being. It is the fourth most prevalent cancer among women worldwide, with approximately 604,000 new cases and 342,000 deaths in 2020, according to the World Health Organization. Early detection and diagnosis of cervical cancer are crucial for reducing mortality and morbidity rates. The Papanicolaou smear test is a widely used screening method that involves the examination of cervical cells under a microscope to identify any abnormalities. However, this method is time-consuming, labor-intensive, subjective, and prone to human errors. Artificial intelligence techniques have emerged as a promising alternative to improve the accuracy and efficiency of Papanicolaou smear diagnosis. Artificial intelligence techniques can automatically analyze Papanicolaou smear images and classify them into normal or abnormal categories, as well as detect the severity and type of lesions. This paper provides a comprehensive review of the recent advances in artificial intelligence diagnostics of the Papanicolaou smear, focusing on the methods, datasets, performance metrics, and challenges. The paper also discusses the potential applications and future directions of artificial intelligence diagnostics of the Papanicolaou smear.

CervixFormer: A Multi-scale swin transformer-Based cervical pap-Smear WSI classification framework.

BACKGROUND AND OBJECTIVES: Cervical cancer affects around 0.5 million women per year, resulting in over 0.3 million fatalities. Therefore, repetitive screening for cervical cancer is of utmost importance. Computer-assisted diagnosis is key for scaling up cervical cancer screening. Current recognition algorithms, however, perform poorly on the whole-slide image (WSI) analysis, fail to generalize for different staining methods and on uneven distribution for subtype imaging, and provide sub-optimal clinical-level interpretations. Herein, we developed CervixFormer-an end-to-end, multi-scale swin transformer-based adversarial ensemble learning framework to assess pre-cancerous and cancer-specific cervical malignant lesions on WSIs. METHODS: The proposed framework consists of (1) a self-attention generative adversarial network (SAGAN) for generating synthetic images during patch-level training to address the class imbalanced problems; (2) a multi-scale transformer-based ensemble learning method for cell identification at various stages, including atypical squamous cells (ASC) and atypical squamous cells of undetermined significance (ASCUS), which have not been demonstrated in previous studies; and (3) a fusion model for concatenating ensemble-based results and producing final outcomes. RESULTS: In the evaluation, the proposed method is first evaluated on a private dataset of 717 annotated samples from six classes, obtaining a high recall and precision of 0.940 and 0.934, respectively, in roughly 1.2 minutes. To further examine the generalizability of CervixFormer, we evaluated it on four independent, publicly available datasets, namely, the CRIC cervix, Mendeley LBC, SIPaKMeD Pap Smear, and Cervix93 Extended Depth of Field image datasets. CervixFormer obtained a fairly better performance on two-, three-, four-, and six-class classification of smear- and cell-level datasets. For clinical interpretation, we used GradCAM to visualize a coarse localization map, highlighting important regions in the WSI. Notably, CervixFormer extracts feature mostly from the cell nucleus and partially from the cytoplasm. CONCLUSIONS: In comparison with the existing state-of-the-art benchmark methods, the CervixFormer outperforms them in terms of recall, accuracy, and computing time.

Exploration of Potential Breath Biomarkers of Chronic Kidney Disease through Thermal Desorption-Gas Chromatography/Mass Spectrometry.

Breath volatile organic compound (VOC) analysis is a non-invasive tool for assessing health status; the compositional profile of these compounds in the breath of patients with chronic kidney disease is believed to change with decreasing renal function. We aimed to identify breath VOCs for recognizing patients with chronic kidney disease. Using thermal desorption-gas chromatography/mass spectrometry, untargeted analysis of breath markers was performed using breath samples of healthy controls (n = 18) versus non-dialysis (n = 21) and hemodialysis (n = 12) patients with chronic kidney disease in this cross-sectional study. A total of 303 VOCs alongside 12 clinical variables were used to determine the breath VOC profile. Metabolomic analysis revealed that age, systolic blood pressure, and fifty-eight breath VOCs differed significantly between the chronic kidney disease group (non-dialysis + hemodialysis) and healthy controls. Thirty-six VOCs and two clinical variables that showed significant associations with chronic kidney disease in the univariate analysis were further analyzed. Different spectra of breath volatile organic compounds between the control and chronic kidney disease groups were obtained. A multivariate model incorporating age, 2-methyl-pentane, and cyclohexanone showed high performance (accuracy, 86%) in identifying patients with chronic kidney disease with odds ratios of 0.18 (95% CI, 0.07-2.49, p = 0.013); 2.10 (0.94-2.24, p = 0.025); and 2.31 (0.88-2.64, p = 0.008), respectively. Hence, this study showed that renal dysfunction induces a characteristic profile of breath VOCs that can be used as non-invasive potential biomarkers in screening tests for CKD.

POEMS Syndrome: Presented as Idiopathic Multicentric Castleman Disease of Plasma Cell Variant for Eight Years and Dramatic Treatment with Siltuximab Followed by Autologous Peripheral Blood Stem Cell Transplantation.

BACKGROUND: POEMS syndrome (POEMS) is a rare plasma cell clonal paraneoplastic syndrome consisting of polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin changes presenting with idiopathic multicentric Castleman disease (iMCD) histology, the treatment of which has not yet been well established. iMCD is also a distinctive rare non-clonal lymphoproliferative disorder, of which dramatic response to Siltuximab, a monoclonal anti-IL-6 antibody, has been reported recently. METHODS: the differential diagnosis between POEMS and iMCD can be very challenging because of the identical histology, overlapping similar symptoms such as polyneuropathy, and vital signs insidiously presented to diagnose POEMS. RESULTS: here, we report the case of a 53-year-old man with iMCD treated for 8 years developing sequential polyneuropathy, monoclonal gammopathy, and bone lytic lesions, all of which were confirmed after his iMCD achieved complete remission resulting from siltuximab administration and finally confirmed as POEMS. CONCLUSIONS: we describe the clinical ambiguity of disease presenting that we can face in the real world between iMCD and POEMS and emphasise careful, enduring observation lasting several years.

Rapid identification of SARS-CoV-2 in the point-of-care using digital PCR-based Dr. PCR™ Di20K COVID-19 Detection Kit without viral RNA extraction.

BACKGROUND: Since COVID-19 was declared the pandemic by the WHO, it has continued to spread. There is a need for rapid, efficient, and accurate diagnostic kits and techniques to control its spread. OBJECTIVE: The diagnostic capability of the qRT-PCR-based Real-Q 2019-nCoV Detection Kit and dPCR-based Dr. PCR™ Di20K COVID-19 Detection Kit was compared and evaluated. METHODS: Diagnostic tests for COVID-19 were performed using two different COVID-19 kits and 301 individual specimens with confirmed COVID-19 positive/negative at the government-accredited medical institution. Assessment of diagnostic capability was measured through diagnostic sensitivity, specificity, Cohen's Kappa coefficient, and dilutional linearity tests. RESULTS: The COVID-19 diagnostic test results using two kits and 301 individual specimens perfectly matched the pre-diagnosis results of the medical institution. In addition, the measurement results of diagnostic sensitivity and specificity were "1", indicating high diagnostic capability. Cohen's Kappa coefficient value is "1", which means that the diagnosis concordance between the two kits is "Almost Perfect". As a result of dilutional linearity tests to evaluate their detection capability, both kits were measured with very high detection reliability. CONCLUSION: Here, we propose that the dPCR-based Dr. PCR™ Di20K COVID-19 Detection Kit has the advantages of the dPCR method reported in the previous study and is suitable for point-of-care testing (POCT) by overcoming the limitations of space, test time, cross-over contamination, and biosafety due to omitting RNA extraction process.

SK1 Inhibitor RB005 Induces Apoptosis in Colorectal Cancer Cells through SK1 Inhibition Dependent and Independent Pathway.

BACKGROUND AND OBJECTIVE: Colorectal cancer (CRC) is the fourth leading cause of cancer- related death globally, with a high incidence rate in economically fast-growing countries. Sphingosine- 1-phosphate (S1P) is a bioactive lipid mediator that plays critical roles in cancer cell proliferation, migration, and angiogenesis converted by the isoforms of sphingosine kinase (SK1 and SK2). SK1 is highly expressed in colorectal cancer; its inhibitors suppress the formation of S1P and increase ceramide levels having a pro-apoptotic function. RB005 is a selective SK1 inhibitor and a structural analog of PP2A activator FTY720. The purpose of this study is to test whether RB005, an SK1 inhibitor, can be used as an anticancer agent by inhibiting the growth of colon cancer cells. METHODS: We performed MTT and colony-forming assay using colon cancer cell lines HT29 and HCT116 cells to examine the cell toxicity effect of RB005. To determine whether apoptosis of RB005 in colon cancer cell line is due to SK1 inhibition or other mechanisms due to its structural similarity with FTY720, we conducted LC/MS, siRNA knockdown, and PP2A activity experiments. RESULTS: RB005 notably inhibited CRC cell growth and proliferation compared to PF543 and ABC294640 by inducing the mitochondria-mediated intrinsic apoptotic pathway. Apoptotic cell death is caused by increased mitochondrial permeability Initiated by the activation of pro-apoptotic protein BAX, increased ceramides, and activation of PP2A. Also, RB005 treatment in HT29 cells did not change the expression level of SK1, but strikingly decreased SK1 activity and S1P levels. All these events of cell death and apoptosis were less effective when SK1 was knocked down by siRNA. CONCLUSION: This result indicates that RB005 shows the in-vitro anti-CRC effect by inhibiting SK1 activity and PP2A activation, increasing proapoptotic ceramide levels following the activation of the intrinsic apoptotic pathway.

A Dansyl-Modified Sphingosine Kinase Inhibitor DPF-543 Enhanced De Novo Ceramide Generation.

Sphingosine-1-phosphate (S1P) synthesized by sphingosine kinase (SPHK) is a signaling molecule, involved in cell proliferation, growth, differentiation, and survival. Indeed, a sharp increase of S1P is linked to a pathological outcome with inflammation, cancer metastasis, or angiogenesis, etc. In this regard, SPHK/S1P axis regulation has been a specific issue in the anticancer strategy to turn accumulated sphingosine (SPN) into cytotoxic ceramides (Cers). For these purposes, there have been numerous chemicals synthesized for SPHK inhibition. In this study, we investigated the comparative efficiency of dansylated PF-543 (DPF-543) on the Cers synthesis along with PF-543. DPF-543 deserved attention in strong cytotoxicity, due to the cytotoxic Cers accumulation by ceramide synthase (CerSs). DPF-543 exhibited dual actions on Cers synthesis by enhancing serine palmitoyltransferase (SPT) activity, and by inhibiting SPHKs, which eventually induced an unusual environment with a high amount of 3-ketosphinganine and sphinganine (SPA). SPA in turn was consumed to synthesize Cers via de novo pathway. Interestingly, PF-543 increased only the SPN level, but not for SPA. In addition, DPF-543 mildly activates acid sphingomyelinase (aSMase), which contributes a partial increase in Cers. Collectively, a dansyl-modified DPF-543 relatively enhanced Cers accumulation via de novo pathway which was not observed in PF-543. Our results demonstrated that the structural modification on SPHK inhibitors is still an attractive anticancer strategy by regulating sphingolipid metabolism.

A solvent-free headspace GC/MS method for sensitive screening of N-nitrosodimethylamine in drug products.

A solvent-free headspace gas chromatography-mass spectrometry (SF-HS-GC/MS) method was developed and validated for screening N-nitrosodimethylamine (NDMA) in various active pharmaceutical ingredients (APIs) and drug products. Experimental parameters such as incubation temperature, incubation time, and sample volume in solvent-free headspace conditions were optimized. The developed SF-HS-GC/MS method was validated in terms of linearity, limit of quantification (LOQ), precision, and accuracy. The results indicated excellent linearity from 5 to 500 ng g-1 with correlation coefficients higher than 0.9999. The LOQ of this method was 5 ng g-1 and matrix effects ranged from 0.97 to 1.11. The accuracy ranged from 92.77 to 106.54% and the precision RSDs were below 5.94%. No significant matrix effect was observed for any of the drug products. Also, artefactual NDMA formation in ranitidine, nizatidine, and metformin was investigated under HS conditions. Adjusted (mild) SF-HS conditions were suggested for precise quantification of NDMA in positive drug products by GC/MS. The present SF-HS-GC/MS method is a promising tool for the screening and determination of toxic NDMA in APIs and drug products.

Chiral polymer hosts for circularly polarized electroluminescence devices.

Polymer electroluminescence devices producing circularly polarized luminescence (CP PLEDs) have valuable photonic applications. The fabrication of a CP PLED requires a polymer host that provides the appropriate chiral environment around the emitting dopant. However, chemical strategies for the design of chiral polymer hosts remain underdeveloped. We have developed new polymer hosts for CP PLED applications. These polymers were prepared through a free-radical polymerization of 3-vinylcarbazole with a chiral N-alkyl unit. This chiral unit forces the carbazole repeat units to form mutually helical half-sandwich conformers with preferred (P)-helical sense along the polymer main chain. Electronic circular dichroism measurements demonstrate the occurrence of chirality transfer from chiral monomers to achiral monomers during chain growth. The (P)-helical-sense-enriched polymer interacts diastereoselectively with an enantiomeric pair of new phosphorescent (R)- and (S)-dopants. The magnitude of the Kuhn dissymmetry factor (g abs) for the (P)-helically-enriched polymer film doped with the (R)-dopant was found to be one order of magnitude higher than that of the film doped with the (S)-dopant. Photoluminescence dissymmetry factors (g PL) of the order of 10-3 were recorded for the doped films, but the magnitude of diastereomeric enhancement decreased to that of g abs. The chiral polymer host permits faster energy transfer to the phosphorescent dopants than the achiral polymer host. Our photophysical and morphological investigations indicate that the acceleration in the chiral polymer host is due to its longer Förster radius and improved compatibility with the dopants. Finally, multilayer CP PLEDs were fabricated and evaluated. Devices based on the chiral polymer host with the (R)- and (S)-dopants exhibit electroluminescence dissymmetry factors (g EL) of 1.09 × 10-4 and -1.02 × 10-4 at a wavelength of 540 nm, respectively. Although challenges remain in the development of polymer hosts for CP PLEDs, our research demonstrates that chiroptical performances can be amplified by using chiral polymer hosts.

Investigation of high correlation with carcass traits of SNPs of the PLCB1, C/EBPα, and TDRKH genes and the combinations of SNPs using the MDR method in the Hanwoo.

BACKGROUND: Recently, many researchers focus on the best way to produce high-quality meat, as the trend in food consumption today is to focus on quality. In general, consumers' preferences in beef differ depending on taste and meatiness. Therefore, researchers are interested in how the marbling score affects the flavors of meat or the various factors that make up the meatiness to captivate the consumers' tastes. OBJECTIVE: This study identifies single nucleotide polymorphisms (SNPs) or gene combinations that affect the carcass traits of Korean cattle (Hanwoo) by using the multifactor dimensionality reduction (MDR) method. METHODS: We collected the candidate SNPs to identify SNPs related to marbling scores from whole-exome sequencing and bovine SNP genotyping data. Using 96 Hanwoo samples, we performed PCR amplification to investigate the polymorphism status. In addition, we investigated genetic relationships between carcass traits and SNPs using 612 Hanwoo samples. Furthermore, each candidate SNP genotype and the combinations of SNP genotypes were verified to improve the accuracy of genetic relationships using MDR method. RESULTS: Twenty-four candidate SNPs associated with carcass trait and marbling scores were identified from SNP genotyping and whole-exome sequencing. Among them, three SNP markers (c.459 T > C of the PLCB1 gene, c.271 A > C of the C/EBPα gene, and g.17257 A > G of the TDRKH gene) were showed statistically significant differences between intramuscular fat and genotypes. Especially, two candidate SNPs, including c.459 T > C located in the PLCB1 gene and c.271 A > C located in the C/EBPα gene, could be highly associated with the intramuscular fat of Hanwoo quality grade. In addition, the combination of SNP genotypes is showed higher significant differences with carcass weight, backfat thickness, and longissimus dorsi muscle area. CONCLUSION: Three SNP genotypes and the combination of SNP genotypes in the PLCB1, C/EBPα, and TDRKH genes may be useful genetic markers for improving beef quality.

Incidental Detection of Ovarian Cancer as a Solitary Peridiaphragmatic Distant Lymph Node Metastasis without Pelvic Lesions on 18F-FDG PET/CT.

The spreading pattern of ovarian carcinoma is unique and unlike most other cancers, because exfoliated ovarian cancer cells primarily disseminate within the abdominal cavity, which are then transported throughout the peritoneum by physiological peritoneal fluid. An initial manifestation of a solitary peridiaphragmatic distant metastatic lymph node without peritoneal involvement is very rare. This study reports a case with an incidentally found single hypermetabolic mass in the peridiaphragmatic space without a pelvic lesion in the baseline staging 18 F-FDG PET/CT that histologically turned out to be metastatic serous papillary carcinoma due to ovarian cancer. 18F-FDG PET/CT may allow the identification of the initial manifestation of unexpected distant oligometastatic statuses of an unknown primary ovarian cancer.

Activation of Hair Cell Growth Factors by Linoleic Acid in Malva verticillata Seed.

Hair loss by excessive stress from work and lifestyle changes has become a growing concern, particularly among young individuals. However, most drugs for alopecia impose a plethora of side effects. We have found the powerful impact of Malva verticillata seed extracts on alleviating hair loss. This study further isolated effective chemicals in M. verticillata seed extracts by liquid silica gel column chromatography. Under the screening for the growth rate (%) of human follicles dermal papilla cells (HFDPCs), we identified linoleic acid (LA) and oleic acid in n-hexane of M. verticillate (MH)2 fraction. LA treatment activated Wnt/ß-catenin signaling and induced HFDPCs growth by increasing the expression of cell cycle proteins such as cyclin D1 and cyclin-dependent kinase 2. LA treatment also increased several growth factors, such as vascular endothelial growth factor, insulin-like growth factor-1, hepatocyte growth factor, and keratinocyte growth factor, in a dose-dependent manner. Besides, LA significantly inhibited Dickkopf-related protein expression (DKK-1), a primary alopecia signaling by dihydrotestosterone. Our findings suggest that LA treatment may alleviate a testosterone-induced signaling molecule and induces HFDPCs growth by activating Wnt/ß-catenin signaling.

Twist to Boost: Circumventing Quantum Yield and Dissymmetry Factor Trade-Off in Circularly Polarized Luminescence.

Circularly polarized luminescence (CPL) enables promising applications in asymmetric photonics. However, the performances of CPL molecules do not yet meet the requirements of these applications. The shortcoming originates from the trade-off in CPL between the photoluminescence quantum yield (PLQY) and the photoluminescence dissymmetry factor (gPL). In this study, we developed a molecular strategy to circumvent this trade-off. Our approach takes advantage of the strong propensity of [Pt(N^C^N)Cl], where the N^C^N ligand is 1-(2-oxazoline)-3-(2-pyridyl)phenylate, to form face-to-face stacks. We introduced chiral substituents, including (S)-methyl, (R)- and (S)-isopropyl, and (S)-indanyl groups, into the ligand framework. This asymmetric control induces torsional displacements that give homohelical stacks of the Pt(II) complexes. X-ray single-crystal structure analyses for the (S)-isopropyl Pt(II) complex reveal the formation of a homohelical dimer with a Pt···Pt distance of 3.48 Å, which is less than the sum of the van der Waals radii of Pt. This helical stack elicits the metal-metal-to-ligand charge-transfer (MMLCT) transition that exhibits strong chiroptical activity due to the electric transition moment making an acute angle to the magnetic transition moment. The PLQY and gPL values of the MMLCT phosphorescence emission of the (S)-isopropyl Pt(II) complex are 0.49 and 8.4 × 10-4, which are improved by factors of ca. 6 and 4, respectively, relative to the values of the unimolecular emission (PLQY, 0.078; gPL, 2.4 × 10-4). Our photophysical measurements for the systematically controlled Pt(II) complexes reveal that the CPL amplifications depend on the chiral substituent. Our investigations also indicate that excimers are not responsible for the enhanced chiroptical activity. To demonstrate the effectiveness of our approach, organic electroluminescence devices were fabricated. The MMLCT emission devices were found to exhibit simultaneous enhancements in the external quantum efficiency (EQE, 9.7%) and the electroluminescence dissymmetry factor (gEL, 1.2 × 10-4) over the unimolecular emission devices (EQE, 5.8%; gEL, 0.3 × 10-4). These results demonstrate the usefulness of using the chiroptically active MMLCT emission for achieving an amplified CPL.

A Bioassay Using a Pentadecanal Derivative to Measure S1P Lyase Activity.

Sphingosine-1-phosphate (S1P) is a unique lipid ligand binding to S1P receptors to transduce various cell survival or proliferation signals via small G proteins. S1P lyase (S1PL) is the specific enzyme that degrades S1P to phosphoethanolamine and (2E)-hexadecenal and therefore regulates S1P levels. S1PL also degrades dihydrosphingosine-1-phosphate (Sa1P), with a higher affinity to produce hexadecanal. Here, we developed a newly designed assay using a C17-Sa1P substrate that degrades into pentadecanal and phosphoethanolamine. For higher sensitivity in pentadecanal analysis, we developed a quantitative protocol as well as a 5,5-dimethyl cyclohexanedione (5,5-dimethyl CHD) derivatization method. The derivatization conditions were optimized for the reaction time, temperature, and concentrations of the 5,5-dimethyl CHD reagent, acetic acid, and ammonium acetate. The S1PL reaction in the cell lysate after spiking 20 µM of C17-Sa1P for 20 min was linear to the total protein concentrations of 50 µg. The S1PL levels (4 pmol/mg/min) were readily detected in this HPLC with fluorescence detection (λex = 366 nm, λem = 455 nm). The S1PL-catalyzed reaction was linear over 30 min and yielded a Km value of 2.68 µM for C17-Sa1P. This new method was validated to measure the S1PL activity of mouse embryonal carcinoma cell lines of the standard cell (F9-0), S1PL knockdown cells (F9-2), and S1PL-overexpressed cells (F9-4). Furthermore, we treated F9-4 cells with different S1PL inhibitors such as FTY720, 4-deoxypyridoxine (DOP), and the deletion of pyridoxal-5-phosphate (P5P), an essential cofactor for S1PL activity, and observed a significant decrease in pentadecanal relative to the untreated cells. In conclusion, we developed a highly sensitive S1PL assay using a C17-Sa1P substrate for pentadecanal quantification for application in the characterization of S1PL activity in vitro.

Molecular subtypes of triple-negative breast cancer: understanding of subtype categories and clinical implication.

BACKGROUND: Triple-negative breast cancer (TNBC) is a heterogeneous entity that encompasses several subtypes with distinct molecular characteristics. The patients with TNBCs show unpredictable response to the chemotherapy, and further there is the lack of effective agents. Thus, many studies have been underway to discover targeted therapy suitable for patients with specific genetic alterations in each molecular subtypes. TNBCs are classified as four major molecular subtypes according to the gene expression patterns. These are luminal androgen receptor (LAR), mesenchymal-like, immunomodulatory (IM), and basal-like types. CONCLUSION: Here, we discuss the unique molecular features of each subtype as well as promising targets for anti-cancer therapy.