Liquid Chromatography-mass Spectrometry Assay Research Articles

Discovery of a Carbamoyl Phosphate Synthetase 1-Deficient HCC Subtype With Therapeutic Potential Through Integrative Genomic and Experimental Analysis.

Metabolic reprogramming plays an important role in tumorigenesis. However, the metabolic types of different tumors are diverse and lack in-depth study. Here, through analysis of big databases and clinical samples, we identified a carbamoyl phosphate synthetase 1 (CPS1)-deficient hepatocellular carcinoma (HCC) subtype, explored tumorigenesis mechanism of this HCC subtype, and aimed to investigate metabolic reprogramming as a target for HCC prevention. A pan-cancer study involving differentially expressed metabolic genes of 7,764 tumor samples in 16 cancer types provided by The Cancer Genome Atlas (TCGA) demonstrated that urea cycle (UC) was liver-specific and was down-regulated in HCC. A large-scale gene expression data analysis including 2,596 HCC cases in 7 HCC cohorts from Database of HCC Expression Atlas and 17,444 HCC cases from in-house hepatectomy cohort identified a specific CPS1-deficent HCC subtype with poor clinical prognosis. In vitro and in vivo validation confirmed the crucial role of CPS1 in HCC. Liquid chromatography-mass spectrometry assay and Seahorse analysis revealed that UC disorder (UCD) led to the deceleration of the tricarboxylic acid cycle, whereas excess ammonia caused by CPS1 deficiency activated fatty acid oxidation (FAO) through phosphorylated adenosine monophosphate-activated protein kinase. Mechanistically, FAO provided sufficient ATP for cell proliferation and enhanced chemoresistance of HCC cells by activating forkhead box protein M1. Subcutaneous xenograft tumor models and patient-derived organoids were employed to identify that blocking FAO by etomoxir may provide therapeutic benefit to HCC patients with CPS1 deficiency. In conclusion, our results prove a direct link between UCD and cancer stemness in HCC, define a CPS1-deficient HCC subtype through big-data mining, and provide insights for therapeutics for this type of HCC through targeting FAO.

Development of Asialoglycoprotein-Mediated Hepatocyte-Targeting Antitumor Prodrugs Triggered by Glutathione.

One antitumor β-elemene derivative W-105 and three novel hepatocyte-targeting prodrugs (W-1-5, W-2-9, and W-3-8) were designed and synthesized. W-105 (IC50 6.107 μM) could cause cell apoptosis through upregulating the activity of caspase-3. The hepatocyte-targeting capacities of the aimed compounds followed the W-105 (parent compound) < W-1-5 (monodentate-galactose) < W-2-9 (bidentate-galactose) < W-3-8 (tridentate-galactose) order, which is attributed to the excellent affinity of the galactose ligand to ASGPR and the galactose-cluster recognition effect. Furthermore, prodrugs W-3-8 exhibited good antitumor activity and low toxic side effects. The liquid chromatography-mass spectrometry (LC-MS) assays revealed that prodrugs (W-1-5, W-2-9, and W-3-8) could release the antitumor pharmacophore in the presence of GSH (mimic the condition of the tumor cell) and maintain the low-toxic structures in the absence of GSH (mimic the condition of the normal cell). The release mechanisms of prodrugs were also proposed. Overall, these prodrugs developed in this study had potential in the treatment of liver cancer.

Risk of breast cancer and prediagnostic urinary excretion of bisphenol A, triclosan and parabens: The Multiethnic Cohort Study.

Exposure to bisphenol A (BPA), triclosan and parabens is widespread but their impact on breast cancer risk remains unclear. This nested case-control study investigated endocrine-disrupting chemicals (EDCs) and breast cancer risk within the Multiethnic Cohort (MEC). We measured prediagnostic urinary BPA, triclosan and parabens in 1032 postmenopausal women with breast cancer (48 African American, 77 Latino, 155 Native Hawaiian, 478 Japanese American and 274 White) and 1030 individually matched controls, using a sensitive and validated liquid chromatography mass spectrometry assay. Conditional logistic regression was used to examine risk with these EDCs with adjustment for creatinine and potential confounders. In all women, breast cancer risk was not associated with BPA (Ptrend =0.53) and was inversely associated with triclosan (ORT3 vs T1 =0.83, 95% CI: 0.66-1.04, Ptrend =0.045) and total parabens (ORT3 vs T1 =0.77, 95% CI: 0.62-0.97, Ptrend =0.03). While risk of hormone receptor positive (HR+) cancer was 20% to 23% lower among women in the upper two tertiles of paraben exposure (Ptrend =0.02), risk of HR negative (HR-) was reduced 27% but only among those in the upper tertile of exposure. Although risk associations did not differ significantly by ethnicity or by body mass index (BMI), the inverse association with triclosan was observed mainly among overweight/obese women (ORT3 vs T1 =0.76, 95% CI: 0.56-1.02, Ptrend =0.02). In summary, breast cancer risk in a multiethnic population was unrelated to BPA and was weakly inversely associated with triclosan and paraben exposures. Studies with multiple urine samples collected before breast cancer diagnosis are needed to further investigate these EDCs and breast cancer risk.

An Aptamer-Based Colorimetric Assay Integrated a Portable Spectrometer for on-Site Detection of Pbtx-2

Introduction Brevetoxins (named using a notation system PbTx 1-n) produced by the algae species Karenia brevis have been increasing in geographical distribution [1]. Humans’ exposure to brevetoxins can occur through consumption of contaminated shellfish as well as aerosol exposure, causing a series of adverse symptoms clinically described as neurologic shellfish poisoning (NSP) including cramps, diarrhea, and coma, etc. Current methods for brevetoxin detection such as mouse bioassay (MBA), high-performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA), etc., feature the advantages of high sensitivity and reliability [2]. However, the dependence on bulk volume laboratory equipment and complicated pretreatment limit the widespread application in on-site brevetoxins detection. Therefore, a sensitive and efficient method for on-site determination of brevetoxin in contaminated shellfish is of great necessity.In this work, an aptamer-based colorimetric assay is designed for sensitive detection of PbTx-2 which is the most predominant form of brevetoxins. The concentration of PbTx-2 can be easily transformed to the absorbance change which can either be directly visualized by a color change or can be quantified using a portable spectrometer. This approach provides an efficient way for PbTx-2 on-site detection combining the low cost and sensitivity of aptamer and the portability of self-designed spectrometer. Method The self-designed portable spectrometer (SDPS) is composed of a spectrometer sensor, a broad-band LED module (wavelength range of 470-850 nm), a micro-control module, and a communication module to contact with portable devices e.g., a personal computer (PC) (Fig.1 B, C). Optic fibers were used for guiding light emitted from the LED or reflected from the sample to reduce the volume of SDPS.Aptamer BT10 was synthesized with high affinity to PbTx-2 according to published literature and it was heat-treatment at 92 ℃ for 10 min, followed by snap-chilling on ice for 5min and bringing it back to the room temperature to get an appropriate secondary structure [3]. Tyrosine-capped AuNPs were synthesized using tyrosine amino acid as a reducing and capping agent. 10 µL BT10 with a concentration of 0.3 µM was incubated with 20 µL different concentrations of PbTx-2 for 30 min to ensure complete binding. Then, 100 µL AuNPs solution was added into the above mixture for 5 min followed by 5 µL positively charged 3,3',5,5'-tetramethylbenzidine (TMB) for 5 min to induce aggregation of AuNPs. Therefore, free aptamers can be adsorbed on the surface of AuNPs and prevent the AuNPs from aggregation while the aptamer binding to PbTx-2 cannot (Fig.1 A). All reactions were performed in a solution chamber and finally the chamber was placed into the SDPS. Through signal processing on a PC, the concentration of PbTx-2 can be determined. Results and Conclusions From the spectrum results, the absorbance at 520 nm is decreasing while the absorbance at 610 nm is increasing with the concentration of PbTx-2 increasing (Fig.1 D). The ratio of absorbance at 610 nm and 520 nm is linear to the concentration of PbTx-2 with a linear range of 0.1 ppm to 10 ppm (Fig.1 E). Moreover, tyrosine-capped instead of citrate-capped AuNPs were chosen for colorimetric assay for experiment results show PbTx-2 can interact with citrate-capped not the tyrosine-capped AuNPs. The positively charged TMB was utilized for inducing AuNPs aggregation not the traditional sodium solution, for we find TMB can be more sensitive for aptamer number change on the surface of AuNPs. This approach allowed a limit of detection of 0.05 ppm PbTx-2 within a detection time of 40 min. Compared with other methods for PbTx-2 detection, this approach features portability and cost-effectiveness, which can be used in resource-limited areas.

Cortisol as biomarker for CYP17 inhibition in mCRPC patients treated with abiraterone acetate.

5035 Background: Abiraterone acetate is an effective metastatic castration resistant prostate cancer (mCRPC) treatment, however, there is a high variability in response. It inhibits CYP17, thereby preventing the production of androgens. Abiraterone trough concentrations (Cmin) &gt; 8.4 ng/mL have been associated with an increased progression free survival (PFS) (Eur J Cancer. 2017;72:54-61; Prostate Cancer Prostatic Dis. 2020;23(2):244-251). However, plasma levels do not directly provide information on the level of CYP17 inhibition. Ideally, testosterone levels should be measured, but these levels are below the detection limit of available assays. The synthesis of cortisol is also inhibited by abiraterone via CYP17 inhibition and might therefore be a biomarker for CYP17 inhibition. The objective of this study was to investigate if cortisol levels are related to abiraterone levels and to clinical response. Methods: An observational study was performed in mCRPC patients treated with abiraterone acetate. At the outpatient clinic, plasma samples were collected for pharmacokinetic (PK) monitoring at each hospital visit. Reference populations of healthy volunteers and mCRPC patients using enzalutamide were included to investigate the influence of mCRPC and abiraterone treatment on the circadian rhythm of cortisol. Abiraterone and cortisol levels were measured using validated liquid chromatography-mass spectrometry (LC-MS/MS) assays. Clinical (prostate specific antigen (PSA) independent) PFS and PSA response were evaluated. Results: In total, 117 mCRPC patients using abiraterone acetate, 100 mCRPC patients using enzalutamide and 12 healthy volunteers were included. A clear circadian rhythm of cortisol was described in healthy volunteers and unaffected in mCRPC patients using enzalutamide. Contrarily, a circadian rhythm of cortisol could not be identified in mCRPC patients using abiraterone acetate, due to continuous suppression throughout the day. Patients with an abiraterone Cmin &gt; 8.4 ng/mL (n = 77) had a median cortisol concentration of 1.03 ng/mL vs. 2.59 ng/mL in patients with an abiraterone Cmin ≤8.4 ng/mL (n = 40) (p = 0.020). The median cortisol concentration in PSA responders (n = 63) was 1.02 ng/mL vs. 2.59 ng/mL for PSA non responders (n = 54) (p = 0.037). Patients in the highest cortisol tertile ( &gt; 3.03 ng/mL) had a median PFS of 3.7 months (n = 39) vs. 13.8 months in patients with cortisol levels ≤3.03 ng/mL (n = 78) (p = 0.007). The median PFS was 16.3 months in patients with an abiraterone Cmin &gt; 8.4 ng/mL and cortisol concentration &lt; 3.03 ng/ml vs. 5.3 months in patients with an abiraterone Cmin &gt; 8.4 ng/mL and cortisol concentration &gt; 3.03 ng/ml (p = 0.02). Conclusions: This study demonstrates that cortisol levels are of additional value to abiraterone concentrations as a marker for abiraterone acetate efficacy. This might help be helpful to assess efficacy of abiraterone treatment.

Stability-indicating LC-MS Method for Determination of Stability of Extemporaneously Compounded Buprenorphine Oral Syringes for Neonatal Abstinence Syndrome.

In the hospital settings, buprenorphine is used for the treatment of patients with neonatal abstinence syndrome. It is extemporaneously compounded and stored in oral plastic syringes. However, limited information exists about the stability of buprenorphine and its compounded formulations when stored under specific conditions. Hence, we developed a stability-indicating high-performance liquid chromatography-mass spectrometry (LC-MS) method to analyze the stability of buprenorphine over time. A stability-indicating LC-MS method was developed to map the potential degradation peaks of buprenorphine when exposed to acidic, basic, and oxidative conditions. This method was used to study the stability of compounded buprenorphine oral syringes stored under refrigeration (2°C-8°C) and room temperature (25°C ± 2°C with 60% relative humidity). Syringes from each storage condition were assessed for stability using pH meter and stability-indicating LC-MS assay for 30 days. Buprenorphine gets completely degraded in the presence of acid at the end of 1 hour of exposure. Various degradation peaks were identified using LC-MS assay for buprenorphine under acidic, basic, and peroxide conditions. Stability study of oral buprenorphine syringes showed no precipitation, cloudiness, or color change during this study at all storage conditions. The LC-MS assay revealed that buprenorphine oral syringes retained greater than 90% of the initial concentrations for 30 days. Highly sensitive stability-indicating LC-MS method was developed for studying the stability of extemporaneously compounded buprenorphine oral syringes. This study demonstrates that buprenorphine extemporaneous formulation prepared according to the manufacturers' recommendations is stable under refrigerated or room temperature conditions for 30 days in oral plastic syringes.

Bioanalysis in the Age of New Drug Modalities

In the absence of regulatory guidelines for the bioanalysis of new drug modalities, many of which contain multiple functional domains, bioanalytical strategies have been carefully designed to characterize the intact drug and each functional domain in terms of quantity, functionality, biotransformation, and immunogenicity. The present review focuses on the bioanalytical challenges and considerations for RNA-based drugs, bispecific antibodies and multi-domain protein therapeutics, prodrugs, gene and cell therapies, and fusion proteins. Methods ranging from the conventional ligand binding assays and liquid chromatography-mass spectrometry assays to quantitative polymerase chain reaction or flow cytometry often used for oligonucleotides and cell and gene therapies are discussed. Best practices for method selection and validation are proposed as well as a future perspective to address the bioanalytical needs of complex modalities.

Urinary phthalate exposures and risk of breast cancer: the Multiethnic Cohort study

BackgroundThe epidemiologic evidence from observational studies on breast cancer risk and phthalates, endocrine disrupting chemicals, has been inconsistent. In the only previous study based on pre-diagnostic urinary phthalates and risk of breast cancer, results were null in mostly white women.MethodsWe examined the association between pre-diagnostic urinary phthalates and breast cancer in a nested case-control study within the Multiethnic Cohort (MEC) study, presenting the first data from five major racial/ethnic groups in the USA. We measured 10 phthalate metabolites and phthalic acid, using a sensitive liquid chromatography mass spectrometry assay on 1032 women with breast cancer (48 African Americans, 77 Latinos, 155 Native Hawaiians, 478 Japanese Americans, and 274 Whites) and 1030 matched controls. Conditional logistic regression was used to examine risk with individual metabolites and ratios of primary (MEHP, mono-2-ethylhexyl-phthalate) to secondary (MEHHP, mono(2-ethyl-5-hydroxyhexyl); MEOHP, mono(2-ethyl-5-oxohexy)) metabolites of di-2-ethylhexyl phthalate (DEHP), a widely used plasticizer. In addition, we investigated risk associations with high (∑HMWP) and low molecular weight (∑LMWP) phthalates, as well as total phthalates which included high and low molecular weight phthalates with phthalic acid (∑LMHMPA) or without phthalic acid in molar ratios (∑LMHMmolar) and adjusted for creatinine and potential confounders.ResultsAmong all women, breast cancer risk was higher for those in tertile 2 and tertile 3 of primary to secondary metabolites of DEHP (MEHP/(MEHHP + MEOHP)) in comparison to those in tertile 1; the respective odds ratios were 1.32 (95% CI 1.04–1.68) and 1.26 (95% CI 0.96–1.66) (Ptrend = 0.05). Risk among Native Hawaiian women increased with exposures to eight of ten individual phthalates and total phthalates (∑LMHMPA ORT3 vs T1 = 2.66, 95% CI 1.39–5.12, Ptrend = 0.001). In analysis by hormone receptor (HR) status, exposure above the median of ∑LMWP was associated with an increased risk of HR-positive breast cancer (OR = 1.30, 95% CI 1.05–1.60) while above the median exposure to phthalic acid was associated with an increased risk of HR-negative breast cancer (ORabove vs below median = 1.59, 95% CI 1.01–2.48).ConclusionsFurther investigations of suggestive associations of elevated breast cancer risk with higher ratios of primary to secondary metabolites of DEHP, and differences in risk patterns by race/ethnicity and HR status are warranted.

An exploratory study on the association of lifestyle factors with serum etonogestrel concentrations among contraceptive implant users

Purpose To explore how diet and exercise habits associate with serum etonogestrel concentrations among contraceptive implant users. Materials and methods We conducted a secondary analysis of healthy, reproductive-age women using etonogestrel implants. This study was registered on ClinicalTrials.gov, NCT03092037. We assessed diet and exercise habits with two validated surveys: Healthy Eating Vital Signs and the Stanford Brief Activity Survey. Participants previously had their serum etonogestrel concentrations measured using a validated liquid-chromatography mass-spectrometry assay. We then used linear modelling to test for associations between survey responses and serum etonogestrel concentrations. Results Among 129 participants, diet and exercise habits had no significant associations with serum etonogestrel concentrations (p = 0.22–0.72), with inconsistent effects found for increased caloric intake and sedentary lifestyle. Conclusion This exploratory study found no significant effect of diet or exercise habits on steady-state pharmacokinetics among contraceptive implant users. Clinical Trial Registration Clinicaltrials.gov: NCT03092037

Orexin-A measurement in narcolepsy: A stability study and a comparison of LC-MS/MS and immunoassays

BackgroundOrexin-A and -B are neuropeptides involved in sleep–wake regulation. In human narcolepsy type 1, this cycle is disrupted due to loss of orexin-producing neurons in the hypothalamus. Cerebrospinal fluid (CSF) orexin-A measurement is used in the diagnosis of narcolepsy type 1. Currently available immunoassays may lack specificity for accurate orexin quantification. We developed and validated a liquid chromatography mass spectrometry assay (LC-MS/MS) for CSF orexin-A and B. MethodsWe used CSF samples from narcolepsy type 1 (n = 22) and type 2 (n = 6) and non-narcoleptic controls (n = 44). Stable isotope-labeled orexin-A and -B internal standards were added to samples before solid-phase extraction and quantification by LC-MS/MS. The samples were also assayed by commercial radioimmunoassay (RIA, n = 42) and enzymatic immunoassay (EIA, n = 72) kits. Stability of orexins in CSF was studied for 12 months. ResultsOur assay has a good sensitivity (10 pmol/L = 35 pg/mL) and a wide linear range (35–3500 pg/mL). Added orexin-A and -B were stable in CSF for 12 and 3 months, respectively, when frozen. The median orexin-A concentration in CSF from narcolepsy type 1 patients was <35 pg/mL (range < 35–131 pg/mL), which was lower than that in CSF from control individuals (98 pg/mL, range < 35–424 pg/mL). Orexin-A concentrations determined using our LC-MS/MS assay were five times lower than those measured with a commercial RIA. Orexin-B concentrations were undetectable. ConclusionsOrexin-A concentrations measured by our LC-MS/MS assay were lower in narcolepsy type 1 patients as compared to controls. RIA yielded on average higher concentrations than LC-MS/MS.

The relationships between microbiota and the amino acids and organic acids in commercial vegetable pickle fermented in rice-bran beds

The microbial community during fermented vegetable production has a large impact on the quality of the final products. Lactic acid bacteria have been well-studied in such processes, but knowledge about the roles of non-lactic acid bacteria is limited. This study aimed to provide useful knowledge about the relationships between the microbiota, including non-lactic acid bacteria, and metabolites in commercial pickle production by investigating Japanese pickles fermented in rice-bran. The samples were provided by six manufacturers, divided into two groups depending on the production conditions. The microbiological content of these samples was investigated by high-throughput sequencing, and metabolites were assessed by liquid chromatography-mass spectrometry and enzymatic assay. The data suggest that Halomonas, halophilic Gram-negative bacteria, can increase glutamic acid content during the pickling process under selective conditions for bacterial growth. In contrast, in less selective conditions, the microbiota consumed glutamic acid. Our results indicate that the glutamic acid content in fermented pickle is influenced by the microbiota, rather than by externally added glutamic acid. Our data suggest that both lactic acid bacteria and non-lactic acid bacteria are positive key factors in the mechanism of commercial vegetable fermentation and affect the quality of pickles.

Sublingual Estradiol Is Associated with Higher Estrone Concentrations than Transdermal or Injectable Preparations in Transgender Women and Gender Nonbinary Adults.

Purpose: Serum hormone profiles among different feminizing gender-affirming hormone therapies (GAHT) are poorly characterized. To address this gap, we described the serum estrogen profiles of three 17β-estradiol preparations, taken with or without an antiandrogen, using a novel liquid chromatography-mass spectrometry (LC-MS/MS) assay in adults taking feminizing GAHT. Methods: This was a secondary analysis of 93 healthy transgender women and gender nonbinary adults taking feminizing GAHT in a prospective cross-sectional study. Eligible participants took 17β-estradiol (sublingual tablet, transdermal patch, or intramuscular/subcutaneous injection) with or without oral spironolactone for ≥12 months before study entry. We determined serum estrone and estradiol concentrations for each hormone preparation and described the association between estrone and (1) clinically relevant estradiol concentration ranges (≤200 and >200 pg/mL) and (2) antiandrogen use. To achieve our objectives, we described our protocol for developing an LC-MS/MS assay to measure estrone and estradiol concentrations. Results: Estrone concentrations were higher among participants taking sublingual 17β-estradiol tablets compared with transdermal or injectable preparations (p < 0.0001). Estradiol concentrations were higher for injectable versus transdermal preparations (p = 0.0201), but both were similar to sublingual tablet concentrations (p > 0.05). Estradiol >200 pg/mL (vs. ≤200 pg/mL) was associated with higher estrone concentrations among participants taking sublingual 17β-estradiol, but not transdermal or injectable 17β-estradiol. We observed no association between spironolactone and estrone concentrations (p > 0.5). Conclusion: Estrone concentrations were higher among transgender women and gender nonbinary adults taking sublingual 17β-estradiol compared with transdermal or injectable preparations. The role of estrone in clinical monitoring and the influence of other antiandrogens (e.g., cyproterone acetate) on the estrogen profile remain to be determined.

Octenidine-based hydrogel shows anti-inflammatory and protease-inhibitory capacities in wounded human skin

Octenidine dihydrochloride (OCT) is a widely used antiseptic molecule, promoting skin wound healing accompanied with improved scar quality after surgical procedures. However, the mechanisms by which OCT is contributing to tissue regeneration are not yet completely clear. In this study, we have used a superficial wound model by tape stripping of ex vivo human skin. Protein profiles of wounded skin biopsies treated with OCT-containing hydrogel and the released secretome were analyzed using liquid chromatography-mass spectrometry (LC–MS) and enzyme-linked immunosorbent assay (ELISA), respectively. Proteomics analysis of OCT-treated skin wounds revealed significant lower levels of key players in tissue remodeling as well as reepithelization after wounding such as pro-inflammatory cytokines (IL-8, IL-6) and matrix-metalloproteinases (MMP1, MMP2, MMP3, MMP9) when compared to controls. In addition, enzymatic activity of several released MMPs into culture supernatants was significantly lower in OCT-treated samples. Our data give insights on the mode of action based on which OCT positively influences wound healing and identified anti-inflammatory and protease-inhibitory activities of OCT.

Steroidogenesis in Peripheral and Transition Zones of Human Prostate Cancer Tissue.

The peripheral zone (PZ) and transition zone (TZ) represent about 70% of the human prostate gland with each zone having differential ability to develop prostate cancer. Androgens and their receptor are the primary driving cause of prostate cancer growth and eventually castration-resistant prostate cancer (CRPC). De novo steroidogenesis has been identified as a key mechanism that develops during CRPC. Currently, there is very limited information available on human prostate tissue steroidogenesis. The purpose of the present study was to investigate steroid metabolism in human prostate cancer tissues with comparison between PZ and TZ. Human prostate cancer tumors were procured from the patients who underwent radical prostatectomy without any neoadjuvant therapy. Human prostate homogenates were used to quantify steroid levels intrinsically present in the tissues as well as formed after incubation with 2 µg/mL of 17-hydroxypregnenolone (17-OH-pregnenolone) or progesterone. A Waters Acquity ultraperformance liquid chromatography coupled to a Quattro Premier XE tandem quadrupole mass spectrometer using a C18 column was used to measure thirteen steroids from the classical and backdoor steroidogenesis pathways. The intrinsic prostate tissue steroid levels were similar between PZ and TZ with dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), pregnenolone and 17-OH-pregnenolone levels higher than the other steroids measured. Interestingly, 5-pregnan-3,20-dione, 5-pregnan-3-ol-20-one, and 5-pregnan-17-ol-3,20-dione formation was significantly higher in both the zones of prostate tissues, whereas, androstenedione, testosterone, DHT, and progesterone levels were significantly lower after 60 min incubation compared to the 0 min control incubations. The incubations with progesterone had a similar outcome with 5-pregnan-3,20-dione and 5-pregnan-3-ol-20-one levels were elevated and the levels of DHT were lower in both PZ and TZ tissues. The net changes in steroid formation after the incubation were more observable with 17-OH-pregnenolone than with progesterone. In our knowledge, this is the first report of comprehensive analyses of intrinsic prostate tissue steroids and precursor-driven steroid metabolism using a sensitive liquid chromatography-mass spectrometry assay. In summary, the PZ and TZ of human prostate exhibited similar steroidogenic ability with distinction in the manner each zone utilizes the steroid precursors to divert the activity towards backdoor pathway through a complex matrix of steroidogenic mechanisms.

Hsa_Circ_0062682 Promotes Serine Metabolism and Tumor Growth in Colorectal Cancer by Regulating the miR-940/PHGDH Axis

Background: Colorectal cancer (CRC) is one of the most common malignancy globally. Increasing evidences indicate that circular RNAs (circRNAs) play a pivotal role in various cancers. Methods: Differential circRNAs were determined by edgeR package. The expression of circ_0062682, miR-940 and PHGDH were analyzed by qRT-PCR. We used in vitro proliferation assays and in vivo xenograft model to evaluate the tumorigenic features of CRC cells. Abundance of serine were measured by liquid chromatography mass spectrometry assay. Findings: Increased expression of circ_0062682 in CRC notably correlated with a poorly prognosis and advanced tumor stage. Functional experiments showed that circ_0062682 knockdown reduced CRC growth both in vitro and in vivo. Mechanistically, we revealed that circ_0062682 could sponge miR-940 and identified that PHGDH, a key oxidoreductase involving in serine biosynthesis, as a novel target of miR-940. The expression of PHGDH was downregulated in circ_0062682–depleted or miR-940 overexpressing CRC cells at both mRNA and protein levels. Interpretation: Circ_0062682 promotes serine metabolism and tumor growth in CRC by regulating the miR-940/PHGDH axis, suggesting circ_0062682 as a potential novel therapeutic target for CRC. Funding: This study was partially supported by grants from the National Natural Science Foundation of China (81972220 and 82002964), Medical Key Professionals Program of Jiangsu Province (ZDRCB2016017), Wuxi Medical Innovation Team (CXTP003), and Medical Leading Talents of Wuxi Taihu Lake Talent Plan. Declaration of Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics Approval Statement: Our study was approved by the Research Ethics Committee of Affiliated Hospital of Jiangnan University.

ACPA decreases non-small cell lung cancer line growth through Akt/PI3K and JNK pathways in vitro

Therapeutic agents used for non-small cell lung cancer (NSCLC) have limited curative efficacy and may trigger serious adverse effects. Cannabinoid ligands exert antiproliferative effect and induce apoptosis on numerous epithelial cancers. We confirmed that CB1 receptor (CB1R) is expressed in NSCLC cells in this study. Arachidonoylcyclopropylamide (ACPA) as a synthetic, CB1R-specific ligand decreased proliferation rate in NSCLC cells by WST-1 analysis and real-time proliferation assay (RTCA). The half-maximal inhibitory concentration (IC50) dose of ACPA was calculated as 1.39 × 10−12 M. CB1 antagonist AM281 inhibited the antiproliferative effect of ACPA. Flow cytometry and ultrastructural analyzes revealed significant early and late apoptosis with diminished cell viability. Nano-immunoassay and metabolomics data on activation status of CB1R-mediated pro-apoptotic pathways found that ACPA inhibited Akt/PI3K pathway, glycolysis, TCA cycle, amino acid biosynthesis, and urea cycle and activated JNK pathway. ACPA lost its chemical stability after 24 hours tested by liquid chromatography-mass spectrometry (LC–MS/MS) assay. A novel ACPA-PCL nanoparticle system was developed by nanoprecipitation method and characterized. Sustained release of ACPA-PCL nanoparticles also reduced proliferation of NSCLC cells. Our results demonstrated that low dose ACPA and ACPA-PCL nanoparticle system harbor opportunities to be developed as a novel therapy in NSCLC patients that require further in vivo studies beforehand to validate its anticancer effect.

Bioanalytical Challenges in Support of Complex Modalities of Antibody-Based Therapeutics.

Antibody-based therapeutic classes are evolving from monoclonal antibodies to antibody derivatives with complex structures to achieve advanced therapeutic effect. These antibody derivatives may contain multiple functional domains and are often vulnerable to in vivo biotransformation. Understanding the pharmacokinetics of these antibody derivatives requires a sophisticated bioanalytical approach to carefully characterize the whole drug and each functional domain with respect to quantity, functionality enabled by biotransformation, and corresponding immune responses. Ligand binding assays and liquid chromatography-mass spectrometry assays are predominantly used in bioanalytical support of monoclonal antibodies and are continuously used for antibody derivatives such as antibody drug conjugate and bispecific antibodies. However, they become increasingly cumbersome in coping with increased complexity of drug modality and associated biotransformation. In this mini-review, we examined the current pharmacokinetic assays in the literature for antibody drug conjugate and bispecific antibodies, and presented our view of promising bioanalytical technologies to address the distinct bioanalytical needs of complex modalities.

Postbiotic and Anti-aflatoxigenic Capabilities of Lactobacillus kunkeei as the Potential Probiotic LAB Isolated from the Natural Honey

The use of probiotic, postbiotic, and anti-aflatoxigenic capabilities of the lactic acid bacteria (LAB) isolated from stressful niches is a major strategy to prepare functional cultures and bio-preservatives for food industries. In the present study, abundant LAB strains isolated from natural honey were screened based on their tolerance to continuous pH and bile salt treatments. Then, the pro-functional properties of the selected LAB were investigated. In accordance with the screening data, a bacilli isolate was selected for further characterization. Sequencing results led to the identification of Lactobacillus kunkeei as the selected LAB isolate. In vitro antibacterial and antifungal activities of the LAB and in situ antifungal activity of the isolate cell-free supernatant (CFS) were verified against food-borne indicators. Accordingly, in vitro antibacterial and antifungal effects of Lact. kunkeei ENH01 on respective Escherichia coli and Aspergillus niger were significantly (P < 0.05) higher than the other indicators. Furthermore, in situ inhibitory effect of Lact. kunkeei CFS on Candida albicans (as the highest in situ effect) was equal to 76.36%. The presence of three antibacterial peptides was also verified in the Lact. kunkeei CFS according to the results of liquid chromatography mass spectrometry (LC/MS) assay. Antibiotic susceptibility profile and auto-aggregation ability of the isolate were noticeable. Anti-mycotoxigenic capabilities of Lact. kunkeei ENH01 as viable and heat-killed cells were also revealed against total aflatoxins according to the HPLC-based analysis. In vivo safety of the isolate was also attested through the evaluation of blood biochemistry and hematological parameters in the Lact. kunkeei ENH01 fed-mice compared with the control. Based on the findings, probiotic properties of Lact. kunkeei ENH01 and postbiotic capabilities of the isolate CFS and its heat-killed cells were approved.

SERS-based immunoassay for monitoring cortisol-related disorders

As a natural response to a stressful situation, the human body produces cortisol. For this reason, cortisol is also called “the stress hormone” and is considered to be the principal stress biomarker. Although cortisol response to stress is essential for survival, abnormal levels in biological fluids may represent serious health risks. In this work, we present a cortisol biosensor which relies on a highly sensitive technique (surface-enhanced Raman spectroscopy, SERS) and a specific recognition (immunoassay). Gold nanostars were used as SERS nanotags, since they provided a better response than nanorods or nanospheres. Using the same concept, two different immunoassay modalities were evaluated, using either magnetic beads or gold-coated glass slides decorated with cortisol antibodies as the capture substrates. The magnetically-assisted SERS immunoassay presented a better performance and was therefore selected to quantify cortisol content in biological fluids (urine and serum). Significant advantages of this assay were found over standard methods such as Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and Enzyme-Linked Immunosorbent Assay (ELISA), including higher sensitivity and repeatability, minimum sample preparation, simplicity, and portability. Therefore, the proposed SERS immunoassay might be implemented as a highly efficient tool for in situ monitoring of human stress levels and cortisol-related disorders (e.g. Cushing's syndrome and Addison's disease).

Routine Therapeutic Drug Monitoring of Dabigatran: Experience at a Tertiary Center.

A liquid chromatography-mass spectrometry assay to determine plasma dabigatran concentrations has been available for routine clinical use at our tertiary institutions since 2017. The aim of the study was to describe (1) the use of the assay over time; (2) the indications for testing; and (3) subsequent dabigatran prescribing decisions. Patients for whom dabigatran concentrations were measured were identified using the laboratory database, and clinical data were extracted from the associated electronic health records. There were 233 samples in 24 months. The use of dabigatran increased over time, with a mean (95% confidence interval) increase of +0.5 (0.3-0.7) samples per month. Dabigatran concentrations ranged from <1 to 1060 mcg/L. The main reasons for testing were uncertainty about impact on renal function and drug interactions (39%), to inform prescribing decisions after thromboembolic or bleeding events (21%), and for investigation following dose-adjustment (16%). Dabigatran dose was changed after 30% (68/233) of assay results. The clinical use of the dabigatran assay has increased, with almost one-third of results associated with a subsequent change in dabigatran prescribing.