Semi-quantitative analysis of serum and cerebrospinal fluid transferrin glycoforms by top-down liquid chromatography mass spectrometry.

Detection of ß-2 transferrin in body fluid could help identify cerebrospinal fluid (CSF) leakage. The most common method, isoelectric focusing, was qualitative and could not provide detailed N-glycan structural information. We presented an alternative method using top-down liquid chromatography-time of flight mass spectrometry (LC-TOF MS). After immunoaffinity enrichment, fluid transferrin glycoforms were analyzed by a high-resolution LC-TOF MS, and the N-glycan structure predicted by accurate mass. The performance was validated with imprecision at 15%, with a cut-off of 0.04 for ß-2 transferrin to tetrasialotransferrin ratio to confirm the presence of CSF in fluid samples.

Optical Enantiodifferentiation of Chiral Nitriles.

Chiroptical sensing of nitriles is achieved with excellent functional group tolerance by hydrozirconation and subsequent transmetalation of the corresponding iminate to a chromophoric palladium complex. A one-pot workflow that uses the Schwartz reagent and [(η3-1-tert-butylindenyl)(µ-Cl)Pd]2 as sensor generates a palladium complex displaying red-shifted CD inductions and characteristic UV changes. These chiroptical responses are accurately correlated to the enantiomeric ratio and total concentration of the original nitrile.

Regioconvergent Nucleophilic Substitutions with Morita-Baylis-Hillman Fluorides.

Lithium iodide enables regioconvergent C-F bond functionalization of isomeric Morita-Baylis-Hillman fluorides with carbon, sulfur, and nitrogen nucleophiles. The defluorinative carbon-carbon and carbon-heteroatom bond formations give multifunctional compounds in excellent yields and with good to high diastereoselectivities at room temperature. The possibility of catalytic enantioselective allylation is also discussed.

Optical Relay Sensing of Cryptochiral Alcohols Displaying α-, ß-, γ- and δ-Stereocenters or Chirality by Virtue of Isotopic Substitution.

A reaction-based optical relay sensing strategy that enables accurate determination of the concentration and enantiomeric ratio (er) of challenging chiral alcohols exhibiting stereocenters at the α-, ß-, γ- or even δ-position or hard-to-detect cryptochirality arising from H/D substitution is described. This unmatched application scope is achieved with a conceptually new sensing approach by which the alcohol moiety is replaced with an optimized achiral sulfonamide chromophore to minimize the distance between the covalently attached chiroptical reporter unit and the stereogenic center in the substrate. The result is a remarkably strong, red-shifted CD induction that increases linearly with the sample er. The CD sensing part of the tandem assay is seamlessly coupled to a redox reaction with a quinone molecule to generate a characteristic UV response that is independent of the enantiopurity of the alcohol and thus allows determination of the total analyte concentration. The robustness and utility of the CD/UV relay are further verified by chromatography-free asymmetric reaction analysis with small aliquots of crude product mixtures, paving the way toward high-throughput chiral compound screening workflows which is a highly sought-after goal in the pharmaceutical industry.

Circular Dichroism Sensing: Strategies and Applications.

The analysis of the absolute configuration, enantiomeric composition, and concentration of chiral compounds are frequently encountered tasks across the chemical and health sciences. Chiroptical sensing methods can streamline this work and allow high-throughput screening with remarkable reduction of operational time and cost. During the last few years, significant methodological advances with innovative chirality sensing systems, the use of computer-generated calibration curves, machine learning assistance, and chemometric data processing, to name a few, have emerged and are now matched with commercially available multi-well plate CD readers. These developments have reframed the chirality sensing space and provide new opportunities that are of interest to a large group of chemists. This review will discuss chirality sensing strategies and applications with representative small-molecule CD sensors. Emphasis will be given to important milestones and recent advances that accelerate chiral compound analysis by outperforming traditional methods, conquer new directions, and pioneering efforts that lie at the forefront of chiroptical high-throughput screening developments. The goal is to provide the reader with a thorough understanding of the current state and a perspective of future directions of this rapidly emerging field.

Chemometric sensing of stereoisomeric compound mixtures with a redox-responsive optical probe.

The analysis of mixtures of chiral compounds is a common task in academic and industrial laboratories typically achieved by laborious and time-consuming physical separation of the individual stereoisomers to allow interference-free quantification, for example using chiral chromatography coupled with UV detection. Current practice thus impedes high-throughput and slows down progress in countless chiral compound development projects. Here we describe a chemometric solution to this problem using a redox-responsive naphthoquinone that enables chromatography-free click chemistry sensing of challenging mixtures. The achiral probe covalently binds amino alcohols within a few minutes at room temperature and generates characteristic UVA and CDA spectra that are intentionally altered via sodium borohydride reduction to provide a second, strikingly different chiroptical data set (UVB and CDB). Chemometric partial least squares processing of the chiroptical outputs then enables spectral deconvolution and accurate determination of individual analyte concentrations. The success of this approach is demonstrated with 35 samples covering considerably varied total analyte amounts and stereoisomeric ratios. All chemicals and machine learning algorithms are readily available and can be immediately adapted by any laboratory.

Effects of internal and external corporate social responsibility on employee job satisfaction during a pandemic: A medical device industry perspective.

The outbreak of the COVID-19 pandemic has dramatically changed human lifestyles and contributed to the creation of a new normal in the business environment. This study examines the direct and indirect impacts of internal and external corporate social responsibility (CSR) practices on employee job satisfaction through organisational identification, conditional on employee age. A total of 236 valid responses were received from eight multinational medical device manufacturers in Malaysia. Partial least squares and PROCESS algorithms were employed to assess the hypothesised interactions between the predictors and criterion variables. The empirical results showed that internal CSR (i.e., CSR to employee) could significantly drive a greater sense of belonging and work satisfaction. Surprisingly, however, external CSR (i.e., CSR to community) negatively affects job fulfilment in the medical devices industry during the pandemic. Nevertheless, the findings also showed that ongoing CSR activities in the community could build organisational identification and subsequently improve job satisfaction. Conversely, CSR to environment did not statistically influence job satisfaction, either directly or indirectly. The mediating effects of organisational identification were not associated with employee age. This study provides a practical framework for effective CSR strategies amid the pandemic that can help organisations align with social responsibility, enhance their reputation, and contribute to society.

Continuous Glucose Monitoring Metrics in the Assessment of Glycemia in Moderate-to-Advanced CKD in Diabetes.

Introduction: Glycated hemoglobin A1c (HbA1c) has reduced reliability in advanced chronic kidney disease (CKD) owing to factors influencing red cell turnover. Recent guidelines support the use of continuous glucose monitoring (CGM) in glycemic assessment in these patients. We evaluated relationships between HbA1c and CGM metrics of average glycemia and glucose variability (GV) in moderate-to-advanced CKD. Methods: There were a total of 90 patients with diabetes in CKD stages G3b (n = 33), G4 (n = 43), and G5 (nondialysis) (n = 14) (age [mean ± SD] 65.4 ± 9.0 years, estimated glomerular filtration rate [eGFR] 26.1 ± 9.6 ml/min per 1.73 m2, and HbA1c 7.4 ± 0.8%). CGM metrics were estimated from blinded CGM (Medtronic Ipro2 with Enlite sensor) and compared with HbA1c in the same week. Results: Correlations between glucose management indicator (GMI) and HbA1c attenuated with advancing CKD (G3b [r = 0.68, P < 0.0001], G4 [r = 0.52, P < 0.001], G5 [r = 0.22, P = 0.44], P = 0.01 for CKD stage). In G3b and G4, HbA1c correlated significantly with time-in-range (TIR) (3.9-10.0 mmol/l) (r = -0.55 and r = -0.54, respectively) and % time > 13.9 mmol/l (r = 0.53 and r = 0.44, respectively), but not in G5. HbA1c showed no correlation with % time <3.0 mmol/l (r = -0.045, P = 0.67) or % coefficient of variation (CV) (r = -0.05, P = 0.64) in any CKD stage. Only eGFR was a significant determinant of bias for the difference between GMI and HbA1c (difference -0.28%, 95% CI [-0.52 to -0.03] per 15 ml/min per 1.73 m2 decrement, P = 0.03). Conclusion: CGM-derived indices might serve as an adjunct to HbA1c monitoring to guide glycemic management, especially in those with eGFR <30 ml/min per 1.73 m2. Time in hypoglycemia and glycemic variability are relevant glycemic targets for optimization not reflected by HbA1c.

Chiroptical Switching and Quantitative Chirality Sensing with (Pseudo)halogenated Quinones.

(Pseudo)halogenated quinones react smoothly with chiral amines, amino alcohols, and amino acids toward push-pull conjugates with optical sensing and switching applications. The chiroptically active conjugates serve as redox switches between two reversibly interconverting states with remarkably different UV and CD signatures. Addition of sodium borohydride generates a hydroquinone derivative that is quantitatively re-oxidized to the original quinone upon exposure to air. This chiroptical quinone/hydroquinone redox switch system combines several attractive features such as simple set-up, use of inexpensive chemicals, short response time, and thermal and photochemical stability. A conceptually new sensing approach that is based on integrated chiroptical amplification and redox switching enables on-the-fly deconvolution of otherwise overlapping CD spectra and is used for quantitative er analysis of challenging samples containing constitutional isomers in varying enantiomeric compositions.

Rapid organocatalytic chirality analysis of amines, amino acids, alcohols, amino alcohols and diols with achiral iso(thio)cyanate probes.

The widespread occurrence and significance of chiral compounds does not only require new methods for their enantioselective synthesis but also efficient tools that allow rapid determination of the absolute configuration, enantiomeric composition and overall concentration of nonracemic mixtures. Although chiral analysis is a frequently encountered challenge in the chemical, environmental, materials and health sciences it is typically addressed with slow and laborious chromatographic or NMR spectroscopic techniques. We now show with almost 40 analytes representing 5 different compound classes, including mono-alcohols which are particularly challenging sensing targets, that this task can be solved very quickly by chiroptical sensing with a single, readily available arylisocyanate probe. The probe reacts smoothly and irreversibly with amino and alcohol groups when an organocatalyst is used at room temperature toward urea or carbamate products exhibiting characteristic UV and CD signals above 300 nm. The UV signal induction is not enantioselective and correlated to the total concentration of both enantiomers, the concomitant generation of a CD band allows determination of the enantiomeric composition from the same sample, and the sense of the induced Cotton effect reveals the absolute configuration by comparison with a reference. This approach eliminates complications that can arise when enantiomerically impure NMR derivatizing agents are used and it outperforms time-consuming HPLC protocols. The generation of distinct UV and CD signals at high wavelengths overcomes issues with insufficient resolution of overlapping signals often encountered with chiral NMR solvating agents that rely on weak binding forces. The broad solvent compatibility is another noteworthy and important characteristic of this assay. It addresses frequently encountered problems with insufficient solubility of polar analytes, for example pharmaceuticals, in standard mobile phase mixtures required for chiral HPLC analysis. We anticipate that the broad application spectrum, ruggedness and practicality of organocatalytic chiroptical sensing with aryliso(thio)cyanate probes together with the availability of automated CD multi-well plate readers carry exceptional promise to accelerate chiral compound development projects at reduced cost and with less waste production.

Comprehensive characterization and resolution of discrepant spectrophotometric bilirubin results in patients on eltrombopag therapy.

Background Eltrombopag is a thrombopoietin receptor agonist used for the treatment of thrombocytopenic conditions. It can cause pH-dependent discoloration of plasma/serum. Eltrombopag is potentially hepatotoxic. It can affect the assessment of hyperbilirubinemia because of its (i) absorbance at ~450 nm (bilirubin), (ii) absorbance at ~550 nm (diazo-bilirubin) and (iii) it can cause yellowish discoloration of the eyes at normal circulating bilirubin levels. Methods We collected 66 samples from patients on a range of eltrombopag dosages up to 150 mg daily. Bilirubin was measured using multiple routine spectrophotometric analyzers, the Doumas reference method and high-performance liquid chromatography (HPLC). Plasma/serum eltrombopag concentrations were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS). Spike-in and admixture experiments delineated the effects of eltrombopag and its metabolites. Results Forty-nine of 52 samples from patients on ≥50 mg daily eltrombopag therapy showed significantly discrepant inter-analyzer total bilirubin results, a difference up to 64 µmol/L (3.7 mg/dL). In one sample, total bilirubin varied from 8 to 65 µmol/L (0.4-3.8 mg/dL) by different routine analyzers, with direct bilirubin ≤4 µmol/L (0.2 mg/dL). There was a positive correlation between total bilirubin difference and plasma eltrombopag concentration (r = 0.679), and spike-in experiments demonstrated that Beckman AU and Doumas reference methods were susceptible to positive interference. HPLC can quantify bilirubin after separating eltrombopag, and results suggest different analyzers are affected to varying degrees by eltrombopag and its metabolites. Conclusions Eltrombopag and its metabolites can cause positive interference to the spectrophotometric measurements of total bilirubin. Accurate measurements of total bilirubin may improve our understanding of the prevalence of hyperbilirubinemia in patients on eltrombopag therapy.

Hyperandrogenism, Elevated 17-Hydroxyprogesterone and Its Urinary Metabolites in a Young Woman with Ovarian Steroid Cell Tumor, Not Otherwise Specified: Case Report and Review of the Literature.

We describe a case of a 24-year-old overweight woman who presented with hirsutism, secondary amenorrhea, clitoromegaly, and symptoms of diabetes mellitus (DM). While a diagnosis of polycystic ovary syndrome (PCOS) with its associated metabolic disturbances was initially considered, serum total testosterone, androstenedione, and 17-hydroxyprogesterone (17-OHP) measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) were significantly increased. As 17-OHP did not increase upon ACTH (Synacthen) stimulation and the urinary steroid profile (USP) was compatible with an ovarian source of 17-OHP excess rather than adrenal, non classical congenital adrenal hyperplasia (NCCAH) was unlikely and an androgen-secreting tumor was suspected. Transabdominal ultrasound revealed the presence of an enlarged right ovary with a polycystic ovary morphology and no discrete mass. Transvaginal ultrasound and [18F]- fluorodeoxyglucose positron emission tomography-computed tomography (FDG PET-CT) enabled the localization of a right ovarian tumor. Laparoscopic right salpingo-oophorectomy was performed and a histological diagnosis of steroid cell tumor, not otherwise specified (SCT-NOS) was made. Hyperandrogenism and menstrual disturbances resolved postoperatively. A literature review revealed that 17-OHP-secreting SCT-NOS may uncommonly show positive responses to ACTH stimulation similar to 21-hydroxylase deficiency. Alternatively, USP might be useful in localizing the source of 17-OHP to the ovaries. Its diagnostic performance should be evaluated in further studies.

Early detection of nasopharyngeal carcinoma by plasma Epstein-Barr virus DNA analysis in a surveillance program.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is prevalent in Southeast Asia. Over the last decade, plasma Epstein-Barr virus (EBV) DNA has been developed as a tumor marker for NPC. In this study, the authors investigated whether plasma EBV DNA analysis is useful for NPC surveillance. METHODS: In total, 1318 volunteers ages 40 to 60 years were prospectively recruited. Plasma EBV DNA and serology for viral capsid antigen immunoglobulin A (IgA) were measured. Participants who had detectable plasma EBV DNA or positive IgA serology underwent nasal endoscopic examination and a follow-up plasma EBV DNA analysis in approximately 2 weeks. All participants were followed for 2 years to record the development of NPC. RESULTS: Three individuals with NPC were identified at enrolment. All of them were positive for EBV DNA and remained positive in follow-up analysis. Only 1 of those patients was positive for EBV serology. In 1 patient who had NPC with a small tumor confined to the mucosa, the tumor was not detectable on endoscopic examination. Because of a 2-fold increase in plasma EBV DNA on the follow-up analysis, that patient underwent magnetic resonance imaging, which revealed the tumor. Among the participants who did not have NPC but had initially positive plasma EBV DNA results, approximately 66% had negative EBV DNA results after a median of 2 weeks. CONCLUSIONS: Plasma EBV DNA analysis proved useful for detecting early NPC in individuals without a clinical suspicion of NPC. Repeating the test in those who had initially positive results differentiated those with NPC from those who had false-positive results. Cancer 2013. © 2013 American Cancer Society.

Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype.

BACKGROUND: Tumours contain hypoxic regions that select for an aggressive cell phenotype; tumour hypoxia induces metastasis-associated genes. Treatment refractory patients with metastatic cancer show increased numbers of circulating tumour cells (CTCs), which are also associated with disease progression. The aim of this study was to examine the as yet unknown relationship between hypoxia and CTCs. METHODS: We generated human MDA-MB-231 orthotopic xenografts and, using a new technology, isolated viable human CTCs from murine blood. The CTCs and parental MDA-MB-231 cells were incubated at 21 and 0.2% (hypoxia) oxygen, respectively. Colony formation was assayed and levels of hypoxia- and anoxia-inducible factors were measured. Xenografts generated from CTCs and parental cells were compared. RESULTS: MDA-MB-231 xenografts used to generate CTCs were hypoxic, expressing hypoxia factors: hypoxia-inducible factor1 alpha (HIF1alpha) and glucose transporter protein type 1 (GLUT1), and anoxia-induced factors: activating transcription factor 3 and 4 (ATF3 and ATF4). Parental MDA-MB-231 cells induced ATF3 in hypoxia, whereas CTCs expressed it constitutively. Asparagine synthetase (ASNS) expression was also higher in CTCs. Hypoxia induced ATF4 and the HIF1alpha target gene apelin in CTCs, but not in parental cells. Hypoxia induced lower levels of carbonic anhydrase IX (CAIX), GLUT1 and BCL2/adenovirus E1B 19-KD protein-interacting protein 3 (BNIP3) proteins in CTCs than in parental cells, supporting an altered hypoxia response. In chronic hypoxia, CTCs demonstrated greater colony formation than parental cells. Xenografts generated from CTCs were larger and heavier, and metastasised faster than MDA-MB-231 xenografts. CONCLUSION: CTCs show an altered hypoxia response and an enhanced aggressive phenotype in vitro and in vivo.

Focal amplification and oncogene dependency of GAB2 in breast cancer.

DNA amplifications in breast cancer are frequent on chromosome 11q, in which multiple driver oncogenes likely reside in addition to cyclin D1 (CCND1). One such candidate, the scaffolding adapter protein, GRB2-associated binding protein 2 (GAB2), functions in ErbB signaling and was recently shown to enhance mammary epithelial cell proliferation, and metastasis of ERBB2 (HER2/neu)-driven murine breast cancer. However, the amplification status and function of GAB2 in the context of amplification remain undefined. In this study, by genomic profiling of 172 breast tumors, and fluorescence in situ hybridization validation in an independent set of 210 scorable cases, we observed focal amplification spanning GAB2 (11q14.1) independent of CCND1 (11q13.2) amplification, consistent with a driver role. Further, small interfering RNA (siRNA)-mediated knockdown of GAB2 in breast cancer lines (SUM52, SUM44PE and MDA468) with GAB2 amplification revealed a dependency on GAB2 for cell proliferation, cell-cycle progression, survival and invasion, likely mediated through altered phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling. GAB2 knockdown also reduced proliferation and survival in a cell line (BT474) with ERBB2 amplification, consistent with the possibility that GAB2 can function downstream of ERBB2. Our studies implicate focal amplification of GAB2 in breast carcinogenesis, and underscore an oncogenic role of scaffolding adapter proteins, and a potential new point of therapeutic intervention.

Basal carcinoma of the breast revisited: an old entity with new interpretations.

The introduction of global gene expression analysis in breast cancer research has focused attention onto a repeatedly described subgroup of invasive breast cancer, the basal-like carcinomas. This subgroup is characterised by the expression of high-molecular weight cytokeratins 5, 14 and 17; using immunohistochemical diagnosis, it represents approximately 7-20% of invasive breast cancers. Some of these tumours fulfil the criteria of grade 3 invasive ductal carcinoma, the so-called triple negative carcinomas. However, other rare subgroups of metaplastic, medullary and myoepithelial carcinomas also belong to this entity. Even though the initial clinical prognostic relevance of basal-like breast cancers may have been overestimated, its distinctive biology generates many questions regarding the pathogenesis, chemosensitivity and optimal clinical management of this subgroup. Physiological progenitor cells within the normal female breast share essential immunohistochemical features with basal-like breast cancers. Although the exact relationship between subgroups of normal breast cells and their respective malignant counterparts is still under investigation, the major hallmarks of physiological progenitor cells are either maintained or reactivated by distinct genetic changes in basal breast cancer cells. This review will discuss the impact of these findings on our global understanding of breast cancer pathogenesis, especially from the perspective of its potential histogenesis. Clinical consequences and potential future research directions driven by the definition of basal breast cancers will also be discussed.

Oxidative stress pathways highlighted in tumor cell immortalization: association with breast cancer outcome.

An improved understanding of cell immortalization and its manifestation in clinical tumors could facilitate novel therapeutic approaches. However, only rare tumor cells, which maintain telomerase expression in vitro, immortalize spontaneously. By expression-profiling analyses of limited-life primary breast tumor cultures pre- and post-hTERT transduction, and spontaneously immortalized breast cancer cell lines, we identified a common signature characteristic of tumor cell immortalization. A predominant feature of this immortalization signature (ImmSig) was the significant overexpression of oxidoreductase genes. In contrast to epithelial cells derived from low histologic grade primary tumors, which required hTERT transduction for the acquisition of ImmSig, spontaneously immortalizing high-grade tumor cultures displayed similar molecular changes independent of exogenous hTERT. Silencing the hTERT gene reversed ImmSig expression, increased cellular reactive oxygen species levels, altered mitochondrial membrane potential and induced apoptotic and proliferation changes in immortalized cells. In clinical breast cancer samples, cell-proliferation-pathway genes were significantly associated with ImmSig. In these cases, ImmSig expression itself was inversely correlated with patient survival (P=0), and was particularly relevant to the outcome of estrogen receptor-positive tumors. Our data support the notion that ImmSig assists in surmounting normal barriers related to oxidative and replicative stress response. Targeting a subset of aggressive breast cancers by reversing ImmSig components could be a practical therapeutic strategy.