Comparative biological activity of palbociclib and ribociclib in hormone receptor-positive breast cancer.

This study examines the biological effects of palbociclib and ribociclib in hormone receptor-positive breast cancer, pivotal to the HARMONIA prospective phase III clinical trial. We explore the downstream impacts of these CDK4/6 inhibitors, focusing on cell lines and patient-derived tumor samples. We treated HR+ breast cancer cell lines (T47D, MCF7, and BT474) with palbociclib or ribociclib (100 nM or 500 nM), alone or combined with fulvestrant (1 nM), over periods of 24, 72, or 144 h. Our assessments included PAM50 gene expression, RB1 phosphorylation, Lamin-B1 protein levels, and senescence-associated ß-galactosidase activity. We further analyzed PAM50 gene signatures from the CORALLEEN and NeoPalAna phase II trials. Both CDK4/6 inhibitors similarly inhibited proliferation across the cell lines. At 100 nM, both drugs partially reduced p-RB1, with further decreases at 500 nM over 144 h. Treatment led to reduced Lamin-B1 expression and increased senescence-associated ß-galactosidase activity. Both drugs enhanced Luminal A and reduced Luminal B and proliferation signatures at both doses. However, the HER2-enriched signature significantly diminished only at the higher dose of 500 nM. Corresponding changes were observed in tumor samples from the CORALLEEN and NeoPalAna studies. At 2 weeks of treatment, both drugs significantly reduced the HER2-enriched signature, but at surgery, this reduction was consistent only with ribociclib. Our findings suggest that while both CDK4/6 inhibitors effectively modulate key biological pathways in HR+/HER2- breast cancer, nuances in their impact, particularly on the HER2-enriched signature, are dose-dependent, influenced by the addition of fulvestrant and warrant further investigation.

O-RADS MRI scoring system: key points for correct application in inexperienced hands.

OBJECTIVES: To evaluate the efficacy of the O-RADS MRI criteria in the stratification of risk of malignancy of solid or sonographically indeterminate ovarian masses and assess the interobserver agreement of this classification between experienced and inexperienced radiologists. METHODS: This single-centre retrospective study included patients from 2019 to 2022 with sonographically indeterminate or solid ovarian masses who underwent MRI with a specific protocol for characterisation according to O-RADS MRI specifications. Each study was evaluated using O-RADS lexicon by two radiologists, one with 17 years of experience in gynaecological radiology and another with 4 years of experience in general radiology. Findings were classified as benign, borderline, or malignant according to histology or stability over time. Diagnostic performance and interobserver agreement were assessed. RESULTS: A total of 183 patients with US indeterminate or solid adnexal masses were included. Fifty-seven (31%) did not have ovarian masses, classified as O-RADS 1. The diagnostic performance for scores 2-5 was excellent with a sensitivity, specificity, PPV, and NPV of 97.4%, 100%, 96.2%, and 100%, respectively by the experienced radiologist and 96.1%, 92.0%, 93.9%, and 94.8% by the inexperienced radiologist. Interobserver concordance was very high (Kappa index 0.92). Almost all the misclassified cases were due to misinterpretation of the classification similar to reports in the literature. CONCLUSION: The diagnostic performance of O-RADS MRI determined by either experienced or inexperienced radiologists is excellent, facilitating decision-making with high diagnostic accuracy and high reproducibility. Knowledge of this classification and use of assessment tools could avoid frequent errors due to misinterpretation. CRITICAL RELEVANCE STATEMENT: Up to 31% of ovarian masses are considered indeterminate by transvaginal US and 32% of solid lesions considered malignant by transvaginal US are benign. The O-RADs MRI accurately classifies these masses, even when used by inexperienced radiologists, thereby avoiding incorrect surgical approaches. KEY POINTS: • O-RADS MRI accurately classifies indeterminate and solid ovarian masses by ultrasound. • There is excellent interobserver agreement between experienced and non-experienced radiologists. • O-RADS MRI is a helpful tool to assess clinical decision-making in ovarian tumours.

Mutational Status of SMAD4 and FBXW7 Affects Clinical Outcome in TP53-Mutated Metastatic Colorectal Cancer.

Next-generation sequencing (NGS) provides a molecular rationale to inform prognostic stratification and to guide personalized treatment in cancer patients. Here, we determined the prognostic and predictive value of actionable mutated genes in metastatic colorectal cancer (mCRC). Among a total of 294 mCRC tumors examined by targeted NGS, 200 of them derived from patients treated with first-line chemotherapy plus/minus monoclonal antibodies were included in prognostic analyses. Discriminative performance was assessed by time-dependent estimates of the area under the curve (AUC). The most recurrently mutated genes were TP53 (64%), KRAS or NRAS (49%), PIK3CA (15%), SMAD4 (14%), BRAF (13%), and FBXW7 (9.5%). Mutations in FBXW7 correlated with worse OS rates (p = 0.036; HR, 2.24) independently of clinical factors. Concurrent mutations in TP53 and FBXW7 were associated with increased risk of death (p = 0.02; HR, 3.31) as well as double-mutated TP53 and SMAD4 (p = 0.03; HR, 2.91). Analysis of the MSK-IMPACT mCRC cohort (N = 1095 patients) confirmed the same prognostic trend for the previously identified mutated genes. Addition of the mutational status of these genes upon clinical factors resulted in a time-dependent AUC of 87%. Gene set enrichment analysis revealed specific molecular pathways associated with SMAD4 and FBXW7 mutations in TP53-defficient tumors. Conclusively, SMAD4 and FBXW7 mutations in TP53-altered tumors were predictive of a negative prognostic outcome in mCRC patients treated with first-line regimens.

A novel gene signature unveils three distinct immune-metabolic rewiring patterns conserved across diverse tumor types and associated with outcomes.

Existing immune signatures and tumor mutational burden have only modest predictive capacity for the efficacy of immune check point inhibitors. In this study, we developed an immune-metabolic signature suitable for personalized ICI therapies. A classifier using an immune-metabolic signature (IMMETCOLS) was developed on a training set of 77 metastatic colorectal cancer (mCRC) samples and validated on 4,200 tumors from the TCGA database belonging to 11 types. Here, we reveal that the IMMETCOLS signature classifies tumors into three distinct immune-metabolic clusters. Cluster 1 displays markers of enhanced glycolisis, hexosamine byosinthesis and epithelial-to-mesenchymal transition. On multivariate analysis, cluster 1 tumors were enriched in pro-immune signature but not in immunophenoscore and were associated with the poorest median survival. Its predicted tumor metabolic features suggest an acidic-lactate-rich tumor microenvironment (TME) geared to an immunosuppressive setting, enriched in fibroblasts. Cluster 2 displays features of gluconeogenesis ability, which is needed for glucose-independent survival and preferential use of alternative carbon sources, including glutamine and lipid uptake/ß-oxidation. Its metabolic features suggest a hypoxic and hypoglycemic TME, associated with poor tumor-associated antigen presentation. Finally, cluster 3 is highly glycolytic but also has a solid mitochondrial function, with concomitant upregulation of glutamine and essential amino acid transporters and the pentose phosphate pathway leading to glucose exhaustion in the TME and immunosuppression. Together, these findings suggest that the IMMETCOLS signature provides a classifier of tumors from diverse origins, yielding three clusters with distinct immune-metabolic profiles, representing a new predictive tool for patient selection for specific immune-metabolic therapeutic approaches.

Development and validation of a nomogram to predict kidney survival at baseline in patients with C3 glomerulopathy.

Background: C3 glomerulopathy is a rare and heterogeneous complement-driven disease. It is often challenging to accurately predict in clinical practice the individual kidney prognosis at baseline. We herein sought to develop and validate a prognostic nomogram to predict long-term kidney survival. Methods: We conducted a retrospective, multicenter observational cohort study in 35 nephrology departments belonging to the Spanish Group for the Study of Glomerular Diseases. The dataset was randomly divided into a training group (n = 87) and a validation group (n = 28). The least absolute shrinkage and selection operator (LASSO) regression was used to screen the main predictors of kidney outcome and to build the nomogram. The accuracy of the nomogram was assessed by discrimination and risk calibration in the training and validation sets. Results: The study group comprised 115 patients, of whom 46 (40%) reached kidney failure in a median follow-up of 49 months (range 24-112). No significant differences were observed in baseline estimated glomerular filtration rate (eGFR), proteinuria or total chronicity score of kidney biopsies, between patients in the training versus those in the validation set. The selected variables by LASSO were eGFR, proteinuria and total chronicity score. Based on a Cox model, a nomogram was developed for the prediction of kidney survival at 1, 2, 5 and 10 years from diagnosis. The C-index of the nomogram was 0.860 (95% confidence interval 0.834-0.887) and calibration plots showed optimal agreement between predicted and observed outcomes. Conclusions: We constructed and validated a practical nomogram with good discrimination and calibration to predict the risk of kidney failure in C3 glomerulopathy patients at 1, 2, 5 and 10 years.

Longitudinal change in proteinuria and kidney outcomes in C3 glomerulopathy.

INTRODUCTION: The association between a change in proteinuria over time and its impact on kidney prognosis has not been analysed in complement component 3 (C3) glomerulopathy. This study aims to investigate the association between the longitudinal change in proteinuria and the risk of kidney failure. METHODS: This was a retrospective, multicentre observational cohort study in 35 nephrology departments belonging to the Spanish Group for the Study of Glomerular Diseases. Patients diagnosed with C3 glomerulopathy between 1995 and 2020 were enrolled. A joint modelling of linear mixed-effects models was applied to assess the underlying trajectory of a repeatedly measured proteinuria, and a Cox model to evaluate the association of this trajectory with the risk of kidney failure. RESULTS: The study group consisted of 85 patients, 70 C3 glomerulonephritis and 15 dense deposit disease, with a median age of 26 years (range 13-41). During a median follow-up of 42 months, 25 patients reached kidney failure. The longitudinal change in proteinuria showed a strong association with the risk of this outcome, with a doubling of proteinuria levels resulting in a 2.5-fold increase of the risk. A second model showed that a ≥50% proteinuria reduction over time was significantly associated with a lower risk of kidney failure (hazard ratio 0.79; 95% confidence interval 0.56-0.97; P < 0.001). This association was also found when the ≥50% proteinuria reduction was observed within the first 6 and 12 months of follow-up. CONCLUSIONS: The longitudinal change in proteinuria is strongly associated with the risk of kidney failure. The change in proteinuria over time can provide clinicians a dynamic prediction of kidney outcomes.

Are Quality of Randomized Clinical Trials and ESMO-Magnitude of Clinical Benefit Scale Two Sides of the Same Coin, to Grade Recommendations for Drug Approval?

The approval of a new drug for cancer treatment by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) is based on positive, well-designed randomized phase III clinical trials (RCTs). However, not all of them are analyzed to support the recommendations. For this reason, there are different scales to quantify and evaluate the quality of RCTs and the magnitude of the clinical benefits of new drugs for treating solid tumors. In this review, we discuss the value of the progression-free survival (PFS) as an endpoint in RCTs and the concordance between it and the overall survival (OS) as a measure of the quality of clinical trial designs. We summarize and analyze the different scales to evaluate the clinical benefits of new drugs such as the The American Society of Clinical Oncology value framework (ASCO-VF-NHB16) and European Society for Medical Oncology Magnitude of Clinical Benefit Scale (ESMO-MCBS) and the concordance between them, focusing on metastatic colorectal cancer (mCRC). We propose several definitions that would help to evaluate the quality of RCT, the magnitude of clinical benefit and the appropriate approval of new drugs in oncology.

Circulating tumor DNA dynamics in advanced breast cancer treated with CDK4/6 inhibition and endocrine therapy.

Circulating tumor DNA (ctDNA) levels may predict response to anticancer drugs, including CDK4/6 inhibitors and endocrine therapy combinations (CDK4/6i+ET); however, critical questions remain unanswered such as which assay or statistical method to use. Here, we obtained paired plasma samples at baseline and week 4 in 45 consecutive patients with advanced breast cancer treated with CDK4/6i+ET. ctDNA was detected in 96% of cases using the 74-gene Guardant360 assay. A variant allele fraction ratio (VAFR) was calculated for each of the 79 detected mutations between both timepoints. Mean of all VAFRs (mVAFR) was computed for each patient. In our dataset, mVAFR was significantly associated with progression-free survival (PFS). Baseline VAF, on-treatment VAF or absolute changes in VAF were not associated with PFS, nor were CA-15.3 levels at baseline, week 4 or the CA-15.3 ratio. These findings demonstrate that ctDNA dynamics using a standardized multi-gene panel and a unique methodological approach predicts treatment outcome. Clinical trials in patients with an unfavorable ctDNA response are needed.

Mycophenolate Mofetil in C3 Glomerulopathy and Pathogenic Drivers of the Disease.

BACKGROUND AND OBJECTIVES: C3 glomerulopathy is a complement-mediated disease arising from abnormalities in complement genes and/or antibodies against complement components. Previous studies showed that treatment with corticosteroids plus mycophenolate mofetil (MMF) was associated with improved outcomes, although the genetic profile of these patients was not systematically analyzed. This study aims to analyze the main determinants of disease progression and response to this therapeutic regimen. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We conducted a retrospective, multicenter, observational cohort study in 35 nephrology departments belonging to the Spanish Group for the Study of Glomerular Diseases. Patients diagnosed with C3 glomerulopathy (n=81) or dense deposit disease (n=16) between January 1995 and March 2018 were enrolled. Multivariable and propensity score matching analyses were used to evaluate the association of clinical and genetic factors with response to treatment with corticosteroids and MMF as measured by proportion of patients with disease remission and kidney survival (status free of kidney failure). RESULTS: The study group comprised 97 patients (84% C3 glomerulopathy, 16% dense deposit disease). Forty-two patients were treated with corticosteroids plus MMF, and this treatment was associated with a higher rate of remission and lower probability of kidney failure (79% and 14%, respectively) compared with patients treated with other immunosuppressives (24% and 59%, respectively), or ecluzimab (33% and 67%, respectively), or conservative management (18% and 65%, respectively). The therapeutic superiority of corticosteroids plus MMF was observed both in patients with complement abnormalities and with autoantibodies. However, patients with pathogenic variants in complement genes only achieved partial remission, whereas complete remissions were common among patients with autoantibody-mediated forms. The main determinant of no remission was baseline proteinuria. Relapses occurred after treatment discontinuation in 33% of the patients who had achieved remission with corticosteroids plus MMF, and a longer treatment length of MMF was associated with a lower risk of relapse. CONCLUSIONS: The beneficial response to corticosteroids plus MMF treatment in C3 glomerulopathy appears independent of the pathogenic drivers analyzed in this study.

Usefulness of Two Independent DNA and RNA Tissue-Based Multiplex Assays for the Routine Care of Advanced NSCLC Patients.

Personalized medicine is nowadays a paradigm in lung cancer management, offering important benefits to patients. This study aimed to test the feasibility and utility of embedding two multiplexed genomic platforms as the routine workup of advanced non-squamous non-small cell lung cancer (NSCLC) patients. Two parallel multiplexed approaches were performed based on DNA sequencing and direct digital detection of RNA with nCounter® technology to evaluate gene mutations and fusions. The results were used to guide genotype-directed therapies and patient outcomes were collected. A total of 224 advanced non-squamous NSCLC patients were prospectively included in the study. Overall, 85% of samples were successfully characterized at DNA and RNA levels and oncogenic drivers were found in 68% of patients, with KRAS, EGFR, METΔex14, BRAF, and ALK being the most frequent (31%, 19%, 5%, 4%, and 4%, respectively). Among all patients with complete genotyping results and follow-up data (n = 156), the median overall survival (OS) was 1.90 years (confidence interval (CI) 95% 1.69-2.10) for individuals harbouring an actionable driver treated with a matched therapy, compared with 0.59 years (CI 95% 0.39-0.79) in those not eligible for any targeted therapy and 0.61 years (CI 95% 0.12-1.10) in patients with no drivers identified (p < 0.001). Integrating DNA and RNA multiplexing technologies into the routine molecular testing of advanced NSCLC patients is feasible and useful and highlights the necessity of widespread integrating comprehensive molecular diagnosis into lung cancer care.

Frequency and spectrum of PIK3CA somatic mutations in breast cancer.

PURPOSE: The therascreen PIK3CA mutation assay and the alpha-specific PI3K inhibitor alpelisib are FDA-approved for identifying and treating patients with advanced PIK3CA-mutated (PIK3CAmut) breast cancer (BC). However, it is currently unknown to what extend this assay detects most PIK3CA mutations in BC. This information is critical as patients and clinicians are using this and other genomic assays to indicate alpelisib. METHODS: Data from 6338 patients with BC was explored across 10 publicly available studies. The primary objective was to evaluate the proportion and distribution of PIK3CA mutations in BC. Secondary objectives were (1) to evaluate in silico the spectrum of PIK3CA mutations in BC that would be captured by the therascreen panel; (2) to evaluate the proportion and distribution of PIK3CA mutations in hormone receptor-positive/HER2-negative (HR+/HER2-), HER2+, and triple-negative BC (TNBC); and (3) to explore the identification of PIK3CA mutations in a cohort of 48 HR+/HER2- advanced BC patients by the Guardant B360 circulating tumor DNA (ctDNA) assay. RESULTS: Patients with PIK3CAmut tumors represented 35.7% (2261/6338). Five PIK3CA mutations comprised 73% of all PIK3CA mutations: H1047R (35%), E545K (17%), E542K (11%), N345K (6%), and H1047L (4%). Therascreen gene list would capture 72% of all PIK3CA mutations and 80% of patients with a known PIK3CAmut BC. Among patients with double PIK3CAmut tumors (12% of all PIK3CAmut), the therascreen panel would capture 78% as harboring 1 single PIK3CA mutation, 17% as PIK3CAmut undetected, and 5% as PIK3CA double-mut. PIK3CA mutation rates were lower in TNBC (16%) compared to HR+/HER2 (42%) and HER2+ (31%) BC; however, the distribution of the 4 main PIK3CA mutations across subtypes was similar. Finally, 28% of PIK3CA mutations identified in ctDNA in 48 patients with advanced HR+/HER2- BC were not part of the therascreen panel. CONCLUSION: PIK3CA mutations in BC are heterogenous and ~ 20% of patients with a known PIK3CA mutation, and 95% with a known double PIK3CAmut tumor, would not be captured by the therascreen panel. Finally, the clinical utility of PIK3CA mutations not present in the therascreen companion diagnostic assay or identified by other sequencing-based assays needs further investigation.

A Pathology-Based Combined Model to Identify PAM50 Non-luminal Intrinsic Disease in Hormone Receptor-Positive HER2-Negative Breast Cancer.

Background: In hormone receptor-positive (HR+)/HER2-negative breast cancer, the HER2-enriched and Basal-like intrinsic subtypes are associated with poor outcome, low response to anti-estrogen therapy and high response to chemotherapy. To date, no validated biomarker exists to identify both molecular entities other than gene expression. Methods: PAM50 subtyping and immunohistochemical data were obtained from 8 independent studies of 1,416 HR+/HER2-negative early breast tumors. A non-luminal disease score (NOLUS) from 0 to 100, based on percentage of estrogen receptor (ER), progesterone receptor (PR) and Ki67 tumor cells, was derived in a combined cohort of 5 studies (training dataset) and tested in a combined cohort of 3 studies. The performance of NOLUS was estimated using Area Under the ROC Curve (AUC). Results: In the training dataset (n = 903) and compared to luminal disease, non-luminal disease had lower percentage of ER-positive cells (median 65.2 vs. 86.2%, p < 0.01) and PR-positive cells (33.2 vs. 56.4%, p < 0.01) and higher percentage of Ki67-positive cells (18.2 vs. 13.1%, p = 0.01). A NOLUS formula was derived: -0.45*ER -0.28*PR +0.27*Ki67 + 73.02. The proportion of non-luminal tumors in NOLUS-positive (≥51.38) and NOLUS-negative (<51.38) groups was 52.6 and 8.7%, respectively. In the testing dataset (n = 514), NOLUS was found significantly associated with non-luminal disease (p < 0.01) with an AUC 0.902. The proportion of non-luminal tumors in NOLUS-positive and NOLUS-negative groups was 76.9% (56.4-91.0%) and 2.6% (1.4-4.5%), respectively. The sensitivity and specificity of the pre-specified cutoff was 59.3 and 98.7%, respectively. Conclusions: In the absence of gene expression data, NOLUS can help identify non-luminal disease within HR+/HER2-negative breast cancer.

Determination of quinolones and fluoroquinolones in hospital sewage water by off-line and on-line solid-phase extraction procedures coupled to HPLC-UV.

In this work, the development of two solid-phase extraction procedures (off-line and on-line formats) for the identification and quantification of several (fluoro)quinolones in hospital sewage water by HPLC-UV is described. Both procedures are based on the use of C18 and anion exchange (SAX) sorbents for the preconcentration and clean-up steps, respectively, and all variables influencing both steps were optimised. In the off-line format, after its pH was adjusted to 2.5, sample was preconcentrated on a C18 cartridge and eluted with 4 mL of methanol/ammonia (94/6). The methanolic extract must be diluted up to 10 mL with water to allow quantitative retention of the analytes on the SAX cartridge. In the on-line format, the addition of 2.5% of NH4Cl to the sewage water sample (pH = 2.5) was necessary to increase the breakthrough volumes of the analytes in the C18 precolumn. Quantitative transfer of the (fluoro)quinolones from the C18 precolumn to the SAX precolumn was accomplished by pumping 2 mL of a mixture methanol/water (40/60, pH = 9.2) at 2 mL min(-1). Elution of the analytes from the SAX precolumn by means of the chromatographic mobile phase required the inclusion of an additional isocratic step at the beginning of the gradient program. Both off-line and on-line solid phase extraction procedures coupled to HPLC-UV were applied to the analysis of a sewage water sample collected in the sewer system at the output of the St Dimphna Hospital (Geel, Belgium). The fluoroquinolone ciprofloxacin was found in this sample and quantified at 5.8 +/- 0.4 microg L(-1) (off-line method) and 5.6 +/- 0.5 microg L(-1) (on-line method). The analysis of spiked samples containing the seven (fluoro)quinolones studied provided quantitative recoveries in all cases with low RSD values (from 6 to 12%), and all the analytes could be identified by means of their UV spectra with match factors varying from 950 to 985 depending on the (fluoro)quinolone.

Study of the evolution and degradation products of ciprofloxacin and oxolinic acid in river water samples by HPLC-UV/MS/MS-MS.

The results of a degradation study of the (fluoro)quinolone antibiotics ciprofloxacin and oxolinic acid in river water samples are presented in this paper. The decomposition of these compounds at ambient temperature was monitored during five months by HPLC-UV, and two consecutive degradation processes (photo- and bio/chemical-degradation) were observed in both cases although with different degradation rates. Ciprofloxacin was completely degraded after 3 months whereas 80% of oxolinic acid remained unaltered after five months of storage. The analysis of the degradation compounds formed was carried out using MS and tandem MS-MS, allowing the identification of four new ciprofloxacin transformation products not previously described in the literature. Possible degradation pathways for this antibiotic in river water are proposed.

Detection of coexisting protein conformations in capillary zone electrophoresis subsequent to transient contact with sodium dodecyl sulfate solutions.

Non-native conformations of proteins were generated by temporary contact with aqueous solutions of sodium dodecyl sulfate (SDS) and separated from the native state with capillary zone electrophoresis (CZE) in alkaline borate buffer deficient of SDS. Nine proteins at concentrations of 2.0 or 3.0 mg.L(-1) were compared in terms of their susceptibility to SDS. For superoxide dismutase and ferritin the tendency of unfolding was modest with < 25% of the protein being transformed to the non-native state at 10 mmol.L(-1) SDS. Highest susceptibility was observed for albumin, myoglobin (Mb), and hemoglobin with > 75% in the non-native state even at 2.0 mmol.L(-1) SDS. The influence of varying SDS concentrations on the conformational state of Mb was tested. Increasing the SDS concentration, circular dichroism revealed a reduction in alpha-helix, an increase in random coil, and an introduction of beta-sheet, which is absent in native structure. Modifications in the secondary structure were in agreement with distinct changes in the shape of the non-native Mb peak in CZE and make a gradual unfolding/refolding process with several coexisting molten globules instead of two-state transition of conformations most plausible for Mb. CZE was found to contribute to a further understanding of holo-Mb transformation towards a population of non-native conformations (i) by means of calculated peak area ratios of native to non-native states, which showed sigmoid transition, (ii) by detecting the release of the prosthetic heme group, and (iii) by changes in the effective electrophoretic mobility of the Mb-SDS peaks. Reconstituted holo-Mb forms differed in the Soret band around 410 nm, indicating diversity in the conformation of the heme pocket.

Determination of macrolide antibiotics in porcine and bovine urine by high-performance liquid chromatography coupled to coulometric detection.

A high-performance liquid chromatography method coupled to coulometric detection has been applied for the determination, in a single run, of up to eight macrolide antibiotics (erythromycin [ERY], tylosin [TYL], tilmicosin [TILM], spiramycin 2 [SPI 2], spiramycin 3 [SPI 3], josamycin [JOS], kitasamycin [KIT], and rosamicin [ROS]) in spiked porcine and bovine urine. Quantification was performed using matrix-matched calibration with roxithromycin (ROX) as the internal standard. The detection limits for each drug were below 3.5 ng injected (equivalent to an initial concentration below 0.07 mg L(-1)) for porcine urine and below 5 ng injected (equivalent to an initial concentration below 0.10 mg L(-1)) for bovine urine. Recoveries from urine samples spiked at three different concentrations within the linear range were not significantly dependent on concentration. The entire procedure provides average macrolide recoveries ranging from 69.7 to 96.6% for bovine urine and from 75.5 and 96.1% for porcine urine.

Stability of fluoroquinolone antibiotics in river water samples and in octadecyl silica solid-phase extraction cartridges.

Intensive use of antibiotics in human and veterinarian medicine and in industrial farming (food additives) has resulted in the transport of important quantities of the active ingredients to environmental waters. Environmental analysis usually requires sample storage for certain periods of time and, consequently, it is of great importance to know the stability of antibiotics in these kinds of sample. Thus, in this work the stability in river water of oxolinic acid (Oxo) and ciprofloxacin (Cip), taken as representatives of fluoroquinolone and quinolone antibiotics respectively, has been evaluated. The stability of these compounds in river water has been studied both in containers and on C(18) solid-phase extraction cartridges (SPE) under different storage conditions (time, light, and temperature). Data analysis revealed that Cip and Oxo have different degradation profiles with different degradation kinetics in river water. It was also concluded that these antibiotics are stable both in the containers and on SPE cartridges for at least 2 weeks at ambient temperature, and stability can be increased substantially if samples are stored at low temperatures (4 and -18 degrees C).

Characterization of lectin aggregates in the hemolymph of freshwater prawn Macrobrachium rosenbergii.

In invertebrates, lectins play relevant roles in innate immunity; however, their regulatory mechanisms have not been identified yet. In this work, we purified, by gel filtration and affinity chromatography, lectin aggregates circulating in the hemolymph of the freshwater prawn Macrobrachium rosenbergii and compared their physicochemical properties with a previously described lectin (MrL). High-molecular weight MrL aggregates (MrL-I) lack hemagglutinating activity and showed bands of 62.1, 67.1 and 81.4 kDa, whereas MrL-III, which corresponds to MrL, showed hemagglutinating activity and is constituted by a single 9.6-kDa band as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. MrL-I and MrL-III showed similar amino acid composition but different carbohydrates concentration. Edman degradation indicated NH2-terminal sequence of five amino acids for the 9.6-kDa MrL-III (DVPLL/A) and eleven for the main 81.4-kDa band identified in MrL-I (DVPLL/AXKQQQD); analysis by MALDI-TOF indicated a different tryptic pattern for MrL-I and MrL-III. MrL-I was recognized by monoclonal antibodies against MrL-III. Circular dichroism indicated that the secondary structure in both proteins is similar and contains 23% of beta-sheet and 24% of alpha-helix. Our results suggest that differential posttranslational processes that favor aggregation are involved in regulating the activity of the lectin.

Capillary zone electrophoresis of metal-binding proteins in formic acid with UV- and mass spectrometric detection using cationic transient capillary isotachophoresis for preconcentration.

A capillary zone electrophoresis (CZE) method with preceding cationic transient capillary isotachophoresis (tCITP-CZE) was developed for uncoated fused-silica capillaries to analyze metal-binding proteins (MBPs) of clinical relevance. UV detection was followed by mass spectrometry (MS). Optimization was done with model proteins of properties similar to relevant human MBPs. Using 1.0 mol x L(-1) formic acid (pH 1.78) as electrolyte resulted in up to 165000 plates m(-1) in CZE and 230000 plates m(-1) in combination with tCITP and analysis time was less than 5 min in uncoupled mode. Cationic tCITP with 125 mmol x L(-1) ammonium formate, buffered to pH 4.00, as leading electrolyte improved sample loadability considerably in comparison with sample stacking without impairing resolution. Following systematic optimization of the electrospray ionization process (ESI) the coupled system ((tCITP)-CZE-UV-ESI-MS) was tested with protein model mixtures and human MBPs. Repeatability of migration times was < 0.64% in pure CZE mode and in tCITP-CZE mode and < 0.83% in CZE-ESI-MS coupled mode. Mass accuracy was < 0.015%. Limits of detection were found to be in the range 50-160 fmol.