[A simple pretreatment of samples for thermal neutron activation analysis--qualitative analysis of trace elements in a pellet of solium cyanide (author's transl)].
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Activation Analysis↗
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[A potentiometric determination of iodide in milk by use of the iron--selective electrode (author's transl)].
A method for quantitative determination of iodide in milk by use of the ion-selective electrode is described. The proposed method is rapid and simple. No sample pretreatment is required exept for sample ionic strength adjustment. It was possible to determine iodide concentrations down to 40 microgram/l with average relative standard deviation of 3,4%. The concentration of iodide in milk samples determined by this method ranged from 61,5 microgram J-/l to 190,8 microgram J-/l.
Animals↗
Colorimetric determination of poly(N-vinyl-2-pyrrolidone) in contact lens solutions.
A rapid colorimetric method is presented for the quantitative determination of poly(N-vinyl-2-pyrrolidone) in contact lens solutions. The method is simple, requires no sample pretreatment, and uses only a small volume of sample. The procedure is based on the measurement of the net absorbance of a poly(N-vinyl-2-pyrrolidone)- Congo red complex at 545 nm. An accuracy of greater than +/- 4% was obtained for the concetnration ranges usually found in contact lens solutions with a minimum detection level of 10 ppm. The method is useful as a screening procedure for solutions of unknown composition and as a quality assurance procedure for routine determinations.
Colorimetry↗
A rare and atypical case of long-distance indirect DNA transfer: Contamination from an investigator never present at the scene.
To maximize the usefulness of DNA obtained from biological samples in forensic genetics, it is crucial to avoid DNA contamination throughout all procedures, from sample collection at crime scenes to STR profile generation in DNA laboratories. This study reports a rare and atypical case of DNA contamination in a forensic setting. During the analysis of biological evidence from a cold case preserved for 18 years, the STR profile obtained from the surface of a plastic bag matched that of an investigator, identified through the DNA elimination database. Case reconstruction confirmed that the investigator-who was located 80 km from the DNA laboratory and had never entered the crime scene or the sample storage room-was not a suspect and that the obtained STR profile originated from contamination. The most plausible explanation for the contamination was indirect transfer: investigator's DNA had adhered to a colleague's clothing and was subsequently dislodged and deposited onto the surface of the plastic bag as the colleague approached the sample pretreatment area. This study integrates trace DNA profiling of challenged samples with rapid contamination investigation and proposes prevention and control measures. This case underscores that, although DNA is widely regarded as the "gold standard" in forensic genetics, its interpretation must be considered within the context of the entire case. Conclusions should not be drawn based solely on a single DNA result.
Humans↗
[Determination of carbamate pesticides in fruits and vegetables by means of high pressure liquid chromatography (author's transl)].
It is shown that carbamate pesticides can be determined at the ppm level in different kinds of fruits and vegetables using high pressure liquid chromatography. The lower detection limit corresponding to a signal to noise ratio of three is between 0,025 and 0,25 ppm depending on the type of carbamate and plant material. In all cases it is below the maximum permissible value for pesticide residues. Due to the high separating power of high pressure liquid chromatography a simple sample pretreatment procedure can be used. The carbamates are extracted from the biological sample by dichloromethane and injected directly into the liquid chromatograph after replacing the extraction solvent by the mobile phase. In many cases a definite identification and quantitation of the carbamate is possible with a single chromatographic column. Some plants with a more complex matrix require a two-column operation in which the effluent fraction of the first column containing the pesticide is transferred by column switching to a second column in order to achieve a complete separation. The improved resolution in the two-column operation is caused by the relative enrichment effect of the fractionation and the increased column length.
Carbamates↗
Comparison of talc-Celite and polyelectrolyte 60 in virus recovery from sewage: development of technique and experiments with poliovirus (type 1, Sabin)-contaminated multilitre samples.
For virus recovery from sewage, a mixture of talc and Celite was tested as a possible inexpensive substitute for polyelectrolyte 60 (PE 60). After adjustment of pH to 6 and the addition of 45-60 plaque forming units (PFU)/ml of poliovirus type I (Sabin) to the sewage sample under test, 100 ml of it was passed through either a PE 60 (400 mg) or a talc (300 mg)-Celite (100 mg) layer; the layer-adsorbed virus was eluted with 10 ml of 10% fetal calf serum (FCS) in saline (pH 7.2). In these experiments, PE 60 layers recovered 73-80% (mean 76%) of the input virus. In comparison, virus recoveries with the talc-Celite layers were 65-70% (mean 68%). Passage of 5 litres of raw sewage (containing 50 to 1.26 X 10(5) PFU/100 ml of the poliovirus) through the talc (15 g)-Celite (5 g) layers and virus elution with 50 ml of 10% FCS in saline gave virus recoveries of 33-63% (mean 49%). Except for pH adjustment and prefiltration through two layers of gauze to remove large solids, no other sample pretreatment was found to be necessary. Application of this technique to recovery of indigenous viruses from field samples of raw sewage and effluents has been highly satisfactory.
Adsorption↗
Aflatoxins and their biosynthetic precursors in lotus seeds: simultaneous UPLC-MS/MS determination, contamination profiling, and matrix-specific accumulation during Aspergillus flavus infection.
Aflatoxin (AF) contamination poses a severe global threat to food and medicinal material safety, yet existing research focuses on terminal AF metabolites while neglecting residual biosynthetic precursors, leading to potential underestimation of contamination risks. In this study, a UPLC-MS/MS method was established for the simultaneous quantification of six AFs and their five precursors in lotus seeds, with optimization of mass spectrum parameters, chromatographic separation conditions, and sample pretreatment. Method validation confirmed linearity (R2 > 0.99), LODs (0.03-0.36 μg/kg), and recoveries (76.53%-120.0%, RSD < 15%). Analysis of 41 natural lotus seed samples revealed a 63.4% AF contamination rate, dominated by B-group AFs, while O-methylsterigmatocystin (OMST) and versicolorin hemiacetal (VOH) were identified as the primary co-residual precursors with co-occurrence rates ≥ 50%. Notably, AFM1 was predominantly detected in natural samples with AFB1 concentrations exceeding 100 μg/kg. Artificial inoculation experiments further demonstrated that sterilization and sealing conditions modulated AF biosynthesis in lotus seeds, with non-sterilized and non-sealed groups showing delayed fungal metabolism and lower toxin accumulation. A significant linear correlation was observed between AFM1 and AFB1 levels (r = 0.94) in infected samples, demonstrating their accumulation levels are coupled with fungal overall metabolic flux. Given the high co-occurrence rate of OMST/VOH with AFB1 in natural samples, their individual and combined toxicities require in-depth investigation. This work deciphers matrix-specific AF dynamics in lotus seeds, supporting regulatory standard refinement (e.g., precursor inclusion) and targeted control (e.g., time-sensitive drying after harvest). Further studies will focus on exploring the molecular mechanisms of substrate-dependent AF synthesis.
Aflatoxins↗
Development and validation of an LC-MS/MS method for the quantification of the KRASG12C inhibitor divarasib.
Divarasib is a newly developed covalent KRASG12C inhibitor, currently under clinical investigation in a phase 3 trial in patients with non-small cell lung cancer (NSCLC). At the moment, very limited pharmacokinetic data are publicly known. However, obtaining more insight into the pharmacokinetic properties of divarasib is important, since this may provide a better understanding of its efficacy and safety risks. Pre-clinical studies have been performed in mouse models to evaluate the effect of drug transporters and drug-metabolizing enzymes on the plasma exposure and tissue distribution of divarasib. Therefore, a reliable quantification method is required. To our knowledge, no bioanalytical assay of divarasib has been published yet. Therefore, in this study we developed and validated an assay to quantify divarasib in human plasma and in eight different mouse-related matrices, and partially in mouse plasma, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was initially evaluated over a concentration range of 1-10,000 nM. However, due to carry-over observed at 10,000 nM, the validated calibration range was established at 1-2000 nM, with matrix-dependent LLOQs of 1-10 nM. Erlotinib was used as an internal standard and acetonitrile was utilized to perform protein precipitation as sample pretreatment. Divarasib demonstrated stability in human plasma and in mouse plasma and tissue homogenates under various experimental conditions. A pilot in vivo study showed the applicability of our validated LC-MS/MS method. Ongoing clinical trials may collect plasma samples, and this developed method enables quantification of divarasib in both mouse and human plasma samples.
Animals↗
Circulating Tumor DNA Profiling Defines Risk Classification in Patients With Ewing Sarcoma: A Report From the Children's Oncology Group and the LEOPARD Study.
PURPOSE: Identification of discrete risk groups remains a high priority for patients with Ewing sarcoma (EWS). We sought to prospectively validate circulating tumor DNA (ctDNA) as a prognostic factor and develop clinical-molecular risk groups. METHODS: We conducted a prospective investigator-initiated biology study for patients with localized EWS (LEOPARD) and embedded ctDNA analysis into the North American frontline metastatic study AEWS1221. Eligible patients were younger than 50 years with newly diagnosed EWS. All patients provided a baseline blood sample for analysis, which was subjected to ultralow-pass whole-genome sequencing and hybrid capture panel sequencing for ctDNA quantification, fusion detection, and characterization of STAG2 and TP53 alterations. Serial ctDNA sequencing was conducted on a subset of patients in each study. We tested for associations between ctDNA burden and secondary genomic events, and clinical features and outcomes. RESULTS: One hundred forty patients with localized disease and 255 with metastatic disease provided evaluable pretreatment samples for ctDNA analysis. Elevated baseline ctDNA was associated with stage, tumor size, primary site, indeterminate pulmonary nodules, and metastatic pattern. Elevated pretreatment ctDNA burden was associated with inferior outcomes in patients with localized (n = 140, hazard ratio [HR] = 2.36, P = .032) and metastatic disease (n = 255, HR = 2.15, P = .001). Patients with metastatic disease and TP53 variants and/or persistent on-therapy ctDNA had dismal outcomes. Patients with localized disease, low ctDNA, small tumors, and favorable genomics had no events and constitute a novel low-risk group. Among patients with metastatic disease, those with lung-only disease, low ctDNA, and favorable genomics represent an intermediate-risk group. CONCLUSION: This study prospectively validates pretreatment ctDNA burden as prognostic in EWS. Risk groups that integrate ctDNA burden with clinical-molecular features differentiate patients with low-, intermediate-, and high-risk disease.
Journal Article↗
A STORM-based protocol for nanoscale imaging and quantitative analysis of protein-associated and phospholipid-associated structures in natural rubber.
Stochastic Optical Reconstruction Microscopy (STORM) enables nanoscale mapping of molecular components beyond the diffraction limit; however, its reproducible implementation in hydrophobic polymer matrices remains challenging because fluorescence-labeling specificity, fluorophore photoswitching, three-dimensional localization, chromatic registration, and quantitative image analysis must be carefully controlled. This protocol presents a standardized experimental workflow for dual-color labeling, astigmatism-based three-dimensional STORM acquisition, and quantitative analysis of protein-associated and phospholipid-associated structures in natural rubber (NR). The workflow covers sample pretreatment, Cy5 NHS ester labeling of protein-associated primary amines, DiI labeling of phospholipid-rich domains, STORM imaging-buffer preparation, three-dimensional single-molecule localization, dual-channel registration, generation of standardized xy projections, aggregate-size analysis, and projected lateral spatial correlation assessment. Reproducibility is supported by defined acquisition and localization criteria, three independent sample preparations with at least five fields of view analyzed per condition, and unlabeled, single-color, dye-only matrix, and processing-associated Cy5 controls. Mean lateral localization precisions of 11.8 ± 2.3 nm for Cy5 and 13.5 ± 2.9 nm for DiI were obtained, while two-dimensional Fourier ring correlation analysis of the xy projections yielded effective lateral image resolutions of approximately 25 and 28 nm, respectively. Image-based particle segmentation and localization-coordinate-based density-based spatial clustering of applications with noise (DBSCAN) were applied to standardized xy projections as complementary quantitative approaches. Application of the protocol to untreated, centrifuged, and protease-treated NR samples demonstrated treatment-associated changes in the detected abundance and projected size distributions of protein- and phospholipid-associated aggregates, together with a non-monotonic change in their projected lateral spatial correlation. These observations describe alterations in nanoscale organization but do not, by themselves, establish stable protein-phospholipid complex formation. Unlike previous studies that primarily demonstrated the feasibility of STORM imaging in rubber materials, the principal contribution of this work is an end-to-end, step-by-step protocol incorporating defined controls, three-dimensional localization, image-quality metrics, chromatic-registration procedures, and complementary quantitative-analysis pipelines for non-expert users. The workflow may be adaptable to other hydrophobic polymers and soft-material systems after appropriate optimization and validation.
Rubber↗
Clindamycin-associated enterocolitis in guinea pigs: evidence for a bacterial toxin.
Experimental enterocolitis was induced in guinea pigs by intraperitoneal injection of clindamycin. Specimens of feces were collected daily in phosphate-buffered saline (pH 7.0) and pooled every second day. The pooled samples were centrifuged to remove solids, and the supernatant was sterilized by membrane filtration. The sterile fecal supernatants were then dialyzed for 48 h against two 15-liter changes of phosphate-buffered saline and subsequently tested for toxicity in cultured monolayers of mouse adrenal cells. A filterable toxin(s) was found in the fecal supernatants on days 2, 4, and 6 postchallenge and not in pretreatment samples. The toxin(s) caused enterocolitis when administered orogastrically to healthy animals and altered the morphology of cultured mouse adrenal cells. The alteration of adrenal cell morphology was neutralized by specific antitoxin to Clostridium histolyticum.
Adrenal Glands↗
Development and clinical validation of a CRISPR/Cas9-engineered reporter phage cocktail for rapid detection of Escherichia coli in urine.
Urinary tract infections are one of the most common infectious diseases, with Escherichia coli as the predominant pathogen. Traditional diagnostic methods fail to meet clinical demands for rapid and specific detection. Here, we developed an efficient urine E. coli detection strategy via a reporter phage cocktail. Four reporter phages (T2::Nluc, T4::Nluc, T5::Nluc, T6::Nluc) were constructed by the CRISPR/Cas9 system combined with homologous recombination. One-step growth curves, optimal multiplicity of infection, and lytic efficiency showed that the Nluc gene block insertion exerted heterogeneous effects on phages. Luminescence assays demonstrated that all five reporter phages (including previously preserved T7::Nluc) and the cocktail offered favorable limits of detection (≥103 CFU/mL), high specificity, and no urine matrix interference. However, single phages exhibited limited coverage among 177 clinical E. coli isolates. But the reporter phage cocktail remedies this limitation. In large-scale clinical validation, the cocktail achieved sensitivity 73.15% (63.76%-81.22%), specificity 100.00% (99.53%-100.00%), positive predictive value (PPV) 100.00% (95.44%-100.00%), and negative predictive value (NPV) 96.42% (95.18%-97.36%) (all 95% confidence interval [CI]), and excellent concordance with the gold-standard method (Kappa = 0.83, 95% CI: 0.77-0.89), greatly outperforming single reporter phages (~40.00% sensitivity). This method requires no sample pretreatment, is simple to operate, and completes detection within 4 h, significantly improving diagnostic efficiency. Accordingly, it provides a novel platform for pathogen detection and supports the clinical translation of reporter phage diagnostics.IMPORTANCEUrinary tract infections impose substantial economic and public health burdens. In this study, we successfully constructed Escherichia coli-specific reporter phages T2::Nluc, T4::Nluc, T5::Nluc, and T6::Nluc. Combined with the previously preserved T7::Nluc, these phages formed a reporter phage cocktail. Co-cultivation of this cocktail with clinical samples enabled rapid and specific detection of E. coli in clinical urine, with a significantly shortened detection time (4 h) and good concordance with the gold-standard detection method (Kappa = 0.83), effectively improving detection efficiency and accuracy. This novel pathogen detection platform, integrating specific recognition and signal amplification, not only provides a new technical approach for the rapid and accurate diagnosis of clinical urinary tract infections but also effectively promotes the coordinated improvement of infectious disease diagnosis and treatment in terms of timeliness-precision-cost.
Escherichia coli↗
Chemical ionization mass spectrometry of trimethylsilylated carbohydrates and organic acids retained in uremic serum.
After appropriate sample pretreatment and derivatization, uremic serum was investigated by combined high resolution gas chromatography and mass spectrometry, using both electron impact and chemical ionization methods. Electron impact and chemical ionization spectra of a number of identified (trimethylsilylated) carbohydrates and organic acids are compared. The utilization of chemical ionization mass spectrometry, with isobutane as the reagent gas, is discussed in detail. The influence of the reagent gas pressure on the total ion current and on the spectral appearance was studied. The identification of compounds, based on electron impact mass spectral data, was confirmed and often aided appreciably by using this technique. The chemical ionization spectra of trimethylsilyated alditols and aldonic acids, as well as of other organic acids showed protonated molecular ions, whereas aldoses did not. Differences with electron impact spectra are found mainly in the high mass region. The loss of one or more trimethylsilanol groups becomes the predominating fragmentation route at higher reagent gas pressures.
Carbohydrates↗
Paradoxical Effect of Myosteatosis on the Immune Checkpoint Inhibitor Response in Metastatic Renal Cell Carcinoma.
BACKGROUND: Treatment for metastatic renal cell carcinoma (mRCC) has shifted from tyrosine kinase inhibitor (TKI) therapy to immune checkpoint inhibitor (ICI)-based therapy, improving outcomes but with variable individual responses. This study investigated the prognostic implications of pretreatment low skeletal muscle mass (LSMM) and myosteatosis in patients with mRCC undergoing first-line ICI-based therapies, comparing outcomes between PD-1 inhibitor + CTLA-4 inhibitor and PD-1 inhibitor + TKI, incorporating single-cell RNA sequencing. METHODS: A retrospective analysis was performed on 90 patients with mRCC treated with ICI-based therapies between November 2019 and March 2023. Patients were grouped based on whether they received PD-1 inhibitor + CTLA-4 inhibitor or PD-1 inhibitor + TKI combinations. LSMM was defined as skeletal muscle index below 40.8 cm2/m2 for men and 34.9 cm2/m2 for women. Myosteatosis was defined using skeletal muscle density, with cut-off values < 41 HU for BMI < 25 kg/m2 and < 33 HU for BMI ≥ 25 kg/m2. Progression-free survival (PFS) and overall survival (OS) were compared using Kaplan-Meier curves and multivariable models. Single-cell RNA sequencing was performed on pretreatment samples to compare the immune microenvironment between patients with and without myosteatosis. RESULTS: The study cohort (26.7% female; median age: 60.5 years) included 59 patients (65.6%) treated with PD-1 inhibitor + CTLA-4 inhibitor and 31 patients (34.4%) treated with PD-1 inhibitor + TKI. LSMM was present in 18.9% of patients, and myosteatosis in 41.1%, with comparable proportions across groups. During follow-up, 29 patients (32.2%) died: 16 in the PD-1 inhibitor + CTLA-4 inhibitor group and 13 in the PD-1 inhibitor + TKI group. The overall 1-year mortality rate was 22.2%, and PFS rate was 53.3%. Myosteatosis predicted poor OS (HR, 5.389; p = 0.008) and PFS (HR, 2.930; p = 0.022) in the PD-1 inhibitor + TKI group but was protective for PFS (HR, 0.461; p = 0.049) in the PD-1 inhibitor + CTLA-4 inhibitor group. LSMM did not significantly affect outcomes in either group. Single-cell RNA sequencing revealed higher CTLA-4 expression in regulatory T cells and more effector memory CD8+ T cells in patients with myosteatosis, whereas patients without myosteatosis had more anti-tumoural non-classical monocytes. CONCLUSIONS: Myosteatosis negatively impacts OS and PFS in patients with mRCC treated with PD-1 inhibitor + TKI therapy but is protective for PFS in those treated with PD-1 inhibitor + CTLA-4 inhibitor therapy. Altered checkpoint expression and immune cell composition associated with myosteatosis may contribute to these differential responses.
Humans↗
Simultaneous determination of niacin, niacinamide, pyridoxine, thiamine, and riboflavin in multivitamin blends by ion-pair high-pressure liquid chromatography.
A high-pressure liquid chromatographic procedure for the simultaneous determination of niacin, niacinamide, pyridoxine, thiamine, and riboflavin was developed and applied to the analysis of multivitamin blends for these water-soluble vitamins. Reversed-phase ion-pair chromatography, using sodium hexanesulfonate as the counterion, was employed. Analysis time is shortened considerably and precision is improved by the application of this analytical technique as compared to the current official methods of analysis. Involved sample pretreatment is not required.
Chromatography, High Pressure Liquid↗
Selective Extraction of Genomic DNA From Animal Tissues Using a Hydrophobic Magnetic Ionic Liquid.
The development of green and efficient methods for genomic DNA extraction from animal tissues is crucial for molecular diagnostics, food traceability, and genetic research. Conventional methods often involve toxic reagents, multiple centrifugation steps, and are time-consuming. In this study, a hydrophobic magnetic ionic liquid (MIL), N-octyl-4-dimethylaminopyridinium hexafluorophosphate MIL ([C8DMAP][PF6]‑Ni MIL), was synthesized and applied for the selective extraction of genomic DNA from various animal tissues. The material exhibited strong paramagnetic behavior, high thermal stability, and excellent hydrophobicity, enabling rapid phase separation under an external magnetic field. A mechanical shaking-assisted extraction method was developed, and key parameters including temperature, time, shaking speed, and [C8DMAP][PF6]-Ni MIL dosage were systematically optimized. The method demonstrated high selectivity for DNA over proteins, RNA, and amino acids, with a maximum recovery rate of 78.06 ± 1.91%. Compared to a commercial DNA extraction kit, the [C8DMAP][PF6]-Ni MIL-based approach provided higher yields from several tissues, including mouse liver, brain, and rabbit lung. Furthermore, the [C8DMAP][PF6]-Ni MIL could be reused for at least six cycles while maintaining extraction efficiency. This work not only provides a high-performance material for DNA extraction, but also demonstrates a sustainable and easily retrievable liquid-phase separation strategy, offering a generalizable platform for complex sample pretreatment.
Animals↗
Toxic trace metals in food. I. A new voltammetric procedure for toxic trace metal control of wines.
A new highly sensitive and particularly reliable analytical procedure for the precise determination of the most relevant toxic trace metals cadmium, lead and copper in wine is presented. It consists of a simple and convenient sample pretreatment by UV irradiation (1,5 h, 500-W-Hg-lamp) to release the toxic trace metals bound by organic substances and subsequent voltammetry (DPASV) of cadmium and lead simultaneously at a mercury film electrode (MFE) formed in situ on a vitreous carbon carrier and of copper on a gold electrode. Particular emphasis has been placed on the efficient exclusion of interference due to contamination. Only special wines, with more than 10% of sugar, require substitution of UV irradiation by a wet digestion also described. The procedure may be easily expanded to include the determination of mercury and zinc.
Electrodes↗
Cardiovascular and metabolic manifestations of heat stroke and severe heat exhaustion.
We prospectively studied the clinical, biochemical (including creatine phosphokinase (CPK) isoenzymes) and electrocardiographic features of exertional heat stroke in 13 patients (group 1) and severe heat exhaustion in 14 patients (group 2). Despite initial presentations with severe hyperthermia, tachycardia and hypotension, only one patient with heat stroke had myocardial ischemia. The CPK isoenzymes were not indicative of myocardial damage in any patient. The patients with heat stroke were somewhat more dehydrated than those with heat exhaustion as measured by differences in serum creatinine, sodium and osmolality, and the former (group 1) had a significantly lower initial glucose level (P less than 0.05). Although significant differences in potassium were not observed in the pretreatment samples, at 12 hours the serum potassium was significantly lower in group 1 (P less than 0.05). This suggests that this group may have been more potassium-depleted at the time of heat stroke. Prompt recognition and vigorous therapy were successful in rapidly lowering high temperatures and in preventing serious complications.
Alanine Transaminase↗