Search Results (452)

Ceftobiprole is a novel and promising antibiotic; however, the direct pharmacological use of its native form is limited by its low water solubility. The first part of this study provides a deeper insight into the physicochemical properties of this drug. One- and two-dimensional nuclear magnetic resonance (NMR) spectra in D₂O were recorded, and a complete assignment of ¹H and ¹³C signals was achieved with the support of quantum mechanical calculations. The combined results from capillary electrophoresis and NMR confirmed the cationic nature of ceftobiprole at pH values well below 3 and the protonation of the secondary amino group, thus supporting the theoretically predicted dominant protonation states. Molecular dynamics simulations revealed that zwitterionic ceftobiprole molecules associate through hydrogen bonding, whereas in the cationic form, the attractive forces involve weaker π-π and stacking interactions. The use of ceftobiprole in its native form in pharmaceutical formulations was made possible through the development of a novel freeze-dried cyclodextrin-based delivery system. Consequently, the second part of this article focuses on evaluating the biological properties of this system (ceftobiprole/maleic acid/sulfobutylether-β-cyclodextrin in a molar ratio of 1:25:4), including its antibacterial activity against the most common pneumonia-causing pathogens and its cytotoxicity towards normal and cancer cells. Full article

Background: Hemoglobin Rothschild (Hb Rothschild), NM_000518.5(HBB):c.112T>C, is an ultra-rare low-oxygen-affinity hemoglobin variant that persistently causes reduced peripheral oxygen saturation on pulse oximetry despite normal arterial oxygenation. Fewer than ten cases have been reported worldwide, and only one involved a child—an asymptomatic carrier identified incidentally. Methods: The patient underwent clinical examination, growth assessment, blood tests, hemoglobin electrophoresis, chest CT, abdominal ultrasound, echocardiography, and pulmonary perfusion scintigraphy. Whole genome sequencing (WGS) of the proband and parents was performed, followed by bioinformatic analysis and ACMG-based variant interpretation. A PRISMA-guided PubMed literature review was conducted. Results: We report on the first pediatric case exhibiting a symptomatic clinical course. A 4-year-old boy was referred for chronically low peripheral oxygen saturation (SpO₂), 78–86%, on pulse oximetry and recurrent lower respiratory tract infections. Early developmental history revealed episodes of apnea in infancy, perioral cyanosis, poor exercise tolerance, and low weight gain. Repeated cardiopulmonary assessments, chest computed tomography (CT), echocardiography, and pulmonary perfusion scintigraphy yielded unremarkable findings. Arterial blood gas analysis consistently showed normal arterial partial pressure of oxygen (PaO₂), excluding true hypoxemia. Hemoglobin electrophoresis revealed an abnormal HbD fraction; WGS identified a heterozygous variant NM_000518.5(HBB):c.112T>C inherited from the patient’s asymptomatic father. This variant increases the partial pressure of oxygen at which hemoglobin is 50% saturated (p50), thereby decreasing hemoglobin’s oxygen affinity and shifting the oxyhemoglobin dissociation curve to the right. These alterations explain the discordance between low peripheral oxygen saturation (SpO₂) and preserved oxygen delivery to tissues. Conclusions: This case expands the clinical spectrum of Hb Rothschild and demonstrates that symptomatic presentation may occur in early childhood. Awareness of low-affinity hemoglobin variants is essential to avoid misdiagnosis and unnecessary cardiopulmonary interventions. Early genetic testing facilitates accurate diagnosis and appropriate counseling. Full article

Fumarate, succinate, maleate, dihydroxyfumarate, D–tartarate, L–tartarate, DL–tartarate, L-malate, D-malate, oxaloacetate, citrate, and DL-isocitrate in the 5–100 μM concentration range were incubated in 12.5 mM HEPES/25 mM TRIS base containing 200 μM Eu³⁺–tetracycline and 60% (v/v) formamide (pH unadjusted). After 30 min of incubation, they were separated at 4 °C by capillary electrophoresis utilizing laser-induced luminescence detection with 12.5 mM HEPES/25 mM TRIS base containing 60% formamide as the running buffer. All analytes yielded peaks, with the exception of fumarate, succinate, and maleate. L-Malate was detected down to 100 nM. The main component of this study was the analysis of malate. The objective was to develop a stereoselective methodology for the detection of L-malate. This was achieved by varying the formamide concentration and separation temperature. When the temperature was increased to 22 °C and the formamide concentration decreased to 40%, the sensitivity for L-malate was diminished about 10-fold, but that for D-malate was eliminated. This combination of conditions allowed for the stereospecific analysis of L-malate. Full article

Compared with capillary electrophoresis (CE), gel electrophoresis (GE) is a traditional method for the analysis of nucleic acids because of its low cost, although the operation process is complicated. The electropherogram from CE can offer more information (e.g., DNA size and its concentration) for researchers. Based on the self-built integrated biochip GE system, we proposed a computational method that converts conventional agarose GE images into CE-like fluorescence profiles for enhanced DNA analysis. The gel images were processed using an image-based algorithm involving median filtering to remove background noise and pixel-wise intensity summation along the migration axis to generate one-dimensional records of electrophoretic separations. Each DNA band in the gel was thereby transformed into a distinct fluorescence peak, reflecting its migration distance and relative intensity. To further enhance resolution and peak separation, Gaussian modeling was applied to fit the fluorescence intensity distribution, providing smoother and more distinguishable spectral peaks. To validate the method, three periodontal pathogens—Porphyromonas gingivalis (P.g), Treponema denticola (T.d), and Tannerella forsythia (T.f)—were amplified using PCR and analyzed by gel electrophoresis. The method successfully identified distinct electrophoretic patterns for the three pathogens by using a 50 bp DNA ladder as an internal calibration reference. The results demonstrate that image-based reconstruction of electrophoretic data provides a reliable, quantitative, and visually interpretable representation of DNA migration, comparable to CE output. This approach bridges a gap between traditional GE and modern capillary systems, allowing for the semi-quantitative analysis of DNA fragments without specialized CE instrument. The proposed method offers a valuable analysis method for the separation of DNA, RNA, protein and polypeptides. Full article

Background: Hypercalcemia is a common and serious complication of malignancy, often contributing to morbidity and mortality. In patients with paraproteinemia, elevated total calcium with normal ionized calcium, termed pseudohypercalcemia, can complicate diagnosis and lead to inappropriate treatment. While this phenomenon has been described in case reports, its prevalence and clinical impact in routine practice remain poorly defined. Methods: We report a case of pseudohypercalcemia in a patient with IgG κ multiple myeloma and conducted a retrospective review of de-identified data to assess the prevalence and biochemical associations of pseudohypercalcemia in paraproteinemia. Available data included serum protein electrophoresis (SPEP), total calcium, albumin, total protein, creatinine, and parathyroid hormone (PTH). Associations between calcium status, paraprotein levels, and the gamma globulin gap were examined. Results: The index case demonstrated pseudohypercalcemia, with elevated total calcium (13.5 mg/dL) but normal ionized calcium (1.22 mmol/L), in the setting of IgG κ paraproteinemia (4.4 g/dL). In the retrospective cohort of 2537 samples, 986 (39%) had a single monoclonal paraprotein. Gamma globulin gap showed a moderate correlation with paraprotein concentration for IgG (r = 0.56, p < 0.0001) and IgA (r = 0.44, p < 0.0001), but a weaker relationship for IgM (r = 0.49, p < 0.0001). In contrast, total calcium showed no significant correlation with paraprotein concentration in the overall cohort. Among samples with elevated calcium (>10.5 mg/dL), the association between calcium and IgG paraprotein levels remained weak (r = 0.34, p = 0.23), and was similar for IgG κ (r = 0.61, p = 0.12) and IgG λ (r = 0.09, p = 0.87). Hypercalcemia was uncommon, occurring in only ~2% of IgG-positive samples, and rarely at paraprotein levels ≥ 1.5 g/dL. Conclusions: Pseudohypercalcemia in paraproteinemia is uncommon but clinically important, as total calcium may be artifactually elevated due to paraprotein-related assay interference, either from assay precipitation effects or calcium binding by paraproteins. Paraprotein burden correlates with gamma globulin gap but not with true calcium status. Reliance on total calcium alone may lead to diagnostic misclassification; ionized calcium should be measured in patients with monoclonal gammopathies to distinguish true hypercalcemia from analytical interference and avoid unnecessary treatment. Full article

We developed and validated a capillary electrophoresis (CE) method for the separation of two opioid–neurotensin hybrid peptides, recently presented as potent analgesics being decapeptides with a hybridic nature (i.e., H-Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH; PK20 and its structural analogue H-Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Ile-Leu-OH; [Ile9]PK20). As these two chimeras differ by only one amino acid, Tle→Ile, and are characterized by possessing the same molecular weight while having different spatial conformations, the aim of the study was to determine their potential separation in terms of the presence of any differences resulting from this structural modification. The separation process was performed using an eCAP fused silica capillary at a detection wavelength of 200 nm in 25 mM phosphate buffer at pH 2.5. The analysis was performed at 25 °C and 10 kV. The developed method was validated by assessing linearity in the concentration range from 50 to 5000 ng/mL. Very good linearity was obtained, with the coefficient of determination (R²) ranging from 0.9991 to 0.9999 for both analyzed derivatives. The method demonstrated baseline resolution (Rs = 1.4). The limit of quantification ranged from 34.72 ng/mL to 34.98 ng/mL. The recoveries of all derivatives ranged from 94.8% to 100%. The total analysis time was only 6 min. The developed method enables the determination of PK20 and [Ile9]PK20 derivatives both in aqueous solutions and in serum. Full article

Background and Clinical Significance: Waldenström macroglobulinemia (WM) is a rare, indolent B-cell non-Hodgkin lymphoma, characterised by the presence of monoclonal immunoglobulin M (IgM) and lymphoplasmacytic infiltration of the bone marrow. It is often associated with various haematological and systemic disorders, including previous cold agglutinin disease (CAD), a condition where cold-sensitive antibodies lead to haemolysis. Case Presentation: A 55-year-old male patient was admitted to the Internal Diseases Ward with symptoms of weakness, reduced effort tolerance, and weight loss, along with life-threatening normoblastic anaemia (haemoglobin [Hb]: 3.90 g/dL). Initial blood tests raised suspicion of CAD due to the presence of multiple blood clots, as well as a decrease in lymphocyte and neutrophil counts. CAD was then confirmed by a cold agglutinin titre of 1:2000 and direct antiglobulin test ([DAT] 4+). Two weeks later, upon transfer to the Haematological Diseases Ward, further investigation revealed elevated IgM levels (up to 31.55 g/L). Additional diagnostic tests, including serum protein electrophoresis, imaging, multiparametric flow cytometry, and bone marrow biopsy, confirmed the diagnosis of WM. The L265P MYD88 mutation test was positive. Treatment with intravenous rituximab was initiated, followed by bendamustine/rituximab (BR) therapy protocol as first-line treatment. After two cycles, the patient’s clinical condition and laboratory results significantly improved, with a marked reduction in IgM (<0.4 g/L). Hb levels steadily rose to 12.60 g/dL, eliminating the need for further blood transfusions. Conclusions: This case highlights the importance of recognising the coexistence of CAD and WM, which may present with overlapping clinical features, including life-threatening anaemia. Extensive diagnostics and prompt treatment with combination therapy can lead to effective clinical improvement. Full article

Autism spectrum disorder (ASD) is a complex group of severe neurodevelopmental disorders characterized by varying degrees of dysfunctional communication and social abilities as well as repetitive and compulsive stereotypic behaviors. We aim to evaluate the genetic predisposition to oxidative response and its relationship with altered oxidative stress markers in ASD patients. Genomic DNA was isolated from peripheral blood lymphocytes of 106 (83 M, 23 F; 7.9 ± 3.2 years) ASD patients and 90 healthy subjects (63 M, 27 F; 21.2 ± 1.8 years). Genotyping was performed by real-time PCR-based allelic discrimination, PCR and electrophoresis of GST deletion variants. Reactive oxygen metabolites (dROMs), the Biological Antioxidant Potential (BAP), and the advanced oxidation protein products (AOPP) were also measured. Furthermore, we assessed oxidative DNA damage by Single Cell Gel Electrophoresis. The evaluation of oxidative stress markers indicated a mild oxidative stress status and a higher level of DNA damage in nuclei of ASD patients’ lymphocytes. We found significant associations between ASD and several polymorphisms of genes involved in the detoxification and the response to oxidative stress. Genetic and environmental factors contribute to the onset of autism spectrum disorder, and ASD patients’ treatment requires a multimodal approach, including behavioral, educational, and pharmacological approaches. Full article

In this paper we study in detail the products formed during the process of water-assisted thermal oxidative decomposition (TOD) of polypropylene in the presence of pressurized oxygen. A set of techniques has shown that the main decomposition product in such a process is acetic acid with small amounts of other carboxylic acids (formic, propionic, succinic). The kinetics of carboxylic acid formation is studied by means of gas chromatography–mass-spectrometry as well as capillary electrophoresis, and the possible mechanisms behind the products formation are discussed. The role of water is considered based on the results obtained from substituting H₂O with D₂O in TOD. Compositions of residual oligomeric fractions as well as gas products are analyzed. Full article

Napier grass (Cenchrus purpureus Schumach) is an important forage crop and livestock feed. However, its yield and quality in Kenya are often limited by Napier grass headsmut and stunt disease. Napier grass genetic improvements through mutation breeding and selection could avail cultivars with increased forage. This study investigated the response of embryogenic calli to different levels of colchicine in inducing polyploidy in the two germplasms of Napier grass; South africa and Bana grass. The experiments were carried out as a factorial experiment in a completely randomized design (CRD). The colchicine concentrations used were 0, 0.05, 0.1, and 0.2%, and the exposure durations were 24, 48, and 72 h. During the shoot regeneration stage, culturing explants on an MS medium (Murashige and Skoog) supplemented with 0.2 mg L⁻¹ Benzyl Adenine (BAP), 0.1 mg L⁻¹ dichlorophenoxyacetic acid (2, 4-D), and 0.1 mg L⁻¹ indole-3-butyric acid (IBA) was more suitable for shoot regeneration. Chromosome doubling was confirmed by genomic DNA and the stomata size and number. Culturing explants on an MS medium supplemented with 1 mg L⁻¹ IBA, 1 mg L⁻¹ 2, 4-D, and 0.5 mg L⁻¹ BAP was more suitable in inducing embryogenic calli in both genotypes. Polyploidy results revealed that a 0.1% concentration of colchicine with two days of treatment established the maximum number of octoploid plantlets induced in vitro, while a 0.2% concentration was very toxic. The stomata size and number of derived octoploid plantlets were bigger with a lower density, a shorter plant height, and a smaller stem diameter, and despite being the first to produce tillers, they were significantly higher than their progenitors. Induced mutants also had a significantly higher number of chromosomes and showed different band patterns and distances during gel electrophoresis. However, we recommend the use of flow cytometry to confirm the ploidy level. The superior mutant plantlets can be selected and recommended for characterization across representative agro-ecologies for large-scale production and used in Cenchrus purpureus breeding programs in Kenya and its environments. Full article

Background: Carrimycin is a mixture of spiramycin derivatives with antibacterial functions. However, recent studies have shown that it possesses certain anticancer properties. The specific mechanism of the anticancer activity is unknown. Methods: To study the anticancer mechanism of carrimycin, we synthesized a derivative of spiramycin, n-hexyl spiramycin (h-SPM), and used a combination of metabolomics and transcriptomics methods. Capillary electrophoresis–mass spectrometry (CE-MS) was used to detect polar small molecule metabolites, and liquid chromatography–mass spectrometry (LC-MS) was used to detect lipid metabolites in cells. Transcriptomics was used to measure mRNA content in cells. Finally, by processing these data using specific bioinformatics methods, the mechanism underlying anticancer effect of carrimycin was determined. Results: Metabolomics and transcriptomic results showed that lipid metabolism and mitochondrial biogenesis pathways in the cells changed after hSPM treatment, NR1D1 genes and ceramide were enriched from these pathways, implicating the involvement of ROS and pro-inflammatory response. Western blotting verified that the protein levels of NR1D1 decreased after h-SPM treatment, and ROS stating and qPCR demonstrated that ROS levels and the mRNA levels of pro-inflammatory genes were greatly induced by h-SPM. Conclusions: h-SPM reduced the protein level of NR1D1, disrupted metabolic regulation, accumulating ceramide, and the subsequent increased ROS generation promoted apoptosis and pro-inflammatory-like response of cells. Our findings unveiled the anticancer mechanism of a potent anticancer derivative of spiramycin and unveiled its mechanism of action. Full article

Tetrahydroprotoberberine alkaloids (THPBs) are bioactive natural products bearing stereogenic centers that frequently exhibit enantiomer-specific pharmacological effects. Xylopinine (XPN), a representative THPB, shows cytotoxic, antimicrobial, and antimalarial activity in vitro, and displays pronounced stereoselectivity in vivo, with the naturally occurring (S)-enantiomer emphasizing the need for reliable enantioselective analysis. In this study, we present the synthesis of racemic XPN from norlaudanosine, and its first comprehensive cyclodextrin-assisted capillary electrophoresis screening dedicated to the enantioseparation of XPN. Sulfated- and sulfobutyl-ether-β-cyclodextrin (S-β-CyD, SBE-β-CyD) provided efficient resolution (R_s > 3), while heptakis-(6-deoxy-6-(2-carboxyethyl)thio)-β-CyD (subetadex, SBX) yielded outstanding separation (R_s > 9). The enantiomer migration order was consistently R,S, except when using SBE-β-CyD, which showed the inverse sequence. Chiral HPLC using a Chiralpak AD column in polar organic mode with methanol modified with 0.1% diethylamine as mobile phase enabled the semi-preparative isolation of XPN enantiomers, with the (S)-enantiomer exceeding 95% purity. The absolute configuration was confirmed by circular dichroism spectroscopy. ¹H NMR titration and 2D rotating-frame nuclear Overhauser effect correlation spectroscopy (ROESY) consistently revealed multi-site recognition of XPN by SBX, supporting the inclusion of both aromatic rings (A and D). Full article

To express and purify staphylococcal enterotoxin M (SEM) using immobilized metal affinity chromatography (IMAC), a signal peptide-truncated (ΔNsp) wild-type SEM (SEM_WT) was N-terminally fused in pET-28a(+) to a polyhistidine tag (His_6×-) and thrombin cleavage site (TCS; LVPR↓GS), generating His_6×-TCS-ΔNspSEM_WT. Unexpectedly, 4 °C desalting reduced the fusion protein’s molecular weight by ~2.0 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequencing and mass spectrometry identified cleavage specifically at the arginine (R) and glycine (G) peptide bond (R–G bond) within the TCS motif. AlphaFold 3 revealed an exposed serine protease catalytic triad: histidine 172, serine 178, and aspartic acid 212 (H₁₇₂/S₁₇₈/D₂₁₂) in the β-grasp domain, suggesting intrinsic thrombin-like activity (TLA). Sequential IMAC and size-exclusion high-performance liquid chromatography (SE-HPLC) purification eliminated contaminant concerns, while chromogenic substrate S-2238 (S-2238) assays demonstrated increasing specific activity and purification fold, supporting intrinsic TLA. Critically, the mutation of serine at position 178 to alanine (His_6×-TCS-ΔNspSEM_S178A) abolished TLA but preserved the secondary/tertiary structure, confirming the activity’s origin within the wild-type construct. Molecular dynamics (MD) simulations probed the atomistic mechanism for specific R–G bond cleavage. This work establishes a foundation for understanding ΔNspSEM_WT’s TLA. Full article

This study compared the yield, percentage solids, electrophoretic profile, gelatinolytic activity, and brine shrimp lethality of Bitis arietans venom prepared using freeze-drying and desiccator drying. Bitis arietans venom was collected from snakes at Bioken snake farm, Kenya, whereafter it was pooled and divided into two parts. Part 1 was desiccator dried venom (DDV) while part 2 was freeze-dried venom (FDV). The yield and percentage solids in DDV and FDV were compared using Welch’s Student’s t-test and the dried venoms were subsequently subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), 2D electrophoresis, gelatin in-gel zymography, and brine shrimp lethality assays. Mean venom yield and percentage solids did not differ between DDV and FDV (p = 0.5647 and p = 0.4676, respectively). SDS-PAGE and two-dimensional (2D) electrophoresis revealed similar protein profiles for DDV and FDV, showing bands and spot clusters within molecular weight ranges of ~16 kDa to >150 kDa and pH ranging from 3.5 to 9.5. Enzyme zymography revealed comparable gelatinolytic activity between DDV and FDV. However, the brine shrimp lethality assay indicated significantly higher toxicity in DDV (LC₅₀: 86.57 μg/mL) compared to FDV (LC₅₀: 460.37 μg/mL). DDV also showed greater lethality than FDV at 100 μg/mL (p = 0.0416) and 1000 μg/mL (p = 0.0008) but not at 10 μg/mL (p = 0.2465). These findings suggest that DDV exhibits higher toxicity in brine shrimp larvae than FDV, although both drying methods result in similar yields, percentage solids, venom profile, and gelatinolytic activity. Further research is necessary to investigate the mechanism behind this difference and its implications for antivenom production and long-term stability of venom. Full article

Understanding Plasmodium falciparum population genetic diversity is crucial to assess the impact of malaria control interventions. This study investigated P. falciparum genetic diversity using merozoite surface protein 1 (msp1), msp2 and glutamate-rich protein (glurp) in Korhogo district, Northern Côte d’Ivoire. DNA was extracted from dried blood spots (DBSs) collected in the health district of Korhogo between 2019 and 2020. The msp1, msp2, and glurp genes were amplified by polymerase chain reaction (PCR), and amplicon sizes were determined by capillary electrophoresis. Out of 179 samples randomly selected and genotyped, 82% were successfully amplified for msp1, 85% for msp2, and 75% for glurp. For msp1, the K1 allelic family had 20 genotypes, MAD20 had 23, and RO33 had only one genotype. For msp2, there were 59 and 33 genotypes for 3D7 and FC27, respectively, and for glurp, 45 genotypes were detected. The parasite population was highly diverse with an expected heterozygosity (H_E) of ≥0.9 for all 3 markers. Our study showed high genetic diversity of msp1, msp2, and glurp in P. falciparum isolates from Korhogo district, Northern Côte d’Ivoire. These data could provide baseline information on P. falciparum genetic diversity for further epidemiological studies, needed to assess interventions implemented in this area. Full article