Fed-batch is the most commonly used cultivation mode for industrial production of recombinant proteins with Pichia pastoris. On a laboratory scale, fed-batch culture provides a way to control the specific biomass growth rates at any pre-set value, allowing the conditions of biomass growth and recombinant product formation to be systematically studied.In this chapter, we present an accessible and versatile approach for designing, performing, and evaluating a fed-batch cultivation in laboratory-scale stirred tank bioreactors.
- Keywords
- Bioprocess design and development, Bioreactor, Data evaluation, Fed-batch culture, Feed rate, Fermentation, Komagataella phaffii, Pichia pastoris, Specific growth rate, Specific productivity,
- MeSH
- Biomass MeSH
- Bioreactors microbiology MeSH
- Fermentation MeSH
- Culture Media MeSH
- Pichia * growth & development MeSH
- Recombinant Proteins genetics biosynthesis MeSH
- Saccharomycetales * growth & development metabolism MeSH
- Batch Cell Culture Techniques * methods MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Culture Media MeSH
- Recombinant Proteins MeSH
The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) system has become a commonly used tool for genome editing and metabolic engineering. For Komagataella phaffii, commercialized as Pichia pastoris, the CRISPR/Cas9 protocol for genome editing was established in 2016 and since then has been employed to facilitate genetic modifications such as markerless gene disruptions and deletions as well as to enhance the efficiency of homologous recombination.In this chapter, we describe a robust basic protocol for CRISPR-based genome editing, demonstrating near 100% targeting efficiency for gene inactivation via a frameshift mutation. As described in other chapters of this volume, CRISPR/Cas9 technologies for use in P. pastoris have been further optimized for various specific purposes.
- Keywords
- CRISPR/Cas9, Gene knockout, Genome editing, Genome engineering, Guide RNA, Homologous recombination, Komagataella phaffii, Pichia pastoris, Synthetic biology,
- MeSH
- CRISPR-Cas Systems * genetics MeSH
- Gene Editing * methods MeSH
- Genome, Fungal MeSH
- Metabolic Engineering * methods MeSH
- Pichia * genetics MeSH
- Saccharomycetales * genetics MeSH
- RNA, Guide, CRISPR-Cas Systems genetics MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- RNA, Guide, CRISPR-Cas Systems MeSH
Colorectal cancer remains a major health burden, and its early detection is crucial for effective treatment. This study investigates the use of a handheld Raman spectrometer in combination with machine learning to classify colorectal tissue samples collected during colonoscopy. A dataset of 330 spectra from 155 participants was preprocessed using a standardized pipeline, and multiple classification models were trained to distinguish between healthy and pathological tissue. Due to the strong class imbalance and limited data size, a custom grid search approach was implemented to optimize both model hyperparameters and preprocessing parameters. Unlike standard GridSearchCV, our method prioritized balanced accuracy on the test set to reduce bias toward the dominant class. Among the tested classifiers, the Decision Tree (DT) and Support Vector Classifier (SVC) achieved the highest balanced accuracy (71.77% for DT and 70.77% for SVC), outperforming models trained using traditional methods. These results demonstrate the potential of Raman spectroscopy as a rapid, non-destructive screening tool and highlight the importance of tailored model selection strategies in biomedical applications. While this study is based on a limited dataset, it serves as a promising step toward more robust classification models and supports the feasibility of this approach for future clinical validation.
- Keywords
- Balanced accuracy, Colorectal cancer, Machine learning, Preprocessing pipeline, Raman spectroscopy, Spectral classification,
- MeSH
- Colorectal Neoplasms * diagnosis diagnostic imaging MeSH
- Middle Aged MeSH
- Humans MeSH
- Spectrum Analysis, Raman * methods MeSH
- Decision Trees MeSH
- Machine Learning * MeSH
- Support Vector Machine MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Although semiconductor materials such as vanadium pentoxide (V2O5) are promising non-plasmonic substrates for surface-enhanced Raman scattering (SERS) spectroscopy, their broader use is still hampered by their relatively low SERS enhancement. One strategy to overcome this limitation is to combine these materials with plasmonic noble metallic nanostructures. In this study, we investigate this approach for thin V2O5 nanostructural film decorated with Au (V2O5/Au) fabricated by sputter deposition and combined with thermal annealing. We found that SERS enhancement arises from both electromagnetic and chemical (charge-transfer) enhancement as well as it benefits from the morphology of the Au layer. The presence of the continuous Au film leads to a tremendous enhancement of the SERS signal of methylene blue (MB) as compared with Au-free V2O5 substrates. Under optimised conditions (an Au deposition time of 240 s), the SERS enhancement caused by the Au layer was found to be 1200-fold. Furthermore, spectral mapping across the V2O5/Au substrates shows that the relative standard deviation (RSD) of the SERS signal is less than 35 %, i.e., still sufficiently low for reliable detection. Finally, we successfully applied V2O5/Au substrates for sensitive detection of 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine (TMPyP), and 2,2'-bipyridine (BPy).
- Keywords
- Charge-transfer, Electromagnetic mechanism, Gold, Plasma-based deposition, SERS, Vanadium pentoxide,
- Publication type
- Journal Article MeSH
Ultra-thin, densely packed polymer brushes (PBs) with ultra-low fouling properties play a crucial role in recent developments of surface modification strategies for biomedical and biosensing technologies. However, micro-defects within PB structures can significantly impair their ultra-low fouling performance, thereby reducing their effectiveness and the application potential. Reliable detection of these micro-defects is essential for further evaluation and optimization of brush-based surfaces. In this study, we present a straightforward spectroscopic method for detection of micro-defects in PBs using Raman mapping. We employed random copolymer brushes of carboxybetaine methacrylamide (CBMAA) and N-(2-hydroxypropyl) methacrylamide (HPMAA) as examples of state-of-the-art ultra-low fouling PB systems prepared on a surface-enhanced Raman scattering (SERS)-active Ag nano-island layer fabricated on Au-coated glass substrates. Methylene blue, drop-deposited on the PB surface, is demonstrated as a suitable reporter molecule, providing spectroscopic information via both Raman and fluorescence signals. Analysis of the spectroscopic maps enabled not only the detection of micro-defects but also the differentiation between "shallow" and "deep" defects, providing detailed insights into the structural integrity of ultra-low fouling PBs.
- Keywords
- Low-fouling coating, Micro-defects, Polymer brush, Surface-enhanced Raman scattering, Surface-enhanced fluorescence,
- Publication type
- Journal Article MeSH
BACKGROUND: The possible clinical utility of endocan, a novel inflammatory biomarker involved in the initiation and progression of atherosclerosis, is largely unknown. We aimed to evaluate its diagnostic and prognostic performance in chest pain patients presenting to the emergency department (ED). METHODS: We prospectively enrolled patients presenting with suspected myocardial infarction (MI) to the ED in an international multicenter study. Endocan, high-sensitivity C-reactive protein (hs-CRP), and high-sensitivity cardiac troponin T (hs-cTnT) were measured in blood samples obtained at presentation. Final diagnoses were centrally adjudicated by two independent cardiologists applying the 4th universal definition of MI and current guidelines. Non-ST-elevation MI (NSTEMI) was the diagnostic endpoint and 5-year cardiovascular death was the primary prognostic endpoint. RESULTS: Among 4728 patients, 843 (17.8 %) had NSTEMI. The diagnostic discrimination of endocan for NSTEMI was low (area under the curve (AUC) 0.585 [95 % CI: 0.563-0.607]. Its combination with hs-cTnT (0.939 [95 % CI: 0.931-0.947]) did not improve the discriminative performance of hs-cTnT alone (0.937 [95 % CI: 0.930-0.950]). Long-term prognostic accuracy of endocan was higher versus hs-CRP, but lower versus hs-cTnT (AUC 0.730 [0.710-0.760] vs 0.650 [0.620-0.680] vs 0.810 [0.790-0.830], respectively). Endocan was associated with an increased 5-year risk for cardiovascular mortality. However, it did not provide relevant incremental prognostic value when added on top of a base model that included SCORE2 risk factors and hs-cTnT. CONCLUSION: Endocan has a low diagnostic accuracy for NSTEMI, and moderate long-term prognostic accuracy for cardiovascular death. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov number, NCT00470587, https://clinicaltrials.gov/ct2/show/NCT00470587https://clinicaltrials.gov/ct2/show/NCT00470587.
- Keywords
- Endocan, Inflammatory biomarkers, Myocardial infarction, Troponin,
- MeSH
- Biomarkers blood MeSH
- Myocardial Infarction * diagnosis blood MeSH
- Cohort Studies MeSH
- Middle Aged MeSH
- Humans MeSH
- Neoplasm Proteins * blood MeSH
- Prognosis MeSH
- Prospective Studies MeSH
- Proteoglycans * blood MeSH
- Aged MeSH
- Troponin T blood MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Multicenter Study MeSH
- Observational Study MeSH
- Names of Substances
- Biomarkers MeSH
- ESM1 protein, human MeSH Browser
- Neoplasm Proteins * MeSH
- Proteoglycans * MeSH
- Troponin T MeSH
Volumetric absorptive microsampling (VAMS) enables accurate collection of low blood volumes, independent of hematocrit. Electromembrane extraction (EME) is a sustainable sample clean-up technique; however, its wider applicability to extract analytes directly from VAMS tips remains unexplored. This study aimed to evaluate applicability of the first commercially available conductive vial EME device (with 2-nitrophenyl octyl ether as liquid membrane) for isolating 41 basic pharmaceuticals (log P 2-6) from 10 μL of blood on VAMS tips. The following extraction parameters were optimized: donor solution composition and volume, conductive vials size, applied voltage, extraction time and agitation speed. It was found that: 1/large conductive vials (600 μL) and 300 μL of donor solution provide higher process efficiency and reproducibility compared to smaller vials (200 μL) or larger donor solution volumes; 2/methanol in donor solution improve reproducibility and 3/sonication of VAMS tips in donor solution within a conductive vial prior to extraction enhances process efficiency. The EME protocol, followed by UHPLC-MS/MS analysis, was evaluated for process efficiency, linearity (1-1000 ng/mL), precision, and accuracy. Eleven analytes met most of the predefined acceptance criteria: process efficiencies 34.9-65.8 %, linearity (R2) 0.9933-0.9995, accuracy 85.9-111.1 % and precision 1.4-13.3 % RSD. The extraction was not impacted by hematocrit variation. EME demonstrated superior reproducibility and reduced matrix effects when compared to conventional VAMS tips treatment. This study confirms the reliability of a commercial conductive vial EME device for isolating basic pharmaceuticals from whole blood on VAMS tips, highlighting its potential for routine bioanalytical applications.
- Keywords
- Conductive vial electromembrane extraction, Microextraction, Sample preparation, Volumetric absorptive microsampling,
- MeSH
- Electric Conductivity MeSH
- Electrochemical Techniques * MeSH
- Pharmaceutical Preparations blood isolation & purification MeSH
- Humans MeSH
- Membranes, Artificial * MeSH
- Blood Specimen Collection * methods instrumentation MeSH
- Reproducibility of Results MeSH
- Tandem Mass Spectrometry MeSH
- Chromatography, High Pressure Liquid MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Pharmaceutical Preparations MeSH
- Membranes, Artificial * MeSH
Pharmaceutical residues in environmental waters continue to raise significant public and ecological concerns, necessitating advanced analytical methodologies for their monitoring. In this work, a novel Lab-In-Syringe automated dispersive micro solid-phase extraction (LIS-DMSPE) method was developed to determine three angiotensin receptor blockers in water samples. The approach was based on the in situ synthesis of Ni/Fe-layered double hydroxides within the void of an automatic syringe pump through controlled pH adjustment following the aspiration of precursor solutions. This enabled the rapid formation of the adsorbent and eliminated the need for pre-synthesized or magnetized sorbents. Enhanced sedimentation speed, achieved by increasing ionic strength with NaNO3, allowed isolating the sedimented sorbent from the sample matrix without centrifugation and filtration. The instrumental setup was successfully coupled online with HPLC-DAD. After in-syringe washing and dissolving the sediment, the low organic solvent content of the extract enabled large-volume injection (200 μL), thereby boosting sensitivity. Parameters including type and volume of precursor solution, NaNO3 addition, buffer volume and pH, stirring rate and time, and the composition of elution/destruction solution were carefully optimized. Recoveries and enrichment factors were in the ranges of 62.0-88.7 % and 13.3-25.8, respectively. The method was linear over a 5-200 μg L-1 concentration range for all analytes. Accuracies ranged from 88.7 % to 105.8 % for real samples spiked at two concentrations with RSDs less than 3.5 %. To our knowledge, this is the first report on a LIS-DMSPE method that does not require pre-synthesis or magnetization of the sorbent for extraction and uses accelerated sedimentation for adsorbent isolation.
- Keywords
- Angiotensin receptor blockers, Automation, Dispersive micro solid-phase extraction, Lab-In-Syringe, Large volume injection, Layered double hydroxides,
- MeSH
- Adsorption MeSH
- Angiotensin Receptor Antagonists * analysis isolation & purification MeSH
- Automation MeSH
- Water Pollutants, Chemical * analysis isolation & purification MeSH
- Solid Phase Extraction MeSH
- Hydroxides * chemistry MeSH
- Syringes MeSH
- Solid Phase Microextraction * methods instrumentation MeSH
- Nickel * chemistry MeSH
- Iron * chemistry MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Angiotensin Receptor Antagonists * MeSH
- Water Pollutants, Chemical * MeSH
- Hydroxides * MeSH
- Nickel * MeSH
- Iron * MeSH
