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

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

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

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 μm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.

• Keywords
• High-throughput lipidomics, Mass spectrometry, Plasma, Quantitation, Reversed-phase, Serum, Ultrahigh-performance liquid chromatography,
• MeSH
• Chromatography, Reverse-Phase * methods   MeSH
• Mass Spectrometry methods   MeSH
• Liquid Chromatography-Mass Spectrometry MeSH
• Humans MeSH
• Lipidomics * methods   MeSH
• Lipids * analysis   MeSH
• High-Throughput Screening Assays methods   MeSH
• Software MeSH
• Tandem Mass Spectrometry methods   MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Lipids * MeSH

The enzymatic degradation of polyethylene terephthalate (PET) by PETases has gained significant attention as a potential solution for plastic waste management. However, the absence of a standardized protocol for PETase production across studies presents a challenge for consistent enzyme characterization and activity comparison. Variations in production methods, including expression systems and purification techniques, may contribute to discrepancies in reported PETase activities. Here, we present the development of a unified protocol for the production of wild-type and engineered IsPETase variants. This protocol comprises standardized expression, purification, and quality control steps to ensure reproducibility and reliability. By enabling more accurate comparisons of PETase variants and addressing inconsistencies in PETase production, this approach facilitates collaborative efforts to advance plastic degradation technologies and lays the groundwork for accelerating research in enzymatic PET degradation and its applications in plastic waste management.

• Keywords
• Enzyme activity assay, IsPETase, Optimization, PET degradation, Standardized protocol,
• MeSH
• Escherichia coli genetics   metabolism   MeSH
• Polyethylene Terephthalates * metabolism   chemistry   MeSH
• Workflow MeSH
• Recombinant Proteins genetics   isolation & purification   chemistry   biosynthesis   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Polyethylene Terephthalates * MeSH
• Recombinant Proteins MeSH

A thin organic film electrode (TOFE) system was employed for the indirect electrochemical investigation of docetaxel (DTX), an anticancer drug from the class of mitotic inhibitors. The TOFE consists of a thin membrane immobilized on a carbon electrode substrate, composed of a water-immiscible organic solvent that forms a stable liquid│liquid interface upon immersion in an aqueous electrolyte. Due to the high oxidation potential of DTX at solid electrodes, an alternative detection strategy was adopted based on the interaction between DTX and the liquid|liquid interface of the TOFE. Systematic optimization of the organic and aqueous phase compositions, as well as the electrode material, was first performed in the absence of DTX to ensure reliable and reproducible conditions at the interface. The optimized TOFE system, based on nitrobenzene as the organic solvent, perchlorate anions as the supporting electrolyte, and an edge-plane pyrolytic graphite as electrode material, provided the best performance. The optimal electrochemical response was also observed when perchlorate anions were present in the aqueous phase, while the nature of the cations in the aqueous phase had negligible influence. The optimized TOFE system was then used to study the effect of DTX on ion transfer processes at the liquid│liquid interface, where a pronounced, concentration-dependent inhibitory effect was observed. Electrochemical investigations using cyclic voltammetry and square-wave voltammetry (SWV), including frequency- and amplitude-dependent analyses, demonstrated that DTX adsorbs to the membrane│water interface, forming blocking layer that markedly slowing ion transfer and displacing the quasi-reversible maxima beyond the instrumentally accessible window. The system exhibited a linear analytical response in the concentration range of 10.0-100.0 μmol L-1 DTX, with low detection and quantification limits when analyzed by SWV. The LOD values were 3.27 μmol L-1 (anodic) and 3.26 μmol L-1 (cathodic), while the LOQ were 9.90 μmol L-1 and 9.89 μmol L-1, respectively. These findings validate the TOFE platform as a robust and versatile tool for the indirect detection of redox-inactive pharmaceutical compounds in biphasic electrochemical systems. Given the key role of mitotic inhibitors like DTX in cancer therapy, studying their behavior at biomimetic interfaces may advance understanding of drug-membrane interactions and support the development of improved delivery and diagnostic strategies.

• Keywords
• Indirect voltammetric detection, Interfacial electrochemistry, Membrane-modified carbon-based electrode, Mitotic inhibitor, Square-wave voltammetry,
• MeSH
• Docetaxel * analysis   MeSH
• Electrochemical Techniques * instrumentation   methods   MeSH
• Electrodes MeSH
• Antineoplastic Agents * analysis   chemistry   MeSH
• Water chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Docetaxel * MeSH
• Antineoplastic Agents * MeSH
• Water MeSH

BACKGROUND: Intraluminal irreversible electroporation (IRE) can be used for recanalizing occluded metal stents. However, optimal IRE parameters for consistent effects across different stent designs remain unclear. The aim of this study was to simulate the process of stent recanalization in silico by employing finite element analysis. METHODS: A virtual model of an occluded biliary stent with an experimental 3-electrode IRE catheter was developed. Electric field distribution, temperature changes, and potential ablation volumes were simulated across various parameters: IRE voltage (300 - 1300 V), stent wire width (0.1 - 0.5 mm) and stent mesh size (0.7 - 5.58 mm). Simulations incorporated five representative stent types commonly used in clinical practice. 685 unique simulations were conducted, analyzing 1162 unique values. RESULTS: Higher voltages generally led to larger ablation zones and increased temperatures. Thinner stent wires and larger mesh sizes also increased the extent of ablation zone. While in-stent ablation was largely independent of stent design, out-of-stent ablation was significantly impacted by mesh size and tissue thickness between the stent and irreversible electroporation electrodes. Voltages above 1000 V produced significant thermal effects, with substantial volumes of tissue heated above 50 °C. Specific stent designs exhibited variations in maximum temperature (72.1 - 83.1 °C) and ablation volume (8.7 - 14.7 mm3). CONCLUSION: Tailored IRE protocols for different stent designs are required due to differences in in- and out-stent ablation volumes. High voltages (>1000 V) induce both thermal and nonthermal ablation mechanisms.

• Keywords
• Irreversible electroporation, ablation, computer simulation, metal stents, temperature distribution,
• MeSH
• Electroporation * methods   MeSH
• Humans MeSH
• Stents * MeSH
• Models, Theoretical * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: CrossFitⓇ sessions and competitions are characterized by high-intensity challenges that combine aerobic and anaerobic activities with short recovery periods. As a result, effective nutritional practices play a crucial role in optimizing performance and enhancing recovery. Therefore, nutritional practices are central to optimizing performance and accelerating recovery. This review aims to summarize current evidence on nutritional and ergogenic aid responses to CrossFitⓇ practice. METHODS: The search was conducted in four electronic databases (PubMed, Web of Science, Scopus and SportDiscus). Gray literature was also extracted for studies exploring the nutritional habits of CrossFitⓇ participants as well as the ingestion of ergogenic aids. In addition, a meta-analysis was conducted to examine the impacts of dietary habits and ergogenic aids on performance. RESULTS: Forty-nine studies met the eligibility criteria and were included in the current review. Carbohydrate intake was below the recommendations for athletes, although protein ingestion remains adequate. High-carbohydrate diets had a positive effect on CrossFitⓇ performance. The evidence concerning the effects of a ketogenic diet on performance is limited. When used prior to or during the workout, the impact of carbohydrates on CrossFitⓇ performance was negligible, whereas the effect of caffeine was significant. Ergogenic aids, particularly creatine and protein, are commonly used by CrossFitⓇ participants. CONCLUSION: The standard diets recommended to CrossFitⓇ participants need to be revised because they are characterized by lower values of carbohydrates. Caffeine should be used prior to or during the CrossFitⓇ sessions. Regarding the impact of ergogenic aids on recovery, future studies are needed.

• Keywords
• Carbohydrates, caffeine, conditioning, energy, performance, strength,
• MeSH
• Diet MeSH
• Dietary Proteins administration & dosage   MeSH
• Dietary Carbohydrates administration & dosage   MeSH
• Sports Nutritional Physiological Phenomena * MeSH
• Diet, Ketogenic MeSH
• Caffeine administration & dosage   MeSH
• Physical Conditioning, Human * MeSH
• Creatine administration & dosage   MeSH
• Performance-Enhancing Substances administration & dosage   MeSH
• Humans MeSH
• Athletic Performance * physiology   MeSH
• Feeding Behavior MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Scoping Review MeSH
• Names of Substances
• Dietary Proteins MeSH
• Dietary Carbohydrates MeSH
• Caffeine MeSH
• Creatine MeSH
• Performance-Enhancing Substances MeSH