SEARCH · Search PubMed

Results for “Indocyanine Green”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

16 recordsLinked to original sources

PubMed2026

Intraoperative indocyanine green near-infrared fluorescence imaging for assessing testicular viability in pediatric testicular torsion: A retrospective study.

OBJECTIVE: To evaluate the clinical efficacy of indocyanine green near-infrared fluorescence (ICG-NIRF) imaging versus conventional surgery for assessing testicular viability and guiding decision-making in pediatric testicular torsion (TT). METHODS: A retrospective analysis was performed on 225 pediatric patients undergoing emergency scrotal exploration for TT between January 2019 and January 2025. Patients were categorized into a conventional surgery group (n = 118) relying on visual grading and an ICG-NIRF imaging group (n = 107). Primary outcomes included intraoperative testicular preservation rates and postoperative success rates. Multivariate Cox regression was utilized to identify factors influencing testicular preservation. RESULTS: Baseline characteristics were comparable between groups. The ICG-NIRF group demonstrated a significantly higher intraoperative preservation rate (74.77% vs. 61.02%, p = 0.028) and postoperative success rate (88.75% vs. 69.44%, p = 0.003) compared to the conventional group. Additionally, the ICG-NIRF group exhibited significantly lower rates of secondary orchiectomy (1.25% vs. 9.72%, p = 0.027) and 6-month testicular atrophy (7.59% vs. 23.08%, p = 0.02). Multivariate analysis confirmed ICG-NIRF application as an independent protective factor for testicular preservation (HR = 0.556, p < 0.001). CONCLUSION: ICG-NIRF imaging provides an objective, real-time assessment of testicular perfusion, significantly improving testicular preservation rates and postoperative outcomes. This technique overcomes the subjectivity of conventional visual methods, offering substantial clinical value for fertility preservation in pediatric TT.

Humans↗

PubMed2026

Aflibercept With Versus Without Reduced-Fluence Photodynamic Therapy for Polypoidal Choroidal Vasculopathy: Optical Coherence Tomography Angiographic Changes From a Randomized Clinical Trial.

OBJECTIVES: To report the longitudinal optical coherence tomography angiography (OCTA) changes in polypoidal choroidal vasculopathy (PCV) treated with intravitreal aflibercept monotherapy or in combination with reduced-fluence PDT. DESIGN: Image analysis of a double-masked, sham-controlled, randomized clinical trial. SUBJECTS: 55 eyes of 55 treatment-naïve participants with symptomatic macular PCV completing 52 weeks of follow-up. METHODS: Participants underwent protocolized, multimodal imaging, including OCT, OCTA, fluorescein angiography, and indocyanine green angiography at baseline, week 12, and week 52. Quantitative OCTA parameters included total lesion area, branching neovascular network (BNN) area, and BNN vessel density (VD). Qualitative features included trunk vessel presence and OCTA signal within the polypoidal lesion (PL). Eyes were categorized by treatment arm and PL closure at week 52. MAIN OUTCOME MEASURES: Longitudinal OCTA changes and predictors of PL closure at week 52. RESULTS: We included 55 eyes (28 combination therapy and 27 monotherapy). Total lesion area decreased at week 12 but returned toward baseline at week 52 (combination: 3.72 ± 3.01mm2 at baseline, 2.86 ± 2.50mm2 at week 12, 3.59 ± 3.26mm2 at week 52; monotherapy: 3.77 ± 2.23mm2 at baseline, 3.27 ± 2.36mm2 at week 12, and 3.47 ± 2.58mm2 at week 52). BNN area decreased at week 12 and remained reduced at week 52 in both treatment arms (combination: 2.29 ± 2.08 mm2 at baseline, 1.46 ± 1.36mm2 at week 12, and 1.53 ± 1.32mm2 at week 52; monotherapy: 2.39 ± 1.85mm2 at baseline, 1.87 ± 1.68mm2 at week 12, and 1.82 ± 1.38mm2 at week 52). BNN VD reduction was greater in the combination arm at week 12 (-10 ± 15% vs - 3 ± 12%, P = .02). The proportion of eyes with trunk vessels increased over time in both arms (combination: 35.7% at baseline, 59.3% at week 12, and 67.8% at week 52; monotherapy: 25.9% at baseline, 44.4% at week 12, and 71.4% at week 52). In multivariable analysis, baseline BCVA predicted BCVA change at week 52 (β=-0.96 [-1.21 to -0.72], P < .01), and baseline CST predicted CST change (β=0.93 [0.75 to 1.10], P < .01). Greater reduction in BNN VD at week 12 was independently associated with PL closure at week 52 (OR 0.62 [0.39 to 0.97], P = .03). CONCLUSIONS: Early reduction in BNN vessel density, rather than reduction in lesion size, was associated with subsequent PL closure. OCTA-derived vascular changes may serve as noninvasive biomarkers for predicting treatment response in PCV.

Humans↗

PubMed2026

Vortex-assisted liquid-liquid microextraction based on natural deep eutectic solvents for the determination of pyrethroid pesticides in urine.

A novel, facile, and environmentally friendly analytical method was developed based on vortex-assisted liquid-liquid microextraction and high-performance liquid chromatography with diode-array detection for detecting pyrethroid pesticides (PPs) in urine. Natural deep eutectic solvents (NADESs) were prepared using plant essential oil-derived monoterpenoids (thymol, carvacrol, and menthol) combined with aromatic primary alcohols (benzyl alcohol, phenethyl alcohol, and phenylpropyl alcohol) as hydrogen bond donors and acceptors. These solvents served as environmentally benign extraction media, thereby avoiding the use of conventional volatile, toxic organic solvents. NADESs are naturally derived, easy to prepare, biodegradable, and environmentally friendly solvents. Hydrophobic and π-π interactions between the NADESs and PPs may contribute to enhancing the affinity of PPs toward the NADESs phase. Vortex technology, accelerating mass transfer between the sample and extractant phases, enables fast extraction of PPs. Under optimized conditions, the method achieved a low detection limit (0.002 mg L-1), satisfactory precision with relative standard deviations (0.3%-2.4%), and acceptable recovery (80.7%-86.2%). The method demonstrated excellent performance in urine analysis and was feasible as a facile and green strategy for monitoring the content of PPs in biological matrices and assessing exposure risk.

Liquid Phase Microextraction↗

PubMed2026

Alginate-based edible coating incorporating green tea extract for preserving postharvest quality and safety of white mushrooms (Agaricus bisporus).

This study aimed to evaluate the effects of a sodium alginate based edible coating incorporated with green tea extract (GTE) (Camellia sinensis) on the postharvest quality attributes and antimicrobial activity against Listeria monocytogenes in white mushrooms during refrigerated storage. The phenolic profile of GTE was characterized, and its minimum inhibitory concentration (MIC) against L. monocytogenes (1.6 mg/mL) was determined. Sodium alginate coatings, with (ALG-GTE) or without GTE (ALG) at MIC (1.6 mg/mL), were characterized (functional groups, solubility in water, moisture, thickness, water contact angle and color) for their chemical and physical properties. The effects of ALG-GTE coatings on quality parameters (firmness, weight loss, color, pH, sugars and organic acids), enzymatic activity [polyphenol oxidase (PPO), peroxidase (POD) and pectin methylesterase (PME)], antimicrobial activity against L. monocytogenes (5 log CFU/g), and surface characteristics (3D optical profilometry) were assessed in white mushrooms (Agaricus bisporus) during refrigerated storage (8 days, 4 ± 1 °C, 90-95% RH). The ALG-GTE coatings preserved sugar composition, particularly rhamnose, reduced organic acids accumulation and delayed weight and firmness loss, reduced color changes, and decreased PME activity in coated white mushrooms. L. monocytogenes counts decreased by 1.4 log CFU/g after 1 day, and no viable cells were detected after 2 days (< 1.5 log CFU/g) in ALG-GTE coated white mushrooms. In addition, ALG-GTE coated white mushrooms exhibited smoother surfaces than uncoated samples. These findings highlight the potential of ALG-GTE coatings as a sustainable alternative capable of improving the microbiological safety and delaying the postharvest changes in fresh mushrooms.

Agaricus↗

PubMed2026

Algae-to-host horizontal gene transfer in Paramecium bursaria is associated with host adaptation during endosymbiosis.

Paramecium bursaria maintains a stable endosymbiosis with green algae, yet the evolutionary consequences of this association remain unclear. Here, we screened the host genome for algal-derived horizontally transferred genes (HTGs) using a lineage-aware workflow designed to detect horizontal gene transfer (HGT) between two defined lineages. We identified 16 candidate HTGs, including four putative newly transferred genes and 12 homologous transferred genes, most of which were functionally associated with redox homeostasis and metabolism. Five HTGs showed symbiosis-dependent expression. RNAi knockdown of GH32s and SATs reduced host proliferation, total cell area, and motility, while GH32s knockdown also reduced endosymbiont load. Duplication patterns suggest that most transfers may have occurred after the P. bursaria lineage diverged from the sampled Paramecium species but before its lineage-specific whole-genome duplication (WGD). The HTGs also showed host-associated shifts in GC content and gene length, while representative HTGs retained conserved domains and functional motifs. Together, our results support algae-to-host HGT in P. bursaria and suggest that some transferred genes may contribute to metabolic integration during endosymbiosis.

Gene Transfer, Horizontal↗

PubMed2026

Efficient rDNA-mediated multi-copy integration of gene clusters in Aureobasidium melanogenum.

Aureobasidium melanogenum is a promising non-conventional yeast chassis for synthetic biology. However, techniques recombining large genetic fragments, such as gene clusters, are still unavailable, hindering further metabolic reprogramming in this chassis. To achieve multi-copy integration of genes, we employed highly repetitive ribosomal DNA (rDNA) sequences in A. melanogenum as homologous recombination sites for large genetic fragments. First, integration efficiency of three different regions of A. melanogenum rDNA were investigated: RNA polymerase I promoter region (rDNA1, 1.0 kb), partial 26S rDNA region (rDNA2, 1.0 kb), and RNA polymerase I terminator region (rDNA3, 1.0 kb). Our findings revealed that the highest copy numbers and expression stability were observed for the short heterologous green fluorescent protein gene (gfp, 0.7 kb) and the long native polyketide synthase gene (pks, 7.0 kb) after rDNA1-mediated integration. Specifically, the copy numbers reached 7.0 and 8.0 for gfp and pks, respectively, and they remained stably expressed in the genome after 120-h subculturing. Furthermore, an 11.0 kb gene cluster (comprising the native pks, phosphopantetheinyl transferase (npg1), and scytalone dehydratase genes (scd) responsible for melanin biosynthesis) was integrated at the rDNA1 site, resulting in stable recombination with 15.0 copies and an approximately 12-fold increase in melanin production. Overall, the convenience and efficiency of the proposed rDNA-mediated multi-copy insertion strategy will facilitate superior metabolic engineering of A. melanogenum chassis cells.

Multigene Family↗

PubMed2026

Comparative effectiveness of torsemide vs furosemide in the management of heart failure patients: Win-ratio reanalysis of the TRANSFORM-HF trial.

BACKGROUND: Loop diuretics are widely used for managing congestion in patients with heart failure (HF). The TRANSFORM-HF trial is a multicenter randomized study that enrolled heart failure patients, comparing a strategy of torsemide vs furosemide. The time-to-event analysis demonstrated neutral effects on all-cause death at 30 months and the composite of all-cause death and first rehospitalization at 12 months. We evaluated whether a hierarchical win-ratio (WR) framework integrating mortality, recurrent hospitalization, and patient-reported health status provides additional interpretive insight. METHODS: This study is a secondary analysis of the pragmatic, multicenter, open-label, randomized TRANSFORM-HF trial, conducted across 60 US hospitals that randomized 2,859 patients hospitalized with HF to torsemide or furosemide. The primary 12-month hierarchical composite outcome was defined as (1) all-cause mortality, (2) recurrent all-cause hospitalizations, and (3) lack of improvement in the Kansas City Cardiomyopathy Questionnaire Clinical Summary Score (KCCQ-CSS). The primary statistical method was a WR analysis adjusting covariates via inverse probability weighting. Subgroup analyses evaluated potential heterogeneity across patient demographics and clinical characteristics. RESULTS: In the primary 12-month intention-to-treat analysis, the adjusted WR was 1.07 (95% CI, 0.98-1.16; P = .13), indicating no significant difference between torsemide and furosemide. A supplementary 30-month analysis with extended mortality follow-up yielded a similar estimate (adjusted WR, 1.06; 95% CI, 0.98-1.16; P = .14); hospitalization and KCCQ-CSS components were assessed through 12 months. As-treated sensitivity analyses were consistent with the neutral primary findings. Exploratory subgroup analyses were not adjusted for multiplicity and should be considered hypothesis-generating. CONCLUSIONS: The overall WR comparison between torsemide and furosemide showed no statistically significant difference in the primary 12-month analysis. The WR framework provided an interpretive decomposition across outcome domains but did not establish superiority of either loop diuretic strategy. All findings should be considered exploratory. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03296813, https://clinicaltrials.gov/study/NCT03296813.

Aged↗

PubMed2026

The journey of fluxapyroxad, mandipropamid and mefentrifluconazole residues in two morphologically distinct chilli peppers: A comprehensive risk assessment from field to processing.

Understanding the residue fate of novel pesticides in crops is crucial for ensuring their safe application and safeguarding public health. This study examined the dissipation, processing factors (PFs), and risk assessment of fluxapyroxad, mandipropamid, and mefentrifluconazole in two morphologically distinct varieties of chilli peppers from field to processing. The half-lives of the three pesticides ranged from 5.42 to 10.05 days, following first-order kinetics. The initial residues were higher in Chaotian chilli peppers (CCP) than in long green chilli peppers (GCP). However, dissipation occurred more rapidly in CCP. Washing notably reduced the residues (PF: 0.60-0.89), whereas sun drying and oven drying concentrated them (PF: 1.92-3.74), with oven drying leading to greater concentrations. Both chronic and acute dietary risk assessments suggested acceptable risk levels for the general population. This study offers reliable guidance for the rational application of these three pesticides in chilli pepper cultivation.

Capsicum↗

PubMed2026

Urban Design Quality and Clinical Mental Health: A Systematic Review.

ObjectivesThis systematic review synthesizes empirical evidence on core urban design dimensions that affect clinical mental health outcomes and examines how environmental exposures mediate or moderate these relationships.BackgroundUrban design has increasingly been recognized as a determinant of psychological well-being, yet a standardized framework to evaluate its mental health impact remains underdeveloped.MethodsFollowing PRISMA 2020 guidelines, we systematically reviewed 19 quantitative empirical studies published through January 2025, examining relationships between outdoor urban design features and validated clinical mental health indicators across four major databases.ResultsFindings reveal that urban design influences clinical mental health outcomes (depression, anxiety, stress, cognitive decline) through two objective spatial scales: street-level features (imageability, enclosure, human scale, complexity) and neighborhood environments (land use mix, density, green infrastructure). Environmental exposures (traffic, noise, air pollution) operate as perceptual and experiential mechanisms that mediate or moderate the mental health effects of these spatial design features.ConclusionsWe propose an integrated three-domain conceptual framework distinguishing objective spatial design scales from subjective exposure mechanisms. This framework provides evidence-based guidance for urban planners and policymakers toward creating mentally healthier urban environments.

Humans↗

PubMed2026

Integrated widely targeted metabolomics and GC-IMS reveal dynamic flavor, nutritional, functional, and metabolic profiles in macadamia kernels during processing.

Different processing stages influence the color, flavor, and antioxidant activities of macadamia kernels. However, the biochemical mechanisms that occur during processing are not well known. This study integrated widely targeted metabolomics (UPLC-MS/MS) with GC-IMS to systematically characterize non-volatile and volatile compounds in macadamia kernels across key three sample groups: fresh kernels (FMN), low-temperature-dried kernels (DMN), and roasted kernels (BMN). A total of 622 non-volatile metabolites and 52 volatile compounds were identified. Low-temperature drying promoted the accumulation of phenolic acids and flavonoids, enhancing antioxidant capacity. Roasting degraded heat-sensitive nutrients but generated flavor compounds via Maillard reaction and lipid oxidation, shifting aroma from green to nutty notes. Nutritional assessment confirmed that roasting significantly reduced antioxidant activities and bile acid binding capacity. Pearson correlation analysis verified the key metabolite-antioxidant relationships. These findings provide critical insights into metabolic dynamics during nut processing and establish a scientific basis for optimizing thermal processing strategies.

Metabolomics↗

PubMed2026

Evolutionary architecture and lineage-specific diversification of Forkhead box transcription factors in Perna viridis.

The Forkhead box (Fox) transcription factors are evolutionarily conserved regulators of development, cell cycle, and apoptosis across metazoans. This study provides the first comprehensive genome-wide analysis of the Fox gene family in the Asian green mussel (Perna viridis). We identified 28 Fox genes distributed across 10 chromosomes. Comparative analysis reveals the absence of the FoxI, FoxQ1, FoxR and FoxS subfamily, consistent with other bivalves and indicative of lineage-specific gene loss during molluscan evolution. Notably, gene duplications in the FoxAB, FoxD, FoxH, FoxN1-4, FoxQ2 and FoxQD subfamilies may reflect functional diversification associated with environmental adaptation. Exon-intron structural variability, including intron loss in several paralogues, suggests structural diversification and potential regulatory variation. Phylogenetic reconstruction confirmed the monophyly of core Fox classes while highlighting divergent expansion patterns in lophotrochozoans. Selection analyses showed strong purifying selection across duplicated Fox paralogs, supporting functional conservation after lineage-specific expansion. Gene Ontology enrichment linked Fox genes to stress response, apoptosis, and transcriptional regulation. By integrating phylogenetic, structural, and transcriptomic analyses, this study provides a genomic framework for understanding Fox gene organisation, evolution, and tissue-associated expression patterns in Perna viridis and establishes a comparative resource for future functional studies in bivalves.

Animals↗

PubMed2026

Branded packaging raises likelihood of cigarette purchasing in an experimental retail setting by increasing craving.

INTRODUCTION: Exposure to branded cigarette packaging in retail settings has been shown to be associated with purchasing behavior, but the mechanisms underlying this effect are unclear. This study tested whether cigarette craving and perceived health harms mediate the effect of branded packaging on cigarette purchasing in a simulated retail environment. METHODS: Young adults aged 21-34 who currently smoke cigarettes (n = 290) completed an experimental shopping task in the RAND StoreLab, a life-sized replica of a convenience store. Participants were randomly assigned to one of two conditions: (1) branding present, in which branded cigarette packages were displayed; and (2) branding absent condition, in which branded elements were removed and packages were standardized in a brown-green color and uniform text. Cigarette purchases were recorded, and participants completed post-shopping measures of cigarette craving and perceived health harms. Causal effect decomposition analyses were used to assess whether these variables mediated the effect of study condition on the likelihood of purchasing cigarettes. RESULTS: Craving, but not perceived health harms, partially mediated the effect of branded packaging on cigarette purchasing. Exposure to branded packs increased the likelihood of purchasing by elevating craving (average mediated effect = 2.4%, 95% CI 0.2% - 5.0%, p = .03). CONCLUSIONS: Branded cigarette packaging appears to increase cigarette purchasing at least in part by increasing cigarette craving at point of sale. Interventions that address craving management in retail settings (e.g., just-in-time interventions, prn nicotine replacement therapy) may help mitigate the impact of branding on young adults' cigarette purchasing.

Humans↗

PubMed2026

Integrated assessment of biocontrol potential and genome analysis of endophytic Bacillus velezensis MGL-B1 against mango stem-end rot.

Mango stem-end rot is a globally significant postharvest disease that severely threatens the mango industry, primarily caused by Botryosphaeria dothidea. However, information on biocontrol agents targeting this pathogen in mango remains limited. In this study, we isolated and identified a strain of Bacillus velezensis MGL-B1 from mango leaf tissues for the first time, which exhibited broad-spectrum antifungal activity. Both in vitro and in vivo assays demonstrated that MGL-B1 effectively inhibited the growth of B. dothidea, with an in vivo biocontrol efficacy reaching 83.72 ± 5.10%, comparable to that of the commonly used chemical fungicide thiabendazole. Further mechanistic analysis revealed that MGL-B1 acts by directly disrupting the integrity of the pathogen's mycelial cell membrane. In addition, its released volatile organic compounds (VOCs) also displayed significant antifungal activity, with components such as 2-nonanone, 2-nonanol, and phenylethyl alcohol being confirmed to exert antifungal effects in in vitro fumigation assays. qPCR analysis showed that MGL-B1 treatment significantly upregulated the transcriptional levels of genes involved in plant-pathogen interaction, phenylpropanoid biosynthesis, and antioxidant defense pathways in mango fruits, with upregulation folds of 16.32, 37.19, and 75.93, respectively; meanwhile, the expression of browning-related genes such as polyphenol oxidase (PPO) was markedly suppressed. Whole-genome sequencing further revealed 14 biosynthetic gene clusters for antimicrobial compounds, including five unknown gene clusters. Collectively, B. velezensis MGL-B1 represents a promising biocandidate strain with multiple antifungal mechanisms and excellent control efficacy, providing a valuable resource for green and sustainable management of mango diseases.

Mangifera↗

PubMed2026

ScRNA-seq analysis reveals the effects of nitrite stress on the endocrine system of the eyestalk in Litopenaeus vannamei.

Nitrite is a harmful substance generated in Litopenaeus vannamei farming systems, largely originating from the inadequate breakdown of surplus feed and shrimp feces. Its accumulation in the water can affect the growth and physiological functions of shrimp, damage the immune system, and even cause mass mortality, thus becoming a key environmental factor restricting the green development of the industry. Under nitrite stress, the eyestalk, as an important neuroendocrine regulatory center in crustaceans, participates in the stress adaptation of the organism and exerts a protective effect by regulating energy metabolism and immune function. However, the molecular regulatory mechanism of the eyestalk in response to nitrite stress remains unclear. In this study, single-cell RNA sequencing (scRNA-seq) technology was used to analyze the heterogeneity of eyestalk cells in L. vannamei under nitrite stress. A total of 18, 394 high-quality cells were obtained, and six major cell subpopulations, including Neurosecretory cell, Motor neuron, Sensory neuron, Interneuron, Neurogliocyte, and Support cell, were identified. Differential expression analysis identified 839 differentially expressed genes, and different cell types showed distinct specific responses to nitrite stress. Functional enrichment analysis indicated that pathways such as glycolysis, oxidative phosphorylation, ribosome function, and endoplasmic reticulum protein processing were significantly activated, while signal transduction and DNA repair-related pathways were inhibited. Further analysis revealed that nitrite stress could induce mitochondrial function changes and trigger oxidative stress, thereby affecting the neuroendocrine system function of the eyestalk. This study provided insights into transcriptomic responses of the eyestalk to nitrite stress at the single-cell level, laying a theoretical foundation for the management of aquaculture environments.

Animals↗

PubMed2026

Unravelling bioanalytical innovations, degradation processes, and impurity landscapes of VEGFR inhibitors.

From pre-formulation studies to clinical trials, VEGFR-targeted small-molecule tyrosine kinase inhibitors (TKIs) require rigorous analytical standards. Bioanalysis, stability-indicating studies, and impurity profiling are used to examine chromatographic advances for VEGFR-targeted TKIs like sunitinib, pazopanib, axitinib, sorafenib, cabozantinib, vandetanib, apatinib, lenvatinib, nintedanib, and regorafenib. An LC-MS/MS and UPLC-MS/MS routinely show sub ng/mL performance, as shown by LLOQs (0.2 ng/mL) for sunitinib and axitinib, 1 ng/mL for pazopanib, 5-7 ng/mL for sorafenib, 0.5-1.5 ng/mL for regorafenib metabolic products, and 0.1-0.5 ng/mL for lenvatinib. These approaches are used for pharmacokinetics and therapeutic drug monitoring due to their good correlation coefficient of 0.1-10,000 ng/mL, accuracy of 95%-108%, and precision of 15% RSD. UPLC-QTOF-MS/MS distinguishes degradants and metabolites during forced degradation studies, enabling structural elucidation following ICH M7 risk evaluation protocol. HPTLC/MLC offers fast, sensitive screenings, while RP-HPLC/DAD or HPLC-UV offer reliable, cost-effective routine quality-control solutions with LOD/LOQ in the μg/mL range and linearity of 10-240 μg/mL. This review lists the structures and CAS numbers of ten VEGFR-2 TKI degradants and metabolites, as well as pharmacopeial impurities in SMILES forms. It will be useful for future method development and regulatory applications. To ensure VEGFR-targeted TKI quality, safety, and therapeutic efficacy, LC-MS/MS for trace quantification and HRMS for structure elucidation provide a robust, future-oriented framework. To improve VEGFR-targeted TKI quality, safety, and regulatory compliance, analytical development should focus on HRMS-based impurity characterization, AI-assisted degradation prediction, green chromatography, and harmonized bioanalytical validation.

Humans↗

PubMed2026

A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

CRISPR-Cas Systems↗