Fluorescence Assay Research Articles

Native Mass Spectrometry-Centric Approaches Revealed That Neuropeptides Frequently Interact with Amyloid-β.

Amyloid-β (Aβ) aggregates are recognized as initiators of Alzheimer's disease, and their interaction with the nervous system contributes to the progression of neurodegeneration. Herein, we investigated the frequency at which neuropeptides interact with Aβ and affect the aggregation kinetics and cytotoxicity of Aβ. To this end, we established a native mass spectrometry (MS)-centric workflow for screening Aβ-interacting neuropeptides, and six out of 12 neuropeptides formed noncovalent complexes with Aβ species in the MS gas phase. Thioflavin-T fluorescence assays and gel separation indicated that leptin and cerebellin decreased Aβ aggregation, whereas kisspeptin increased this process. In addition, leptin and cerebellin attenuated Aβ-induced cytotoxicity, which was independent of the influence of metal ions. Leptin can chelate copper from copper-bound Aβ species, reducing the cytotoxicity caused by the aggregation of Aβ and metal ion complexes. Overall, our study demonstrated that neuropeptides frequently interact with Aβ and revealed that leptin and cerebellin are potential inhibitors of Aβ aggregation, providing great insight into understanding the molecular mechanism of Aβ interacting with the nervous system and facilitating drug development.

In Vitro Antiviral Activity of Rhodiola crenulata Extract against Zika Virus and Japanese Encephalitis Virus: Viral Binding and Stability.

Zika virus (ZIKV) and Japanese encephalitis virus (JEV) can cause permanent neurological damage and death, yet no approved drugs exist for these infections. Rhodiola crenulate, an herb used in traditional Chinese medicine for its antioxidation and antifatigue properties, was studied for its antiviral activity against ZIKV and JEV in vitro. The cytotoxicity of Rhodiola crenulata extract (RCE) was evaluated using the CCK-8 reagent. Antiviral effects of RCE were assessed in ZIKV-infected or JEV-infected Vero cells via quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, fluorescent focus assay (FFA), and immunofluorescence assay (IFA). The cell-free antiviral effects of RCE were evaluated using an inactivation assay. To determine the stage of the viral life cycle affected by RCE, time-of-addition, binding, and entry assays were conducted. Three bioactive constituents of RCE (salidroside, tyrosol, and gallic acid) were tested for antiviral activity. RCE exhibited dose-dependent anti-ZIKV and anti-JEV activities at non-cytotoxic concentrations, which were likely achieved by disrupting viral binding and stability. Gallic acid exhibited antiviral activity against ZIKV and JEV. Our findings indicate that RCE disrupts viral binding and stability, presenting a potential strategy to treat ZIKV and JEV infections.

A Diterpenoid of the Medicinal Plant Andrographis paniculata Targets Cutaneous TRPV3 Channel and Relieves Itch.

Transient receptor potential vanilloid subtype 3 (TRPV3) is an ion channel implicated in skin physiology and itch. TRPV3 inhibitors can present a novel strategy for combating debilitating itch conditions, and medicinal plants are a natural pool of such compounds. Here, we report the isolation of a TRPV3-inhibiting compound from Andrographis paniculata, a medicinal plant with anti-inflammatory properties whose bioactive components are poorly characterized in terms of molecular targets. Using 1H and 13C NMR and high-resolution mass spectrometry, the compound was identified as a labdane-type diterpenoid, 14-deoxy-11,12-didehydroandrographolide (ddA). The activity of the compound was evaluated by fluorescent calcium assay and manual whole-cell patch-clamp technique. ddA inhibited human TRPV3 in stably expressing CHO and HaCaT keratinocytes, acting selectively among other TRP channels implicated in itch and inflammation and not showing toxicity to HaCaT cells. Antipruritic effects of the compound were evaluated in scratching behavior models on ICR mice. ddA suppressed itch induced by the TRPV3 activator carvacrol. Additionally, ddA potently suppressed histamine-induced itch with efficacy comparable to loratadine, a clinically used antihistamine drug. These results suggest the potential of ddA as a possible safe and efficacious alternative for antipruritic therapy.

A sensitive intelligent point-of-care test method for tert-butylhydroquinone in edible oil via a test strip with a smartphone

Tert-butylhydroquinone (TBHQ) is easily overused or illegally added to edible oil and attracts a growing concern because of its cytotoxic, liver-damaging, and carcinogenic effects. Thus, a sensitive and intelligent point-of-care testing (iPOCT) method is developed to fulfill the on-site monitoring. This iPOCT method depended on a fluorescent immunochromatographic assay within 15 min. Under optimization, the limit of quantification (LOQ) was calculated as 0.03 μg mL-1. The iPOCT method provided a low limit of detection (LOD) of 0.02 μg mL−1, a wide linear range of 0.03–100 μg mL−1, and great selectivity. Recoveries by the spiking experiments ranged from 97.4% to 103.5% with relative standard deviations (RSDs) of 2.4%–4.9% in soybean, peanut, rapeseed, and corn oil samples. The results showed that the iPOCT method is highly consistent with the high-performance liquid chromatography (HPLC) method.

A sensitive fluorescence assay of serum dipeptidyl peptidase IV activity to predict the suitability of its inhibitors in patients with type 2 diabetes mellitus

DPP-IV inhibitors, which are close to the natural hypoglycemic pathway of human physiology and have few side effects, have been extensively employed in the management of type 2 diabetes mellitus (T2DM). However, there are currently no specific blood indicators that can indicate or predict a patient's suitability for DPP-IV inhibitors. In this study, based on the self-developed high-specificity fluorescent substrate glycyl-prolyl-N-butyl-4-amino-1, 8-naphthimide (GP-BAN), a detection method of human serum DPP-IV activity was established and optimized. The method demonstrates a favorable lower limit of detection (LOD) at 0.32 ng/mL and a satisfactory lower limit of quantification (LOQ) of 1.12 ng/mL, and can be used for the detection of DPP-IV activity in trace serum (2 μL). In addition, Vitalliptin and Sitagliptin showed similar IC50 values when human recombinant DPP-IV and human serum were used as enzyme sources, and the intra-day and inter-day precision obtained by the microplate analyzer were less than 15 %. These results indicate that the microplate reader based detection technique has good accuracy, repeatability and reproducibility. A total of 700 volunteers were recruited, and 646 serum samples were tested for DPP-IV activity. The results showed that serum DPP-IV activity was higher in patients with T2DM than in controls (P < 0.01). However, the statistical data of family history of diabetes, gender and age of diabetic patients showed no statistical significance, and there was no contrast difference. The DPP-IV activity of serum in T2DM patients ranged from 2.4 μmol/min/L to 78.6 μmol/min/L, with a huge difference of up to 32-fold. These results suggest that it is necessary to test DPP-IV activity in patients with T2DM when taking DPP-IV inhibitors to determine the applicability of DPP-IV inhibitors in T2DM patients. These results suggest that it is necessary to detect the activity of DPP-IV in blood before taking DPP-IV inhibitors in patients with T2DM to judge the applicability of DPP-IV inhibitors in patients with T2DM.

Rapid, sensitive, and visual detection of swine Japanese encephalitis virus with a one-pot RPA-CRISPR/EsCas13d-based dual readout portable platform

Japanese encephalitis (JE), a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV), poses a serious threat to global public health. The low viremia levels typical in JEV infections make RNA detection challenging, necessitating early and rapid diagnostic methods for effective control and prevention. This study introduces a novel one-pot detection method that combines recombinant enzyme polymerase isothermal amplification (RPA) with CRISPR/EsCas13d targeting, providing visual fluorescence and lateral flow assay (LFA) results. Our portable one-pot RPA-EsCas13d platform can detect as few as two copies of JEV nucleic acid within 1 h, without cross-reactivity with other pathogens. Validation against clinical samples showed 100 % concordance with real-time PCR results, underscoring the method's simplicity, sensitivity, and specificity. This efficacy confirms the platform's suitability as a novel point-of-care testing (POCT) solution for detecting and monitoring the JE virus in clinical and vector samples, especially valuable in remote and resource-limited settings.

Rapid and Highly Sensitive Detection of Mycobacterium tuberculosis Utilizing the Recombinase Aided Amplification-Based CRISPR-Cas13a System.

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (MTB) infection, remains a major threat to global public health. To facilitate early TB diagnosis, an IS6110 gene-based recombinase aided amplification (RAA) assay was coupled to a clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas13a fluorescence assay to create a rapid MTB detection assay (named RAA-CRISPR-MTB). Its diagnostic efficacy was evaluated for sensitivity and specificity through sequential testing of recombinant plasmids, mycobacterium strains, and clinical specimens. RAA-CRISPR detected IS6110 genes at levels approaching 1 copy/μL with pUC57-6110 as the template and 10 copies/μL with H37Rv as the template. There was no observed cross detection of non-tuberculosis mycobacteria (NTM) with either template. Furthermore, RAA-CRISPR testing of 151 clinical specimens yielded a diagnostic specificity rate of 100% and a diagnostic sensitivity rate of 69% that exceeded the corresponding Xpert MTB/RIF assay rate (60%). In conclusion, we established a novel RAA-CRISPR assay that achieved highly sensitive and specific MTB detection for use as a clinical TB diagnostic tool in resource-poor settings.

Plate-Based High-Throughput Fluorescence Assay for Assessing Enveloped Virus Integrity.

Viruses are a considerable threat to global health and place major burdens on economies worldwide. Manufactured viruses are also being widely used as delivery agents to treat (gene therapies) or prevent diseases (vaccines). Therefore, it is vital to study and fully understand the infectious state of viruses. Current techniques used to study viruses are often slow or nonexistent, making the development of new techniques of paramount importance. Here we present a high-throughput and robust, cell-free plate-based assay (FAIRY: Fluorescence Assay for vIRal IntegritY), capable of differentiating intact from nonintact enveloped viruses, i.e, infectious from noninfectious. Using a thiazole orange-terminated polymer, a 99% increase in fluorescence was observed between treated (heat or virucide) and nontreated. The FAIRY assay allowed for the rapid determination of the infectivity of a range of enveloped viruses, highlighting its potential as a valuable tool for the study of viruses and interventions against them.

Expression mechanisms of mir-486-5p and its host gene sANK1 in porcine muscle.

MiR-486-5p has been identified as a crucial regulator of the PI3K/AKT signalling pathway, which plays a significant role in skeletal muscle development. Its host gene, sANK1, is also essential for skeletal muscle development. However, the understanding of porcine miR-486-5p and sANK1 has been limited. In this study, PCR analyses revealed a positive correlation between the expression of miR-486-5p and sANK1 in the longissimus dorsi muscle of the Bama mini-pig and Landrace-pig, as well as during myoblast differentiation. Furthermore, the expression of miR-486-5p/sANK1 was higher in the Bama mini-pig compared to the Landrace-pig. There was a total of 18 single nucleotide polymorphisms (SNP) present in the sANK1 promoter region. Among these SNPs, 14 of them resulted in alterations in transcription factor binding sites (TFBs). Additionally, the promoter fluorescence assay demonstrated that the activity of the sANK1 promoter derived from the Bama mini-pig was significantly higher compared to Landrace-pig. It is worth noting that ten regulatory SNPs have the potential to influence the activity of the sANK1 promoter. A nuclear mutation A-G located at position - 401 (relative to the transcription start site) in the Bama mini-pig was identified, which creates a putative TFB motif for MyoD. The findings presented in this study offer fundamental molecular knowledge and expression patterns of miR-486-5p/sANK1, which can be valuable for gaining a deeper understanding of the gene's involvement in porcine skeletal muscle development, and meat quality.

Graphene Quantum Dots Inhibit Lipid Peroxidation in Biological Membranes.

Excessive reactive oxygen species (ROS) in cellular environments leads to oxidative stress, which underlies numerous diseases, including inflammatory diseases, neurodegenerative diseases, cardiovascular diseases, and cancer. Oxidative stress can be particularly damaging to biological membranes such as those found in mitochondria, which are abundant with polyunsaturated fatty acids (PUFAs). Oxidation of these biological membranes results in concomitant disruption of membrane structure and function, which ultimately leads to cellular dysfunction. Graphene quantum dots (GQDs) have garnered significant interest as a therapeutic agent for numerous diseases that are linked to oxidative stress. Specifically, GQDs have demonstrated an ability to protect mitochondrial structure and function under oxidative stress conditions. However, the fundamental mechanisms by which GQDs interact with membranes in oxidative environments are poorly understood. Here, we used C11-BODIPY, a fluorescent lipid oxidation probe, to develop quantitative fluorescence assays that determine both the extent and rate of oxidation that occurs to PUFAs in biological membranes. Based on kinetics principles, we have developed a generalizable model that can be used to assess the potency of antioxidants that scavenge ROS in the presence of biological membranes. By augmenting our fluorescence assays with 1H NMR spectroscopy, the results demonstrate that GQDs scavenge nascent hydroxyl and peroxyl ROS that interact with membranes and that GQDs are potent inhibitors of ROS-induced lipid oxidation in PUFA-containing biological membranes. The antioxidant potency of GQDs is comparable to or even greater than established antioxidant molecules, such as ascorbic acid and Trolox. This work provides mechanistic insights into the mitoprotective properties of GQDs under oxidative stress conditions, as well as a quantitative framework for assessing antioxidant interactions in biological membrane systems.

Design of blue-emitting adenosine monophosphate-copper nanocluster: Detection of Vitamin B2 in real samples

We demonstrate a simple preparation protocol for the synthesis of novel adenosine monophosphate-stabilized copper nanoclusters (AMP-Cu NCs). Synthesis parameters are optimized to reach the best fluorometric signal. The prepared Cu NCs show characteristic blue emission at 430 nm, which is related to the presence of Cu13 metal cores. Three distinct lifetime components are identified and the metallic feature of the NCs is proven by X-ray photoelectron spectroscopy. The stability and the long-term storage of the AMP-Cu NCs were investigated by pH- and time-dependent measurements. Besides the synthesis and characterization, a possible liquid fluorescent assay method is also developed for the detection of the biologically important Vitamin B2 in complex real samples by with 2.01 ± 0.31 μM detection limit. For the real samples, the yeast and dietary supplements were selected, where the detected concentration of Riboflavin was in good agreement with the expected amount in these complex chemical systems.

Interrogating a Compound Library in Search of an Inhibitor for TREM-like Transcript-1 to Fibrinogen Binding.

Cardiovascular disease (CVD) remains one of leading causes of death worldwide. Aberrant platelet function mediate fibrin(ogen) rich thrombi that lead to occlusive thrombi associated with mortality. The receptor, TREM-like transcript-1 (TLT-1), stored in the platelet a-granules and released upon platelet activation, binds fibrinogen and von Willebrand factor. Once it is released from platelets TLT-1 is a potential therapeutic target to prevent the thrombosis associated with CVD. Here we design an assay to screen a compound library of small molecules inhibitors. HEK-293 cells stably transfected with a full length human treml-1 construct were used to screen library of 800 compounds, for inhibition of TLT-1 to fibrinogen binding in an attachment assay using crystal violet staining. The possible cytotoxicity of the best compounds was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide MTT and calcein AM staining assays. Here we demonstrate that the addition of TLT-1 to HEK-293 cells increases cell adhesion by more than 2-fold. We identified ~80 compounds that inhibit binding by more than 80%. We further tested the top compounds and confirmed that reduction of hTLT-1 to fibrinogen bound in the top compounds was not caused by cytotoxicity, as per colorimetric and fluorescent viability assays. Four compounds were identified as potential small molecule inhibitors one of which, BM-8372, demonstrated significant effect in platelet aggregation assays. Significance Statement TLT-1 is a key platelet receptor that binds fibrinogen and mediates clot formation The developed assay successfully screens 800 small molecules, pinpointing ~80 potent inhibitors that reduce TLT-1 binding by over 80%. Importantly, the study rigorously rules out cytotoxicity concerns, affirming the therapeutic potential of the identified compounds. By elucidating TLT-1's role and presenting promising inhibitors, this research offers a significant stride toward developing novel strategies to combat CVD-related thrombosis.

Characterizing the amyloid core region of the tumor suppressor protein p16INK4a using a limited proteolysis and peptide-based approach

The human tumor suppressor p16INK4a is a small monomeric protein that can form amyloid structures. Formation of p16INK4a amyloid fibrils is induced by oxidation which creates an intermolecular disulfide bond. The conversion into amyloid is associated with a change from an all α-helical structure into β-sheet fibrils. Currently, structural insights into p16INK4a amyloid fibrils are lacking. Here, we investigate the amyloid-forming regions of this tumor suppressor using isotope-labeling limited-digestion mass spectrometry analysis. We discover two key regions that likely form the structured core of the amyloid. Further investigations using thioflavin-T fluorescence assays, electron microscopy and solution nuclear magnetic resonance spectroscopy of shorter peptide regions confirm the self-assembly of the identified sequences that include methionine and leucine repeat regions. This work describes a simple approach for studying protein motifs involved in the conversion of monomeric species into aggregated fibril structures. It provides first insights into the polypeptide sequence underlying the core structure of amyloid p16INK4a formed after a unique oxidation-driven structural transition.

Zinc-Binding Oligonucleotide Backbone Modifications for Targeting a DNA-Processing Metalloenzyme.

A series of chemically-modified oligonucleotides for targeting the DNA repair nuclease SNM1A have been designed and synthesised. Each oligonucleotide contains a modified internucleotide linkage designed to both mimic the native phosphodiester backbone and chelate to the catalytic zinc ion(s) in the SNM1A active site. Dinucleoside phosphoramidites containing urea, squaramide, sulfanylacetamide, and sulfinylacetamide linkages were prepared and employed successfully in solid-phase oligonucleotide synthesis. All the modified oligonucleotides were found to interact with SNM1A in a gel electrophoresis-based assay, demonstrating the first examples of inhibition of DNA damage repair enzymes for many of these groups in oligonucleotides. One strand containing a sulfinylacetamide-linkage was found to have the strongest interaction with SNM1A and was further tested in a real-time fluorescence assay. This allowed an IC50 value of 231 nM to be determined, significantly lower than previously reported substrate-mimics targeting this enzyme. It is expected that these modified oligonucleotides will serve as a scaffold for the future development of fluorescent or biotin-labelled probes for the in vivo study of DNA repair processes.

Single-mode termination of phage transcriptions, disclosing bacterial adaptation for facilitated reinitiations.

Bacterial and bacteriophage RNA polymerases (RNAPs) have divergently evolved and share the RNA hairpin-dependent intrinsic termination of transcription. Here, we examined phage T7, T3 and SP6 RNAP terminations utilizing the single-molecule fluorescence assays we had developed for bacterial terminations. We discovered the phage termination mode or outcome is virtually single with decomposing termination. Therein, RNAP is displaced forward along DNA and departs both RNA and DNA for one-step decomposition, three-dimensional diffusion and reinitiation at any promoter. This phage displacement-mediated decomposing termination is much slower than readthrough and appears homologous with the bacterial one. However, the phage sole mode of termination contrasts with the bacterial dual mode, where both decomposing and recycling terminations occur compatibly at any single hairpin- or Rho-dependent terminator. In the bacterial recycling termination, RNA is sheared from RNA·DNA hybrid, and RNAP remains bound to DNA for one-dimensional diffusion, which enables facilitated recycling for reinitiation at the nearest promoter located downstream or upstream in the sense or antisense orientation. Aligning with proximity of most terminators to adjacent promoters in bacterial genomes, the shearing-mediated recycling termination could be bacterial adaptation for the facilitated reinitiations repeated at a promoter for accelerated expression and coupled at adjoining promoters for coordinated regulation.

Fast, portable and sensitive detection of group B streptococcus DNA using one-pot MIRA-CRISPR system with suboptimal PAM

The group B Streptococcus (GBS) can generate vertical transmission to infants during delivery, has been seriously threatening the health of infants. Rapid and accurate prenatal GBS diagnosis for pregnant women is a deterministic blueprint to avoid infant viruses. Here, we developed an extraction-free nucleic acid isothermal amplification/CRISPR-Cas12a cutting one-pot system for GBS diagnostic assay by using suboptimal protospacer adjacent motifs, effectively avoiding multiple handling steps and uncapping contamination. The GBS diagnosis assay based on a one-pot system was validated by using fluorescent technique and lateral flow assay strips, exhibited fantastic specificity, accuracy and sensitivity with a limit of detection of 32 copies per reaction (0.64 copies/μL). Moreover, a portable device was constructed and integrated with the one-pot system to realize the GBS detection without professional and scene restrictions, it showed excellent performance in clinical sample detection, which achieved optical and portable GBS detection for point-of-care testing or home-self testing.

Design, synthesis, and evaluation of antitumor activity of 2-arylmethoxy-4-(2-fluoromethyl-biphenyl-3-ylmethoxy) benzylamine derivatives as PD-1/PD-l1 inhibitors

A series of novel 2-arylmethoxy-4-(2-fluoromethyl-biphenyl-3-ylmethoxy) benzylamine derivatives was designed, synthesized, and evaluated for their antitumor effects as PD-1/PD-L1 inhibitors both in vitro and in vivo. Firstly, the ability of these compounds to block the PD-1/PD-L1 immune checkpoint was assessed using the homogeneous time-resolved fluorescence (HTRF) assay. Two of the compounds can strongly block the PD-1/PD-L1 interaction, with IC50 values of less than 10 nM, notably, compound HD10 exhibited significant clinical potential by inhibiting the PD-1/PD-L1 interaction with an IC50 value of 3.1 nM. Further microscale thermophoresis (MST) analysis demonstrated that HD10 had strong interaction with PD-L1 protein. Co-crystal structure (2.7 Å) analysis of HD10 in complex with the PD-L1 protein revealed a strong affinity between the compound and the target PD-L1 dimer. This provides a solid theoretical basis for further in vitro and in vivo studies. Next, a typical cell-based experiment demonstrated that HD10 could remarkably prevent the interaction of hPD-1 293 T cells from human recombinant PD-L1 protein, effectively restoring T cell function, and promoting IFN-γ secretion in a dose-dependent manner. Moreover, HD10 was effective in suppressing tumor growth (TGI = 57.31 %) in a PD-1/PD-L1 humanized mouse model without obvious toxicity. Flow cytometry, qPCR, and immunohistochemistry data suggested that HD10 inhibits tumor growth by activating the immune system in vivo. Based on these results, it seems likely that HD10 is a promising clinical candidate that should be further investigated.

Combination of an enzyme-linked immunosorbent assay and an indirect fluorescence assay for a nationwide sero-survey of Toxoplasma gondii infection in pig herds in Taiwan

Toxoplasma gondii is a zoonotic pathogen that can infect farm animals, companion animals, and humans, sometimes causing public health issues. In Taiwan, the pig industry is a vital agricultural industry, with a self-sufficiency rate of 91 %, and pigs are also food-producing animal reservoirs of Toxoplasma gondii. Infected pigs are usually asymptomatic, and abortions and death may occur in severe cases. We combined an enzyme-linked immunosorbent assay (ELISA) and an indirect fluorescence assay (IFA) to investigate the seroprevalence of Toxoplasma gondii among pig populations in Taiwan. A stratified sampling approach to determine the number of sample farms proportional to the number of pig farms in each county was employed, with 15 blood samples collected at each farm between July and September 2017. With the tested results, empirical Bayesian smoothing was utilized to assess the proportion of Toxoplasma-positive farms at the county level. Bayesian mixed-effects logistic regression models, incorporating farm and county as random effects, were employed to investigate associations between Toxoplasma test results and potential risk factors. A total of 930 serum samples from 62 pig farms were collected and tested. An overall herd prevalence of 27.4 % was shown with the seroprevalence in northern Taiwan being greater than that in southern Taiwan. The sampling month and companion dog density in 2017 were significantly associated with Toxoplasma infections in pigs. With every increase in the number of companion dogs per km² at the county level, the odds of Toxoplasma infection in pigs increased by 4.7 % (95 % CI: 1.7–8.9 %). This study demonstrated that combining ELISA for screening with IFA for confirmation is a cost-effective and time-saving method for conducting a large-scale sample investigation. This was also the first nationwide, cross-sectional study in Taiwanese pig herds to investigate Toxoplasma gondii infection.

From yeast screening for suitability as single cell protein to fed-batch cultures.

Fed-batch cultures have rarely been used in single cell protein (SCP) research. This work evaluated multiple yeast species for suitability as SCP cultivated using glucose- and sucrose-based substrate and performed in-depth studies of fed-batch SCP cultivation kinetics for selected yeasts, including determination of specific crude nitrogen-to-protein conversion factors. SCP was cultivated using fully synthetic media in flask batch or bioreactor fed-batch cultures. Crude nitrogen and nucleic acid content were determined using the Dumas method and fluorescence assay kits, respectively. C. utilis compared favorably to other yeasts in flask batch cultures in terms of process yield (0.52 ± 0.01 gxgs-1) and crude nitrogen content (10.0 ± 0.5 and 9.9 ± 0.5%CDW for glucose and sucrose, respectively). This is the first time biomass composition data was reported for SCP cultivated in fed-batch mode. C. utilis crude nitrogen content was consistent across the tested conditions (protein content stabilized around 50%CDW in fed-batch), while that of the benchmark yeast S. cerevisiae was higher in batch cultures and at the beginning of fed-batch relative to the end (protein content decreased over time and stabilized around 43%CDW). Total nucleic acid content of the yeasts was similar (6.8%CDW and 6.3%CDW, for C. utilis and S. cerevisiae, respectively), with crude nitrogen-to-protein conversion factors of 4.97 and 5.80. This study demonstrated the suitability of C. utilis as SCP, notably the robustness of its crude nitrogen content (as an indicator of protein content) across batch and fed-batch conditions, compared to that of the benchmark yeast S. cerevisiae.

Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids.

Aim: This study aimed to explore using peripheral blood mononuclear cell (PBMC)-derived chimeric antigen receptor (CAR) NK cells targeting ROBO1 as a personalized medicine approach for ovarian cancer. Methods: A two-step strategy generated ROBO1-targeted CAR NK cells from PBMCs of ovarian cancer patients. Efficacy was evaluated using xCELLigence RTCA, CCK-8 and Live/Dead fluorescence assays. Results: ROBO1-NK cells exhibited higher efficiency in eradicating primary ovarian cancer cells and lysing ovarian tumor organoids compared with primary NK cells without ROBO1-CAR modification. Conclusion: These findings highlight the potential of developing ROBO1-targeted CAR-NK cells from patients' PBMCs as a personalized treatment option for ovarian cancer.