Modification of mesenchymal stromal cells with silibinin-loaded PLGA nanoparticles improves their therapeutic efficacy for cutaneous wound repair.

The use of mesenchymal stromal cells (MSCs) for treating chronic inflammatory disorders, wounds, and ischemia-reperfusion injuries has shown improved healing efficacy. However, the poor survival rate of transplanted cells due to oxidative stress in injured or inflamed tissue remains a significant concern for MSC-based therapies. In this study, we developed a new approach to protect MSCs from oxidative stress, thereby improving their survival in a wound microenvironment and enhancing their therapeutic effect. We produced PLGA nanoparticles loaded with the cytoprotective phytochemical silibinin (SBN), and used them to modify MSCs. Upon internalization, these nanoformulations released SBN, activating the Nrf2/ARE signaling pathway, resulting in threefold reduction in intracellular ROS content and improved cell survival under oxidative stress conditions. Modification of MSCs with SBN-loaded PLGA nanoparticles increased their survival upon transplantation to full-thickness cutaneous wounds and improved wound healing. This study suggests that MSC modification with cytoprotective nanoparticles could be a promising approach for improving wound healing.

Evolution of cisplatin resistance through coordinated metabolic reprogramming of the cellular reductive state.

BACKGROUND: Cisplatin (CDDP) is a mainstay treatment for advanced head and neck squamous cell carcinomas (HNSCC) despite a high frequency of innate and acquired resistance. We hypothesised that tumours acquire CDDP resistance through an enhanced reductive state dependent on metabolic rewiring. METHODS: To validate this model and understand how an adaptive metabolic programme might be imprinted, we performed an integrated analysis of CDDP-resistant HNSCC clones from multiple genomic backgrounds by whole-exome sequencing, RNA-seq, mass spectrometry, steady state and flux metabolomics. RESULTS: Inactivating KEAP1 mutations or reductions in KEAP1 RNA correlated with Nrf2 activation in CDDP-resistant cells, which functionally contributed to resistance. Proteomics identified elevation of downstream Nrf2 targets and the enrichment of enzymes involved in generation of biomass and reducing equivalents, metabolism of glucose, glutathione, NAD(P), and oxoacids. This was accompanied by biochemical and metabolic evidence of an enhanced reductive state dependent on coordinated glucose and glutamine catabolism, associated with reduced energy production and proliferation, despite normal mitochondrial structure and function. CONCLUSIONS: Our analysis identified coordinated metabolic changes associated with CDDP resistance that may provide new therapeutic avenues through targeting of these convergent pathways.

Applications of covalent chemistry in targeted protein degradation.

Proteolysis-targeting chimeras (PROTACs) and targeted covalent inhibitors (TCIs) are currently two exciting strategies in the fields of chemical biology and drug discovery. Extensive research in these two fields has been conducted, and significant progress in these fields has resulted in many clinical candidates, some of which have been approved by FDA. Recently, a novel concept termed covalent PROTACs that combine these two strategies has emerged and gained an increasing interest in the past several years. Herein, we briefly review and highlight the mechanism and advantages of TCIs and PROTACs, respectively, and the recent development of covalent PROTACs using irreversible and reversible covalent chemistry.

Tissue-Oxygen-Adaptation of Bone Marrow-Derived Mesenchymal Stromal Cells Enhances Their Immunomodulatory and Pro-Angiogenic Capacity, Resulting in Accelerated Healing of Chemical Burns.

Transplantation of mesenchymal stromal cells (MSCs) provides a powerful tool for the management of multiple tissue injuries. However, poor survival of exogenous cells at the site of injury is a major complication that impairs MSC therapeutic efficacy. It has been found that tissue-oxygen adaptation or hypoxic pre-conditioning of MSCs could improve the healing process. Here, we investigated the effect of low oxygen tension on the regenerative potential of bone-marrow MSCs. It turned out that incubation of MSCs under a 5% oxygen atmosphere resulted in increased proliferative activity and enhanced expression of multiple cytokines and growth factors. Conditioned growth medium from low-oxygen-adapted MSCs modulated the pro-inflammatory activity of LPS-activated macrophages and stimulated tube formation by endotheliocytes to a much higher extent than conditioned medium from MSCs cultured in a 21% oxygen atmosphere. Moreover, we examined the regenerative potential of tissue-oxygen-adapted and normoxic MSCs in an alkali-burn injury model on mice. It has been revealed that tissue-oxygen adaptation of MSCs accelerated wound re-epithelialization and improved the tissue histology of the healed wounds in comparison with normoxic MSC-treated and non-treated wounds. Overall, this study suggests that MSC adaptation to 'physiological hypoxia' could be a promising approach for facilitating skin injuries, including chemical burns.

Comparison of EMT-Related and Multi-Drug Resistant Gene Expression, Extracellular Matrix Production, and Drug Sensitivity in NSCLC Spheroids Generated by Scaffold-Free and Scaffold-Based Methods.

Multicellular 3D tumor models are becoming a powerful tool for testing of novel drug products and personalized anticancer therapy. Tumor spheroids, a commonly used 3D multicellular tumor model, more closely reproduce the tumor microenvironment than conventional 2D cell cultures. It should be noted that spheroids can be produced using different techniques, which can be subdivided into scaffold-free (SF) and scaffold-based (SB) methods. However, it remains unclear, to what extent spheroid properties depend on the method of their generation. In this study, we aimed to carry out a head-to-head comparison of drug sensitivity and molecular expression profile in SF and SB spheroids along with a monolayer (2D) cell culture. Here, we produced non-small cell lung cancer (NSCLC) spheroids based on human lung adenocarcinoma cell line A549. Drug sensitivity analysis of the tested cell cultures to five different chemotherapeutics resulted in IC50 (A549-SB) > IC50 (A549-SF) > IC50 (A549-2D) trend. It was found that SF and SB A549 spheroids displayed elevated expression levels of epithelial-to-mesenchymal transition (EMT) markers and proteins associated with drug resistance compared with the monolayer A549 cell culture. Enhanced drug resistance of A549-SB spheroids can be a result of larger diameters and elevated deposition of extracellular matrix (ECM) that impairs drug penetration into spheroids. Thus, the choice of the spheroid production method can influence the properties of the generated 3D cell culture and their drug resistance. This fact should be considered for correct interpretation of drug testing results.

Terpenoids from stigma maydis (Zea mays L.) alleviate hydrogen peroxide-induced SH-SY5Y cell injury by activating Nrf2.

Five terpenoids (1-5), including three new ent-kaurane diterpenoids (1-3), one new ent-rosane type diterpenoid (4) and one known triterpenoid (5), were isolated from stigma maydis (Zea mays L.). The structures of the compounds were elucidated by comprehensive spectroscopic analyses. The relative configurations of stigmanes A-D (1-4) were determined by NOESY experiments. In addition, the relative configuration of stigmane D (4) was further established by NMR calculations. The absolute configurations of these compounds were identified by a comparison of experimental and calculated specific rotations. The neuroprotective effects of these compounds against H2O2-induced injury in human neuroblastoma SH-SY5Y cells were evaluated, and the results showed that among the compounds, 2 exhibited the most significant neuroprotection. Further study demonstrated that 2 could activate nuclear factor E2-related factor (Nrf2), downregulate apoptosis and reactive oxygen species (ROS) generation, and increase antioxidant enzyme activities in SH-SY5Y cells. However, the neuroprotective effect was reversed when Nrf2 was silenced. In conclusion, this study suggested that terpenoids from stigma maydis exerted neuroprotective effects through Nrf2 activation.

School-based epidemiology study of myopia in Tianjin, China.

PURPOSE: To study the epidemiology of myopia in school-aged children in Tianjin and the relationship between visual acuity-based screening and refraction-based screening. METHOD: This school-based prospective cohort study was performed on children from 42 elementary schools and 17 middle schools in Tianjin, China. Totally 14,551 children, ages ranging from 5 to 16 years, were included in this study. Uncorrected visual acuity (UCVA) was determined by logarithmic tumbling E chart. Non-cycloplegic photorefraction was examined by the Spot (v2.1.4) photoscreener. The relationship between the UCVA and refractive error was investigated for different age groups. RESULTS: The overall prevalence of myopia at this school based screen is 78.2%, ranged from 10% at age of 5 to 95% at age of 16. The most dramatic increase in prevalence is from age of 6 (14.8%) to age of 7 (38.5%). The overall prevalence of high myopia is 2.5%. UCVA is found corresponding to spherical equivalent refraction (SER) in a manner of normal distribution and is significantly affected by age. When using UCVA to estimate the prevalence of myopia, the overall sensitivity and specificity are 0.824 and 0.820, respectively. Age-dependent optimal cutoff points and 95% confident intervals of such estimation are reported. CONCLUSIONS: Myopia is heavily affecting school-aged children in Tianjin, China. The refraction screening is preferable for myopia screening, whereas the UCVA screening results need to be interpreted in an age-dependent manner for myopia estimation.

HPV16+ -miRNAs in cervical cancer and the anti-tumor role played by miR-5701.

BACKGROUND: The early diagnosis of cervical cancer is difficult, resulting in an unsatisfactory prognosis. The present study aimed to explore the expression of HPV16-related microRNAs (miRNAs) in the serum of Uygur cervical cancer in Sinkiang and analyze the miRNAs showing different expression. METHODS: The serum of 30 HPV16 positive (HPV16Pos) and 30 negative patients (HPV16Neg) was collected and then the differentially expressed miRNAs were screened out by function and pathway enrichment analyses. Next, the cervical cancer cells were cultured. miR-5701-mimic and inhibitor were transfected into cells. The level of miR-5701 was detected by a quantitative reverse transcriptase-polymerase chain reaction. The proliferation of cells was detected using a Cell Counting Kit-8 assay. Western blotting was used to measure the expression of THBS4. RESULTS: We identified three up-regulated miRNAs (hsa-miR-1291, hsa-miR-144-5p and hsa-miR-5701) and seven down-regulated known-miRNAs (hsa-miR-21-5p, hsa-miR-101-3p, hsa-miR-370-3p, hsa-miR-151a-3p, hsa-miR-144-3p, hsa-miR-199a-3p and hsa-miR-199b-3p) relative to HPV16Neg. The mitogen-activated protein kinase signal pathway is predicted to be a key mechanism of HPV16-related cervical cancer. Furthermore, miR-5701 inhibits the proliferation of cervical cancer cells and suppresses the expression of THBS4 (P < 0.05), which was confirmed as a target gene of miR-5701. CONCLUSIONS: In the present study, we confirm 10 differentially expressed miRNAs that could be potential markers or therapeutic targets of cervical cancer. miR-5701 inhibits the proliferation of cervical cancer cells and the expression of its target gene THBS4.

Pseudoesotropia in Chinese Children: A Triphasic Development of the Interepicanthal Folds Distance-to-Interpupillary Distance Ratio and Its Changing Perception.

PURPOSE: To delineate the development of the interepicanthal fold distance (IEFD) to interpupillary distance (IPD) in Chinese children, and to quantify how their ratio (EFDPD ratio) affects parent's judgment on whether a child's two eyes appear misaligned. METHODS: The values of IPD and IEFD were measured in 750 children, aged between 3 and 17 years. The developmental trend of EFDPD ratio was established. Two hundred parents were shown a series of pictures of children with varying EFDPD ratios and asked to judge whether the child in each picture demonstrated misaligned eyes. Based on the parent's responses, psychometric functional associations with EFDPD ratios were established. RESULTS: The EFDPD ratios were significantly higher (0.63 ± 0.027) and showed little change among children from 3 to 6 years of age (p = 0.704). During the age of seven to 12 years, however, the EFDPD ratio significantly decreased (p < 0.001) before stabilizing at 0.59 ± 0.023 by the ages of 13 to 17 years (p = 0.376). Children with EFDPD ratios > 0.65 were more likely to be perceived as strabismic by the parents, while children with an EFDPD ratio < 0.55 were rarely perceived as so. As many as 30% of the children aged between 3 and 6 years demonstrated EFDPD ratios > 0.65, and this number reduced to 5% by the age of 12 years. CONCLUSIONS: The development of the EFDPD ratio in Chinese children shows a triphasic pattern, with a large value before the age of 6 years, a quick drop between 7 and 12 years, and little change after 13 years of age. As the EFDPD ratio declines, fewer children appear as strabismic. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Retraction Note to: An assessment of transgenomics as a tool for gene discovery in Populus euphratica Oliv.

This article (Zhou et al. 2018) has been retracted by the authors because the sequence BIBAC 002A111F06 was incorrectly assigned to the wrong bacterial species. The BIBAC 002A111F06 sequence (GenBank Accession KC129717) reported in the paper was attributed to Populus euphratica Oliv. The BLAST search of this KC129717 sequence against the nr database at NCBI showed that it has very high similarity to a genomic sequence from the gram-negative bacteria Stenotrophomonas maltophilia. The bacterium associates with Populus euphratica Oliv. and DNA isolated from Populus euphratica Oliv. for the construction of the BIBAC clone library inlcuded DNA from Stenotrophomonas maltophilia. Therefore, the phenotype of the transgenic Arabidopsis line carrying the KC129717 sequence cannot be attributed to genes from Populus euphratica Oliv. The authors apologize for the confusion and misinterpretation of our data resulting from the incorrect sequence assignment. All authors agree to this retraction.

An assessment of transgenomics as a tool for gene discovery in Populus euphratica Oliv.

KEY MESSAGE: Transgenomics for gene discovery in Populus euphratica. Transgenomics, a member of the omics family of methodologies, is characterized as the introduction of DNA from one organism into another on a genome-wide scale followed by the identification of recipients with altered phenotypes. This strategy allows investigators to identify the gene(s) involved in these phenotypic changes. It is particularly promising for woody plants that have a long life cycle and for which molecular tools are limited. In this study, we constructed a large-insert binary bacterial artificial chromosome library of Populus euphratica, a stress-tolerant poplar species, which included 55,296 clones with average insert sizes of about 127 kb. To date, 1077 of the clones have been transformed into Arabidopsis thaliana via Agrobacterium by the floral dip method. Of these, 69 transgenic lines showed phenotypic changes represented by diverse aspects of plant form and development, 22 of which were reproducibly associated with the same phenotypic change. One of the clones conferring transgenic plants with increased salt tolerance, 002A1F06, was further analyzed and the 127,284 bp insert in this clone harbored eight genes that have been previously reported to be involved in stress resistance. This study demonstrates that transgenomics is useful in the study of functional genomics of woody plants and in the identification of novel gene(s) responsible for economically important traits. Thus, transgenomics can also be used for validation of quantitative trait loci mapped by molecular markers.

Graphite nanoparticle as nanoquencher for 17ß-estradiol detection using shortened aptamer sequence.

A graphite nanoparticle (GN) with a spherical shape and stacked by a few layered graphene sheets is an ideal fluorescent nanoquencher for the fluorophore. We developed a novel aptasensor based on fluorescence resonance energy transfer for the sensitive and specific detection of 17ß-estradiol (E2) by using the GN as a fluorescent nanoquencher and the shorter E2 specific aptamer as the sensing probe. The physiological and chemical properties of the aptasensor in response to the E2 capture were investigated with an atomic force microscopic analysis and an E2 detection principle was clarified accordingly. Also, it was demonstrated that the sensitivity of the aptasensor was affected by the length of aptamer and the particle size of the nanoquencher, and the highest sensitivity for E2 detection was achieved with a shorter aptamer of 35 base sequences and a smaller GN with a particle size of around 5 nm. And the detection limit was 1.02 ng mL-1. Moreover, this presented no cross reaction with E2 analogs and was successfully utilized for the real environmental water monitoring.

ent-Kaurane Diterpenoids with Neuroprotective Properties from Corn Silk ( Zea mays).

Thirteen new ent-kaurane diterpenoids, stigmaydenes A-M (1-13), together with two known compounds (14, 15), were isolated from the crude extract of corn silk ( Zea mays). The structures of the compounds were confirmed by comprehensive spectroscopic analyses. The absolute configuration of compound 1 was defined by single-crystal X-ray diffraction. The absolute configurations of the compounds were also confirmed by comparison of experimental and calculated specific rotations. The compounds were evaluated for their neuroprotective effects against H2O2-induced SH-SY5Y cell injury, and compound 8 was active at 100 µM, as determined by flow cytometry (annexin V-FITC/PI staining) and Hoechst 33258 staining. The results suggested that compound 8 could protect neuronal cells from H2O2-induced injury by inhibiting apoptosis in SH-SY5Y cells.

Prevalence of refractive errors in Tibetan adolescents.

BACKGROUND: The prevalence of adolescent eye disease in remote areas of the Qinghai-Tibet Plateau has rarely been reported. To understand the prevalence of common eye diseases in Tibet, we performed ocular-disease screening on students from primary and secondary schools in Tibet, and compared the prevalence to that in the Central China Plain (referred to here as the "plains area"). METHODS: The refractive status of students was evaluated with a Spot™ vision screener. The test was conducted three or fewer times for both eyes of each student and results with best correction were recorded. RESULTS: A total of 3246 students from primary and secondary schools in the Tibet Naidong district were screened, yielding a refractive error rate of 28.51%, which was significantly lower than that of the plains group (28.51% vs. 56.92%, p < 0.001). In both groups, the prevalence of refractive errors among females was higher than that among males. CONCLUSIONS: We found that Tibetan adolescents had a lower prevalence of refractive errors than did adolescents in the plains area, which may be related to less intensive schooling and greater exposure to sunlight.

Direct Electrochemical Detection of Bisphenol A Using a Highly Conductive Graphite Nanoparticle Film Electrode.

We developed an accurate and sensitive sensor for electrochemical detection of bisphenol A (BPA) with a high-conductivity graphite nanoparticle (GN) film electrode. The GNs consisted of several stacked graphene sheets and showed a homogenous spherical shape, high conductivity, large surface area and good adsorption properties to BPA. The constructed GN film electrode exhibited improved amperometric current responses such as decreased impedance and lowered BPA oxidation potential compared with those of a pristine electrode, and also possessed a large surface area to allow fast electron transfer and BPA accumulation. A pre-accumulation process with BPA adsorption resulted in considerable current signal enhancement during BPA detection. The loading amount of GNs on the film electrode and the time for target BPA enrichment were optimized. The GN film electrode-based sensor showed high reproducibility and high selectivity for BPA over other reagents. Differential pulse voltammetry experiments revealed that the concentrations of BPA were linearly correlated with the current changes, and the lowest limit of detection of the sensor was 35 nM. Furthermore, the sensor showed great accuracy and reliability, as confirmed by high-performance liquid chromatography measurements. The sensor was also successfully used for BPA determination in groundwater samples, demonstrating its potential for real environmental analysis.

Bioactive norditerpenoids and neolignans from the roots of salvia miltiorrhiza.

Two new norditerpenoids, miltiolactones A and B (1a, 1b); seven new neolignans, miltiolignanolides A-G (2a, 2b and 3a, 3b and 4a, 4b, and 5) were obtained from the root extract of Salvia miltiorrhiza. Using HPLC separation with a chiral column, compounds 1-4 were found to exist as four pairs of enantiomers. Compounds 2-5 are novel neolignans with a dibenzocycloheptatriene ring system, which form an unprecedented 6/7/6 carbon skeleton. The structures were established via extensive spectroscopic analysis, and experimental and calculated electronic circular dichroism (ECD) spectra, and compound 1 was also elucidated by X-ray diffraction experiments. Compounds 2a and 2b significantly increased the viability of H9c2 cells from H2O2-induced cell death, and 3a showed significant antioxidant activity in an ABTS assay. In addition, compounds 2a, 2b and 3a, 3b display moderate inhibitory effects on nitric oxide (NO) production in LPS-induced microglial cells.

A critical role for the melanocortin 4 receptor in stress-induced relapse to nicotine seeking in rats.

Tobacco addiction is characterized by a lack of control over smoking and relapse after periods of abstinence. Smoking cessation leads to a dysphoric state that contributes to relapse to smoking. After the acute withdrawal phase, exposure to stressors increases the risk for relapse. Blockade of melanocortin 4 (MC4 ) receptors has anxiolytic and antidepressant-like effects in animal models. The aim of these studies was to investigate the role of MC4 receptors in the dysphoria associated with nicotine withdrawal and stress-induced reinstatement of nicotine seeking. To study stress-induced reinstatement, rats self-administered nicotine for 16 days and then nicotine seeking was extinguished by substituting saline for nicotine. Nicotine seeking was reinstated by intermittent footshock stress. The intracranial self-stimulation (ICSS) procedure was used to assess the negative mood state associated with nicotine withdrawal. Elevations in the ICSS thresholds are indicative of a dysphoric state. The selective MC4 receptor antagonists HS014 and HS024 prevented stress-induced reinstatement of extinguished nicotine seeking. Drug doses that prevented stress-induced relapse did not affect responding for food pellets, which indicates that the drugs did not induce sedation or motor impairments. In the ICSS experiments, the nicotinic acetylcholine receptor antagonist mecamylamine elevated the ICSS thresholds of the nicotine-dependent rats. Pre-treatment with HS014 or HS024 did not prevent the elevations in ICSS thresholds. These studies indicate that MC4 receptors play a critical role in stress-induced reinstatement of nicotine seeking, but these receptors may not play a role in the dysphoria associated with acute nicotine withdrawal.

A fatty acid condensing enzyme from Physaria fendleri increases hydroxy fatty acid accumulation in transgenic oilseeds of Camelina sativa.

MAIN CONCLUSION: Co-expression of a lesquerella fatty acid elongase and the castor fatty acid hydroxylase in camelina results in higher hydroxy fatty acid containing seeds with normal oil content and viability. Producing hydroxy fatty acids (HFA) in oilseed crops has been a long-standing goal to replace castor oil as a renewable source for numerous industrial applications. A fatty acid hydroxylase, RcFAH, from Ricinus communis, was introduced into Camelina sativa, but yielded only 15 % of HFA in its seed oil, much lower than the 90 % found in castor bean. Furthermore, the transgenic seeds contained decreased oil content and the germination ability was severely affected. Interestingly, HFA accumulation was significantly increased in camelina seed when co-expressing RcFAH with a fatty acid condensing enzyme, LfKCS3, from Physaria fendleri, a native HFA accumulator relative to camelina. The oil content and seed germination of the transgenic seeds also appeared normal compared to non-transgenics. LfKCS3 has been previously characterized to specifically elongate the hydroxylated ricinoleic acid to lesquerolic acid, the 20-carbon HFA found in lesquerella oil. The elongation reaction may facilitate the HFA flux from phosphatidylcholine (PC), the site of HFA formation, into the acyl-CoA pool for more efficient utilization in triacylglycerol (TAG) biosynthesis. This was demonstrated by increased HFA accumulation in TAG concurrent with reduced HFA content in PC during camelina seed development, and increased C20-HFA in HFA-TAG molecules. These effects of LfKCS3 thus may effectively relieve the bottleneck for HFA utilization in TAG biosynthesis and the feedback inhibition to fatty acid synthesis, result in higher HFA accumulation and restore oil content and seed viability.

Uncovering the relationship between floating marine litter and human activities in watersheds.

Rivers serve as the primary pathway for transporting floating marine litter (FML) from land to sea. However, the complex dynamics of transboundary rivers pose a significant obstacle when examining the impact of watershed-based human activities on FML distribution. This study conducts year-long monthly monitoring of FML using trawl and visual surveys in the coastal water of a peninsula dominated by indigenous rivers in south China. Overall, small pieces debris dominates FML in the nearshore waters of the peninsula, with meso-sized (0.5 cm-2.5 cm) FML accounting for 73.93 % of the total. The density of FML is more profoundly influenced by human activities within watersheds rather than its composition. Moreover, the association between human activity and FML density exhibits greater significant compared to variations based on geography and seasonality. This study provides a scientific basis for coastal protection and contributes for understanding of the mechanisms of marine litter transfer from land to sea.

Lysineless HiBiT and NanoLuc Tagging Systems as Alternative Tools Monitoring Targeted Protein Degradation.

Target protein degradation (TPD) has emerged as a revolutionary approach in drug discovery, leveraging the cell's intrinsic machinery to selectively degrade disease-associated proteins. Proteolysis-Targeting Chimeras (PROTACs) exemplify this strategy, exploiting heterobifunctional molecules to induce ubiquitination and subsequent degradation of target proteins. The clinical advancement of PROTACs underscores their potential in therapeutic intervention, with numerous projects progressing through clinical stages. However, monitoring subtle changes in protein abundance induced by TPD molecules demands highly sensitive assays. Nano-luciferase (nLuc) fusion proteins, or the NanoBiT technology derived from it, offer a robust screening platform due to their high sensitivity and stability. Despite these advantages, concerns have arisen regarding potential degradation artifacts introduced by tagging systems due to the presence of lysine residues on them, prompting the development of alternative tools. In this study, we introduce HiBiT-RR and nLuc K0 , variants devoid of lysine residues, to mitigate such artifacts. Our findings demonstrate that HiBiT-RR maintains similar sensitivity and binding affinity with the original HiBiT. Moreover, the comparison between nLuc WT and nLuc K0 constructs reveals variations in degradation patterns induced by certain PROTAC molecules, emphasizing the importance of choosing appropriate tagging systems to ensure the reliability of experimental outcomes in studying protein degradation processes.