Camouflaging nanoreactor traverse the blood-brain barrier to catalyze redox cascade for synergistic therapy of glioblastoma.

The blood-brain barrier (BBB) is a complex and highly restrictive barrier that prevents most biomolecules and drugs from entering the brain. However, effective strategies for delivering drugs to the brain are urgently needed for the treatment of glioblastoma. Based on the efficient BBB penetration properties of exosomes derived from brain metastatic breast cancer cells (EB), this work prepared a nanoreactor (denoted as MAG@EB), which was constructed by self-assembly of Mn2+, arsenate and glucose oxidase (GOx) into nanoparticles wrapped with EB. MAG@EB can enhance the efficiency of traversing the BBB, target and accumulate at in situ glioblastoma sites. The GOx-driven glycolysis effectively cuts off the glucose supply while also providing an abundance of H2O2 and lowering pH. Meanwhile, the released Mn2+ mediated Fenton-like reaction converts elevated H2O2 into highly toxic ·OH. Besides, AsV was reduced to AsIII by glutathione, and the tumor suppressor gene P53 was activated by AsIII to kill glioblastoma cells. Glioblastoma succumbed to the redox cascade triggered by MAG@EB, as the results demonstrated in vivo and in vitro, yielding a remarkable therapeutic effect. This work provides a promising therapeutic option mediated by cascaded nanoreactors for the future treatment of glioblastoma.

Psychometric validation of a patient-reported experience measure for older adults attending the emergency department: the PREM-ED 65 study.

INTRODUCTION: Optimising emergency department (ED) patient experience is vital to ensure care quality. However, there are few validated instruments to measure the experiences of specific patient groups, including older adults. We previously developed a draft 82-item Patient Reported Experience Measure (PREM-ED 65) for adults ≥65 attending the ED. This study aimed to derive a final item list and provide initial validation of the PREM-ED 65 survey. METHODS: A cross-sectional study involving patients in 18 EDs in England. Adults aged 65 years or over, deemed eligible for ED discharge, were recruited between May and August 2021 and asked to complete the 82-item PREM at the end of the ED visit and 7-10 days post discharge. Test-retest reliability was assessed 7-10 days following initial attendance. Analysis included descriptive statistics, including per-item proportions of responses, hierarchical item reduction, exploratory factor analysis (EFA), reliability testing and assessment of criterion validity. RESULTS: Five hundred and ten initial surveys and 52 retest surveys were completed. The median respondent age was 76. A similar gender mix (men 47.5% vs women 50.7%) and reason for attendance (40.3% injury vs 49.0% illness) was observed. Most participants self-reported their ethnicity as white (88.6%).Hierarchical item reduction identified 53/82 (64.6%) items for exclusion, due to inadequate engagement (n=33), ceiling effects (n=5), excessive inter-item correlation (n=12) or significant differential validity (n=3). Twenty-nine items were retained.EFA revealed 25 out of the 29 items demonstrating high factor loadings (>0.4) across four scales with an Eigenvalue >1. These scales were interpreted as measuring 'relational care', 'the ED environment', 'staying informed' and 'pain assessment'. Cronbach alpha for the scales ranged from 0.786 to 0.944, indicating good internal consistency. Test-retest reliability was adequate (intraclass correlation coefficient 0.67). Criterion validity was fair (r=0.397) when measured against the Friends and Families Test question. CONCLUSIONS: Psychometric testing demonstrates that the 25-item PREM-ED 65 is suitable for administration to adults ≥65 years old up to 10 days following ED discharge.

Integrated oral microgel system ameliorates renal fibrosis by hitchhiking co-delivery and targeted gut flora modulation.

BACKGROUND: Renal fibrosis is a progressive process associated with chronic kidney disease (CKD), contributing to impaired kidney function. Active constituents in traditional Chinese herbs, such as emodin (EMO) and asiatic acid (AA), exhibit potent anti-fibrotic properties. However, the oral administration of EMO and AA results in low bioavailability and limited kidney accumulation. Additionally, while oral probiotics have been accepted for CKD treatment through gut microbiota modulation, a significant challenge lies in ensuring their viability upon administration. Therefore, our study aims to address both renal fibrosis and gut microbiota imbalance through innovative co-delivery strategies. RESULTS: In this study, we developed yeast cell wall particles (YCWPs) encapsulating EMO and AA self-assembled nanoparticles (NPYs) and embedded them, along with Lactobacillus casei Zhang, in chitosan/sodium alginate (CS/SA) microgels. The developed microgels showed significant controlled release properties for the loaded NPYs and prolonged the retention time of Lactobacillus casei Zhang (L. casei Zhang) in the intestine. Furthermore, in vivo biodistribution showed that the microgel-carried NPYs significantly accumulated in the obstructed kidneys of rats, thereby substantially increasing the accumulation of EMO and AA in the impaired kidneys. More importantly, through hitchhiking delivery based on yeast cell wall and positive modulation of gut microbiota, our microgels with this synergistic strategy of therapeutic and modulatory interactions could regulate the TGF-ß/Smad signaling pathway and thus effectively ameliorate renal fibrosis in unilateral ureteral obstruction (UUO) rats. CONCLUSION: In conclusion, our work provides a new strategy for the treatment of renal fibrosis based on hitchhiking co-delivery of nanodrugs and probiotics to achieve synergistic effects of disease treatment and targeted gut flora modulation.

Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase.

DNA base editors enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no base editor for direct thymine (T) editing currently. Here we develop two deaminase-free glycosylase-based base editors for direct T editing (gTBE) and C editing (gCBE) by fusing Cas9 nickase (nCas9) with engineered human uracil DNA glycosylase (UNG) variants. By several rounds of structure-informed rational mutagenesis on UNG in cultured human cells, we obtain gTBE and gCBE with high activity of T-to-S (i.e., T-to-C or T-to-G) and C-to-G conversions, respectively. Furthermore, we conduct parallel comparison of gTBE/gCBE with those recently developed using other protein engineering strategies, and find gTBE/gCBE show the outperformance. Thus, we provide several base editors, gTBEs and gCBEs, with corresponding engineered UNG variants, broadening the targeting scope of base editors.

Strategies to Regulate the Degradation and Clearance of Mesoporous Silica Nanoparticles: A Review.

Mesoporous silica nanoparticles (MSNs) have attracted extensive attention as drug delivery systems because of their unique meso-structural features (high specific surface area, large pore volume, and tunable pore structure), easily modified surface, high drug-loading capacity, and sustained-release profiles. However, the enduring and non-specific enrichment of MSNs in healthy tissues may lead to toxicity due to their slow degradability and hinder their clinical application. The emergence of degradable MSNs provided a solution to this problem. The understanding of strategies to regulate degradation and clearance of these MSNs for promoting clinical trials and expanding their biological applications is essential. Here, a diverse variety of degradable MSNs regarding considerations of physiochemical properties and doping strategies of degradation, the biodistribution of MSNs in vivo, internal clearance mechanism, and adjusting physical parameters of clearance are highlighted. Finally, an overview of these degradable and clearable MSNs strategies for biosafety is provided along with an outlook of the encountered challenges.

Clinical coding of long COVID in primary care 2020-2023 in a cohort of 19 million adults: an OpenSAFELY analysis.

Background: Long COVID is the patient-coined term for the persistent symptoms of COVID-19 illness for weeks, months or years following the acute infection. There is a large burden of long COVID globally from self-reported data, but the epidemiology, causes and treatments remain poorly understood. Primary care is used to help identify and treat patients with long COVID and therefore Electronic Health Records (EHRs) of past COVID-19 patients could be used to help fill these knowledge gaps. We aimed to describe the incidence and differences in demographic and clinical characteristics in recorded long COVID in primary care records in England. Methods: With the approval of NHS England we used routine clinical data from over 19 million adults in England linked to SARS-COV-2 test result, hospitalisation and vaccination data to describe trends in the recording of 16 clinical codes related to long COVID between November 2020 and January 2023. Using OpenSAFELY, we calculated rates per 100,000 person-years and plotted how these changed over time. We compared crude and adjusted (for age, sex, 9 NHS regions of England, and the dominant variant circulating) rates of recorded long COVID in patient records between different key demographic and vaccination characteristics using negative binomial models. Findings: We identified a total of 55,465 people recorded to have long COVID over the study period, which included 20,025 diagnoses codes and 35,440 codes for further assessment. The incidence of new long COVID records increased steadily over 2021, and declined over 2022. The overall rate per 100,000 person-years was 177.5 cases in women (95% CI: 175.5-179) and 100.5 in men (99.5-102). The majority of those with a long COVID record did not have a recorded positive SARS-COV-2 test 12 or more weeks before the long COVID record. Interpretation: In this descriptive study, EHR recorded long COVID was very low between 2020 and 2023, and incident records of long COVID declined over 2022. Using EHR diagnostic or referral codes unfortunately has major limitations in identifying and ascertaining true cases and timing of long COVID. Funding: This research was supported by the National Institute for Health and Care Research (NIHR) (OpenPROMPT: COV-LT2-0073).

Generation of rat forebrain tissues in mice.

Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.

Impact of vaccination on the association of COVID-19 with cardiovascular diseases: An OpenSAFELY cohort study.

Infection with SARS-CoV-2 is associated with an increased risk of arterial and venous thrombotic events, but the implications of vaccination for this increased risk are uncertain. With the approval of NHS England, we quantified associations between COVID-19 diagnosis and cardiovascular diseases in different vaccination and variant eras using linked electronic health records for ~40% of the English population. We defined a 'pre-vaccination' cohort (18,210,937 people) in the wild-type/Alpha variant eras (January 2020-June 2021), and 'vaccinated' and 'unvaccinated' cohorts (13,572,399 and 3,161,485 people respectively) in the Delta variant era (June-December 2021). We showed that the incidence of each arterial thrombotic, venous thrombotic and other cardiovascular outcomes was substantially elevated during weeks 1-4 after COVID-19, compared with before or without COVID-19, but less markedly elevated in time periods beyond week 4. Hazard ratios were higher after hospitalised than non-hospitalised COVID-19 and higher in the pre-vaccination and unvaccinated cohorts than the vaccinated cohort. COVID-19 vaccination reduces the risk of cardiovascular events after COVID-19 infection. People who had COVID-19 before or without being vaccinated are at higher risk of cardiovascular events for at least two years.

A self-assembly active nanomodulator based on berberine for photothermal immunotherapy of breast cancer via dual regulation of immune suppression.

Breast cancer (BC) remains a significant global health concern, especially affecting women, necessitating the development of effective treatment strategies. Photothermal immunotherapy has holds promise for addressing BC by eradicating tumors, preventing metastasis, and reducing recurrence rates. However, the dynamic amplification of indoleamine 2,3-dioxygenase 1 (IDO-1) and programmed cell death-ligand 1 (PD-L1) triggered by photothermal therapy (PTT) poses presents a significant barrier to immune cell infiltration, thus promoting immune evasion. To enhance overall efficiency, a hyaluronic acid (HA)-coated berberine (BBR)-indocyanine green self-assembly active nano modulator (HBI NDs) was successfully developed. This nano modulator aims to reverse immune resistance and further contribute to the synergistic anti-tumor effects. The prepared HBI NDs demonstrated a uniform spherical morphology, high drug loading, and favorable optical properties. The results based on in vitro cell experiments and tumor animal models confirmed that HBI NDs selectively accumulated in tumor tissues, downregulated PD-L1 and IDO-1 protein expression, and induced elevated cell apoptosis. Consequently, these effects result in efficient immune infiltration and positive anti-tumor outcomes. In conclusion, the HBI NDs nanodrug exhibits considerable potential as a novel agent for enhancing anticancer efficacy and promoting immune infiltration.

Enhanced C-To-T and A-To-G Base Editing in Mitochondrial DNA with Engineered DdCBE and TALED.

Mitochondrial base editing with DddA-derived cytosine base editor (DdCBE) is limited in the accessible target sequences and modest activity. Here, the optimized DdCBE tools is presented with improved editing activity and expanded C-to-T targeting scope by fusing DddA11 variant with different cytosine deaminases with single-strand DNA activity. Compared to previous DdCBE based on DddA11 variant alone, fusion of the activation-induced cytidine deaminase (AID) from Xenopus laevis not only permits cytosine editing of 5'-GC-3' sequence, but also elevates editing efficiency at 5'-TC-3', 5'-CC-3', and 5'-GC-3' targets by up to 25-, 10-, and 6-fold, respectively. Furthermore, the A-to-G editing efficiency is significantly improved by fusing the evolved DddA6 variant with TALE-linked deoxyadenosine deaminase (TALED). Notably, the authors introduce the reported high-fidelity mutations in DddA and add nuclear export signal (NES) sequences in DdCBE and TALED to reduce off-target editing in the nuclear and mitochondrial genome while improving on-target editing efficiency in mitochondrial DNA (mtDNA). Finally, these engineered mitochondrial base editors are shown to be efficient in installing mtDNA mutations in human cells or mouse embryos for disease modeling. Collectively, the study shows broad implications for the basic study and therapeutic applications of optimized DdCBE and TALED.

Physicochemical Index Analyses of the Egg White in Blue-Shelled Eggs and Commercial Brown-Shelled Eggs during Storage.

To compare the physical and chemical changes in egg whites during storage, assisting in the evaluation of differences in egg freshness between various chicken breeds, we chose 240 blue-shelled eggs (Blue group) and 240 commercial brown-shelled eggs (Brown group) that 28-week-old hens had laid. In this study, all eggs were stored at 25 °C. The egg weight, egg components' weight and proportion, Haugh Unit value and the contents of S-ovalbumin, ovomucin and lysozyme in the thick albumen (KA) and thin albumen (NA) were measured at eight time points every 3 days until the 21st day of storage. The eggshell, yolk and KA proportions in the Brown group were significantly lower, whereas the NA proportion was significantly higher than that in the Blue group (p < 0.001). The Haugh Unit value and S-ovalbumin in the Brown group were significantly higher, whereas KA ovomucin and NA lysozyme were significantly lower than those in the Blue group (p < 0.001). There existed significant negative correlations between the KA and NA, irrespective of weight or proportion. The Haugh Unit value was significantly positively correlated with lysozyme and ovomucin, but significantly negatively correlated with S-ovalbumin. During storage, the KA weight (proportion), Haugh Unit value, lysozyme and ovomucin decreased, whereas the NA weight (proportion) and S-ovalbumin increased. At each time point, the NA lysozyme in the Brown group was lower than that in the Blue group (p < 0.05). After storage for 6 days, the KA ovomucin in the Brown group began to be lower than that in the Blue group (p < 0.05). The study showed that the weight (proportion) differences in egg components between blue-shelled eggs and commercial brown-shelled eggs are mainly due to the NA. The Haugh Unit value and albumin protein indexes of blue-shelled eggs were better than those of brown-shelled eggs, and showed mild changes during storage, indicating the better storage performance of blue-shelled eggs.

An external validation of the Kidney Donor Risk Index in the UK transplant population in the presence of semi-competing events.

BACKGROUND: Transplantation represents the optimal treatment for many patients with end-stage kidney disease. When a donor kidney is available to a waitlisted patient, clinicians responsible for the care of the potential recipient must make the decision to accept or decline the offer based upon complex and variable information about the donor, the recipient and the transplant process. A clinical prediction model may be able to support clinicians in their decision-making. The Kidney Donor Risk Index (KDRI) was developed in the United States to predict graft failure following kidney transplantation. The survival process following transplantation consists of semi-competing events where death precludes graft failure, but not vice-versa. METHODS: We externally validated the KDRI in the UK kidney transplant population and assessed whether validation under a semi-competing risks framework impacted predictive performance. Additionally, we explored whether the KDRI requires updating. We included 20,035 adult recipients of first, deceased donor, single, kidney-only transplants between January 1, 2004, and December 31, 2018, collected by the UK Transplant Registry and held by NHS Blood and Transplant. The outcomes of interest were 1- and 5-year graft failure following transplantation. In light of the semi-competing events, recipient death was handled in two ways: censoring patients at the time of death and modelling death as a competing event. Cox proportional hazard models were used to validate the KDRI when censoring graft failure by death, and cause-specific Cox models were used to account for death as a competing event. RESULTS: The KDRI underestimated event probabilities for those at higher risk of graft failure. For 5-year graft failure, discrimination was poorer in the semi-competing risks model (0.625, 95% CI 0.611 to 0.640;0.611, 95% CI 0.597 to 0.625), but predictions were more accurate (Brier score 0.117, 95% CI 0.112 to 0.121; 0.114, 95% CI 0.109 to 0.118). Calibration plots were similar regardless of whether the death was modelled as a competing event or not. Updating the KDRI worsened calibration, but marginally improved discrimination. CONCLUSIONS: Predictive performance for 1-year graft failure was similar between death-censored and competing event graft failure, but differences appeared when predicting 5-year graft failure. The updated index did not have superior performance and we conclude that updating the KDRI in the present form is not required.

Bivariate copula regression models for semi-competing risks.

Time-to-event semi-competing risk endpoints may be correlated when both events occur on the same individual. These events and the association between them may also be influenced by individual characteristics. In this article, we propose copula survival models to estimate hazard ratios of covariates on the non-terminal and terminal events, along with the effects of covariates on the association between the two events. We use the Normal, Clayton, Frank and Gumbel copulas to provide a variety of association structures between the non-terminal and terminal events. We apply the proposed methods to model semi-competing risks of graft failure and death for kidney transplant patients. We find that copula survival models perform better than the Cox proportional hazards model when estimating the non-terminal event hazard ratio of covariates. We also find that the inclusion of covariates in the association parameter of the copula models improves the estimation of the hazard ratios.

Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase.

Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 fold. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e. G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate.

Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor.

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.

Engineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and expanded target range for genome editing.

The type V-F CRISPR-Cas12f system is a strong candidate for therapeutic applications due to the compact size of the Cas12f proteins. In this work, we identify six uncharacterized Cas12f1 proteins with nuclease activity in mammalian cells from assembled bacterial genomes. Among them, OsCas12f1 (433 aa) from Oscillibacter sp. and RhCas12f1 (415 aa) from Ruminiclostridium herbifermentans, which respectively target 5' T-rich Protospacer Adjacent Motifs (PAMs) and 5' C-rich PAMs, show the highest editing activity. Through protein and sgRNA engineering, we generate enhanced OsCas12f1 (enOsCas12f1) and enRhCas12f1 variants, with 5'-TTN and 5'-CCD (D = not C) PAMs respectively, exhibiting much higher editing efficiency and broader PAMs, compared with the engineered variant Un1Cas12f1 (Un1Cas12f1_ge4.1). Furthermore, by fusing the destabilized domain with enOsCas12f1, we generate inducible-enOsCas12f1 and demonstate its activity in vivo by single adeno-associated virus delivery. Finally, dead enOsCas12f1-based epigenetic editing and gene activation can also be achieved in mammalian cells. This study thus provides compact gene editing tools for basic research with remarkable promise for therapeutic applications.

Dexmedetomidine alleviates oxidative stress and mitochondrial dysfunction in diabetic peripheral neuropathy via the microRNA-34a/SIRT2/S1PR1 axis.

OBJECTIVE: Dexmedetomidine (Dex) is a highly selective α2-adrenoceptor agonist with sedative, analgesic, sympatholytic, and hemodynamic-stabilizing properties, which plays a neuroprotective role in diabetic peripheral neuropathy (DPN) and diabetes-induced nerve damage. However, the related molecular mechanisms are not fully understood. Therefore, our study explored the mechanism of Dex in DPN using rat and RSC96 cell models. METHODS: Sciatic nerve sections were observed under an optical microscope and the ultrastructure of the sciatic nerves was observed under a transmission electron microscope. Oxidative stress was assessed by detecting MDA, SOD, GSH-Px, and ROS levels. The motor nerve conduction velocity (MNCV), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL) of rats were measured. Cell viability, apoptosis, and the changes in the expression of related genes and proteins were examined. Furthermore, the relationship between microRNA (miR)-34a and SIRT2 or SIRT2 and S1PR1 was analyzed. RESULTS: Dex reversed DPN-induced decreases in MNCV, MWT, and TWL. Dex alleviated oxidative stress, mitochondrial damage, and apoptosis in both the rat and RSC96 cell models of DPN. Mechanistically, miR-34a negatively targeted SIRT2, and SIRT2 inhibited S1PR1 transcription. The overexpression of miR-34a or S1PR1 or the inhibition of SIRT2 counteracted the neuroprotective effects of Dex in DPN in vivo and in vitro. CONCLUSION: Dex alleviates oxidative stress and mitochondrial dysfunction associated with DPN by downregulating miR-34a to regulate the SIRT2/S1PR1 axis.

The pigments in eggshell with different colour and the pigment regulatory gene expression in corresponding chicken's shell gland.

Eggshell colour is the unique appearance and economically valuable trait of eggs, whereas the colour is often short of uniformity, especially in the blue-shelled breeds, hence, their pigment differences and molecular mechanism need clarity. To investigate the relationship between the pigment content of eggshells and related gene expression in the eggshell glands of chickens, four subtypes of blue-shelled eggs ('Olive', 'Green', 'Blue', and 'Light') from the same blue-eggshell chicken line were selected; Hy-Line 'White' and 'Brown'-shelled eggs were used as control groups. The L*, a*, b* values, and protoporphyrin-IX and biliverdin contents in each group of eggshells were measured. In addition, the shell glands of the corresponding hens were collected to detect SLCO1B3 genotype and mRNA expression, and ABCG2 and HMOX1 transcription and protein expression. Eggshell colour L* values were negatively correlated with protoporphyrin-IX, b* values were positively correlated with total pigment content (P < 0.001), and a* values were positively correlated with protoporphyrin-IX (P < 0.001) but negatively with biliverdin. Moreover, all four blue-eggshell subtypes were SLCO1B3 homozygous, with SLCO1B3 mRNA expression in shell glands being significantly higher than in the White and Brown groups. ABCG2 and HMOX1 mRNA expression were highest in the Brown and Green groups, respectively (P < 0.05), and were positively correlated with protoporphyrin-IX (P < 0.001) and biliverdin contents in eggshells, respectively. Western blot and immunohistochemical results demonstrated that the Brown group had the highest ABCG2 expression (P < 0.05), followed by the Green and Olive groups. HMOX1 protein expression was higher in the Olive and Green groups (P < 0.05), and lowest in the White group. This study suggests that ABCG2 and HMOX1 have important regulatory roles in the production and transport of protoporphyrin-IX and biliverdin in blue-shelled chicken eggs, respectively.