Recessively Inherited Deficiency of Secreted WFDC2 (HE4) Causes Nasal Polyposis and Bronchiectasis.

Rationale: Bronchiectasis is a pathological dilatation of the bronchi in the respiratory airways associated with environmental or genetic causes (e.g., cystic fibrosis, primary ciliary dyskinesia, and primary immunodeficiency disorders), but most cases remain idiopathic. Objectives: To identify novel genetic defects in unsolved cases of bronchiectasis presenting with severe rhinosinusitis, nasal polyposis, and pulmonary Pseudomonas aeruginosa infection. Methods: DNA was analyzed by next-generation or targeted Sanger sequencing. RNA was analyzed by quantitative PCR and single-cell RNA sequencing. Patient-derived cells, cell cultures, and secretions (mucus, saliva, seminal fluid) were analyzed by Western blotting and immunofluorescence microscopy, and mucociliary activity was measured. Blood serum was analyzed by electrochemiluminescence immunoassay. Protein structure and proteomic analyses were used to assess the impact of a disease-causing founder variant. Measurements and Main Results: We identified biallelic pathogenic variants in WAP four-disulfide core domain 2 (WFDC2) in 11 individuals from 10 unrelated families originating from the United States, Europe, Asia, and Africa. Expression of WFDC2 was detected predominantly in secretory cells of control airway epithelium and also in submucosal glands. We demonstrate that WFDC2 is below the limit of detection in blood serum and hardly detectable in samples of saliva, seminal fluid, and airway surface liquid from WFDC2-deficient individuals. Computer simulations and deglycosylation assays indicate that the disease-causing founder variant p.Cys49Arg structurally hampers glycosylation and, thus, secretion of mature WFDC2. Conclusions: WFDC2 dysfunction defines a novel molecular etiology of bronchiectasis characterized by the deficiency of a secreted component of the airways. A commercially available blood test combined with genetic testing allows its diagnosis.

Impact of ERAS on Hysteromyomectomy: A Study on Anxiety, Depression, and Ovarian Function.

Objective: To analyze the application of the Enhanced Recovery After Surgery (ERAS) nursing mode in patients undergoing radical cystectomy with urinary diversion. Methods: A retrospective analysis was conducted on clinical data of 72 patients with bladder cancer who underwent "robot-assisted laparoscopic radical cystectomy + urinary diversion" in Nanjing University Medical College Affiliated Gulou Hospital between January 2021 and January 2023. All patients met the complete inclusion criteria. They were divided into a control group (n=35) and a observation group (n=37). Patients in the control group received routine rehabilitation nursing intervention, while patients in the study group received ERAS nursing mode intervention. The outcomes include time to first intake, time to first defecation, duration of enteral nutrition, duration of antibiotic use, duration of drainage tube placement, length of hospital stay, psychological status Self-rating Depression Scale (SDS), Self-rating Anxiety Scale (SAS), quality of life (SF-36) scores, sexual function assessment Arizona Sexual Experience Scale (ASEX), International Index of Erectile Function-5 (IIEF-5), and occurrence of complications were compared between the two groups. Results: In the observation group, perioperative indicators, psychological status, quality of life, sexual function, and complication rates were notably improved compared to the control group (all P < .05). Conclusion: ERAS nursing mode intervention in bladder cancer patients exhibited significant effectiveness, enhancing postoperative recovery, reducing anxiety and depression, improving quality of life and sexual function, and lowering complication risks. These findings support the clinical merit and applicability of ERAS nursing in urinary diversion for bladder cancer patients.

Parkin Regulates Mitosis and Genomic Stability through Cdc20/Cdh1.

Mutations in the E3 ubiquitin ligase Parkin have been linked to familial Parkinson's disease. Parkin has also been implicated in mitosis through mechanisms that are unclear. Here we show that Parkin interacts with anaphase promoting complex/cyclosome (APC/C) coactivators Cdc20 and Cdh1 to mediate the degradation of several key mitotic regulators independent of APC/C. We demonstrate that ordered progression through mitosis is orchestrated by two distinct E3 ligases through the shared use of Cdc20 and Cdh1. Furthermore, Parkin is phosphorylated and activated by polo-like kinase 1 (Plk1) during mitosis. Parkin deficiency results in overexpression of its substrates, mitotic defects, genomic instability, and tumorigenesis. These results suggest that the Parkin-Cdc20/Cdh1 complex is an important regulator of mitosis.

Three-dimensional bioprinted hepatorganoids prolong survival of mice with liver failure.

OBJECTIVE: Shortage of organ donors, a critical challenge for treatment of end-stage organ failure, has motivated the development of alternative strategies to generate organs in vitro. Here, we aim to describe the hepatorganoids, which is a liver tissue model generated by three-dimensional (3D) bioprinting of HepaRG cells and investigate its liver functions in vitro and in vivo. DESIGN: 3D bioprinted hepatorganoids (3DP-HOs) were constructed using HepaRG cells and bioink, according to specific 3D printing procedures. Liver functions of 3DP-HOs were detected after 7 days of differentiation in vitro, which were later transplanted into Fah-deficient mice. The in vivo liver functions of 3DP-HOs were evaluated by survival time and liver damage of mice, human liver function markers and human-specific debrisoquine metabolite production. RESULTS: 3DP-HOs broadly acquired liver functions, such as ALBUMIN secretion, drug metabolism and glycogen storage after 7 days of differentiation. After transplantation into abdominal cavity of Fah-/-Rag2-/- mouse model of liver injury, 3DP-HOs further matured and displayed increased synthesis of liver-specific proteins. Particularly, the mice acquired human-specific drug metabolism activities. Functional vascular systems were also formed in transplanted 3DP-HOs, further enhancing the material transport and liver functions of 3DP-HOs. Most importantly, transplantation of 3DP-HOs significantly improved the survival of mice. CONCLUSIONS: Our results demonstrated a comprehensive proof of principle, which indicated that 3DP-HO model of liver tissues possessed in vivo hepatic functions and alleviated liver failure after transplantation, suggesting that 3D bioprinting could be used to generate human liver tissues as the alternative transplantation donors for treatment of liver diseases.

Ferric ion crosslinking-based 3D printing of a graphene oxide hydrogel and its evaluation as a bio-scaffold in tissue engineering.

As a precursor of graphene, graphene oxide (GO) exhibits excellent mechanical, thermal, and electrical properties, besides appreciable biocompatibility in tissue engineering applications. However, the current GO-3D fabrication technology is still in need of optimization and simplification to ensure fine architecture and reasonable mechanical properties, which would further promote the performance of GO as bio-scaffolds in cell or microorganism attachment and in material transformation. To address this issue, we proposed a GO ink, with appreciable rheological properties and excellent printing performance via high-speed centrifugation and ferric ion-assisted cross-linking. A woodpile structure with controllable micro-pores was produced by micro-extrusion-based 3D printing technology followed by an optimized freeze-drying process. Cellular adhesion and viability were verified by inoculation and culture of HepaRG cells using the fabricated GO 3D structure, thus suggesting ferric ion-assisted cross-linking and controllable pore distribution for improving the performance of the GO construct as a bio-scaffold for in vitro liver tissue models.

[Effect of occupational stress on hypertension in desert oilfield workers].

OBJECTIVE: To explore influence of occupational stress on hypertension in Xinjiang Uygur Autonomous Region desert oilfield workers. METHODS: Cluster sampling was applied. A total of 1280 petroleum workers from 3 oil fields were used in Karamay City, Xinjiang. Occupational Stress Scale(OSI-R) was used to evaluate occupational stress and analyze the impact of occupational stress on hypertension. RESULTS: With the increase of occupational stress, the prevalence rate of hypertension is increasing(χ~2=21. 078, P<0. 001). Multivariate analysis showed that the risk of high blood pressure in the occupational task was 1. 562 times(95%CI 1. 072-2. 277)as high as that of the less occupational group, and the risk of high blood pressure in the group with strong individual tension reaction was 1. 701 times(95%CI 1. 158-2. 498)as much as that of the weak group(P<0. 05). Analysis of influencing factors of hypertension showed that the risk of high blood pressure in the shift was 1. 389 times(95%CI 1. 115-1. 730)as high as those without the shift, in the frequent drinkers was 1. 877 times(95%CI 1. 300-2. 710)that of the non drinkers, in the high salt patients was 1. 286 times(95%CI 1. 107-1. 691)that of the low salt, in the obese was 1. 564 times(95%CI 1. 249-2. 216)that of the normal people, and in the highly occupational stress was 1. 976 times(95%CI 1. 641-2. 336)as high as the low occupational stress. CONCLUSION: Heavy occupational tasks and strong individual strain can increase the risk of hypertension in desert oilfield workers. Shift, drinking history, salt consumption, BMI and occupational stress were the influencing factors of hypertension in desert oilfield workers.

Influenza A(H7N9) Virus Antibody Responses in Survivors 1 Year after Infection, China, 2017.

Avian influenza A(H7N9) virus has caused 5 epidemic waves in China since its emergence in 2013. We investigated the dynamic changes of antibody response to this virus over 1 year postinfection in 25 patients in Suzhou City, Jiangsu Province, China, who had laboratory-confirmed infections during the fifth epidemic wave, October 1, 2016-February 14, 2017. Most survivors had relatively robust antibody responses that decreased but remained detectable at 1 year. Antibody response was variable; several survivors had low or undetectable antibody titers. Hemagglutination inhibition titer was >1:40 for <40% of the survivors. Measured in vitro in infected mice, hemagglutination inhibition titer predicted serum protective ability. Our findings provide a helpful serologic guideline for identifying subclinical infections and for developing effective vaccines and therapeutics to counter H7N9 virus infections.

Plasma butyrylcholinesterase regulates ghrelin to control aggression.

Ongoing mouse studies of a proposed therapy for cocaine abuse based on viral gene transfer of butyrylcholinesterase (BChE) mutated for accelerated cocaine hydrolysis have yielded surprising effects on aggression. Further investigation has linked these effects to a reduction in circulating ghrelin, driven by BChE at levels ∼ 100-fold above normal. Tests with human BChE showed ready ghrelin hydrolysis at physiologic concentrations, and multiple low-mass molecular dynamics simulations revealed that ghrelin's first five residues fit sterically and electrostatically into BChE's active site. Consistent with in vitro results, male BALB/c mice with high plasma BChE after gene transfer exhibited sharply reduced plasma ghrelin. Unexpectedly, such animals fought less, both spontaneously and in a resident/intruder provocation model. One mutant BChE was found to be deficient in ghrelin hydrolysis. BALB/c mice transduced with this variant retained normal plasma ghrelin levels and did not differ from untreated controls in the aggression model. In contrast, C57BL/6 mice with BChE gene deletion exhibited increased ghrelin and fought more readily than wild-type animals. Collectively, these findings indicate that BChE-catalyzed ghrelin hydrolysis influences mouse aggression and social stress, with potential implications for humans.

How fast fast-folding proteins fold in silico.

In reported microcanonical molecular dynamics simulations, fast-folding proteins CLN025 and Trp-cage autonomously folded to experimentally determined native conformations. However, the folding times of these proteins derived from the simulations were more than 4-10 times longer than their experimental values. This article reports autonomous folding of CLN025 and Trp-cage in isobaric-isothermal molecular dynamics simulations with agreements within factors of 0.69-1.75 between simulated and experimental folding times at different temperatures. These results show that CLN025 and Trp-cage can now autonomously fold in silico as fast as in experiments, and suggest that the accuracy of folding simulations for fast-folding proteins begins to overlap with the accuracy of folding experiments. This opens new prospects of developing computer algorithms that can predict both ensembles of conformations and their interconversion rates for a protein from its sequence for artificial intelligence on how and when a protein acts as a receiver, switch, and relay to facilitate various subcellular-to-tissue communications. Then the genetic information that encodes proteins can be better read in the context of intricate biological functions.

On the use of the experimentally determined enzyme inhibition constant as a measure of absolute binding affinity.

Defined as a state function representing an inhibitor's absolute affinity for its target enzyme, the experimentally determined enzyme inhibition constant (Ki) is widely used to rank order binding affinities of different inhibitors for a common enzyme or different enzymes for a common inhibitor and to benchmark computational approaches to predicting binding affinity. Herein, we report that adsorption of bis(7)-tacrine to the glass container surface increased its Ki against Electrophorus electricus acetylcholinesterase (eeAChE) to 3.2 ± 0.1 nM (n = 5) compared to 2.9 ± 0.4 pM (n = 5) that was determined using plastic containers with other assay conditions kept the same. We also report that, due to binding or "adsorption" of bis(7)-tacrine to the inactive eeAChE, the bis(7)-tacrine Ki increased from 2.9 ± 0.4 pM (n = 5) to 734 ± 70 pM (n = 5) as the specific eeAChE activity decreased from 342 U/mg to 26 U/mg while other assay conditions were kept the same. These results caution against using Kis to rank order binding potencies, define selectivity, or benchmark computational methods without knowing detailed assay conditions.

Prime, Shock, and Kill: Priming CD4 T Cells from HIV Patients with a BCL-2 Antagonist before HIV Reactivation Reduces HIV Reservoir Size.

UNLABELLED: Understanding how some HIV-infected cells resist the cytotoxicity of HIV replication is crucial to enabling HIV cure efforts. HIV killing of CD4 T cells that replicate HIV can involve HIV protease-mediated cleavage of procaspase 8 to generate a fragment (Casp8p41) that directly binds and activates the mitochondrial proapoptotic protein BAK. Here, we demonstrate that Casp8p41 also binds with nanomolar affinity to the antiapoptotic protein Bcl-2, which sequesters Casp8p41 and prevents apoptosis. Further, we show that central memory CD4 T cells (TCM) from HIV-infected individuals have heightened expression of BCL-2 relative to procaspase 8, possibly explaining the persistence of HIV-infected TCMdespite generation of Casp8p41. Consistent with this hypothesis, the selective BCL-2 antagonist venetoclax induced minimal killing of uninfected CD4 T cells but markedly increased the death of CD4 T cells and diminished cell-associated HIV DNA when CD4 T cells from antiretroviral therapy (ART)-suppressed HIV patients were induced with αCD3/αCD28 to reactivate HIVex vivo Thus, priming CD4 T cells from ART suppressed HIV patients with a BCL-2 antagonist, followed by HIV reactivation, achieves reductions in cell-associated HIV DNA, whereas HIV reactivation alone does not. IMPORTANCE: HIV infection is incurable due to a long-lived reservoir of HIV(+)memory CD4 T cells, and no clinically relevant interventions have been identified that reduce the number of these HIV DNA-containing cells. Since postintegration HIV replication can result in HIV protease generation of Casp8p41, which activates BAK, causing infected CD4 T cell death, we sought to determine whether this occurs in memory CD4 T cells. Here, we demonstrate that memory CD4 T cells can generate Casp8p41 and yet are intrinsically resistant to death induced by diverse stimuli, including Casp8p41. Furthermore, BCL-2 expression is relatively increased in these cells and directly binds and inhibits Casp8p41's proapoptotic effects. Antagonizing BCL-2 with venetoclax derepresses this antagonism, resulting in death, preferentially in HIV DNA containing cells, since only these cells generate Casp8p41. Thus, BCL-2 antagonism is a clinically relevant intervention with the potential to reduce HIV reservoir size in patients.

FF12MC: A revised AMBER forcefield and new protein simulation protocol.

Specialized to simulate proteins in molecular dynamics (MD) simulations with explicit solvation, FF12MC is a combination of a new protein simulation protocol employing uniformly reduced atomic masses by tenfold and a revised AMBER forcefield FF99 with (i) shortened CH bonds, (ii) removal of torsions involving a nonperipheral sp(3) atom, and (iii) reduced 1-4 interaction scaling factors of torsions ϕ and ψ. This article reports that in multiple, distinct, independent, unrestricted, unbiased, isobaric-isothermal, and classical MD simulations FF12MC can (i) simulate the experimentally observed flipping between left- and right-handed configurations for C14-C38 of BPTI in solution, (ii) autonomously fold chignolin, CLN025, and Trp-cage with folding times that agree with the experimental values, (iii) simulate subsequent unfolding and refolding of these miniproteins, and (iv) achieve a robust Z score of 1.33 for refining protein models TMR01, TMR04, and TMR07. By comparison, the latest general-purpose AMBER forcefield FF14SB locks the C14-C38 bond to the right-handed configuration in solution under the same protein simulation conditions. Statistical survival analysis shows that FF12MC folds chignolin and CLN025 in isobaric-isothermal MD simulations 2-4 times faster than FF14SB under the same protein simulation conditions. These results suggest that FF12MC may be used for protein simulations to study kinetics and thermodynamics of miniprotein folding as well as protein structure and dynamics. Proteins 2016; 84:1490-1516. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Evaluation of the BH3-only protein Puma as a direct Bak activator.

Interactions among Bcl-2 family proteins play critical roles in cellular life and death decisions. Previous studies have established the BH3-only proteins Bim, tBid, and Noxa as "direct activators" that are able to directly initiate the oligomerization and activation of Bak and/or Bax. Earlier studies of Puma have yielded equivocal results, with some concluding that it also acts as a direct activator and other studies suggesting that it acts solely as a sensitizer BH3-only protein. In the present study we examined the interaction of Puma BH3 domain or full-length protein with Bak by surface plasmon resonance, assessed Bak oligomerization status by cross-linking followed by immunoblotting, evaluated the ability of the Puma BH3 domain to induce Bak-mediated permeabilization of liposomes and mitochondria, and determined the effect of wild type and mutant Puma on cell viability in a variety of cellular contexts. Results of this analysis demonstrate high affinity (KD = 26 ± 5 nM) binding of the Puma BH3 domain to purified Bak ex vivo, leading to Bak homo-oligomerization and membrane permeabilization. Mutations in Puma that inhibit (L141E/M144E/L148E) or enhance (M144I/A145G) Puma BH3 binding to Bak also produce corresponding alterations in Bak oligomerization, Bak-mediated membrane permeabilization and, in a cellular context, Bak-mediated killing. Collectively, these results provide strong evidence that Puma, like Bim, Noxa, and tBid, is able to act as a direct Bak activator.

At least 10% shorter C-H bonds in cryogenic protein crystal structures than in current AMBER forcefields.

High resolution protein crystal structures resolved with X-ray diffraction data at cryogenic temperature are commonly used as experimental data to refine forcefields and evaluate protein folding simulations. However, it has been unclear hitherto whether the C-H bond lengths in cryogenic protein structures are significantly different from those defined in forcefields to affect protein folding simulations. This article reports the finding that the C-H bonds in high resolution cryogenic protein structures are 10-14% shorter than those defined in current AMBER forcefields, according to 3709 C-H bonds in the cryogenic protein structures with resolutions of 0.62-0.79 Å. Also, 20 all-atom, isothermal-isobaric, 0.5-µs molecular dynamics simulations showed that chignolin folded from a fully-extended backbone formation to the native ß-hairpin conformation in the simulations using AMBER forcefield FF12SB at 300 K with an aggregated native state population including standard error of 10 ± 4%. However, the aggregated native state population with standard error reduced to 3 ± 2% in the same simulations except that C-H bonds were shortened by 10-14%. Furthermore, the aggregated native state populations with standard errors increased to 35 ± 3% and 26 ± 3% when using FF12MC, which is based on AMBER forcefield FF99, with and without the shortened C-H bonds, respectively. These results show that the 10-14% bond length differences can significantly affect protein folding simulations and suggest that re-parameterization of C-H bonds according to the cryogenic structures could improve the ability of a forcefield to fold proteins in molecular dynamics simulations.

Use of 1-4 interaction scaling factors to control the conformational equilibrium between α-helix and ß-strand.

1-4 interaction scaling factors are used in AMBER forcefields to reduce the exaggeration of short-range repulsion caused by the 6-12 Lennard-Jones potential and a nonpolarizable charge model and to obtain better agreements of small-molecule conformational energies with experimental data. However, the effects of these scaling factors on protein secondary structure conformations have not been investigated until now. This article reports the finding that the 1-4 interactions among the protein backbone atoms separated by three consecutive covalent bonds are more repulsive in the α-helix conformation than in two ß-strand conformations. Therefore, the 1-4 interaction scaling factors of protein backbone torsions ϕ and ψ control the conformational equilibrium between α-helix and ß-strand. Molecular dynamics simulations confirm that reducing the ϕ and ψ scaling factors readily converts the α-helix conformation of AcO-(AAQAA)3-NH2 to a ß-strand conformation, and the reverse occurs when these scaling factors are increased. These results suggest that the ϕ and ψ scaling factors can be used to generate the α-helix or ß-strand conformation in situ and to control the propensities of a forcefield for adopting secondary structure elements.

Regulation of error-prone translesion synthesis by Spartan/C1orf124.

Translesion synthesis (TLS) employs low fidelity polymerases to replicate past damaged DNA in a potentially error-prone process. Regulatory mechanisms that prevent TLS-associated mutagenesis are unknown; however, our recent studies suggest that the PCNA-binding protein Spartan plays a role in suppression of damage-induced mutagenesis. Here, we show that Spartan negatively regulates error-prone TLS that is dependent on POLD3, the accessory subunit of the replicative DNA polymerase Pol δ. We demonstrate that the putative zinc metalloprotease domain SprT in Spartan directly interacts with POLD3 and contributes to suppression of damage-induced mutagenesis. Depletion of Spartan induces complex formation of POLD3 with Rev1 and the error-prone TLS polymerase Pol ζ, and elevates mutagenesis that relies on POLD3, Rev1 and Pol ζ. These results suggest that Spartan negatively regulates POLD3 function in Rev1/Pol ζ-dependent TLS, revealing a previously unrecognized regulatory step in error-prone TLS.

Modified method of recording and reproducing natural head position with a multicamera system and a laser level.

INTRODUCTION: As computer-assisted surgical design becomes increasingly popular in maxillofacial surgery, recording patients' natural head position (NHP) and reproducing it in the virtual environment are vital for preoperative design and postoperative evaluation. Our objective was to test the repeatability and accuracy of recording NHP using a multicamera system and a laser level. METHODS: A laser level was used to project a horizontal reference line on a physical model, and a 3-dimensional image was obtained using a multicamera system. In surgical simulation software, the recorded NHP was reproduced in the virtual head position by registering the coordinate axes with the horizontal reference on both the frontal and lateral views. The repeatability and accuracy of the method were assessed using a gyroscopic procedure as the gold standard. RESULTS: The interclass correlation coefficients for pitch and roll were 0.982 (0.966, 0.991) and 0.995 (0.992, 0.998), respectively, indicating a high degree of repeatability. Regarding accuracy, the lack of agreement in orientation between the new method and the gold standard was within the ranges for pitch (-0.69°, 1.71°) and for roll (-0.92°, 1.20°); these have no clinical significance. CONCLUSIONS: This method of recording and reproducing NHP with a multicamera system and a laser level is repeatable, accurate, and clinically feasible.

Low-mass molecular dynamics simulation: a simple and generic technique to enhance configurational sampling.

CLN025 is one of the smallest fast-folding proteins. Until now it has not been reported that CLN025 can autonomously fold to its native conformation in a classical, all-atom, and isothermal-isobaric molecular dynamics (MD) simulation. This article reports the autonomous and repeated folding of CLN025 from a fully extended backbone conformation to its native conformation in explicit solvent in multiple 500-ns MD simulations at 277K and 1atm with the first folding event occurring as early as 66.1ns. These simulations were accomplished by using AMBER forcefield derivatives with atomic masses reduced by 10-fold on Apple Mac Pros. By contrast, no folding event was observed when the simulations were repeated using the original AMBER forcefields of FF12SB and FF14SB. The results demonstrate that low-mass MD simulation is a simple and generic technique to enhance configurational sampling. This technique may propel autonomous folding of a wide range of miniature proteins in classical, all-atom, and isothermal-isobaric MD simulations performed on commodity computers-an important step forward in quantitative biology.

[Comparison of genetic damage in mice exposed to black carbon and ozone-oxidized black carbon].

OBJECTIVE: To make an assessment on the genotoxicity caused by black carbon (BC) and ozonized black carbon (O3-BC). METHODS: In this study, 74 healthy male ICR mice [weighed (28 ± 1.5) g] were randomly divided into 7 groups, including one phosphate buffer solution (PBS) control group and six particles exposed groups by intratracheal instillation with either BC or O3-BC at the doses of 50, 100, 200 µg/mouse, respectively. There were 12 mice in the groups of 200 µg/mouse and 10 mice in others. The mice were sacrificed 24 h after four intratrachealinstillations. The activities of catalase (CAT) in serum and the levels of malondialdehyde (MDA) in lung tissue homogenate were measured. As the DNA damage mark, 8-hydroxyguanosine (8-OHdG) in urine and serum were quantified with ELISA method. Micronucleus test was used for potential genotoxicity of BC and O3-BC. Hematoxylin and eosin staining was used to stain lung paraffin section. RESULTS: The mice were in good condition during instillation, and the liver coefficient of the test groups was significantly lower than that of the control group (P<0.05). The activities of CAT in serum significantly increased in the 100 µg/mouse and 200 µg/mouse groups after being exposed to these two kinds of particles. The micronucleus rate in allthe BC and O3-BC exposed groups increased (P<0.05), but there was no statistically significant difference among the groups in the levels of 8-OHdG in serum and urine and MDA in lung tissue homogenate. Inflammatory response was found in the lung tissue under the microscope after exposure to BC and O3-BC. CONCLUSION: Intratracheal instillation of BC and O3-BC induced increasing of oxidative stress and genetic damage in mice. But there was no significant difference between these two particles in toxicity. Whether the genotoxicity of O3-BC is higher than that of BC or not is uncertain. Further research is needed.

3D Printing Cervical Implant Scaffolds Incorporated with Drug-Loaded Carboxylated Chitosan Microspheres for Long-Term Anti-HPV Protein Delivery.

As attempting personalized medicine, 3D-printed tissue engineering scaffolds are employed to combine with therapeutic proteins/peptides especially in the clinical treatment of infectious diseases, genetic diseases, and cancers. However, current drug-loading methods, such as immersion and encapsulation, usually lead to the burst release of the drugs. To address these issues, we proposed an integrated strategy toward the long-term controlled release of protein. In this study, patient-customized 3D scaffolds incorporated with drug-loaded microspheres were printed to realize the effective delivery of the anti-human papillomavirus (anti-HPV) protein after cervical conization in the treatment of cervical cancer. The 3D-printed scaffold could provide mechanical support to the defect site and ensure local release of the drug to avoid systemic administration. Meanwhile, microspheres serve as functional components to prevent the inactivation of proteins, as well as regulate their release period to meet the time requirement of different treatment courses. The research also utilized bovine serum albumin as a model protein to validate the feasibility of these scaffolds as a generic technology platform. The bioactivity of the released anti-HPV protein was validated using a pseudovirus model, which demonstrated that the microsphere encapsulation would not cause protein denaturation during the scaffold fabrication process. Besides, 3D-printed scaffolds incorporated with carboxylated chitosan microspheres were biocompatible and beneficial for cell attachment, which have been demonstrated by favorable cell viability and better coverage results for HeLa and HFF-1. This study highlights the great potential of scaffolds incorporated with microspheres to serve as tissue engineering candidate products with the function of effective protein delivery in a long-term controlled manner and personalized shapes for clinical trials.