ObjectiveTo construct a stable monoclonal human embryonic kidney 293 （HEK293） cell line expressing recombinant human coagulation factor Ⅶ （rhFⅦ） and evaluate the expression level and procoagulant bioactivity of rhFⅦ. MethodsThe plasmid pCDNA3.1-EGFP-FⅦ was transfected into HEK293 cells to verify the effectiveness of the transfection system. The plasmid pCDNA3.1-FⅦ was transfected into HEK293 cells， and monoclonal stable transfected cell lines were selected using geneticin （G418）. The transcription of the FⅦ gene was identified by reverse transcription polymerase chain reaction （RT-PCR）. The expression level of rhFⅦ in the supernatant of the monoclonal stable transfected cell line was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. The concentration of rhFⅦ was determined by enzyme-linked immunosorbent assay （ELISA）， and the procoagulant activity of rhFⅦ was measured by human coagulation factor Ⅶ potency assay. ResultsHEK293 cells transfected with pcDNA3.1-EGFP-FⅦ showed green fluorescence， indicating that rhFⅦ was successfully expressed in the supernatant of HEK293 cells after transient transfection with pcDNA3.1-FⅦ. The monoclonal stable transfected cell line was obtained by G418 screening. RT-PCR identified that the FⅦ gene was integrated into the genome of the monoclonal stable transfected cell line. The cell viability was good as detected by Cell Counting Kit-8， and a single band of rhFⅦ was obtained by purification of the cell supernatant. The highest rhFⅦ expression was （1.27±0.09） mg/L， and the highest procoagulant activity was （380.29±13.80）%. ConclusionThe monoclonal HEK293 cell lines which can express rhFⅦ protein efficiently and stably with excellent procoagulant bioactivity is successfully screened.

Objective: To explore the application and clinical efficacy of red blood cell therapeutic apheresis in erythropoietic protoporphyria (EPP) and hereditary hemochromatosis (HH). Methods: 1) The EPP patient was hospitalized twice for "abdominal pain, nausea, vomiting, and brown urine". One and two sessions of red blood cell exchange/therapeutic plasma exchange (RCE/TPE) were respectively performed during the two hospitalizations. During each session, one RCE with 6-8 units of leukoreduced RBCs and 3-4 TPE procedures with 1 800-2 000 mL of frozen plasma was conducted. Biochemical parameters were monitored before and after treatment. 2) The HH patient was hospitalized for “repeatedly elevated aminotransferases”. Erythrocytapheresis was performed once, removing 550 mL of red blood cells, and venous phlebotomy was conducted once every 2 months subsequently. Blood routine and ferritin levels were assessed before and after treatment. Results: 1) During the first hospitalization, the EPP patient was relieved of the abdominal pain and brown urine after therapeutic apheresis. The total bilirubin level decreased from 141.8 μmol/L on admission to 68.6 μmol/L at discharge, with a symptom remission duration of 10 months. During the second hospitalization, the EPP patient still had recurrent abdominal pain after therapeutic apheresis. He developed psychiatric symptoms and gastrointestinal bleeding subsequently, accompanied by elevated bilirubin levels. Liver function deteriorated and the patient went into the state of the end-stage liver disease (ESLD). 2) For the HH patient, the hemoglobin level prior to erythrocytapheresis and vein phlebotomy was 150-160 g/L, with the lowest value occurring two days after erythrocytapheresis, decreasing to 107 g/L. The ferritin level before erythrocytapheresis was 2 428.08 ng/mL and it declined gradually after theraphy, with the lowest value occurring two months after erythrocytapheresis, decreasing to 1 094 ng/mL. The ferritin level was 1 114 ng/mL two months following the first vein phlebotomy, however it increased to 1 472 ng/mL two months after the second vein phlebotomy. Conclusion: RCE/TPE may alleviate protoporphyrin liver disease and help patients with bridging liver transplantation before EPP developments to ESLD. For HH patients with significantly elevated ferritin levels, erythrocytapheresis reduces serum ferritin more quickly and maintains its level longer relative to phlebotomy.

Objective: To explore the application and clinical efficacy of red blood cell therapeutic apheresis in erythropoietic protoporphyria (EPP) and hereditary hemochromatosis (HH). Methods: 1) The EPP patient was hospitalized twice for "abdominal pain, nausea, vomiting, and brown urine". One and two sessions of red blood cell exchange/therapeutic plasma exchange (RCE/TPE) were respectively performed during the two hospitalizations. During each session, one RCE with 6-8 units of leukoreduced RBCs and 3-4 TPE procedures with 1 800-2 000 mL of frozen plasma was conducted. Biochemical parameters were monitored before and after treatment. 2) The HH patient was hospitalized for “repeatedly elevated aminotransferases”. Erythrocytapheresis was performed once, removing 550 mL of red blood cells, and venous phlebotomy was conducted once every 2 months subsequently. Blood routine and ferritin levels were assessed before and after treatment. Results: 1) During the first hospitalization, the EPP patient was relieved of the abdominal pain and brown urine after therapeutic apheresis. The total bilirubin level decreased from 141.8 μmol/L on admission to 68.6 μmol/L at discharge, with a symptom remission duration of 10 months. During the second hospitalization, the EPP patient still had recurrent abdominal pain after therapeutic apheresis. He developed psychiatric symptoms and gastrointestinal bleeding subsequently, accompanied by elevated bilirubin levels. Liver function deteriorated and the patient went into the state of the end-stage liver disease (ESLD). 2) For the HH patient, the hemoglobin level prior to erythrocytapheresis and vein phlebotomy was 150-160 g/L, with the lowest value occurring two days after erythrocytapheresis, decreasing to 107 g/L. The ferritin level before erythrocytapheresis was 2 428.08 ng/mL and it declined gradually after theraphy, with the lowest value occurring two months after erythrocytapheresis, decreasing to 1 094 ng/mL. The ferritin level was 1 114 ng/mL two months following the first vein phlebotomy, however it increased to 1 472 ng/mL two months after the second vein phlebotomy. Conclusion: RCE/TPE may alleviate protoporphyrin liver disease and help patients with bridging liver transplantation before EPP developments to ESLD. For HH patients with significantly elevated ferritin levels, erythrocytapheresis reduces serum ferritin more quickly and maintains its level longer relative to phlebotomy.

Objective: The CT/MRI Liver Imaging Reporting and Data System (LI-RADS) demonstrates high specificity with relatively limited sensitivity for diagnosing hepatocellular carcinoma (HCC) in high-risk patients. This study aimed to explore the possibility of improving sensitivity by combining CT/MRI LI-RADS v2018 with second-line contrast-enhanced ultrasound (CEUS) LI-RADS v2017 using sulfur hexafluoride (SHF) or perfluorobutane (PFB). Materials and Methods: This retrospective analysis of prospectively collected multicenter data included high-risk patients with treatment-naive hepatic observations. The reference standard was pathological confirmation or a composite reference standard (only for benign lesions). Each participant underwent concurrent CT/MRI, SHF-enhanced US, and PFB-enhanced US examinations. The diagnostic performances for HCC of CT/MRI LI-RADS alone and three combination strategies (combining CT/ MRI LI-RADS with either LI-RADS SHF, LI-RADS PFB, or a modified algorithm incorporating the Kupffer-phase findings for PFB [modified PFB]) were evaluated. For the three combination strategies, apart from the CT/MRI LR-5 criteria, HCC was diagnosed if CT/MRI LR-3 or LR-4 observations met the LR-5 criteria using LI-RADS SHF, LI-RADS PFB, or modified PFB. Results: In total, 281 participants (237 males; mean age, 55 ± 11 years) with 306 observations (227 HCCs, 40 non-HCC malignancies, and 39 benign lesions) were included. Using LI-RADS SHF, LI-RADS PFB, and modified PFB, 20, 23, and 31 CT/MRI LR-3/4 observations, respectively, were reclassified as LR-5, and all were pathologically confirmed as HCCs. Compared to CT/MRI LI-RADS alone (74%, 95% confidence interval [CI]: 68%–79%), the three combination strategies combining CT/MRI LI-RADS with either LI-RADS SHF, LI-RADS PFB, or modified PFB increased sensitivity (83% [95% CI: 77%–87%], 84% [95% CI: 79%–89%], 88% [95% CI: 83%–92%], respectively; all P < 0.001), while maintaining the specificity at 92% (95% CI: 84%–97%). Conclusion The combination of CT/MRI LI-RADS with second-line CEUS using SHF or PFB improved the sensitivity of HCC diagnosis without compromising specificity.

Background Air pollution remains a critical public health issue, with persistent exposure to air pollutants continuing to pose significant health risks. Currently, research investigating the association between air pollution and myocardial infarction mortality in Shenzhen remains inadequate. Objective To quantitatively assess the association between air pollutants and myocardial infarction mortality in residents. Methods Based on the mortality surveillance system of Shenzhen Center for Disease Control and Prevention, we conducted a time-stratified case-crossover study of 10089 permanent residents who died from myocardial infarction in Shenzhen between 2013 and 2022. Using residential address information, we obtained individual-level exposure data for air pollutants from the China High Air Pollutants dataset and meteorological factors from the China Meteorological Administration Land Data Assimilation System. A time-stratified case-crossover study design was employed to construct a conditional logistic regression model to assess the association between short-term exposure to air pollutants and myocardial infarction mortality. The exposure-response relationship was visualized, and a comprehensive health risk assessment was performed. Results This study included a total of 10 089 cases of myocardial infarction deaths in Shenzhen. The median [interquartile range (IQR)] concentrations of fine particulate matter (PM_2.5), inhalable particulate matter (PM₁₀), sulfur dioxide (SO₂), nitrogen dioxide (NO₂), carbon monoxide (CO), and ozone (O₃) in Shenzhen from 2013 to 2022 were 24.59 (20.19) μg·m⁻³, 42.85 (28.42) μg·m⁻³, 8.53 (3.39) μg·m⁻³, 29.47 (13.56) μg·m⁻³, 0.77 (0.27) mg·m⁻³, and 86.53 (51.39) μg·m⁻³, respectively. The moving average concentrations of PM_2.5 and PM₁₀ over the current day and the previous 2 days (lag02) showed the highest risk of myocardial infarction mortality, with an odds ratio (OR) and 95% confidence interval (95%CI) of 1.004 (1.001, 1.007) and 1.004 (1.002, 1.006), respectively. At lag05, SO₂ demonstrated the strongest association with the risk of myocardial infarction mortality, with an OR (95%CI) of 1.042 (1.019, 1.065). The exposure-response relationships of PM_2.5, PM₁₀, and SO₂ with myocardial infarction mortality were approximately linear, whereas NO₂ exhibited a nonlinear relationship. At lag05, the highest risk of myocardial infarction mortality associated with NO₂ exposure was observed when the NO₂ concentration was ≤21.92 μg·m⁻³, with an OR (95% CI) of 1.024 (1.003, 1.046). The health risk assessment indicated that, using the WHO Air Quality Guidelines as the reference, the local PM_2.5 and NO₂ exposure led to an excess mortality of 398 and 298 cases, respectively. The sensitivity analysis revealed that after adjusting for O₃ and restricting the study period to 2013—2019, the effect estimates for PM_2.5, PM₁₀, NO₂, and SO₂ on myocardial infarction mortality slightly increased. Conclusion Short-term exposure to air pollutants, including PM_2.5, PM₁₀, NO₂, and SO₂, could increase the risk of myocardial infarction mortality. This study provides important scientific evidence for environmental health risk assessment and environmental management in Shenzhen.

Objective: To perform transient transfection of recombinant human Factor X(rhFX) into HEK293 cells,to optimize transfection parameters,to develop a high-yield in vitro expression system for rhFX production,and to assess the biological activity of expressed rhFX. Methods: The eukaryotic expression vector pcDNA3. 1-EGFP-FX was constructed by inserting the F10 gene into pcDNA3. 1-EGFP and subsequently transfected into HEK293cells to validate the transfection system efficiency. The recombinant expression vector pcDNA3. 1-FX was generated through ligation of the F10 gene fragment with pcDNA3. 1, followed by liposome-mediated transfection into HEK293 cells. The expression of rhFX in the cell supernatant was analyzed by Western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Transfection conditions were systematically optimized,and rhFX concentration was quantified by enzyme-linked immunosorbent assay(ELISA). The coagulation bioactivity of rhFX was evaluated through prothrombin time(PT) assay and chromogenic substrate method. Results: rhFX was successfully expressed in the supernatant of HEK293 cells. rhFX was successfully expressed in the supernatant of HEK293 cells. The highest expression level of rhFX was achieved on the third day after transfection when the cell density was 80% and the ratio of plasmid DNA to polyethyleneimine(PEI) transfection reagent was 1 ∶ 2.Triplicate ELISA measurements demonstrated a maximum rhFX concentration of 5. 20 ng/mL in the supernatant.The prothrombin time(PT) of rhFX-containing supernatant was significantly shorter(P 50) of etoxaban was determined to be 1. 449 nmol/L using the chromogenic substrate method based on rhFX,which was in the same order of magnitude as the reported 0. 78 nmol/L in the literature. Conclusion HEK293cells successfully express biologically active recombinant human Factor X(rhFX) protein,laying a foundation for advancing the development of rhFX-based therapeutics.

The prevalence of Class III malocclusion varies among different countries and regions. The populations from Southeast Asian countries (Chinese and Malaysian) showed the highest prevalence rate of 15.8%, which can seriously affect oral function, facial appearance, and mental health. As anterior crossbite tends to worsen with growth, early orthodontic treatment can harness growth potential to normalize maxillofacial development or reduce skeletal malformation severity, thereby reducing the difficulty and shortening the treatment cycle of later-stage treatment. This is beneficial for the physical and mental growth of children. Therefore, early orthodontic treatment for Class III malocclusion is particularly important. Determining the optimal timing for early orthodontic treatment requires a comprehensive assessment of clinical manifestations, dental age, and skeletal age, and can lead to better results with less effort. Currently, standardized treatment guidelines for early orthodontic treatment of Class III malocclusion are lacking. This review provides a comprehensive summary of the etiology, clinical manifestations, classification, and early orthodontic techniques for Class III malocclusion, along with systematic discussions on selecting early treatment plans. The purpose of this expert consensus is to standardize clinical practices and improve the treatment outcomes of Class III malocclusion through early orthodontic treatment.
Humans ; Malocclusion, Angle Class III/classification* ; Orthodontics, Corrective/methods* ; Consensus ; Child

Instrument separation is a critical complication during root canal therapy, impacting treatment success and long-term tooth preservation. The etiology of instrument separation is multifactorial, involving the intricate anatomy of the root canal system, instrument-related factors, and instrumentation techniques. Instrument separation can hinder thorough cleaning, shaping, and obturation of the root canal, posing challenges to successful treatment outcomes. Although retrieval of separated instrument is often feasible, it carries risks including perforation, excessive removal of tooth structure and root fractures. Effective management of separated instruments requires a comprehensive understanding of the contributing factors, meticulous preoperative assessment, and precise evaluation of the retrieval difficulty. The application of appropriate retrieval techniques is essential to minimize complications and optimize clinical outcomes. The current manuscript provides a framework for understanding the causes, risk factors, and clinical management principles of instrument separation. By integrating effective strategies, endodontists can enhance decision-making, improve endodontic treatment success and ensure the preservation of natural dentition.
Humans ; Root Canal Therapy/adverse effects* ; Consensus ; Root Canal Preparation/adverse effects*

Peri-implant keratinized mucosa (PIKM) augmentation refers to surgical procedures aimed at increasing the width of PIKM. Consensus reports emphasize the necessity of maintaining a minimum width of PIKM to ensure long-term peri-implant health. Currently, several surgical techniques have been validated for their effectiveness in increasing PIKM. However, the selection and application of PIKM augmentation methods may present challenges for dental practitioners due to heterogeneity in surgical techniques, variations in clinical scenarios, and anatomical differences. Therefore, clear guidelines and considerations for PIKM augmentation are needed. This expert consensus focuses on the commonly employed surgical techniques for PIKM augmentation and the factors influencing their selection at second-stage surgery. It aims to establish a standardized framework for assessing, planning, and executing PIKM augmentation procedures, with the goal of offering evidence-based guidance to enhance the predictability and success of PIKM augmentation.
Humans ; Consensus ; Dental Implants ; Mouth Mucosa/surgery* ; Keratins

With the growing emphasis on maternal and child oral health, the significance of managing oral health across preconception, pregnancy, and infancy stages has become increasingly apparent. Oral health challenges extend beyond affecting maternal well-being, exerting profound influences on fetal and neonatal oral development as well as immune system maturation. This expert consensus paper, developed using a modified Delphi method, reviews current research and provides recommendations on maternal and child oral health management. It underscores the critical role of comprehensive oral assessments prior to conception, diligent oral health management throughout pregnancy, and meticulous oral hygiene practices during infancy. Effective strategies should be seamlessly integrated across the life course, encompassing preconception oral assessments, systematic dental care during pregnancy, and routine infant oral hygiene. Collaborative efforts among pediatric dentists, maternal and child health workers, and obstetricians are crucial to improving outcomes and fostering clinical research, contributing to evidence-based health management strategies.
Humans ; Pregnancy ; Female ; Infant ; Consensus ; Mouth Diseases/therapy* ; Pregnancy Complications/therapy* ; Oral Health ; Infant, Newborn ; Delphi Technique ; Oral Hygiene