OBJECTIVE: To investigate the clinical characteristics, steroid hormone profiles, and genetic variants in two female patients with Non-classic adrenal hyperplasia (NCAH). METHODS: Clinical data and samples were collected from two patients who had visited Huaian Maternal and Child Health Care Hospital Affiliated to Medical College of Yangzhou University on September 27, 2022 and June 25, 2023, respectively, with an initial diagnosis of Polycystic ovary syndrome (PCOS) and suspected NCAH. Seven steroid hormones in dried blood spots were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Single base variants and repeat/deletions in the CYP21A2 gene were analyzed by using a classic congenital adrenal hyperplasia (CAH) gene assay, and 10 related genes were analyzed by third-generation sequencing (TGS) should the variants be unclear. This study has been approved by the Medical Ethics Committee of the hospital (Ethics No.: 2025003). RESULTS: Patient 1 was a 14-year-old girl, and patient 2 was a 23-year-old woman with insulin resistance. Both patients had hirsutism, acne, bilateral polycystic ovarian morphology, in addition with significantly elevated serum testosterone by chemiluminescence. The steroid hormone profiles of both patients suggested a significant increase in 17-hydroxyproesterone, normal cortisol and 11-deoxycortisol. Patient 2 additionally showed a significant rise in 21-deoxycortisol. The presentation of both patients was indicative of NCAH, which was also evidenced by their respective medical histories. Sanger sequencing of long fragment PCR amplification combined with multiplex ligation-dependent probe amplification (MLPA) revealed that patient 1 harbored a mild c.92C>T (p.P31L) variant and a severe variant with a large segmental deletion in CYP21A2. Patient 2 was finally confirmed by TGS to carry mild CYP21A2 variants in the 5' untranslated region (5' UTR) promotor region (c.-126C>T, c.-113G>A, c.-110T>C) and a severe c.293-13C/A>G variant. The promotor region variants had resulted in decompression of the long fragment P1X/P2 amplification, leading to homozygous result of Sanger sequencing for c.293-13C/A>G, which in turn halved the amplification signal for the wt-113 SNP probe. In addition, the wtI2G-A probe was enhanced by interference in the MLPA assay. CONCLUSION This study demonstrated that NCAH should be excluded when PCOS is accompanied by a significant increase in serum testosterone, that mass spectrometry of steroid hormone profiles containing 17-hydroxyprogesterone is useful for the detection of NCAH, and that TGS is advantageous in confirming the diagnosis of NCAH when compared with conventional genetic testing methods.
Humans ; Female ; Adrenal Hyperplasia, Congenital/blood* ; Adolescent ; Steroid 21-Hydroxylase/genetics* ; Young Adult ; Genetic Variation ; Adult

ObjectiveTo explore the clinical efficacy and mechanism of Bupi Qingfei prescription （BPQF） in treating stable bronchiectasis in the patients with syndromes of lung-spleen Qi deficiency and phlegm-heat accumulation in the lungs. MethodsA randomized， double-blind， placebo-controlled trial was conducted. Patients were randomized into BPQF and placebo control （PC） groups. On the basis of conventional Western medicine treatment， the BPQF granules and placebo were respectively administered at 10 g each time， twice a day， for a course of 24 weeks. The TCM symptom scores， Quality of Life Questionnaire for Bronchiectasis （QOL-B） scores， lung function indicators， T lymphocyte subsets， level of inflammatory factors in the sputum， level of neutrophil elastase （NE） in the sputum， and occurrence of adverse reactions were observed before and after treatment in the two groups. ResultsA total of 64 patients completed the study， encompassing 32 in the BPQF group and 32 in the PC group. After treatment， the BPQF group showed decreased TCM symptom scores （P<0.01）， increased QOL-B scores （P<0.01）， and declined levels of tumor necrosis factor （TNF）-α and NE （P<0.05， P<0.01）. The PC group showed decreased TCM symptom （except spleen deficiency） scores （P<0.01）， increased the QOL-B health cognition and respiratory symptom domain scores （P<0.05， P<0.01）， and a declined TNF-α level （P<0.01）. Moreover， the BPQF group had lower TCM symptom （except chest tightness） scores （P<0.05， P<0.01）， higher QOL-B （except treatment burden） scores （P<0.05， P<0.01）， and lower levels of interleukin-6 and TNF-α （P<0.05） than the PC group. Neither group showed serious adverse reactions during the treatment process. ConclusionBPQF can ameliorate the clinical symptoms of stable bronchiectasis patients who have lung-spleen Qi deficiency or phlegm-heat accumulation in the lungs by regulating the immune balance and inhibiting airway inflammatory responses.

Objective To establish an in vitro enzymatic activity evaluation system for OTULIN （OTU domain DUB with linear linkage specificity）and provide an experimental basis for OTULIN inhibitor screening. Methods The linear ubiquitin substrate protein His-Ub-Ub-Flag was designed, expressed, and purified. Using OTULIN^80-352 as the target enzyme and PR619 as a positive control, the enzyme-to-substrate ratio and reaction time were optimized to establish an in vitro OTULIN activity evaluation system. Results The linear ubiquitin substrate protein His-Ub-Ub-Flag was successfully obtained. OTULIN activity was significantly inhibited by PR619, which confirmed the feasibility of the assay. The optimal enzyme-to-substrate ratio of OTULIN^80-352 to His-Ub-Ub-Flag was 1∶16, and the optimal reaction condition was incubation at 37℃ for 20 min. Conclusion An in vitro enzymatic activity evaluation system for OTULIN was successfully established, which could be used to evaluate the inhibitory effects of candidate compounds on OTULIN activity and provide technical support for subsequent screening of OTULIN small-molecule inhibitors.

CRISPR-Cas-based biosensing systems have become important platforms for rapid nucleic acid detection because of their programmable sequence recognition and high specificity. In many diagnostic workflows, the CRISPR reaction is coupled with polymerase chain reaction or isothermal pre-amplification to enrich low-abundance targets before readout. This design improves sensitivity, but it also introduces additional primers and enzymes, requirements for temperature control and reaction compatibility, and a higher demand for contamination management. These factors make the whole assay less convenient for integrated and field-deployable point-of-care testing. Developing pre-amplification-free CRISPR assays is therefore not simply a pursuit of shorter protocols, but a way to simplify molecular diagnosis at the system level, especially in settings where rapid decisions, closed workflows, and minimal manual operation are required. Microfluidic devices provide an important engineering route for this purpose. Their small reaction volumes, precise fluid manipulation, high-throughput partitioning, and compatibility with portable readout make it possible to integrate target confinement, reaction control, signal acquisition, and quantitative analysis on a chip. Droplet microfluidics and microwell arrays can divide a sample into large numbers of independent microreactors, enabling digital counting of rare recognition events. Continuous-flow, centrifugal, and paper-based microfluidic formats further provide options for automated operation, low-cost fabrication, and on-site use. In this context, microfluidics is not only a signal-enhancement method, but also a platform that connects CRISPR molecular recognition with practical assay implementation. This review summarizes recent progress in microfluidic-based pre-amplification-free CRISPR-Cas biosensing for rapid detection. We focus on four related technical directions. crRNA engineering, including spacer-length tuning, multi-crRNA design, chemical modification, and allosteric regulation, can improve recognition kinetics, nuclease stability, and mismatch discrimination. Digital microfluidic detection based on droplets or microwell arrays converts single-molecule recognition events into countable positive partitions, thereby improving quantitative capability without target pre-amplification. Physical signal transduction interfaces couple CRISPR activity to electrochemical, electrochemiluminescent, fluorescent, or surface-enhanced Raman scattering readouts, making weak molecular signals easier to detect with portable instruments. On-chip cascade signal amplification strategies further enhance output intensity through enzymatic reactions, DNA circuits, nanomaterials, or cross-domain amplification modules while avoiding direct amplification of the target nucleic acid. Together, these strategies show that the performance of a pre-amplification-free assay depends not only on biochemical recognition, but also on how the reaction is confined, amplified, transduced, and operated. In addition to describing these mechanisms, this review compares different strategies in terms of sensitivity improvement, operational complexity, cost, and suitability for point-of-care testing. Representative applications in food authenticity identification and rapid pathogen screening are also discussed to illustrate how these platforms perform in practical scenarios. Finally, we analyze the remaining challenges, including the detection of low-abundance targets in complex matrices, on-chip sample preparation, device-to-device reproducibility, long-term reagent storage, and standardization. Future development will likely depend on tighter integration of sample processing, CRISPR reaction, signal readout, and data interpretation, ultimately moving pre-amplification-free CRISPR diagnostics toward automated and user-friendly sample-to-answer systems.

The Expert Consensus on Clinical Application of Qinbaohong Zhike Oral Liquid in Treatment of Acute Bronchitis and Acute Attack of Chronic Bronchitis （GS/CACM 337-2023） was released by the China Association of Chinese Medicine on December 13^th， 2023. This expert consensus was developed by experts in methodology， pharmacy， and Chinese medicine in strict accordance with the development requirements of the China Association of Chinese Medicine （CACM） and based on the latest medical evidence and the clinical medication experience of well-known experts in the fields of respiratory medicine （pulmonary diseases） and pediatrics. This expert consensus defines the application of Qinbaohong Zhike oral liquid in the treatment of cough and excessive sputum caused by phlegm-heat obstructing lung， acute bronchitis， and acute attack of chronic bronchitis from the aspects of applicable populations， efficacy evaluation， usage， dosage， drug combination， and safety. It is expected to guide the rational drug use in medical and health institutions， give full play to the unique value of Qinbaohong Zhike oral liquid， and vigorously promote the inheritance and innovation of Chinese patent medicines.

Kirsten rat sarcoma viral oncogene homolog(KRAS)protein inhibitors are a promising class of thera-peutics,but research on molecules that effectively penetrate the blood-brain barrier(BBB)remains limited,which is crucial for treating central nervous system(CNS)malignancies.Although molecular generation models have recently advanced drug discovery,they often overlook the complexity of bio-logical and chemical factors,leaving room for improvement.In this study,we present a structure-constrained molecular generation workflow designed to optimize lead compounds for both drug effi-cacy and drug absorption properties.Our approach utilizes a variational autoencoder(VAE)generative model integrated with reinforcement learning for multi-objective optimization.This method specifically aims to enhance BBB permeability(BBBp)while maintaining high-affinity substructures of KRAS in-hibitors.To support this,we incorporate a specialized KRAS BBB predictor based on active learning and an affinity predictor employing comparative learning models.Additionally,we introduce two novel metrics,the knowledge-integrated reproduction score(KIRS)and the composite diversity score(CDS),to assess structural performance and biological relevance.Retrospective validation with KRAS inhibitors,AMG510 and MRTX849,demonstrates the framework's effectiveness in optimizing BBBp and highlights its potential for real-world drug development applications.This study provides a robust framework for accelerating the structural enhancement of lead compounds,advancing the drug development process across diverse targets.

Malignant tumors(commonly referred to as cancer)represent a major global public health challenge and contribute significantly to the worldwide disease burden.Early screening plays a critical role in improving detection rates,enabling timely intervention,and enhancing pa-tient survival rates.However,current cancer screening guidelines primarily focus on site-specific screening,which may not fully address the need for comprehensive early detection.A scientifical-ly rational,multi-cancer screening approach offers several advantages:it optimizes the use of bio-logical samples,reduces time costs for participants,enhances the efficiency and comprehensive-ness of screening,and minimizes overall expenses.Such an approach also facilitates the rational allocation of healthcare resources,ultimately helping to reduce the societal burden of cancer.To address this need,the Cancer Epidemiology Committee of the Chinese Anti-Cancer Association has developed the Expert Consensus on Combined Screening for Common Cancers in China.This consensus integrates multidisciplinary expertise and synthesizes the latest domestic and interna-tional researches on cancer screening,early detection,and treatment for prevalent malignancies.Drawing upon China's unique demographic and healthcare context,as well as practical screening experiences,the consensus provides evidence-based recommendations on target populations,screening technologies,and procedural workflows for multi-cancer screening.These guidelines align with the principles and methodologies established by the World Health Organization(WHO),aiming to:enhance the effectiveness of combined cancer screening in China,improve early detec-tion rates,and provide a scientific foundation for national cancer prevention and control strategies.

Objective:To explore the effect of horizontal Baduanjin guided by interactive standard reaching theory(ISRT)on cardiac function and quality of life in patients with chronic heart failure(CHF).Methods:This random-ized controlled study enrolled 114 patients diagnosed with CHF in Bozhou People's Hospital between September 2024 and February 2025.Patients were randomly divided into control group(n=57,routine nursing care)and interven-tion group(n=57,horizontal Baduanjin under the guidance of interactive standard theory).Both groups were con-tinuously treated for 3 months.Cardiac function indexes,N-terminal pro-brain natriuretic peptide(NT-proB-NP),self-care ability,quality of life and incidence of major adverse cardiovascular events(MACE)were com-pared between the two groups.Results:Compared to patients in the control group,those in the intervention had significant higher left ventricular ejection fraction(LVEF)[(56.34±0.86)％vs.(46.69±0.91)％],6min walking distance(6MWD)[(477.16±6.62)m vs.(451.39±4.17)m]and the proportion of New York Heart Association(NYHA)class Ⅱ(70.18％vs.35.09％),and significant lower NT-proBNP[(237.76±6.41)pg/ml vs.(377.46±6.85)pg/ml],scores of the European heart failure self-care behavior scale 9(EHFSCB-9)[(12.61±1.81)points vs.(25.51±1.71)points],the physical domain[(3.96±0.91)points vs.(11.05±0.79)points],emotional domain[(4.00±0.85)points vs.(11.95±0.81)points]and other domain[(4.05±0.83)points vs.(13.47±1.15)points]of MLHFQ(P＜0.001 all).Compared to those in the control group,patients in intervention group had significant lower incidence of MACE(7.02％vs.22.81％,P=0.035).Conclusion:Under the guidance of the interactive standard theory,the horizontal Baduanjin exercise may enhance the cardiac function,improve self-care ability and quality of life in patients with chronic heart failure.

Aim To evaluate the continuous flow exposure sys-tem at the air-liquid interface(ALI)in vitro to provide reference data for in vitro studies on inhalation toxicology,and to conduct a preliminary evaluation of the inhalation toxicity of the com-pound limonene by using the system in conjunction with an ALI culture model of Calu-3 cells.Methods Fluorescein sodium(Na-flu)dosimetry supplemented with quartz microbalance(QCM)was used to evaluate the deposition volume and pore-to-pore homogeneity of the ALI continuous flow exposure system;limonene aerosol was exposed to an ALI-cultured model of Calu-3 cells for 3 h using the ALI continuous flow exposure system at exposure doses of high(0.213 μg·cm-2),medium(0.104μg·cm-2),low(0.064 5 μg·cm-2),clean air exposure was used as a negative control group,and the activity,lactate dehydrogenase(LDH)release,trans-epithelial electrical resist-ance(TEER),mucin MUC5AC and inflammatory factor gene expression of the exposed cells were detected after 24 h to evalu-ate the inhalation toxicity of limonene.Results The deposition of sodium fluorescein in the ALI continuous flow exposure system was 0.085±0.007 μg/30 min/well,and inter-well homogeneity was optimized from the initial 26％to less than 10％after sever-al debugging sessions;compared with the control group,there was no significant change in cellular activity and IL-8 gene ex-pression,but cellular IL-6 gene expression increased after limo-nene aerosol exposure;the mid-exposure dose of limonene pro-moted cellular release of LDH and inflammatory factor gene ex-pression.The medium exposure dose of limonene induced the cells to release LDH＞10％and decreased the expression of cel-lular tumor necrosis factor TNF-α gene;the high exposure dose of limonene decreased the cellular TEER value,impaired the cellular barrier function,and increased the expression of cellular mucin MUC5AC gene.Conclusions The ALI continuous flow exposure system can be used for inhalation toxicity in vitro stud-ies after commissioning;high and medium exposure doses of limonene are inhalation toxic.

Malignant tumors (commonly referred to as cancer) represent a major global public health challenge and contribute significantly to the worldwide disease burden. Early screening plays a critical role in improving detection rates, enabling timely intervention, and enhancing patient survival rates. However, current cancer screening guidelines primarily focus on site-specific screening, which may not fully address the need for comprehensive early detection. A scientifically rational, multi-cancer screening approach offers several advantages: it optimizes the use of biological samples, reduces time costs for participants, enhances the efficiency and comprehensiveness of screening, and minimizes overall expenses. Such an approach also facilitates the rational allocation of healthcare resources, ultimately helping to reduce the societal burden of cancer. To address this need, the Cancer Epidemiology Committee of the Chinese Anti-Cancer Association has developed the Expert Consensus on Combined Screening for Common Cancers in China. This consensus integrates multidisciplinary expertise and synthesizes the latest domestic and international researches on cancer screening, early detection, and treatment for prevalent malignancies. Drawing upon China's unique demographic and healthcare context, as well as practical screening experiences, the consensus provides evidence-based recommendations on target populations, screening technologies, and procedural workflows for multi-cancer screening. These guidelines align with the principles and methodologies established by the World Health Organization (WHO), aiming to enhance the effectiveness of combined cancer screening in China, improve early detection rates, and provide a scientific foundation for national cancer prevention and control strategies.