The emergence of clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) system represents a revolutionary paradigm shift in molecular diagnostics, offering transformative potential for RNA analysis within the rigorous demands of forensic science. Conventional forensic RNA detection methodologies, such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or microarray analysis, are significantly hampered by inherent limitations including complex, multi-step protocols requiring sophisticated laboratory infrastructure, pronounced susceptibility to inhibitors prevalent in complex forensic matrices (e.g., humic acids, heme, indigo dyes), and often inadequate sensitivity for trace or degraded samples typical of crime scenes, thereby failing to meet the critical operational imperatives of forensic practice: rapidity, high specificity, sensitivity, portability, and robustness against interference. This review posits that CRISPR-Cas-based RNA detection technology provides a groundbreaking solution by leveraging the programmable, sequence-specific recognition conferred by the synergistic interaction between a designed guide RNA (gRNA) and Cas effector proteins (e.g., Cas12a, Cas13a, Cas14). Upon target RNA binding, specific Cas enzymes undergo conformational activation, exhibiting collateral cleavage activity―a unique catalytic amplification mechanism where the enzyme non-specifically cleaves surrounding reporter molecules, enabling ultra-high sensitivity. To further enhance detection limits, CRISPR-Cas systems are strategically integrated with isothermal pre-amplification techniques like recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP), which efficiently amplify target RNA at constant temperatures, eliminating the need for thermal cyclers. This powerful cascade―isothermal pre-amplification followed by CRISPR-mediated sequence-specific recognition and collateral signal amplification―achieves exceptional sensitivity, often down to the single-molecule (attomolar) level, while drastically reducing analysis time to potentially 30-60 min. Crucially, the compatibility of CRISPR-Cas detection with simple, equipment-free readout systems, such as lateral flow strips (LFS) for visual colorimetric results or portable fluorescence/electrochemical sensors, facilitates true point-of-need (PON) forensic analysis directly at crime scenes, morgues, or field labs. This enables rapid applications like specific body fluid identification (e.g., distinguishing menstrual blood via miRNA, identifying saliva via mRNA), post-mortem interval (PMI) estimation through RNA degradation/expression patterns, donor age inference via age-related RNA markers, tissue identification, and microbial forensics, thereby accelerating investigative leads, minimizing sample degradation risks, and optimizing resource allocation. However, significant challenges impede widespread adoption, including persistent environmental interference inhibiting enzymes, fluctuations in Cas/amplification enzyme activity affecting reproducibility, a critical lack of standardized protocols and validated quality assurance/quality control (QA/QC) frameworks essential for forensic reliability and court admissibility, and current limitations in multiplex detection capability. Consequently, future research must prioritize overcoming multiplexing bottlenecks for comprehensive analysis, enhancing system robustness through Cas protein engineering and optimized reagents, developing fully integrated, sample-to-answer microfluidic or lateral flow devices for user-friendly field deployment, and collaboratively establishing universally accepted validation guidelines, performance standards, and stringent QA/QC procedures. Furthermore, the urgent development of clear ethical guidelines governing the use of this highly sensitive technology, particularly concerning RNA data privacy and potential misuse, is imperative. This review systematically outlines the principles, forensic applications, current limitations, and future trajectories of CRISPR-RNA detection, with the authors’ conviction that focused efforts addressing these challenges will translate this technology into a cornerstone of next-generation forensic practice, driving unprecedented efficiency and innovation in field investigations and laboratory analysis to enhance justice delivery.

BACKGROUND: Generalized pustular psoriasis (GPP), a rare and recurrent autoinflammatory disease, imposes a substantial burden on patients and society. Awareness of GPP in China remains limited. METHODS: This cross-sectional survey, conducted between September 2021 and May 2023 across 14 hospitals in China, included GPP patients of all ages and disease phases. Data collected encompassed demographics, clinical characteristics, economic impact, disease severity, quality of life, and treatment-related complications. Risk factors for GPP recurrence were analyzed. RESULTS: Among 127 patients (female/male ratio = 1.35:1), the mean age of disease onset was 25 years (1st quartile [Q1]-3rd quartile [Q3]: 11-44 years); 29.2% had experienced GPP for more than 10 years. Recurrence occurred in 75.6% of patients, and nearly half reported no identifiable triggers. Younger age at disease onset ( P  = 0.021) and transitioning to plaque psoriasis ( P  = 0.022) were associated with higher recurrence rates. The median diagnostic delay was 8 months (Q1-Q3: 2-41 months), and 32.3% of patients reported misdiagnoses. Comorbidities were present in 53.5% of patients, whereas 51.1% experienced systemic complications during treatment. Depression and anxiety affected 84.5% and 95.6% of patients, respectively. During GPP flares, the median Dermatology Life Quality Index score was 19.0 (Q1-Q3: 13.0-23.5). This score showed significant differences between patients with and without systemic symptoms; it demonstrated correlations with both depression and anxiety scores. Treatment costs caused financial hardship in 55.9% of patients, underscoring the burden associated with GPP. CONCLUSIONS The substantial disease and economic burdens among Chinese GPP patients warrant increased attention. Patients with early onset disease and those transitioning to plaque psoriasis require targeted interventions to mitigate the high recurrence risk.
Humans ; Male ; Female ; Psoriasis/pathology* ; Adult ; Cross-Sectional Studies ; Adolescent ; Child ; Young Adult ; Quality of Life ; Middle Aged ; China/epidemiology* ; Recurrence ; Risk Factors ; Surveys and Questionnaires ; East Asian People

The chemical components of Carthami Flos were investigated by using macroporous resin, silica gel column chromatography, reversed-phase octadecylsilane(ODS) column chromatography, Sephadex LH-20, and semi-preparative high-performance liquid chromatography(HPLC). The planar structures of the compounds were established based on their physicochemical properties and ultraviolet-visible(UV-Vis), infrared(IR), high-resolution electrospray ionization mass spectrometry(HR-ESI-MS), and nuclear magnetic resonance(NMR) spectroscopic technology. The absolute configurations were determined by comparing the calculated and experimental electronic circular dichroism(ECD). Six flavonoid C-glycosides were isolated from the 30% ethanol elution fraction of macroporous resin obtained from the 95% ethanol extract of Carthami Flos, and identified as saffloquinoside F(1), 5-hydroxysaffloneoside(2), iso-5-hydroxysaffloneoside(3), isosafflomin C(4), safflomin C(5), and vicenin 2(6). Among these, the compounds 1 to 3 were new chalcone C-glycosides. The compounds 1, 2, 4, and 5 could significantly increase the viability of H9c2 cardiomyocytes damaged by oxygen-glucose deprivation/reoxygenation(OGD/R) at a concentration of 50 μmol·L~(-1), showing their good cardioprotective activity.
Glycosides/pharmacology* ; Flowers/chemistry* ; Drugs, Chinese Herbal/pharmacology* ; Carthamus tinctorius/chemistry* ; Chalcones/pharmacology* ; Animals

OBJECTIVES: To investigate the clinical characteristics and prognosis of chronic disseminated candidiasis (CDC) in children with acute leukemia (AL) following chemotherapy. METHODS: A retrospective analysis was conducted on children diagnosed with CDC (including confirmed, clinically diagnosed, and suspected cases) after AL chemotherapy from January 2015 to December 2023 at Fujian Medical University Union Hospital, Zhangzhou Municipal Hospital, and Quanzhou First Hospital Affiliated to Fujian Medical University. Clinical characteristics and prognosis were analyzed. RESULTS: The incidence of CDC in children with AL following chemotherapy was 1.92% (32/1 668). Among the children with acute lymphoblastic leukemia, the incidence of CDC in the high-risk group was significantly higher than in the low-risk group (P=0.002). All patients presented with fever unresponsive to antibiotics during the neutropenic period, with 81% (26/32) involving the liver. C-reactive protein (CRP) levels were significantly elevated (≥50 mg/L) in 97% (31/32) of the patients. The efficacy of combined therapy with liposomal amphotericin B and caspofungin or posaconazole for CDC was 66% (19/29), higher than with caspofungin (9%, 2/22) or liposomal amphotericin B (18%, 2/11) monotherapy. The overall cure rate was 72% (23/32). The proportion of patients with CRP ≥50 mg/L and/or a positive β-D-glucan test for more than 2 weeks and breakthrough infections during caspofungin treatment was significantly higher in the treatment failure group compared to the successful treatment group (P<0.05). CONCLUSIONS CDC in children with AL after chemotherapy may be associated with prolonged neutropenia due to intensive chemotherapy. Combination antifungal regimens based on liposomal amphotericin B have a higher cure rate, while persistently high CRP levels and positive β-D-glucan tests may indicate poor prognosis.
Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Antifungal Agents/therapeutic use* ; Candidiasis/diagnosis* ; Chronic Disease ; Leukemia/complications* ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/complications* ; Prognosis ; Retrospective Studies

OBJECTIVE: To investigate the clinical features and risk factors associated with cutaneous chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children. METHODS: A retrospective analysis was conducted on the clinical data of children who underwent allo-HSCT in the Wuhan Children's Hospital from August 1, 2016, to December 31, 2023, and were regularly followed up for 1 year or more. The differences in clinical features between children with and without cutaneous cGVHD were compared, and the risk factors affecting the occurrence of cutaneous cGVHD were analyzed. RESULTS: During the study period, 296 children received allo-HSCT. Until December 31, 2024, follow-up showed that 20 children (6.8%) developed cutaneous cGVHD, which manifested as cutaneous lichenification, hyperpigmentation, keratosis pilaris, sclerotic changes, and hair or nail involvement. According to their skin lesion area and degree of grading, 5 cases were mild, 10 cases were moderate, and 5 cases were severe. Multivariate logistic regression analysis revealed that female donors and previous acute GVHD were risk factors for the development of cutaneous cGVHD after allo-HSCT. All 20 children were treated with glucocorticoid ± calcineurin inhibitors (tacrolimus/cyclosporine) as first-line therapeutic agents. Only 1 child improved after first-line treatment. The remaining 19 children treated with a second-line regimen of combination interventions based on individualized status, including 10 children who could not tolerate hormonotherapy or first-line treatment, and showed no significant improvement after 3 months, as well as 9 children with multi-organ cGVHD. After comprehensive second-line treatment, 17 children showed improvement in cutaneous symptoms. There were 3 deaths, including 1 due to primary disease recurrence and 2 due to pulmonary infections. CONCLUSION The skin is the first manifestation and most common organ involved in cGVHD in children. Cutaneous cGVHD severely affects the daily activities of transplanted children and requires prolonged immunosuppressive therapy, but has a favorable prognosis. First-line treatments for adults are not applicable to children who usually require a combination treatment with multiple drugs.
Humans ; Graft vs Host Disease/etiology* ; Hematopoietic Stem Cell Transplantation/adverse effects* ; Retrospective Studies ; Risk Factors ; Female ; Child ; Skin Diseases/etiology* ; Chronic Disease ; Transplantation, Homologous ; Male ; Child, Preschool ; Adolescent

OBJECTIVE: To investigate the protective effects of stir-fried Semen Armeniacae Amarum (SAA) against aristolochic acid I (AAI)-induced nephrotoxicity and DNA adducts and elucidate the underlying mechanism involved for ensuring the safe use of Asari Radix et Rhizoma. METHODS: In vitro, HEK293T cells overexpressing Flag-tagged multidrug resistance-associated protein 3 (MRP3) were constructed by Lentiviral transduction, and inhibitory effect of top 10 common pairs of medicinal herbs with Asari Radix et Rhizoma in clinic on MRP3 activity was verified using a self-constructed fluorescence screening system. The mRNA, protein expressions, and enzyme activity levels of NAD(P)H quinone dehydrogenase 1 (NQO1) and cytochrome P450 1A2 (CYP1A2) were measured in differentiated HepaRG cells. Hepatocyte toxicity after inhibition of AAI metabolite transport was detected using cell counting kit-8 assay. In vivo, C57BL/6 mice were randomly divided into 5 groups according to a random number table, including: control (1% sodium bicarbonate), AAI (10 mg/kg), stir-fried SAA (1.75 g/kg) and AAI + stir-fried SAA (1.75 and 8.75 g/kg) groups, 6 mice in each group. After 7 days of continuous gavage administration, liver and kidney damages were assessed, and the protein expressions and enzyme activity of liver metabolic enzymes NQO1 and CYP1A2 were determined simultaneously. RESULTS: In vivo, combination of 1.75 g/kg SAA and 10 mg/kg AAI suppressed AAI-induced nephrotoxicity and reduced dA-ALI formation by 26.7%, and these detoxification effects in a dose-dependent manner (P<0.01). Mechanistically, SAA inhibited MRP3 transport in vitro, downregulated NQO1 expression in vivo, increased CYP1A2 expression and enzymatic activity in vitro and in vivo, respectively (P<0.05 or P<0.01). Notably, SAA also reduced AAI-induced hepatotoxicity throughout the detoxification process, as indicated by a 41.3% reduction in the number of liver adducts (P<0.01). CONCLUSIONS Stir-fried SAA is a novel drug candidate for the suppression of AAI-induced liver and kidney damages. The protective mechanism may be closely related to the regulation of transporters and metabolic enzymes.
Aristolochic Acids/toxicity* ; Animals ; Humans ; NAD(P)H Dehydrogenase (Quinone)/genetics* ; HEK293 Cells ; Kidney/pathology* ; Cytochrome P-450 CYP1A2/genetics* ; Mice, Inbred C57BL ; DNA Adducts/drug effects* ; Male ; Kidney Diseases/drug therapy* ; Drugs, Chinese Herbal/therapeutic use* ; Mice ; Prunus armeniaca ; Plant Extracts

Objective:To investigate the expression and role of DEAD-box RNA helicases 5(DDX5 helicases)in head and neck squamous carcinoma(HNSCC).Methods:Tissue microarray microarray was used to assess relevant mRNA expression profile data,and R software was used to screen differential mRNAs(DEGs).The expression level of DDX5 was predicted using GEPIA 2,TCGA databases,and detected by immunohistochemistry,western blot and RT-qPCR in the HNSCC tissue and cell lines.Based on high-throughput sequencing data of DECs,differentially expressed miRNAs(DEMIs)relevant DDX5 competitive endogenous RNA network(ceRNA)was constructed.The software cytoscape was used to visualize the ceRNA network map and further screen the regulatory ax-is.Results:The results of microarray screening revealed that DDX5 expression in HNSCC was upregulated.Immunohistochemistry ver-ified that DDX5 was stronger expressed in the nuclei of squamous carcinoma cells.qPCR results suggested that significant expression of DDX5 mRNA at the tissue and cellular levels(P＜0.05).Western blot results showed high expression of DDX5 protein in the tissues.The ceRNA network was constructed,from which the relevant HNSCC axis circRNA-039626-miR-222-5p-DDX5 was identified.Con-clusion:DDX5 is highly expressed in HNSCC,and the circRNA-039626-miR-222-5p-DDX5 axis may be a potential regulatory axis for the development of HNSCC.

This study investigates whether compounds in Salvia miltiorrhiza Bunge can bind to the Toll like receptor 4/myeloid differentiation protein 2 (TLR4/MD2) protein complex and exhibit anti-inflammatory activity. Virtual screening of reported chemical components of Salvia miltiorrhiza Bunge against TLR4/MD2 was conducted in this study. The selected compound, neoprzewaquinone A (Neo A), was tested for its impact on the binding of lipopolysaccharide (LPS) to receptors on the cell membrane, its affinity for the protein, its influence on the dimerization of TLR4 and MD2 in LPS-induced cells, and its effects on the phosphorylation of nuclear factor-κB (NF-κB) p65 protein and the secretion of inflammatory cytokines in cells. Results indicate that Neo A in Salvia miltiorrhiza Bunge exhibited the highest virtual binding affinity with TLR4/MD2, with a value of -12.8 kcal·mol^-1. Neo A significantly inhibited the binding of LPS to receptors on the cell membrane (P < 0.01). Moreover, Neo A demonstrated affinity for rhTLR4/MD2, rhTLR4, and rhMD2, with K_D values of 267, 534, and 228 nmol·L^-1, respectively. Amino acid residues like TYR131 and PHE121 in TLR4/MD2 might play a role in the alkyl and π-alkyl hydrophobic interactions with Neo A. Neo A also significantly inhibited the dimerization of TLR4 and MD2 in LPS-mediated cells (P < 0.01) and markedly suppressed the phosphorylation of NF-κBp65 protein (P < 0.05). Furthermore, Neo A significantly or markedly inhibited the secretion of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in LPS-induced cells (P < 0.05, P < 0.01). In conclusion, Neo A exerts its anti-inflammatory effects by binding TLR4/MD2 then disrupting the binding of LPS to TLR4/MD2. It may serve as a TLR4/MD2 inhibitor with the potential to treat inflammation-related diseases targeting TLR4/MD2.

Objective To predict whether antiviral therapy is required in patients with chronic hepatitis B virus(HBV)infection and an age of≤30 years by establishing a noninvasive model,and to investigate the diagnostic value of this model.Methods A retrospective analysis was performed for the clinical data of 175 patients with chronic HBV infection who were admitted to Shenzhen Third People's Hospital from January 2017 to January 2023 and met the inclusion criteria,and according to the results of liver biopsy,they were divided into treatment group with 41 patients(with indications for antiviral therapy)and observation group with 134 patients(without indications for antiviral therapy).The two groups were analyzed in terms of the indicators including clinical data,imaging examinations,and serum biochemical parameters.The univariate and multivariate Logistic regression analyses were used to investigate the parameters affecting the indication for antiviral therapy,and different models for predicting the need for antiviral therapy were constructed based on related parameters.The receiver operating characteristic(ROC)curve was used to compare the diagnostic value of different models.The independent-samples t test was used for comparison of normally distributed continuous variables between groups,and the Mann-Whitney U rank sum test was used for comparison of non-normally distributed continuous variables between groups;the chi-square test or the Fisher's exact test was used for comparison of categorical data between groups.Results There were significant differences between the treatment group and the observation group in alanine aminotransferase,ferritin,total cholesterol(CHOL),triglyceride,platelet count,liver stiffness measured by sound touch elastography(STE),and procollagen Ⅲ N-terminal propeptide(PIIIP)(all P<0.05).The multivariate Logistic regression analysis showed that CHOL(odds ratio[OR]=0.4,95％confidence interval[CI]:0.2—1.0),STE(OR=1.5,95％CI:1.0—2.1),and PIIIP(OR=1.1,95％CI:1.0—1.1)were independent predictive factors for the indications for antiviral therapy.Model 1(STE+PIIIP+CHOL),model 2(STE+PIIIP),model 3(STE+CHOL),model 4(PIIIP+CHOL)had an area under the ROC curve of 0.908,0.848,0.725,and 0.725,respectively,while STE,PIIIP,and CHOL used alone had an AUC of 0.836,0.725,and 0.634,respectively,suggesting that model 1 had the largest AUC,with a specificity of 77.34％and a sensitivity of 96.36％,and had a significant difference compared with STE,PIIIP,CHOL,and the models 2,3,and 4(Z=0.21,3.08,3.06,3.23,0.89,and 0.88,all P<0.05).Conclusion The noninvasive model established based on CHOL,STE,and PIIIP has a good value in predicting the need for antiviral therapy in patients with chronic HBV infection and an age of≤30 years.

Objective To compare the role and importance of fibular fixation in tibiofibular fractures by Meta-analysis.Methods The literature related to the comparison of the efficacy of fixation of the fibula with or without fixation on the treatment of tibiofibular fractures was searched through the databases of China Knowledge Network,Wipu,Wanfang,The Cochrane Li-brary,Web of science and Pubmed,and statistical analysis was performed using RevMan 5.3 software.The rates of malrotation,rotational deformity,internal/external deformity,anterior/posterior deformity,non-union,infection,secondary surgery and op-erative time were compared between the fibula fixation and non-fixation groups.Results A total of 11 publications were includ-ed,six randomised controlled trials and five case-control trials,eight of which were of high quality.A total of 813 cases were in-cluded,of which 383 were treated with fibula fixation and 430 with unfixed fibulae.Meta-analysis results showed that fixation of the fibulae in the treatment of tibiofibular fractures reduced the rates of postoperative rotational deformity[RR=0.22,95％CI(0.10,0.45),P＜0.000 1]and internal/external deformity[RR=0.34,95％CI(0.14,0.84),P=0.02]and promoted fracture heal-ing[RR=0.76,95％CI(0.58,0.99),P=0.04].In contrast,the rates of poor reduction[RR=0.48,95％CI(0.10,2.33),P=0.36],anterior/posterior deformity[RR=1.50,95％CI(0.76,2.96),P=0.24],infection[RR=1.43,95％CI(0.76,2.72),P=0.27],sec-ondary surgery[RR=1.32,95％CI(0.82,2.11),P=0.25],and operative time[MD=10.21,95％CI(-17.79,38.21),P=0.47]were not statistically significant(P＞0.05)for comparison.Conclusion Simultaneous fixation of the tibia and fibula is clinically more effective in the treatment of tibiofibular fractures.