ObjectiveTo verify the association between the Ddit3-Trib3-Akt signaling pathway and rat spermatogenesis by constructing an in vitro co-culture system of testis. MethodsTesticular tissue blocks from 20-25-day-old male rats were placed in an in vitro culture system， and the culture medium was replaced every 2 to 3 days. PCR was used to verify the expression of marker genes of various spermatogenic cells. RNA interference technology was employed to verify the correlation between the Ddit3-Trib3-Akt signaling pathway and rat spermatogenesis. ResultsThe co-culture system could be continuously cultured for more than 2.5 months in vitro. RT-PCR showed that specific marker genes of spermatogonia， spermatocyte and spermoblast were expressed. The RNA and protein expression of Trib3 and Akt changed after the knocking down of Ddit3 and Trib3， respectively. It demonstrated the existence of Ddit3-Trib3-Akt signaling pathway in rat spermatogenesis. ConclusionThe culture time of more than 2.5 months indicates that the culture system can temporarily maintain the proliferation and differentiation of stem cells， and simultaneously maintain and stabilize spermatogenesis in a simple system. The successful validation of the Ddit3-Trib3-Akt signaling pathway also confirms that this culture system can be used to study possible molecular mechanisms of spermatogenesis in vitro.

Objective: To investigate the clinical characteristics, the types of unexpected antibodies, and their impacts on immunological risks among patients with different frequencies of cross-matching incompatibility, so as to propose corresponding solutions. Methods: Data of cross-matching incompatibility samples from 92 medical institutions during 2022 to 2024 were collected and divided into three groups based on the frequency of cross-matching. Statistical analysis was performed on disease types, distribution of hematologic diseases, alloantibody detection rates, and proportions of alloantibody types. Results: The 858 patients were divided into three groups based on the frequency of blood cross-matching incompatibility: ≥5 times (8.28%, 71/858), 2 to 4 times (28.21%, 242/858); 1 time (63.52%, 545/858). There was a clustered distribution of disease types in the ≥5 cross-matchings group, with 71.83% (51/71) of patients having tumors or hematologic and hematopoietic diseases. In contrast, the disease types in the 2 to 4 cross-matchings and 1 cross-matching groups were more diverse. An analysis of 249 patients with hematologic diseases found that multiple myeloma was the most common disease in all three groups, accounting for 31.43% (11/35), 35.37% (29/82), and 37.88% (50/132) respectively. In the ≥5 cross-matchings group, myelodysplastic syndrome (14.29%, 5/35) and thalassemia (14.29%, 5/35) were the second most common diseases. In contrast, in the 2 to 4 cross-matchings group and 1 cross-matching group, autoimmune hemolytic anemia was the second most common disease, with prevalence rates of 20.73% (17/82) and 24.24% (32/132), respectively. Alloantibodies were detected in 54.66% of the patients, with antibodies against Rh blood group being most frequent (>50%) in all three groups. The detection rates of alloantibodies/alloantibodies with coexisting autoantibodies decreased across groups: the ≥5 cross-matchings group (70.42%, 50/71) > the 2 to 4 cross-matchings group (54.96%, 133/242) > the 1 cross-matching group (52.48%, 286/545). Conclusion: The risk of alloantibody production increases in patients with multiple cross-matching incompatibilities, especially in those with tumors or hematologic diseases. For handling of cross-matching incompatibility cases, it is recommended to optimize the cross-matching process, implement individualized transfusion plans, and enhance the technical capabilities of clinical transfusion departments and blood group reference laboratories to ensure the safety and effectiveness of transfusions.

This study was aimed at exploring the interaction between the nucleoprotein(NP)of influenza A virus(IAV)and TRIM25.The physicochemical properties and protein structure of IAV NP protein were analyzed through bioinformatics methods.The interaction between IAV NP and TRIM25 proteins was simulated with molecular docking techniques,and the in-teraction sites were predicted.With the cDNA of the A/Puerto Rico/8/1934(H1N1)PR8 strain as the template,the NP pro-tein was cloned into the eukaryotic expression vector pCMV-C-Flag through PCR amplification,the eukaryotic expression re-combinant plasmid pCMV-Flag-NP was constructed,and the expression was further verified.The protein expression levels of pCMV-Flag-NP and pCMV-HA-TRIM25 were detected at various time periods.The interaction between NP protein and TRIM25 protein was verified by co-immunoprecipitation.The co-localization of NP protein and TRIM25 protein in cells was ob-served with laser confocal microscopy.Bioinformatics analysis revealed that the NP protein consists of 498 amino acids and 20 amino acids,and is an unstable hydrophilic protein.The NP protein has multiple phosphorylation sites,as well as N-glycosyla-tion and O-glycosylation sites,but no transmembrane domain or signal peptide domain.Additionally,the NP protein's second-ary structure consists of a high proportion of alpha-helices and random coils.The molecular docking prediction results indicated that IAV NP interacts with TRIM25 protein and has multiple potential interaction sites,including the 233rd alanine,234th ala-nine,236th lysine,and 440th alanine of the NP protein.After successfully constructing and expressing the IAV NP protein,we verified the interaction between IAV NP and TRIM25 protein by immunoprecipitation and laser confocal microscopy obser-vations.Our results together suggested that the structure of the IAV NP protein is closely related to its function,and its im-portance to the virus is clear.In addition,the interaction between IAV NP and TRIM25 protein may be associated with TRIM25's anti-influenza virus mechanism.Further in-depth research may provide new ideas for anti-influenza virus strategies.

The pregnant woman was 30 years old,G2P0.This singleton pregnancy at 22 weeks of gestation was screened for second-trimester ultrasound malformations,suggesting fetal aortic valve atresia,aortic stenosis with reverse blood flow,mitral valve atresia,and markedly enlarged left ventricle,which was considered for the diagnosis of hypoplastic left heart syndrome(HLHS).The pregnancy was terminated at our hospital and subsequently underwent genetic testing with results of heterozygous variants in the NOTCH1 gene,which can cause aortic valve disease type 1.The findings of the fetal autopsy were aortic valve atresia,mitral valve widening and thickening,and left ventricular enlargement with myocardial infarction.This report focuses on the ultrasound characteristics of HLHS with left ventricular enlargement and its hemodynamic changes in order to improve clinicians'understanding of the progressive changes in the disease phenotype of HLHS.

Objective:To construct and evaluate a risk warning model for enteral nutrition associated diarrhea(ENAD)in acute stroke patients based on logistic regression and Nomogram.Methods:This study was a retrospective study,a total of 172 patients with acute stroke who were admitted to Yancheng First People's Hospital from January 2022 to May 2024 were selected,and clinical data of patients were collected.Multivariate logistic regression analysis was used to identify the influencing factors of ENAD occurrence,based on the results of the multivariate logistic regression analysis,a Nomogram warning model was constructed,the receiver operating characteristic(ROC)curve was applied to analyze the predictive value of the Nomogram warning model for the risk of ENAD occurrence in patients with acute stroke.Results:Univariate analysis showed that,the occurrence of ENAD in patients with acute stroke was related to fasting time,mechanical ventilation,oral potassium preparations,albumin,number of types of antibiotics used,acute physiology and chronic health status Ⅱ(APACHE Ⅱ)score,enteral nutrition infusion rate,length of stay in the intensive care unit(ICU),use of proton pump inhibitors,duration of antibiotic use,use of gastrointestinal prokinetic drugs,and daily enteral nutrition infusion volume(P＜0.05).The results of multiple logistic regression analysis showed that,high APACHE Ⅱ score,prolonged use of antibiotics,low albumin,use of gastrointestinal prokinetic drugs,oral potassium preparations,prolonged fasting time,and rapid enteral nutrition infusion were risk factors for ENAD in patients with acute stroke(P＜0.05).Construct and validate a Nomogram warning model based on the results of multiple logistic regression analysis.After evaluation,the model fits well with an area under the curve(AUC)of 0.823,indicating high predictive value for ENAD occurrence in patients with acute stroke.Conclusion:High APACHE Ⅱ score,prolonged use of antibiotics,low albumin,use of gastrointestinal prokinetic drugs,oral potassium preparations,prolonged fasting time,and rapid enteral nutrition infusion are risk factors for ENAD in patients with acute stroke.The Nomogram warning model constructed based on the above indicators has high predictive value for the occurrence of ENAD in patients with acute stroke.

ObjectiveTo investigate the mechanisms by which the combination of berberine （BBR） and baicalin （BAI） ameliorates type 2 diabetes mellitus （T2DM） due to internal accumulation of dampness-heat from the perspectives of gut microbiota and metabolomics. MethodsAntibiotics were used to induce pseudo-sterile mice. Thirty pseudo-sterile mice were randomized into a normal fecal microbiota transplantation group （n=10） and a T2DM （syndrome of internal accumulation of dampness-heat） fecal microbiota transplantation group （n=20）. The mice were then administrated with suspensions of fecal microbiota from healthy volunteers and a patient with T2DM due to internal accumulation of dampness-heat by gavage， respectively. Each mouse received 200 µL suspension every other day for a total of 15 times to reshape the gut microbiota. The T2DM model mice were then assigned into a model group （n=8） and a BBR-BAI group （n=11）. BBR was administrated at a dose of 200 mg·kg^-1， and BAI was administrated in a ratio of BBR-BAI 10∶1 based on preliminary research findings. The administration lasted for 8 consecutive weeks. Fasting blood glucose （FBG）， glycated hemoglobin （HbA1c）， insulin （INS）， triglycerides （TG）， total cholesterol （CHOL）， low-density lipoprotein cholesterol （LDL-C）， high-density lipoprotein cholesterol （HDL-C）， aspartate aminotransferase （AST）， and alanine aminotransferase （ALT） levels were measured to evaluate the effects of the BBR-BAI combination on glucose and lipid metabolism and liver function in T2DM mice. Hematoxylin-eosin staining was employed to observe pathological changes in the colon tissue. The expression of claudin-1， zonula occludens-1 （ZO-1）， and occludin in the colon tissue was determined by Western blot. Real-time quantitative polymerase chain reaction（Real-time PCR） was employed to assess the levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） in the colon tissue. The fecal microbiota composition and differential metabolites were analyzed by 16S rRNA sequencing and ultra-high performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry （UPLC-Q-TOF-MS）， respectively. ResultsThe BBR-BAI combination lowered the FBG， HbA1c， and INS levels （P<0.05， P<0.01） and alleviated insulin resistance （P<0.01） in T2DM mice. Additionally， BBR-BAI elevated the levels of ZO-1， occludin， and claudin-1 （P<0.05， P<0.01） and down-regulated the expression levels of TNF-α， IL-1β， and IL-6 in the colon （P<0.05， P<0.01）. The results of 16S rRNA sequencing showed that BBR-BAI increased the relative abundance of Ligilactobacillus， Phascolarctobacterium， and Akkermansia （P<0.05）， while significantly decreasing the relative abundance of Alistipes， Odoribacter， and Colidextribacter （P<0.05）. UPLC-Q-TOF-MS identified 28 differential metabolites， which were primarily involved in arachidonic acid metabolism and α-linolenic acid metabolism. ConclusionBBR-BAI can ameliorate T2DM due to internal accumulation of dampness-heat by modulating the relative abundance of various bacterial genera in the gut microbiota and the expression of fecal metabolites.

Patients suffering from nerve injury often experience exacerbated pain responses and complain of memory deficits. The dorsal hippocampus (dHPC), a well-defined region responsible for learning and memory, displays maladaptive plasticity upon injury, which is assumed to underlie pain hypersensitivity and cognitive deficits. However, much attention has thus far been paid to intracellular mechanisms of plasticity rather than extracellular alterations that might trigger and facilitate intracellular changes. Emerging evidence has shown that nerve injury alters the microarchitecture of the extracellular matrix (ECM) and decreases ECM rigidity in the dHPC. Despite this, it remains elusive which element of the ECM in the dHPC is affected and how it contributes to neuropathic pain and comorbid cognitive deficits. Laminin, a key element of the ECM, consists of α-, β-, and γ-chains and has been implicated in several pathophysiological processes. Here, we showed that peripheral nerve injury downregulates laminin β1 (LAMB1) in the dHPC. Silencing of hippocampal LAMB1 exacerbates pain sensitivity and induces cognitive dysfunction. Further mechanistic analysis revealed that loss of hippocampal LAMB1 causes dysregulated Src/NR2A signaling cascades via interaction with integrin β1, leading to decreased Ca²⁺ levels in pyramidal neurons, which in turn orchestrates structural and functional plasticity and eventually results in exaggerated pain responses and cognitive deficits. In this study, we shed new light on the functional capability of hippocampal ECM LAMB1 in the modulation of neuropathic pain and comorbid cognitive deficits, and reveal a mechanism that conveys extracellular alterations to intracellular plasticity. Moreover, we identified hippocampal LAMB1/integrin β1 signaling as a potential therapeutic target for the treatment of neuropathic pain and related memory loss.
Animals ; Laminin/genetics* ; Hippocampus/metabolism* ; Neuralgia/metabolism* ; Cognitive Dysfunction/etiology* ; Male ; Peripheral Nerve Injuries/metabolism* ; Extracellular Matrix/metabolism* ; Integrin beta1/metabolism* ; Pyramidal Cells/metabolism* ; Signal Transduction

"Prescriptions being modified according to the syndrome and processing following prescription" is one of the characteristics of clinical medication in traditional Chinese medicine(TCM), and it is also an important sign that distinguishes TCM from other traditional medicine. The processing methods of TCM decoctions originate from the ingenious combination of medicinal materials, the mutual restraint of seven emotions, the harmony of four properties, and the pairing and combining of medicinal materials in prescriptions. They are the concrete embodiment of the essence and characteristics of "prescriptions being modified according to the syndrome and processing following prescription". However, due to insufficient inheritance and innovation, many characteristic varieties and pharmaceutical experience have been lost or forgotten. There is an urgent need to systematically explore and organize the processing theory and characteristic varieties of TCM decoctions, delve into the scientific connotation of the processing principles, and optimize the processing technology. Therefore, this article systematically organizes and summarizes the historical evolution and modern research progress of TCM decoction processing, conducts in-depth reflection on the current problems, and puts forward reasonable suggestions, with the aim of further inheriting, enriching, and developing the processing theory of TCM decoctions and providing support for ensuring the clinical efficacy of prescriptions.
Drugs, Chinese Herbal/isolation & purification* ; Humans ; Medicine, Chinese Traditional/methods*

This study established the HPLC fingerprints, characteristic chromatograms, and a multi-component content determination method for Bidens bipinnata and B. biternata. The chemical pattern recognition analysis was then employed to clarify the characteristic indexes of quality differences between the two original plants of Bidentis Herba, providing a reference for establishing the quality standards of Bidentis Herba. HPLC was launched on an Agilent Poroshell 120 EC-C_(18) chromatographic column(4.6 mm×250 mm, 4 μm) by gradient elution with a mobile phase of 0.1% aqueous phosphoric acid-acetonitrile at a flow rate of 0.7 mL·min~(-1), detection wavelength of 270 nm, column temperature of 25 ℃, and an injection volume of 5 μL. The similarity between the fingerprints of 18 batches of Bidentis Herba samples and the common pattern(R) ranged from 0.572 to 0.933. A total of 23 chromatographic peaks were calibrated. Through comparison with the reference substances, six components(neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, rutin, and hyperoside) were identified and subjected to quantitative analysis. The characteristic fingerprints of B. bipinnata and B. biternata were calibrated with 20 and 17 characteristic peaks, respectively. Among them, peaks 8, 9, 22, and 23 were the characteristic peaks of B. bipinnata, and peak 7 was the characteristic peak of B. biternata, which can be used to distinguish the two original plants of Bidentis Herba. The relative standard deviation of the content of the above-mentioned six components ranged from 36% to 123%. The cluster analysis, principal component analysis, and orthogonal partial least squares-discriminant analysis(OPLS-DA) classified the 18 batches of Bidentis Herba samples into two categories. Additionally, through the analysis of variable importance in projection(VIP) under OPLS-DA, three characteristic indexes, rutin, isochlorogenic acid A, and isochlorogenic acid B, were identified. The analytical method established in this study can comprehensively evaluate the consistency of Bidentis Herba samples derived from different original plants, specifically identify the differential components between them, and effectively distinguish the two original plants of Bidentis Herba, providing a basis for the differentiation between different original plants and the quality control of Bidentis Herba.
Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal/chemistry* ; Quality Control ; Bidens/chemistry*