Objective: To perform serological identification and molecular analysis of four samples with antibodies against high-frequency antigens, and to track the condition of newborns after delivery. Methods: Blood group serological tests were conducted using tube method, and unexpected antibody screening and identification were performed using polybrene and human globulin card. Gene haplotype analysis of blood group system was performed using PacBio third-generation sequencing. The DNA mutations were confirmed through Sanger sequencing. Results: Four samples showed normal blood types in common blood type systems. However, they were positive in all unexpected antibody screening and identification, with negative direct antiglobulin tests results. Third-generation sequencing revealed 3 cases of c.376C>T homozygous mutation and 1 case of c.421C>A homozygous mutation in the ABCG2 gene. Three pregnant women gave birth to four children, all of whom developed hyperbilirubinemia, accompanied by decreased red blood cell count and normal or low hemoglobin concentration. Conclusion: Four samples were obtained from individuals with the rare Jr(a-) blood group. Immunization during pregnancy led to the production of anti-Jr antibody, which may contribute to hyperbilirubinemia in newborns.

Objective:To explore the inhibitory mechanism of herb cake-partitioned moxibustion on tumor growth in colitis-associated colorectal cancer(CAC)based on histone lysine demethylase 4D(KDM4D). Methods:Inbred male Sprague-Dawley rats were randomly divided into a normal group,a CAC group,a herb cake-partitioned moxibustion group,and an inhibitor group.Except the normal group,rats in the other three groups were treated with azoxymethane(AOM)combined with dextran sulfate sodium(DSS)to make CAC rat models.Rats in the normal group and the CAC group did not receive interventions;rats in the herb cake-partitioned moxibustion group received moxibustion at Qihai(CV6)and bilateral Tianshu(ST25),2 cones for one point each time,once a day for 30 d with 1-day rest every week;rats in the inhibitor group received intraperitoneal injection of KDM4D inhibitor,5-chloro-8-hydroxyquinoline(5-c-8HQ),once a day for 30 d.After intervention,the general condition,colon length,tumor number and volume,and histopathological colon changes were observed.The expression of adenomatous polyposis coli(APC),axis inhibitor(Axin),cyclin D1,matrix metalloproteinase(MMP)-7 and MMP-9 mRNAs were detected by real-time quantitative polymerase chain reaction.The proliferating cell nuclear antigen(PCNA),cleaved caspase3,KDM4D,APC,and Axin proteins were detected by immunohistochemistry. Results:Compared with the normal group,the general condition was poor,the colon length was significantly shortened(P<0.01),the number and volume of colonic tumors were increased(P<0.01),the structure of glandular duct was obviously disordered with"back-to-back"and cowall phenomenon,and also high-grade adenocarcinoma formed;the protein expression levels of PCNA and KDM4D were significantly increased(P<0.01),while cleaved caspase3,APC,and Axin were significantly reduced(P<0.01);the mRNA expression levels of cyclin D1,MMP-7,and MMP-9 were significantly increased(P<0.01),while APC and Axin were significantly reduced(P<0.01)in the CAC group.Compared with the CAC group,the general condition was improved,the length of colon was significantly increased(P<0.01),the number and volume of the colonic tumors were reduced(P<0.05),and the colon tissues showed epithelial cell proliferation with enlarged and deep staining nuclei,dysplasia and inflammatory cell infiltration;the protein expression levels of PCNA and KDM4D were significantly reduced(P<0.01),while the cleaved caspase3,APC,and Axin were significantly increased(P<0.01);the mRNA expression levels of cyclin D1,MMP-7,and MMP-9 were reduced(P<0.05),while the APC and Axin were increased(P<0.05)in the colon tissues of rats in the herb cake-partitioned moxibustion group and the inhibitor group. Conclusion:Herb cake-partitioned moxibustion regulated abnormally expressed KDM4D in CAC rats,activated APC and Axin,the upstream molecules of Wnt/β-catenin pathway,inhibited abnormally activated downstream molecules of Wnt/β-catenin pathway.This may be a key mechanism of herb cake-partitioned moxibustion in inhibiting CAC tumor growth.

Objective To investigate whether cisplatin induces tumor necrosis factor-α(TNF-α)secretion in human head and neck squamous cell carcinoma(HNSCC)cells to trigger RIP1/RIP3/MLKL-dependent necroptosis of the cells.Methods HNSCC cell lines HN4 and SCC4 treated with cisplatin(CDDP)or the combined treatment with CDDP and z-VAD-fmk(a caspase inhibitor)or Nec-1(a necroptosis inhibitor)for 24 h were examined for changes in cell viability using CCK8 assay and expressions of caspase-8 and necroptosis pathway proteins(RIP1/RIP3/MLKL)using Western blotting.The changes in migration of the cells were assessed with cell scratch assay,and the expressions of epithelial-mesenchymal transition(EMT)marker proteins N-cadherin,vimentin,and E-cadherin as well as the expressions of NF-κB(p65)and TNF-α were detected with Western blotting.Results The IC50 of cisplatin was 10 μg/mL in HN4 cells and 15 μg/mL in SCC4 cells.Cisplatin treatment significantly decreased the expressions of caspase-8,N-cadherin and vimentin and increased the expressions of E-cadherin,the necroptosis pathway proteins(RIP1/RIP3/MLKL),TNF-α,and NF-κB(p65),and these changes were obviously inhibited by treatment with Nec-1.Cisplatin stimulation also significantly lowered migration of the cells,and this inhibitory effect was strongly attenuated by Nec-1 treatment.Conclusion Cisplatin activates nuclear factor-κB signaling in HNSCCs to promote TNF-α autocrine and induce RIP1/RIP3/MLKL-dependent necroptosis,thus leading to inhibition of cell proliferation.

Objective:To investigate the dynamic changes of diaphragm and limb skeletal muscle in patients with sepsis by bedside ultrasound and their correlation with the ratio of blood urea/creatinine ratio (UCR) in 7 days after intensive care unit (ICU) admission.Methods:A prospective observational study was conducted. A total of 55 patients with sepsis admitted to ICU of General Hospital of Ningxia Medical University from June 2022 to February 2023 were selected as the research objects. General information, laboratory indicators [urea, serum creatinine (SCr), and UCR] on days 1, 4, and 7 of ICU admission, and prognostic indicators were observed. Bedside ultrasound was used to assess the dynamic changes of diaphragm morphology [including diaphragmatic excursion (DE), end-inspiratory diaphragm thickness (DTei), and end-expiratory diaphragm thickness (DTee)] on days 1, 4, and 7 of ICU admission, as well as limb skeletal muscle (quadriceps femoris) morphology [including rectus femoris-muscle layer thickness (RF-MLT), vastus intermedius-muscle layer thickness (VI-MLT), and rectus femoris-cross sectional area (RF-CSA)]. Diaphragm thickening fraction (DTF) and RF-CSA atrophy rate were calculated, and the incidence of diaphragm and limb skeletal muscle dysfunction was recorded. The correlation between ultrasound morphological parameters of diaphragm and quadriceps and UCR at each time points in 7 days after ICU admission was analyzed by Pearson correlation.Results:A total of 55 patients with sepsis were included, of which 29 were in septic shock. As the duration of ICU admission increased, the incidence of diaphragm dysfunction in patients with sepsis increased first and then decreased (63.6%, 69.6%, and 58.6% on days 1, 4, and 7 of ICU admission, respectively), while the incidence of limb skeletal muscle dysfunction showed an increasing trend (54.3% and 62.1% on days 4 and 7 of ICU admission, respectively), with a probability of simultaneous occurrence on days 4 and 7 of ICU admission were 32.6% and 34.5%, respectively. The UCR on day 7 of ICU admission was significantly higher than that on day 1 [121.77 (95.46, 164.55) vs. 97.00 (70.26, 130.50)], and RF-CSA atrophy rate on day 7 was significantly higher than that on day 4 [%: -39.7 (-52.4, -22.1) vs. -26.5 (-40.2, -16.4)]. RF-CSA was significantly lower on day 7 compared to day 1 [cm 2: 1.3 (1.0, 2.5) vs. 2.1 (1.7, 2.9)], with all differences being statistically significant (all P < 0.05). Pearson correlation analysis showed that RF-CSA on day 7 of ICU admission was negatively associated with the UCR on the same day ( r = -0.407, P = 0.029). Conclusions:Diaphragmatic dysfunction in patients with sepsis occurred early and can be improved. Limb skeletal muscle dysfunction occurred relatively later and progresses progressively. The RF-CSA on day 7 of ICU admission may be a reliable measure of limb skeletal muscle dysfunction in patients with sepsis, can be an indicator of early identification and diagnosis of ICU-acquired weakness (ICU-AW). Continuous loss of muscle mass occurring in septic patients is mainly associated with persistent organismal catabolism, and undergoes significant changes around a week in ICU.

Objective To investigate whether cisplatin induces tumor necrosis factor-α(TNF-α)secretion in human head and neck squamous cell carcinoma(HNSCC)cells to trigger RIP1/RIP3/MLKL-dependent necroptosis of the cells.Methods HNSCC cell lines HN4 and SCC4 treated with cisplatin(CDDP)or the combined treatment with CDDP and z-VAD-fmk(a caspase inhibitor)or Nec-1(a necroptosis inhibitor)for 24 h were examined for changes in cell viability using CCK8 assay and expressions of caspase-8 and necroptosis pathway proteins(RIP1/RIP3/MLKL)using Western blotting.The changes in migration of the cells were assessed with cell scratch assay,and the expressions of epithelial-mesenchymal transition(EMT)marker proteins N-cadherin,vimentin,and E-cadherin as well as the expressions of NF-κB(p65)and TNF-α were detected with Western blotting.Results The IC50 of cisplatin was 10 μg/mL in HN4 cells and 15 μg/mL in SCC4 cells.Cisplatin treatment significantly decreased the expressions of caspase-8,N-cadherin and vimentin and increased the expressions of E-cadherin,the necroptosis pathway proteins(RIP1/RIP3/MLKL),TNF-α,and NF-κB(p65),and these changes were obviously inhibited by treatment with Nec-1.Cisplatin stimulation also significantly lowered migration of the cells,and this inhibitory effect was strongly attenuated by Nec-1 treatment.Conclusion Cisplatin activates nuclear factor-κB signaling in HNSCCs to promote TNF-α autocrine and induce RIP1/RIP3/MLKL-dependent necroptosis,thus leading to inhibition of cell proliferation.

BACKGROUND: The hepatitis B virus (HBV) vaccine has been efficiently used for decades. However, hepatocellular carcinoma caused by HBV is still prevalent globally. We previously reported that interferon (IFN)-induced tripartite motif-containing 25 (TRIM25) inhibited HBV replication by increasing the IFN expression, and this study aimed to further clarify the anti-HBV mechanism of TRIM25. METHODS: The TRIM25-mediated degradation of hepatitis B virus X (HBx) protein was determined by detecting the expression of HBx in TRIM25-overexpressed or knocked-out HepG2 or HepG2-NTCP cells via Western blotting. Co-immunoprecipitation was performed to confirm the interaction between TRIM25 and HBx, and colocalization of TRIM25 and HBx was identified via immunofluorescence; HBV e-antigen and HBV surface antigen were qualified by using an enzyme-linked immunosorbent assay (ELISA) kit from Kehua Biotech. TRIM25 mRNA, pregenomic RNA (pgRNA), and HBV DNA were detected by quantitative real-time polymerase chain reaction. The retinoic acid-inducible gene I (RIG-I) and pgRNA interaction was verified by RNA-binding protein immunoprecipitation assay. RESULTS: We found that TRIM25 promoted HBx degradation, and confirmed that TRIM25 could enhance the K90-site ubiquitination of HBx as well as promote HBx degradation by the proteasome pathway. Interestingly, apart from the Really Interesting New Gene (RING) domain, the SPRY domain of TRIM25 was also indispensable for HBx degradation. In addition, we found that the expression of TRIM25 increased the recognition of HBV pgRNA by interacting with RIG-I, which further increased the IFN production, and SPRY, but not the RING domain is critical in this process. CONCLUSIONS The study found that TRIM25 interacted with HBx and promoted HBx-K90-site ubiquitination, which led to HBx degradation. On the other hand, TRIM25 may function as an adaptor, which enhanced the recognition of pgRNA by RIG-I, thereby further promoting IFN production. Our study can contribute to a better understanding of host-virus interaction.
Humans ; Hepatitis B virus ; DEAD Box Protein 58/metabolism* ; RNA ; Liver Neoplasms ; Virus Replication ; Tripartite Motif Proteins/genetics* ; Transcription Factors ; Ubiquitin-Protein Ligases/genetics*

The nucleus tractus solitarii (NTS) is one of the morphologically and functionally defined centers that engage in the autonomic regulation of cardiovascular activity. Phenotypically-characterized NTS neurons have been implicated in the differential regulation of blood pressure (BP). Here, we investigated whether phenylethanolamine N-methyltransferase (PNMT)-expressing NTS (NTS^PNMT) neurons contribute to the control of BP. We demonstrate that photostimulation of NTS^PNMT neurons has variable effects on BP. A depressor response was produced during optogenetic stimulation of NTS^PNMT neurons projecting to the paraventricular nucleus of the hypothalamus, lateral parabrachial nucleus, and caudal ventrolateral medulla. Conversely, photostimulation of NTS^PNMT neurons projecting to the rostral ventrolateral medulla produced a robust pressor response and bradycardia. In addition, genetic ablation of both NTS^PNMT neurons and those projecting to the rostral ventrolateral medulla impaired the arterial baroreflex. Overall, we revealed the neuronal phenotype- and circuit-specific mechanisms underlying the contribution of NTS^PNMT neurons to the regulation of BP.
Solitary Nucleus/metabolism* ; Blood Pressure/physiology* ; Phenylethanolamine N-Methyltransferase/metabolism* ; Neurons/metabolism* ; Paraventricular Hypothalamic Nucleus/metabolism*

Leptin, an adipocyte-derived peptide hormone, has been shown to facilitate breathing. However, the central sites and circuit mechanisms underlying the respiratory effects of leptin remain incompletely understood. The present study aimed to address whether neurons expressing leptin receptor b (LepRb) in the nucleus tractus solitarii (NTS) contribute to respiratory control. Both chemogenetic and optogenetic stimulation of LepRb-expressing NTS (NTS^LepRb) neurons notably activated breathing. Moreover, stimulation of NTS^LepRb neurons projecting to the lateral parabrachial nucleus (LPBN) not only remarkably increased basal ventilation to a level similar to that of the stimulation of all NTS^LepRb neurons, but also activated LPBN neurons projecting to the preBötzinger complex (preBötC). By contrast, ablation of NTS^LepRb neurons projecting to the LPBN notably eliminated the enhanced respiratory effect induced by NTS^LepRb neuron stimulation. In brainstem slices, bath application of leptin rapidly depolarized the membrane potential, increased the spontaneous firing rate, and accelerated the Ca²⁺ transients in most NTS^LepRb neurons. Therefore, leptin potentiates breathing in the NTS most likely via an NTS-LPBN-preBötC circuit.
Leptin/pharmacology* ; Membrane Potentials ; Neurons/metabolism* ; Solitary Nucleus/metabolism*

Objective:To evaluate the risk factors for diaphragmatic dysfunction of patients with sepsis and septic shock, and the application value of bedside ultrasound.Methods:Patients with sepsis and septic shock in the Intensive Care Unit (ICU), General Hospital of Ningxia Medical University from January 2020 to May 2021 were prospectively recruited as the research subjects, general postoperative patients and healthy volunteers were admitted as postoperative control and normal control groups. General clinical data were collected, patients with sepsis and septic shock were dynamically observed high sensitive c-reactive protein (hs-CRP), interleukin-6 (IL-6), serum albumin, transferrin, prealbumin levels, blood lactate, Pcv-aCO 2, ScvO 2, etc.; and indirect calorimetry was used to measure the resting energy level of the patient to calculate the missing energy value. Bedside ultrasound was used to dynamically evaluate the changes of diaphragm excursion (DE),inspiratory diaphragm thickness, and expiratory diaphragm thickness, to calculate relevant parameters. DE<10 mm or diaphragmatic thickness fraction (DTF) < 20% was diagnosed as diaphragmatic dysfunction. Results:(1) On day 1 in the ICU, the DE of the septic shock group, sepsis group and postoperative control group were significantly lower than that in the normal control group [10.3 (9.0, 13.6) mm, 12.3 (9.1, 15.0) mm, 12.9 (10.5, 15.7) mm vs. 22.0 (16.0, 24.6) mm, all P<0.05], and the incidence of DTF<20% was significantly higher than in the normal control group (32.7%, 41.9%, 33.3% vs. 0 %, all P<0.05), and the incidence of DE<10 mm in the septic shock group and sepsis group was significantly higher than that of postoperative control group and normal control group (36.7%, 35.5% vs. 10.0%, 0%, respectively, all P<0.05). On day 7, the DE in the septic shock group was significantly lower than that in the sepsis group [10.5 (6.8, 13.5) mm vs. 14.4 (10.6, 18.6) mm, P<0.05].(2) Correlation analysis of each index: The DE of patients with sepsis and septic shock on day 1, 3, and 7 was negatively correlated with the hs-CRP ( r=-0.253, -0.436, -0.455, all P<0.05); On day 3, DE was also negatively correlated with IL-6 ( r=-0.338, P=0.009); and DTF was negatively correlated with hs-CRP ( r=-0.375, P=0.004). On day 1, there was a positive correlation between DTF and serum transferrin levels in patients with sepsis and septic shock ( r=0.221, P=0.049). On day 3 and 7, the DE was positively correlated with serum prealbumin levels ( r=0.318, 0.408, both P<0.05). Conclusions:Patients with sepsis and septic shock have developed diaphragmatic dysfunction on day 1 in the ICU, which is mainly manifested as decreased in diaphragm mobility and diaphragmatic thickness fraction, and is related to inflammation and high protein catabolism.

Objective:To explore the value of current indications for fetal pulmonary valvuloplasty (FPV) by summarizing the postnatal diagnosis, treatment, and prognosis of fetuses with pulmonary atresia with intact ventricular septum (PA/IVS) and right ventricular hypoplasia (RVH).Methods:This prospective study was conducted at the Heart Center of Women and Children's Hospital, Qingdao University from September 2018 to March 2021, which included pregnant women who were (1) with fetal PA/IVS and RVH; (2) unable to receive FPV due to fetal position or gestational age despite the indications; (3) given integrated pre- and postnatal management. Prenatal fetal echocardiography assessment, postnatal diagnosis, treatment, and follow-up were summarized using Wilcoxon matched-pair signed-rank test.Results:A total of 35 singleton pregnant women were diagnosed with fetal PA/IVS and RVH by ultrasonic cardiogram and admitted during the study period. Among the 28 fetuses meeting the FPV indications, 18 underwent FPV, while the other 10 did not due to inappropriate fetal position or gestational age. After excluding four terminated pregnancies, the rest six cases were enrolled. The median gestational age at the initial prenatal fetal echocardiography diagnosis was 28.9 weeks (28.3-30.4 weeks). Compared with the initial evaluation, the fetal right ventricular to left ventricular length/diameter ratio [0.8 (0.6-0.9) vs 0.6 (0.5-0.8)] and tricuspid regurgitation velocity [4.7 m/s (3.2-5.1 m/s) vs 4.1 m/s (3.3-4.8 m/s)] were increased, while tricuspid valve Z value [－0.8(－1.6-0.8) vs 0.4 (－0.3-1.9)] and single-ventricular predictive score [0.5 (0.0-2.0) vs 2.0 (1.0-3.0)] were decreased when re-evaluated six weeks later ( T were－2.21, 2.00,－2.20, and 2.00; all P<0.05). All of the six fetuses were born alive with a median gestational age of 38.9 weeks (37.3-40.1 weeks). The median weight was 3 425 g (3 100-4 160) g after being transferred to cardiac intensive care unit. The median age was 12.5 d (0.0-20.0 d) at the first surgical intervention. The median follow-up duration was 15 months (11.8-18.5 months). At initial diagnosis, the single-ventricular predictive score was 1-2 points in four fetuses, and =3 points in two fetuses. There was no death during follow-up. Four patients achieved anatomical biventricular circulation, one achieved clinical biventricular circulation, and one still needed further follow-up, with single-ventricular predictive score at initial diagnosis of 1-3, 3, and 2 points, respectively. Conclusions:The prognosis is good in fetuses with PA/IVS and RVH who have FPV indications but do not receive intrauterine intervention, which suggests that the current FPV indications may be too broad, and a more suitable FPV indication need to be further explored given the difficulty of implementing FPV.