BACKGROUND:Human periodontal ligament stem cells are potential functional cells for periodontal tissue engineering.However,long-term in vitro culture may lead to reduced stemness and replicative senescence of periodontal ligament stem cells,which may impair the therapeutic effect of human periodontal ligament stem cells. OBJECTIVE:To investigate the effect of oxymatrine on the stemness maintenance and osteogenic differentiation of periodontal ligament stem cells in vitro,and to explore the potential mechanism. METHODS:Periodontal ligament stem cells were isolated from human periodontal ligament tissues by tissue explant enzyme digestion and cultured.The surface markers of mesenchymal cells were identified by flow cytometry.Periodontal ligament stem cells were incubated with 0,2.5,5,and 10 μg/mL oxymatrine.The effect of oxymatrine on the proliferation activity of periodontal ligament stem cells was detected by CCK8 assay.The appropriate drug concentration for subsequent experiments was screened.Western blot assay was used to detect the expression of stem cell non-specific proteins SOX2 and OCT4 in periodontal ligament stem cells.qRT-PCR and western blot assay were used to detect the expression levels of related osteogenic genes and proteins in periodontal ligament stem cells. RESULTS AND CONCLUSION:(1)The results of CCK8 assay showed that 2.5 μg/mL oxymatrine significantly enhanced the proliferative activity of periodontal stem cells,and the subsequent experiment selected 2.5 μg/mL oxymatrine to intervene.(2)Compared with the blank control group,the protein expression level of SOX2,a stem marker of periodontal ligament stem cells in the oxymatrine group did not change significantly(P>0.05),and the expression of OCT4 was significantly up-regulated(P<0.05).(3)Compared with the osteogenic induction group,the osteogenic genes ALP,RUNX2 mRNA expression and their osteogenic associated protein ALP protein expression of periodontal ligament stem cells were significantly down-regulated in the oxymatrine+osteogenic induction group(P<0.05).(4)The oxymatrine up-regulated the expression of stemness markers of periodontal ligament stem cells and inhibited the bone differentiation of periodontal ligament stem cells,and the results of high-throughput sequencing showed that it may be associated with WNT2,WNT16,COMP,and BMP6.

Objective To compare the effect of discrete trial training(DTT),pivotal response treatment(PRT)and a combination of DTT and PRT on joint attention in children aged three to six years with mild to moderate autism spectrum disor-der(ASD). Methods From January,2023 to March,2024,39 children with ASD aged 36 to 72 months in Tiger Children's Rehabili-tation Center in Shanghai were randomly divided into DTT,PRT and combination groups,who received DTT,PRT and a combination of DTT and PRT,respectively,for ten weeks.They were assessed with Joint Attention As-sessment Scale for children with ASD before and after intervention. Results Two cases in DTT group and one case in PRT group dropped down,resulting in a final sample of 36 cases.The main effects of group(F=11.225,P<0.001)and time(F=416.935,P<0.001)were significant,as well as the interaction(F=10.501,P<0.001),and the combination group was the best during intervention and follow-up(P<0.05). Conclusion Both DTT and PRT may improve joint attention in children with ASD,and the combination of DTT and PRT is the best.

The traditional diagnosis of neuropsychiatric disorders mainly depends on the subjective evaluation of specialists,neuropsychological test,biochemical examination and other methods,which lacks objective,accurate and intelligent biomarkers.With the rapid development of neuroimaging and artificial intelligence technology,unsupervised learning has been widely used in the auxiliary diagnosis of neuropsychiatric disorders for it has the advantages of independence of external labels,high model generalization,and automatic feature extraction.Compared with the traditional supervised learning methods,unsupervised learning is more capable of achieving objective,accurate and intelligent diagnosis of neuropsychiatric disorders.Herein an overview on the applications of unsupervised learning in the auxiliary diagnosis of neuropsychiatric disorders is provided,summarizing the findings of unsupervised learning in Alzheimer's disease,schizophrenia,major depressive disorder,and autism spectrum disorder,and discussing the research challenges such as insufficient image processing capability,small sample size,insufficient biochemical index data.The corporation with neural network,multi-site large sample size,and deep fusion of multidimensional data are the development trends of unsupervised learning method.

Objective:To observe the effects of acupuncture and moxibustion on ubiquitin-like containing PHD and RING finger domains 1(UHRF1)and DNA methyltransferase 1(DNMT1)in ectopic endometrium of rats with endometriosis(EMS). Methods:Forty Sprague-Dawley rats were randomly divided into a sham operation group with 10 rats and a model-building group with 30 rats according to body mass.EMS rat models were established in the model-building group and then were divided into a model group,an acupuncture and moxibustion group,and a progesterone group,with 10 rats in each group.All rats were fixed by a fixator.The sham operation group and the model group were given normal saline by gavage.The acupuncture and moxibustion group received acupuncture at Xuehai(SP10)and Sanyinjiao(SP6),moxibustion at Guanyuan(CV4),and gavage of normal saline.The progesterone group was given the mixed liquid made of dydrogesterone and normal saline by gavage.After 28 d of treatments,the three diameters(length,width,and height)of EMS rats'ectopic cysts were measured,the cyst volumes were calculated,the volumes before intervention were subtracted,and the difference values were used to evaluate the growth of ectopic cysts.UHRF1 and DNMT1 mRNA and protein levels in normal endometrium,eutopic endometrium,and ectopic endometrium were detected by real-time polymerase chain reaction and immunohistochemistry. Results:There was no significant difference in the ectopic cyst volume difference between the acupuncture and moxibustion group and the progesterone group(P>0.05),but they were smaller than that of the model group(P<0.05).The levels of UHRF1 and DNMT1 mRNA and protein in the ectopic endometrium of the model group were lower than those in the normal endometrium(P<0.05).The levels of DNMT1 mRNA and UHRF1 protein in the eutopic endometrium of the model group were lower than those in the normal endometrium(P<0.05).The levels of UHRF1 mRNA and protein and the level of DNMT1 protein in the ectopic endometrium of the acupuncture and moxibustion group were higher than those in the model group(P<0.05),and the level of UHRF1 mRNA was higher than that in the progesterone group(P<0.05).The level of DNMT1 mRNA in the eutopic endometrium of the acupuncture and moxibustion group was higher than that in the model group(P<0.05).The levels of UHRF1 and DNMT1 mRNA and protein in the acupuncture and moxibustion group were insignificantly different from those in the normal endometrium(P>0.05). Conclusion:Acupuncture and moxibustion may up-regulate the levels of UHRF1 mRNA and UHRF1 and DNMT1 proteins in the ectopic endometrium to the normal level so as to reduce the volume of ectopic cysts and cure EMS in rats.

Objective To explore the causal relationship between social stress and tinnitus onset using a two-sample Mendelian randomization approach. Methods Genetic data pertaining to social stress and tinnitus were extracted from genome-wide association studies (GWAS) databases. Single nucleotide polymorphisms (SNP) that were independent and strongly correlated with social stress were selected as instrumental variables, with tinnitus serving as the outcome variable. Mendelian randomization analyses were performed using inverse-variance weighted (IVW) method, weighted median method, and Mendelian randomization-Egger regression. The intercept term from Mendelian randomization-Egger regression was utilized to assess horizontal pleiotropy, Cochran's Q statistic was employed to evaluate heterogeneity, and leave-one-out analysis was conducted for sensitivity assessment. Results The social stress dataset encompassed 459, 742 samples, while the tinnitus dataset comprised 117, 882 samples. A total of 10 SNPs tightly associated with social stress were identified as instrumental variables. The analysis results of random-effects inverse variance weighting (IVW), weighted median method, and Mendelian randomization-Egger regression were similar (OR=1.251, 1.274, 1.438), indicating that social stress was a risk factor for tinnitus, and there was a positive causal effect between them, with no heterogeneity or pleiotropy in the results. Conclusion There is a causal relationship between social stress and tinnitus onset, which may offer novel perspectives for the clinical prevention and treatment of tinnitus in the future.

There is a certain relationship between chemotherapy and stroke in cancer patients. Its mechanism may be associated with the increase of the prevalence of traditional vascular factors, the promotion of coagulation dysfunction, the induction of anemia, the impairment of cardiac function, and vascular inflammation. The pathophysiological mechanism of chemotherapy-associated stroke is still in the exploratory stage. This article reviews the pathophysiological mechanism, monitoring indicators, and diagnosis and treatment progress of stroke in cancer patients during chemotherapy.

RAS, a member of the small GTPase family, functions as a binary switch by shifting between inactive GDP-loaded and active GTP-loaded state. RAS gain-of-function mutations are one of the leading causes in human oncogenesis, accounting for ∼19% of the global cancer burden. As a well-recognized target in malignancy, RAS has been intensively studied in the past decades. Despite the sustained efforts, many failures occurred in the earlier exploration and resulted in an 'undruggable' feature of RAS proteins. Phosphorylation at several residues has been recently determined as regulators for wild-type and mutated RAS proteins. Therefore, the development of RAS inhibitors directly targeting the RAS mutants or towards upstream regulatory kinases supplies a novel direction for tackling the anti-RAS difficulties. A better understanding of RAS phosphorylation can contribute to future therapeutic strategies. In this review, we comprehensively summarized the current advances in RAS phosphorylation and provided mechanistic insights into the signaling transduction of associated pathways. Importantly, the preclinical and clinical success in developing anti-RAS drugs targeting the upstream kinases and potential directions of harnessing allostery to target RAS phosphorylation sites were also discussed.

With the advancements of stem cells and regenerative medicine, interspecies chimera has become a hot topic and will pave a new way of providing donor sources in organ transplantation. However, the interspecies chimera is confronted with a number of scientific questions and technical obstacles, including selections of appropriate embryonic stage and appropriate culture medium; those factors will deeply influence the developmental balance between donor cells and receptor embryos. Due to its relatively rapid reproductive cycle and similar organ size to human's, porcine is a very potential donor candidate to study these questions. To compare the development and chimeric efficiency of interspecies embryos, we tested and evaluated three different culture systems, PZM-3 (Porcine zygotic medium), culture medium for iPSCs (N2B27) and 3.5 h of N2B27 before PZM-3 (N2B27(3.5 h)), and two different embryonic stages, 8-cell and blastocyst in mouse-porcine chimeric embryos using parthenogenetically activated porcine embryos and mouse induced pluripotent stem cells (miPS). The results showed that, PZM-3 was beneficial for both development of chimeric embryos and miPSCs proliferation in porcine embryos in the 8-cell injection group. After early blastocyst injection, the chimeric efficiency did not appear significantly different among the three culture systems but was lower than 8-cell injection. In summary, the results suggest that 8-cell injection and PZM-3 culture medium are more beneficial to the in vitro development and chimeric efficiency of mouse-porcine chimeric embryos.
Animals ; Blastocyst ; Chimera ; Culture Media ; Embryo Culture Techniques ; veterinary ; Embryo, Mammalian ; Embryonic Development ; Induced Pluripotent Stem Cells ; cytology ; Mice ; Swine

Objective To investigate the production and mechanism of chemokine (C-C motif) ligand 5 (CCL5) by macrophages in U14 cervical cancer-bearing mice during infection. Methods The U14 cervical cancer cells were injected in C57BL/6 mice to induce tumor-bearing condition. Lipopolysaccharide (LPS) was injected into C57BL/6 mice to induce infection. The protein expression of CCL5 in the serum and the CCL5 mRNA expression in inflammatory cells were measured by ELISA and fluorescence quantitative-PCR in four groups. Macrophages were induced in the tumor conditioned medium (TCM) which extracted from mice serum. The protein expression levels of CCL5, prostaglandin E2 (PGE2) and cyclic adenosine monophosphate (cAMP) in the medium and CCL5, PGE2 and cAMP mRNA expression in the macrophages were detected in different groups. In order to determine whether the inhibition was related to PGE2, selective cyclooxygenase 2(COX-2) inhibitor NS398 was used to reverse this phenomenon and protein kinase A (PKA) inhibitor H89 demonstrated the mechanism through blocking cAMP/PKA signaling pathway. Results (1) The protein and mRNA level of CCL5 in tumor-bearing mice were respectively (151±35) pg/ml and 1.0, which were lower than those in the tumor-free mice (691 ± 85) pg/ml and 4.5 ± 0.8, there were significant difference between them (all P<0.05). The protein and mRNA level of PGE2 in tumor-bearing mice were (1 198±83) pg/ml and 5.8±0.8, which were higher than those in the tumor-free mice (187±25) pg/ml and 1.0, the difference were significant (all P<0.05). The protein and mRNA level of CCL5 in tumor-free+LPS mice were (4 049±141) pg/ml and 31.5±2.0, which were higher than those in the tumor-bearing+LPS mice (1 951±71) pg/ml and 12.1±2.8, the difference were also significant (P<0.05). The protein and mRNA level of PGE2 in tumor-free+LPS mice were (676±70) pg/ml and 3.4±0.4, which were lower than those in tumor-bearing+LPS mice (2 550±382) pg/ml and 11.6±0.9, the difference were also significant (all P<0.05). (2) Macrophages were cultured in vitro using TCM derived from mice. The protein and mRNA level of CCL5 in tumor-bearing mice TCM were respectively (1 626 ± 177) pg/ml and 28.6 ± 1.2, which were higher than those in the tumor-free mice TCM [(27 ± 3) pg/ml and 1.0], there were significant difference (P<0.05). The protein and mRNA level of PGE2 in tumor-bearing mice TCM were (790 ± 156) pg/ml and 1.7 ± 0.3, which were higher than those in the tumor-free mice TCM [(448 ± 115) pg/ml, 1.0], the difference were significant (all P<0.05). The protein and mRNA level of cAMP in tumor-bearing mice TCM were (164 ± 30) pg/ml and 1.6 ± 0.3, which weres higher than those in the tumor-free mice TCM [(118 ± 25) pg/ml,1.0], the difference were significant (all P<0.05). The protein and mRNA level of CCL5 in tumor-free + LPS mice TCM were (10 475 ± 742) pg/ml and 212.0 ± 5.7, which were higher than those in the tumor-bearing+LPS mice TCM [(6 375±530) pg/ml, 142.3±2.5], the difference were significant (all P<0.05). The protein and mRNA level of PGE2 in tumor-free+LPS mice TCM were (2 438±95) pg/ml and 4.3±0.7, which weres lower than those in the tumor-bearing + LPS mice TCM [(3 441 ± 163) pg/ml, 5.9 ± 0.3], the difference were significant (all P<0.05). The protein and mRNA level of cAMP in tumor-free+LPS mice TCM were (340 ± 13) pg/ml and 4.1 ± 0.4, which were lower than those in the tumor-bearing + LPS mice TCM [(542 ± 42) pg/ml, 5.4 ± 0.5], the difference were significant (all P<0.05). (3) Using COX-2 inhibitor NS398 in the tumor-bearing+LPS mice, the protein and mRNA level of CCL5, PGE2 and cAMP were (7 691±269) pg/ml and 159.0±8.9, (2 820±152) pg/ml and 4.9 ± 0.3, (465 ± 8) pg/ml and 4.3 ± 0.4, respectively, and there were significant difference (all P<0.05), compared to before treatment. Using PKA inhibitor H89 in the tumor-bearing+LPS mice, the protein and mRNA level of CCL5, PGE2 and cAMP were (8 375±520) pg/ml and 177.0±8.8, (2 650±35) pg/ml and 4.7 ± 0.4, (368 ± 13) pg/ml and 3.1 ± 0.7, respectively, and there were significant difference (all P<0.05), compared to before treatment. Conclusion TCM of U14 cells activated macrophages to release PGE2 could inhibit the expression of CCL5 levels by cAMP/PKA signaling pathway.

Insufficient epigenetic reprogramming of donor nuclei is believed to be one of the most important causes of low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Previous studies have shown that both the in vitro and in vivo development of mouse SCNT embryos could be increased significantly by treatment with various histone deacetylase inhibitors (HDACi), including Trichostatin A, Scriptaid, and m-carboxycinnamic acid bishydroxamide (CBHA), in which only the effect of CBHA has not yet been tested in other species. In this paper we examine the effect of CBHA treatment on the development of porcine SCNT embryos. We have discovered the optimum dosage and time for CBHA treatment: incubating SCNT embryos with 2 μmol/L CBHA for 24 h after activation could increase the blastocyst rate from 12.7% to 26.5%. Immunofluorescence results showed that the level of acetylation at histone 3 lysine 9 (AcH3K9), acetylation at histone 3 lysine 18 (AcH3K18), and acetylation at histone 4 lysine 16 (AcH4K16) was raised after CBHA treatment. Meanwhile, CBHA treatment improved the expression of development relating genes such as pou5f1, cdx2, and the imprinted genes like igf2. Despite these promising in vitro results and histone reprogramming, the full term development was not significantly increased after treatment. In conclusion, CBHA improves the in vitro development of pig SCNT embryos, increases the global histone acetylation and corrects the expression of some developmentally important genes at early stages. As in mouse SCNT, we have shown that nuclear epigenetic reprogramming in pig early SCNT embryos can be modified by CBHA treatment.
Acetylation ; Animals ; Blastocyst ; cytology ; Cell Nucleus ; metabolism ; Cinnamates ; pharmacology ; Embryo, Mammalian ; drug effects ; metabolism ; Embryonic Development ; drug effects ; Epigenesis, Genetic ; Female ; Gene Expression ; Histone Deacetylase Inhibitors ; pharmacology ; Histones ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; In Vitro Techniques ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Nuclear Transfer Techniques ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Swine