Perilla frutescens (L.) Britt. is a characteristic oil crop rich in polyunsaturated fatty acids, particularly α-linolenic acid, which has important development and utilization value. The Dof transcription factor is one of the plant-specific transcription factor families, which is widely involved in important biological processes such as plant growth, development, and metabolic regulation. In order to explore the key Dof transcription factors involved in the oil biosynthesis and systematically analyze their regulatory mechanisms of P. frutescens seeds, a total of 56 PfDof gene family members were identified from the genome and transcriptome data of P. frutescens and classified into four subfamilies according to sequence characteristics. All PfDofs contained highly conserved C₂-C₂ zinc finger domains, with gene duplication being the primary mechanism driving their evolution and expansion. Genes within the same subgroup exhibited similar gene structures and conserved motifs. The 56 PfDofs were predicted as unstable hydrophilic proteins, with α-helixes and random coils as their predominant structural components. The RNA-seq results revealed that 11 PfDofs exhibited differential expression during different developmental stages of P. frutescens seeds. RT-qPCR was performed to further validate the expression patterns of these 11 members across various tissue samples (root, stem, leaf, and flower) of P. frutescens and at different developmental stages of its seeds. The results showed that PfDof29 exhibited the highest expression level in seeds, which was consistent with the transcriptome data. Subcellular localization studies demonstrated that PfDof29 was localized to the nucleus and had a transcriptional activation activity. Overexpression of PfDof29 in Nicotiana tabacum resulted in a significant increase in total oil content of tobacco leaves, accompanied by reductions in starch and soluble sugar content, while the protein content remained unchanged. Additionally, the metabolic balance between saturated and unsaturated fatty acids in the transgenic tobacco leaves was altered, with a significant increase in α-linolenic acid content. The expression levels of the fatty acid desaturase genes NtFAD2, NtFAD3, and NtFAD8 were significantly upregulated. A yeast one-hybrid assay revealed that PfDof29 could directly bind to the promoter region of PfFAD8, thereby regulating its expression. This study provides an initial understanding of the regulatory mechanisms of PfDof transcription factors in the synthesis and accumulation of oil in P. frutescens. These findings offer new insights into the enhancement of oil content and quality of P. frutescens seeds.
Transcription Factors/physiology* ; Perilla frutescens/metabolism* ; Plant Proteins/metabolism* ; Gene Expression Regulation, Plant ; alpha-Linolenic Acid/biosynthesis* ; Lipids/biosynthesis* ; Seeds/genetics*

Environmental Microbiology, as a compulsory foundational course for the major of environmental and ecological engineering. Its knowledge system not only lays a profound theoretical foundation but also evinces a close-knit nexus to practical applications in environmental protection and ecological restoration. This course thus serves as an outstanding platform facilitating both teaching reforms and practical innovations of professional courses. With Environmental Microbiology as a paradigmatic illustration, this paper concentrates on addressing issues like archaic course contents and enervating teaching approaches, with the aspiration of augmenting students' professional acuities, practical dexterities, and innovation and entrepreneurial aptitudes. In light of the aforesaid aim, we initiated an all-encompassing probe into the teaching reform and practicable paradigms from the perspective of amalgamating professional education with innovation-steered stratagems. Across multiple fronts, comprehensive reformative undertakings were implemented, subsuming the optimization and reconfiguration of course contents, the exploration of sundry teaching methodologies, and the scientific erection of an evaluation system. Ultimately, a nascent teaching model dubbed "three-dimensional, six-loop and five-collaborative" was formed. This series of comprehensive and innovative reform measures has not only substantially augmented the profundity and extensiveness of professional knowledge, permeating into the core of disciplinary essence, but also remarkably bolstered students' practical operational capabilities and innovative thinking patterns, thereby catalyzing their transformation into well-rounded individuals with a competitive edge. By virtue of these efforts, a solid foundation has been firmly laid for the teaching reform of Environmental Microbiology and the cultivation of high-caliber environmental professionals in the contemporary era, heralding a new chapter in the development of environmental education.
Environmental Microbiology ; Teaching ; Education, Professional/methods* ; Curriculum

Objective:To discuss the effect of KH-type splicing regulatory protein(KHSRP)on the malignant biological behaviors of colorectal cancer(CRC)by activating the Janus kinase(JAK)/signal transducer and activator of transcription(STAT)signaling pathway,and to clarify its possible mechanism.Methods:The CRC tissue and adjacent normal tissue from 64 CRC patients were selected.The human CRC cells(HT29,SW620,SW480,DLD-1,LOVO,and RKO)and normal human colorectal mucosal FHC cells were cultured in vitro.The total RNA from CRC tissue and cells were extracted,real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression levels of KHSRP in the CRC tissue,adjacent normal tissue and all kinds of cells.The HT29 and SW620 cells were divided into sh-NC group(lentiviral plasmid inserted with non-targeting nucleotide sequence)and sh-KHSRP group(transfected with KHSRP knockdown lentivirus).The SW480 and DLD-1 cells were divided into oe-NC group(lentiviral plasmid inserted with non-targeting nucleotide sequence)and oe-KHSRP group(transfected with KHSRP overexpression lentivirus).Immunohistochemistry(IHC)staing in method was used to analyze the expressions of KHSRP in CRC tissue and adjacent normal tissue;CCK-8 method was used to detect the proliferation activities of the CRC cells in various groups;Transwell assay was used to detect the numbers of migration and invasion cells in various groups;Western blotting method was used to detect the expression levels of KHSRP,JAK1,phosphorylated JAK1(p-JAK1),JAK2,phosphorylated JAK2(p-JAK2),STAT1,STAT2,STAT3,and STAT5 proteins in the CRC cells in various groups.The subcutaneous xenograft tumor models in the nude mice were used to measure the tumor volumes and weights of the mice in various groups.Results:Compared with adjacent normal tissue,the expression level of KHSRP in the CRC tissue was increased(P<0.05).Compared with FHC cells,the expression levels of KHSRP in the CRC cells were increased(P<0.05).Therefore,the HT29 and SW620 cells were selected for knockdown of KHSRP,while the SW480 and DLD-1 cells were selected for over-expression of KHSRP.The Western blotting results showed that the expression amounts of KHSRP protein in the CRC tissue and cells were higher than those in adjacent normal tissue and FHC cells.The IHC results showed that compared with adjacent normal tissue,the expression level of KHSRP protein in CRC tissue was increased(P<0.01).The RT-qPCR results showed that compared with sh-NC group,the expression levels of KHSRP mRNA in the HT29 and SW620 cells in sh-KHSRP group were decreased(P<0.01);compared with oe-NC group,the expression levels of KHSRP mRNA in the SW480 and DLD-1 cells in oe-KHSRP group were increased(P<0.01),indicating successful transfection.The CCK-8 results showed that compared with sh-NC group,the proliferation activities of the HT29 and SW620 cells in sh-KHSRP group after knockdown of KHSRP were decreased(P<0.05 or P<0.01);compared with oe-NC group,the proliferation activities of the SW480 and DLD-1 cells in oe-KHSRP group after over-expression of KHSRP were increased(P<0.05 or P<0.01).Compared with sh-NC group,the numbers of migration and invasion cells in the HT29 and SW620 cells in sh-KHSRP group after knockdown of KHSRP were decreased(P<0.05);compared with oe-NC group,the numbers of migration and invasion cells in the CRC cells in oe-KHSRP group after over-expression of KHSRP were increased(P<0.05).After knockdown of KHSRP,the tumor volume and weight in sh-KHSRP group were smaller than those in sh-NC group(P<0.05 or P<0.01),while the tumor volume andweight in oe-KHSRP group were larger than those in oe-NC group after over-expression of KHSRP(P<0.05 or P<0.01).Compared with sh-NC group,the expression levels of JAK1,p-JAK1,and STAT3 proteins in the CRC cells in sh-KHSRP group were significantly decreased(P<0.05 or P<0.01);compared with oe-NC group,the expression levels of JAK1,p-JAK1,and STAT3 proteins in the CRC cells in oe-KHSRP group were significantly increased(P<0.05 or P<0.01).Conclusion:High expression of KHSRP promotes the proliferation,migration,and invasion of the CRC cells and enhances the growth of subcutaneous xenograft tumors in the mice;its mechanism may be associated with its activation of the JAK1/STAT3 signaling pathway.

Objective To investigate the effects of sleep deprivation on the firing activity of hippocampal parvalbumin-expressing interneuron(PV-IN)of mice during trace eyeblink conditioning(tEBC)training and during recovery sleep.Methods A total of 15 PV-Cre mice were randomly assigned into a sleep deprivation group(n=8)and a control group(n=7).After daily tEBC training,the sleep deprivation group experienced 1.5 h of sleep deprivation followed by 1.5 h of recovery sleep,and the control group mice were allowed to sleep freely,for 5 consecutive days.Multichannel recording and optogenetic techniques was applied to identify the hippocampal PV-IN in vivo.Results The sleep deprived mice failed to show significant increase in the incidence of conditioned eyeblink response(CR)across 5 training days(P>0.05).On training days 3～5,the CR incidence was significantly lower in the sleep deprived mice than the control mice(P<0.05).The enhancement of conditioned stimulus(CS)-evoked firing activity in hippocampal PV-INs during tEBC training was significantly lower in the sleep deprived mice when compared to the control mice(P<0.05).The sleep deprived mice obtained notably strengthened firing activity of hippocampal PV-INs peri sharp wave ripple events during recovery sleep period when compared with the pre-training sleep period(P<0.05),and the enhancement was remarkably greater than that in the control mice(P<0.05).Sleep deprivation had no obvious effects on the firing activity of hippocampal non PV-IN during either tEBC training or recovery sleep(P>0.05).Conclusion Sleep deprivation produces dual effects on the firing activities of hippocampal PV-INs during tEBC training and recovery sleep.These effects may be the cellular mechanisms of tEBC memory formation and consolidation disorders caused by sleep deprivation.

Objective To systematically identify the key genes regulating the exit from na?ve pluripotency in embryonic stem cells(ESCs)in order to provide novel targets and theoretical insights into the mechanisms for pluripotency transition and early cell fate determination.Methods Nanog-green fluorescent protein(Nanog-GFP)reporter-labeled ESCs were infected with a genome-wide Brie knockout library,and further cultured under leukemia inhibitory factor/serum(LIF/S)conditions for 14 d.Flow cytometry was used to sort Nanog-GFP?(na?ve-state)and Nanog-GFP-(primed state)cell populations,followed by genomic DNA extraction and high-throughput sequencing.Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout(MAGeCK)was applied to identify differential genes between GFP?/Input,GFP?/Input,and GFP?/GFP? groups.Metascape and Gene Set Enrichment Analysis(GSEA)were conducted for functional enrichment analysis.Then the obtained candidate genes were employed to construct knockout models,and their roles were assessed through cell morphology observation,Nanog-positive rate detection,colony formation assays,and pluripotency gene expression analysis.Results The GFP?/Input screening revealed 2 921 negatively regulated genes(mainly enriched in basic life processes,such as RNA metabolism and cell cycle)and 1 393 positively regulated genes(enriched in the processes of nervous system development,carbohydrate metabolism,and vascular system development).In the GFP?/Input screening,2 765 negatively regulated genes(enriched in RNA metabolism,cell cycle,and other fundamental processes)and 1 303 positively regulated genes(enriched in neural development,cell survival,and endothelial migration)were identified.The GFP?/GFP? comparison identified 1 001 negatively regulated genes[involved in stress response and inhibition of mitogen-activated protein kinase(MAPK)signaling]and 983 positively regulated genes[related to fibroblast growth factor/extracellular signal-regulated kinase(FGF/ERK)signaling pathway and glucose metabolism).These genes,were not only known pluripotency regulators(e.g.,Nanog,Nr5a2,Klf2,Klf4)and exit-associated genes(e.g.,Gata6,Grb2,Zeb1,Fgfr1),but also some novel candidates(e.g.,Dmrt1,Rxra,Zbtb14 and Tmem41b).Functional validation showed that transient knockout of Dmrt1,Tmem41b,and Hic2 significantly increased the proportion of Nanog? cells(P<0.01),suggesting their role in suppressing ground-state exit.ESCs with stable Dmrt1 knockout exhibited a more na?ve-state phenotype,presenting compact,dome-shaped colonies,with increased ratio of undifferentiated colonies(P<0.01),up-regulation of ground-state markers(Nanog,Nr5a2,Dppa3,P<0.01),and down-regulation of primed-state markers(Fgf5,Lefty1,Dnmt3b,P<0.01).Rescue experiments for Dmrt1 expression reversed these above phenotypes.Conclusion A candidate gene set regulating exit from na?ve pluripotency in ESC is screened out and identified with genome-wide CRISPR.Our findings implicate Dmrt1 plays a critical role in promoting the exit.

BACKGROUND: Currently, lung cancer is one of the malignant tumors with a high morbidity and mortality all over the world. However, the exact mechanisms underlying lung cancer progression remain unclear. The tumor necrosis factor receptor associated factor (TRAF) family members are cytoplasmic adaptor proteins, which function as both adaptor proteins and ubiquitin ligases to regulate diverse receptor signalings, leading to the activation of nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase (MAPK) and interferon regulatory factor (IRF) signaling. The aim of this study was to investigate the expression of TRAFs in different tissues and cancer types, as well as its mRNA expression, protein expression, prognostic significance and functional enrichment analysis in non-small cell lung cancer (NSCLC), in order to provide new strategies for the diagnosis and treatment of NSCLC. METHODS: RNA sequencing data from the The Genotype-Tissue Expression database was used to analyze the expression patterns of TRAF family members in different human tissues. RNA sequencing data from the Cancer Cell Line Encyclopedia database was used to analyze the expression patterns of TRAF family members in different types of cancer cell lines. RNA sequencing data from the The Cancer Genome Atlas (TCGA) database was used to analyze the mRNA levels of TRAF family members across different types of human cancers. Immunohistochemistry (IHC) analyses from HPA database were used to analyze the TRAF protein levels in NSCLC [lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC)]. Overall survival analysis was performed by Log-rank test using original data from Kaplan-Meier Plotter database to evaluate the correlation between TRAF expressions and prognosis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on the TRAF family-related genes using RNA sequencing data from the TCGA database for NSCLC. The correlation between the expression levels of TRAF family members and the tumor immune microenvironment was analyzed using the ESTIMATE algorithm based on RNA sequencing data from the TCGA database. RESULTS: The TRAF family members exhibited significant tissue-specific expression heterogeneity. TRAF2, TRAF3, TRAF6 and TRAF7 were widely expressed in most tissues, while the expressions of TRAF1, TRAF4 and TRAF5 were restricted to specific tissues. The expressions of TRAF family members were highly specific among different types of cancer cell lines. In mRNA database of LUAD and LUSC, the expressions of TRAF2, TRAF4, TRAF5 and TRAF7 were significantly upregulated; while TRAF6 did the opposite; moveover, TRAF1 and TRAF3 only displayed a significant upregulation in LUAD and LUSC, respectively. Except for TRAF3, TRAF4 and TRAF7, other TRAF proteins displayed an obviously deeper IHC staining in LUAD and LUSC tissues compared with normal tissues. Additionally, patients with higher expression levels of TRAF2, TRAF4 and TRAF7 had shorter overall survival; while patients with higher expression levels of TRAF3, TRAF5 and TRAF6 had significantly longer overall survival; however, no significant difference had been observed between TRAF1 expression and the overall survival. TRAF family members differentially regulated multiple pathways, including NF-κB, immune response, cell adhesion and RNA splicing. The expression levels of TRAF family members were closely associated with immune cell infiltration and stromal cell content in the tumor immune microenvironment, with varying positive and negative correlations among different members. CONCLUSIONS TRAF family members exhibit highly specific expression differences across different tissues and cancer types. Most TRAF proteins exhibit upregulation at both mRNA and protein levels in NSCLC, whereas, only upregulated expressions of TRAF2, TRAF4 and TRAF7 predict worse prognosis. The TRAF family members regulate processes such as inflammation, immunity, adhesion and splicing, and influence the tumor immune microenvironment.
Humans ; Carcinoma, Non-Small-Cell Lung/pathology* ; Lung Neoplasms/mortality* ; Prognosis ; Gene Expression Regulation, Neoplastic ; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/metabolism*

AIM: To compare the visual quality in patients with low-to-moderate myopia after 0.05 D interval spherical lens optometry-guided small-incision lenticule extraction(SMILE)and conventional 0.25 D interval spherical lens optometry-guided SMILE.METHODS: Retrospective study. A total of 400 cases(400 eyes)with low-to-moderate myopia that underwent SMILE in the ophthalmology department of 989th Hospital of Joint Logistic Support Force from August 2021 to August 2023 were enrolled and the data from the right eyes were collected for analysis. According to the method of optometry test modality, they were divided into 0.05 D group and 0.25 D group, with 200 eyes in each group. The differences were compared between the two groups of patients in intraoperative corneal ablation thickness, uncorrected distance visual acuity(UDVA), high-order corneal aberrations(HOA), spherical aberrations, vertical coma, horizontal coma and trefoil aberrations before and at 1, 3 and 6 mo after surgery. Additionally, the percentage of eyes with residual spherical equivalent(SE)≤±0.25 D, postoperative visual symptoms and scores on the quality of visual(Qov)were compared between the two groups at 6 mo after surgery.RESULTS: The corneal ablation thickness in the 0.05 D group was 92.78±16.56 μm, which was slightly higher than that in the 0.25 D group(83.24±17.33 μm; P<0.001). The UDVA at each postoperative time point in the 0.05 D group was superior to that in the 0.25 D group(all P<0.001). The HOA, spherical aberration, horizontal coma and vertical coma in the two groups at 1, 3 and 6 mo after operation were higher than those before operation(all P<0.05). The spherical aberration in the 0.05 D group at each time point after surgery were higher than those in the 0.25 D group, and vertical coma were lower than those in the 0.25 D group(all P<0.05). At 6 mo postoperatively, the percentage of eyes with residual SE ≤±0.25 D in the 0.05 D group was 97.5%(195 eyes), which was higher than 87.5%(175 eyes)in the 0.25 D group(P<0.05). The most common adverse visual symptoms after SMILE in both groups were hazy vision and glare. The total Qov score in the 0.05 D group was 0.35(0.24, 0.55), which was lower than [0.62(0.32, 0.89)] in the 0.25 D group(P<0.05).CONCLUSION: Compared with conventional 0.25 D interval spherical lens optometry-guided SMILE, the 0.05 D interval spherical lens optometry-guided SMILE for the correction of low-to-moderate myopia has better predictability and can achieve better vision and visual quality.

Objective: To investigate the hematological characteristics of the rare McLeod phenotype associated with X-linked chronic granulomatous disease, KEL and XK gene analysis, identification of unexpected antibodies, serological characteristics of high-frequency antigen antibodies, and transfusion strategies. Methods: Serological methods were employed to determine the ABO, Rh, and other blood group system antigen phenotypes of the child, along with screening and identification of unexpected antibodies. The titers of high-frequency antigen antibodies were measured using tube antihuman globulin and microcolumn gel card techniques. Kell blood group typing was performed using serological and genotyping methods, while XK gene sequencing was conducted via next-generation sequencing. Peripheral blood smears from the child's mother were examined for erythrocyte morphology. Results: The child's serological results were as follows: blood group O, ccDEE, MM, Le(a-b+), JK(a+b+), Fy(a+b-), and Kell phenotype K-k+, Kp(a-b+). Plasma analysis revealed alloantibodies anti-C、e, as well as a high-frequency antigen antibody anti-KL, with titers of 512 (tube method) and 2 048 (microcolumn gel method). Genotyping results showed KEL genotype K-k+, Kp(a-b+), Js(a-b+), while XK gene NGS identified a hemizygous deletion of exons 1-3 (XK N. 01), consistent with XK: -1 or Kx-(McLeod). The mother's peripheral blood smear exhibited prominent acanthocytes. Conclusion: The hematological features of this rare McLeod phenotype with X-CGD include weakened Kell antigen expression and a complete exon deletion in the XK gene. Early clinical attention should be given to the symptoms and laboratory diagnosis of X-linked chronic granulomatous disease in pediatric patients. XK genotyping for McLeod phenotype should be prioritized to guide cautious transfusion strategies, preventing life-threatening complications due to incompatible blood products.

The development of malignant tumors is influenced by multiple factors,including genetics,environment,lifestyle,and pathogen infection.Viral infection plays a significant role in tumor development and progression.This article integrates the common pathways of seven oncogenic viruses(HPV,EBV,HBV,HCV,KSHV,HTLV-1,and HIV)based on the theory of cancer toxin pathomechanism.The findings reveal that cancer toxin formation is influenced by both internal factors(healthy qi deficiency,viscus dysfunction,and qi,blood,and body fluid stagnation)and external factors(viral infection,environmental factors,and the six exogenous pathogens),driving tumor development,metastasis,and recurrence.Viral infection not only provides the material foundation for cancer toxin but also accelerates its maturation and spread through chronic inflammation and immune imbalance,forming a complex pathogen-esis of"viral toxicity-endogenous cancer toxin",which translates into a three-stage strategy of"prevention,treatment,and post-recovery prevention".This article systematically discusses the pathogenesis of major cancer toxins and their associated tumors.Inte-grating the cancer toxin theory,the article explores preventive and therapeutic strategies for virus-associated malignancies,providing a theoretical basis and practical reference for the integrated prevention and treatment of tumors using traditional Chinese and Western medicine.

Objective To investigate the combined value of multimodal functional magnetic resonance imaging(fMRI)and magnetic resonance spectroscopy(MRS)in the differential diagnosis of postoperative recur-rence and pseudoprogression(PsP)of glioma.Methods One hundred and four patients with glioma confirmed by surgical pathology were selected from our hospital from September 2021 to March 2024,and the patients were divided into recurrence group(n=70)and PsP group(n=34)according to the revised glioma treatment response assessment criteria.Another group of 73 patients with glioma were selected for model validation.The general data,apparent diffusion coefficient(ADC)of multimodal fMRI parameters,standardized cerebral blood volume(CBV)and MRS indexes of the two groups were compared.The influencing factors of postoperative glioma recurrence were analyzed by logistic regression,and clinical efficacy of the combined treatment in the diagnosis of postoperative glioma recurrence and PsP was analyzed by receiver operating curve(ROC).Results The level of ADC in the recurrence group was lower than that in the PsP group(P<0.05),and the levels of CBV,choline(Cho)/creatine(Cr)and Cho/N-acetylaspartic acid(NAA)were higher(P<0.05).Logistic regression analysis showed that the levels of ADC,CBV,Cho/Cr and Cho/NAA were risk factors for postoperative recurrence of glioma(P<0.05).ROC showed that the combined use of multimodal fMRI and MRS in the differential diagnosis of postoperative glioma recurrence and PsP had an AUC of 0.916,outperforming the diagnostic accuracy of each individual modality.Validation of the combined model constructed with multimodal fMRI and MRS yielded an AUC of 0.929,95%CI(0.844～0.976),with a sensitivity of 88.46%and specificity of 91.49%.There was no statistical difference when compared to the AUC of the combined predictive model established in the earlier phase.Conclusion Multi-mode fMRI combined with MRS demonstrate high clinical value in the differential diagnosis of postoperative glioma recur-rence and PsP,and it is worth to be popularized.