Objective:To investigate the relationship between the morphology of bone marrow plasma cells, the identification of M protein, and the detection of minimal residual disease(MRD)by flow cytometry in elderly patients with multiple myeloma(MM).Additionally, the impact on progression-free survival(PFS)is analyzed.Methods:A total of 60 elderly MM patients with bone marrow morphology reports and corresponding flow MRD detection, collected from February 1, 2017, to January 31, 2022, at Beijing Chaoyang Hospital Affiliated to Capital Medical University, were included in this study.We collected data on plasma cells from morphological examination and flow cytometry-based MRD detection. By combining these findings with M-protein identification results from 35 cases, we analyzed the correlations among these three parameters. Patients were stratified into two groups based on median values of: flow cytometry MRD(0.246% of nucleated cells), morphological plasma cell percentage(3.5%), and M-protein quantification(2.5 g/dl).This stratification enabled evaluation of their prognostic value for PFS.Results:A total of 60 elderly MM patients were enrolled, including 41 males with age of 65.0(63.0, 68.5)years and 19 females with age of 67.0(64.0, 72.0)years The morphology of bone marrow in 60 elderly patients with MM revealed 10 cases of primitive plasma cells(16.67%), 48 cases of immature plasma cells(80.00%), and 2 cases with no plasma cells(3.33%).A positive correlation was observed between the proportion of bone marrow plasma cells and the corresponding flow MRD in terms of both the proportion of nuclear cells and the proportion of plasma cells.Specifically, the proportion of the morphological protoplasma group showed a strong correlation with flow MRD(proportion of plasma cells)( r=0.82, P<0.01), while the proportion of the morphological immature plasma group exhibited moderate correlations with flow MRD(proportion of nuclear cells)( r=0.74, P<0.05)and flow MRD(proportion of plasma cells)( r=0.70, P<0.01).No significant correlation was found between the type and quantity of M protein and flow MRD( P>0.05).The PFS time for the flow MRD ≥0.246%(nucleated cells)group was shorter than that of the<0.246% group( P<0.05).There was no significant difference in PFS between the groups with plasma cell ratios ≥3.5% and<3.5%( P=0.15).Additionally, no significant difference in PFS was observed between patients with M protein quantitation ≥2.5 g/dl and those with M protein quantitation<2.5 g/dl( P=0.94). Conclusions:The proportion of bone marrow plasma cells correlates with flow MRD in elderly patients with MM, and a high MRD load indicates a poor prognosis.However, no significant correlation was found between M protein levels and flow MRD or PFS.Clinical attention should focus on the dynamic monitoring of plasma cell morphology and flow MRD.Nevertheless, the morphological detection of plasma cells remains crucial for auxiliary diagnosis due to its intuitiveness, cost-effectiveness, and broad applicability.

Immune checkpoint inhibitor-related pneumonitis(CIP)is a relatively common immune-related adverse event.The current treatment for CIP mainly relies on glucocorticoids,with 70％～80％of patients being controlled by conventional glucocorticoid therapy.However,steroid-unresponsive CIP is often se-vere and can be life-threatening.There is no standard treatment protocol for steroid-unresponsive CIP,highlighting a significant unmet clinical need.This paper reviews the diagnosis,treatment progress,and exploratory research of steroid-unresponsive CIP to provide evidence-based guidelines and directions for clinical and translational research.

Immune checkpoint inhibitor-related pneumonitis(CIP)is a relatively common immune-related adverse event.The current treatment for CIP mainly relies on glucocorticoids,with 70％～80％of patients being controlled by conventional glucocorticoid therapy.However,steroid-unresponsive CIP is often se-vere and can be life-threatening.There is no standard treatment protocol for steroid-unresponsive CIP,highlighting a significant unmet clinical need.This paper reviews the diagnosis,treatment progress,and exploratory research of steroid-unresponsive CIP to provide evidence-based guidelines and directions for clinical and translational research.

Objective:To investigate the relationship between the morphology of bone marrow plasma cells, the identification of M protein, and the detection of minimal residual disease(MRD)by flow cytometry in elderly patients with multiple myeloma(MM).Additionally, the impact on progression-free survival(PFS)is analyzed.Methods:A total of 60 elderly MM patients with bone marrow morphology reports and corresponding flow MRD detection, collected from February 1, 2017, to January 31, 2022, at Beijing Chaoyang Hospital Affiliated to Capital Medical University, were included in this study.We collected data on plasma cells from morphological examination and flow cytometry-based MRD detection. By combining these findings with M-protein identification results from 35 cases, we analyzed the correlations among these three parameters. Patients were stratified into two groups based on median values of: flow cytometry MRD(0.246% of nucleated cells), morphological plasma cell percentage(3.5%), and M-protein quantification(2.5 g/dl).This stratification enabled evaluation of their prognostic value for PFS.Results:A total of 60 elderly MM patients were enrolled, including 41 males with age of 65.0(63.0, 68.5)years and 19 females with age of 67.0(64.0, 72.0)years The morphology of bone marrow in 60 elderly patients with MM revealed 10 cases of primitive plasma cells(16.67%), 48 cases of immature plasma cells(80.00%), and 2 cases with no plasma cells(3.33%).A positive correlation was observed between the proportion of bone marrow plasma cells and the corresponding flow MRD in terms of both the proportion of nuclear cells and the proportion of plasma cells.Specifically, the proportion of the morphological protoplasma group showed a strong correlation with flow MRD(proportion of plasma cells)( r=0.82, P<0.01), while the proportion of the morphological immature plasma group exhibited moderate correlations with flow MRD(proportion of nuclear cells)( r=0.74, P<0.05)and flow MRD(proportion of plasma cells)( r=0.70, P<0.01).No significant correlation was found between the type and quantity of M protein and flow MRD( P>0.05).The PFS time for the flow MRD ≥0.246%(nucleated cells)group was shorter than that of the<0.246% group( P<0.05).There was no significant difference in PFS between the groups with plasma cell ratios ≥3.5% and<3.5%( P=0.15).Additionally, no significant difference in PFS was observed between patients with M protein quantitation ≥2.5 g/dl and those with M protein quantitation<2.5 g/dl( P=0.94). Conclusions:The proportion of bone marrow plasma cells correlates with flow MRD in elderly patients with MM, and a high MRD load indicates a poor prognosis.However, no significant correlation was found between M protein levels and flow MRD or PFS.Clinical attention should focus on the dynamic monitoring of plasma cell morphology and flow MRD.Nevertheless, the morphological detection of plasma cells remains crucial for auxiliary diagnosis due to its intuitiveness, cost-effectiveness, and broad applicability.

Objective:To discuss the effect of microRNA(miR)-30c-5p on the proliferation,migration,and invasion of the human prostate cancer cells(LNCap),and to clarify its possible mechanism.Methods:The LNCap cells were divided into LNCap group(without plasmid transfection),miR-30c-5p mimic group(transfected with miR-30c-5p mimic),mimic NC group(transfected with miR-30c-5p mimic NC),sh-DNA damage inducible transcript 4(DDIT4)group(transfected with sh-DDIT4),sh-NC group(transfected with sh-DDIT4 NC),miR-30c-5p mimic+pc-DNA3.1-NC group(co-transfected with miR-30c-5p mimic and pc-DNA3.1 empty vector),and miR-30c-5p mimic+pc-DNA3.1-DDIT4 group(co-transfected with miR-30c-5p mimic and pc-DNA3.1-DDIT4 over-expression plasmid).The RWPE-1 cells were cultured normally.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of miR-30c-5p and DDIT4 mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of DDIT4 protein in the cells in various groups;CCK-8 method was used to detect the proliferation rates of the LNCap cells in various groups;Transwell assay was used to detect the numbers of the invasion LNCap cells in various groups;Scratch assay was used to detect the scratch healing rates of LNCap cells in various groups;dual-luciferase reporter assay was used to detect the targeting relationship between miR-30c-5p and DDIT4.In the in vivo tumor formation experiment,18 male BALB/c nude mice were divided randomly into blank group,agomiR-NC group(transfected with agomiR-30c-5p NC),and agomiR-30c-5p group(transfected with agomiR-30c-5p);there were six mice in each group.The mice in agomiR-NC group and agomiR-30c-5p group were subcutaneously injected with LNCap cells,while the mice in blank group were given an equal volume of physiological saline.The volumes of tumor of the mice in various groups were detected.HE staining was used to observe the morphology of prostate cancer tissue the mice of in various groups;RT-qPCR method and immunofluorescence staining were used to detect the expression levels of miR-30c-5p and DDIT4 mRNA and the fluorescence intensities of DDIT4 protein in prostate cancer tissue of the mice in various groups.Results:The In vitro prostate cancer cell experiment results showed that compared with RWPE-1 cells,the expression level of miR-30c-5p in the prostate cancer LNCap cells was decreased(P<0.01),and the expression levels of DDIT4 mRNA and protein were increased(P<0.05 or P<0.01).After 48 of transfection,compared with LNCap group and mimic NC group,the expression level of miR-30c-5p in the LNCap cells in miR-30c-5p mimic group was increased(P<0.01).Compared with LNCap group and sh-NC group,the expression level of DDIT4 mRNA in the LNCap cells in sh-DDIT4 group was decreased(P<0.01).Compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group,the expression level of miR-30c-5p in The LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was decreased(P<0.01);compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group,the expression level of DDIT4 mRNA in the LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased(P<0.01);compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group,the expression level of DDIT4 protein in the LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased(P<0.05).The CCK-8 method results showed that compared with LNCap group and mimic NC group,the proliferation rate of the LNCap cells in miR-30c-5p mimic group was decreased(P<0.01);compared with LNCap group and sh-NC group,the proliferation rate of the LNCap cells in sh-DDIT4 group was decreased(P<0.01);compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group,the proliferation rate of the LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased(P<0.01).The Transwell assay results showed that compared with LNCap group and mimic NC group,the number of the invasion LNCap cells in miR-30c-5p mimic group was decreased(P<0.01);compared with LNCap group and sh-NC group,the number of invasion LNCap cells in sh-DDIT4 group was decreased(P<0.01);compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group,the number of the invasion LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased(P<0.01).The scratch assay results showed that compared with LNCap group and mimic NC group,the scratch healing rate of the LNCap cells in miR-30c-5p mimic group was decreased(P<0.01);compared with LNCap group and sh-NC group,the scratch healing rate of the LNCap cells in sh-DDIT4 group was decreased(P<0.01);compared with miR-30c-5p mimic group and miR-30c-5p mimic+pcDNA3.1 NC group,the scratch healing rate of the LNCap cells in miR-30c-5p mimic+pc-DNA3.1-DDIT4 group was increased(P<0.01).The dual-luciferase reporter assay results showed that compared with the LNCap cells co-transfected with WT-DDIT4 and mimic NC,the luciferase activity of the LNCap cells co-transfected with WT-DDIT4 and miR-30c-5p mimic was decreased(P<0.01).The in vivo nude mouse tumor formation experiment results showed that on the 3 rd,6 th,9 th,12 th,and 15th days after cell injection,compared with blank group and agomiR-NC group,the tumor volumes of the nude mice in agomiR-30c-5p group were decreased(P<0.05).The HE staining results showed that in prostate cancer tissue of the mice in blank group and agomiR-NC group,the cell nuclei were enlarged,and nucleoli were prominent and deformed.In the mice in agomiR-30c-5p group,some regions of prostate cancer tissues results showed neatly arranged cells with normally shaped nuclei.The RT-qPCR and immunofluorescence staining showed that compared with agomiR-NC group,the expression level of miR-30c-5p in prostate cancer tissue of the mice in agomiR-30c-5p group was increased(P<0.01).Compared with blank group and agomiR-NC group,the expression level of DDIT4 mRNA in prostate cancer tissue of the mice in agomiR-30c-5p group was decreased(P<0.01).DDIT4 protein was mainly expressed in the cytoplasm.Compared with blank group and agomiR-NC group,the fluorescence intensity of DDIT4 protein in prostate cancer tissue of the mice in agomiR-30c-5p group was decreased(P<0.01).Conclusion:The expression level of miR-30c-5p in the prostate cancer LNCap cells is decreased,and it inhibits the proliferation,migration,and invasion of the prostate cancer cells by targeting downregulation of DDIT4,thereby participating in the occurrence and development of prostate cancer.

Due to good mechanical properties and biocompatibility, tissue engineering scaffolds have become the vital method for repairing and regenerating articular cartilage defects. With the continuous development of tissue engineering technology, many scaffolds preparation and formation methods have been developed and tested in the past decade, however, the preparation of ideal regenerative scaffolds remain controversial. As load-bearing tissue inside the body joints, the matrix structure and cell composition of articular cartilage are hierarchical, and there are several smooth natural gradients from the cartilage surface to the subchondral bone layer, including cell phenotype and number, specific growth factors, matrix composition, fiber arrangement, mechanical properties, nutrient and oxygen consumption. Therefore, in the design of regenerative scaffolds, it is necessary to achieve these gradients to regenerate articular cartilage in situ. In recent studies, many new biomimetic gradient scaffolds have been used to simulate the natural gradient of articular cartilage. These scaffolds show different mechanical, physicochemical or biological gradients in the structure, and have achieved good repair effects. The related articles on tissue engineering for the treatment of articular cartilage defects were retrieved by searching databases with key wordsarticular cartilage injury, cartilage repair and gradient scaffolds. In this work,the structural, biochemical, biomechanical and nutrient metabolism gradients of natural articular cartilage were studied and summarized firstly. Then, the latest design and construction of articular cartilage gradient scaffolds were classified. Besides that, the material composition (such as hydrogels, nanomaterials, etc.) and the preparation process (such as electrospinning, 3D printing, etc.) of grandient scaffolds were further enhanced. Finally, the prospect and challenge of biomimetic gradient scaffolds in cartilage engineering are discussed, which provides a theoretical basis for the successful application of gradient scaffolds in clinical transformation.

In the present study,we examined the codon usage bias between pseudorabies virus (PRV) US1 gene and the US1-like genes of 20 reference alphaherpesviruses.Comparative analysis showed noticeable disparities of the synonymous codon usage bias in the 21 alphaherpesviruses,indicated by codon adaptation index,effective number of codons (ENc) and GC3s value.The codon usage pattern of PRV US1 gene was phylogenetically conserved and similar to that of the US1-like genes of the genus Varicellovirus of alphaherpesvirus,with a strong bias towards the codons with C and G at the third codon position.Cluster analysis of codon usage pattern of PRV US1 gene with its reference alphaherpesviruses demonstrated that the codon usage bias of US1-like genes of 21 alphaherpesviruses had a very close relation with their gene functions.ENc-plot revealed that the genetic heterogeneity in PRV US1 gene and the 20 reference alphaherpesviruses was constrained by G+C content,as well as the gene length.In addition,comparison of codon preferences in the US1 gene of PRV with those of E.coli,yeast and human revealed that there were 50 codons showing distinct usage differences between PRV and yeast,49 between PRV and human,but 48 between PRV and E.coli.Although there were slightly fewer differences in codon usages between E.coli and PRV,the difference is unlikely to be statistically significant,and experimental studies are necessary to establish the most suitable expression system for PRV US1.In conclusion,these results may improve our understanding of the evolution,pathogenesis and functional studies of PRV,as well as contributing to the area of herpesvirus research or even studies with other viruses.