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Introduction: The Vel– phenotype is a rare blood group, and it is challenging for identifying this phenotype due to limited available reagents. Moreover, there are relatively few studies on genomic editing of erythroid antigens and generation of knockout (KO) cell lines at present. Methods: To identify the high-efficiency small-guiding RNA (sgRNA) sequence, candidate sgRNAs were transfected into HEK 293T cells and analyzed using Sanger sequencing. Following this, the high-efficiency sgRNA was transfected into K562 cells using lentivirus transduction to generate KO Vel blood group gene cells. The expression of the Vel protein was detected using Western blot on single-cell clones. Additionally, flow cytometry was used to detect the erythroid markers CD235a and CD71. Hemoglobin quantification and Giemsa staining were also performed to evaluate the erythroid differentiation of KO clones induced by hemin. Results: The high-efficiency sgRNA was successfully obtained and used for CRISPR-Cas9 editing in K562 cells. After limiting dilution and screening, two KO clones had either deleted 2 or 4 bases and showed no expression of the Vel protein. In the hemin-induced KO clone, there was a significant difference in erythroid marker and hemoglobin quantification compared to untreated cells. The morphological changes were also observed for the hemin-induced KO clone. Conclusion: In this study, a highly efficient sgRNA was screened out and used to generate Vel erythroid antigen KO single-cell clones in K562 cells. The edited cells could then be induced to undergo erythroid differentiation with the use of hemin. [ABSTRACT FROM AUTHOR]

Objectives: This study aimed to investigate the molecular mechanism of the Jk(a‐b‐) phenotype in a Chinese transfusion patient. Background: Many different mutation types relating to Jk(a‐b‐) phenotype have been reported. However, the splice‐site mutation is relatively rare and the related functional verification is lacking. Materials and Methods: In this study, the blood sample was collected from a transfusion patient with the Jk(a‐b‐) phenotype. Serotyping was performed using routine serological methods. The exons sequences and coding regions of the JK gene were amplified using polymerase chain reaction and directly sequenced. To perform a minigene splicing assay, the intronic mutation sequences were cloned into a pSPL3 splice reporting vector. The splicing reporter minigene assay was performed in HEK 293T cells. Results: The Jk(a‐b‐) phenotype of the blood sample was identified through serological testing. Sequencing results revealed that the sample had a novel homozygous splice‐site mutation JK*02N (NM_015865.7: c.663+3A>C). Further analysis, including cDNA sequencing and minigene splicing assay, confirmed that the novel splice‐site mutation resulted in exon skipping. Interestingly, different numbers of exons being skipped were obtained by the two methods. Conclusion: This study revealed a novel homozygous splicing‐site mutation associated with the Jk(a‐b‐) phenotype in Chinese population. Our results emphasise the importance of the in vitro functional method minigene splicing assay, while also acknowledging its potential limitations when compared to cDNA sequencing. [ABSTRACT FROM AUTHOR]

The antitumor effects of a whey peptide‐based enteral diet, whose main components are whey peptides and yogurt fermented by Lactobacillus delbureckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, were investigated in mice. Our results indicated that the tumor weight in C26 carcinoma‐transplanted mice was significantly smaller at day 16 post‐implantation in the whey peptide‐based enteral diet group (1.36 ± 0.54 g) than in the control group (1.83 ± 0.89 g) (p < 0.05). The whey peptide‐based enteral diet group exhibited higher tumor cell apoptosis, lower cell proliferation, and inactive angiogenesis indicating by higher degree of TUNEL, lower positive rates of Ki‐67, VEGF, and CD34 than control group. It also attenuated inflammatory cell infiltration of spleen and liver as indicated by the decreased spleen index (10.89 ± 2.06 vs. 12.85 ± 2.92, p < 0.05) and increased liver index (58.09 ± 11.37 vs. 53.19 ± 6.67, p < 0.05) in the whey peptide‐based enteral diet group than the control diet group. These results proved the inhibitory effect of the whey peptide‐based enteral diet on tumor growth, which might be attributed to the whey peptides component. Practical Application: A whey peptide‐based enteral diet (MEIN®), containing cheese whey and multiple nutrients, was selected to verify the anti‐tumor effect by animal experiments. The tumor weight growth, tumor cell proliferation, inflammatory cell infiltration of spleen and liver in tumor model mice was significantly attenuated by the whey peptide‐based enteral diet, that might be attributed to its whey peptides component. These results provided an additive direction for cancer therapy and need a further study including clinical trials. [ABSTRACT FROM AUTHOR]

Objective: Investigation of a Jr(a‐) family samples, identification of the mutant and assessment of the differences of Jr antigen density of the Jr(a‐) family members, random adult and newborn individuals' RBCs. Background: The anti‐Jra antibody is generated when a Jr(a‐) individual pregnant or transfused with Jr(a+) blood unit, which can lead to mild‐to‐moderate hemolytic disease of the foetus and newborn (HDFN) or hemolytic transfusion reaction (HTR). Several mutations had been identified. The anti‐Jra caused HDFN is not rare in East Asia, but due to the lack of antibody and molecular background, it is likely to lead missed detection. Methods and Materials: One G4P1 woman had been detected as IAT positive during prenatal examination. Suspected as anti‐Jra after the laboratory serological testing, the maternal sample was further assessed by molecular analysis. The antigen density was detected by flow cytometry after reacting with anti‐Jra serum in family members and the normal individuals. Results: One novel frameshift mutation c.717delC and one previously identified mutation c.706C > T in ABCG2 was identified on proband. The infant haemoglobin(Hb) and bilirubin increased significantly after exchange transfusion and the severe HDFN was relieved. Flow cytometry results showed that the Jra antigens on adult RBCs were significantly less than those on the infant. Conclusion: The c.717delC mutation can lead to the shortening of protein ABCG2 in the site of p.Leu307Stop, result in the loss of Jra antigen. The difference in antigen density between adult and infant RBCs may be a possible reason that leads to severe HDFN but not transfusion reaction. Breastfeeding may lead to slower recovery from HDFN. [ABSTRACT FROM AUTHOR]

Background: Angiogenesis and tissue repair in chronic non-healing diabetic wounds remain critical clinical problems. Engineered MSC-derived exosomes have significant potential for the promotion of wound healing. Here, we discuss the effects and mechanisms of eNOS-rich umbilical cord MSC exosomes (UCMSC-exo/eNOS) modified by genetic engineering and optogenetic techniques on diabetic chronic wound repair. Methods: Umbilical cord mesenchymal stem cells were engineered to express two recombinant proteins. Large amounts of eNOS were loaded into UCMSC-exo using the EXPLOR system under blue light irradiation. The effects of UCMSC-exo/eNOS on the biological functions of fibroblasts and vascular endothelial cells in vitro were evaluated. Full-thickness skin wounds were constructed on the backs of diabetic mice to assess the role of UCMSC-exo/eNOS in vascular neogenesis and the immune microenvironment, and to explore the related molecular mechanisms. Results: eNOS was substantially enriched in UCMSCs-exo by endogenous cellular activities under blue light irradiation. UCMSC-exo/eNOS significantly improved the biological functions of cells after high-glucose treatment and reduced the expression of inflammatory factors and apoptosis induced by oxidative stress. In vivo, UCMSC-exo/eNOS significantly improved the rate of wound closure and enhanced vascular neogenesis and matrix remodeling in diabetic mice. UCMSC-exo/eNOS also improved the inflammatory profile at the wound site and modulated the associated immune microenvironment, thus significantly promoting tissue repair. Conclusion: This study provides a novel therapeutic strategy based on engineered stem cell-derived exosomes for the promotion of angiogenesis and tissue repair in chronic diabetic wounds. [ABSTRACT FROM AUTHOR]

The leaf of Nelumbo nucifera (Family Nelumbonaceae) has been widely included in the diet of Chinese people from the time of the Min Dynasty. In this study, a randomized double-blind trial (n = 60) was performed to determine the effects of extract from sun dried Nelumbo nucifera leaves (NnEx), which included quercetin-3-glucuronide (Q3GA) as the main components, in overweight patients (24 kg/m22) during 12 weeks. For both men and women, compared with those in the non-intervention control groups, the whole body fat was significantly decreased after NnEx ingestion, and men also significantly reduced visceral fat and waist circumference after NnEx ingestion compared with those in the control group. [ABSTRACT FROM AUTHOR]

Background: Recently, basigin (BSG), which carries OK antigens on red blood cells (RBCs), was reported to be the receptor of the Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRh5). BSG-PfRh5 is the only essential receptor-ligand pair in P. falciparum invasion that is known to date. However, the kind of OK/BSG polymorphism involved in the selection pressure caused by P. falciparum malaria has not been determined.Study Design and Methods: Blood samples were collected to detect the expression of OK/BSG. The coding region of PfRh5 was cloned and expressed. Enzyme-linked immunosorbent assay-based erythrocyte binding assay was used to measure the recombinant PfRh5 (rPfRh5) binding of RBCs with different OK/BSG expressions. Sequencing of the BSG gene and quantification of the BSG mRNA were performed for selected samples. The candidate microRNAs (miRNAs), which might target the BSG gene, were obtained by miRNA sequencing. Dual-Luciferase reporter assay and overexpression of identified miRNAs were performed in K562 cells.Results: The rPfRh5 was successfully expressed and verified. The OK/BSG expression levels varied among blood donors and were strongly associated with rPfRh5 binding. No single-nucleotide polymorphism was related to the OK/BSG expression. A potential BSG regulator, miR-501-3p, was identified by miRNA sequencing and Dual-Luciferase assay, but was not proven to regulate the expression of BSG in K562 cells.Conclusion: Although the mechanism of OK/BSG expression and regulation on RBCs has not been fully clarified, our findings suggest that the OK/BSG expression levels on RBCs might be related to P. falciparum invasion. Moreover, posttranscriptional regulation might play a role in controlling the OK/BSG expression. [ABSTRACT FROM AUTHOR]

Introduction: The Vel- phenotype is a rare blood group, and it is challenging for identifying this phenotype due to limited available reagents. Moreover, there are relatively few studies on genomic editing of erythroid antigens and generation of knockout (KO) cell lines at present., Methods: To identify the high-efficiency small-guiding RNA (sgRNA) sequence, candidate sgRNAs were transfected into HEK 293T cells and analyzed using Sanger sequencing. Following this, the high-efficiency sgRNA was transfected into K562 cells using lentivirus transduction to generate KO Vel blood group gene cells. The expression of the Vel protein was detected using Western blot on single-cell clones. Additionally, flow cytometry was used to detect the erythroid markers CD235a and CD71. Hemoglobin quantification and Giemsa staining were also performed to evaluate the erythroid differentiation of KO clones induced by hemin., Results: The high-efficiency sgRNA was successfully obtained and used for CRISPR-Cas9 editing in K562 cells. After limiting dilution and screening, two KO clones had either deleted 2 or 4 bases and showed no expression of the Vel protein. In the hemin-induced KO clone, there was a significant difference in erythroid marker and hemoglobin quantification compared to untreated cells. The morphological changes were also observed for the hemin-induced KO clone., Conclusion: In this study, a highly efficient sgRNA was screened out and used to generate Vel erythroid antigen KO single-cell clones in K562 cells. The edited cells could then be induced to undergo erythroid differentiation with the use of hemin., Competing Interests: The authors have no conflicts of interest to declare., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

This study is an attempt to evaluate the therapeutic effect of the ethanolic extract of Lindera aggregata on the liver and intestinal microbiota in rats with alcohol-induced liver injury (ALI). Rats were treated with 70 mg probiotics, 1 g/kg, 2 g/kg, and 3 g/kg ethanolic extract of Lindera aggregata , respectively, for 10 days. We found that Lindera aggregata could significantly reduce the biochemical parameters in the serum of ALD rats. Lindera aggregata alleviates oxidative stress and inflammation by upregulating SIRT1 and Nrf2 and downregulating COX2 and NF- κ B. The results of 16S rRNA gene sequencing showed that the medium dose of Lindera aggregata had the best effect on the growth of beneficial bacteria. Diversity analysis and LEfSe analysis showed that beneficial bacteria gradually occupied the dominant niche. The relative abundance of potential pathogens in the gut decreased significantly. We demonstrated that the ethanolic extract of Lindera aggregata can alleviate the oxidative stress and inflammation induced by alcohol through the SIRT1/Nrf2/NF- κ B pathway and can modulate the disturbance of gut microbiota induced by alcohol intake., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xin Zhao et al.)

Objective: To investigate whether miR-492 is involved in the post-transcriptional regulation of OK blood group antigen expression on red blood cells., Methods: Two 3'-UTR fragments of the BSG gene were synthesized with a chemical method, which respectively encompassed the BSG rs8259 TT or BSG rs8259 AA sites. The fragments were added with Xho I and Not I restriction enzyme cutting sites at both ends and cloned into a pUC57 vector, which in turn was constructed into a psiCHECK-2 vector and verified by sequencing. K562 cells were transfected with various combinations of miR-492 mimic and constructed psiCHECK2-BSG-T or psiCHECK2-BSG-A recombinant plasmid. A blank control group was set up. Each transfection experiment was repeated three times. The activity of Renilla reniformis luciferase was determined and normalized with that of firefly luciferase, and detected with a dual-luciferase reporter assay system. The data were subjected to statistical analysis., Results: The sequencing results confirmed that the recombinant psiCHECK2 plasmids containing the BSG rs8259 TT or rs8259 AA sites were constructed successfully. The results of dual-luciferase report gene detection showed that the miR-492 mimic could significantly inhibit psiCHECK2-BSG-T at a concentration over 100 nmol/L. However, it could not inhibit psiCHECK-BSG-A., Conclusion: miR-492 may be involved in the regulation of OK antigen expression on red blood cells with the BSG rs8259 TT genotype.