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Introduction: The approximately 70% 12-month relapse in children experiencing the initial episode of steroid-sensitive nephrotic syndrome (SSNS) is a significant concern, with over 50% developing frequent relapses or steroid-dependent nephrotic syndrome (FRNS/SDNS). There is a lack of strategies to reduce relapse after the onset. Whether early administration of rituximab, which effectively reduces relapses in FRNS/SDNS, may be a solution has not been evaluated., Methods: A prospective, multicenter, open-label, single-arm trial was conducted in China, with a 12-month follow-up. Children aged 1 to 18 years with the first episode of nephrotic syndrome (NS) were screened for eligibility. Proteinuria was evaluated daily using dipsticks. A dose of 375 mg/m 2 of rituximab was intravenously infused within 1 week after achieving corticosteroid-induced remission. The main outcome was 12-month relapse-free survival., Results: Out of the initially 66 children screened, 44 were enrolled and received rituximab, with all but 1 participant completing the 12-month follow-up. The median age at diagnosis was 4.3 years (interquartile range [IQR]: 3.4-5.9), and 33 (77%) of the participants were male. In the rituximab group, the 12-month relapse-free survival was significantly higher compared to historical controls (32 of 43 [74.4%] vs. 10 of 33 [30.3%]; P  < 0.001; hazard ratio [HR], 3.76; 95% confidence interval [CI], 1.80-7.81). The post hoc analysis revealed a higher 24-month relapse-free survival and a lower incidence of FRNS/SDNS at the 12-month follow-up. Treatment with rituximab was well-tolerated., Conclusion: Our findings support that early administration of rituximab may be associated with a higher 12-month relapse-free survival and a reduced incidence of FRNS/SDNS in children experiencing the initial episode of SSNS., (© 2024 International Society of Nephrology. Published by Elsevier Inc.)

Tumor hypoxia is the most common feature of radioresistance to the radiotherapy (RT) of lung cancer and results in poor clinical outcomes. High-linear energy transfer (LET) radiation is a novel RT technique to overcome this problem. However, a limited number of studies have been elucidated on the underlying mechanism(s) of RIBE and RISBE in cancer cells exposed to high-LET radiation under hypoxia. Here, we developed a new method to investigate the RIBE and RISBE under hypoxia using the SPICE-QST proton microbeams and a layered tissue co-culture system. Normal lung fibroblast (WI-38) and lung cancer (A549) cells were exposed in the range of 06 Gy of proton microbeams, wherein only ~0.04-0.15% of the cells were traversed by protons. Subsequently, primary bystander A549 cells were co-cultured with secondary bystander A549 cells in the presence or absence of a GJIC and NO inhibitor using co-culture systems. Studies show that there are differences in RIBE in A549 and WI-38 primary bystander cells under normoxia and hypoxia. Interestingly, treatment with a GJIC inhibitor showed an increase in the toxicity of primary bystander WI-38 cells but a decrease in A549 cells under hypoxia. Our results also show the induction of RISBE in secondary bystander A549 cells under hypoxia, where GJIC and NO inhibitors reduced the stressful effects on secondary bystander A549 cells. Together, these preliminary results, for the first time, represented the involvement of intercellular communications through GJIC in propagation of RIBE and RISBE in hypoxic cancer cells.

Following carbon ion beam irradiation in mammalian cells, such as used in carbon ion radiotherapy (CIRT), it has been suggested that the balance between whether nonhomologous end joining (NHEJ) or homologous recombination (HR) is utilized depends on the DNA double-strand break (DSB) complexity. Here, we quantified DSB distribution and identified the importance of each DSB repair pathway at increasing depths within the carbon ion spread-out Bragg peak (SOBP) beam range. Chinese hamster ovary (CHO) cell lines were irradiated in a single biological system capable of incorporating the full carbon ion SOBP beam range. Cytotoxicity and DSB distribution/repair kinetics were examined at increasing beam depths using cell survival as an endpoint and γ-H2AX as a surrogate marker for DSBs. We observed that proximal SOBP had the highest number of total foci/cell and lowest survival, while distal SOBP had the most dense tracks. Both NHEJ- and HR-deficient CHO cells portrayed an increase in radiosensitivity throughout the full carbon beam range, although NHEJ-deficient cells were the most radiosensitive cell line from beam entrance up to proximal SOBP and demonstrated a dose-dependent decrease in ability to repair DSBs. In contrast, HR-deficient cells had the greatest ratio of survival fraction at entrance depth to the lowest survival fraction within the SOBP and demonstrated a linear energy transfer (LET)-dependent decrease in ability to repair DSBs. Collectively, our results provide insight into treatment planning and potential targets to inhibit, as HR was a more beneficial pathway to inhibit than NHEJ to enhance the cell killing effect of CIRT in targeted tumor cells within the SOBP while maintaining limited unwanted damage to surrounding healthy cells., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Objectives: Juvenile dermatomyositis (JDM) is a chronic autoimmune disease. Some patients remain in an active state even though they were administrated with a combination of corticosteroid and methotrexate. Existing research has suggested that interferon and Janus kinase played an important role in pathogenesis. Existing research has suggested the efficacy of JAK inhibitors (JAKi). Our retrospective study aimed to investigate the efficacy of tofacitinib in refractory JDM patients., Methods: A total of eighty-eight patients in China who had been diagnosed with JDM and subjected to tofacitinib therapy for over 3 months were retrospectively analyzed. Skin and muscle manifestations were assessed using the Cutaneous Assessment Tool-binary method (CAT-BM), Childhood Myositis Assessment Scale (CMAS), and kinase. Pulmonary function was assessed using a high-resolution CT (computerized tomography) scan and pulmonary symptoms. All patients were subjected to regular follow-up, and core measures were assessed every 3 months after initiation. Furthermore, the data were analyzed using the Wilcoxon single test, Mann-Whitney U test, and chi-square test., Results: Compared with the baseline data, skin and muscle manifestations were found significantly improved during the respective follow-up visit. At the most recent follow-up, nearly 50% of patients achieved a clinical complete response and six patients received tofacitinib monotherapy. Sixty percent of patients suffering from interstitial lung disease well recovered on high-resolution CT. Seventy-five percent of patients showed a reduction in the size or number of calcinosis, and 25% of patients showed completely resolved calcinosis., Conclusion: In this study, the result suggested that tofacitinib therapy exerted a certain effect on skin manifestations, muscle manifestations, interstitial lung disease (ILD), calcinosis, as well as downgrade of medication. In-depth research should be conducted to focus on the correlation between the pathogenesis of JDM and JAKi., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: WDR35 variants are known to cause a rare autosomal recessive disorder-Cranioectodermal dysplasia (CED). The CED patients are commonly present with facial dysmorphisms (frontal bossing and low-set ears), sagittal craniosynostosis, growth retardation, dolichocephaly, skeletal deformities (brachydactyly, terminal hypoplasia of the fingers and narrow thorax), ectodermal abnormalities (sparse hair, and finger/toe nail dysplasia), nephronophthisis, retinal dystrophy and hepatic fibrosis. Diagnosis of CED can be difficult because it presents with high genetic heterogeneity. However, our understanding of the phenotype of CED caused by WDR35 variants could be more explicit, and the correlation between genotype and phenotype needs further improvement., Case Presentation: We report a case of the first Chinses patient of CED caused by WDR35 variants, a 3-year-and-3-month-old patient, who was admitted to our hospital with frontal bossing, growth retardation, low set ears, dolichocephaly, sparse hair, and small limbs, abnormal renal function, and moderate anemia. The child showed a novel phenotype of the ectopic testis except for presenting typical CED characteristics, and he was identified with novel compound heterozygous WDR35 variants (c.2590 C > T, p.Gln864* and c.2408&#95;2416del, p.Asn803&#95;Ala805del; NM&#95;001006657). He was given iron succinate and erythropoietin to improve anemia and to inhibit repeated metabolic acidosis and hyperkalemia through acid correction, diuretic, and potassium-lowering treatments. The parents refused to accept renal replacement therapy for their child and were discharged voluntarily., Conclusions: This is the first reported case of the WDR35 variants that can lead to CED and ectopic testis, which is also the first Chinese patient associated with WDR35 variants. This study expands our understanding of genotype-phenotype association in patients with WDR35 variants and provides genetic counseling for prevention and intervention in this genetic disorder. Neonatal carriers should be followed up for kidney and CED-related diseases to detect warning signs., (© 2023. BioMed Central Ltd., part of Springer Nature.)

OX40 (CD134), a member of the TNF receptor superfamily, is a widely studied costimulatory immune checkpoint. Several OX40 agonistic antibodies are in the clinical stage for cancer treatment, among which PF-04518600 is the leader and currently in phase II trial. It has been recognized that one potential mode of action for anti-OX40 antibodies is the deletion of intratumoral Tregs. Thus, a novel human anti-OX40 antibody, BAT6026, was generated with enhanced antibody dependent cellular cytotoxicity (ADCC) via fucose deletion to strengthen its Treg depletion activity. This characteristic of BAT6026 differentiates it from other previously reported anti-OX40 antibodies in the field of tumor therapy. The affinity of BT6026 to OX40 was 0.28nM, approximately 8 times stronger than that of PF-04518600. BAT6026 effectively competed for the binding of ligand OX40L to OX40, whereas PF-04518600 only partially competed. Moreover, compared to PF-04518600, BAT6026 activated T cells more effectively when clustered by FcγRs engagement and stimulated SEB-pretreated PBMCs to secrete IL-2 cytokines in vitro . In addition, BAT6026 demonstrated stronger anti-tumor activity than PF-04518600 in an OX40-humanized mouse MC38 tumor model. BAT6026 also showed a significantly synergistic effect on tumor inhibition when combined treatment with PD-1 antibody. Analysis of tumor-infiltrating T cells revealed that BAT6026 treatment significantly reduced Treg cells and increased CD8+ T cells in tumor. Preclinical safety assessment in non-human primates demonstrated a good safety profile for BAT6026. Together these data warrant further development of BAT6026 into clinical trials for patients with cancer., Competing Interests: All authors are employees of Bio-Thera Solutions Guangzhou, China. The authors declare that this study received funding from Bio-Thera Solutions. The funder had the following involvement in the study: study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication., (Copyright © 2023 Liang, Zheng, Liu, Mei, Zhou, Xiao, Qin, Yue, Lin, Liu, Li and Yu.)

Background: The effect of recombinant human GH (rhGH) in Chinese children with chronic kidney disease (CKD) is unclear., Methods: This was a 52-week, multicenter, randomized, open-label, negative-controlled phase 3 study. Prepubertal subjects were randomized 1:1 to either daily subcutaneous injections of rhGH 0.05 mg/kg/day or no treatment for 52 weeks., Results: A total of 68 subjects with a mean age of 7.8 ± 3.27 years were enrolled. At week 52, the height standard deviation score (HT-SDS) in the treated group increased by 0.75 ± 0.58, which was significantly higher compared with 0.17 ± 0.47 in the untreated group (least squares mean 0.58, 95% confidence interval, 0.32-0.84; P < 0.001). At week 52, significant improvements were observed in other growth parameters (height velocity [P < 0.001]), insulin-like growth factor 1 (IGF-1) SDS [P < 0.001], IFG-1/insulin-like growth factor binding protein-3 molar ratio [P < 0.001], and height [P < 0.001]) compared with the untreated control. Seven patients reported treatment-related adverse events (TRAEs) and most TRAEs were mild in severity. Most subjects recovered without further intervention., Conclusions: Daily rhGH for 52 weeks in children with CKD-induced growth retardation significantly improved HT-SDS and other growth parameters without compromising safety., Impact: The efficacy and safety of growth hormone (GH) therapy in Chinese children with chronic kidney disease (CKD) are unclear. This study found that giving short stature Chinese children with CKD daily recombinant human growth hormone (rhGH) for 52 weeks improved growth parameters without compromising safety. This study's information can give physicians the confidence to treat these patients in their clinical practice., (© 2022. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Volatile esters are major aromas contributing to the organoleptic quality of apple fruit. However, the molecular mechanisms underlying the regulation of volatile ester biosynthesis in apple remain elusive. This study investigated the volatile profiles and transcriptomes of 'Qinguan' (QG) apple fruit during development and/or postharvest storage. Although the constitution of volatiles varied widely between the peel and flesh, the volatile profiles of the peel and flesh of ripening QG fruit were dominated by volatile esters. WGCNA results suggested that 19 genes belonging to ester biosynthesis pathways and 11 hub transcription factor genes potentially participated in the biosynthesis and regulation of esters. To figure out key regulators of ester biosynthesis, correlation network analysis, dual-luciferase assays, and yeast one-hybrid assay were conducted and suggested that MdMYB94 trans-activated the MdAAT2 promoter and participated in the regulation of ester biosynthesis. This study provides a framework for understanding ester biosynthesis and regulation in apple.

Cardiovascular diseases (CVD), with high morbidity and mortality, seriously affect people's life and social development. Clinically, reperfusion therapy is typically used to treat ischemic cardiomyopathy, such as severe coronary heart disease and acute myocardial infarction. However, reperfusion therapy can lead to myocardial ischemia reperfusion injury (MIRI), which can affect the prognosis of patients. Studying the mechanisms of MIRI can help us improve the treatment of MIRI. The pathological process of MIRI involves many mechanisms such as ferroptosis and mitophagy. Ferroptosis can exacerbate MIRI, and regulation of mitophagy can alleviate MIRI. Both ferroptosis and mitophagy are closely related to ROS, but there is no clear understanding of the relationship between ferroptosis and mitophagy. In this review, we analyzed the relationship between ferroptosis and mitophagy according to the role of mTOR, NLPR3 and HIF. In addition, simultaneous regulation of mitophagy and ferroptosis may be superior to single therapy for MIRI. We summarized potential drugs that can regulate mitophagy and/or ferroptosis, hoping to provide reference for the development of drugs and methods for MIRI treatment., Competing Interests: The authors declare there are no competing interests., (©2023 Liu et al.)

Objective To investigate the impact of miR-181c on migration and angiogenesis of lung cancer cells. Methods The Oncomine platform, UALCAN was used to analyze the differential expression of miR-181c and reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in lung cancer obtained from the Cancer Genome Atlas (TCGA) database. The targeting relationship between miR-181c on RECK gene was predicted using Targetscan software. miR-181c mimic, inhibitor and negative control were introduced into A549 cells respectively. After transfection, the real-time quantitative PCR was used to detect the relative expressions of miR-181c and RECK mRNA, and Western blot analysis was used to detect the expression levels of RECK, matrix metalloproteinase 2 (MMP2) and MMP9 proteins. Transwell TM assay was performed to analyze the cell migration ability. The secretion of vascular endothelial growth factor (VEGF)-A in the cell culture supernatant was analyzed by using ELISA. Human umbilical vein endothelial cells (HUVECs) were treated with the culture supernatant, then in vitro tubule formation assay was carried out to evaluate the angiogenesis ability. The targeting correlation between miR-181c and RECK was validated by double luciferase reporter gene assay. Results UALCAN analysis displayed that the expression of miR-181c was significantly higher and RECK expression was significantly lower in lung cancer tissues compared to that in normal tissues. Targetscan prediction showed that there was a miR-181c binding site in the 3'-untranslated region (3' UTR) of RECK gene. miR-181c could downregulate the expression of RECK, increase the expressions of MMP2 and MMP9, and promote the A549 cell migration. ELISA and tubule formation assay showed that miR-181c could induce the secretion of VEGF-A in A549 cells and enhance the ability of HUVECs differentiae into tubules. The double luciferase reporter gene assay confirmed that RECK was the direct regulation target of miR-181c. Conclusion miR-181c promotes the migration and angiogenesis of human A549 cells by directly targeting RECK.

Primary Coenzyme Q10 (CoQ 10 ) deficiency is an ultra-rare disorder caused by defects in genes involved in CoQ 10 biosynthesis leading to multidrug-resistant nephrotic syndrome as the hallmark kidney manifestation. Promising early results have been reported anecdotally with oral CoQ 10 supplementation. However, the long-term efficacy and optimal prescription remain to be established. In a global effort, we collected and analyzed information from 116 patients who received CoQ 10 supplements for primary CoQ 10 deficiency due to biallelic pathogenic variants in either the COQ2, COQ6 or COQ8B genes. Median duration of follow up on treatment was two years. The effect of treatment on proteinuria was assessed, and kidney survival was analyzed in 41 patients younger than 18 years with chronic kidney disease stage 1-4 at the start of treatment compared with that of an untreated cohort matched by genotype, age, kidney function, and proteinuria. CoQ 10 supplementation was associated with a substantial and significant sustained reduction of proteinuria by 88% at 12 months. Complete remission of proteinuria was more frequently observed in COQ6 disease. CoQ 10 supplementation led to significantly better preservation of kidney function (5-year kidney failure-free survival 62% vs. 19%) with an improvement in general condition and neurological manifestations. Side effects of treatment were uncommon and mild. Thus, our findings indicate that all patients diagnosed with primary CoQ 10 deficiency should receive early and life-long CoQ 10 supplementation to decelerate the progression of kidney disease and prevent further damage to other organs., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Introduction: High-quality bowel preparation is an essential precondition for colonoscopy. Few studies have evaluated the smartphone WeChat application as a means of improving the quality of bowel preparation., Aim: To assess the effect of patient education by using smartphone WeChat application aids on the quality of bowel preparation., Material and Methods: A multicenter prospective, endoscopist-blinded, randomized, controlled study was conducted. Patients were randomly assigned to three groups. A total of 478 patents in groups A were accepted for smartphone WeChat application, 477 in groups B were accepted for conventional education plus smartphone WeChat application while group C (473 patients) was a control group. The primary outcome was the quality of the bowel preparation according to the Boston Bowel Preparation Scale (BBPS). The secondary outcomes included polyp detection rate (PDR), cecal intubation rate, insertion and withdrawal time, anxiety score, self-rated sleep quality, and willingness to undergo another colonoscopy., Results: Total BBPS score was significantly higher in groups B and C than in the control group (7.5 ±1.2, 7.5 ±1.3 vs. 6.5 ±1.2, p < 0.001). PDR in group A (40.2%, 192/478) and group B (41.7%, 199/477) was higher than that in the control group (p = 0.003) and mean number of polyps per patient was higher too (p = 0.015). Moreover, a shorter cecal insertion time was recorded in group A and group B than in the control group (8.8 ±3.9, 8.9 ±3.8 vs. 10.5 ±4.2 min, p < 0.001). Intervention groups showed lower anxiety scores and better quality sleep and were more likely to be willing to repeat colonoscopy., Conclusions: Patient instruction via smartphone WeChat application efficiently improved bowel preparation for colonoscopy., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2022 Fundacja Videochirurgii.)

Lipid phosphate phosphatases (LPPs) are a key enzyme in the production and degradation of phosphatidic acid (PA), which plays an important role in plant growth, development, stress resistance and plant hormone response. Thus far, little is known about the LPP family genes in kiwifruit ( Actinidia spp.). According to this study, 7 members in the AcLPP family were identified from the whole genome of kiwifruit, the subcellular localization predictions were mainly on the plasma membrane. Chromosomal localization analysis showed that the AcLPP genes were unevenly distributed on 5 chromosomes, it was determined to have undergone strong purifying selection pressure. There were 5 duplicate gene pairs and all underwent segmental duplication events. The LPP genes of kiwifruit were conserved when compared with other plants, especially in terms of evolutionary relationships, conserved motifs, protein sequences, and gene structures. Cis -regulatory elements mainly included hormone response elements and abiotic response elements. Functional annotation of GO revealed that AcLPP genes were closely related to phosphatase/hydrolase activity, phosphorus metabolism and dephosphorylation. AcLPP genes family were predicted to be targets of miRNA. Transcript level analysis revealed that the AcLPP family played diverse functions in different tissues and during growth, development, and postharvest storage stages. qPCR analysis showed that the members of AcLPP gene family might be regulated by ETH, ABA, GA 3 , and IAA hormone signals. The family members were regulated by the stress of salt stress, osmotic stress, cold stress, and heat stress. These results would provide a basis and reference for studying the agricultural characteristics of kiwifruit and improving its stress resistance., 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 Yang, Chen, Su, Liu, Zeng, Li, Cha, Liu, Xing, Ren and Ding.)

Steroid-resistant nephrotic syndrome (SRNS) is one of the major causes of end-stage kidney disease (ESKD) in children and young adults. For approximately 30% of children with SRNS results from a genetic cause. In this study, genotype-phenotype correlations in a cohort of 283 pediatric patients with SRNS or early-onset NS (nephrotic syndrome presenting within the first year of life) from 23 major pediatric nephrology centers in China were analyzed. All patients were performed with next-generation sequencing and Sanger sequencing. The overall mutation detection rate was 37.5% (106 of 283 patients). WT1 was the most frequently detected mutation, followed by NPHS1, NPHS2, and ADCK4, and these four major causative genes (WT1, NPHS1, NPHS2, and ADCK4) account for 73.6% of patients with monogenic SRNS. Thirteen of 106 individuals (12.3%) carried mutations in ADCK4 that function within the coenzyme Q10 biosynthesis pathway. In the higher frequently ADCK4-related SRNS, two mutations, c.737G>A (p.S246N) and c.748G>C (p.D250H), were the most prevalent. Our study provides not only definitive diagnosis but also facilitate available targeted treatment for SRNS, and prediction of prognosis and renal outcome. Our indications for genetic testing are patients with FSGS, initial SRNS, cases of positive family history or those with extra-renal manifestations., 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 Zhu, Zhang, Yu, Yu, Huang, Sun, Li, Wang, Li, Sun, Yang, Deng, Shao, Liu, Liu, Qin, Feng, Zhu, Yang, Zheng, Zheng, Zhong, Hou, Mao, Wang and Ding.)

In this study, we aimed to investigate how homologous recombinant (HR)-related genomic instability is involved in ionizing radiation (IR)-induced thymic lymphoma in mice. We divided five-week-old Rosa26 Direct Repeat-GFP (RaDR-GFP) transgenic mice into non-IR control and IR groups and exposed the mice in the IR group to a 7.2 Gy dose of γ-rays, delivered in 1.8 Gy fractions, once a week for four weeks. We then estimated mouse survival and recorded their body, thymus, and spleen weights. The frequency of HR events in the chromosomes of the thymus, bone marrow, and spleen cells and the phenotype of thymic lymphoma cells were analyzed using fluorescence-activated cell sorting (FACS). We found that most mice in the IR group developed thymic lymphoma, their survival rate decreasing to 20% after 180 days of IR exposure, whereas no mice died in the non-IR control group until day 400. The thymus and spleen weighed significantly more in the IR-4-month group than that in the non-IR group; however, we observed no significant differences between the body weights of the control and IR mice. FACS analysis indicated that the frequency of HR events significantly increased at two and four months after the last IR dose in the bone marrow and thymus cells, but not in the spleen cells of RaDR-GFP transgenic mice, suggesting that recombinant cells accumulated in the thymus upon IR exposure. This suggests that IR induces genome instability, revealed as increased HR, that drives the development of thymic lymphoma. Additionally, phenotypic analysis of lymphoma cells showed an increase in the CD4 - /CD8 + (CD8SP) cell population and a decrease in the CD4 + /CD8 - (CD4SP) cell population in the IR-4-month group compared to that in the non-IR group, indicating that IR induces an aberrant cell phenotype characteristic of lymphoma. In conclusion, we observed a significant increase in HR events and abnormal phenotype in thymic lymphoma cells at two and four months after IR exposure in both the thymus and bone marrow tissues, suggesting that genomic instability is involved in the early stages of thymic lymphomagenesis. Our study indicates that HR-visualizing RaDR-GFP transgenic mice can help explore the links between the molecular mechanisms of genome instability and IR-induced tumorigenesis., Competing Interests: None., (AJCR Copyright © 2022.)

Radiation-induced bystander effect (RIBE) has been identified as an important contributing factor to tumor resistance and normal tissue damage. However, the RIBE in cancer and normal cells under hypoxia remain unclear. In this study, confluent A549 cancer and WI-38 normal cells were subjected to condition of hypoxia or normoxia, before exposure to high-LET protons microbeam. After 6 h incubation, cells were harvested and assayed for colony formation, micronucleus formation, chromosome aberration and western blotting. Our results show that there were differences of RIBE in bystander A549 and WI-38 cells under hypoxia and normoxia. The differences were also observed in the roles of HIF-1α expression in bystander A549 and WI-38 cells under both conditions. Furthermore, inhibition of gap junction intercellular communication (GJIC) showed a decrease in toxicity of hypoxia-treated bystander A549 cells, but increased in bystander WI-38 cells. These findings clearly support that GJIC protection of bystander normal cells from toxicity while enhancing in bystander cancer cells. Together, the data show a promising strategy for high-LET radiation in designing an entire new line of drugs, either increase or restore GJIC in bystander cancer cells which in turn leads to enhancement of radiation accuracy for treatment of hypoxic tumors., (©2022 by Radiation Research Society. All rights of reproduction in any form reserved.)

Aquaporins, integral membrane proteins widely distributed in organisms, facilitate the transport of water, glycerol, and other small uncharged solutes across cellular membranes and play important physiological roles in eukaryotes. However, characterizations and physiological functions of the prokaryotic aquaporins remain largely unknown. Here, we report that Streptococcus pneumoniae (pneumococcus) AqpC (Pn-AqpC), representing a new aquaporin subfamily possessing a distinct substrate-selective channel, functions as an oxygen porin by facilitating oxygen movement across the cell membrane and contributes significantly to pneumococcal virulence. The use of a phosphorescent oxygen probe showed that Pn-AqpC facilitates oxygen permeation into pneumococcal and Pn-AqpC-expressing yeast cells. Reconstituting Pn-AqpC into liposomes prepared with pneumococcal and Escherichia coli cellular membranes further verified that Pn-AqpC transports O 2 but not water or glycerol. Alanine substitution showed that Pro232 in the substrate channel is key for Pn-AqpC in O 2 transport. The deletion of Pn- aqpC significantly reduced H 2 O 2 production and resistance to H 2 O 2 and NO of pneumococci, whereas low-H 2 O 2 treatment helped the ΔPn -aqpC mutant resist higher levels of H 2 O 2 and even NO, indicating that Pn-AqpC-facilitated O 2 permeation contributes to pneumococcal resistance to H 2 O 2 and NO. Remarkably, the lack of Pn -aqpC alleviated cell autolysis, thus reducing pneumolysin (Ply) release and decreasing the hemolysis of pneumococci. Accordingly, the ΔPn- aqpC mutant markedly reduced survival in macrophages, decreased damage to macrophages, and significantly reduced lethality in mice. Therefore, the oxygen porin Pn-AqpC, through modulating H 2 O 2 production and pneumolysin release, the two major pneumococcal virulence factors, controls the virulence of pneumococcus. Pn-AqpC orthologs are widely distributed in various pneumococcal serotypes, highlighting that the oxygen porin is important for pneumococcal pathogenicity. IMPORTANCE Pneumococcus is the leading cause of community-acquired pneumonia, bacteremia, and meningitis. This work reports that a novel aquaporin subfamily represented by pneumococcal Pn-AqpC functions as an oxygen porin facilitating O 2 influx into cells. Importantly, by mediating O 2 influx, Pn-AqpC controls the production and release of H 2 O 2 and Ply, the two major pneumococcal virulence factors. Moreover, by enhancing endogenous H 2 O 2 production, Pn-AqpC significantly increases pneumococcal resistance to H 2 O 2 and even NO, the major bactericidal chemical produced by macrophages. Consequently, the deletion of Pn- aqpC markedly decreased pneumococcal survival in macrophages and reduced damage to macrophages. Accordingly, the ΔPn- aqpC mutant displays significantly attenuated virulence in a murine pneumonia model. Given that Pn-AqpC orthologs are widely distributed in all pneumococcal serotypes, this new subfamily of aquaporins is identified as novel virulence-related proteins.

Lycopene is the main pigment in red-flesh citrus fruits, and its formation is a research hotspot. To explore the basis of lycopene accumulation in red-flesh mutants, we profiled the terpenoid metabolites. Compared with their respective wild types, Cara Cara (Cara) [and Red-Anliu (R-An)] oranges showed increased carotenoid and limonoid aglycone contents and decreased contents of abscisic acid (ABA) catabolites, monoterpenoid volatiles, and sesquiterpenoid volatiles. Cara contained less than half of the amount of ABA glucose ester (ABAGE), the main ABA derivative in oranges. Parallel lower transcript levels of NCED and ABA glucosyltransferase in Cara were detected at the mature green stage. These results document the changes in terpenoid profiles in Cara and show that the red flesh of citrus color mutants is related to weak ABA catabolism, especially ABAGE, and decreased transcript levels of two genes encoding uridine diphosphate (UDP)-glycosyltransferases that catalyze ABAGE biosynthesis.

Kidney disease is manifested in a wide variety of phenotypes, many of which have an important hereditary component. To delineate the genotypic and phenotypic spectrum of pediatric nephropathy, a multicenter registration system is being implemented based on the Chinese Children Genetic Kidney Disease Database (CCGKDD). In this study, all the patients with kidney and urological diseases were recruited from 2014 to 2020. Genetic analysis was conducted using exome sequencing for families with multiple affected individuals with nephropathy or clinical suspicion of a genetic kidney disease owing to early-onset or extrarenal features. The genetic diagnosis was confirmed in 883 of 2256 (39.1%) patients from 23 provinces in China. Phenotypic profiles showed that the primary diagnosis included steroid-resistant nephrotic syndrome (SRNS, 23.5%), glomerulonephritis (GN, 32.2%), congenital anomalies of the kidney and urinary tract (CAKUT, 21.2%), cystic renal disease (3.9%), renal calcinosis/stone (3.6%), tubulopathy (9.7%), and chronic kidney disease of unknown etiology (CKDu, 5.8%). The pathogenic variants of 105 monogenetic disorders were identified. Ten distinct genomic disorders were identified as pathogenic copy number variants (CNVs) in 11 patients. The diagnostic yield differed by subgroups, and was highest in those with cystic renal disease (66.3%), followed by tubulopathy (58.4%), GN (57.7%), CKDu (43.5%), SRNS (29.2%), renal calcinosis /stone (29.3%) and CAKUT (8.6%). Reverse phenotyping permitted correct identification in 40 cases with clinical reassessment and unexpected genetic conditions. We present the results of the largest cohort of children with kidney disease in China where diagnostic exome sequencing was performed. Our data demonstrate the utility of family-based exome sequencing, and indicate that the combined analysis of genotype and phenotype based on the national patient registry is pivotal to the genetic diagnosis of kidney disease., Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-021-00014-1., Competing Interests: Conflict of interestNo conflicts of interest to disclose., (© The Author(s) 2021.)

Astronauts can develop psychological stress (PS) during space flights due to the enclosed environment, microgravity, altered light-dark cycles, and risks of equipment failure or fatal mishaps. At the same time, they are exposed to cosmic rays including high atomic number and energy (HZE) particles such as iron-56 (Fe) ions. Psychological stress or radiation exposure can cause detrimental effects in humans. An earlier published pioneering study showed that chronic restraint-induced psychological stress (CRIPS) could attenuate Trp53 functions and increase carcinogenesis induced by low-linear energy transfer (LET) γ rays in Trp53-heterozygous (Trp53+/-) mice. To elucidate possible modification effects from CRIPS on high-LET HZE particle-induced health consequences, Trp53+/- mice were received both CRIPS and accelerated Fe ion irradiation. Six-week-old Trp53+/- C57BL/6N male mice were restrained 6 h per day for 28 consecutive days. On day 8, they received total-body Fe-particle irradiation (Fe-TBI, 0.1 or 2 Gy). Metaphase chromosome spreads prepared from splenocytes at the end of the 28-day restraint regimen were painted with the fluorescence in situ hybridization (FISH) probes for chromosomes 1 (green), 2 (red) and 3 (yellow). Induction of psychological stress in our experimental model was confirmed by increase in urinary corticosterone level on day 7 of restraint regimen. Regardless of Fe-TBI, CRIPS reduced splenocyte number per spleen at the end of the 28-day restraint regimen. At 2 Gy, Fe-TBI alone induced many aberrant chromosomes and no modifying effect was detected from CRIPS on induction of aberrant chromosomes. Notably, neither Fe-TBI at 0.1 Gy nor CRIPS alone induced any increase in the frequency of aberrant chromosomes, while simultaneous exposure resulted in a significant increase in the frequency of chromosomal exchanges. These findings clearly showed that CRIPS could enhance the frequency of chromosomal exchanges induced by Fe-TBI at a low dose of 0.1 Gy., (©2021 by Radiation Research Society. All rights of reproduction in any form reserved.)

Previous work pointed to a critical role of excessive production of reactive oxygen species (ROS) in increased radiation hematopoietic death in GFP mice. Meanwhile, enhanced antioxidant capability was not demonstrated in the mouse model of radio-induced adaptive response (RAR) using rescue of radiation hematopoietic death as the endpoint. ROS induction by ex vivo X-irradiation at a dose ranging from 0.1 to 7.5 Gy in the nucleated bone marrow cells was comparatively studied using GFP and wild type (WT) mice. ROS induction was also investigated in the cells collected from mice receiving a priming dose (0.5 Gy) efficient for RAR induction in WT mice. Significantly elevated background and increased induction of ROS in the cells from GFP mice were observed compared to those from WT mice. Markedly lower background and decreased induction of ROS were observed in the cells collected from WT mice but not GFP mice, both receiving the priming dose. GFP overexpression could alter background and induction of ROS by X-irradiation in hematopoietic cells. The results provide a reasonable explanation to the previous study on the fate of cells and mice after X-irradiation and confirm enhanced antioxidant capability in RAR. Investigations involving GFP overexpression should be carefully interpreted.

Background: In mainland China, dialysis for children with end-stage renal disease (ESRD) was not introduced until the 1980s. To describe the development of pediatric dialysis in different regions of China, a national pediatric dialysis network, namely, International Pediatric Dialysis Network-China (IPDN-China) ( www.pedpd.org.cn ), was launched in 2012., Methods: Original and updated information from the renal centers registered with the IPDN-China was collected between 2012 and 2016 from two sources, namely, the registry and the survey, and demographic features were analyzed., Results: Due to promotion by the IPDN-China, the number of registered renal centers increased from 12 to 39 between 2012 and 2016, with a significant increase in the coverage of the Chinese administrative divisions (from 26.5 to 67.6%) (p < 0.01); and the coverage of the pediatric (0~14 years old) population increased to nearly 90% in 2016. The distribution of renal centers indicated that East China had the highest average number of registered centers per million population (pmp) 0~14-year-old age group. Seventeen relatively large dialysis centers were distributed across 14 divisions. Various modalities of renal replacement therapy (RRT) were available in most centers. The IPDN-China has promoted collaborations between dieticians, psychologists, and social workers on dialysis teams to provide better service to children with ESRD and their families. The proportion of centers with all three types of paramedic support (i.e., dieticians, psychologists, and social workers) as well as the proportion of centers with a partial paramedic team significantly increased between 2012 (25.0%) and 2016 (69.2%) (p < 0.05). In terms of the point prevalent cases of patients (aged < 18 years), data from the survey of 39 registered centers revealed that the number of children with ESRD who were on RRT was 578 (49% received a kidney transplant) at the end of 2016, which was more than that reported in previous surveys. Data from the registry showed that 349 dialysis patients had been enrolled as of the end of 2016. The median age at RRT start was 9.5 years, and the leading cause of ESRD was congenital abnormalities of the kidney and urinary tract (CAKUT)., Conclusions: The IPDN-China has helped to promote the development of pediatric dialysis for ESRD in China by improving the organization of care for dialysis patients and increasing the availability and the quality of RRT for patients who need it. To improve knowledge about the epidemiology and outcomes of pediatric RRT around the country, a sustained effort needs to be made by the IPDN-China to increase the enrollment of dialysis patients and increase the number of registered centers in the future.

Application of green fluorescent protein (GFP) in a variety of biosystems as a unique bioindicator or biomarker has revolutionized biological research and made groundbreaking achievements, while increasing evidence has shown alterations in biological properties and physiological functions of the cells and animals overexpressing transgenic GFP. In this work, response to total body irradiation (TBI) was comparatively studied in GFP transgenic C57BL/6-Tg (CAG-EGFP) mice and C57BL/6 N wild type mice. It was demonstrated that GFP transgenic mice were more sensitive to radiation-induced bone marrow death, and no adaptive response could be induced. In the nucleated bone marrow cells of GFP transgenic mice exposed to a middle dose, there was a significant increase in both the percentage of cells expressing pro-apoptotic gene Bax and apoptotic cell death. While in wild type cells, lower expression of pro-apoptotic gene Bax and higher expression of anti-apoptotic gene Bcl-2, and significant lower induction of apoptosis were observed compared to GFP transgenic cells. Results suggest that presence of GFP could alter response to TBI at whole body, cellular and molecular levels in mice. These findings indicate that there could be a major influence on the interpretation of the results obtained in GFP transgenic mice., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2020.)

Dunaliella salina is well known for its ability to accumulate large amounts of β-carotene. Myo-inositol (MI) enhances the biomass production of D. salina, but the underlying mechanisms were unclear. The present study showed that the concentration of exogenous MI decreased gradually and reached a constant level at the 4th day of cultivation. MI enhanced the contents of total colored carotenoids and the activity of photosystem II. Metabolic profiles were significantly changed after the addition of exogenous MI, as revealed by multivariate statistical analysis. The metabolites could be categorized into four groups based on the relative levels in different samples. Exogenous MI increased the levels of most detected sugars, amino acids, and total saturated and unsaturated fatty acids. Based on the physiological and metabolic analyses, a hypothetical growth-promoting model that MI promotes the growth of D. salina TG by increasing the levels of key metabolites and possibly enhancing photosynthesis, was proposed. This study provides valuable information for understanding the growth-promoting mechanisms of MI in D. salina from the metabolic perspective., (© 2020 Phycological Society of America.)

VQ motif-containing proteins (VQ proteins) are transcriptional regulators that work independently or in combination with other transcription factors (TFs) to control plant growth and development and responses to biotic and abiotic stresses. VQ proteins contain a conserved FxxhVQxhTG amino acid motif that is the main element of its interaction with WRKY TFs. We identified 59 members of the tobacco ( Nicotiana tabacum L.) NtVQ gene family by in silico analysis and examined their differential expression in response to phytohormonal treatments and following exposure to biotic and abiotic stressors. NtVQ proteins clustered into eight groups based upon their amino acid sequence and presence of various conserved domains. Groups II, IV, V, VI, and VIII contained the largest proportion of NtVQ gene family members differentially expressed in response to one or more phytohormone, and NtVQ proteins with similar domain structures had similar patterns of response to different phytohormones. NtVQ genes differentially expressed in response to temperature alterations and mechanical wounding were also identified. Over half of the NtVQ genes were significantly induced in response to Ralstonia solanacearum infection. This first comprehensive characterization of the NtVQ genes in tobacco lays the foundation for further studies of the NtVQ -mediated regulatory network in plant growth, developmental, and stress-related processes.

To explore the approaches and diagnostic yield of genetic testing for renal disease in children, we describe the genotype and phenotype of the national cohort of children with renal disease from 13 different regions of China recruited from 2014 to 2018 by building up the multicenter registration system (Chinese Children Genetic Kidney Disease Database, CCGKDD). Genetic diagnosis was confirmed in 42.1% of our cohort of 1001 pediatric patients with clinical suspicion of a genetic renal disease. Of the 106 distinct monogenetic disorders detected, 15 accounted for 60.7% of genetic diagnoses. The diagnostic yield was 29.1% in steroid resistant nephritic syndrome (SRNS), 61.4% in cystic renal disease, 17.0% in congenital anomalies of the kidney and urinary tract (CAKUT), 62.3% in renal tubular disease/renal calcinosis, and 23.9% for chronic kidney disease (CKD) 3 to 5 stage with unknown origin. Genetic approaches of target gene sequence (TGS), singleton whole-exome sequencing (WES) and trio-WES were performed with diagnostic rates of 44.8%, 36.2%, and 42.6%, respectively. The early use of trio-WES could improve the diagnostic rate especially in renal tubular disease and calcinosis. We report the genetic spectrum of Chinese children with renal disease. Establishment of the CCGKDD will improve the genetic work on renal disease., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Psoriasis is a common inflammatory skin disease. Current treatment for psoriasis relies on conventional immunosuppressive agents. However, long-term treatment with global immunosuppression may cause various side effects. Thus, it is compelling to seek alternative drugs for treating psoriasis with potentially less side effects. Betulinic acid (BA) is a naturally occurring pentacyclic triterpene, an ingredient that originally exists in natural plants and lacks systemic toxicity. BA can regulate immunity with anti-fibrotic, anti-inflammatory and antioxidant properties. However, it's unknown whether BA has a therapeutic effect on psoriasis. The objectives of this study were to investigate whether BA attenuates psoriatic skin inflammation and to identify its mechanisms of action. A murine model of imiquimod-induced psoriasis was utilized to evaluate skin lesion while flow cytometry, immunohistochemistry, quantitative RT-PCR and Western blotting analyses were performed for immunoassays. We found that BA treatment alleviated psoriatic symptoms and inflammatory skin lesion. BA lowered the PASI scores, decreased epidermal thickness and reduced T cell infiltration in the skin lesion. Moreover, BA reduced the frequency of IL-17A-expressing CD4 + and γδ T cells in psoriatic mice, but did not alter CD4 + FoxP3 + Treg frequency. BA also reduced IL-17A production but increased anti-inflammatory cytokine IL-10 level in serum of the psoriatic mice. Furthermore, BA inhibited gene expression of pro-inflammatory mediators in skin lesions, including RORγt, IL-17A, IL-6 and TNFα. Importantly, it suppressed NFκB signaling in the skin lesion. Finally, BA inhibited T cell proliferation and IL-17A production by CD4 + T-Cells in vitro. Thus, BA attenuates psoriasis and inhibits Th17 development., (Copyright © 2019. Published by Elsevier B.V.)

In this study, a novel poly (vinyl alcohol) (PVA)/poly (ethylene glycol) (PEG) scaffold was carefully designed via thermal processing and subsequent supercritical fluid (SCF) foaming. Interestingly, a bimodal open-celled structure with interconnected networks was successfully created in the plasticized PVA (WPVA)/PEG scaffold. Large cells were produced from the nucleation sites generated in the PVA phase during rapid depressurization, while plenty of small pores generate in the cell walls of the big cells. The formation mechanism of this cellular structure was studied by considering the various phase morphologies and the diffusion behaviour of the carbon dioxide (CO 2 ) in individual phases. In addition, the intermolecular interactions of the WPVA/PEG blend were studied using X-ray diffraction and FTIR analysis. The results demonstrate that various types of hydrogen bonds among the hydroxyl groups on the PVA chains, PEG and water molecules are formed in the blend system. The realization of thermoplastic foaming of the PVA/PEG blend benefits from the interactions of complexation and plasticization between water and PEG molecules. The SEM images also revealed that L929 fibroblast cells were able to attach and spread on surfaces of the WPVA/PEG samples. Thus the WPVA/PEG scaffold with unique bimodal cellular structure is nontoxic and favours the attachment and proliferation of cells, making it promising for use as the candidate for tissue engineering applications.

A transplanted organ is usually rejected without any major immunosuppressive treatment because of vigorous alloimmune responsiveness. However, continuous global immunosuppression may cause severe side effects, including nephrotoxicity, tumors, and infections. Therefore, it is necessary to seek novel immunosuppressive agents, especially natural ingredients that may provide sufficient efficacy in immunosuppression with minimal side effects. Shikonin is a bioactive naphthoquinone pigment, an ingredient originally extracted from the root of Lithospermum erythrorhizon . Previous studies have shown that shikonin regulates immunity and exerts anti-inflammatory effects. In particular, it can ameliorate arthritis in animal models. However, it is unclear whether shikonin inhibits alloimmunity or allograft rejection. In this study and for the first time, we demonstrated that shikonin significantly prolonged the survival of skin allografts in wild-type mice. Shikonin increased the frequencies of CD4 + Foxp3 + regulatory T cells (Tregs) post-transplantation and induced CD4 + Foxp3 + Tregs in vitro as well. Importantly, depleting the Tregs abrogated the extension of skin allograft survival induced by shikonin. It also decreased the frequencies of CD8 + CD44 high CD62L low effector T cells and CD11c + CD80 + /CD11c + CD86 + mature DCs after transplantation. Moreover, we found that shikonin inhibited the proliferation of T cells in vitro and suppressed their mTOR signaling. It also reduced the gene expression of pro-inflammatory cytokines, including IFNγ, IL-6, TNFα, and IL-17A, while increasing the gene expression of anti-inflammatory mediators IL-10, TGF-β1, and indoleamine-2, 3-dioxygenase (IDO) in skin allografts. Further, shikonin downregulated IDO protein expression in skin allografts and DCs in vitro . Taken together, shikonin inhibits allograft rejection via upregulating CD4 + Foxp3 + Tregs. Thus, shikonin is a novel immunosuppressant that could be potentially used in clinical transplantation.

Premature chromosome condensation (PCC) is a sensitive and unique way to detect interphase chromosome damage and its recovery in mammalian cells irradiated with ionizing radiation. In this chapter, we describe G1 PCC assay with which one can measure immediate chromosome breaks in G1 type chromosomes and their repair/rejoining. In order to induce G1 PCC, one needs to fuse mitotic cells with G1 cells to be tested. There are two methods to fuse cells; one is to use Sendai virus or its equivalent, and another method needs polyethylene glycol (PEG) as a fusing agent. The date obtained with PCC assay can bridge the gap between radiation-induced DNA damage (mainly double strand breaks) and chromosome aberrations observable at metaphase stage.

The anti-hyperglycemic effect of metformin is believed to be caused by its direct action on signaling processes in hepatocytes, leading to lower hepatic gluconeogenesis. Recently, metformin was reported to alter the gut microbiota community in humans, suggesting that the hyperglycemia-lowering action of the drug could be the result of modulating the population of gut microbiota. However, the critical microbial signaling metabolites and the host targets associated with the metabolic benefits of metformin remained elusive. Here, we performed metagenomic and metabolomic analysis of samples from individuals with newly diagnosed type 2 diabetes (T2D) naively treated with metformin for 3 d, which revealed that Bacteroides fragilis was decreased and the bile acid glycoursodeoxycholic acid (GUDCA) was increased in the gut. These changes were accompanied by inhibition of intestinal farnesoid X receptor (FXR) signaling. We further found that high-fat-diet (HFD)-fed mice colonized with B. fragilis were predisposed to more severe glucose intolerance, and the metabolic benefits of metformin treatment on glucose intolerance were abrogated. GUDCA was further identified as an intestinal FXR antagonist that improved various metabolic endpoints in mice with established obesity. Thus, we conclude that metformin acts in part through a B. fragilis-GUDCA-intestinal FXR axis to improve metabolic dysfunction, including hyperglycemia.

Much of the diversity of anthocyanin pigmentation in plant tissues is due to the action of glycosyltransferases, which attach sugar moieties to the anthocyanin aglycone. This step can increase both their solubility and stability. We investigated the pigmentation of the outer and inner pericarps of developing fruits of the red-fleshed kiwifruit Actinidia chinensis cv. 'Hongyang'. The results show that the red color of the inner pericarp is due to anthocyanin. Based on expression analyses of structural genes, AcUFGT was shown to be the key gene involved in the anthocyanin biosynthetic pathway. Expression of AcUFGT in developing fruit paralleled changes in anthocyanin concentration. Thirteen putative UFGT genes, including different transcripts, were identified in the genome of 'Hongyang'. Among these, only the expression of AcUFGT3a was found to be highly consistent with anthocyanin accumulation. Fruit infiltrated with virus-induced gene silencing showed delayed red colorations, lower anthocyanin contents and lower expressions of AcUFGT3a. At the same time, transient overexpression of AcUFGT3a in both Actinidia arguta and green apple fruit resulted in higher anthocyanin contents and deeper red coloration. In vitro biochemical assays revealed that recombinant AcUFGT3a recognized only anthocyanidins as substrate but not flavonols. Also, UDP-galactose was used preferentially as the sugar donor. These results indicate AcUFGT3a is the key enzyme regulating anthocyanin accumulation in red-fleshed kiwifruit., (© 2017 Scandinavian Plant Physiology Society.)

We developed an in vitro DNA detection system using a pair of dCas9 proteins linked to split halves of luciferase. Luminescence was induced upon colocalization of the reporter pair to a ∼44 bp target sequence defined by sgRNAs. We used the system to detect Mycobacterium tuberculosis DNA with high specificity and sensitivity. The reprogrammability of dCas9 was further leveraged in an array design that accesses sequence information across the entire genome.

Coloration in apple (Malus×domestica) flesh is mainly caused by the accumulation of anthocyanin. Anthocyanin is biosynthesized through the flavonoid pathway and regulated by MYB, bHLH, and WD40 transcription factors (TFs). Here, we report that the HD-Zip I TF MdHB1 was also involved in the regulation of anthocyanin accumulation. MdHB1 silencing caused the accumulation of anthocyanin in 'Granny Smith' flesh, whereas its overexpression reduced the flesh content of anthocyanin in 'Ballerina' (red-fleshed apple). Moreover, flowers of transgenic tobacco (Nicotiana tabacum 'NC89') overexpressing MdHB1 showed a remarkable reduction in pigmentation. Transient promoter activation assays and yeast one-hybrid results indicated that MdHB1 indirectly inhibited expression of the anthocyanin biosynthetic genes encoding dihydroflavonol-4-reductase (DFR) and UDP-glucose:flavonoid 3-O-glycosyltransferase (UFGT). Yeast two-hybrid and bimolecular fluorescence complementation determined that MdHB1 acted as a homodimer and could interact with MYB, bHLH, and WD40 in the cytoplasm, consistent with its cytoplasmic localization by green fluorescent protein fluorescence observations. Together, these results suggest that MdHB1 constrains MdMYB10, MdbHLH3, and MdTTG1 to the cytoplasm, and then represses the transcription of MdDFR and MdUFGT indirectly. When MdHB1 is silenced, these TFs are released to activate the expression of MdDFR and MdUFGT and also anthocyanin biosynthesis, resulting in red flesh in 'Granny Smith'., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The anchorless fibronectin-binding proteins (FnBPs) are a group of important virulence factors for which the structures are not available and the functions are not well defined. In this study we performed comprehensive studies on a prototypic member of this group: the fibronectin-/fibrinogen-binding protein from Streptococcus suis (FBPS). The structures of the N- and C-terminal halves (FBPS-N and FBPS-C), which together cover the full-length protein in sequence, were solved at a resolution of 2.1 and 2.6 Å, respectively, and each was found to be composed of two domains with unique folds. Furthermore, we have elucidated the organization of these domains by small-angle X-ray scattering. We further showed that the fibronectin-binding site is located in FBPS-C and that FBPS promotes the adherence of S suis to host cells by attaching the bacteria via FBPS-N. Finally, we demonstrated that FBPS functions both as an adhesin, promoting S suis attachment to host cells, and as a bacterial factor, activating signaling pathways via β1 integrin receptors to induce chemokine production., Competing Interests: The authors declare no conflict of interest.

DNA double strand break (DSB) repair pathway choice following ionizing radiation (IR) is currently an appealing research topic, which is still largely unclear. Our recent paper indicated that the complexity of DSBs is a critical factor that enhances DNA end resection. It has been well accepted that the RPA-coated single strand DNA produced by resection is a signaling structure for ATR activation. Therefore, taking advantage of high linear energy transfer (LET) radiation to effectively produce complex DSBs, we investigated how the complexity of DSB influences the function of ATR pathway on the G2/M checkpoint regulation. Human skin fibroblast cells with or without ATM were irradiated with X rays or heavy ion particles, and dual-parameter flow cytometry was used to quantitatively assess the mitotic entry at early period post radiation by detecting the cells positive for phosphor histone H3. In ATM-deficient cells, ATR pathway played a pivotal role and functioned in a dose- and LET-dependent way to regulate the early G2/M arrest even as low as 0.2Gy for heavy ion radiation, which indicated that ATR pathway could be rapidly activated and functioned in an ATM-independent, but DSB complexity-dependent manner following exposure to IR. Furthermore, ATR pathway also functioned more efficiently in ATM-proficient cells to block G2 to M transition at early period of particle radiation exposure. Accordingly, in contrast to ATM inhibitor, ATR inhibitor had a more effective radiosensitizing effect on survival fraction following heavy ion beams as compared with X ray radiation. Taken together, our results reveal that the complexity of DSBs is a crucial factor for the activation of ATR pathway for G2/M checkpoint regulation, and ATM-dependent end resection is not essential for the activation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: This study aims to evaluate the safety and efficacy of Xuezhikang in the treatment of unstable angina pectoris (UAP) in patients with elevated liver enzymes caused by statins., Methods: Ninety UAP patients with elevated liver enzymes (higher than 3-fold the upper limits of normal caused by statins) were randomly divided into 3 groups: withdrawal of simvastatin (group A), continued taking of simvastatin (group B) and switching to Xuezhikang capsules (group C)., Results: Blood lipids and high-sensitivity C-reactive protein (hs-CRP) were measured before and after treatment, and liver enzymes were measured every two weeks. As the liver enzymes of the patients in group A returned to normal, they could again undertake administration of simvastatin. If the liver enzyme levels remained abnormal at four weeks, polyene phosphatidylcholine would be added. The endpoint events of each group were followed-up. After treatment for 4 weeks, the liver enzymes decreased in all of the three groups and there was a significant difference (p < 0.05). However, at 8 weeks the liver enzymes showed no significant difference among the three groups (p > 0.05). The blood lipids and hs-CRP were both reduced in group B and C. The decreased triglyceride and hs-CRP levels and increased high-density lipoprotein cholesterol level in group C were more remarkable than those in group B (p < 0.05). The incidence rate of endpoint events in group A was the highest (p < 0.05) among the three groups., Conclusions: Xuezhikang is safe and effective in the treatment for UAP patients with elevated liver enzymes caused by statins., Key Words: Liver enzymes; Simvastatin; Unstable angina pectoris; Xuezhikang capsules.

We have studied the induction of chromosome aberrations in human fibroblasts exposed in G0/G1 to X-rays or heavy ions to study the influence of G1 cell cycle arrest. Confluent normal fibroblasts were exposed to X-rays or accelerated particles with different LET values and chromosome aberrations were investigated in the first G0/G1 and G2//M phase. The particles used here were 490MeV/nucleon Si, 500MeV/nucleon Fe, and 200MeV/nucleon Fe ions. Cells were subcultured 24h after exposure and premature chromosome condensation (PCC) was performed by fusion-induced method for analysis of G0/G1 cells, and chemically-induced method for analysis of G2 and metaphase cells. Chromosome damage was assessed in chromosomes 1 and 3 using whole chromosome fluorescence in situ hybridization (FISH). Cell cycle was analyzed by flow cytometry at different incubation times following subculture. After irradiation with 2Gy of high-LET particles, the yields of chromosome aberrations and fragments were significantly higher in G0/G1 phase than in G2/M phase, whereas similar yields of damage were measured in both phases after exposure to X-rays. In contrast, the yield of misrepair, assessed by the number of color junctions, was similar in the G0/G1 and G2/M phases after exposure to either X-rays or high-LET particles. The yields of chromosome aberrations, fragments, and color junctions in both the G0/G1 and the G2/M phases, increased with LET up to 200keV/μm, then decreased for 440keV/μm Fe particles. A good correlation was found between chromosome aberrations in both G0/G1 and G2/M cells and survival fractions after 2Gy of different LET radiations, although the slopes were steeper for the G0/G1 cells. Flow cytometry analysis indicated that high-LET particles induce more non cycling G0/G1 cells within 48h of subculture than X-rays, suggesting that chromosome aberrations scored at the G2/M phase may not accurately describe the true radiation effect., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: Gene knock out technique is very important for gene function study. We developed a simple and efficient method to knock out chromosomal genes in Escherichia coli., Methods: Using the Escherichia coli Keio single-mutant library, we combined Red homology recombination with P1 phage transduction and developed a method to knock out the genes in Escherichia coli MG1655., Results: We obtained beta-oxidation mutants deltafadD, deltafadE and deltafadD-deltafadE and fatty acid synthesis mutants deltafabH, deltafabF and deltafabH-deltafabF. There were no obvious growth changes between deltafadD or deltafadE mutant strains and MG1655. However, deltafabH and deltafabH-deltafabF mutant strains grew much slower than the wild type strain. The fatty acid contents in deltafadD, deltafadE and deltafadD-deltafadE were 18.2 mg/L, 20.0 mg/L and 19.2 mg/L respectively, higher than 17.5 mg/L in wild type. The fatty acid contents in deltafabH, deltafabF and deltafabH-deltafabF were 12.6 mg/L, 15.2 mg/L and 11.2mg/L respectively, lower than that in wild type., Conclusion: Using Keio mutant library, P1 phage transduction and resistant gene elimination, we have established a simple and efficient method for gene knock-out in Escherichia coli.

Brucella melitensis and Brucella suis are intracellular pathogens of livestock and humans. Here we report four genome sequences, those of the virulent strain B. melitensis M28-12 and vaccine strains B. melitensis M5 and M111 and B. suis S2, which show different virulences and pathogenicities, which will help to design a more effective brucellosis vaccine.

Biological drugs represent an important and rapidly growing class of therapeutics useful in the treatment of a variety of disorders ranging from cancer to inflammation to infectious diseases. Unlike single chemical entities, the recombinant production of these drugs in living cells confers considerable structural and chemical heterogeneity to the biologically derived protein product that constitutes the active pharmaceutical ingredient (API). In mammalian based expression systems, much of this diversity is conferred through heterogeneous protein glycosylation. These post-translational modifications can have significant effects on the structure, biological function, and pharmacological properties of the API. In addition, the bulk proteins that comprise the API are further formulated through the use of multiple excipients designed to ensure product stability, solubility, and lot-to-lot consistency. Unfortunately, these matrices can interfere with commonly available analytical methods used in the thorough chemical characterization of the biological drug product. At the same time, a demonstration of the suitable extraction of the bulk drug substance in a manner and form that does not destabilize the active ingredient or introduce any structural bias with direct reference to the original drug product is both critical and necessary. Here, we use recombinant human follicle stimulating hormone (follitropin alpha for injection) from a pharmaceutical source as an example to illustrate a suitable purification strategy to effectively extract the bulk drug substance from the formulated drug product with high purity and yield. We assess the suitability of this extraction method in preserving the structural integrity and overall quality of the drug substance relative to the formulated drug product, placing a particular emphasis on glycosylation as a key product attribute. In so doing, we demonstrate that it is possible to effectively extract the active pharmaceutical ingredient from a formulated biological drug product in a manner that is consequently sufficient for its use in comparability studies., (Copyright © 2010 Elsevier B.V. All rights reserved.)

T7 RNA polymerase is known to induce bending of its promoter DNA upon binding, as evidenced by gel-shift assays and by recent end-to-end fluorescence energy transfer distance measurements. Crystal structures of promoter-bound and initially transcribing complexes, however, lack downstream DNA, providing no information on the overall path of the DNA through the protein. Crystal structures of the elongation complex do include downstream DNA and provide valuable guidance in the design of models for the complete melted bubble structure at initiation. In the current study, we test a specific structural model for the initiation complex, obtained by alignment of the C-terminal regions of the protein structures from both initiation and elongation and then simple transferal of the downstream DNA from the elongation complex onto the initiation complex. Fluorescence resonance energy transfer measurement of distances from a point upstream on the promoter DNA to various points along the downstream helix reproduce the expected helical periodicity in the distances and support the model's orientation and phasing of the downstream DNA. The model also makes predictions about the extent of melting downstream of the active site. By monitoring fluorescent base analogues incorporated at various positions in the DNA, we have mapped the downstream edge of the bubble, confirming the model. The initially melted bubble, in the absence of substrate, encompasses 7-8 bases and is sufficient to allow synthesis of a three base transcript before further melting is required. The results demonstrate that despite massive changes in the N-terminal portion of the protein and in the DNA upstream of the active site, the DNA downstream of the active site is virtually identical in both initiation and elongation complexes.

To study the technology for establishing DNA microarrays for the diagnosis of HPV. HPV6, 11, 16 and 18 gene fragments were isolated and printed onto aminosilane-coated glass slides by PixSys 5500 microarray printer as probes to prepare the HPV. HPV samples, after labeled with Cy3 or Cy5, were hybridaized with the microarray followed by scanning for analysis. The experimental condition for preparing the HPV gene chips was investigated and the possibility of HPV genotying using DNA microarrays was discussed. The technique established in this study for preparing HPV DNA microarrays is applicable and has potential clinical application significance.