Co-expression of IL-21-Enhanced NKG2D CAR-NK cell therapy for lung cancer.

BACKGROUND: Adoptive cell therapy has achieved great success in treating hematological malignancies. However, the production of chimeric antigen receptor T (CAR-T) cell therapy still faces various difficulties. Natural killer (NK)-92 is a continuously expandable cell line and provides a promising alternative for patient's own immune cells. METHODS: We established CAR-NK cells by co-expressing natural killer group 2 member D (NKG2D) and IL-21, and evaluated the efficacy of NKG2D-IL-21 CAR-NK cells in treating lung cancer in vitro and in vivo. RESULTS: Our data suggested that the expression of IL-21 effectively increased the cytotoxicity of NKG2D CAR-NK cells against lung cancer cells in a dose-dependent manner and suppressed tumor growth in vitro and in vivo. In addition, the proliferation of NKG2D-IL-21 CAR-NK cells were enhanced while the apoptosis and exhaustion of these cells were suppressed. Mechanistically, IL-21-mediated NKG2D CAR-NK cells function by activating AKT signaling pathway. CONCLUSION: Our findings provide a novel option for treating lung cancer using NKG2D-IL-21 CAR-NK cell therapy.

Doxorubicin downregulates autophagy to promote apoptosis-induced dilated cardiomyopathy via regulating the AMPK/mTOR pathway.

The broad-spectrum antineoplastic drug doxorubicin (DOX) has one of the most serious chronic side effects on the heart, dilated cardiomyopathy, but the precise molecular mechanisms underlying disease progression subsequent to long latency periods remain puzzling. Here, we established a model of DOX-induced dilated cardiomyopathy. In a cardiac cytology exploration, we found that differentially expressed genes in the KEGG signaling pathway enrichment provided a novel complex network of mTOR bridging autophagy and oxidative stress. Validation results showed that DOX caused intracellular reactive oxygen species accumulation in cardiomyocytes, disrupted mitochondria, led to imbalanced intracellular energy metabolism, and triggered cardiomyocyte apoptosis. Apoptosis showed a negative correlation with DOX-regulated cardiomyocyte autophagy. To evaluate whether the inhibition of mTOR could upregulate autophagy to protect cardiomyocytes, we used rapamycin to restore autophagy depressed by DOX. Rapamycin increased cardiomyocyte survival by easing the autophagic flux blocked by DOX. In addition, rapamycin reduced oxidative stress, prevented mitochondrial damage, and restored energy metabolic homeostasis in DOX-treated cardiomyocytes. In vivo, we used metformin (Met) which is an AMPK activator to protect cardiac tissue to alleviate DOX-induced dilated cardiomyopathy. In this study, Met significantly attenuated the oxidative stress response of myocardial tissue caused by DOX and activated cardiomyocyte autophagy to maintain cardiomyocyte energy metabolism and reduce cardiomyocyte apoptosis by downregulating mTOR activity. Overall, our study revealed the role of autophagy and apoptosis in DOX-induced dilated cardiomyopathy and demonstrated the potential role of regulation of the AMPK/mTOR axis in the treatment of DOX-induced dilated cardiomyopathy.

First report of root rot caused by Fusarium nygamai on foxtail millet (Setaria italica) in China.

Foxtail millet [Setaria italica (L.) P. Beauv.] is one of the most important nutritious food crops in China. In August 2020, plants of the foxtail millet cultivar Xiao Huang Miao were found that were wilted and root rot symptoms of 25-75% incidence in a field production area of about 3000 m2 near Tongliao of Inner Mongolia and Chaoyang cities of Liaonning province. The wilted plants showed yellowing, stunting, and the lower stalk became straw colored, softened, with gray-white mould on the surface of the stem nodes. The root system was poorly developed, brown and rotted. Symptomatic roots were surface-disinfested with 70% ethanol for 1 min and in 2% sodium hypochlorite (NaOCl) for 3 min, rinsed with sterilized water three times, and placed on potato dextrose agar (PDA) and incubated at 26ºC for 5 days. Ten pure cultures were obtained from single conidia with an inoculation needle under stereomicroscope. The cultures were transferred to carnation leaf agar (CLA) medium and incubated two weeks in the dark at 26ºC for microscopic observation. Macroconidia had one to four septa (three septa dominated), and were slender and straight with curved apical cell and foot-shaped basal cell, 25.5 - 30.5 × 2.5 - 4.5 µm (n=50). Microconidia were non-septate, oval, and were formed in short chains or false heads on monophialides, 2.5 - 15 × 2.75 - 4.0 µm (n=50). Chlamydospores were singly or in chains, circular or subcircular, 5.25 - 11.5 µm in diameter (n=50). Morphologically, the fungus was identified as Fusarium nygamai Burgess & Trimboli (Klaasen and Nelson,1998; Leslie and Summerell, 2006,). To validate this identification, rDNA internal transcribed spacer (ITS), partial translation elongation factor 1 alpha (TEF-á) gene, and RNA polymerase II second largest subunit (rpb2) of the ten isolates were amplified and sequenced (White et al.1990;O'Donnell K. et al. 2015,2010). Identical sequences were obtained and the sequence of the isolate GZGF23 was submitted to GenBank. BLASTn analysis of the ITS (OL964384), TEF-á (OL961517) and RPB2(ON756204) sequence of isolate GZGF23 revealed 99.86% (MH862671, 557/565bp), 100% (MT011009, 713/1770bp) and 100% (MT010976, 1002/3907bp) sequence similarity respectively with F. nygamai (CBS749.97). Pathogenicity studies were conducted on outdoor potted ground and with the foxtail millet cultivar "Xiao Huang miao". Five 12-L pots were filled with sterilized field soil mixed with 300ml conidial suspension at 3 × 105 spores/ml. Another five 12-L pots were filled with sterilized field soil mixed with 300ml sterilized water that served as controls. About twenty seeds per pot were surface disinfected in 2% NaOCl for 3 min, and rinsed with sterilized water. The foxtail millet seeds were sown the same day as soil inoculation and 6 plants were left in each pot when seedling emerged. Five weeks after seedling emergence, all inoculated plants exhibited symptoms similar to the syptoms observed in the field but control plants had no symptoms. The same results were obtained when pathogenicity tests were repeated two times in the same manner. Fusarium nygamai was reisolated from inoculated plants and its morphological and molecular characteristics matched the original isolate, but the fungus was not reisolated from control plants. This is the first report of root rot caused by F. nygamai on foxtail millet in China. The disease might bring a threat to foxtail millet production and effective control measures should be identified to reduce losses. References: Klaasen J. A. and Nelson P. E. 1998. Mycopathologia 140: 171-176. Leslie J. F. and Summerell B. A. 2006. Blackwell Publishing, Oxford, U.K. O'Donnell K., et al. 2015. Phytoparasitica 43:583-595. White T. J., et al. 1990. Academic Press, San Diego, CA, pp 315-322. O'Donnell K et al. 2010. J.Clin.Microbiol. 48:3708.

Domestication-associated PHYTOCHROME C is a flowering time repressor and a key factor determining Setaria as a short-day plant.

Phytochromes play vital roles in the regulation of flowering time, but little is known in Panicoideae species, especially the C4 model Setaria. Here, genomic variations of PHYTOCHROME C (PHYC) between wild and cultivated Setaria gene pools were analysed and three SiphyC mutants were identified. The function of SiPHYC was verified by CRISPR-Cas9 approach and transcriptome sequencing. Furthermore, efficiency of indoor cultivation of SiphyC mutants were systematically evaluated. An extreme purified selection of PHYC was detected in wild to cultivated domestication process of Setaria. SiphyC mutants and knockout transgenic plants showed an early heading date and a loss of response to short-day photoperiod. Furthermore, variable expression of SiFTa, SiMADS14 and SiMADS15 might be responsible for promoting flowering of SiphyC mutants. Moreover, SiphyC mutant was four times that of the indoor plot ratio of wild-type and produced over 200 seeds within 45 d per individual. Our results suggest that domestication-associated SiPHYC repressed flowering and determined Setaria as a short-day plant, and SiphyC mutants possess the potential for creating efficient indoor cultivation system suitable for research on Setaria as a model, and either for maize or sorghum as well.

Key circular RNAs identified in male osteoporosis patients by whole transcriptome sequencing.

BACKGROUND: Osteoporosis (OP) is a systemic disease with bone loss and microstructural deterioration. Numerous noncoding RNAs (ncRNAs) have been proved to participate in various diseases, especially circular RNAs (circRNAs). However, the expression profile and mechanisms underlying circRNAs in male osteoporosis have not yet been explored. METHODS: The whole transcriptome expression profile and differences in mRNAs, circRNAs, and microRNAs (miRNAs) were investigated in peripheral blood samples of patients with osteoporosis and healthy controls consisting of males ≥ 60-years-old. RESULTS: A total of 398 circRNAs, 51 miRNAs, and 642 mRNAs were significantly and differentially expressed in osteoporosis compared to healthy controls. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the host genes of significantly differentially expressed circRNAs were mainly enriched in the regulation of cell cycle process: biological process (BP), organelle part cellular components (CC), protein binding molecular function (MF), Toll-like receptor signaling pathway, tumor necrosis factor (TNF) signaling pathway, and thyroid hormone signaling pathway. circRNA-miRNA-mRNA regulatory network was constructed using the differentially expressed RNAs. Moreover, key circRNAs (hsa_circ_0042409) in osteoporosis were discovered and validated by qPCR. CONCLUSIONS: The key cicrRNAs plays a major role in the pathogenesis of osteoporosis and could be used as potential biomarkers or targets in the diagnosis and treatment of osteoporosis.

Genetic control and phenotypic characterization of panicle architecture and grain yield-related traits in foxtail millet (Setaria italica).

KEY MESSAGE: Multi-environment QTL mapping identified 23 stable loci and 34 co-located QTL clusters for panicle architecture and grain yield-related traits, which provide a genetic basis for foxtail millet yield improvement. Panicle architecture and grain weight, both of which are influenced by genetic and environmental factors, have significant effects on grain yield potential. Here, we used a recombinant inbred line (RIL) population of 333 lines of foxtail millet, which were grown in 13 trials with varying environmental conditions, to identify quantitative trait loci (QTL) controlling nine agronomic traits related to panicle architecture and grain yield. We found that panicle weight, grain weight per panicle, panicle length, panicle diameter, and panicle exsertion length varied across different geographical locations. QTL mapping revealed 159 QTL for nine traits. Of the 159 QTL, 34 were identified in 2 to 12 environments, suggesting that the genetic control of panicle architecture in foxtail millet is sensitive to photoperiod and/or other environmental factors. Eighty-eight QTL controlling different traits formed 34 co-located QTL clusters, including the triple QTL cluster qPD9.2/qPL9.5/qPEL9.3, which was detected 23 times in 13 environments. Several candidate genes, including Seita.2G388700, Seita.3G136000, Seita.4G185300, Seita.5G241500, Seita.5G243100, Seita.9G281300, and Seita.9G342700, were identified in the genomic intervals of multi-environmental QTL or co-located QTL clusters. Using available phenotypic and genotype data, we conducted haplotype analysis for Seita.2G002300 and Seita.9G064000,which showed high correlations with panicle weight and panicle exsertion length, respectively. These results not only provided a basis for further fine mapping, functional studies and marker-assisted selection of traits related to panicle architecture in foxtail millet, but also provide information for comparative genomics analyses of cereal crops.

QTL mapping for foxtail millet plant height in multi-environment using an ultra-high density bin map.

KEY MESSAGE: Using a fixed RIL population derived from a widely used foxtail millet backbone breeding line and an elite cultivar, we constructed a high-density bin map and identified six novel multi-environment effect QTLs and seven candidate genes for dwarf phenotype. Plant height is an important trait that determines tradeoffs between competition and resource allocation, which is crucial for yield potential. To improve the C4 model plant foxtail millet (Setaria italica) productivity, it is necessary to isolate plant height-related genes that contribute to ideal plant architecture in breeding. In the present study, we generated a foxtail millet population of 333 recombinant inbred lines (RILs) derived from a cross between a backbone line Ai 88 and an elite cultivar Liaogu 1. We evaluated plant height in 13 environmental conditions across 4 years, the mean plant height of the RIL population ranged from 89.5 to 149.9 cm. Using deep re-sequencing data, we constructed a high-density bin map with 3744 marker bins. Quantitative trait locus (QTL) mapping identified 26 QTLs significantly associated with plant height. Of these, 13 QTLs were repeatedly detected under multiple environments, including six novel QTLs that have not been reported before. Seita.1G242300, a gene encodes gibberellin 2-oxidase-8, which was detected in nine environments in a 1.54-Mb interval of qPH1.3, was considered as an important candidate gene. Moreover, other six genes involved in GA biosynthesis or signaling pathways, and fifteen genes encode F-box domain proteins which might function as E3 ligases, were also considered as candidate genes in different QTLs. These QTLs and candidate genes identified in this study will help to elucidate the genetic basis of foxtail millet plant height, and the linked markers will be useful for marker-assistant selection of varieties with ideal plant architecture and high yield potential.

Overexpression of Dnmt3a ameliorates diabetic muscle atrophy by modulating the Pten/Akt pathway.

NEW FINDINGS: What is the central question of this study? Does Dnmt3a play a crucial role in regulating diabetic muscle atrophy? What is the main finding and its importance? Muscle atrophy is one of the major long-term complications of diabetes mellitus. However, little is known about the molecular mechanism involved. In this paper, we demonstrated that Dnmt3a overexpression effectively improves the diabetic muscle health in mice and documented the underlying mechanisms. DNMT3A might become a promising target to prevent muscle atrophy in patients with diabetes. ABSTRACT: Muscle atrophy is one of the major long-term complications of diabetes mellitus, which greatly affects the mobility of patients. Epigenetic processes mediated by DNA methyltransferases (DNMTs) play crucial roles in the locomotor system, but little is known about the functions of DNMTs in diabetic muscle atrophy. Here, we investigated the function of Dnmt3a in diabetic muscle atrophy and explored the mechanisms involved. Adeno-associated virus AAV2 overexpressing Dnmt3a or its vector control was injected into the tibialis anterior muscle of streptozotocin-induced diabetic mice. Muscle mass and muscle cross-sectional area were used to evaluate muscle atrophy. In vitro, adeno-associated virus AAV2 overexpressing Dnmt3a or its vector control was transfected into C2C12 myoblasts. Horse serum was used to induce differentiation and palmitate to stimulate the C2C12 myoblasts. The expressions of myogenic regulatory factors were examined by real-time PCR and western blot analysis. Overexpression of Dnmt3a attenuated muscle atrophy in diabetic mice and promoted myotube formation of C2C12 myoblasts. Overexpression of Dnmt3a restored the expressions of myogenic regulatory factors atrogin-1, MuRF1, Pax7, Myod1 and myogenin, both in vivo and in vitro. Moreover, overexpression of Dnmt3a activated the phosphorylation of Akt by inhibiting the activation of Pten. This study demonstrates that overexpression of Dnmt3a prevents diabetic muscle atrophy by modulating the Pten/Akt pathway.

Downregulation of miR­16 protects H9c2(2­1) cells against hypoxia/reoxygenation damage by targeting CIAPIN1 and regulating the NF­κB pathway.

The aim of the present study was to determine the function of microRNA­16 (miR­16) in myocardial hypoxia/reoxygenation (H/R)­induced cardiomyocyte injury and the possible mechanism underlying its involvement. An H/R model was constructed using H9c2(2­1) cells in vitro. The results of reverse transcription­quantitative PCR demonstrated that the expression levels of miR­16 were significantly upregulated in H9c2(2­1) cells in the H/R group compared with the sham group (1.53±0.09 vs. 1.0±0.08; P=0.0019). Cell Counting Kit­8 assays revealed that the relative proliferative ability of H9c2(2­1) cells was significantly decreased in the H/R + negative control (NC) group compared with the sham group (0.53±0.05 vs. 1.0±0.08; P=0.00005). Upregulation of miR­16 using miR­16 mimics further decreased the proliferative ability of cells (0.31±0.03 vs. 0.53±0.05; P=0.0097), whereas downregulation of miR­16 using an miR­16 inhibitor increased the proliferative ability of cells compared with the H/R+NC group (0.89±0.08 vs. 0.53±0.05; P=0.000385). Flow cytometric analysis found that the apoptotic rate of H9c2(2­1) cells was increased significantly following H/R compared with the sham group (25.86±2.62% vs. 9.29±0.82%, P=0.000014). Upregulation of miR­16 further increased the apoptotic rate (38.62±2.04% vs. 25.86±2.62%; P=0.000099), whereas downregulation of miR­16 decreased the apoptotic rate compared with the H/R+NC group (15.14±0.92% vs. 25.86±2.62%; P=0.000343). miR­16 directly bound to the 3'­untranslated region of cytokine­induced apoptosis inhibitor 1 (CIAPIN1) and negatively modulated CIAPIN1 expression. Overexpression of CIAPIN1 reversed the changes in the expression of apoptosis­associated proteins caused by H/R. Western blot analysis revealed that the levels of phospho­(p­)nuclear factor­κB (NF­κB) and p­NF­κB inhibitor α (IκBα) were upregulated following H/R (1.82±0.11 vs. 1.0±0.08; P=0.000152; and 1.77±0.07 vs. 1.0±0.00; P=0.000024, respectively), and these changes were further enhanced when miR­16 expression levels were increased (3.10±0.14 vs. 1.82±0.11; P=0.000006; and 2.19±0.10 vs. 1.77±0.07; P=0.0017, respectively). Downregulation of miR­16 exhibited the opposite effect on p­NF­κB and p­IκBα expression levels. The present study illustrates that downregulation of miR­16 may protect against H/R­induced injury partially by targeting CIAPIN1 and the NF­κB signaling pathway.

[Application of CT scan in diagnosis of pathological type and origin of metastatic ovarian tumors].

OBJECTIVE: To evaluate the application of CT scan in diagnosis of pathological types and origins of metastatic ovarian tumors. METHODS: Clinical data, histopathological results and CT images of 43 patients with pathologically-proved metastatic ovarian tumor were retrospectively analyzed. Diagnostic values of CT imaging for pathological type and origin of metastatic ovarian tumors were evaluated. RESULTS: The pathological types of metastatic ovarian tumor were related to the size of the lesion (P<0.01), while not related to the sites of lesion (unilateral or bilateral), the cystic-solid and mixed lesions with or without separation (all P>0.05). Metastatic ovarian tumors of colorectal origin were usually unilateral lesions, and showed cystic or cystic-solid masses, while those of gastric origin were usually bilateral lesions, and showed solid or solid-based masses. CONCLUSIONS: CT imaging may be of value in diagnosis of pathological types and origin of metastatic ovarian tumor.

Effect of vitamin E succinate on the expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor in gastric cancer cells and CD4(+) T cells.

Gastric malignancy, which shows poor prognosis, is one of the most frequent causes of cancer-associated deaths. Vitamin E succinate (VES) inhibits cell proliferation and induces apoptosis in a concentration- and time-dependent manner. We explored the effect of VES on the expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor in gastric cancer cells and CD4(+) T cells. On one hand, VES dose-dependently regulated the expression of the TRAIL receptor in gastric cancer cells. Moreover, the activation of the TRAIL receptor, death receptor 4 (DR4), and death receptor 5 (DR5) in gastric cancer cells increased for up to 12 h. On the other hand, the expression of TRAIL protein in human CD4(+) T cells was obviously upregulated in the presence of VES. On the basis of these findings, we combined VES and human CD4(+) T cells to induce apoptosis of MKN28 human gastric cancer cells. The results showed that VES induced higher gastric cancer cell apoptosis when combined with human CD4(+) T cells than when applied alone. We conclude that VES can induce the expression of TRAIL receptor in gastric cancer cells, as well as the expression of TRAIL in CD4(+) T cells. Overall, our results provide a theoretical basis for future immunotherapy studies.

[Effects of elevated atmospheric CO2 on paddy soil nitrogen content during rice season].

Taking the China rice/wheat FACE (Free-Air Carbon Dioxide Enrichment) as a platform, this paper studied the effects of elevated CO2 on the NH4(+)-N and NO3(-)-N contents at different depths of paddy soil in rice season. Under elevated CO2, the NH4(+)-N content in plough layer increased at early growth stage but decreased at late growth stage, and the soil NO3(-)-N content at the depths 5, 15, 30, 60, and 90 cm increased by 46.5%, 36.8%, 23.3%, 103.7%, and 42.7%, respectively, with a significant increase occurred at the depths 60 cm (P < 0.01) and 90 cm (P < 0.05), compared with the control.

Bid integrates intrinsic and extrinsic signaling in apoptosis induced by alpha-tocopheryl succinate in human gastric carcinoma cells.

The underlying mechanisms of alpha-tocopheryl succinate (alpha-TOS)-mediated apoptosis are not understood in detail, although the redox-silent vitamin E analog is a potent apoptogen and anti-cancer agent. Our previous studies showed the important role of Fas signaling in apoptosis induced by the mitocan. The objective of the present study was to investigate whether apoptosis triggered by alpha-TOS in gastric carcinomas cells involves both mitochondria- and death receptor-dependent pathways. alpha-TOS induced apoptosis and mitochondrial permeability transition in a concentration- and time-dependent manner. As a consequence, cytochrome c and the apoptosis-inducing factor were released and caspases were activated. Bax was translocated from the cytosol to mitochondria and Bid was cleaved into its truncated form, tBid. Knocking down Bid by RNAi and Fas antisense oligodeoxynucleotides resulted in a decreased release and cleavage. The results imply that Bid may serve as a critical integrating factor of the death receptor and mitochondrial pathway in alpha-TOS-mediated apoptosis.

Diode-pumped passively Q-switched Nd:LuVO4 laser at 1.34 microm with a V3+:YAG saturable absorber.

We report a diode-pumped passively Q-switched Nd:LuVO4 laserat 1.34 microm using V3+:YAG as saturable absorber. The characteristics of a-cut and c-cut Nd:LuVO4 passive Q-switching operation were studied. The average output power of 1.02 W was obtained under the pump power of 12.88 W, corresponding to the optical conversion efficiency of 8% and slope efficiency of 10% in c-cut Nd:LuVO4 laser. The maximum pulse energy of 17.6 microJ and the highest peak power of 820 W were obtained at pulse width of 21 ns and pulse repetition rate of 22.4 kHz in a-cut Nd:LuVO4 laser.