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X70 pipeline steel is butt welded by fully automatic welding at 25 ° slope, and the characteristic information of welding process is collected. A 3D transient heat and mass transfer model of weld pool is established. This model was verified by means of the shape of the weld pool and the two-dimensional shape of the weld, and then the flow field of the fully automatic welding process under steep slope conditions was simulated to clarify the mechanism of the lack of fusion defect. The results show that the welding arc is deflected when it stays on both side walls of the groove. When the arc stays at the bottom of the lower groove, the overflow phenomenon occurs at the structural mutation position of the molten pool located at the bottom of the side wall, and some liquid metal overflows to the front of the groove, forming a "void area" with gas-liquid interface. In the welding process, the liquid metal tends to flow to the side walls for heat transfer under various thermal effects, and forms a large range of eddy current at the side walls. This has a positive effect on sidewall melting. In addition, under the influence of the eddy current, it is difficult for the high-temperature liquid metal in the arc center to flow to the "void area" located at the bottom of the side wall of lower groove. Therefore, the bottom of the lower groove is more likely to cause lack of fusion defect.

Plastid engineering has several unique advantages such as high expression of transgenes due to high polyploidy of plastid genomes and environmental biosafety because of maternal inheritance of transgenes, and has become a promising tool for molecular farming, metabolic engineering, and genetic improvement. However, there are no standard vectors available for plastid transformation. Moreover, the construction of plastid transformation vectors containing long operons or genes encoding proteins that are toxic to Escherichia coli was tedious or difficult. Here, we developed a simple plastid transformation technology without the need for in vitro vector construction by using multiple linear DNA fragments which share homologous sequences (HSs) at their ends. The strategy is based on homologous recombination between HSs of DNA fragments via endogenous recombination machinery in plastids, which subsequently are integrated into the plastid genome. We found that HSs of 200 bp or longer were sufficient for mediating the integration into the plastid genome with at least similar efficiency to that of plasmid DNA-based plastid transformation. Furthermore, we successfully used this method to introduce a phage lysin-encoding gene and a long operon into a tobacco plastid genome. The establishment of this technology simplifies the plastid transformation procedure and provides a novel solution for expressing proteins, which are either toxic to the cloning host or large operons in plastids, without need of vector cloning.

The transformation relationships between three new crystal forms of all-biobased nylon 4F salts.

Almost all salt forms of nucleotides exist as crystalline hydrates. In this study, the hydrates of adenosine-3′,5′-cyclic phosphate sodium (cAMPNa·nH2O, n = 2, 4, 5, including its methanol solvate of n = 3) were systematically investigated for the crystal structures, their humidity sensibility, the transformation behaviors mediated by humidity, and the relationship between crystal water behaviors and specific structures. It was found that there are some major differences in crystal morphologies, molecular conformation, bonding profiles, and crystal water distribution in the lattices of different hydrates. The difference in the desorption dynamics of crystal water for the different hydrates was mainly attributed to their steric effects in the crystal lattice despite the relation to the interaction of water molecules with sodium ions and cAMP anions. The reversible conversions between the hydrates and their corresponding low hydrates or even anhydrates occur along with desorption/uptake of crystal water mediated by humidity, yet the reversible transformation capability would be disabled once the host structures were destroyed due to excessive loss of crystal water.

Plant-mediated RNA interference (RNAi) has emerged as a promising technology for pest control through expression of double-stranded RNAs (dsRNAs) targeted against essential insect genes. However, little is known about the underlying molecular mechanisms and whether long dsRNA or short interfering RNAs (siRNAs) are the effective triggers of the RNAi response. Here we generated transplastomic and nuclear transgenic tobacco plants expressing dsRNA against the Helicoverpa armigera ATPaseH gene. We showed that expression of long dsRNA of HaATPaseH was at least three orders of magnitude higher in transplastomic plants than in transgenic plants. HaATPaseH-derived siRNAs are absent from transplastomic plants, while they are abundant in transgenic plants. Feeding transgenic plants to H. armigera larvae reduced gene expression of HaATPaseH and delayed growth. Surprisingly, no effect of transplastomic plants on insect growth was observed, despite efficient dsRNA expression in plastids. Furthermore, we found that dsRNA ingested by H. armigera feeding on transplastomic plants was rapidly degraded in the intestinal fluid. In contrast, siRNAs are relatively stable in the digestive system. These results suggest that plant-derived siRNAs may be more effective triggers of RNAi in Lepidoptera than dsRNAs, which will aid the optimization of the strategies for plant-mediated RNAi to pest control.

Background RNA interference (RNAi) has emerged as a promising technology for insect pest control. Because of the accumulation of high levels of long double-stranded RNAs (dsRNAs) in plastids, it was previously shown that expression of dsRNAs from plastid genome led to higher mortality of some insect pests with chewing mouthparts than dsRNAs expression from nuclear genome. However, whether plastid-expressed dsRNAs have effects on phloem sap-sucking pests is unknown. In this study, we compared the RNAi effects of nuclear transgenic and transplastomic plants on the whitefly Bemisia tabaci, a serious sap-sucking pest. Results Nuclear transgenic and transplastomic tobacco plants were developed for the expression of dsRNA against BtACTB gene of Bemisia tabaci, respectively. Feeding nuclear transgenic plants to Bemisia tabaci resulted in reduced gene expression of BtACTB and survival rate, and impaired fecundity of Bemisia tabaci. We did not observe any effects of transplastomic plants on Bemisia tabaci fitness. Furthermore, we found that the inability of B. tabaci to obtain dsRNAs from plastids might restrict its RNAi responses. Conclusion Our study indicated that the expression of dsRNAs in nuclear transgenic plants was more effective than that in transplastomic plants for the control of Bemisia tabaci. The inaccessibility of Bemisia tabaci to plastids contributes to the inefficiency of plastid-mediated RNAi. Our findings are of great significance to future optimization of transgenically delivered RNAi approaches for efficient controlling of sap-sucking pests. © 2020 Society of Chemical Industry.

Nylon 5I is one of the new bio-based nylon materials. Its raw material 1,5-pentanediamine (PDA) is prepared by biological methods using biomass as the raw material. The high-performance polymer materials require the original high-quality monomers. 1,5-Pentanediamine-isophthalate (PDA-IPA) was taken as the direct monomer for the preparation of nylon 5I, and the crystallization was a valuable and essential approach to preparing the good-performance monomer salt. In this report, we found and obtained two crystal forms of PDA-IPA, monohydrate and an anhydrous form. Their crystal structures were determined and analyzed by single crystal X-ray diffraction (SCXRD), powder X-ray diffraction (PXRD), and Fourier transform infrared spectroscopy (FTIR). Hirshfeld surface maps were employed to capture the differences in the interactions present in the two forms. The thermal behaviors were characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Moreover, the monohydrate and anhydrous phase can transform to each other through solid-solid transformation or solution-mediated phase transformation, and the critical values of the phase transformation were determined. Finally, the relative stability of the two forms under different thermodynamic conditions was discussed, especially the influence of temperature and water activity on the stability.

Expression of double-stranded RNAs in plastids offers great potential for the efficient control of chewing insects. However, many insect pests do not consume plant tissue but rather feed on the host plant by sucking sap from the vascular system. Whether or not plastid-mediated RNA interference (RNAi) can be employed to control sap-sucking insects is unknown. Here, we show that five species of sap-sucking hemipteran insects acquire plastid RNA upon feeding on plants. We generated both nuclear transgenic and transplastomic tobacco plants expressing double-stranded RNAs targeting the MpDhc64C gene, a newly identified efficient target gene of RNAi whose silencing causes lethality to the green peach aphid Myzus persicae. In a whole-plant bioassay, transplastomic plants exhibited significant resistance to aphids, as evidenced by reduced insect survival, impaired fecundity, and decreased weight of survivors. The protective effect was comparable with that conferred by the best-performing nuclear transgenic plants. We found that the proportion of aphids on mature leaves of transplastomic plants was significantly lower compared with that of nuclear transgenic plants. When aphids were allowed to infest only the mature leaves, transplastomic plants grew significantly faster and were overall better protected from the pest compared with nuclear transgenic plants. When monitored by electrical-penetration-graph analyses and aphid avoidance response experiments, the insects displayed remarkable alterations in feeding behavior, which was different in nuclear transgenic and transplastomic plants, likely reflecting specific avoidance strategies to toxic RNA molecules. Taken together, our study demonstrates that plastid-mediated RNAi provides an efficient strategy for controlling at least some sap-sucking insect pests, even though there is most likely no or only very little chloroplast RNA in the sap.

Importation of taxadiene synthase into chloroplasts is important for the efficient heterologous production of taxadiene. Taxadiene, the first committed precursor to taxol, is synthesized from geranylgeranyl pyrophosphate (GGPP) by action of taxadiene synthase (TS). Heterologous production of taxadiene could potentially rely on both cytosolic mevalonic acid (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. We suggest the compartmentalized engineering in chloroplast as an efficient approach for taxadiene production. In this study, we directly introduced the TS gene from Taxus brevifolia into the tobacco chloroplast genome and found that the transplastomic plants accumulated a low content of taxadiene, ~ 5.6 μg/g dry weight (DW). Moreover, we tried a combination of MEP and MVA pathways for taxadiene synthesis by nuclear transformation with a truncated version of TS (without encoding a transit peptide) into the transplastomic plants. However, this did not further improve the taxadiene production. In contrast, we found that taxadiene could be produced up to 87.8 μg/g DW in leaves of transgenic plants expressing TS with a chloroplast transit peptide, which was significantly higher than that in leaves of transplastomic plants. Thus, this study highlights the importance of TS importation into chloroplast for production of taxadiene.

Although ~80% of newly diagnosed pediatric patients with acute lymphoblastic leukemia (ALL) become disease‑free following appropriate treatment, relapses frequently occur, with dismal prognosis. Therefore, it is urgent to develop novel therapeutic modalities. Resistance to chemotherapy is a major obstacle for the treatment of relapsed ALL. It has been indicated that Wnt pathway is potentially associated with leukemia recurrence. In the current study, a vincristine (VCR)‑resistant variant of the human ALL cell line BALL‑1 (BALL‑1/VCR) that also had relatively specific resistance to both doxorubicin and etoposide was generated. Over‑activation of the Wnt/β‑catenin signaling pathway was observed in BALL‑1/VCR cells, whereas Dickkopf‑related protein 1 selectively suppressed the Wnt signaling pathway and sensitized the response of BALL‑1/VCR to anticancer agents. In addition, prednisolone exposure in combination with Wnt inhibition restored chemo‑sensitivity in relapsed ALL blasts. Since the resistance of BALL‑1/VCR cells is potentially attributed to the overexpression of MDR‑associated protein 1 (MRP1), the development of drug resistance in relapsed ALL may associated with the overexpression of MRP1 and P‑glycoprotein. The results of this study demonstrated that, as a potential candidate to mimic relapsed ALL, BALL‑1/VCR could be used in further research, while Wnt‑inhibition may become a promising therapeutic approach for treating ALL.

Cord blood transplantation (CBT) can be a life-saving procedure in the treatment of a broad variety of disorders, including hematologic, immune, and genetic diseases. However, delayed platelet recovery hinders the application of CBT.The aim of this study was to determine the optimal combination of cytokines to amplify megakaryocyte (Mk).CB CD34+ cells were obtained by immunomagnetic isolation and amplified under four different cytokine combinations. CD34+ cells of the group with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (FL), and interleukin-6 (IL-6) were collected on days 0, 3, 7, 10, and 14. Immunophenotype was analyzed by flow cytometry (FCM). Polyploidic Mk cultured cells were collected on days 7 and 14 for colony-forming unit-Mk assay. The NOD/SCID mice were injected with expanded CD34+ cells, and the peripheral blood (PB) and bone marrow (BM) were tested on 3, 7, and 14 days.The group with TPO, SCF, FL, and IL-6 reached the maximal total expansion fold and Mk population at day 7, which was slightly reduced later. After transplantation into NOD/SCID mice with expanded CD34+ cells, the human CD41+ cells were detected in mice PB on day 3 and in BM on day 7, then disappeared after 14 days. The expressing of activated platelet CD 42b+/CD62P+ increased gradually after transplantation.Platelets can recover rapidly in vivo by means of expanded CD34+ cells with various cytokines. In our system, a group of TPO, SCF, FL, and IL-6 represents the best cytokine combination for expansion of Mk progenitor cells from CB CD34+ cells.

Owing to their immunosuppressive properties mesenchymal stem cells (MSCs) are widely applicable in the treatment of autoimmune disease. The aim of this study was to investigate whether the indoleamine 2,3-dioxygenase-1 (IDO-1) and cyclooxygenase-2 (COX-2) genes enhanced the immunosuppressive functional ability of MSCs following stable transfection. To strengthen the immunomodulatory ability of MSCs, IDO-1 and COX-2 were overexpressed in umbilical cord progenitor cell-derived MSCs using recombinant plasmids and electroporation. RT-qPCR analysis and western blotting confirmed the expression of IDO-1 and COX-2 in transfected MSCs. Further functional assays in co-culture experiments, including lymphocyte proliferation and cyto-toxicity assays showed that COX-2-transfected MSCs possessed more potent immunomodulatory cells than the untreated MSCs, or MSCs transfected with IDO-1. Additionally, synthesis of interferon-γ and tumor necrosis factor-α (TNF-α) was significantly inhibited in lymphocytes co-cultured with COX-2-transfected MSCs, which was consistent with changes in immune-related genes in MSCs. An enhanced expression of IDO-1, COX-2, heme-oxygenase-1, inducible nitric-oxide synthase, TNF-α-stimulated gene/protein-6, transforming growth factor-β (TGF-β), human leukocyte antigen molecule 5 (HLA-G5) and interleukin-10 (IL-10) was identified following COX-2 transfection. We showed that the overexpression of COX-2 enhanced the immunosuppressive function of MSCs. COX-2-modified MSCs more potently inhibited the activation and proliferation of peripheral blood mononuclear cells.

The aim of the study was to investigate the effect of exosomes derived from bone marrow stromal cells (BM-SCs) on the chemoresistant characteristics of nalm-6 cells treated with etoposide (VP16). The present study isolated exosomes from BM-SC-conditioned medium by using standard differential centrifugation steps and detected the expression of 70 kilodalton heat shock proteins (HSP70) and lysosomal-associated membrane protein 3 (CD63) in exosomes by western blot analysis. Nalm-6 cells were co-cultured with exosomes in the presence of VP16. Cell viability and apoptosis were then detected using the Cell Counting Kit-8 method and Annexin-V/propidium iodide, respectively. Finally, protein levels of B-cell lymphoma 2 (BCL-2), BCL-2-like protein 4 (BAX), caspase-3, and poly ADP-ribose polymerase (PARP) were examined by western blot analysis. Exosomes were successfully isolated from the conditioned medium and confirmed by the expression of HSP70 and CD63. BM-SC-derived exosomes increased the viability of nalm-6 cells in the presence of VP16 and inhibited the apoptosis induced by VP16. Western blot analysis results showed that exosomes can block the significant reduction of BCL-2, full-length caspase-3 and full-length PARP, while preventing the increase of BAX, cleaved caspase-3 and cleaved PARP induced by VP16. Exosomes derived from BM-SCs can protect nalm-6 cells from VP16-induced apoptosis to maintain their survival and induce resistance to VP16. In addition, BCL-2/BAX, caspase-3, and PARP may be involved in the mechanism of exosome-induced drug resistance.

Activated platelet-specific autoreactive T cells play a key role in the pathophysiology of immune thrombocytopenia (ITP). Tolerogenic dendritic cells (tDCs) that induce T cells tolerance to platelet autoantigens are a promising strategy for specific cellular therapy in autoimmunity. In this study, we generated three kinds of GPIIb-specific tDCs stimulated by IL-10 (10-DCs), TGFβ1 (T-DCs), or a combination of IL-10 and TGFβ1 (10T-DCs), respectively, and compared their phenotypes and biological function. Our data demonstrate that GPIIb-specific 10T-DCs induced the weakest memory lymphocytes responses and exhibited stronger tolerogenic potential than others, making them suitable for tolerance inducing therapies. Furthermore, in ITP mice model we found that 10T-DCs abrogated the decrease in platelet counts and the increase in serum IFN-γ level, confirming the in vivo tolerogenic potential of 10T-DCs. Our study provides a promising strategy for ITP intervention in the clinic by the induction of platelet-specific immune tolerance.

Background: Cord blood transplantation (CBT) can be a life-saving procedure in the treatment of a broad variety of disorders, including hematologic, immune, and genetic diseases. However, delayed platelet recovery hinders the application of CBT. Purpose: The aim of this study was to determine the optimal combination of cytokines to amplify megakaryocyte (Mk). Methods: CB CD34+ cells were obtained by immunomagnetic isolation and amplified under four different cytokine combinations. CD34+ cells of the group with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (FL), and interleukin-6 (IL-6) were collected on days 0, 3, 7, 10, and 14. Immunophenotype was analyzed by flow cytometry (FCM). Polyploidic Mk cultured cells were collected on days 7 and 14 for colony-forming unit-Mk assay. The NOD/SCID mice were injected with expanded CD34+ cells, and the peripheral blood (PB) and bone marrow (BM) were tested on 3, 7, and 14 days. Results: The group with TPO, SCF, FL, and IL-6 reached the maximal total expansion fold and Mk population at day 7, which was slightly reduced later. After transplantation into NOD/SCID mice with expanded CD34+ cells, the human CD41+ cells were detected in mice PB on day 3 and in BM on day 7, then disappeared after 14 days. The expressing of activated platelet CD 42b+/CD62P+ increased gradually after transplantation. Conclusion: Platelets can recover rapidly in vivo by means of expanded CD34+ cells with various cytokines. In our system, a group of TPO, SCF, FL, and IL-6 represents the best cytokine combination for expansion of Mk progenitor cells from CB CD34+ cells.

Dendritic cells (DCs) play important roles in initiation of the pathogenic processes of autoimmune disorders, such as rheumatoid arthritis (RA). Tolerogenic dendritic cells (tolDCs) are generated from naive DCs and induce T cell tolerance; thus, they represent a promising strategy for specific cellular therapy for autoimmune diseases. In this study, we generated green fluorescent protein (GFP)-labeled tolDCs and confirmed their phenotypes and biological functions. We found that tolDCs suppressed the memory lymphocyte response and exhibited strong tolerogenic potential; thus, these cells show promise for the treatment of autoimmune diseases. Additionally, a collagen-induced arthritis (CIA) mouse model was used to test the role of tolDCs in vivo. The results of a further mechanistic experiment revealed that tolDCs suppressed inflammatory arthritis at least partially by up-regulating regulatory T (Treg) cells. Collectively, our data suggest that tolDCs may be used as a promising alternative therapy for inflammatory arthritis.

The AIOLOS gene is important in the control of mature B-lymphocyte differentiation and proliferation. Previous research has shown that deregulated AIOLOS expression is associated with adult B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia in human patients. However, the function of AIOLOS in childhood B-cell precursor (BCP)-ALL is not fully understood. In the present study, Nalm-6 cells were divided into three groups: the untransfected control (UT), the lentiviral vector control (Lenti-Mock) and the AIOLOS-overexpressing (Lenti-AIOLOS) group. Lenti-AIOLOS Nalm-6 cells were constructed by lentiviral transduction, followed by cell proliferation assay, cell-cycle analysis and apoptosis assay, to evaluate the effects of AIOLOS on proliferation, cell cycle distribution and apoptosis of Nalm-6 cells in vitro. Moreover, the expression levels of genes associated with apoptosis and the cell cycle, as well as the transcription factors IKZF1 and NF-κB, were investigated by quantitative reverse transcription-polymerase chain reaction and western blot analysis. The results showed that the proliferation of Nalm-6 cells in the Lenti-AIOLOS group was reduced by 16% on day 8 compared with cells in the UT group (P>0.05). The reduction peaked at 29% on day 10 (P