Cloning, expression of Aspergillus niger JL-15 endo-polygalacturonase A gene in Pichia pastoris and oligo-galacturonates production.

The endo-galacturonase A gene (pgaA) was cloned using the cDNAs synthesized from total RNA of Aspergillus niger JL-15 by reverse transcription as template. The open reading frame (ORF) of pgaA was 1113bp, encoding a peptide of 370 amino acids with the predicted molecular mass of 38.8kDa. The pgaA was successfully expressed in Pichia pastoris GS115 under the control of AOX1 promoter. After induction by methanol for 96h, the activity of the recombinant endo-galacturonase A (rePgaA) in culture supernatant was 2091.0U/mg. SDS-PAGE analysis showed that the molecular mass of rePgaA was about 40.0kDa. Enzymatic properties assays showed that the optimum temperature and pH for rePgaA were 50°C and pH 5.0, respectively. The Michaelis-Menten constant (Km) and maximal velocity (Vmax) of rePgaA for citrus pectin were 3.20mgml(-1) and 40.97µmolmin(-1)ml(-1), respectively. The rePgaA mediated a rapid decrease in viscosity of pectin solution with release of small amount of reducing sugar. High performance liquid chromatography (HPLC) analysis revealed that digalacturonate (G2) and trigalalcturonate (G3) were the main hydrolysis products released from pectin by rePgaA. The rePgaA showed very low activity on G2 and G3, which suggested it was a typical endo-acting enzyme.

Combined Application of Leguminous Green Manure and Straw Determined Grain Yield and Nutrient Use Efficiency in Wheat-Maize-Sunflower Rotations System in Northwest China.

Leguminous green manure (LGM) has a reputation for improving crop productivity. However, little is known about the beneficial interactions with straw on crop yield and nutrient (N, P, K) use efficiency. Herein, a 9-year field experiment (from 2015 to 2023) containing three treatments-(1) chemical fertilizer as the control (CK), (2) NPK + straw return (Straw) and (3) NPK + straw return with LGM (Straw + LGM)-was conducted to investigate whether the combined application of LGM and straw can increase productivity and nutrient use efficiency in the wheat-maize-sunflower diversified cropping rotation. The results showed that in the third rotation (2021-2023), Straw + LGM significantly increased wheat yield by 10.2% and maize yield by 19.9% compared to CK. The total equivalent yield under Straw + LGM was the highest (26.09 Mg ha-1), exceeding Straw and CK treatments by 2.7% and 12.3%, respectively. For each 2 Mg ha-1 increase in straw returned to the field, sunflower yield increased by 0.2 Mg ha-1, whereas for each 1 Mg ha-1 increase in LGM yield from the previous crop, sunflower yield increased by 0.45 Mg ha-1. Compared to CK, the co-application of LGM and straw increased the N use efficiency of maize in the first and third rotation cycle by 70.6% and 55.8%, respectively, and the P use efficiency by 147.8% in the third rotation cycle. Moreover, Straw treatment led to an increase of net income from wheat and sunflower by 14.5% and 44.6%, while Straw + LGM increased the net income from maize by 15.8% in the third rotation cycle. Combining leguminous green manure with a diversified cropping rotation has greater potential to improve nutrient use efficiency, crop productivity and net income, which can be recommended as a sustainable agronomic practice in the Hetao District, Northwest China.

Increasing serum half-life and extending cholesterol lowering in vivo by engineering antibody with pH-sensitive binding to PCSK9.

Target-mediated clearance and high antigen load can hamper the efficacy and dosage of many antibodies. We show for the first time that the mouse, cynomolgus, and human cross-reactive, antagonistic anti-proprotein convertase substilisin kexin type 9 (PCSK9) antibodies J10 and the affinity-matured and humanized J16 exhibit target-mediated clearance, resulting in dose-dependent pharmacokinetic profiles. These antibodies prevent the degradation of low density lipoprotein receptor, thus lowering serum levels of LDL-cholesterol and potently reducing serum cholesterol in mice, and selectively reduce LDL-cholesterol in cynomolgus monkeys. In order to increase the pharmacokinetic and efficacy of this promising therapeutic for hypercholesterolemia, we engineered pH-sensitive binding to mouse, cynomolgus, and human PCSK9 into J16, resulting in J17. This antibody shows prolonged half-life and increased duration of cholesterol lowering in two species in vivo by binding to endogenous PCSK9 in mice and cynomolgus monkeys, respectively. The proposed mechanism of this pH-sensitive antibody is that it binds with high affinity to PCSK9 in the plasma at pH 7.4, whereas the antibody-antigen complex dissociates at the endosomal pH of 5.5-6.0 in order to escape from target-mediated degradation. Additionally, this enables the antibody to bind to another PCSK9 and therefore increase the antigen-binding cycles. Furthermore, we show that this effect is dependent on the neonatal Fc receptor, which rescues the dissociated antibody in the endosome from degradation. Engineered pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load.

Changes in the Microbial Community in Maize (Zea mays L.) Root Spatial Structure Following Short-Term Nitrogen Application.

The beneficial interactions between crop roots and microbiomes play a key role in crop nutrient availability, growth promotion, and disease suppression. Recent research, however, rarely reported the effects of nitrogen (N) application rate on microbial community composition at different spatial structures in the maize root zone. Therefore, one experiment was conducted to examine the influence of three N-application levels (0, 180, and 360 kg N ha-1) on microbial community composition in three root-associated compartments of maize (bulk soil, rhizoplane, and endosphere). The microbial diversity and community composition differed significantly among the various compartments. The effects of N application on fungal composition decreased in the order bulk soil > rhizosphere > endosphere at different sampling positions. Also, the fungal composition was more sensitive to the N-fertilizer rate in the bulk soil and the rhizosphere than the bacterial community. A total of 14.42, 9.46, and 3.55% of all taxonomic groups were sensitive to N fertilizer, respectively. The keystone species fungal groups were Humicola (bulk soil), Gibberella (rhizosphere soil), and Humicola (endosphere). Together, our results demonstrate that compared with that of the bacterial community, the fungal community composition was more susceptible to different N-application rates. N fertilization affected the distribution of microflora by changing soil physicochemical properties and enzyme activities. There were strong correlations between microbial communities in maize under the N180 treatment. Moreover, the N180 treatment had the maximum fresh yield and biomass at 64.5 and 24.3 kg·ha-1, respectively.

Character variation of root space microbial community composition in the response of drought-tolerant spring wheat to drought stress.

Drought is the most prevalent environmental stress in crop production, posing a significant danger to food security. Microorganisms in the crop root zone affect crop growth and development, enhance effective nutrient use, and resist adversity hazards. To analyze the changes and functional differences of root space microbial (endosphere-rhizosphere-bulk soil) communities in spring wheat under drought stress. In this study, the root, rhizosphere, and bulk soil of the drought-tolerant group (DTG, three varieties) and drought-sensitive group (DSG, three varieties) were collected. The control (CK, 25-28%), moderate drought (MD, 15-18%), and severe drought (SD, 9-12%) were analyzed by high-throughput sequencing and bioinformatics. The results showed significant differences in the diversity of Bacteria and Fungi in the root space of spring wheat under drought stress (P < 0.05), with the drought-tolerant group exhibiting higher microbial diversity. The microbial community change in spring wheat root space was mainly determined by the niche differentiation of endosphere, rhizosphere, and bulk soil and declined from endosphere to bulk soil due to drought. The antagonism between microbial and root-space species increased, and the community's complexity and stability deteriorated. Enriching drought-resistant preference groups like Actinobaciota, Variovorax, Streptomyces, and Conocybe altered the structure and function of the microbial community in the root space of spring wheat. Spring wheat's root space Bacteria and Fungi have different strategies to respond to drought.

Simultaneous targeting of Src kinase and receptor tyrosine kinase results in synergistic inhibition of renal cell carcinoma proliferation and migration.

There have been recent improvements in the treatment for metastatic renal cell carcinoma (RCC) with receptor tyrosine kinase (RTK) inhibitors being one of newer treatment options. We hypothesized that simultaneous targeting of Src kinase and the RTK may have synergistic effects to further improve therapies on metastatic RCC. The effects of Src kinase inhibitor saracatinib and multiple RTK inhibitor sunitinib on RCC cell line (ACHN) and Caki-1 were studied. Saracatinib alone or in combination with sunitinib inhibited the migration of ACHN and Caki-1 cells in vitro. Activation of migration related components FAK, P130Cas and Paxillin were blocked by saracatinib at 0.05- to 3-µM concentrations. Combined treatment resulted in improved growth inhibition, greater loss of the S phase cell population and decreased clonogenic colony formation compared to sunitinib alone in the metastatic Caki-1 line. Molecular studies in Caki-1 showed that saracatinib alone and in combination with sunitinib inhibited phosphorylation of the cell progression regulator c-Myc in a dose-dependent manner. Sunitinib alone or in combination suppressed cyclin-D1 expression with the combination showing greater dose-dependent effect. Sunitinib inhibited vascular endothelial growth factor (VEGF) secretion through the inhibition of STAT3 signaling and VEGF biosynthesis. HIF1-α expression in normoxic and hypoxic conditions in Caki-1 cells was inhibited by either saracatinib or sunitinib when administered alone, however, a greater reduction occurred when these compounds were given in combination. Targeting Src kinase and RTK simultaneously with saracatinib and sunitinib resulted in 70-80% blockade of RCC cell migration, synergistic inhibition of cell growth and reduction of acquired drug resistance in Caki-1 cells. The results show promise for combination targeted therapy of RCC.

Proprotein convertase substilisin/kexin type 9 antagonism reduces low-density lipoprotein cholesterol in statin-treated hypercholesterolemic nonhuman primates.

Proprotein convertase substilisin/kexin type 9 (PCSK9) promotes the degradation of low-density lipoprotein (LDL) receptor (LDLR) and thereby increases serum LDL-cholesterol (LDL-C). We have developed a humanized monoclonal antibody that recognizes the LDLR binding domain of PCSK9. This antibody, J16, and its precursor mouse antibody, J10, potently inhibit PCSK9 binding to the LDLR extracellular domain and PCSK9-mediated down-regulation of LDLR in vitro. In vivo, J10 effectively reduces serum cholesterol in C57BL/6 mice fed normal chow. J16 reduces LDL-C in healthy and diet-induced hypercholesterolemic cynomologous monkeys, but does not significantly affect high-density lipoprotein-cholesterol. Furthermore, J16 greatly lowered LDL-C in hypercholesterolemic monkeys treated with the HMG-CoA reductase inhibitor simvastatin. Our data demonstrate that anti-PCSK9 antibody is a promising LDL-C-lowering agent that is both efficacious and potentially additive to current therapies.

Hsp90-Akt phosphorylates ASK1 and inhibits ASK1-mediated apoptosis.

Hsp90 client protein Akt has been shown to inhibit cell apoptosis in part by inhibiting proapoptotic kinase ASK1 (apoptosis signal-regulating kinase 1) activity. In the present study, we show that Hsp90 inhibits hydrogen peroxide (H(2)O(2))-induced ASK1-p38 activation in endothelial cells (EC). The inhibitory effect of Hsp90 on ASK1-p38 activities is diminished when the Akt phosphorylation site on ASK1 (pSer83) is absent or when Akt is genetically deleted in cells, suggesting that Hsp90 and Akt function together to inhibit ASK1-p38 signaling. Thus, inhibition of Hsp90 by 17-allyamino-17-demethoxygeldanamycin (17-AAG) or phosphatidylinositol 3-kinase (PI3K) LY294002 induced and synergized ASK1 activation and ASK1-mediated EC apoptosis. Furthermore, we show that in resting EC Hsp90, Akt and ASK1 form a ternary complex in which both Akt and ASK1 bind to the middle domain of Hsp90, suggesting that Hsp90 may hold Akt and ASK1 in close proximity. The N-terminal domain of ASK1 containing the Akt phosphorylation site (pSer83) associates with Akt in resting state. However, Akt is released from the N-terminal domain concomitant with binding to the C-terminal domain of ASK1 in response to ASK1 activator H(2)O(2), inhibitor of Hsp90 17-AAG and Akt inhibitor LY294002, leading to a more stable Hsp90-Akt-ASK1 complex. We conclude that Hsp90-Akt forms a complex with ASK1 and protect EC from stress-induced apoptosis.

Thioredoxin-2 inhibits mitochondria-located ASK1-mediated apoptosis in a JNK-independent manner.

Apoptosis signal-regulating kinase 1 (ASK1) mediates cytokines and oxidative stress (ROS)-induced apoptosis in a mitochondria-dependent pathway. However, the underlying mechanism has not been defined. In this study, we show that ASK1 is localized in both cytoplasm and mitochondria of endothelial cells (ECs) where it binds to cytosolic (Trx1) and mitochondrial thioredoxin (Trx2), respectively. Cys-250 and Cys-30 in the N-terminal domain of ASK1 are critical for binding of Trx1 and Trx2, respectively. Mutation of ASK1 at C250 enhanced ASK1-induced JNK activation and apoptosis, whereas mutation of ASK1 at C30 specifically increased ASK1-induced apoptosis without effects on JNK activation. We further show that a JNK-specific inhibitor SP600125 completely blocks TNF induced JNK activation, Bid cleavage, and Bax mitochondrial translocation, but only partially inhibits cytochrome c release and EC death, suggesting that TNF induces both JNK-dependent and JNK-independent apoptotic pathways in EC. Mitochondria-specific expression of a constitutively active ASK1 strongly induces EC apoptosis without JNK activation, Bid cleavage, and Bax mitochondrial translocation. These data suggest that mitochondrial ASK1 mediates a JNK-independent apoptotic pathway induced by TNF. To determine the role of Trx2 in regulation of mitochondrial ASK1 activity, we show that overexpression of Trx2 inhibits ASK1-induced apoptosis without effects on ASK1-induced JNK activation. Moreover, specific knockdown of Trx2 in EC increases TNF/ASK1-induced cytochrome c release and cell death without increase in JNK activation, Bid cleavage, and Bax translocation. Our data suggest that ASK1 in cytoplasm and mitochondria mediate distinct apoptotic pathways induced by TNF, and Trx1 and Trx2 cooperatively inhibit ASK1 activities.

Effect of the specific Src family kinase inhibitor saracatinib on osteolytic lesions using the PC-3 bone model.

The hematogenous metastatic spread of prostate cancer is preferentially to bone and can result in significant patient morbidity. Although these metastatic lesions are typically osteoblastic, bone resorption is believed to have a prerequisite role in their development. Src kinase has been identified to contribute to prostate cancer tumor growth and metastasis. In addition, Src is also essential in bone metabolism, especially in bone resorption. We hypothesized that inhibiting Src activity with the specific Src family kinase inhibitor saracatinib (AZD0530) would inhibit tumor cell growth and osteoclast differentiation in the tumor-bone interface, thus providing a new approach for advanced prostate cancer. We found that saracatinib inhibited PC-3 cell growth and invasion in a dose-dependent manner. Phosphorylation of Src, focal adhesion kinase, and P38 kinases was inhibited by saracatinib at the submicromolar range. Saracatinib also inhibited the expression and secretion of invasion-related molecules interlukin-8, urokinase-type plasminogen activator, and matrix metalloprotease-9. Receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis and signaling were inhibited by saracatinib in both macrophages and PC-3 cells. In in vivo studies, control mice developed more severe osteolytic lesions compared with the treatment group. Immunohistochemical and biochemical assays of bone metabolites confirmed that saracatinib preserved bone architecture in the presence of prostate cancer tumor cells. In summary, we have shown the inhibition of PC3 cell growth and invasion by saracatinib. Src inhibition also blocked the RANKL stimulatory pathway in osteoclasts and PC3 cells. The inhibition of Src thus targets multiple sites involved in prostate cancer bone metastasis, which may offer a therapeutic advantage in treating advanced prostate cancer.