Construction of an alternative NADPH regeneration pathway improves ethanol production in Saccharomyces cerevisiae with xylose metabolic pathway.

Full conversion of glucose and xylose from lignocellulosic hydrolysates is required for obtaining a high ethanol yield. However, glucose and xylose share flux in the pentose phosphate pathway (PPP) and glycolysis pathway (EMP), with glucose having a competitive advantage in the shared metabolic pathways. In this work, we knocked down ZWF1 to preclude glucose from entering the PPP. This reduced the [NADPH] level and disturbed growth on both glucose or xylose, confirming that the oxidative PPP, which begins with Zwf1p and ultimately leads to CO2 production, is the primary source of NADPH in both glucose and xylose. Upon glucose depletion, gluconeogenesis is necessary to generate glucose-6-phosphate, the substrate of Zwf1p. We re-established the NADPH regeneration pathway by replacing the endogenous NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene TDH3 with heterogenous NADP + -GAPDH genes GDH, gapB, and GDP1. Among the resulting strains, the strain BZP1 (zwf1Δ, tdh3::GDP1) exhibited a similar xylose consumption rate before glucose depletion, but a 1.6-fold increased xylose consumption rate following glucose depletion compared to the original strain BSGX001, and the ethanol yield for total consumed sugars of BZP1 was 13.5% higher than BSGX001. This suggested that using the EMP instead of PPP to generate NADPH reduces the wasteful metabolic cycle and excess CO2 release from oxidative PPP. Furthermore, we used a copper-repressing promoter to modulate the expression of ZWF1 and optimize the timing of turning off the ZWF1, therefore, to determine the competitive equilibrium between glucose-xylose co-metabolism. This strategy allowed fast growth in the early stage of fermentation and low waste in the following stages of fermentation.

Oligomerization of a plant helper NLR requires cell-surface and intracellular immune receptor activation.

Plant disease resistance involves both detection of microbial molecular patterns by cell-surface pattern recognition receptors and detection of pathogen effectors by intracellular NLR immune receptors. NLRs are classified as sensor NLRs, involved in effector detection, or helper NLRs required for sensor NLR signaling. TIR-domain-containing sensor NLRs (TNLs) require helper NLRs NRG1 and ADR1 for resistance, and helper NLR activation of defense requires the lipase-domain proteins EDS1, SAG101, and PAD4. Previously, we found that NRG1 associates with EDS1 and SAG101 in a TNL activation-dependent manner [X. Sun et al., Nat. Commun. 12, 3335 (2021)]. We report here how the helper NLR NRG1 associates with itself and with EDS1 and SAG101 during TNL-initiated immunity. Full immunity requires coactivation and mutual potentiation of cell-surface and intracellular immune receptor-initiated signaling [B. P. M. Ngou, H.-K. Ahn, P. Ding, J. D. G. Jones, Nature 592, 110-115 (2021), M. Yuan et al., Nature 592, 105-109 (2021)]. We find that while activation of TNLs is sufficient to promote NRG1-EDS1-SAG101 interaction, the formation of an oligomeric NRG1-EDS1-SAG101 resistosome requires the additional coactivation of cell-surface receptor-initiated defense. These data suggest that NRG1-EDS1-SAG101 resistosome formation in vivo is part of the mechanism that links intracellular and cell-surface receptor signaling pathways.

Both levoglucosan kinase activity and transport capacity limit the utilization of levoglucosan in Saccharomyces cerevisiae.

Manufacturing fuels and chemicals from cellulose materials is a promising strategy to achieve carbon neutralization goals. In addition to the commonly used enzymatic hydrolysis by cellulase, rapid pyrolysis is another way to degrade cellulose. The sugar obtained by fast pyrolysis is not glucose, but rather its isomer, levoglucosan (LG). Here, we revealed that both levoglucosan kinase activity and the transportation of levoglucosan are bottlenecks for LG utilization in Saccharomyces cerevisiae, a widely used cell factory. We revealed that among six heterologous proteins that had levoglucosan kinase activity, the 1,6-anhydro-N-acetylmuramic acid kinase from Rhodotorula toruloides was the best choice to construct levoglucosan-utilizing S. cerevisiae strain. Furthermore, we revealed that the amino acid residue Q341 and W455, which were located in the middle of the transport channel closer to the exit, are the sterically hindered barrier to levoglucosan transportation in Gal2p, a hexose transporter. The engineered yeast strain expressing the genes encoding the 1,6-anhydro-N-acetylmuramic acid kinase from R. toruloides and transporter mutant Gal2pQ341A or Gal2pW455A consumed ~ 4.2 g L-1 LG in 48 h, which is the fastest LG-utilizing S. cerevisiae strain to date.

Highly efficient rDNA-mediated multicopy integration based on the dynamic balance of rDNA in Saccharomyces cerevisiae.

Engineered Saccharomyces cerevisiae strains are good cell factories, and developing additional genetic manipulation tools will accelerate construction of metabolically engineered strains. Highly repetitive rDNA sequence is one of two main sites typically used for multicopy integration of genes. Here, we developed a simple and high-efficiency strategy for rDNA-mediated multicopy gene integration based on the dynamic balance of rDNA in S. cerevisiae. rDNA copy number was decreased by pre-treatment with hydroxyurea (HU). Then, heterologous genes were integrated into the rDNA sequence. The copy number of the integrated heterologous genes increased along with restoration of the copy number of rDNA. Our results demonstrated that HU pre-treatment doubled the number of integrated gene copies; moreover, compared with removing HU stress during transformation, removing HU stress after selection of transformants had a higher probability of resulting in transformants with high-copy integrated genes. Finally, we integrated 18.0 copies of the xylose isomerase gene into the S. cerevisiae genome in a single step. This novel rDNA-mediated multicopy genome integration strategy provides a convenient and efficient tool for further metabolic engineering of S. cerevisiae.

Pathogen effector recognition-dependent association of NRG1 with EDS1 and SAG101 in TNL receptor immunity.

Plants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 "helper" NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). The mechanism of RNL/EDS1 family protein cooperation is not understood. Here, we present genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent interaction of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex interacts with NRG1, and EDS1-PAD4 with ADR1, in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling.

Bacterial effector targeting of a plant iron sensor facilitates iron acquisition and pathogen colonization.

Acquisition of nutrients from different species is necessary for pathogen colonization. Iron is an essential mineral nutrient for nearly all organisms, but little is known about how pathogens manipulate plant hosts to acquire iron. Here, we report that AvrRps4, an effector protein delivered by Pseudomonas syringae bacteria to plants, interacts with and targets the plant iron sensor protein BRUTUS (BTS) to facilitate iron uptake and pathogen proliferation in Arabidopsis thaliana. Infection of rps4 and eds1 by P. syringae pv. tomato (Pst) DC3000 expressing AvrRps4 resulted in iron accumulation, especially in the plant apoplast. AvrRps4 alleviates BTS-mediated degradation of bHLH115 and ILR3(IAA-Leucine resistant 3), two iron regulatory proteins. In addition, BTS is important for accumulating immune proteins Enhanced Disease Susceptibility1 (EDS1) at both the transcriptional and protein levels upon Pst (avrRps4) infections. Our findings suggest that AvrRps4 targets BTS to facilitate iron accumulation and BTS contributes to RPS4/EDS1-mediated immune responses.

A Coevolved EDS1-SAG101-NRG1 Module Mediates Cell Death Signaling by TIR-Domain Immune Receptors.

Plant nucleotide binding/leucine-rich repeat (NLR) immune receptors are activated by pathogen effectors to trigger host defenses and cell death. Toll-interleukin 1 receptor domain NLRs (TNLs) converge on the ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) family of lipase-like proteins for all resistance outputs. In Arabidopsis (Arabidopsis thaliana) TNL-mediated immunity, AtEDS1 heterodimers with PHYTOALEXIN DEFICIENT4 (AtPAD4) transcriptionally induced basal defenses. AtEDS1 uses the same surface to interact with PAD4-related SENESCENCE-ASSOCIATED GENE101 (AtSAG101), but the role of AtEDS1-AtSAG101 heterodimers remains unclear. We show that AtEDS1-AtSAG101 functions together with N REQUIRED GENE1 (AtNRG1) coiled-coil domain helper NLRs as a coevolved TNL cell death-signaling module. AtEDS1-AtSAG101-AtNRG1 cell death activity is transferable to the Solanaceous species Nicotiana benthamiana and cannot be substituted by AtEDS1-AtPAD4 with AtNRG1 or AtEDS1-AtSAG101 with endogenous NbNRG1. Analysis of EDS1-family evolutionary rate variation and heterodimer structure-guided phenotyping of AtEDS1 variants and AtPAD4-AtSAG101 chimeras identify closely aligned ɑ-helical coil surfaces in the AtEDS1-AtSAG101 partner C-terminal domains that are necessary for reconstituted TNL cell death signaling. Our data suggest that TNL-triggered cell death and pathogen growth restriction are determined by distinctive features of EDS1-SAG101 and EDS1-PAD4 complexes and that these signaling machineries coevolved with other components within plant species or clades to regulate downstream pathways in TNL immunity.

AcERF2, an ethylene-responsive factor of Atriplex canescens, positively modulates osmotic and disease resistance in Arabidopsis thaliana.

Ethylene-responsive factors (ERFs) comprise a large family of transcription factors in plants and play important roles in developmental processes and stress responses. Here, we characterized a novel AP2/ERF transcription factor, AcERF2, from the halophyte Atriplex canescens (four-wing saltbush, Chenopodiaceae). AcERF2 was proved to be a transcriptional activator in yeast and localized to the nucleus upon transient expression in Nicotiana benthamiana, indicating its potential role as a transcription factor. Overexpression of AcERF2 driven by a CaMV35S promoter led to decreased accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), and increased antioxidant enzymatic activities, as well as rapid stomatal closure under osmotic treatment in Arabidopsis. Arabidopsis plants overexpressing AcERF2 were hypersensitive to abscisic acid (ABA) during germination, seedling establishment, and primary root elongation, and exhibited significant tolerance to osmotic stress. Furthermore, overexpression of AcERF2 induced transcript accumulation of plant defense-related genes (PR1, PR2, PR5, ERF1 and ERF3) and increased Arabidopsis resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungal pathogen Botrytis cinerea. These results suggest that AcERF2 may play a positive modulation role in response to osmotic stress and pathogen infection in plants.

Baicalein suppresses non small cell lung cancer cell proliferation, invasion and Notch signaling pathway.

BACKGROUND: Baicalein is an important Chinese herbal medicine and has multiple pharmacological activities. However, the biological mechanisms of the anti-tumor effects of Baicalein on non small cell lung cancer (NSCLC) still need to be understood. METHODS: Human NSCLC A549 and H1299 cells were pretreated with Baicalein or DMSO. Cells viability and transwell cell invasion assays were performed to assess cell proliferation and invasion. QRT-PCR assay was used to analyze mRNA expression levels of Twist1, E-cadhertin, Vimentin, Notch1 and hes-1. Western blot analysis was also performed to determine protein expression. RESULTS: In the study, we found that Baicalein had a significantly inhibited effect on proliferation ability of A549 and H1299 cells. Cells treated with Baicalein showed a down-regulated expression of CyclinD1 and CDK1 in A549 and H1299 cells. Furthermore, we found that Baicalein significantly inhibited cell invasion and Epithelial-Mesenchymal Transition (EMT) by up-regulating the mRNA and protein expression of E-cadherin and down-regulated the Twist1 and Vimentin expression, Moreover, Treatment of Baicalein down-regulated Notch1 and hes-1 expression in A549 and H1299 cells, which indicated that Baicalein could suppress the Notch signaling pathway. CONCLUSION: Our studies suggest that Baicalein may be a potential phytochemical flavonoid for therapeutics of NSCLC and serve as a molecular target for NSCLC.

Exploration for the Salinity Tolerance-Related Genes from Xero-Halophyte Atriplex canescens Exploiting Yeast Functional Screening System.

Plant productivity is limited by salinity stress, both in natural and agricultural systems. Identification of salt stress-related genes from halophyte can provide insights into mechanisms of salt stress tolerance in plants. Atriplex canescens is a xero-halophyte that exhibits optimum growth in the presence of 400 mM NaCl. A cDNA library derived from highly salt-treated A. canescens plants was constructed based on a yeast expression system. A total of 53 transgenic yeast clones expressing enhanced salt tolerance were selected from 105 transformants. Their plasmids were sequenced and the gene characteristics were annotated using a BLASTX search. Retransformation of yeast cells with the selected plasmids conferred salt tolerance to the resulting transformants. The expression patterns of 28 of these stress-related genes were further investigated in A. canescens leaves by quantitative reverse transcription-PCR. In this study, we provided a rapid and robust assay system for large-scale screening of genes for varied abiotic stress tolerance with high efficiency in A. canescens.

Kallikrein-related peptidase 4 contributes to the tumor metastasis of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a disfiguring malignancy and significantly impacts the quality of patient's life. Kallikrein-related peptidase 4 (KLK4), which is closely related to cancers, is highly expressed in OSCC. To explore the biological function of KLK4 in OSCC, a KLK4-specific shRNA was used to silence its endogenous expression, and then the migration and invasion of OSCC cells were explored. Results of our study showed that silencing KLK4 inhibited the migration and invasion of OSCC cells. The protein levels of epithelial mesenchymal transition-associated markers and proteases were also altered by KLK4 silencing. Further study showed that the phosphatidylinositol 3-kinase (PI3 K)/protein kinase B (AKT) signaling pathway was involved in the function of KLK4. Treatment with a PI3 K/AKT activator reversed the migration-inhibitory effect of KLK4 shRNA. Our study suggests that KLK4 may contribute to the metastasis of OSCC through the PI3 K/AKT signaling pathway.

KLK4 silencing inhibits the growth of oral squamous cell carcinoma through Wnt/ß-catenin signaling pathway.

Oral squamous cell carcinoma (OSCC) is a malignancy that largely impacts the quality of people's daily life. Kallikrein-related peptidase 4 (KLK4) is highly expressed in OSCC; however, its roles in OSCC cells are unclear. In the present study, the effect of KLK4 silencing on the growth of OSCC cells was investigated. Our study showed that the proliferation and colony formation of OSCC cells was inhibited by KLK4 silencing and their cell cycle was arrested. Additionally, apoptosis of OSCC cells was enhanced by KLK4 silencing, with increased protein levels of cleaved PARP, cleaved caspase-3, Bax and decreased levels of Bcl-2. KLK4 silencing inhibited the Wnt/ß-catenin signaling pathway, as evidence by decreased protein levels of Wnt1, ß-catenin, reduced GSK-3ß phosphorylation as well as decreased levels of cyclinD1 and c-myc proteins. We further showed that Wnt/ß-catenin activator reversed the effects of KLK4 silencing on the proliferation and apoptosis of OSCC cells. We concluded that KLK4 silencing inhibited the growth of OSCC cells through Wnt/ß-catenin signaling pathway, suggesting that KLK4 may become a promising therapeutic target for the treatment of OSCC.

N-AC-l-Leu-PEI-mediated miR-34a delivery improves osteogenic differentiation under orthodontic force.

Rare therapeutic genes or agents are reported to control orthodontic bone remodeling. MicroRNAs have recently been associated with bone metabolism. Here, we report the in vitro and in vivo effects of miR-34a on osteogenic differentiation under orthodontic force using an N-acetyl-L-leucine-modified polyethylenimine (N-Ac-l-Leu-PEI) carrier. N-Ac-l-Leu-PEI exhibited low cytotoxicity and high miR-34a transfection efficiency in rat bone mineral stem cells and local alveolar bone tissue. After transfection, miR-34a enhanced the osteogenic differentiation of Runx2 and ColI, Runx2 and ColI protein levels, and early osteogenesis function under orthodontic strain in vitro. MiR-34a also enhanced alveolar bone remodeling under orthodontic force in vivo, as evidenced by elevated gene and protein expression, upregulated indices of alveolar bone anabolism, and diminished tooth movement. We determined that the mechanism miR-34a in osteogenesis under orthodontic force may be associated with GSK-3ß. These results suggested that miR-34a delivered by N-Ac-l-Leu-PEI could be a potential therapeutic target for orthodontic treatment.

Isoindolone-Containing Meroperpenoids from the Endophytic Fungus Emericella nidulans HDN12-249.

Six isoindolone containing meroterpenoids, emericellolides A-C (1-3) and emeriphenolicins E-G (4-6), were isolated from a plant endophytic fungus Emericella nidulans HDN12-249. Emericellolides A-C (1-3) feature the unprecedented macrolide skeleton composed of an unusual l-glutamate fragment, an isoindolone unit, and a sesquiterpene moiety, while structures of emeriphenolicins E-G (4-6) with two farnesyl groups attached to one isoindolone unit are rare in isoindolone-derived meroterpenoids. These structures including the absolute configurations were established on the basis of MS, NMR, Mo2(AcO)4-induced ECD, Marfey's method, and chemical conversion. Compound 4 exhibited cytotoxicity against HeLa cells with IC50 value of 4.77 µM.

AcEBP1, an ErbB3-Binding Protein (EBP1) from halophyte Atriplex canescens, negatively regulates cell growth and stress responses in Arabidopsis.

An ErbB-3-binding protein gene AcEBP1, also known as proliferation-associated 2G4 gene (PA2G4s) belonging to the M24 superfamily, was obtained from the saltbush Atriplex canescens. Subcellular localization imaging showed the fusion protein AcEBP1-eGFP was located in the nucleus of epidermal cells in Nicotiana benthamiana. The AcEBP1 gene expression levels were up-regulated under salt, osmotic stress, and hormones treatment as revealed by qRT-PCR. Overexpression of AcEBP1 in Arabidopsis demonstrated that AcEBP1 was involved in root cell growth and stress responses (NaCl, osmotic stress, ABA, low temperature, and drought). These phenotypic data were correlated with the expression patterns of stress responsive genes and PR genes. The AcEBP1 transgenic Arabidopsis plants also displayed increased sensitivity under low temperature and evaluated resistance to drought stress. Together, these results demonstrate that AcEBP1 negatively affects cell growth and is a regulator under stress conditions.

Relationships of sagittal skeletal discrepancy, natural head position, and craniocervical posture in young Chinese children.

OBJECTIVE: The aim of this study was to investigate the relationships of sagittal skeletal discrepancy, natural head position (NHP), and craniocervical posture in young Chinese children with average vertical facial pattern. METHODS: Ninety patients with average Frankfort mandibular plane angle (FH/ML) were classified into skeletal class I, II, and III relationships according to their ANB angle. Cephalometric radiographs in NHP were taken. Variables representing sagittal and vertical craniofacial morphology, head posture, and craniocervical posture were measured and compared. RESULTS: Subjects in the skeletal class II group showed the largest craniovertical angles and craniocervical angles, while subjects in the skeletal class III group exhibited the smallest craniovertical angles and craniocervical angles, though not all the measurements showed significant differences. The angle formed by the nasion-sella line and the tangent to the posterior border of the mandibular ramus (NSL/RL) was largest in the skeletal class II group and smallest in the skeletal class III group (p = 0.05). DISCUSSION: Significant differences exist in NHP and craniocervical posture among skeletal class I, II, and III relationships in young Chinese children. Subjects with skeletal class II relationship tended to exhibit more extended head, and children with skeletal class III relationship often exhibited flexed head.

Versixanthones A-F, Cytotoxic Xanthone-Chromanone Dimers from the Marine-Derived Fungus Aspergillus versicolor HDN1009.

Six unusual xanthone-chromanone dimers, versixanthones A-F (1-6), featuring different formal linkages of tetrahydroxanthone and 2,2-disubstituted chroman-4-one monomers, were isolated from a culture of the mangrove-derived fungus Aspergillus versicolor HDN1009. The absolute configurations of 1-6, representing the central and axial chirality elements or preferred helicities, were established by a combination of X-ray diffraction analysis, chemical conversions, and TDDFT-ECD calculations. The interconversion of different biaryl linkages between 1 and 4 and between 2 and 3 in DMSO by a retro-oxa-Michael mechanism provided insight into the formation of the xanthone-chromanone dimers and supported the assignments of their absolute configurations. Compounds 1-6 exhibited cytotoxicities against the seven tested cancer cell lines, with the best IC50 value of 0.7 µM. Compound 5 showed further inhibitory activity against topoisomerase I.

Susceptibility to corrosion of laser welding composite arch wire in artificial saliva of salivary amylase and pancreatic amylase.

In this study, laser-welded composite arch wire (CAW) with a copper interlayer was exposed to artificial saliva containing salivary amylase or pancreatic amylase, and the resultant corrosion behavior was studied. The purpose was to determine the mechanisms by which salivary amylase and pancreatic amylase contribute to corrosion. The effects of amylase on the electrochemical resistance of CAW were tested by potentiodynamic polarization measurements. The dissolved corrosion products were determined by ICP-OES, and the surfaces were analyzed by SEM, AFM and EDS. The results showed that both exposure to salivary amylase and pancreatic amylase significantly improved the corrosion resistance of CAW. Even isozyme could have different influences on the alloy surface. When performing in vitro research of materials to be used in oral cavity, the effect of α-amylase should be taken into account since a simple saline solution does not entirely simulate the physiological situation.

AcPIP2, a plasma membrane intrinsic protein from halophyte Atriplex canescens, enhances plant growth rate and abiotic stress tolerance when overexpressed in Arabidopsis thaliana.

KEY MESSAGE: An aquaporin protein AcPIP2 from Atriplex canescens was involved in plant growth rate, abiotic stress tolerance in Arabidopsis. Under limited water condition, AcPIP2 leaded to the sensitivity to drought stress. An aquaporin protein (AcPIP2) was obtained from the saltbush Atriplex canescens, which was in PIP2 subgroup belonging to the PIP subfamily, MIP superfamily. The subcellular localization of AcPIP2 showed the fusion protein AcPIP2-eGFP located at the plasma membrane in Nicotiana benthamiana. Overexpression of AcPIP2 in Arabidopsis fully proved that AcPIP2 was involved in plant growth rate, transpiration rate and abiotic stress tolerance (NaCl, drought and NaHCO3) in Arabidopsis, which is mostly in correspondence to gene expression pattern characterized by qRT-PCR performed in A. canescens. And under limited water condition, AcPIP2 overexpression leaded to the sensitivity to drought stress. In the view of the resistant effect in transgenic Arabidopsis overexpressing AcPIP2, the AcPIP2 may throw some light into understanding how the A. canescens plants cope with abiotic stress, and could be used in the genetic engineering to improve plant growth or selective tolerance to the abiotic stress.