Cell factory design with advanced metabolic modelling empowered by artificial intelligence.

Advances in synthetic biology and artificial intelligence (AI) have provided new opportunities for modern biotechnology. High-performance cell factories, the backbone of industrial biotechnology, are ultimately responsible for determining whether a bio-based product succeeds or fails in the fierce competition with petroleum-based products. To date, one of the greatest challenges in synthetic biology is the creation of high-performance cell factories in a consistent and efficient manner. As so-called white-box models, numerous metabolic network models have been developed and used in computational strain design. Moreover, great progress has been made in AI-powered strain engineering in recent years. Both approaches have advantages and disadvantages. Therefore, the deep integration of AI with metabolic models is crucial for the construction of superior cell factories with higher titres, yields and production rates. The detailed applications of the latest advanced metabolic models and AI in computational strain design are summarized in this review. Additionally, approaches for the deep integration of AI and metabolic models are discussed. It is anticipated that advanced mechanistic metabolic models powered by AI will pave the way for the efficient construction of powerful industrial chassis strains in the coming years.

Approximate solution of diffraction integral equation of resonator.

Utilizing the diffraction integral equation and the principle of slow amplitude approximation, we obtain a novel approximate solution of the transverse mode including the cavity parameters a (a is the section size of the resonator) and g = 1-L/R (L is the cavity length, R is the radius of curvature of the cavity). With this approximate solution, we can explore the influence of the resonator parameters a and g on the transverse mode. The theoretical analysis demonstrates that a and g have a certain influence on the shape and quality of the transverse mode, and selecting the appropriate a and g can effectively improve the quality of the transverse pattern. Moreover, laser experiments are conducted to validate analysis conclusion.

Rapid measurement method of intracavity phase retardation based on laser frequency splitting.

We present a method for phase retardation measurement of intracavity optical elements which is based on frequency splitting caused by weak phase anisotropy of Nd: YAG. The measurement range covers 0-π and the measurement uncertainty is less than 0.0300 rad. A theoretical analysis is provided to obtain the phase retardation of intracavity optical elements by using the phase difference or frequency difference of two eigenmodes. The minimum error is 0.0036 rad by using the composite wave plate to verify various phase retardation conditions. This work provides a rapid and accurate intracavity method for measuring the phase retardation of optical elements.

Comparison of guidelines for biological ancillary materials used for the manufacture of gene and cellular therapy products in Asia.

BACKGROUND AIMS: Although biologiocal ancillay materials (AMs) have specific risk associated with their derivations, it plays key role to manufature cell and gene therapy (CGT) products. It is important to understand the regulation relevant to AMs for developers. METHODS: The authors investigated the guidelines and pharmacopeia (hereinafter referred to as "guidelines") for biological AMs used for the manufacture of CGT products in Asia (China, India, Japan, Korea and Taiwan). In addition, the authors benchmarked the relevant guidelines in the United States (US) and European Union (EU). RESULTS AND DISCUSSIONS: The guidelines could be classified into two types based on whether specific AMs are scoped: (i) general guidelines for risk assessment of AMs and (ii) guidelines for specific AMs. The authors compared the risk categories for each type of AM provided in the general guidelines between the US and China and the specific requirements for bovine serum and trypsin in the guidelines of China, Japan, Taiwan, US and EU. The authors further compiled in-depth descriptions of the respective regulations in China, India, Japan, Korea and Taiwan. There is limited availability of some guidelines for specific AMs. Moreover, there are no common requirements established across the surveyed countries and regions. Therefore, the authors suggest a risk assessment approach for AMs with consideration of their biological origin and traceability, production steps applied and ability to control or remove AMs from the final medicinal product over the CGT manufacturing process.

MeSPL9 attenuates drought resistance by regulating JA signaling and protectant metabolite contents in cassava.

KEY MESSAGE: Analysis of drought-related genes in cassava shows the involvement of MeSPL9 in drought stress tolerance and overexpression of a dominant-negative form of this gene demonstrates its negative roles in drought stress resistance. Drought stress severely impairs crop yield and is considered a primary threat to food security worldwide. Although the SQUAMOSA promoter binding protein-like 9 (SPL9) gene participates extensively in numerous developmental processes and in plant response to abiotic stimuli, its role and regulatory pathway in cassava (Manihot esculenta) response to the drought condition remain elusive. In the current study, we show that cassava SPL9 (MeSPL9) plays negative roles in drought stress resistance. MeSPL9 expression was strongly repressed by drought treatment. Overexpression of a dominant-negative form of miR156-resistant MeSPL9, rMeSPL9-SRDX, in which a 12-amino acid repressor sequence was fused to rMeSPL9 at the C terminus, conferred drought tolerance without penalizing overall growth. rMeSPL9-SRDX-overexpressing lines not only exhibited increased osmoprotectant metabolites including proline and anthocyanin, but also accumulated more endogenous jasmonic acid (JA) and soluble sugars. Transcriptomic and real-time PCR analysis suggested that differentially expressed genes were involved in sugar or JA biosynthesis, signaling, and metabolism in transgenic cassava under drought conditions. Exogenous application of JA further confirmed that JA conferred improved drought resistance and promoted stomatal closure in cassava leaves. Taken together, our findings suggest that MeSPL9 affects drought resistance by modulating protectant metabolite levels and JA signaling, which have substantial implications for engineering drought tolerant crops.

Natural variation MeMYB108 associated with tolerance to stress-induced leaf abscission linked to enhanced protection against reactive oxygen species in cassava.

KEY MESSAGE: Natural variation of the MeMYB108 exon was associated with reactive oxygen scavengers led to alleviate leaf abscission under drought in cassava. The reactive oxygen scavengers play important roles in regulating the cassava (Manihot esculenta Crantz) leaf abscission induced by stresses. To date, the relationship between natural variations of MYB genes and reactive oxygen scavengers under drought in cassava genotypes remains unclear. Here, we reported the transcription factor MeMYB108 played an important role in regulating leaf abscission exposed to drought in cassava. The expression levels of MeMYB108 in abscission zones of cassava leaf pulvinus were higher in cassava genotype SC124, which were less easy to shed leaves under stress than cassava genotype SC8 when the leaf abscission induced by the same drought condition. Compared with wild type and interference expression plants, overexpression of MeMYB108 significantly reduced the drought-induced leaf abscission rate under drought. The consecutively 2-year analysis of reactive oxygen scavengers showed significant differences among different cassava genotypes under drought-induced leaf abscission, indicating the relevance between reactive oxygen scavengers and leaf abscission. Correlation analysis revealed the natural variation of the MeMYB108 exon was associated with reactive oxygen scavengers during drought-induced leaf abscission. Association analysis between pairwise LD of DNA polymorphism indicated the MeMYB108 allele enhanced the tolerance of cassava to drought-induced leaf abscission. Complementation transgenic lines containing the elite allele of MeMYB108 SC124 decreased the leaf abscission rate induced by drought conditions, demonstrating natural variation in MeMYB108 contributed to leaf abscission tolerance induced by drought in cassava. Further studies showed MeMYB108 played an active role in the tolerance of cassava to drought-induced leaf abscission by inducing scavenging of reactive oxygen species.

Improved U-Net3+ with stage residual for brain tumor segmentation.

BACKGROUND: For the encoding part of U-Net3+,the ability of brain tumor feature extraction is insufficient, as a result, the features can not be fused well during up-sampling, and the accuracy of segmentation will reduce. METHODS: In this study, we put forward an improved U-Net3+ segmentation network based on stage residual. In the encoder part, the encoder based on the stage residual structure is used to solve the vanishing gradient problem caused by the increasing in network depth, and enhances the feature extraction ability of the encoder which is instrumental in full feature fusion when up-sampling in the network. What's more, we replaced batch normalization (BN) layer with filter response normalization (FRN) layer to eliminate batch size impact on the network. Based on the improved U-Net3+ two-dimensional (2D) model with stage residual, IResUnet3+ three-dimensional (3D) model is constructed. We propose appropriate methods to deal with 3D data, which achieve accurate segmentation of the 3D network. RESULTS: The experimental results showed that: the sensitivity of WT, TC, and ET increased by 1.34%, 4.6%, and 8.44%, respectively. And the Dice coefficients of ET and WT were further increased by 3.43% and 1.03%, respectively. To facilitate further research, source code can be found at: https://github.com/YuOnlyLookOne/IResUnet3Plus . CONCLUSION: The improved network has a significant improvement in the segmentation task of the brain tumor BraTS2018 dataset, compared with the classical networks u-net, v-net, resunet and u-net3+, the proposed network has smaller parameters and significantly improved accuracy.

Effects of different concentrations of mesenchymal stem cells treatment on LPS-induced acute respiratory distress syndrome rat model.

PURPOSE: This study was prospectively designed to investigate the effects of different concentrations of mesenchymal stem cells treatment on respiratory mechanics, oxygenation, hemodynamics and inflammatory response in LPS-induced acute respiratory distress syndrome (ARDS) rat model. Methods: One hundred and twenty six LPS-induced ARDS model rats (weighted 200-220 g) were randomly divided into three groups: 1) Control group (N = 42); 2) low-dose hUC-MSC treatment group (MSC group 1, 1x107 cell/kg, N = 42); 3) high-dose hUC-MSC treatment group (MSC group 2, 2x107 cell/kg, N = 42), sham operation group as healthy group (N = 15). The rats were observed closely for 24 hours after hUC-MSC treatment, and the survival rate was calculated. At 24 hours, all rats were tested for hemodynamics, blood gas analysis, heart, lung, liver and kidney functions, inflammatory factors detection in blood samples and broncho-alveolar lavage fluid (BALF). The lung tissue of the rats was collected for HE staining analysis. Results: After LPS injection, ARDS was obvious in all LPS-infused rat groups, consistent with severe acute lung injury and high death rate. However, compared with the control group, a single intravenous injection hUC-MSC at dose of 1 × 107 cells/kg (low dose group) and 2 × 107 cells/kg (high dose group) reduced the mortality of rats with LPS-induced ARDS, as well as improving the lung function, increased the arterial oxygen pressure, improved the heart function, and reduced the levels of inflammatory factors including IL-1ß, IL-6, and TNF-α. In addition, the high dose MSC group showed better lung injury therapeutic effects than the low dose MSC group. Data from this study demonstrated that injection of hUC-MSC had a significant therapeutic effect in treating the rat model of LPS-induced ARDS and multiple organ function injury.

Coherent combination of two intracavity eigenmodes producing linearly polarized emission in an isotropic laser.

The behavior of directly linearly polarized emission was investigated in an isotropic Nd:YAG laser with a weakly anisotropic Fabry-Perot cavity. In order to explore the polarization mechanism, the intracavity eigenmodes were analyzed theoretically. A theoretical model was developed and shown to be suitable for describing the orientation-dependent beam profiles of the polarizer-specific features observed in experiments. This model indicates that the linear polarization emission results from the coherently combined state of intracavity eigenmodes as opposed to an intrinsic polarization state. The coherent combination is attributed to the frequency degeneracy and spontaneous phase locking of polarized eigenmodes. For the first time, the polarization coupling mechanism based on the coherent combination of eigenmodes in an isotropic solid-state laser was demonstrated. Moreover, this study also contributes an effective method to judge different types of linear polarization states.

Cavity-dumped Q-switched Er:Yb:YAl3(BO3)4 pulse laser.

We reported an electro-optically cavity-dumped Q-switched Er:Yb:YAl3(BO3)4 pulse laser for the first time. A 1531.1 nm pulse laser with an average output power of 521 mW, energy of 10 µJ, and a duration of 3.1 ns was achieved at a repetition rate of 100 kHz under the quasi-continuous-wave pumping. The pulse characteristics of the laser were investigated in detail. The result shows that the depolarization effects and the length of high voltage (HV) time applied to the EO-switch had significant impacts on the pulse characteristics.

Transplantation of stem cells from umbilical cord blood as therapy for type I diabetes.

In recent years, human umbilical cord blood has emerged as a rich source of stem, stromal and immune cells for cell-based therapy. Among the stem cells from umbilical cord blood, CD45+ multipotent stem cells and CD90+ mesenchymal stem cells have the potential to treat type I diabetes mellitus (T1DM), to correct autoimmune dysfunction and replenish ß-cell numbers and function. In this review, we compare the general characteristics of umbilical cord blood-derived multipotent stem cells (UCB-SCs) and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and introduce their applications in T1DM. Although there are some differences in surface marker expression between UCB-SCs and UCB-MSCs, the two cell types display similar functions such as suppressing function of stimulated lymphocytes and imparting differentiation potential to insulin-producing cells (IPCs) in the setting of low immunogenicity, thereby providing a promising and safe approach for T1DM therapy.

Tau GSTs involved in regulation of leaf abscission by comparison the gene profiling of MeGSTs in various abscission-promoting treatments in cassava abscission zones.

BACKGROUND: Glutathione S-transferases (GSTs) have been reported to regulate the plant tolerance to environmental stresses. Many plant GSTs exhibited the roles on promoting tolerance to drought stress, oxidative stress and plant hormones. The biological function of GSTs has been well characterized in Arabidopsis thaliana in response to exogenous environmental stresses. However, their regulation function under exogenous environmental stresses regulating leaf abscission in cassava (Manihot esculenta Crantz) remained unknown. RESULTS: Here, 83 GSTs were identified from tropical plant cassava. The amino acid motifs and phylogenetic analyses indicated that MeGSTs were divided into 9 classes. The global expression analyses were carried out to analyze the gene expression patterns of MeGST in cassava abscission zones by comparing the MeGST genes expression patterns in both ethylene and drought induced cassava leaf abscission. Totally, 34 GSTs were detected to express in both ethylene and drought induced leaf abscission in cassava abscission zones. Comparison of GST expression profiling between ethylene and drought induced leaf abscission suggested that Tau GST genes showed with the similar expression in both treatments induced leaf abscission in cassava abscission zone. GO annotation indicated that all 17 Tau GST genes participated in the pathway of toxin catabolism (GO: 0009407). The expression levels of 17 Tau MeGST genes were analyzed in two cassava cultivars, 'SC124' and 'Arg7', the two cultivars exhibit different levels of leaf abscission when suffered from the same environmental stress. Higher expression levels of Tau MeGSTs were detected in the precocious abscission Arg7 cultivar, while lower expression levels in delayed abscission SC124 cultivar. All the results indicated that Tau MeGSTs have the function in regulation the cassava leaf abscission under environmental stresses. CONCLUSION: Analysis of the expression patterns of GSTs in various abscission-promoting treatments in cassava abscission zones helps us to understand the possible roles of GSTs in cassava leaf abscission.

Exposure to various abscission-promoting treatments suggests substantial ERF subfamily transcription factors involvement in the regulation of cassava leaf abscission.

BACKGROUND: Cassava plants (Manihot esculenta Crantz) have obvious abscission zone (AZ) structures in their leaf pulvinus-petioles. Cassava leaf abscission can be triggered by either 17 days of water-deficit stress or 4 days of ethylene treatment. To date, little is known about cassava AP2/ERF factors, and less is known regarding their roles in regulating abscission zone development. RESULTS: Here, the cassava and Arabidopsis AP2/ERF genes were compared, finding that the cassava genome contains approximately 1.54-fold more ERF subfamily than the Arabidopsis genome. Microarray analysis was used to identify the AP2/ERF genes that are expressed in cassava leaf pulvinus-petiole abscission zones by comparing the AP2/ERF gene expression profiles of ethylene- and water-deficit stress-induced leaf abscission. In total, 99 AP2/ERF genes were identified as expressed in AZs across six time points during both ethylene- and water-deficit stress-induced leaf abscission. Comparative expression profile analysis of similar SOTA (Self Organizing Tree Algorithm) clusters at six time points during ethylene- and water-deficit stress-induced leaf abscission demonstrated that 20 ERF subfamily genes had similar expression patterns in response to both treatments. GO (Gene Ontology) annotation confirmed that all 20 ERF subfamily genes participate in ethylene-mediated signalling. Analysis of the putative ERF promoter regions shown that the genes contained primarily ethylene- and stress-related cis-elements. Further analysis of ACC oxidase activity in AZs across six time points during abscission shown increased ethylene production in response to both ethylene and water-deficit stress; however, the difference was more dramatic for water-deficit stress. Finally, the expression ratios of 20 ERF subfamily genes were analysed in two cassava cultivars, 'KU50' and 'SC5', that exhibit different levels of leaf abscission when challenged with the same water-deficit stress. The analysis indicated that most of the ERF genes were expressed at higher levels in the precocious abscission 'KU50' cultivar than in the delayed abscission 'SC5' cultivar. CONCLUSION: Ccomparative analysis of both ethylene- and water-deficit stress-induced leaf abscission shown that the ERF subfamily functions in the regulation of cassava abscission zone development.

Efficient orthogonally polarized dual-wavelength Nd:LaMgB5O10 laser.

We reported an efficient, continuous-wave, diode-pumped, orthogonally polarized dual-wavelength laser working at 1051.8 and 1081.4 nm in a new neodymium-doped borate crystal, Nd:LaMgB5O10, which was grown by the top-seeded solution growth method. A maximum output power of 5.1 W was obtained with an absorbed pump power of 14.8 W, corresponding to an optical-to-optical conversion efficiency of 34.5% and a slope efficiency of 42.5%. By slightly tilting the laser cavity output mirror, the balanced dual-wavelength emissions were obtained with the total output power as high as 4.2 W. This new efficient dual-wavelength laser may be a promising light source for terahertz generation with a rarely large frequency difference of 7.8 THz through difference frequency generation.

Clinical Evaluation of Coronary In-Stent Restenosis Using Dual-Source Computed Tomography.

OBJECTIVE: To explore the feasibility of dual-source computed tomography (DSCT) in the evaluation of coronary in-stent restenosis (ISR) by comparing the results of DSCT and selective coronary angiography (CAG). METHODS: In-stent restenosis examination results from DSCT were compared with those obtained using CAG. RESULTS: Among 173 stents studied, 156 yielded good quality images when evaluated with DSCT. CAG identified 38 ISR cases, while DSCT found 40. Among the 112 stents in the study with an inner diameter ≥3.0 mm, CAG identified 29 as having ISR, while DSCT reported the same finding in 30; among the 44 stents with inner diameter <3.0 mm, CAG identified ISR in 9, while DSCT found ISR in 10. CONCLUSIONS: Stent inner diameter is a key factor influencing the imaging of the stent lumen. DSCT demonstrated a higher negative predictive value in ISR assessment, suggesting that it could replace CAG for assessing the patency of stents with a larger inner diameter (≥3 mm).

Histone lysine-specific demethylase 1 (LSD1) protein is involved in Sal-like protein 4 (SALL4)-mediated transcriptional repression in hematopoietic stem cells.

The stem cell protein SALL4 plays a critical role in hematopoiesis by regulating the cell fate. In primitive hematopoietic precursors, it activates or represses important genes via recruitment of various epigenetic factors such as DNA methyltransferases, and histone deacylases. Here, we demonstrate that LSD1, a histone lysine demethylase, also participates in the trans-repressive effects of SALL4. Based on luciferase assays, the amine oxidase domain of LSD1 is important in suppressing SALL4-mediated reporter transcription. In freshly isolated adult mouse bone marrows, both SALL4 and LSD1 proteins are preferentially expressed in undifferentiated progenitor cells and co-localize in the nuclei. Further sequential chromatin immunoprecipitation assay confirmed that these two factors share the same binding sites at the promoter regions of important hematopoietic regulatory genes including EBF1, GATA1, and TNF. In addition, studies from both gain- and loss-of-function models revealed that SALL4 dynamically controls the binding levels of LSD1, which is accompanied by a reversely changed histone 3 dimethylated lysine 4 at the same promoter regions. Finally, shRNA-mediated knockdown of LSD1 in hematopoietic precursor cells resulted in altered SALL4 downstream gene expression and increased cellular activity. Thus, our data revealed that histone demethylase LSD1 may negatively regulate SALL4-mediated transcription, and the dynamic regulation of SALL4-associated epigenetic factors cooperatively modulates early hematopoietic precursor proliferation.

SALL4 is a robust stimulator for the expansion of hematopoietic stem cells.

HSCs are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The lack of donors and current inability to rapidly and efficiently expand HSCs are roadblocks in the development of successful cell therapies. Thus, the challenge of ex vivo human HSC expansion remains a fertile and critically important area of investigation. Here, we show that either SALL4A- or SALL4B-transduced human HSCs obtained from the mobilized peripheral blood are capable of rapid and efficient expansion ex vivo by >10 000-fold for both CD34(+)/CD38(-) and CD34(+)/CD38(+) cells in the presence of appropriate cytokines. We found that these cells retained hematopoietic precursor cell immunophenotypes and morphology as well as normal in vitro or vivo potential for differentiation. The SALL4-mediated expansion was associated with enhanced stem cell engraftment and long-term repopulation capacity in vivo. Also, we demonstrated that constitutive expression of SALL4 inhibited granulocytic differentiation and permitted expansion of undifferentiated cells in 32D myeloid progenitors. Furthermore, a TAT-SALL4B fusion rapidly expanded CD34(+) cells, and it is thus feasible to translate this study into the clinical setting. Our findings provide a new avenue for investigating mechanisms of stem cell self-renewal and achieving clinically significant expansion of human HSCs.

Reversal of hyperglycemia in diabetic mouse models using induced-pluripotent stem (iPS)-derived pancreatic beta-like cells.

Diabetes mellitus is characterized by either the inability to produce insulin (type 1 diabetes) or as insensitivity to insulin secreted by the body (type 2 diabetes). In either case, the body is unable to move blood glucose efficiently across cell membranes to be used. This leads to a variety of local and systemic detrimental effects. Current treatments for diabetes focus on exogenous insulin administration and dietary control. Here, we describe a potential cure for diabetes using a cellular therapy to ameliorate symptoms associated with both reduced insulin secretion and insulin sensitivity. Using induced pluripotent stem (iPS) cells, we were able to derive beta-like cells similar to the endogenous insulin-secreting cells in mice. These beta-like cells secreted insulin in response to glucose and corrected a hyperglycemic phenotype in two mouse models of type 1 and 2 diabetes via an iPS cell transplant. Long-term correction of hyperglycemia was achieved, as determined by blood glucose and hemoglobin A1c levels. These data provide an initial proof of principle for potential clinical applications of reprogrammed somatic cells in the treatment of diabetes type 1 or 2.

Role of SALL4 in hematopoiesis.

PURPOSE OF REVIEW: Stem cell gene SALL4 has been well characterized for its essential role in developmental events as well as embryonic stem cell pluripotency maintenance. Several current reports now shed new light on its functions in regulating hematopoietic cell self-renewal and differentiation. In this review we attempt to summarize SALL4 roles for normal hematopoiesis, and how the knowledge obtained can be used to develop advanced cell therapies. RECENT FINDINGS: SALL4 may act as a critical controller to regulate the fate of hematopoietic cells. In normal bone marrow, SALL4 is selectively expressed in primitive hematopoietic precursors and rapidly downregulated following differentiation. Of particular interest, SALL4 isoforms are able to stimulate large scale ex-vivo expansion of hematopoietic stem/progenitor cells (HSCs/HPCs). The SALL4 expanded HSCs/HPCs retain multilineage repopulation and long-term engraftment activities, which are clinically meaningful. The stem cell self-renewal mediated by SALL4 is linked to epigenetic machinery. SUMMARY: The emerging knowledge about how SALL4 regulates HSC behavior may be used in the near future to develop advanced cell therapies, for example, through large-scale stem cell expansion ex vivo.

The cassava (Manihot-esculenta Crantz)'s nitrate transporter NPF4.5, expressed in seedling roots, involved in nitrate flux and osmotic stress.

AtNPF4.5/AIT2, which was predicted to be a low-affinity transporter capable for nitrate uptake, was screened by ABA receptor complex in Arabidopsis ten years ago. However, the molecular and biochemical characterizations of AtNPF4.5 in plants remained largely unclear. In this study, the function of a plasma-membrane-localized and root-specifically-expressed gene MeNPF4.5 (Manihot-esculenta NITRATE TRANSPORTER 1 PTR FAMILY4.5), an ortholog of the Arabidopsis thaliana NPF4.5, was investigated in cassava roots as a nitrate efflux transporter on low nitrate medium and an influx transporter following exposure to high concentration of external nitrates. Moreover, RNA interference (RNAi) of MeNPF4.5 reduced the nitrate efflux capacity but the overexpressing cassava seedlings increased the ability of efflux from the elongation to the mature zone of root under low nitrate treatments. Besides, MeNPF4.5-RNAi expression reduced the nitrate influx capacity but enhanced nitrate absorption in parts of overexpressing plants from the meristem, elongation to mature zone of roots under high nitrate conditions. Furthermore, MeNPF4.5-RNAi seedlings survived owing to roots that could grow normally, but the MeNPF4.5-over-expressors showed adverse growth under 7% PEG6000 stress, suggesting that MeNPF4.5 negatively regulated the osmotic stress and was involved in nitrate flux through cassava seedlings.