Nanoassemblies of heptamethine cyanine dye-initiated poly(amino acid) enhance ROS generation for effective antitumour phototherapy.

Phototherapy shows great potential for pinpoint tumour treatment. Heptamethine cyanine dyes like IR783 have high potential as agents for antitumour phototherapy due to their inherent tumour targeting ability, though their effectiveness in vivo is unsatisfactory for clinical translation. To overcome this limitation, we present an innovative strategy involving IR783-based polymeric nanoassemblies that improve the dye's performance as an antitumoural photosensitizer. In the formulation, IR783 is modified with cysteamine and used to initiate the ring-opening polymerization (ROP) of the N-carboxyanhydride of benzyl-L-aspartate (BLA), resulting in IR783-installed poly(BLA). Compared to free IR783, the IR783 dye in the polymer adopts a twisted molecular conformation and tuned electron orbital distribution, remarkably enhancing its optical properties. In aqueous environments, the polymers spontaneously assemble into nanostructures with 60 nm diameter, showcasing surface-exposed IR783 dyes that function as ligands for cancer cell and mitochondria targeting. Moreover, the nanoassemblies stabilized the dyes and enhanced the generation of reactive oxygen species (ROS) upon laser irradiation. Thus, in murine tumor models, a single injection of the nanoassemblies with laser irradiation significantly inhibits tumour growth with no detectable off-target toxicity. These findings highlight the potential for improving the performance of heptamethine cyanine dyes in antitumor phototherapy through nano-enabled strategies.

Selective Intracellular Delivery of Antibodies in Cancer Cells with Nanocarriers Sensing Endo/Lysosomal Enzymatic Activity.

The differential enzymatic activity in the endo/lysosomes of particular cells could trigger targeted endosomal escape functions, enabling selective intracellular protein delivery. However, this strategy may be jeopardized due to protein degradation during endosomal trafficking. Herein, using custom made fluorescent probes to assess the endosomal activity of cathepsin B (CTSB) and protein degradation, we found that certain cancer cells with hyperacidified endosomes grant a spatiotemporal window where CTSB activity surpass protein digestion. This inspired the engineering of antibody-loaded polymeric nanocarriers having CTSB-activatable endosomal escape ability. The nanocarriers selectively escaped from the endo/lysosomes in the cells with high endosomal CTSB activity and delivered active antibodies to intracellular targets. This study provides a viable strategy for cell-specific protein delivery using stimuli-responsive nanocarriers with controlled endosomal escape.

Revitalizing Cytokine-Based Cancer Immunotherapy through Advanced Delivery Systems.

Cytokines can coordinate robust immune responses, holding great promise as therapeutics against infections, autoimmune diseases, and cancers. In cancer treatment, numerous pro-inflammatory cytokines have displayed promising efficacy in preclinical studies. However, their clinical application is hindered by poor pharmacokinetics, significant toxicity and unsatisfactory anticancer efficacy. Thus, while IFN-α and IL-2 are approved for specific cancer treatments, other cytokines still remain subject of intense investigation. To accelerate the application of cytokines as cancer immunotherapeutics, strategies need to be directed to improve their safety and anticancer performance. In this regard, delivery systems could be used to generate innovative therapies by targeting the cytokines or nucleic acids, such as DNA and mRNA, encoding the cytokines to tumor tissues. This review centers on these innovative delivery strategies for cytokines, summarizing key approaches, such as gene delivery and protein delivery, and critically examining their potential and challenges for clinical translation.

Block catiomers with flanking hydrolyzable tyrosinate groups enhance in vivo mRNA delivery via π-π stacking-assisted micellar assembly.

Messenger RNA (mRNA) therapeutics have recently demonstrated high clinical potential with the accelerated approval of SARS-CoV-2 vaccines. To fulfill the promise of unprecedented mRNA-based treatments, the development of safe and efficient carriers is still necessary to achieve effective delivery of mRNA. Herein, we prepared mRNA-loaded nanocarriers for enhanced in vivo delivery using biocompatible block copolymers having functional amino acid moieties for tunable interaction with mRNA. The block copolymers were based on flexible poly(ethylene glycol)-poly(glycerol) (PEG-PG) modified with glycine (Gly), leucine (Leu) or tyrosine (Tyr) via ester bonds to generate block catiomers. Moreover, the amino acids can be gradually detached from the block copolymers after ester bond hydrolyzation, avoiding cytotoxic effects. When mixed with mRNA, the block catiomers formed narrowly distributed polymeric micelles with high stability and enhanced delivery efficiency. Particularly, the micelles based on tyrosine-modified PEG-PG (PEG-PGTyr), which formed a polyion complex (PIC) and π-π stacking with mRNA, displayed excellent stability against polyanions and promoted mRNA integrity in serum. PEG-PGTyr-based micelles also increased the cellular uptake and the endosomal escape, promoting high protein expression both in vitro and in vivo. Furthermore, the PEG-PGTyr-based micelles significantly extended the half-life of the loaded mRNA after intravenous injection. Our results highlight the potential of PEG-PGTyr-based micelles as safe and effective carriers for mRNA, expediting the rational design of polymeric materials for enhanced mRNA delivery.

An IL-12-Based Nanocytokine Safely Potentiates Anticancer Immunity through Spatiotemporal Control of Inflammation to Eradicate Advanced Cold Tumors.

Treatment of immunologically cold tumors is a major challenge for immune checkpoint inhibitors (ICIs). Interleukin 12 (IL-12) can invigorate ICIs against cold tumors by establishing a robust antitumor immunity. However, its toxicity and systemic induction of counteracting immunosuppressive signals have hindered translation. Here, IL-12 activity is spatiotemporally controlled for safely boosting efficacy without the stimulation of interfering immune responses by generating a nanocytokine that remains inactive at physiological pH, but unleashes its full activity at acidic tumor pH. The IL-12-based nanocytokine (Nano-IL-12) accumulate and release IL-12 in tumor tissues, eliciting localized antitumoral inflammation, while preventing systemic immune response, counteractive immune reactions, and adverse toxicities even after repeated intravenous administration. The Nano-IL-12-mediated spatiotemporal control of inflammation prompt superior anticancer efficacy, and synergize with ICIs to profoundly inflame the tumor microenvironment and completely eradicate ICI-resistant primary and metastatic tumors. The strategy could be a promising approach toward safer and more effective immunotherapies.

Auxin plays a role in the adaptation of rice to anaerobic germination and seedling establishment.

Auxin is well known to stimulate coleoptile elongation and rapid seedling growth in the air. However, its role in regulating rice germination and seedling establishment under submergence is largely unknown. Previous studies revealed that excessive levels of indole-3-acetic acid(IAA) frequently cause the inhibition of plant growth and development. In this study, the high-level accumulation of endogenous IAA is observed under dark submergence, stimulating rice coleoptile elongation but limiting the root and primary leaf growth during anaerobic germination (AG). We found that oxygen and light can reduce IAA levels, promote the seedling establishment and enhance rice AG tolerance. miRNA microarray profiling and RNA gel blot analysis results show that the expression of miR167 is negatively regulated by submergence; it subsequently modulates the accumulation of free IAA through the miR167-ARF-GH3 pathway. The OsGH3-8 encodes an IAA-amido synthetase that functions to prevent free IAA accumulation. Reduced miR167 levels or overexpressing OsGH3-8 increase auxin metabolism, reduce endogenous levels of free IAA and enhance rice AG tolerance. Our studies reveal that poor seed germination and seedling growth inhibition resulting from excessive IAA accumulation would cause intolerance to submergence in rice, suggesting that a certain threshold level of auxin is essential for rice AG tolerance.

Polymeric micelles effectively reprogram the tumor microenvironment to potentiate nano-immunotherapy in mouse breast cancer models.

Nano-immunotherapy improves breast cancer outcomes but not all patients respond and none are cured. To improve efficacy, research focuses on drugs that reprogram cancer-associated fibroblasts (CAFs) to improve therapeutic delivery and immunostimulation. These drugs, however, have a narrow therapeutic window and cause adverse effects. Developing strategies that increase CAF-reprogramming while limiting adverse effects is urgent. Here, taking advantage of the CAF-reprogramming capabilities of tranilast, we developed tranilast-loaded micelles. Strikingly, a 100-fold reduced dose of tranilast-micelles induces superior reprogramming compared to free drug owing to enhanced intratumoral accumulation and cancer-associated fibroblast uptake. Combination of tranilast-micelles and epirubicin-micelles or Doxil with immunotherapy increases T-cell infiltration, resulting in cures and immunological memory in mice bearing immunotherapy-resistant breast cancer. Furthermore, shear wave elastography (SWE) is able to monitor reduced tumor stiffness caused by tranilast-micelles and predict response to nano-immunotherapy. Micellar encapsulation is a promising strategy for TME-reprogramming and SWE is a potential biomarker of response.

Nanocarriers escaping from hyperacidified endo/lysosomes in cancer cells allow tumor-targeted intracellular delivery of antibodies to therapeutically inhibit c-MYC.

Intracellular protein delivery is a powerful strategy for developing innovative therapeutics. Nanocarriers present great potential to deliver proteins inside cells by promoting cellular uptake and overcoming entrapment and degradation in acidic endo/lysosomal compartments. Thus, because cytosolic access is essential for eliciting the function of proteins, significant efforts have been dedicated to engineering nanocarriers with maximal endosomal escape regardless of the cell type. On the other hand, controlling the ability of nanocarriers to escape from the endo/lysosomal compartments of particular cells may offer the opportunity for enhancing delivery precision. To test this hypothesis, we developed pH-sensitive polymeric nanocarriers with adjustable endosomal escape potency for selectively reaching the cytosol of defined cancer cells with dysregulated endo/lysosomal acidification. By loading antibodies against nuclear pore complex in the nanocarriers, we demonstrated the selective delivery into the cytosol and subsequent nucleus targeting of cancer cells rather than non-cancerous cells both in vitro and in vivo. Systemically injected nanocarriers loading anti-c-MYC antibodies suppressed c-MYC in solid tumors and inhibit tumor growth without side effects, confirming the therapeutic potential of our approach. These results indicated that regulating the ability of nanocarriers to escape from endo/lysosomal compartments in particular cells is a practical approach for gaining delivery specificity.

Polymeric Micelles with pH-Responsive Cross-Linked Core Enhance In Vivo mRNA Delivery.

Messenger RNA (mRNA) is emerging as a promising therapeutic modality for a variety of diseases. Because of the fragility and limited intracellular access of mRNA, the development of delivery technologies is essential for promoting the applicability of mRNA-based treatments. Among effective nanocarriers, polymeric micelles loading mRNA by polyion complex (PIC) formation with block catiomers have the potential to meet the delivery needs. Since PICs are relatively unstable in in vivo settings, herein, we constructed mRNA-loaded micelles having pH-responsive cross-linked cores by complexing mRNA with cis-aconitic anhydride-modified poly(ethylene glycol)-poly(l-lysine) (PEG-pLL(CAA)) block copolymers. The micelles were stable at physiological pH (pH 7.4) but achieved the complete release of the mRNA at endosomal pH (pH 5.5-4.5). The cross-linking also enhanced the stability of the micelles against disassembly from polyanions and protected the loaded mRNA from degradation by nucleases. Thus, the cross-linked micelles increased the delivery of mRNA to cancer cells, promoting protein expression both in vitro and in vivo. Our results highlight the potential of PEG-pLL(CAA)-based micelles for mRNA delivery.

Effects of Hypoxia-Hyperoxia Preconditioning on Indicators of Muscle Damage After Acute Resistance Exercise in Male Athletes.

Purpose: The purpose of this study was to investigate the effects of acute repeated hypoxia-hyperoxia preconditioning on resistance exercise (RE)-induced muscle damage in male athletes. Methods: Eleven young male athletes participated in this randomized double-blind counter-balanced crossover study, and were divided into Normoxia (N) and Hypoxia-Hyperoxia (HH) trials. Subjects of the respective trials were supplied with normoxic (FiO2 = 0.21), or alternating hypoxic/hyperoxic air (FiO2 = 0.10/0.99, 5 min each) for 60 min. Thirty minutes after preconditioning, subjects performed acute bouts of RE consisting of bench press, deadlift, and squats. Each exercise included 6 sets of 10 repetitions at 75% one-repetition maximum (1RM) with 2 min rest between sets. After a 2-week washout period, subjects changed trials and completed the same study procedure after the alternate preconditioning. Muscle soreness, maximal voluntary contraction (MVC), and circulating biochemical markers were tested before preconditioning (baseline) and during recovery at 0, 24, and 48 h after exercise. Results: Acute RE significantly increased levels of muscle soreness, creatine kinase (CK) and myoglobin (Mb), and decreased levels of peak knee extension torque in the N trial. Muscle soreness, CK, and Mb levels of the HH trial were significantly lower than that of the N trial after exercise. Interestingly, interleukin-6 (IL-6) levels of the HH trial increased significantly 0 h after exercise compared to baseline and were significantly higher than that of the N trial 0 and 24 h after exercise. However, no significant differences of thiobarbituric acid reactive substances (TBARS), cortisol, testosterone, peak torque, and average power levels were found between N and HH trials during recovery. Conclusion: Our data suggest that pre-exercise treatment of alternating hypoxic/hyperoxic air could attenuate muscle damage and pain after acute RE, but has no effect on muscle strength recovery in young male athletes.

Toward automated segmentation for acute ischemic stroke using non-contrast computed tomography.

PURPOSE: Non-contrast computed tomography (NCCT) is a first-line imaging technique for determining treatment options for acute ischemic stroke (AIS). However, its poor contrast and signal-to-noise ratio limit the diagnosis accuracy for radiologists, and automated AIS lesion segmentation using NCCT also remains a challenge. In this paper, we propose R2U-RNet, a novel model for AIS lesion segmentation using NCCT. METHODS: We used an in-house retrospective NCCT dataset with 261 AIS patients with manual lesion segmentation using follow-up diffusion-weighted images. R2U-RNet is based on an R2U-Net backbone with a novel residual refinement unit. Each input image contains two image channels from separate preprocessing procedures. The proposed model incorporates multiscale focal loss to mitigate the class imbalance problem and to leverage the importance of different levels of details. A proposed noisy-label training scheme is utilized to account for uncertainties in the manual annotations. RESULTS: The proposed model outperformed several iconic segmentation models in AIS lesion segmentation using NCCT, and our ablation study demonstrated the efficacy of the proposed model. Statistical analysis of segmentation performance revealed significant effects of regional stroke occurrence and side of the stroke, suggesting the importance of region-specific information for automated segmentation, and the potential influence of the hemispheric difference in clinical data. CONCLUSION: This study demonstrated the potentials of R2U-RNet model for automated NCCT AIS lesion segmentation. The proposed model can serve as a tool for accelerating AIS diagnoses and improving the treatment quality of AIS patients.

Block catiomer with flexible cationic segment enhances complexation with siRNA and the delivery performance in vitro.

RNA interference (RNAi) by small interfering RNAs (siRNAs) is a promising therapeutic approach. Because siRNA has limited intracellular access and is rapidly cleared in vivo, the success of RNAi depends on efficient delivery technologies. Particularly, polyion complexation between block catiomers and siRNA is a versatile approach for constructing effective carriers, such as unit polyion complexes (uPIC), core-shell polyion complex (PIC) micelles and vesicular siRNAsomes, by engineering the structure of block catiomers. In this regard, the flexibility of block catiomers could be an important parameter in the formation of PIC nanostructures with siRNA, though its effect remains unknown. Here, we studied the influence of block catiomer flexibility on the assembly of PIC structures with siRNA using a complementary polymeric system, i.e. poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) and PEG-poly(glycidylbutylamine) (PEG-PGBA), which has a relatively more flexible polycation segment than PEG-PLL. Mixing PEG-PGBA with siRNA at molar ratios of primary amines in polymer to phosphates in the siRNA (N/P ratios) higher than 1.5 promoted the multimolecular association of uPICs, whereas PEG-PLL formed uPIC at all N/P ratios higher than 1. Moreover, uPICs from PEG-PGBA were more stable against counter polyanion exchange than uPICs from PEG-PLL, probably due to a favorable complexation process, as suggested by computational studies of siRNA/block catiomer binding. In in vitro experiments, PEG-PGBA uPICs promoted effective intracellular delivery of siRNA and efficient gene knockdown. Our results indicate the significance of polycation flexibility on assembling PIC structures with siRNA, and its potential for developing innovative delivery systems.

Near-infrared boosted ROS responsive siRNA delivery and cancer therapy with sequentially peeled upconversion nano-onions.

RNA interference (RNAi) therapy has become an appealing approach for cancer treatment, while the specificity and efficiency of controlled small interference RNA (siRNA) release remain challenging due to the heterogeneity of tumor environment. Herein, upconversion nano-onions (UCNOs) with stacked polymer coating layers are constructed to decompose sequentially in response to extracellular environment and NIR stimulation. The UCNOs (UCNPs-PEIRB-PEISeSe/siRNA-R8-HA) are composed of upconversion nanoparticles (UCNPs) core functionalized with inner coating layer of photosensitizer rose bengal (RB) conjugated PEI 600, middle coating layer of singlet oxygen (1O2) sensitive diselenide linked PEI 600 with therapeutic siRNA loading and cell-penetrating peptide R8 modification, and outer coating layer of negatively charged hyaluronic acid (HA). HA prevents siRNA leakage during delivery process and specifically targets tumor cells with overexpressed CD44 membrane receptors, and digested by cell secreted hyaluronidase (HAase). Upon the subsequent irradiation at 808 nm, UCNPs core generates emissions around 540 nm, which activate RB to boost ROS generation for complete PEI-SeSe decompose. The NIR boosted decompose of UCNOs induces a fast and efficient siRNA release, which effectively improves the gene silencing efficiency in vitro and suppresses tumor growth in vivo. The proposed sequentially responsive UCNOs have promising potential application in precision medicine.

Influence of the Intention to Lean the Body Forward on Kinematics and Kinetics of Sprinting for Active Adults.

This study investigated the influence of the intention to lean the body forward on spatiotemporal and ground reaction force variables during the acceleration phase of a sprint. Fourteen active adults performed two 50 m sprints (with and without the intention to lean), during which spatiotemporal variables and impulses were obtained using a long force platform system. Effect size (Cohen's d) was used to examine the differences between the two trials. We found that running speed and net anteroposterior impulse did not change by the intention for all steps. However, step frequency increased in the initial two steps through decreases in support time and flight time by the intention. Moreover, these shorter support and flight times were caused by a decrease in the vertical impulse. The propulsive impulse did not change during the initial part of acceleration phase, but the braking impulse decreased at the first step. This study demonstrates that an intention to lean the body forward leads to a smaller braking impulse and a higher step frequency through shorter support and flight times and a smaller vertical impulse during the initial part of the acceleration phase of a sprint.

Identification of sugar response complex in the metallothionein OsMT2b gene promoter for enhancement of foreign protein production in transgenic rice.

KEY MESSAGE: A 146-bp sugar response complex MTSRC is identified in the promoter of rice metallothionein OsMT2b gene conferring high-level expression of luciferase reporter gene and bioactive recombinant haFGF in transgenic rice. A rice subfamily type 2 plant metallothionein (pMT) gene, OsMT2b, encoding a reactive oxygen species (ROS) scavenger protein, has been previously shown to exhibit the most abundant gene expression in young rice seedling. Expression of OsMT2b was found to be regulated negatively by ethylene and hydrogen peroxide in rice stem node under flooding stress, but little is known about its response to sugar depletion. In this study, transient expression assay and transgenic approach were employed to characterize the regulation of the OsMT2b gene expression in rice. We found that the expression of OsMT2b gene is induced by sugar starvation in both rice suspension cells and germinated embryos. Deletion analysis and functional assay of the OsMT2b promoter revealed that the 5'-flanking region of the OsMT2b between nucleotides - 351 and - 121, which contains the sugar response complex (- 266 to - 121, designated MTSRC) is responsible for high-level promoter activity under sugar starvation. It was also found that MTSRC significantly enhances the Act1 promoter activity in transgenic rice cells and seedlings. The modified Act1 promoter, Act1-MTSRC, was used to produce the recombinant human acidic fibroblast growth factor (haFGF) in rice cells. Our result shows that the bioactive recombinant haFGF is stably produced in transformed rice cell culture and yields are up to 2% of total medium proteins. Our studies reveal that MTSRC serves as a strong transcriptional activator and the Act1-MTSRC promoter can be applicable in establishing an efficient expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings.

Design, synthesis and evaluation of novel diaryl-1,5-diazoles derivatives bearing morpholine as potent dual COX-2/5-LOX inhibitors and antitumor agents.

In this paper, 41 hybrid compounds containing diaryl-1,5-diazole and morpholine structures acting as dual COX-2/5-LOX inhibitors have been designed, synthesized and biologically evaluated. Most of them showed potent antiproliferative activities and COX-2/5-LOX inhibitory in vitro. Among them, compound A33 displayed the most potency against cancer cell lines (IC50 = 6.43-10.97 µM for F10, HeLa, A549 and MCF-7 cells), lower toxicity to non-cancer cells than celecoxib (A33: IC50 = 194.01 µM vs.celecoxib: IC50 = 97.87 µM for 293T cells), and excellent inhibitory activities on COX-2 (IC50 = 0.17 µM) and 5-LOX (IC50 = 0.68 µM). Meanwhile, the molecular modeling study was performed to position compound A33 into COX-2 and 5-LOX active sites to determine the probable binding models. Mechanistic studies demonstrated that compound A33 could block cell cycle in G2 phase and subsequently induced apoptosis of F10 cells. Furthermore, compound A33 could significantly inhibit tumor growth in F10-xenograft mouse model, and pharmacokinetic study of compound A33 indicated that it showed better stability in vivo. In general, compound A33 could be a promising candidate for cancer therapy.

Fourier phase retrieval with a single mask by Douglas-Rachford algorithms.

The Fourier-domain Douglas-Rachford (FDR) algorithm is analyzed for phase retrieval with a single random mask. Since the uniqueness of phase retrieval solution requires more than a single oversampled coded diffraction pattern, the extra information is imposed in either of the following forms: 1) the sector condition on the object; 2) another oversampled diffraction pattern, coded or uncoded. For both settings, the uniqueness of projected fixed point is proved and for setting 2) the local, geometric convergence is derived with a rate given by a spectral gap condition. Numerical experiments demonstrate global, power-law convergence of FDR from arbitrary initialization for both settings as well as for 3 or more coded diffraction patterns without oversampling. In practice, the geometric convergence can be recovered from the power-law regime by a simple projection trick, resulting in highly accurate reconstruction from generic initialization.

Suppressive effect of microRNA319 expression on rice plant height.

KEY MESSAGE: miR319 was identified as a dwarf-inducing gene from Shiokari and its dwarf near isogenic line, and its transgenic rice showed a reduced plant height. This finding reveals the potential application of miR319 in future molecular breeding. It is well known that microRNAs (miRNAs) play important roles in plant physiology, especially in development and stress responses. However, little is known about the role of miRNAs in plant height. In this study, the rice cultivar Shiokari and its dwarf near isogenic line Shiokari-d6 were analysed to identify and characterize plant height-associated miRNAs. This anatomic and morphological investigation revealed that the major cause of the shorter height of Shiokari-d6 is the significantly dis-elongated internodes, particularly the second internode and those underneath it. The results of miRNA microarray profiling and real-time RT-PCR indicated that miR319 is expressed at a significantly higher level in Shiokari-d6 than in Shiokari. Transgenic rice overexpressing miR319 in Oryza sativa L. cv. Tainung 67 generated through Agrobacterium-mediated transformation had a stable dwarf phenotype regardless of whether the plants were from the T1 or T2 generation. We also found that the internodes of miR319-overexpressing rice are shortened, particularly the third internode and those underneath it. Furthermore, we identified three putative miR319 target genes that were previously uncharacterized with expression levels that were negatively correlated with the expression of miR319. In conclusion, miR319 is the first miRNA proposed to be involved in plant height regulation, and its function may influence the elongation of internodes, which leads to decreased plant height.

The modified rice αAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings.

Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA) but also by O2 deficiency. Promoters of two rice α-amylase genes, αAmy3 and αAmy8, have been shown to direct high-level production of recombinant proteins in rice suspension cells and germinated seeds. In the present study, we modified the cis-acting DNA elements within the sugar/GA response complex (SRC/GARC) of αAmy8 promoter. We found that addition of a G box and duplicated TA box leads to high-level expression of αAmy8 SRC/GARC and significantly enhances αAmy8 promoter activity in transformed rice cells and germinated transgenic rice seeds. We also show that these modifications have drastically increased the activity of αAmy8 promoter in rice seedlings under hypoxia. Our results reveal that the G box and duplicated TA box may play important roles in stimulating promoter activity in response to hypoxia in rice. The modified αAmy8 promoter was used to produce the recombinant human epidermal growth factor (hEGF) in rice cells and hypoxic seedlings. We found that the bioactive recombinant hEGF are stably produced and yields are up to 1.8% of total soluble protein (TSP) in transformed rice cells. The expression level of synthetic hEGF containing preferred rice codon usage comprises up to 7.8% of TSP in hypoxic transgenic seedlings. Our studies reveal that the modified αAmy8 promoter can be applicable in establishing a novel expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings under hypoxia.

The promoter and the 5'-untranslated region of rice metallothionein OsMT2b gene are capable of directing high-level gene expression in germinated rice embryos.

KEY MESSAGE: Critical regions within the rice metallothionein OsMT2b gene promoter are identified and the 5'-untranslated region (5'-UTR) is found essential for the high-level promoter activity in germinated transgenic rice embryos. Many metallothionein (MT) genes are highly expressed in plant tissues. A rice subfamily p2 (type 2) MT gene, OsMT2b, has been shown previously to exhibit the most abundant gene expression in young rice seedling. In the present study, transient expression assays and a transgenic approach were employed to characterize the expression of the OsMT2b gene in rice. We found that the OsMT2b gene is strongly and differentially expressed in germinated rice embryos during seed germination and seedling development. Histochemical staining analysis of transgenic rice carrying OsMT2b::GUS chimeric gene showed that high-level GUS activity was detected in germinated embryos and at the meristematic part of other tissues during germination. Deletion analysis of the OsMT2b promoter revealed that the 5'-flanking region of the OsMT2b between nucleotides -351 and -121 relative to the transcriptional initiation site is important for promoter activity in rice embryos, and this region contains the consensus sequences of G box and TA box. Our study demonstrates that the 5'-untranslated region (5'-UTR) of OsMT2b gene is not only necessary for the OsMT2b promoter activity, but also sufficient to augment the activity of a minimal promoter in both transformed cell cultures and germinated transgenic embryos in rice. We also found that addition of the maize Ubi intron 1 significantly enhanced the OsMT2b promoter activity in rice embryos. Our studies reveal that OsMT2b351-ubi(In) promoter can be applied in plant transformation and represents potential for driving high-level production of foreign proteins in transgenic rice.