Daurisoline suppress glioma progression by inhibiting autophagy through PI3K/AKT/mTOR pathway and increases TMZ sensitivity.

Glioma is one of the most common primary malignant tumors of the central nervous system. Temozolomide (TMZ) is the only effective chemotherapeutic agent, but it easily develops resistance and has unsatisfactory efficacy. Consequently, there is an urgent need to develop safe and effective compounds for glioma treatment. The cytotoxicity of 30 candidate compounds to glioma cells was detected by the CCK-8 assay. Daurisoline (DAS) was selected for further investigation due to its potent anti-glioma effects. Our study revealed that DAS induced glioma cell apoptosis through increasing caspase-3/6/9 activity. DAS significantly inhibited the proliferation of glioma cells by inducing G1-phase cell cycle arrest. Meanwhile, DAS remarkably suppressed the migration and invasion of glioma cells by regulating epithelial-mesenchymal transition. Mechanistically, our results revealed that DAS impaired the autophagic flux of glioma cells at a late stage by mediating the PI3K/AKT/mTOR pathway. DAS could inhibit TMZ-induced autophagy and then significantly promote TMZ chemosensitivity. Nude mice xenograft model revealed that DAS could restrain glioma proliferation and promote TMZ chemosensitivity. Thus, DAS is a potential anti-glioma drug that can improve glioma sensitivity to TMZ and provide a new therapeutic strategy for glioma in chemoresistance.

NPS-2143 inhibit glioma progression by suppressing autophagy through mediating AKT-mTOR pathway.

Gliomas are the most common tumours in the central nervous system. In the present study, we aimed to find a promising anti-glioma compound and investigate the underlying molecular mechanism. Glioma cells were subjected to the 50 candidate compounds at a final concentration of 10 µM for 72 h, and CCK-8 was used to evaluate their cytotoxicity. NPS-2143, an antagonist of calcium-sensing receptor (CASR), was selected for further study due to its potent cytotoxicity to glioma cells. Our results showed that NPS-2143 could inhibit the proliferation of glioma cells and induce G1 phase cell cycle arrest. Meanwhile, NPS-2143 could induce glioma cell apoptosis by increasing the caspase-3/6/9 activity. NPS-2143 impaired the immigration and invasion ability of glioma cells by regulating the epithelial-mesenchymal transition process. Mechanically, NPS-2143 could inhibit autophagy by mediating the AKT-mTOR pathway. Bioinformatic analysis showed that the prognosis of glioma patients with low expression of CASR mRNA was better than those with high expression of CASR mRNA. Gene set enrichment analysis showed that CASR was associated with cell adhesion molecules and lysosomes in glioma. The nude mice xenograft model showed NPS-2143 could suppress glioma growth in vivo. In conclusion, NPS-2143 can suppress the glioma progression by inhibiting autophagy.

[Influence of Artificial Root Exudates and Actual Root Exudates on the Microbial Community in Pyrene-contaminated Soil].

The high-throughput Illumina NovaSeq sequencing method was adopted to study the effect of artificial root exudates and Lolium perenne L. root exudates on the community structure, α and ß diversity, and gene function of the bacterial communities in pyrene-contaminated soils to understand the impact of root exudates on microbial communities. The results showed that root exudates did not significantly change the composition of pyrene-contaminated soil bacterial communities. The main dominant bacterial phyla were Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, etc. The main dominant bacterial genera were Sphingomonas, Lactobacillus, Bacillus, etc. Root exudates changed the relative abundance of dominant species to a different extent and resulted in discriminating bacteria. The genus Lachnospiraceae belonging to Proteobacteria and Ruminiclostridium belonging to Firmicutes were the biomarkers in the artificial root exudates group and the actual root exudate group, respectively. The common discriminating bacteria in both root exudate groups compared to those in the control group were polycyclic aromatic hydrocarbon (PAHs)-degrading bacteria. Root exudates selectively promoted the growth of PAHs-degrading bacteria. Root exudates had little effect on the richness and diversity of the bacterial communities in pyrene-contaminated soil. However, they significantly influenced the soil bacterial community structure, which resulted from significant changes in low-abundance species. The bacterial community structures of the two root exudate groups were similar. Root exudates decreased pyrene concentration in the soil by 14.0% (artificial root exudates) and 8.7% (actual root exudates). The promotion of pyrene degradation affected by root exudates was due to the growth promotion of PAHs-degrading bacteria and the significant increase in the abundance of some functional genes. This research can supply data for the exploration of a rhizoremediation mechanism in PAHs-contaminated soils.

Which sites better represent the sensory function of hands in convalescent stroke patients? A study based on electrophysiological examination.

Background: Assessing hand sensation in stroke patients is necessary; however, current clinical assessments are time-consuming and inaccurate. Objective: This study aimed to explore the nature of light touch sensation and two-point discrimination (2-PD) of different hand sites in convalescent stroke patients based on somatosensory evoked potentials (SEP). Methods: Light touch sensation and 2-PD of the thumb, the index finger, the little finger, thenar, and hypothenar were measured (n = 112) using sensory measurement tools. Sensory differences among the hand sites were then compared. The correlation analysis between SEP and the hemiplegic hand function was made. Sensory functions were divided into three levels: sensory intactness, sensory impairment, and sensory loss. Results: Light touch sensations were mainly associated with sensory impairment in the finger and palm region. The 2-PD of the finger region was mainly sensory loss and that of the palm region was mainly sensory impairment. There was no statistical difference in the light touch sensation among the sites of the hand. The correlation coefficients between the 2-PD and SEP N20 amplitudes differed. The correlation coefficients of the thenar and hypothenar were the smallest, and that of the finger was the largest. Light touch sensation and 2-PD in patients with stroke were related to the hemiplegic hand function. Conclusion: Any site on the hand could be selected as the measurement site for light touch sensation. The little finger and hypothenar may be appropriate sites when screening for 2-PD. To improve the patient's recovery they could receive more sensory stimulation of the hand.

A Meta-Analysis: Whether Repetitive Transcranial Magnetic Stimulation Improves Dysfunction Caused by Stroke with Lower Limb Spasticity.

OBJECTIVE: To evaluate the efficacy of repetitive transcranial magnetic stimulation (rTMS) in improving lower limb spasticity after stroke. METHODS: The PubMed, Web of Science, Cochrane Library, EMBASE, China National Knowledge Infrastructure (CNKI), China Biology Medicine (CBM) disc, China Science and Technology Journal Database (VIP), and Wanfang databases were searched online from their inception to May 2021 for randomized controlled trials (RCTs) involving repetitive transcranial magnetic stimulation for lower extremity spasticity after stroke. Valid data were extracted from the included literature, and the quality evaluation was conducted with the Cochrane Handbook for Systematic Reviews of Interventions along with the Physiotherapy Evidence Database scale (PE-Dro scale). The data that met the quality requirements were systematically analysed using Review Manager 5.4 software. RESULTS: A total of 554 patients from seven articles (nine studies) were quantitatively analysed. Outcomes included the Modified Ashworth Scale (MAS), Fugl-Meyer Assessment of Lower Extremity (FMA-LE), Modified Barthel Index (MBI), and Timed Up and Go (TUG), measured as the effect of rTMS compared with controls conditions after treatment. The systematic review showed that rTMS reduced MAS and increased MBI scores, respectively (SMD = -0.24, 95% CI [-0.45, -0.03], P = 0.02; MD = 6.14, 95% CI [-3.93,8.35], P ＜ 0.00001), compared with control conditions. Low-frequency rTMS (LF-rTMS) significantly improved FMA-LE scores (SMD = 0.32, 95% CI [0.13, 0.51], P = 0.001). However, there was no significant difference in FMA-LE scores when using high-frequency rTMS (HF-rTMS) (P > 0.1) and in TUG times (P > 0.1) between the treatment and control groups. CONCLUSIONS: rTMS was effective in improving spasticity and activities of daily living. LF-rTMS has positive clinical effects on enhancing motor function in patients who experience lower extremity spasticity after stroke. To better validate the above conclusions, more multicentre, high-quality, and double-blind randomized controlled trials are needed.

Saponins from Panax japonicus attenuate cognitive impairment in ageing rats through regulating microglial polarisation and autophagy.

CONTEXT: Panax japonicus is the dried rhizome of Panax japonicus C.A. Mey. (Araliaceae). Saponins from Panax japonicus (SPJ) exhibit anti-inflammatory and antioxidative effects. OBJECTIVE: To explore the neuroprotective effect of SPJ on natural ageing of rat. MATERIALS AND METHODS: Sprague-Dawley (SD) rats 18-month-old were divided into ageing control, ageing treated with SPJ 10 or 30 mg/kg (n = 8). Five-month-old rats were taken as the adult control (n = 8). Rats were fed regular feed or feed containing SPJ for 4 months. Cognitive level was evaluated by Morris water maze (MWM) test. The mechanisms of SPJ's neuroprotection were evaluated by transmission electron microscope, western blot analysis, and immunofluorescence in vivo and in vitro. RESULTS: SPJ attenuated ageing-induced cognitive impairment as indicated by elevated number of times crossing the target platform (from 1.63 to 3.5) and longer time spent in the target platform quadrant (from 1.33 to 1.98). Meanwhile, SPJ improved the morphology of microglia and synapse, and activated M2 microglia polarisation including increased hippocampus levels of CD206 (from 0.98 to 1.47) and YM-1 (from 0.67 to 1.1), and enhanced autophagy-related proteins LC3B (from 0.48 to 0.82), Beclin1 (from 0.32 to 0.51), Atg5 (from 0.22 to 0.89) whereas decreased p62 level (from 0.71 to 0.45) of ageing rats. In vitro study also showed that SPJ regulated the microglial polarisation and autophagy. DISCUSSION AND CONCLUSIONS: SPJ improved cognitive deficits of ageing rats through attenuating microglial inflammation and enhancing microglial autophagy, which could be used to treat neurodegenerative disorders.

Probing nanoscale spatial distribution of plasmonically excited hot carriers.

Surface plasmons (SPs) of metals enable the tight focusing and strong absorption of light to realize an efficient utilization of photons at nanoscale. In particular, the SP-generated hot carriers have emerged as a promising way to efficiently drive photochemical and photoelectric processes under moderate conditions. In situ measuring of the transport process and spatial distribution of hot carriers in real space is crucial to efficiently capture the hot carriers. Here, we use electrochemical tip-enhanced Raman spectroscopy (EC-TERS) to in situ monitor an SP-driven decarboxylation and resolve the spatial distribution of hot carriers with a nanometer spatial resolution. The transport distance of about 20 nm for the reactive hot carriers is obtained from the TERS imaging result. The hot carriers with a higher energy have a shorter transport distance. These conclusions can be guides for the design and arrangement of reactants and devices to efficiently make use of plasmonic hot carriers.

Probing the Local Generation and Diffusion of Active Oxygen Species on a Pd/Au Bimetallic Surface by Tip-Enhanced Raman Spectroscopy.

Active oxygen species (AOS) play key roles in many important catalytic reactions relevant to clean energy and environment. However, it remains challenging to characterize the active sites for producing AOS and to image the surface properties of AOS, especially on multicomponent metallic catalyst surfaces. Herein, we utilize tip-enhanced Raman spectroscopy (TERS) to probe the local generation and diffusion of OH radicals on a Pd/Au(111) bimetallic catalyst surface. The reactive OH radicals can be catalytically generated from hydrogen peroxide (H2O2) at the metal surface, which then oxidizes the surface adsorbed thiolate, a reactant that is used as the TERS probe. By TERS imaging of the spatial distribution of unreacted thiolate molecules, we demonstrate that the Pd surface is active for generation of OH radicals and the Pd step edge shows much higher activity than the Pd terrace, whereas the Au surface is inactive. Furthermore, we find that the locally generated OH radicals at the Pd step edge could diffuse to both the Au and the Pd surface sites to induce oxidative reactions, with a diffusion length estimated to be about 5.4 nm. Our TERS imaging with few-nanometer spatial resolution not only unravels the active sites but also characterizes in real space the diffusion behavior of OH radicals. The results are highly valuable to understand AOS-triggered catalytic reactions. The strategy of using reactants with large Raman cross sections as TERS probes may broaden the application of TERS for studying catalysis with reactive small molecules.

Cyclic GMP-AMP synthase is essential for cytosolic double-stranded DNA and fowl adenovirus serotype 4 triggered innate immune responses in chickens.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a predominant DNA sensor inducing the activation of the innate immune responses that produce proinflammatory cytokines and type I interferons, which has been well-investigated in mammals. However, chicken cGAS (chcGAS), which participates in avian innate immunity, has not been well-investigated. Here, we cloned the complete open reading frame sequence of chcGAS. Multiple sequence alignment and phylogenetic analysis revealed that chcGAS was homologous to mammalian cGAS. The chcGAS mRNA was highly expressed in the bone marrow and ileum. The subcellular localization of chcGAS was mainly in the cytoplasm, and partial co-localization was observed in the endoplasmic reticulum. Through overexpression and RNA interference, we demonstrated that chcGAS responded to exogenous dsDNA, HS-DNA, and poly(dA:dT), and to self dsDNA from the DNA damage response, thereby triggering the activation of STING/TBK1/IRF7-mediated innate immunity in both chicken embryonic fibroblasts and chicken liver cancer cells. Furthermore, downregulation of chcGAS enhanced the infection of fowl adenovirus serotype 4 in LMH cells. Our results demonstrated that chcGAS was an important cytosolic DNA sensor activating innate immune responses and may shed light on a strategy for preventing infectious diseases in the poultry industry.

Gene expression and methylation profiles identified CXCL3 and CXCL8 as key genes for diagnosis and prognosis of colon adenocarcinoma.

Colon adenocarcinoma (COAD) is one of the most common malignant tumors with high morbidity and mortality rates worldwide. Due to the poor clinical outcomes, it is indispensable to investigate novel biomarkers for the diagnosis and prognosis of COAD. The aim of this study is to explore key genes as potential biomarkers for the diagnosis and prognosis of COAD for clinical utility. Gene expression profiles (GSE44076 and GSE44861) and gene methylation profile (GSE29490) were analyzed to identify the aberrantly methylated-differentially expressed genes by R language and Perl software. Function enrichments were performed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Moreover, hub genes were identified through protein-protein interaction (PPI) network. Besides, key genes were found by the module analysis and The Cancer Genome Atlas (TCGA) survival analysis. Finally, TCGA data and quantitative real-time polymerase chain reaction (RT-qPCR) was used to validate the key genes involved in COAD. Our study found two hypomethylation-high-expression genes (CXCL3 and CXCL8) in COAD tissues compared with the adjacent normal tissues. These results were also confirmed by RT-qPCR with 25 pairs of COAD and adjacent normal tissues. Meanwhile, low expression of the two genes was associated with poor survival in patients with COAD. CXCL3 and CXCL8 may serve as key genes in the diagnosis and prognosis for COAD.

Diagnostic potential of ionomic profile in the plasma of cervical cancer patients receiving neoadjuvant chemoradiotherapy.

BACKGROUND AND AIM: Major and trace elements play an important role in human body, and it has been reported that ionomic distribution differ greatly in tumor patients. The aim of the present study was to investigate the effects of cisplatin-based neoadjuvant chemoradiotherapy on the ionomic profile in human plasma as a potential biomarker for the therapeutic effects of cervical cancer. METHOD: Thirty-seven patients with cervical cancer receiving neoadjuvant chemoradiotherapy were included in this study, pretherapy and post-treatment blood samples were collected and concentrations of 24 ions were analyzed by inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: The results showed that after cisplatin chemotherapy and radiotherapy, patients' plasma Pt level significantly increased, Na, Mg, P, K, Ca, Se, Cu, Zn, Se, Sr, Ba levels significantly decreased (P < 0.01), and Al, Cu ions were significantly correlated with the treatment effect (P < 0.05). In addition, the pattern of elemental correlations changed dramatically after the neoadjuvant chemoradiotherapy. CONCLUSION: The results indicated that the plasma ionomic profile may serve as a quick and convenient tool to reflect the therapeutic effect of cisplatin-based chemoradiotherapy in cervical cancer patients, and supplement of certain essential elements may be of great importance for the maintenance of ion homeostasis in human body and for the reduction of adverse effect of chemotherapy and radiotherapy.

Electrochemical Tip-Enhanced Raman Spectroscopy with Improved Sensitivity Enabled by a Water Immersion Objective.

Electrochemical tip-enhanced Raman spectroscopy (EC-TERS) appears as a promising in situ nanospectroscopic tool for characterization and understanding of the electrochemical interfacial processes at the nanometer scale and molecular level. However, the wide application of EC-TERS is hampered by its low sensitivity as a result of the optical path distortion due to the refractive index mismatch of the multilayer media (air, glass, and electrolyte). Here, we propose a new side-illumination EC-TERS setup by coupling a water immersion objective with a high numerical aperture to a scanning tunneling microscope scanning head customized with a large open space and a compact spectroelectrochemical cell. It not only effectively eliminates the optical distortion but also increases the sensitivity remarkably, which allows sensitive monitoring of the electrochemical redox processes of anthraquinone molecules. More importantly, EC-TERS is able to independently control the tip position and laser illumination position. By utilizing this feature, we reveal that the irreversible reduction reaction of anthraquinone observed in EC-TERS is induced by the synergistic effect of the negative potential and laser illumination rather than the localized surface plasmon. The highly improved sensitivity and the flexibility to control the tip and laser illumination position on the nanometer scale endows EC-TERS as an important tool for the fundamental understanding of the photo- or plasmon electrochemistry and the interfacial structure-activity relationship of important electrochemical systems.

Review on antibacterial characteristics of bridge engineering biomaterials.

This review summarizes the research on timber construction materials used in bridge construction. It focuses on the application of antiseptic treatments and the use of timber engineering materials in decks and bridges. This review also provides an overview on the future research and prospects of engineered timber materials.

[Applicability analysis of spatially explicit model of leaf litter in evergreen broad-leaved forests].

The spatially explicit model of leaf litter can help to understand its dispersal process, which is very important to predict the distribution pattern of leaves on the surface of the earth. In this paper, the spatially explicit model of leaf litter was developed for 20 tree species using litter trap data from the mapped forest plot in an evergreen broad-leaved forest in Tiantong, Zhejiang Pro- vince, eastern China. Applicability of the model was analyzed. The model assumed an allometric equation between diameter at breast height (DBH) and leaf litter amount, and the leaf litter declined exponentially with the distance. Model parameters were estimated by the maximum likelihood method. Results showed that the predicted and measured leaf litter amounts were significantly correlated, but the prediction accuracies varied widely for the different tree species, averaging at 49.3% and ranging from 16.0% and 74.0%. Model qualities of tree species significantly correlated with the standard deviations of the leaf litter amount per trap, DBH of the tree species and the average leaf dry mass of tree species. There were several ways to improve the forecast precision of the model, such as installing the litterfall traps according to the distribution of the tree to cover the different classes of the DBH and distance apart from the parent trees, determining the optimal dispersal function of each tree species, and optimizing the existing dispersal function.

Gene-carried hepatoma targeting complex induced high gene transfection efficiency with low toxicity and significant antitumor activity.

BACKGROUND: The success of gene transfection is largely dependent on the development of a vehicle or vector that can efficiently deliver a gene to cells with minimal toxicity. METHODS: A liver cancer-targeted specific peptide (FQHPSF sequence) was successfully synthesized and linked with chitosan-linked polyethylenimine (CP) to form a new targeted gene delivery vector called CPT (CP/peptide). The structure of CPT was confirmed by (1)H nuclear magnetic resonance spectroscopy and ultraviolet spectrophotometry. The particle size of CPT/ DNA complexes was measured using laser diffraction spectrometry and the cytotoxicity of the copolymer was evaluated by methylthiazol tetrazolium method. The transfection efficiency evaluation of the CP copolymer was performed using luciferase activity assay. Cellular internalization of the CP/DNA complex was observed under confocal laser scanning microscopy. The targeting specificity of the polymer coupled to peptide was measured by competitive inhibition transfection study. The liver targeting specificity of the CPT copolymer in vivo was demonstrated by combining the copolymer with a therapeutic gene, interleukin-12, and assessed by its abilities in suppressing the growth of ascites tumor in mouse model. RESULTS: The results showed that the liver cancer-targeted specific peptide was successfully synthesized and linked with CP to form a new targeted gene delivery vector called CPT. The composition of CPT was confirmed and the vector showed low cytotoxicity and strong targeting specificity to liver tumors in vitro. The in vivo study results showed that interleukin-12 delivered by the new gene vector CPT/DNA significantly enhanced the antitumor effect on ascites tumor-bearing imprinting control region mice as compared with polyethylenimine (25 kDa), CP, and other controls, which further demonstrate the targeting specificity of the new synthesized polymer. CONCLUSION: The synthesized CPT copolymer was proven to be an effective liver cancer-targeted vector for therapeutic gene delivery, which could be a potential candidate for targeted cancer gene therapy.

Gene-carried chitosan-linked polyethylenimine induced high gene transfection efficiency on dendritic cells.

A dendritic cell (DC) networking system has become an attractive approach in cancer immunotherapy. Successful DC gene engineering depends on the development of transgene vectors. A cationic polymer, chitosan-linked polyethylenimine (PEI) (CP), possessing the advantages of both PEI and chitosan, has been applied in nonviral transfection of DCs. Physicochemical evaluation showed that CP/DNA complexes could form cationic nanoparticles. Compared with DCs transfected with commercial reagent, Lipofectamine2000, it showed higher transfection efficiency and lower cytotoxicity when DCs were transfected with CP/DNA complexes. A nuclear trafficking observation of CP/DNA complexes by a confocal laser scanning microscope further revealed that the CP could help DNA enter into the cytoplasm and finally into the nucleus of a DC. Finally, vaccination of DCs transfected with CP/DNA encoding gp100 slightly improved resistance to the B16BL6 melanoma challenge. This is the first report that CP polymer is used as a nonviral vector for DC gene delivery and DC vaccine. Essentially, these results might be helpful to design a promising nonviral vector for DC gene delivery.

Gene-carried chitosan-linked-PEI induced high gene transfection efficiency with low toxicity and significant tumor-suppressive activity.

PEI and chitosan are considered to be promising non-viral gene delivery vectors. To improve the transfection efficiency of chitosan, we linked chitosan with polyethylenimine (PEI, Mw=1.8 kDa) by 1,1'-carbonyldiimidazole to form a complex. The composition, particle size, as well as the zeta potential of this chitosan-linked-PEI (CP) complex were measured. And the DNA binding ability, cytotoxicity, and gene transfection efficiency of CP complex were also investigated in cancer cells. In HepG2, A549 and HeLa cells, CP complex exhibited lower cytotoxicity as compared with PEI25KDa (Mw=25 kDa), a positive control proved to be an efficient gene transfection polymer. Likewise, it showed good transfection efficiency in these cancer cell lines. Specifically, the long-term transfection efficiency of CP was higher than PEI25KDa as demonstrated by the in vitro cancer cell model. The confocal laser scanning microscopy data showed the time for CP to enter the nucleus was 4h, which was longer than that of PEI25KDa but shorter than that of chitosan. Furthermore, CP complexes were used as a gene carrier to deliver the CCL22 gene into H22 cells. When these gene-altered cells were inoculated in mice, the tumor growth rate was significantly decreased, indicating the CP copolymer was a promising vector for the therapeutic gene delivery.

N/P ratio significantly influences the transfection efficiency and cytotoxicity of a polyethylenimine/chitosan/DNA complex.

For designing a complex vector that has the advantages of both polyethylenimine (PEI) and chitosan for gene delivery, a PEI/chitosan/DNA complex was constructed at various N/P ratios (the ratios of moles of the amine groups of cationic polymers to those of the phosphate ones of DNA) and both the cytotoxicity and the transfection efficiency of the vector were evaluated. The results demonstrated that the chitosan/DNA binding degree was depended on the N/P ratio. The mean size of the complex vector was between 100 nm and 150 nm. Compared with PEI/DNA, the complex vector (PEI/chitosan/DNA with chitosan/DNA N/P=4, PEI/DNA N/P=10) appeared to have low cytotoxicity, which maintained the cell survival rate at greater than 80%, and showed higher transfection efficiency of nearly 1000 fold compared with that using chitosan/DNA alone. Furthermore, the expression efficiency of the complex vector carrying enhanced green fluorescent protein was not inhibited in the presence of serum in both HeLa cells and A549 cells. The PEI/chitosan complex may be a promising gene carrier that has high transfection efficiency as well as low cytotoxicity.

Combination of poly(ethylenimine) and chitosan induces high gene transfection efficiency and low cytotoxicity.

In this study, the cytotoxicity and transfection efficiency of using chitosan/DNA complex combined with poly(ethylenimine) (PEI) were investigated. The combination of PEI with the chitosan/DNA complex markedly enhanced the gene expression of HeLa cells to 1000-fold of that induced by chitosan alone. PEI's cytotoxicity was considerably decreased upon combination with the chitosan/DNA complex. Furthermore, the PEI/chitosan/DNA could maintain the gene expression efficiency in the presence of serum.