Alpha-synuclein Affects Certain Iron Transporters of BV2 Microglia Cell through its ferric reductase activity.

OBJECTIVE: Alpha-synuclein (α-syn) is a major component of lewy bodies, which is biomarker of Parkinson's disease (PD). It accumulates in substantia nigra pars compacts (SNpc) to form insoluble aggregates and cause neurotoxicity, which is often accompanied by iron deposition. METHOD: We compared the iron reductase activity between monomeric α-syn (M-α-syn) and oligomeric α-syn (O-α-syn), investigated the effect of α-syn on iron metabolism of BV2 microglia cells as well. RESULTS: α-syn had ferric reductase activity, and O-α-syn had stronger enzyme activity than M-α-syn. M-α-syn upregulated iron uptake protein, divalent metal transporter1 (DMT1) expression and iron influx, but did not regulate iron release protein, ferroportin1 (FPN1) expression and iron efflux. O-α-syn elevated the expression of both DMT1 and FPN1, thus increased the iron influx and efflux in BV2 microglial cells, but the expressions of iron regulatory protein1 (IRP1) and hypoxia inducible factor2α (HIF-2α) had no significant change. Moreover, both M-α-syn and O-α-syn could increase the mRNA expressions of TNF-α and IL-1ß in BV2 microglia cells. CONCLUSION: Both types of α-syn can activate microglia, which leads to increased expressions of pro-inflammatory factors. α-syn can affect DMT1 and FPN1 expressions in BV2 microglia cells, which might be through its ferric reductase activity.

Multiplex Assay of Cytokines with Tunable Detection Ranges for the Precise Diagnosis of Breast Cancer.

Precise profiling of the cytokine panel consisting of different levels of cytokines can provide personalized information about several diseases at certain stages. In this study, we have designed and fabricated an "all-in-one" diagnostic tool kit to bioassay multiple inflammatory cytokines ranging from picograms per milliliter to µg/mL in a small cytokine panel. Taking advantage of the kit fabricated by the DNA-encoded assembly of nanocatalysts in dynamic regulation and signal amplification, we have demonstrated the multiplex, visual, and quantitative detection of C-reactive protein (CRP), procalcitonin (PCT), and interleukin-6 (IL-6) with limits of detection of 1.6 ng/mL (61.54 pM), 20 pg/mL (1.57 pM), and 4 pg/mL (0.19 pM), respectively. This diagnostic tool kit can work well with commercial kits for detecting serum cytokines from breast cancer patients treated with immunotherapies. Furthermore, a small cytokine panel composed of CRP, PCT, and IL-6 is revealed to be significantly heterogeneous in each patient and highly dynamic for different treatment courses, showing promise as a panel of quantitative biomarker candidates for individual treatments. So, our work may provide a versatile diagnostic tool kit for the visual detection of clinical biomarkers with an adjustable broad detection range.

Acylcarnitines promote gallbladder cancer metastasis through lncBCL2L11-THOC5-JNK axis.

BACKGROUND: The progression of gallbladder cancer (GBC) is accompanied by abnormal fatty acid ß-oxidation (FAO) metabolism. Different types of lipids perform various biological functions. This study aimed to determine the role of acyl carnitines in the molecular mechanisms of GBC progression. METHODS: Distribution of lipids in GBC was described by LC-MS-based lipidomics. Cellular localization, expression level and full-length of lncBCL2L11 were detected using fluorescence in situ hybridization (FISH) assays, subcellular fractionation assay and 5' and 3' rapid amplification of the cDNA ends (RACE), respectively. In vitro and in vivo experiments were used to verify the biological function of lncBCL2L11 in GBC cells. Methylated RNA Immunoprecipitation (MeRIP) was performed to detect the methylation levels of lncBCL2L11. RNA pull-down assay and RNA immunoprecipitation (RIP) assay were used to identify lncBCL2L11 interacting proteins. Co-Immunoprecipitation (Co-IP) and Western blot assay were performed to validate the regulatory mechanism of lncBCL2L11 and THO complex. RESULTS: Acylcarnitines were significantly up-regulated in GBC tissues. High serum triglycerides correlated to decreased survival in GBC patients and promoted tumor migration. LncBCL2L11 was identified in the joint analysis of highly metastatic cells and RNA sequencing data. LncBCl2L11 prevented the binding of THOC6 and THOC5 and causes the degradation of THOC5, thus promoting the accumulation of acylcarnitines in GBC cells, leading to the malignant progression of cancer cells. In addition, highly expressed acylcarnitines stabilized the expression of lncBCL2L11 through N6-methyladenosine methylation (m6A), forming a positive feedback regulation in tumor dissemination. CONCLUSIONS: LncBCL2L11 is involved in gallbladder cancer metastasis through FAO metabolism. High lipid intake is associated with poor prognosis of GBC. Therefore, targeting lncBCL2L11 and its pathway-related proteins or reducing lipid intake may be significant for the treatment of GBC patients.

Contrasting Iron Metabolism in Undifferentiated Versus Differentiated MO3.13 Oligodendrocytes via IL-1ß-Induced Iron Regulatory Protein 1.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characterized by the loss of dopaminergic neurons and the accumulation of iron in the substantia nigra. While iron accumulation and inflammation are implicated in PD pathogenesis, their impact on oligodendrocytes, the brain's myelin-forming cells, remains elusive. This study investigated the influence of interleukin-1ß (IL-1ß), an elevated proinflammatory cytokine in PD, on iron-related proteins in MO3.13 oligodendrocytes. We found that IL-1ß treatment in undifferentiated MO3.13 oligodendrocytes increased iron regulatory protein 1 and transferrin receptor 1 (TfR1) expression while decreasing ferroportin 1 (FPN1) expression. Consequently, iron uptake was enhanced, and iron release was reduced, leading to intracellular iron accumulation. Conversely, IL-1ß treatment in differentiated MO3.13 oligodendrocytes exhibited the opposite effect, with decreased TfR1 expression, increased FPN1 expression, and reduced iron uptake. These findings suggest that IL-1ß-induced dysregulation of iron metabolism in oligodendrocytes may contribute to the pathological processes observed in PD. IL-1ß can increase the iron content in undifferentiated oligodendrocytes, thus facilitating the differentiation of undifferentiated MO3.13 oligodendrocytes. In differentiated oligodendrocytes, IL-1ß may facilitate iron release, providing a potential source of iron for neighboring dopaminergic neurons, thereby initiating a cascade of deleterious events. This study provides valuable insights into the intricate interplay between inflammation, abnormal iron accumulation, and oligodendrocyte dysfunction in PD. Targeting the IL-1ß-mediated alterations in iron metabolism may hold therapeutic potential for mitigating neurodegeneration and preserving dopaminergic function in PD.

UBA3 promotes the occurrence and metastasis of intrahepatic cholangiocarcinoma through MAPK signaling pathway.

Intrahepatic cholangiocarcinoma (ICC) accounts for approximately 15% of primary liver cancers, and the incidence rate has been increasing in recent years. Surgical resection is the best treatment for ICC, but the 5-year survival rate is less than 30%. ICC signature genes are crucial for the early diagnosis of ICC, so it is especially important to identify signature genes. The aim of this study is to screen the signature genes of ICC and find the potential target for the treatment of ICC. We find that UBA3 is highly expressed in ICC, and knockdown of UBA3 inhibits ICC proliferation, invasion and migration. Mechanistic experiments show that UBA3 promotes ICC proliferation, invasion and migration by affecting ANXA2 through the MAPK signaling pathway. UBA3 is a target of bufalin, and bufalin targeting UBA3 inhibits ICC development and progression through the MAPK signaling pathway. In conclusion, our study shows that bufalin inhibits ICC by targeting UBA3, which has emerged as a new biomarker and potential therapeutic target for ICC.

Alpha-synuclein Affects Certain Iron Transporters of BV2 Microglia Cell through its ferric reductase activity.

Alpha-synuclein (α-syn) is a major component of lewy bodies, which is biomarker of Parkinson's disease (PD). It accumulates in substantia nigra pars compacts (SNpc) to form insoluble aggregates and cause neurotoxicity, which is often accompanied by iron deposition. In this study, we compared the iron reductase activity between monomeric α-syn (M-α-syn) and oligomeric α-syn (O-α-syn), investigated the effect of α-syn on iron metabolism of BV2 microglia cells as well. We found that α-syn had ferric reductase activity, and O-α-syn had stronger enzyme activity than M-α-syn. M-α-syn upregulated iron uptake protein, divalent metal transporter1 (DMT1) expression and iron influx, but did not regulate iron release protein, ferroportin1 (FPN1) expression and iron efflux. O-α-syn elevated the expression of both DMT1 and FPN1, thus increased the iron influx and efflux in BV2 microglial cells, but the expressions of iron regulatory protein1 and hypoxia inducible factor2α have no significant change. Moreover, both M-α-syn and O-α-syn could increase the mRNA expressions of TNF-α and IL-1ß in BV2 microglia cells. Taken together, our data suggest that both types of α-syn can activate microglia, which leads to increased expressions of pro-inflammatory factors. α-syn can affect DMT1 and FPN1 expressions in BV2 microglia cells, which might be through its ferric reductase activity.

Heme oxygenase-1: The roles of both good and evil in neurodegenerative diseases.

Heme oxygenase-1 (HO-1) is the only way for cells to decompose heme. It can cleave heme to produce carbon monoxide (CO), ferrous iron (Fe2+ ), and biliverdin (BV). BV is reduced to bilirubin (BR) by biliverdin reductase(BVR). In previous studies, HO-1 was considered to have protective effects because of its anti-inflammatory, anti-apoptosis, and antiproliferation functions. However, emerging experimental studies have found that the metabolites derived from HO-1 can cause increase iin intracellular oxidative stress, mitochondrial damage, iron death, and autophagy. Because of its particularity, it is very meaningful to understand its exact mechanism. In this review, we summarized the protective and toxic effects of HO-1, its potential mechanism, its role in neurodegenerative diseases and related drug research. This knowledge may be beneficial to the development of new therapies for neurodegenerative diseases and is crucial to the development of new therapeutic strategies and biomarkers.

Bufalin reverses cancer-associated fibroblast-mediated colorectal cancer metastasis by inhibiting the STAT3 signaling pathway.

At present, recurrence and metastasis are still important factors that lead to a poor prognosis among colorectal cancer (CRC) patients. Cancer-associated fibroblasts (CAFs) can promote tumorigenesis and development. Bufalin is the main active monomer of the clinical drug cinobufacini, which exhibits antitumor activity in various cancers. But few research have investigated the effect of bufalin in inhibiting metastasis from the perspective of the tumor microenvironment. We first isolated CAFs from freshly resected colorectal cancer patient specimens and observed the effect of CAFs on CRC cell invasion through a series of experiments. We explored the effect of bufalin on the physiological activity of CRC mediated by CAFs through experiments. In our study, we found that CAFs could promote CRC cell activity through the STAT3 pathway. Bufalin reversed CAF-mediated CRC invasion and metastasis by inhibiting the STAT3 pathway. Overexpression of STAT3 attenuated the inhibitory function of bufalin on invasion and metastasis. Taken together, bufalin can reverse CAF-mediated colorectal cancer metastasis based on inhibiting the STAT3 signaling pathway.

Iron Overload Causes Ferroptosis But Not Apoptosis in MO3.13 Oligodendrocytes.

Oligodendrocytes are the most iron-rich cells in the brain. Studies have shown that oligodendrocytes are very sensitive to oxidative stress, and iron overload is more likely to cause damage to oligodendrocytes. The purpose of this experiment was to investigate the damaging effect and mechanism of ferric ammonium citrate (FAC) on MO3.13 oligodendrocytes. In FAC treatment group, the intracellular iron concentration and intracellular reactive oxygen species were increased. There were no obvious changes in nucleus and chromatin, but increased mitochondrial membrane density, decreased mitochondrial cristae and mitochondrial length were observed. Glutathione peroxidase 4 (GPX4) expression was decreased, but the ratio of Bcl-2/Bax protein levels and cleaved caspase-3 expression did not change. Moreover, the iron chelator deferoxamine (DFO) and the ferroptosis inhibitor ferrostatin-1(Fer-1) could inhibit the upregulation of GPX4, which indicating that DFO and Fer-1 could inhibit ferroptosis in MO3.13 oligodendrocytes induced by iron overload. Furthermore, the phosphorylation level of p53 was not changed, while the ratio of protein expressions of p-Erk1/2/Erk1/2 were markedly increased. Taken together, our data suggest that iron overload induces ferroptosis but not apoptosis in oligodendrocytes. The mechanism may be related to mitogen-activated protein kinase pathway activation rather than p53 pathway activation.

An electrochemical biosensor for SARS-CoV-2 detection via its papain-like cysteine protease and the protease inhibitor screening.

The persistent coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still infecting hundreds of thousands of people every day. Enriching the kits for SARS-CoV-2 detection and developing the drugs for patient treatments are still urgently needed for combating the spreading virus, especially after the emergence of various mutants. Herein, an electrochemical biosensor has been fabricated in this work for the detection of SARS-CoV-2 via its papain-like cysteine protease (PLpro) and the screening of protease inhibitor against SARS-CoV-2 by using our designed chimeric peptide-DNA (pDNA) nanoprobes. Utilizing this biosensor, the sensitive and specific detection of SARS-CoV-2 PLpro can be conducted in complex real environments including blood and saliva. Five positive and five negative patient throat swab samples have also been tested to verify the practical application capability of the biosensor. Moreover, we have obtained a detection limit of 27.18 fM and a linear detection range from 1 pg mL-1 to 10 µg mL-1 (I = 1.63 + 4.44 lgC). Meanwhile, rapid inhibitor screening against SARS-CoV-2 PLpro can be also obtained. Therefore, this electrochemical biosensor has the great potential for COVID-19 combating and drug development.

The Effectiveness of CDC's Rx Awareness Campaigns on Reducing Opioid Stigma: Implications for Health Communication.

Often health communication campaigns addressing misusing prescription opioids and opioid use disorder (OUD) do not pay enough attention to the associated stigma. This study investigated the effectiveness of a well-designed opioid awareness campaign on reducing stigma and provided evidence for future health communication design. CDC's Rx Awareness videos were used as the experiment material. 137 college students participated in this online experiment, and audience characteristics and video features were considered and tested. The results showed that Rx Awareness videos significantly reduced participants' stigmatizing attitudes and perceived public stigma and increased their empathy toward people with OUD. Empathy is a promising strategy to reduce opioid stigma. People with an opioid prescription history expressed more empathy. Recovery information, prescription history, and narrators' race influenced the audience's perceived public stigma. Implications for health communications and limitations of the study are discussed.

A Reasoned Action Approach to Social Connection and Mental Health: Racial Group Differences and Similarities in Attitudes, Norms, and Intentions.

This study employed a Reasoned Action Approach to investigate two communication behaviors that were being built into a statewide behavioral health campaign: initiating a conversation about one's own mental health struggles, and starting a conversation to discuss someone else's mental health difficulties. We examined whether the extent of attitudes, perceived norms, and perceived behavioral control regarding intent to perform these behaviors varied by racial identity. Using original survey data from Texans (N = 2,033), we conducted regression analyses for the two communication behaviors and found that intention to seek help was primarily explained by instrumental attitude, injunctive norm, descriptive norm, and perceived capacity; and intention to start a conversation to help someone else was primarily explained by instrumental attitude, injunctive norm, and perceived capacity. Additionally, we identified important common and distinct determinants of the two behaviors across different racial groups. Implications for health communication campaign message development and audience segmentation are discussed.

Effect of fermentation methods on properties of dough and whole wheat bread.

BACKGROUND: Whole wheat bread is high in nutritional value but poor in technological quality; therefore, research on how to improve its technological quality has attracted extensive attention. The effects of fermentation methods, including straight dough(STD), sourdough (SOD), sponge dough (SPD), and refrigerated SPD (RSD) methods, on the dough and bread quality of whole wheat bread were investigated, focusing on pasting properties, rheological properties, thermal properties, microstructure, basic quality, and starch digestibility. RESULTS: The rapid viscosity analysis and rheological results demonstrated that SOD had the highest pasting temperature and the lowest viscosity, indicating an inhibition of starch pasting and partial protein hydrolysis, whereas the opposite trend presented by SPD and RSD indicated a greater starch hydration and a stronger gluten network. Thermal gravimetric analysis and differential scanning calorimetry results indicated reduced starch thermal degradation and increased starch pasting enthalpy in SOD and RSD. Scanning electron microscopy images revealed that the starch granules of SOD and RSD were tightly wrapped by a gluten network. SOD and RSD breads had the largest specific volume, the softest texture, and the lowest glycemic index. CONCLUSION: The effects of different fermentation methods on dough and bread structure can provide instructive information for future studies on their applications in whole wheat bread production. © 2023 Society of Chemical Industry.

tRF-3013b inhibits gallbladder cancer proliferation by targeting TPRG1L.

BACKGROUND: tRNA-derived fragments (tRFs) are newly discovered noncoding RNAs and regulate tumor progression via diverse molecular mechanisms. However, the expression and biofunction of tRFs in gallbladder cancer (GBC) have not been reported yet. METHODS: The expression of tRFs in GBC was detected by tRF and tiRNA sequencing in GBC tissues and adjacent tissues. The biological function of tRFs was investigated by cell proliferation assay, clonal formation assay, cell cycle assay, and xenotransplantation model in GBC cell lines. The molecular mechanism was discovered and verified by transcriptome sequencing, fluorescence in situ hybridization (FISH), target gene site prediction, and RNA binding protein immunoprecipitation (RIP). RESULTS: tRF-3013b was significantly downregulated in GBC compared with para-cancer tissues. Decreased expression of tRF-3013b in GBC patients was correlated with poor overall survival. Dicer regulated the production of tRF-3013b, and its expression was positively correlated with tRF-3013b in GBC tissues. Functional experiments demonstrated that tRF-3013b inhibited GBC cell proliferation and induced cell-cycle arrest. Mechanically, tRF-3013b exerted RNA silencing effect on TPRG1L by binding to AGO3, and then inhibited NF-κB. TPRG1L overexpression could rescue the effects of tRF-3013b on GBC cell proliferation. CONCLUSIONS: This study indicated that Dicer-induced tRF-3013b inhibited GBC proliferation by targeting TPRG1L and repressed NF-κB, pointing to tRF-3013b as a novel potential therapeutic target of GBC.

Talking About Prescription Opioid Misuse: The Effect of Family Communication Patterns and Messages Advocating Direct and Indirect Communication.

Families play an important role in addressing substance misuse and addiction. Extant literature suggests patterns of communication within families influence the ways in which they engage loved ones who may be misusing substances like prescription opioids. However, little is known regarding how strategic health messages about family communication influence individuals' intentions to engage in conversations about substance misuse. Applying a normative approach, we conducted a (2 × 2) between-participants experiment examining whether messages advocating indirect (versus direct) communication are more effective for individuals (n = 613) who describe their family as having a low (versus high) conversation orientation. Univariate analysis of variance tests show match effects for message attitudes and message elaboration. For intentions to talk with a loved one about the risks of OUD, there was only evidence of a matching effect between the message advocating indirect communication with low conversation audiences. Both message types were equally effective at influencing intentions for high conversation participants. These findings suggest message designers should consider the kinds of communication behaviors and actions advocated in appeals targeting family members. Messages that are inclusive of the conversation dynamics of particular audiences may have greater effect. In particular, for low conversation audiences, messages advocating an indirect approach may be more effective at motivating intentions to engage someone who is misusing opioids.

Effect of peripherally inserted central catheter (PICC) parenteral nutrition on immune function and nutritional support after radical gastrectomy for gastric cancer.

Objective of the present study was to investigate the effects of peripherally inserted central catheter (PICC) parenteral nutrition support on immune function and nutritional support in patients undergoing radical gastrectomy for gastric cancer. 140 patients who underwent radical gastrectomy for gastric cancer were selected as participants and were divided into study group and the control group by random number table, with 70 cases in each group. Patients in the two groups underwent standard gastrectomy under general anesthesia by the same group of doctors. The study group received postoperative PICC catheter parenteral nutrition, and the control group received central venous catheter (CVC) nutrition support. Comparative study was done using t test and Chi-square test. The serum levels of ALB, TFN, PA, Hb, CD4+, CD8+, CD4+/CD8+, IgA, IgG, IgM and CD3+ in the two groups were observed before and after treatment, and the postoperative complications of the two groups were compared. After treatment, the levels of ALB, TFN, PA and Hb in the two groups were significantly increased (P<0.05). Levels of CD3+, CD4+, CD4+/CD8+, IgA, IgG and IgM also amplified significantly after treatment in both the groups, while CD8+ decreased significantly (P<0.05). What's more, the improvement degree of the study group was significantly greater than that of the control group (P<0.05). The time of drawing drainage tube, recovering intestinal function, getting off bed and the length of hospital stay in the study group were significantly shorter than those in the control group (P<0.05). The incidence of postoperative complications in the study group and control group were 8.6% (6/70 cases) and 11.4% (8/70 cases) respectively, and there was no significant difference (P>0.05). PICC catheter parenteral nutrition support and improve the nutritional status of patients, it was proved a safe and effective nutritional support which improve the cellular immune function and accelerated the recovery of gastrointestinal function.

Fiber-Shaped Perovskite Solar Cells with High Power Conversion Efficiency.

A perovskite solar cell fiber is created with a high power conversion efficiency of 7.1% through a controllable deposition method. A combination of aligned TiO2 nanotubes, a uniform perovskite layer, and transparent aligned carbon nanotube sheet contributes to the high photovoltaic performance. It is flexible and stable, and can be woven into smart clothes for wearable applications.

miR-132 upregulation promotes gastric cancer cell growth through suppression of FoxO1 translation.

Gastric carcinoma (GC) is a prevalent malignant cancer worldwide and is highly lethal due to its fast growth. Hence, treatments to suppress GC cell growth may be applied together with surgery and chemotherapy to increase therapeutic outcome. Previous studies have shown the involvement of some microRNAs (miRNAs or miRs) in the carcinogenesis of GC, whereas a role of miR-132 in regulating the growth of GC has not been reported. Here, we report that overexpression of miR-132 in GC cells decreased FoxO1 protein levels, whereas depletion of miR-132 increased FoxO1 protein levels, without altering FoxO1 transcripts. Bioinformatics analyses showed that miR-132 bound to 3'-untranslated region (3'-UTR) of FoxO1 messenger RNA (mRNA) to prevent its translation, which was confirmed by luciferase reporter assay. Moreover, miR-132-mediated suppression of FoxO1 in GC cells resulted in a significant increase in GC cell growth in vitro and in vivo, while increases in FoxO1 by expression of antisense of miR-132 significantly decreased GC cell growth in vitro and in vivo. Finally, miR-132 levels were found significantly increased in GC specimens, compared to those in paired non-tumor gastric tissue. Together, our data suggest that miR-132 upregulation in GC cells may promote cell growth through suppression of FoxO1 translation.

Effects of Hydroxy Groups in the A-Ring on the Anti-proteasome Activity of Flavone.

The ubiquitin-proteasome pathway plays an important role in regulating apoptosis and the cell cycle. Recently, proteasome inhibitors have been shown to have antitumor effects and have been used in anticancer therapy for several cancers such as multiple myeloma. Although some flavones, such as apigenin, chrysin and luteolin, have a specific role in the inhibition of proteasome activity and induced apoptosis in some reports, these findings did not address all flavone types. To further investigate the proteasome-inhibitory mechanism of flavonoids, we examined the inhibitory activity of 5,6,7-trihydroxyflavone, baicalein and 5,6,7,4'-tetrahydroxyflavone, scutellarein on extracted proteasomes from mice and cancer cells. Unlike the other flavones, baicalein and scutellarein did not inhibit proteasome activity or accumulate levels of ubiquitinated proteins. These results indicate that flavones with hydroxy groups at positions 5, 6 and 7 of the A-ring lack the anti-proteasome function.

Three-step method for systematic lymphadenectomy in gastric cancer surgery using the 'curettage and aspiration dissection technique' with Peng's multifunctional operative dissector.

BACKGROUND: Gastric cancer is one of the most common malignancies and is a leading cause of cancer death worldwide. Surgery is the most effective and successful method of treatment for gastric cancer, and systematic lymph node (LN) dissection is unquestionably the most effective procedure for treating LN metastases of gastric cancer. Systematic lymphadenectomy is the most important part of curative resection, but lymphadenectomy is also the most difficult procedure in gastric cancer surgery. The aim of this study is to report our three-step method for lymphadenectomy in gastric cancer. METHODS: In this study, the lymph node stations and groups were defined according to the 13th edition of the Japanese Classification for Gastric Carcinoma. The authors' novel, simplified method consists of three steps: (1) the Kocher maneuver and dissection of the greater omentum together with the anterior sheet of the mesocolon, (2) dissection of the lesser omentum, and (3) lymphadenectomy following the main vessels. We primarily used Peng's multifunctional operative dissector, which combines four different functions (cutting, separating, aspirating and coagulating). Our systematic lymphadenectomy included three steps, and the main procedure started from right to left and in the caudal to cranial direction. RESULTS: A total of 830 consecutive patients underwent our three-step-method systematic lymphadenectomy in advanced gastric cancer surgery. The mean operation time was 146 minutes, and the mean blood loss was 248 ml. The median postoperative hospital stay was 10.9±4.8 days. The median number of examined LN was 31.6 (range 17 to 72) per patient, and the median number of metastatic LN was 5.6 (range 0 to 42) per patient. The overall incidence of postoperative complications was 10.6%, and the rate of hospital death was 0.9%. The overall three-year survival rate was 52.6%. CONCLUSIONS: Our three-step method for lymphadenectomy is easy to perform and is a useful procedure for gastric cancer surgery.