Effects of occupational exposure to dust, gas, vapor and fumes on chronic bronchitis and lung function.

Background: Chronic obstructive pulmonary disease (COPD) is one of the leading causes of mortality worldwide, and therefore the identification of the modifiable risk factors [such as exposure to vapors, gases, dust and fumes (VGDF)] for accelerate disease progression has important significance. Methods: We conducted COPD surveillance in six cities of southern China between 2014 and 2019. We recorded the diagnosis of chronic bronchitis, respiratory symptoms, occupational exposure to VGDF and other covariates by using a structured questionnaire. Logistic regression and multivariate linear regression model were adopted for analysis. We performed sensitivity analyses based on two methods of propensity score (PS) methods to evaluate the robustness of our results. Results: A total of 7,418 participants were included. Cough [odds ratios (ORs): 1.60, 95% confidence interval (CI): 1.22 to 2.08] and phlegm (OR: 1.49, 95% CI: 1.19 to 1.85) correlated significantly with exposure to dust. There was an increased risk of cough (OR: 1.53, 95% CI: 1.11 to 2.07) for occupational exposure to gas/vapor/fume. Dual exposure to dust and gas/vapor/fume was associated with a significantly increased risk of chronic bronchitis (OR: 1.74, 95% CI: 1.20 to 2.52), cough (OR: 1.43, 95% CI: 1.15 to 1.79) and phlegm (OR: 1.49, 95% CI: 1.24 to 1.79). In 5,249 participants with complete data of spirometry, gas/vapor/fume was associated with a decreased ratio of forced expiratory volume in one second and forced vital capacity (FEV1/FVC) (ß: -1.05, 95% CI: -1.85 to -0.26) and maximal mid-expiratory flow (MMEF) (ß: -0.15, 95% CI: -0.23 to -0.07). Dual exposure to dust and gas/vapor/fume was significantly associated with decreased FEV1/FVC (ß: -0.74, 95% CI: -1.28 to -0.20) and MMEF (ß: -0.06, 95% CI: -0.12 to -0.01). Results of sensitivity analysis were not materially changed. Conclusions: VGDF exposure is associated with chronic bronchitis, respiratory symptoms and decreased lung function, suggesting that VGDF contributes to the pathogenesis and progression of COPD.

Topical combined Phyllanthus emblica Linn. and simvastatin improves wound healing in diabetic mice by enhancing angiogenesis and reducing neutrophil infiltration.

The present study aimed to investigate the effects of combined Phyllanthus emblica Linn. (PE) and simvastatin (SIM) on diabetic wounds in male BALB/C mice. Bilateral full thickness wound excisions were performed in the control and diabetic groups (45 mg/kg streptozotocin, intraperitoneally injected daily for 5 days). The diabetic mice received daily treatment with four different types of cream: Vehicle [diabetes mellitus (DM) + Vehicle group], 100% PE (DM + PE group), 5% SIM (DM + SIM group) and combined 100% PE + 5% SIM (DM + Combination group) for 4, 7 and 14 days. The tissue malondialdehyde (MDA) and IL-6 protein levels, the number of infiltrated neutrophils, and the percentages of wound closure (%WC), capillary vascularity (%CV) and re-epithelialization (%RE) were subsequently measured. The results indicated that in the DM + Combination group, %CV and %WC were significantly increased when compared with the DM + Vehicle group on days 7 and 14. The tissue MDA content on day 14, and the number of infiltrated neutrophils on days 4 and 7 were significantly reduced in the DM + Combination group compared with those in the DM + Vehicle group. Furthermore, a strong positive correlation was revealed between %CV and %WC in the five groups on day 7 (r=0.736; P=0.0003). These findings indicated that topical application of combined PE and SIM could enhance wound healing by upregulating angiogenesis and reducing neutrophil infiltration in mice with diabetic wounds.

[Characteristic Analysis and Source Apportionment of VOCs in Urban Areas of Beijing in Summer].

Refined characterization of volatile organic compound (VOCs) components and source apportionment can provide scientific and effective support for ozone (O3) pollution prevention and control. Using hourly-resolution VOCs online data monitored at urban sites in Beijing from July to August in 2020, the chemical characteristics of VOCs and ozone formation potential (OFP) in environmental receptors during high and low ozone concentration periods were analyzed, and refined source apportionment was conducted with a positive matrix factorization (PMF) model. The results showed that the average φ[total volatile organic compounds (TVOCs)] at the monitoring sites during the observation period was 12.65×10-9, and the φ(TVOCs) during the high and low ozone concentration periods were 13.44×10-9 and 12.33×10-9, respectively, with an OFP of 107.6 µg·m-3and 99.2 µg·m-3, respectively. Ozone production was controlled by VOCs, with the highest reactivity of aromatic hydrocarbons and the top three species contributing to OFP being isoprene, toluene, and m/p-xylene. The main sources of VOCs in environmental receptors during low O3 periods included vehicular emissions (26.4%), background emissions (15.7%), solvent using (13.0%), auto repair (12.8%), secondary generation sources (9.7%), biomass combustion (6.1%), printing industry (5.7%), LNG-fueled vehicles (5.5%), and vegetation emissions (5.0%), of which background emissions, secondary generation, and printing industry sources have been little discussed in recent studies of VOCs source apportionment in Beijing. The contribution of auto repair sources and secondary generation sources increased by 3.4% and 2.6%, respectively, during the high O3 periods compared to those during the low O3 periods, and vehicular emissions remained the most significant source of VOCs contribution in the urban area of Beijing. Vegetation emissions rose from 07:00 pm and reach a maximum in the late afternoon. The contribution of background emission sources was less variable; vehicular emissions and LNG-fueled vehicle sources showed a morning and evening peak, with a relatively low contribution in the afternoon.

Impact of polluting fuels for cooking on diabetes mellitus and glucose metabolism in south urban China.

We hypothesized that exposure to polluting fuels for cooking was associated with abnormality of glucose metabolism and diabetes mellitus (DM) in south urban China. 3414 residents were surveyed in 14 urban areas of Guangdong Province in 2018. We recorded polluting fuels for cooking exposure, different DM status (DM, prediabetes), fasting blood glucose (FBG), oral glucose tolerance test (OGTT), glycated hemoglobin (HbA1c ), and other covariates by using a structured questionnaire. We conducted logistic regression model and multivariate linear regression model based on propensity-score method (inverse probability of weighting) to examine the effect of polluting fuels for cooking exposure on DM and glucose metabolism. Exposure to polluting fuels for cooking was associated with DM (odds ratio: 2.57, 95% confidence interval: 1.71 to 3.86) and prediabetes (odds ratio: 1.98, 95% confidence interval: 1.52 to 2.58) in both the adjusted and unadjusted models (all p < 0.05). Exposure to polluting fuels for cooking was significantly associated with an increase of FBG (ß: 0.30 mmol/L, 95% confidence interval: 0.22 to 0.38 mmol/L). Sensitivity analysis showed that the results were not substantially changed. There was an increased risk of DM, prediabetes and high levels of FBG, OGTT, and HbA1c among participants aged ≥ 40 years with exposure to polluting fuels for cooking. We demonstrated that exposure to polluting fuels for cooking was associated with higher levels of FBG, which contributed to the increased risk of DM and prediabetes in middle-aged elderly Chinese population living in urban areas.

Role of AMPK in the expression of tight junction proteins in heat-treated porcine Sertoli cells.

Hyperthermia can cause dysfunction of the tight junctions (TJs) in testes. Adenosine 5'-monophosphate-activated protein kinase (AMPK) participates in the regulation of TJs in testis. However, whether AMPK regulates the expression of TJ proteins in the response of Sertoli cells to heat treatment remains unknown. We subjected Sertoli cells from 3-week-old piglets to heat treatment (43 °C, 30 min), which decreased cell viability, and increased the early apoptosis rate. These effects were reversible and the cells gradually recovered to normal viability at 48 h post-heat treatment. Expression of TJ proteins (claudin 11, JAMA, occludin, and ZO1) was detected in immature porcine Sertoli cells. The mRNA and protein levels of TJ proteins significantly decreased at 1 h after heat exposure, but recovered with increasing recovery time. Additionally, the expression of claudin 11 in the cytoplasm was also markedly decreased by heat treatment. AMPK phosphorylation, the cellular ATP level, and Ca2+/calmodulin-dependent protein kinase kinase B (CaMKKB) level, but not the liver kinase B1 (LKB1) level, were downregulated by heat treatment. More importantly, activation or overexpression of AMPK, which is a regulator of the assembly of TJs, partially rescued the heat treatment-induced downregulation of TJ proteins. By contrast, AMPK knockdown using small interfering RNA (siRNA) further decreased the expression levels of TJ proteins. In addition, claudin 11 was almost undetectable post heat treatment. Collectively, this study demonstrated that heat treatment could reversibly perturb the expression of TJ proteins in immature porcine Sertoli cells by inhibiting the AMPK signaling pathway.

ERK1/2 regulates heat stress-induced lactate production via enhancing the expression of HSP70 in immature boar Sertoli cells.

Lactate produced by Sertoli cells plays an important role in spermatogenesis, and heat stress induces lactate production in immature boar Sertoli cells. Extracellular signaling regulated kinase 1 and 2 (ERK1/2) participates in heat stress response. However, the effect of ERK1/2 on heat stress-induced lactate production is unclear. In the present study, Sertoli cells were isolated from immature boar testis and cultured at 32 °C. Heat stress was induced in a 43 °C incubator for 30 min. Proteins and RNAs were detected by western blotting and RT-PCR, respectively. Lactate production and lactate dehydrogenase (LDH) activity were detected using commercial kits. Heat stress promoted ERK1/2 phosphorylation, showing a reducing trend with increasing recovery time. In addition, heat stress increased heat shock protein 70 (HSP70), glucose transporter 3 (GLUT3), and lactate dehydrogenase A (LDHA) expressions, enhanced LDH activity and lactate production at 2-h post-heat stress. Pretreatment with U0126 (1 × 10-6 mol/L), a highly selective inhibitor of ERK1/2 phosphorylation, reduced HSP70, GLUT3, and LDHA expressions and decreased LDH activity and lactate production. Meanwhile, ERK2 siRNA1 reduced the mRNA level of ERK2 and weakened ERK1/2 phosphorylation. Additionally, ERK2 siRNA1 reduced HSP70, GLUT3, and LHDA expressions decreased LDH activity and lactate production. Furthermore, HSP70 siRNA3 downregulated GLUT3 and LDHA expressions and decreased LDH activity and lactate production. These results show that activated ERK1/2 increases heat stress-induced lactate production by enhancing HSP70 expression to promote the expressions of molecules related to lactate production (GLUT3 and LDHA). Our study reveals a new insight in reducing the negative effect of heat stress in boars.

Retinoic Acid Receptor-Related Orphan Receptors: Critical Roles in Tumorigenesis.

Retinoic acid receptor-related orphan receptors (RORs) include RORα (NR1F1), RORß (NR1F2), and RORγ (NR1F3). These receptors are reported to activate transcription through ligand-dependent interactions with co-regulators and are involved in the development of secondary lymphoid tissues, autoimmune diseases, inflammatory diseases, the circadian rhythm, and metabolism homeostasis. Researches on RORs contributing to cancer-related processes have been growing, and they provide evidence that RORs are likely to be considered as potential therapeutic targets in many cancers. RORα has been identified as a potential therapeutic target for breast cancer and has been investigated in melanoma, colorectal colon cancer, and gastric cancer. RORß is mainly expressed in the central nervous system, but it has also been studied in pharyngeal cancer, uterine leiomyosarcoma, and colorectal cancer, in addition to neuroblastoma, and recent studies suggest that RORγ is involved in various cancers, including lymphoma, melanoma, and lung cancer. Some studies found RORγ to be upregulated in cancer tissues compared with normal tissues, while others indicated the opposite results. With respect to the mechanisms of RORs in cancer, previous studies on the regulatory mechanisms of RORs in cancer were mostly focused on immune cells and cytokines, but lately there have been investigations concentrating on RORs themselves. Thus, this review summarizes reports on the regulation of RORs in cancer and highlights potential therapeutic targets in cancer.

Heat stress-induced autophagy promotes lactate secretion in cultured immature boar Sertoli cells by inhibiting apoptosis and driving SLC2A3, LDHA, and SLC16A1 expression.

This study aimed to determine whether heat stress (HS) could induce autophagy in immature boar Sertoli cells (SCs) and test whether HS-induced autophagy could regulate lactate secretion by SCs. Cultured immature boar SCs were incubated at 43 °C for 30 minutes. The ratio of LC3B-II to LC3B-I and the mRNA transcript levels of LC3B showed time-dependent changes 0 to 48 hours after HS treatment, which peaked at 24 hours and increased by 30.25% or 260%, respectively, compared with control SCs. The density of autolysosomes, which were labeled with a red dye, was higher at 24 hours than at any other time point. However, the apoptosis rate, cleavage of caspase-3, and mRNA transcript levels of CASP3 (caspase-3) at 24 hours after HS were lower than at 12 hours. Furthermore, lactate secretion, and mRNA transcript levels of SLC2A3 (GLUT3), LDHA (LDHA), and SLC16A1 (MCT1) also showed time-dependent changes with a peak at 24 hours. In addition, LY294002 (20 µM) significantly inhibited changes in ratio of LC3B-II to LC3B-I, LC3B mRNA transcript levels, and autolysosome formation. It also resulted in significantly less lactate secretion and increased apoptosis but showed no effect on B-cell lymphoma-2 expression in heat-treated immature SCs. These findings indicated that HS-induced autophagy regulates lactate secretion by inhibiting apoptosis and increasing mRNA transcript and protein levels of SLC2A3, LDHA, and SLC16A1, which suggests that HS-induced autophagy may enhance lactate secretion by SCs.

[Characteristics of N2, N2O, NO, CO2 and CH4 Emissions in Anaerobic Condition from Sandy Loam Paddy Soil].

Understanding the characteristics of the production of nitrogen gases (N2, N2O and NO), CO2 and CH4 in anaerobic paddy soils is not only a prerequisite for an improved mechanistic understanding of key microbial processes involved in the production of atmospheric greenhouse gases (GHG), but might also provide the basis for designing greenhouse gas mitigation strategies. Moreover, quantifying the composition fractions of denitrification gaseous products is of key importance for improving parameterization schemes of microbial processes in process-oriented models which are increasingly used for assessing soil GHG emissions at site and national scales. In our experiments we investigated two sandy loam soils from two paddy fields. The initial concentrations of soil nitrate and dissolved organic carbon (DOC) were set at approximately 50 mg.kg-1 and mg.kg-1, respectively, by adding a mixture solution of KNO3 and glucose. The emissions of N2, N2O NO, CO2 and CH4, as well as concentrations of carbon and nitrogen substrates for each soil sample were measured simultaneously, using a gas-flow-soil-core technique and a paralleling substrate monitoring system. The results showed that the accumulative emissions of N2, N2O and NO of the two soil samples for the entire incubation period were 6 - 8, 20, and 15 - 18 mg.kg-1, respectively. By measuring the cumulative emissions of denitrification gases (N, = N2 + N2O + NO) we were able to explain 95% to 98% of observed changes in s1ifr nilrate concentrations. The mass fractions of N2, N2O and NO emissions to Nt were approximately 15% -19%, 47% -49%, and 34% -36%, respectively. Thus, in our experiments N2O and NO were the main products of denitrification for the entire incubation period. However, as the temporal courses of hourly or daily production of the denitrification gases showed, NO production dominated and peaked firstly, and then N2O, before finally N2 became the dominant product. Our results show the high temporal dynamic of denitrification end products and this knowledge is of crucial importance for model development, since so far existing models assume a fixed fraction of denitrification end products.

[Effect of carbon substrate concentration on N2, N2O, NO, CO2, and CH4 emissions from a paddy soil in anaerobic condition].

Understanding the effects of carbon and nitrogen substrates concentrations on the emissions of denitrification gases including nitrogen (N2) , nitrous oxide (N2O) and nitric oxide (NO), carbon dioxide (CO2) and methane (CH4) from anaerobic paddy soils is believed to be helpful for development of greenhouse gas mitigation strategies. Moreover, understanding the quantitative dependence of denitrification products compositions on carbon substrate concentration could provide some key parameters or parameterization scheme for developing process-oriented model(s) of nitrogen transformation. Using a silt loam soil collected from a paddy field, we investigated the influence of carbon substrate concentration on the emissions of the denitrification gases, CO2 and CH4 from anaerobically incubated soils by setting two treatments: control (CK) with initial soil nitrate and dissolved organic carbon (DOC) concentrations of ~ 50 mg.kg-1 and -28 mg kg-1 , respectively; and DOC added (C + ) with initial soil nitrate and DOC concentrations of ~50 mg.kg-1 and ~300 mg.kg-1 , respectively. The emissions of denitrification gases, CO2 and CH4, as well as concentrations of carbon and nitrogen substrates for each treatment were dynamically measured, using the gas-flow-soil-core technique and a paralleling substrate monitoring system. The results showed that CH4 emission was not observed in CK treatment while observed in C treatment. Aggregate emission of greenhouse gases for C + treatment was significantly higher comparing with the CK treatment (P <0. 01). The mass fractions of NO, N20 and N2 emissions in total nitrogen gases emissions were approximately 9% , 35% and 56% for CK treatment, respectively; and approximately 31% , 50% and 19% for C+ treatment, respectively, with significant differences between these two treatments (P < 0.01). The results indicated that carbon substrate concentrations can significantly change the composition of nitrogen gas emissions. The results also implicated that organic fertilizer should not be applied to nitrate-rich paddy soils prior to or during flooding so as to mitigate greenhouse gases emissions.

[Preparation and identification of a monoclonal antibody against recombinant human L-type pyruvate kinase N].

OBJECTIVE: To prepare and identify the monoclonal antibody (mAb) against pyruvate kinase N terminal (PK-N). METHODS: BALB/C mice were immunized with immunogen PK-N-GST-tag. Then the spleen cells were isolated and fused with SP2/0 cells. After several rounds of detecting and cloning, the hybridoma cell strains secreting anti-PK-N mAb were obtained. Its specificity was evaluated with ELISA and Western blot, and the titer, immunoglobulin subtype and affinity of the mAb were measured. RESULTS: Two cell strains of hybridoma, 2B2E4G and 2C6F5, were obtained. The hybridoma cell strains secreting anti-PK-N mAb belonged to IgG2b subtype, with a mAb titer in ascetic fluid of 1 : 409600 and 1 : 102400, respectively. Their affinity reached 3.54 x 10(8) L/mol and 2.72 x 10(8) L/mol, respectively, as determined by ELISA. Western blot demonstrated that the mAb could specifically recognize the immunogen and the natural cell lysis protein. The cell immunohistochemistry proved that the antibody could recognize human L type pyruvate kinase expressed in the plasma of HL-7702 cell strain and paraffin slice of hepatoma. CONCLUSION: The success in anti-PK-N mAb preparation provides a foundation for further studies into glycolysis in normal condition and metabolic diseases.

Sensitivity of different cytotoxic responses of Vero cells exposed to organic chemical pollutants and their reliability in the bio-toxicity test of trace chemical pollutants.

OBJECTIVE: To find a sensitive cytotoxic response to reflect the bio-toxicity of trace organic pollutants, the sensitivity and reliability of morphological change and proliferation inhibition of Vero cells exposed to 2, 4, 6-trichlorophenol (TCP) and the leachate from products related to drinking water (PRDW) were compared, and the mechanism of the morphological change in Vero cells exposed to chemical pollutants was studied. METHODS: Vero cells were treated by different concentration of TCP and the leachate from PRDW. Methylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT) assay was carried out for proliferation inhibition. Bioluminescence method was carried out as another method to test the toxicity of TCP. Flow Cytometry assay was used to test cell Apoptosis and damage of cell-membrane. RESULTS: 0.25 mg/L TCP had an effect on cell morphology, and the proportion of morphologically changed cells increased with increasing TCP concentration. At low TCP concentrations, inhibition of cell proliferation did not seem to correlate to TCP concentration, and was negative when TCP concentration was <1.0 mg/L. After exposure to leachate from PRDW extracted at different temperatures, the percentage of morphologically changed cells increased with extracting temperature, but the inhibition of cell proliferation failed to reflect the correlation between extracting temperature and proliferation inhibition of Vero cells. Although the Sensitivity of bioluminescence method seems to be similar to morphological change in Vero cells, the bacterial in this method is not homologous enough with human body cells to reflect the toxicity to human body. These imply cell morphological change is a more sensitive and reliable method to reflect bio-toxicity of organic pollutants than proliferation inhibition. Flow cytometry analysis and cell rejuvenation experiments indicated cell membrane damage, which results in cell morphological change, was an early and sensitive cytotoxic response comparing with necrosis. CONCLUSION: These results indicated that the cell membrane toxicity represented by morphological changes is a more sensitive and reliable method to indicate the composite bio-toxicity of trace chemicals than proliferation inhibition, inhibition on bioluminescence and necrosis. Nevertheless, the quantification of morphological change should be studied further.

Sensitivity of morphological change of Vero cells exposed to lipophilic compounds and its mechanism.

To find a sensitive cytotoxic response to reflect the toxicity of trace organic pollutants, the sensitivity and reliability of morphological change and proliferation inhibition of Vero cells exposed to lipophilic compounds and the leachate from products related to drinking water (PRDW) were compared, and the mechanism of the morphological change in Vero cells was studied. Results showed the proportion of morphologically changed cells increased with increasing 2,4,6-trichlorophenol (TCP)/perfluorooctane sulfonate (PFOS) concentration. However, at low TCP concentrations, inhibition of cell proliferation did not correlate to TCP concentration. After exposure to the leachate from PRDW extracted at different temperatures, the percentage of morphologically changed cells increased with extracting temperature, but the inhibition of cell proliferation failed to reflect the correlation to extracting temperature. These imply cell morphological change is a more sensitive and reliable method to reflect toxicity of trace organic pollutants than proliferation inhibition. Flow cytometry analysis indicated cell membrane damage was an early and sensitive cytotoxic response comparing with necrosis, resulting in cell morphological change, which may be due to the interference of lipophilic compounds. Lipophilic compound accumulated in cell membrane to interfere the assembly process of membrane protein and phospholipid.

Evaluation of x-inactivation status and cytogenetic stability of human dermal fibroblasts after long-term culture.

Human primary fibroblasts are a popular type of somatic cells for the production of induced pluripotent stem (iPS) cells. Here we characterized biological properties of primary fibroblasts in terms of cell-growth rate, cytogenetic stability, and the number of inactive X chromosomes during long-term passaging. We produced eight lines of female human dermal fibroblasts (HDFs) and found normal karyotype and expected pattern of X chromosome inactivation (XCI) at low passages (Passage P1-5). However, four out of the eight HDF lines at high passage numbers (≥ P10) exhibited duplicated hallmarks of inactive X chromosome including two punctuate signals of histone H3 lysine 27 trimethylation (H3K27me3) and X inactive-specific transcript (XIST) RNA signals in approximately 8.5-18.5% of the cells. Our data suggest that the copy number of inactive X chromosomes in a subset of female HDF is increased by a two-fold. Consistently, DNA fluorescent in situ hybridization (FISH) identified 3-4 copies of X chromosomes in one nucleus in this subset of cells with two inactive Xs. We conclude that female HDF cultures exhibit a higher risk of genetic anomalies such as carrying an increased number of X chromosomes including both active and inactive X chromosomes at a high passage (≥ P10).

Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry.

Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round-headed and normal spermatozoa. Two-dimensional (2-D) fluorescence difference gel electrophoresis (DIGE) coupled with mass spectrometry (MS) was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry (MS/MS) exhibited significant changes (paired t-test, P < 0.05) in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility.

Establishment of a high-resolution 2-D reference map of human spermatozoal proteins from 12 fertile sperm-bank donors.

AIM: To extend the analysis of the proteome of human spermatozoa and establish a 2-D gel electrophoresis (2-DE) reference map of human spermatozoal proteins in a pH range of 3.5-9.0. METHODS: In order to reveal more protein spots, immobilized pH gradient strips (24 cm) of broad range of pH 3-10 and the narrower range of pH 6-9, as well as different overlapping narrow range pH immobilized pH gradient (IPG) strips, including 3.5-4.5, 4.0-5.0, 4.5-5.5, 5.0-6.0 and 5.5-6.7, were used. After 2-DE, several visually identical spots between the different pH range 2-D gel pairs were cut from the gels and confirmed by mass spectrometry and used as landmarks for computer analysis. RESULTS: The 2-D reference map with pH value from 3.5 to 9.0 was synthesized by using the ImageMaster analysis software. The overlapping spots were excluded, so that every spot was counted only once. A total of 3872 different protein spots were identified from the reference map, an approximately 3-fold increase compared to the broad range pH 3-10 IPG strip (1306 spots). CONCLUSION: The present 2-D pattern is a high resolution 2-D reference map for human fertile spermatozoal protein spots. A comprehensive knowledge of the protein composition of human spermatozoa is very meaningful in studying dysregulation of male fertility.