BRD7 improves glucose homeostasis independent of IRS proteins.

Bromodomain-containing protein 7 (BRD7) has emerged as a player in the regulation of glucose homeostasis. Hepatic BRD7 levels are decreased in obese mice, and the reinstatement of hepatic BRD7 in obese mice has been shown to establish euglycemia and improve glucose homeostasis. Of note, the upregulation of hepatic BRD7 levels activates the AKT cascade in response to insulin without enhancing the sensitivity of the insulin receptor (InsR)-insulin receptor substrate (IRS) axis. In this report, we provide evidence for the existence of an alternative insulin signaling pathway that operates independently of IRS proteins and demonstrate the involvement of BRD7 in this pathway. To investigate the involvement of BRD7 as a downstream component of InsR, we utilized liver-specific InsR knockout mice. Additionally, we employed liver-specific IRS1/2 knockout mice to examine the requirement of IRS1/2 for the action of BRD7. Our investigation of glucose metabolism parameters and insulin signaling unveiled the significance of InsR activation in mediating BRD7's effect on glucose homeostasis in the liver. Moreover, we identified an interaction between BRD7 and InsR. Notably, our findings indicate that IRS1/2 is not necessary for BRD7's regulation of glucose metabolism, particularly in the context of obesity. The upregulation of hepatic BRD7 significantly reduces blood glucose levels and restores glucose homeostasis in high-fat diet-challenged liver-specific IRS1/2 knockout mice. These findings highlight the presence of an alternative insulin signaling pathway that operates independently of IRS1/2 and offer novel insights into the mechanisms of a previously unknown insulin signaling in obesity.

Understanding the Function and Mechanism of Zebrafish Tmem39b in Regulating Cold Resistance.

Autophagy and endoplasmic reticulum (ER) stress response are among the key pathways regulating cold resistance of fish through eliminating damaged cellular components and facilitating the restoration of cell homeostasis upon exposure to acute cold stress. The transmembrane protein 39A (TMEM39A) was reported to regulate both autophagy and ER stress response, but its vertebrate-specific paralog, the transmembrane protein 39B (TMEM39B), has not been characterized. In the current study, we generate tmem39b-knockout zebrafish lines and characterize their survival ability under acute cold stress. We observed that the dysfunction of Tmem39b remarkably decreased the cold resilience of both the larval and adult zebrafish. Gene transcription in the larvae exposed to cold stress and rewarming were characterized by RNA sequencing (RNA-seq) to explore the mechanisms underlying functions of Tmem39b in regulating cold resistance. The results indicate that the deficiency of Tmem39b attenuates the up-regulation of both cold- and rewarming-induced genes. The cold-induced transcription factor genes bif1.2, fosab, and egr1, and the rewarming-activated immune genes c3a.3, il11a, and sting1 are the representatives influenced by Tmem39b dysfunction. However, the loss of tmem39b has little effect on the transcription of the ER stress response- and autophagy-related genes. The measurements of the phosphorylated H2A histone family member X (at Ser 139, abbreviated as γH2AX) demonstrate that zebrafish Tmem39b protects the cells against DNA damage caused by exposure to the cold-warming stress and facilitates tissue damage repair during the recovery phase. The gene modules underlying the functions of Tmem39b in zebrafish are highly enriched in biological processes associated with immune response. The dysfunction of Tmem39b also attenuates the up-regulation of tissue C-reactive protein (CRP) content upon rewarming. Together, our data shed new light on the function and mechanism of Tmem39b in regulating the cold resistance of fish.

Mitochondria Dysfunction and Cell Apoptosis Limit Resistance of Nile Tilapia (Oreochromis niloticus) to Lethal Cold Stress.

Inability of Nile tilapia (Oreochromis niloticus) to withstand cold stress represents a major economic concern, which restricts the culture area, limits the growing period and even results in mass mortality in cold seasons. However, the cellular and molecular mechanisms determining cold susceptibility of Nile tilapia remain largely unknown. In this study, we characterized the ability of juvenile Nile tilapia to survive lethal cold stress (12 °C) and the median survival time (LT50) of the experimental fish under exposure to 12 °C cold stress was estimated as 3.14 d. After being exposed to 12 °C for 3 d, the survivors that lost equilibrium (LE) and those that swam normally (NO) were regarded as cold-sensitive and cold-tolerant, respectively. The untreated (Ctrl), NO and LE fish were subjected to histological, biochemical and gene expression analyses to explore the cellular and molecular events underlying cold susceptibility of Nile tilapia. Exposure of Nile tilapia to lethal cold stress caused systemic tissue structure changes, mitochondrial swelling and dysfunction, induction of apoptosis and endoplasmic reticulum (ER) stress-related genes and cell apoptosis. The extent of these adverse cellular and molecular events determines an individual's ability to survive cold stress. Our data indicate that mitochondria dysfunction and mitochondria-mediated cell apoptosis are the main factors limiting Nile tilapia's cold resistance.

STING agonism enhances anti-tumor immune responses and therapeutic efficacy of PARP inhibition in BRCA-associated breast cancer.

Poly (ADP-ribose) polymerase (PARP) inhibitors exert their efficacy via synthetic lethal effects and by inducing cGAS/STING-mediated immune responses. We demonstrate that compared to monotherapies, combined PARP inhibition and STING agonism results in increased STING pathway activation, greater cytotoxic T-cell recruitment and enhanced dendritic cell activation in BRCA1-deficient breast cancer models. The combination markedly improved anti-tumor efficacy in vivo, with evidence of complete tumor clearance, prolongation of survival and induction of immunologic memory.

Acute toxicity of tire wear particles, leachates and toxicity identification evaluation of leachates to the marine copepod, Tigriopus japonicus.

Tire wear particles (TWPs) have been characterized as microplastics in recent years, and many of these TWPs will be eventually deposited in coastal areas, leading to adverse effects to marine organisms. Results of the acute toxicity test in this study showed that the 96-h LC50 values of the particles and leachate were 771.4 mg/L (95% CI = 684.4-869.6 mg/L) and 5.34 g/L (95% CI = 4.75-6.07 g/L), respectively. The chemical constituents of TWP and the leachate are very complex, and little research has been conducted to determine which of these constituents contribute to the toxicity of TWP leachate to marine organisms. Therefore, the composition of the TWP and leachate was analyzed, and a variety of chemicals were identified, including metals (Mn, Zn, etc.) and organic compounds (cyclohexanthiol, 4-ethyl-1,2-dimethylbenzene, benzothiazole, stearic acid, N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine, etc.). In addition, the marine copepod Tigriopus japonicus was applied as a model species in the toxicity identification evaluation study to characterize, identify and confirm the toxicity-causing substances in the TWP leachate. Zn was identified and confirmed as the main toxicant contributing to the toxicity. Furthermore, Zn concentrations in the leachate over time were investigated. The release of Zn from TWPs to the aquatic environment was slow, and conformed to a parabolic model with a release constant k of 2.06. The organic component, benzothiazole, exhibited an antagonistic effect with zinc in the acute toxicity of the TWP leachate.

The regulatory subunits of PI3K, p85α and p85ß, differentially affect BRD7-mediated regulation of insulin signaling.

Bromodomain-containing protein 7 (BRD7) has been shown to interact with the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), p85, in the insulin signaling pathway. Here, we show that upregulation of hepatic BRD7 improves glucose homeostasis even in the absence of either p85 isoform, p85α or p85ß. However, BRD7 leads to differential activation of downstream effector proteins in the insulin signaling pathway depending on which isoform of p85 is present. In the presence of only p85α, BRD7 overexpression increases phosphorylation of insulin receptor (IR) upon insulin stimulation, without increasing the recruitment of p85 to IR substrate. Overexpression of BRD7 also increases activation of Akt in response to insulin, but does not affect basal phosphorylation levels of Akt. Meanwhile, the phosphorylation of glycogen synthase kinase 3ß (GSK3ß) is increased by overexpression of BRD7. On the other hand, in the presence of only p85ß, BRD7 overexpression does not affect phosphorylation levels of IR, and Akt phosphorylation is not affected by insulin stimulation following BRD7 upregulation. However, BRD7 overexpression leads to increased basal phosphorylation levels of Akt and GSK3ß. These data demonstrate that BRD7's action on glucose homeostasis does not require the presence of both p85 isoforms, and p85α and p85ß have unique roles in insulin signaling in the liver.

Cosmc deficiency causes spontaneous autoimmunity by breaking B cell tolerance.

Factors regulating the induction and development of B cell­mediated autoimmunity are not well understood. Here, we report that targeted deletion in murine B cells of X-linked Cosmc, encoding the chaperone required for expression of core 1 O-glycans, causes the spontaneous development of autoimmune pathologies due to a breakdown of B cell tolerance. BC-CosmcKO mice display multiple phenotypic abnormalities, including severe weight loss, ocular manifestations, lymphadenopathy, and increased female-associated mortality. Disruption of B cell tolerance in BC-CosmcKO mice is manifested as elevated self-reactive IgM and IgG autoantibodies. Cosmc-deficient B cells exhibit enhanced basal activation and responsiveness to stimuli. There is also an elevated frequency of spontaneous germinal center B cells in BC-CosmcKO mice. Mechanistically, loss of Cosmc confers enhanced B cell receptor (BCR) signaling through diminished BCR internalization. The results demonstrate that Cosmc, through control of core 1 O-glycans, is a previously unidentified immune checkpoint gene in maintaining B cell tolerance.

Molecular mechanisms of zooplanktonic toxicity in the okadaic acid-producing dinoflagellate Prorocentrum lima.

Prorocentrum lima is a dinoflagellate that forms hazardous blooms and produces okadaic acid (OA), leading to adverse environmental consequences associated with the declines of zooplankton populations. However, little is known about the toxic effects and molecular mechanisms of P. lima or OA on zooplankton. Here, their toxic effects were investigated using the brine shrimp Artemia salina. Acute exposure of A. salina to P. lima resulted in lethality at concentrations 100-fold lower than densities observed during blooms. The first comprehensive results from global transcriptomic and metabolomic analyses in A. salina showed up-regulated mRNA expression of antioxidant enzymes and reduced non-enzyme antioxidants, indicating general detoxification responses to oxidative stress after exposure to P. lima. The significantly up-regulated mRNA expression of proteasome, spliceosome, and ribosome, as well as the increased fatty acid oxidation and oxidative phosphorylation suggested the proteolysis of damaged proteins and induction of energy expenditure. Exposure to OA increased catabolism of chitin, which may further disrupt the molting and reproduction activities of A. salina. Our data shed new insights on the molecular responses and toxicity mechanisms of A. salina to P. lima or OA. The simple zooplankton model integrated with omic methods provides a sensitive assessment approach for studying hazardous algae.

Monoglyceride lipase mediates tumor-suppressive effects by promoting degradation of X-linked inhibitor of apoptosis protein.

We have previously reported that Monoglyceride Lipase (MGL) expression is absent or reduced in various human malignancies and MGL-deficient mice develop tumors in multiple organs. Evidence also suggests MGL to be a tumor suppressor, however, the mechanisms underlying its tumor-suppressive actions remain to be investigated. Here, we report a novel function of MGL as a negative regulator of XIAP, an important inhibitor of apoptosis. We found that MGL directly interacted with XIAP and enhanced E3-ligase activity and proteasomal degradation of XIAP. MGL overexpression induced cell death that was coupled with caspase activation and reduced XIAP levels. N-terminus of MGL was found to mediate interactions with XIAP and induce cell death. MGL-deficient cells exhibited elevated XIAP levels and exhibited resistance to anticancer drugs. XIAP expression was significantly elevated in tissues of MGL-deficient animals as well as human lung cancers exhibiting reduced MGL expression. Thus, MGL appears to mediate its tumor-suppressive actions by inhibiting XIAP to induce cell death.

Transcriptomic responses of Artemia salina exposed to an environmentally relevant dose of Alexandrium minutum cells or Gonyautoxin2/3.

Toxicities of the marine algae Alexandrium minutum and its excreted gonyautoxins (GTXs) to the marine crustacean Artemia salina were investigated. Mortality was observed for neither larvae nor adult A. salina exposed to A. minutum at a density of 5000 cells/mL or 0.5 µM GTX2/3. After exposure, the full transcriptome of adult A. salina was assembled and functionally annotated. A total of 599,286 transcripts were obtained, which were clustered into 515,196 unigenes. Results of the transcriptional effect level index revealed that direct exposure to the toxic algae A. minutum caused greater alterations in the transcriptome than did exposure to the extracellular product GTX2/3. Mechanisms of effects were different between exposure of A. salina to A. minutum cells or GTX2/3. Exposure to A. minutum modulated formation of the ribonucleoprotein complex and metabolism of amino acids and lipids in A. salina. Exposure to GTX2/3 exposure inhibited expression of genes related to metabolism of chitin, which might result in disruption of molting process or disturbed sheath morphogenesis. Overall, effects on transcription observed in this study represent the first report based on application of next generation sequencing techniques to investigate the transcriptomic response of A. salina exposed to an environmentally realistic level of A. minutum or GTX2/3.

Geographical distribution and seasonal variation in paralytic shellfish toxins in the coastal water of the South China Sea.

Paralytic shellfish toxins (PSTs) are a group of the most harmful neurotoxins distributed worldwide. Marine organism samples, including mollusks, crustaceans and fish, collected from 11 sites around the coastal water of the South China Sea, were analyzed in terms of the PST and toxicity via high-performance liquid chromatography. The PST geographical distribution, detectable rate for PST and toxin content all increased slightly from 2006 to 2008 to 2015. High PST content with more than 2 nmol g-1 appeared in Shantou (ST), Shanwei (SW), Zhanjiang (ZJ), Beihai (BH), Xuwen (XW), Haikou (HK), Dongfang (DF), Wanning (WN) and Sanya (SY). Low PST content with less than 2 nmol g-1 appeared in Shenzhen (SZ) and Yangjiang (YJ). High PST toxicity over the safety limit 800 µg STXeq/kg appeared in ST, SW, ZJ, BH, DF, SY and XW. PST has significant seasonal characteristics and was mostly detected in spring, early summer (March-June) and autumn (September-November) in the coastal water of the South China Sea. PST was usually detected in some specific species of scallop, mussel, bloody clam, hard clam, razor clam, oyster, crab, shrimp and fish. Toxin profile variation of marine organism samples was influenced greatly by species, sampling time and site.

Small Cell Neuroendocrine Carcinoma of the Cervix in Pregnancy: A Case Report and Review.

Small cell neuroendocrine carcinoma of the cervix is a rare subtype of cervical cancer. Here we report a case in which a 27-year-old female patient presented at 34-week gestation with abnormal vaginal bleeding, underwent normal labor, and gave birth to a healthy neonate. Her pregnancy was complicated with a cervical tumor which turned out to be small cell neuroendocrine cervical carcinoma. We reviewed and discussed the features, diagnosis, and prognosis of small cell neuroendocrine carcinoma of the cervix.

BRD7 deficiency leads to the development of obesity and hyperglycemia.

Obesity is a debilitating disease that has become a global epidemic. Although progress is being made, the underlying molecular mechanism by which obesity develops still remains elusive. Recently, we reported that the expression levels of bromodomain-containing protein 7 (BRD7) are significantly reduced in the liver of obese mice. However, it is not clear whether decreased levels of hepatic BRD7 are directly associated with the development of obesity and disturbance in glucose homeostasis. Here, using heterozygous BRD7 knockout and liver-specific BRD7 knockout mouse models, we report that reduced BRD7 levels lead to increased weight gain with little effect on glucose metabolism. On the other hand, upregulating BRD7 in the liver starting at an early age protects mice from gaining excessive weight and developing glucose intolerance and insulin resistance when challenged with a high-fat diet.

CRISPR/Cas9­mediated hypoxia inducible factor­1α knockout enhances the antitumor effect of transarterial embolization in hepatocellular carcinoma.

Transarterial embolization (TAE) is a palliative option commonly used for the treatment of advanced, unresectable hepatocellular carcinoma (HCC). However, patient prognosis in regards to overall survival has not improved with this method, mainly due to hypoxia­inducible factor­1α (HIF­1α)­induced angiogenesis and invasiveness. Thus, it is hypothesized that HIF­1α may be an ideal knockout target for the treatment of HCC in combination with TAE. Thus, in the present study, HIF­1α knockout was conducted in human liver cancer SMMC­7721 cells and a xenograft HCC model was established using a lentivirus­mediated CRISPR/Cas system (LV­Cas) with small guide RNA­721 (LV­H721). Furthermore, hepatic artery ligation (HAL) was used to mimic human transarterial chemoembolization in mice. The results revealed that HIF­1α was highly expressed in both HCC patient tissues and SMMC­7721­induced tumor tissues. The HIF­1α knockout in SMMC­7721 cells significantly suppressed cell invasiveness and migration, and induced cell apoptosis under CoCl2­mimicking hypoxic conditions. Compared with the control groups, HAL + LV­H721 inhibited SMMC­7721 tumor growth in orthotopic HCC and markedly prolonged the survival of HCC­bearing mice, which was accompanied by a lower CD31 expression (tumor angiogenesis) and increased apoptosis in the tumor cells. These findings demonstrated a valuable antitumor synergism in combining CRISPR/Cas9­mediated HIF­1α knockout with TAE in mice and highlighted the possibility that HIF­1α may be an effective therapeutic knockout target in combination with TAE for HCC treatment.

Monoglyceride lipase gene knockout in mice leads to increased incidence of lung adenocarcinoma.

Monoglyceride lipase (MGL) is a recently discovered cancer-related protein. The role of MGL in tumorigenesis remains to be fully elucidated. We have previously shown that MGL expression was reduced or absent in multiple human malignancies, and overexpression of MGL inhibited cancer cell growth. Here, we have generated the MGL knockout mice to further investigate the role of MGL in tumorigenesis in vivo. Our results indicate that MGL-deficient (MGL+/-, MGL-/-) mice exhibited a higher incidence of neoplasia in multiple organs, including the lung, spleen, liver and lymphoid tissues. Interestingly, lung neoplasms were the most common neoplastic changes in the MGL-deficient mice. Importantly, MGL-deficient animals developed premalignant high-grade dysplasia and adenocarcinomas in their lungs. Investigation of the MGL expression status in lung cancer specimens from patients also revealed that MGL expression was significantly reduced in the majority of primary human lung cancers when compared to corresponding matched normal tissues. Furthermore, mouse embryonic fibroblasts (MEFs) from MGL-deficient animals showed characteristics of cellular transformation including increased cell proliferation, foci formation and anchorage-independent growth. Our results also indicate that MGL deficiency was associated with activation of EGFR and ERK. In addition, pro-inflammatory molecules COX-2 and TNF-α were also activated in the MGL-deficient lung tissues. Thus, our results provide new insights into the novel role of MGL as an important negative regulator of EGFR, COX-2 and TNF-α. Accordingly, EGFR and COX-2/TNF-α activation/induction is expected to play important roles in MGL deficiency-driven lung tumors. Collectively, our results implicate the tumor suppressive role of MGL in preventing tumor development in vivo, particularly in context to the lung cancer, and highlight its role as a potential tumor suppressor.

PancanQTL: systematic identification of cis-eQTLs and trans-eQTLs in 33 cancer types.

Expression quantitative trait locus (eQTL) analysis, which links variations in gene expression to genotypes, is essential to understanding gene regulation and to interpreting disease-associated loci. Currently identified eQTLs are mainly in samples of blood and other normal tissues. However, no database comprehensively provides eQTLs in large number of cancer samples. Using the genotype and expression data of 9196 tumor samples in 33 cancer types from The Cancer Genome Atlas (TCGA), we identified 5 606 570 eQTL-gene pairs in the cis-eQTL analysis and 231 210 eQTL-gene pairs in the trans-eQTL analysis. We further performed survival analysis and identified 22 212 eQTLs associated with patient overall survival. Furthermore, we linked the eQTLs to genome-wide association studies (GWAS) data and identified 337 131 eQTLs that overlap with existing GWAS loci. We developed PancanQTL, a user-friendly database (http://bioinfo.life.hust.edu.cn/PancanQTL/), to store cis-eQTLs, trans-eQTLs, survival-associated eQTLs and GWAS-related eQTLs to enable searching, browsing and downloading. PancanQTL could help the research community understand the effects of inherited variants in tumorigenesis and development.

Seasonal variability of Protoceratium reticulatum and yessotoxins in Japanese scallop Patinopecten yessoensis in northern Yellow Sea of China.

This paper reports a toxic strain of Protoceratium reticulatum, its morphology, phylogeny, yessotoxins (YTXs) production and abundance in northern Yellow Sea of China from 2011 to 2015 was investigated. YTXs in hepatopancreas and edible parts of bottom sowing cultured Japanese scallop Patinopecten yessoensis in this sea area were determined weekly for 5 years. Other potential producers of YTXs, Gonyaulax spinifera and Lingulodinium polyedrum, were also investigated. Results revealed that Protoceratium reticulatum strain from the northern Yellow Sea belongs to a geographically widely distributed species. Motile cells of Protoceratium reticulatum contribute to YTXs in Japanese scallop, and G. spinifera may also be a potential contributor. Resting cysts of Protoceratium reticulatum, G. spinifera, and L. polyedrum in sediments were possibly important origins of YTXs in scallop cultured at sea bottom. YTXs in scallop decreased from 2011 to 2015, most toxins were concentrated in hepatopancreas, while a small portion in edible parts which was safe for consumption the whole year around.

Lipid Signaling in Tumorigenesis.

Lipids are important cellular building blocks and components of signaling cascades. Deregulation of lipid metabolism or signaling is frequently linked to a variety of human diseases such as diabetes, cardiovascular diseases, and cancer. It is widely believed that lipid molecules or their metabolic products are involved in tumorigenic inflammation and thus, lipids are implicated as significant contributors or even primary triggers of tumorigenesis. Lipids are believed to directly or indirectly activate growth promoting signals such as those involving LPA, insulin, IGF-1 and EGF to promote cancer cell growth. Cellular levels of certain lipids, including sphingosine-1-phosphate and ceramide, maintain a delicate balance between cell death and survival and alterations in their levels lead to unfavorable consequences including tumorigenesis. This article provides an overview of current knowledge that implicates lipids in tumorigenesis and explores the potential mechanisms that support a positive link between obesity and cancer.

First report on the detection of pectenotoxin groups in Chinese shellfish by LC-MS/MS.

Chinese shellfish samples were harvested from different locations along the Chinese coast. These shellfish were analyzed by liquid chromatography in combination with mass spectrometry to detect the following toxins: okadaic acid (OA), dinophysistoxins (DTXs), petenotoxins (PTXs), azaspiracids (AZAs), yessotoxins (YTXs), spirlides (SPXs) and gymnodimines (GYM). The results revealed the lipophilic toxin profiles varied with shellfish sampling locations. In addition to OA, GYM and YTX derivatives, PTX-2 and its derivatives were found for the first time in the following Chinese shellfish: Crassostrea gigas, Mactra chinensis and Mytilus galloprovincialis. The presence of GYM, YTXs, OA and PTXs in Chinese shellfish collected from regions where no previous record of DSP-neutral toxic compounds was reported. Serious efforts should therefore be made to conduct a phycotoxin monitoring program to detect the presence of lipophilic toxins in biological materials of marine origin, which may ensure that Chinese seafood products do not present a health risk. With respect to suspected carcinogenicity, further research on the distribution and concentrations of toxic compounds are needed, in order to carry out long-term risk assessments, particularly sub-acute and chronic toxicity tests associated with of lower doses.

[Characteristics of nitrogen cycling in farm systems in a small watershed of Three Gorges Reservoir Area, China].

In China, the agricultural activities are often carried out by single family, and the balance of nitrogen in every farm may have great effect on agricultural production and environment. A farm survey was carried out on the amounts of chemical fertilizers consumed, the amounts of food and feed purchased, and the livestock manures used in Zhangjiachong watershed of Three Gorges Reservoir Area for each farmer. According to the characteristics of nitrogen flow, farms in this watershed could be divided into 5 types: breeding specialized farms, orange-growing farms, tea-growing farms, vegetable-growing farms and traditional farms. Differences in environmental nitrogen load were observed among the five farm types in the following order: breeding specialized farms > vegetable-growing farms > orange-growing farms > tea-growing farms approximately = traditional farms, being (363 +/- 129), (355 +/- 127), (345 +/- 107), (152 +/- 60) and (151 +/- 73) kg x (hm2 x a)(-1) respectively by nitrogen. Nitrogen cycle for different farm fields showed that field surplus nitrogen ranged from (102 +/- 68) to (303 +/- 134) kg x (hm2 x a)(-1) among different farms. Among the five farm types, the field surplus nitrogen followed the order of vegetable-growing farms > citrus-growing farms > tea-growing farms > breeding specialized farms approximately = traditional farms, being (303 +/- 134), (262 +/- 100), (111 +/- 46), (102 +/- 68) and (103 +/- 67) kg x (hm2 x a)(-1). The nitrogen from farm life activities accounted for 28% of the total N load, while the field surplus nitrogen was 72%. It was estimated that field nitrogen consumption was the predominated nitrogen pollution source in the watershed. However, the farmer's daily life pollution should not to be neglected. Breeding specialized farms, vegetable-growing farms and orange-growing farms were the high potential pollution sources for the water environment in the watershed, and the major attention should be paid to these types of farms. Field survey on farm nitrogen cycle is of significance in evaluating the nitrogen flows in the agro-ecosystems and revealing the characteristics of nitrogen cycling in the region.