Integrated Bioinformatic Analysis and Validation Identifies Immune Microenvironment-Related Potential Biomarkers in Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, accompanied by cognitive and memory impairment, accounting for about 60% - 80% of dementia types. The pathogenesis of AD has not been clarified, and there is no effective therapy to prevent or treat AD. In this study, we aimed to identify the potential biomarkers involved in the brain immune microenvironment in AD. METHODS: AD datasets from GEO database were obtained to identify the differentially expressed disease-related genes (DEDRGs) in AD through weighted gene co-expression network analysis (WGCNA) and differential expression analysis. Functional Enrichment analysis was performed to explore the potential biological function of DEDRGs. The hub DEDRGs were identified through the protein-protein interaction (PPI) network. Furthermore, the CIBERSORT algorithm was employed to bulk gene expression profiles of AD to depict the immune microenvironment characteristics in AD. Pearson's correlation analysis was utilized to depict the correlation between each of immune cells and hub DEDRGs. RESULTS: A total of 27 DEDRGs were identified through WGCNA and differential expression analysis. Functional enrichment analysis of 27 DEDRGs indicated that chemokine signaling pathway was the most significantly enriched KEGG pathway, response to biotic stimulus was the most significantly enriched GO term, and most of DEDRGs were enriched into urinary system cancer in DO analysis. 6 hub DEDRGs, ANGPT1, CCL2, CD44, CXCR4, GJA1 and VCAM1, were screened through PPI network and all of them were up-regulated in AD. Immune infiltration analysis revealed that there were higher infiltration levels of T cells CD4 memory activated, T cells gamma delta, NK cells resting and macrophages M0, and lower infiltration level of NK cell activated in AD, and macrophages M2 owned the highest positively association with VCAM1 and CXCR4, but VCAM1 was statistically and negatively correlated to T cells CD8. CONCLUSION: Our study identified 6 hub DEDRGs, ANGPT1, CCL2, CD44, CXCR4, GJA1 and VCAM1, were statistically associated with immune infiltrating cells, and were significantly related to the pathological development of AD, which may provide a theoretical basis for developing potential biomarkers and implementing effective therapies against AD.

Probing

The isomer depletion of ^{93m}Mo was recently reported [Chiara et al., Nature (London) 554, 216 (2018)NATUAS0028-083610.1038/nature25483] as the first direct observation of nuclear excitation by electron capture (NEEC). However, the measured excitation probability of 1.0(3)% is far beyond the theoretical expectation. In order to understand the inconsistency between theory and experiment, we produce the ^{93m}Mo nuclei using the ^{12}C(^{86}Kr,5n) reaction at a beam energy of 559 MeV and transport the reaction residues to a detection station far away from the target area employing a secondary beam line. The isomer depletion is expected to occur during the slowdown process of the ions in the stopping material. In such a low γ-ray background environment, the signature of isomer depletion is not observed, and an upper limit of 2×10^{-5} is estimated for the excitation probability. This is consistent with the theoretical expectation. Our findings shed doubt on the previously reported NEEC phenomenon and highlight the necessity and feasibility of further experimental investigations for reexamining the isomer depletion under low γ-ray background.

Roles of circ_0000135/miR-140-3p/PDZK1 network in cervical cancer.

PURPOSE: To explore the effect of circ_0000135/miR-140-3p/PDZ domain containing 1 (PDZK1) on the occurrence and development of cervical cancer. METHODS: Clinical data were collected to verify circ_0000135/miR-140-3p/PDZK1 expression in cervical cancer. mRNA expressions of circ_0000135 and miR-140-3p were detected by real-time quantitative PCR. Correlation between circ_0000135 and miR-140-3p/miR-140-3p and PDZK1 was analyzed in vitro. Protein expression detection in cells was conducted by Western blot; while cell proliferation, invasion and cycle distribution by CCK8 assay, Transwell chamber assay and flow cytometry, respectively. Rescue and animal experiment were performed to verify the effect of circ_0000135/miR-140-3p/PDZK1 on cervical cancer. RESULTS: circ_0000135 and PDZK1 expressions were increased, while those of miR-140-3p were decreased in cervical cancer tissues and cells (both P < 0.05). sh-circ_0000135 group had decreased cell viability, arrested cells in G0/G1 phase, decreased CyclinD1 expression, inhibited cell migration and invasion; sh-circ_0000135 group showed reduced tumor volume, weight, and lower Ki67 expression (all P < 0.05). circ_0000135 had conserved target of miR-140-3p. There was a direct interaction between circ_0000135 and miR-140-3p. miR-140-3p might have direct interaction with PDZK1. sh-circ_0000135 and/or miR-140-3p treatment showed obviously decreased PDZK1 expression, decreased cell activity, arrested cells in G0/G1 phase, downregulated cell migration and invasion; sh-circ_0000135 and/or miR-140-3p mimic treatment showed obviously decreased tumor volume, tumor weight, and Ki67 expression (all P < 0.05). CONCLUSION: circ_0000135 may play an anti-tumor role on the progression of cervical cancer by sponging miR-140-3p to suppress the expression of PDZK1, providing a promising therapeutic target.

Advancement of Photospheric Radius Expansion and Clocked Type-I X-Ray Burst Models with the New

We report the first (in)elastic scattering measurement of ^{25}Al+p with the capability to select and measure in a broad energy range the proton resonances in ^{26}Si contributing to the ^{22}Mg(α,p) reaction at type I x-ray burst energies. We measured spin-parities of four resonances above the α threshold of ^{26}Si that are found to strongly impact the ^{22}Mg(α,p) rate. The new rate advances a state-of-the-art model to remarkably reproduce light curves of the GS 1826-24 clocked burster with mean deviation <9% and permits us to discover a strong correlation between the He abundance in the accreting envelope of the photospheric radius expansion burster and the dominance of ^{22}Mg(α,p) branch.

Direct Measurement of the Astrophysical

Fluorine is one of the most interesting elements in nuclear astrophysics, where the ^{19}F(p,α)^{16}O reaction is of crucial importance for Galactic ^{19}F abundances and CNO cycle loss in first generation Population III stars. As a day-one campaign at the Jinping Underground Nuclear Astrophysics experimental facility, we report direct measurements of the essential ^{19}F(p,αγ)^{16}O reaction channel. The γ-ray yields were measured over E_{c.m.}=72.4-344 keV, covering the Gamow window; our energy of 72.4 keV is unprecedentedly low, reported here for the first time. The experiment was performed under the extremely low cosmic-ray-induced background environment of the China JinPing Underground Laboratory, one of the deepest underground laboratories in the world. The present low-energy S factors deviate significantly from previous theoretical predictions, and the uncertainties are significantly reduced. The thermonuclear ^{19}F(p,αγ)^{16}O reaction rate has been determined directly at the relevant astrophysical energies.

Influences of low level of dietary calcium on bone characters in laying hens.

Cage layer fatigue (CLF), which is commonly caused by calcium deficiency in the feed, leads to loss of structural bone and increase of bone fragility. In order to investigate the influence of low-calcium diets on bone quality and strength, histopathology, and egg quality, 72 laying hens were randomly allocated to 2 groups at 22 wk of age and received low calcium and control calcium until 34 wk, respectively. Egg production, feed consumption, BW, and egg quality were measured throughout. Bone mineral density, bone biomechanical properties, and histomorphology of femurs and tibias were assessed after birds were sacrificed in 26, 30, and 34 wk. The results showed that low-calcium (1.5%) diets decreased BW, feed consumption, and egg production. The broken eggs rate increased, and the eggshell strength and thickness were lower in treated birds than those in control birds at 30 wk and 34 wk. Femoral and tibial bone index and bone mineral density were lower, cortical thicknesses were thinner, and bone length were shorter over time when birds are in a low-calcium diet than those in control birds. In biomechanical properties, the values of stiffness, Young's modulus, and breaking strength were lower in both femurs and tibias in low-calcium hens at 30 wk and 34 wk than those in bones of control hens. In histomorphology of bone, the cortex turned thinner and there were more cavities in cortex and cancellous bone; the trabecular bone network was fewer, thinner, less cohesive, and generally fragmented; and trabeculae were less well-connected in low-calcium birds. Some cell nuclei in cancellous bone disappeared, and vacuolation was observed in bone cells. There appeared osteoid in cortex bone and cancellous bone in tibias. It was concluded that low-calcium diets could facilitate the development of osteoporosis characterized by an increase of osteoid and loss of structural bone and decrease the values of bone quality and strength, accompanied with a decrease in egg production and egg qualities, which may elucidate the developing mechanism of CLF.

Dietary taurine supplementation enhances antioxidative capacity and improves breast meat quality of broiler chickens.

1. The aim of this study was to determine the effect of dietary taurine supplementation on growth performance, meat quality and antioxidant responses in breast muscle of broiler chickens.2. A total of 72 Arbor Acres broiler chickens (28-day-old) with similar body weight were randomly allocated into two groups, and fed either 0 g/kg (control; C) or 5 g/kg taurine-supplemented diets (TS) for 14 days.3. The results showed that TS had no effect on growth performance or chemical composition of breast muscle in broilers. The drip and cooking losses were significantly decreased (P < 0.05), and the pH24h of breast muscle were increased (P < 0.05) in the TS group. Meanwhile, broilers in the TS group exhibited significantly higher (P < 0.05) scavenging activities of superoxide and 2,2-diphenyl-1-picrylhydrazyl radicals, and lower (P < 0.05) contents of carbonyl, malondialdehyde, and 4-hydroxynonenal. TS increased (P < 0.05) total antioxidant capacity and superoxide dismutase activities. Moreover, TS significantly upregulated (P < 0.05) the mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1, NAD(P)H quinone dehydrogenase 1, superoxide dismutase, catalase, and glutathione peroxidase.4. These findings suggested that TS enhanced antioxidative capacity and improved breast meat quality of broilers via activating the Nrf2 pathway.

Impact of ketamine intervention for acute lung injury on RAGE and TLR9.

OBJECTIVE: The purpose of this study was to explore the benefits of ketamine intervention for acute lung injury (ALI) and its effects on the receptor for advanced glycation end-product (RAGE) and toll-like receptor 9 (TLR9). MATERIALS AND METHODS: Lipopolysaccharide (LPS, 3 mg/kg) was used to induce ALI rat model. Forty healthy Sprague-Dawley rats (6-8 weeks) were assigned into control, model, low ketamine (5 mg/kg), and high ketamine (50 mg/kg) groups. After 24 h, these rats were sacrificed and lungs were collected. RESULTS: The pathological score, lung W/D ratio, the percentage of leukocytes and epithelial in bronchoalveolar lavage fluids (BALF), the expression levels of RAGE, TLR9, and other inflammation markers in serum and lungs were significantly higher in the Model group, indicating a good ALI model. Ketamine intervention restored all these parameters, with more benefits in the High dose group. CONCLUSIONS: The high dose ketamine decreased the degree of ALI by inhibiting the expression of RAGE, TLR9, TNF-α, NF-κB, IL-6 and MPO in tissues.

[The safety and effect of transhepatic hilar approach for the treatment of bismuth type â ¢ and â £ hilar cholangiocarcinoma].

Objective: To compare the efficiency between the transhepatic hilar approach and conventional approach for the surgical treatment of Bismuth type Ⅲ and Ⅳ hilar cholangiocarcinoma. Methods: There were 42 consecutive patients with hilar cholangiocarcinoma of Bismuth type Ⅲ and Ⅳ who underwent surgical treatment at Department of Biliary-Pancreatic Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University from January 2008 to December 2013.The transhepatic hilar approach was used in 19 patients and conventional approach was performed in 23 patients.There were no differences in clinical parameters between the two groups(all P>0.05). The t-test was used to analyze the measurement data, and the χ(2) test was used to analyze the count data.Kaplan-Meier analysis was used to analyze the survival period.Multivariate COX regression analysis was used to analyze the prognosis factors. Results: Among the 19 patients who underwent transhepatic hilar approach, 3 patients changed the operative planning after reevaluated by exposing the hepatic hilus.The intraoperative blood was 300(250-400)ml in the transhepatic hilar approach group, which was significantly less than the conventional approach group, 800(450-1 300)ml(t=4.276, P=0.00 1), meanwhile, the R0 resection rate was significantly higher in the transhepatic hilar approach group than in the conventional approach group(89.4% vs. 52.2; χ(2)=6.773, P=0.009) and the 3-year and 5-year cumulative survival rate was better in the transhepatic hilar approach group than in the conventional approach group(63.2% vs. 47.8%, 26.3% vs. 0; χ(2)=66.363, 127.185, P=0.000). On univariate analysis, transhepatic hilar approach, intraoperative blood loss, intraoperative blood transfusion, R0 resection and lymph node metastasis were significant risk factors for patient survival(all P<0.05). On multivariate analysis, use of transhepatic hilar approach, intraoperative blood loss, R0 resection and lymph node metastasis were significant independent risk factors for patient survival(all P<0.05). Conclusion: The transhepatic hilar approach is the preferred technique for surgical treatment for hilar cholangiocarcinoma because it can improve accuracy of surgical planning, safety of operation, R0 resection rate and survival rate compared with the conventional approach.

[Effect of sodium thiosulfate on coronary artery calcification in maintenance hemodialysis patients].

Objective: To investigate the factors correlated to coronary artery calcification (CAC)in maintenance hemodialysis (MHD) patients and observe the effect of sodium thiosulfate (STS) on the progression of vascular calcification and its safety. Methods: Thirty-eight subjects from Fuzhou Genernal Hospital who underwent coronary artery CT scan using Philip's spiral CT were enrolled and the calcification degree was evaluated by CAC scores from December 2013 to December 2014. The hemodialysis patients were divided into CAC group (CAC scores>10, 27 cases) and non-CAC group (CAC scores≤10, 11 cases)according to the CT scan results.The differences of age, duration of dialysis, blood pressure and other hematological indices between the two groups were analyzed to investigate the factors correlated to CAC. Next, those with CAC (CAC scores≥50) received intravenous 0.18 g/kg STS (dissolved in 100 ml saline) in 30 minutes after each dialysis for 3 months (n=17, only 15 patients completed STS treatment) or received conventional treatment (n=10). Baseline data between the two groups before treatment had no significant statistical difference. All examination indices were evaluated before and after the treatment course. The changes of vascular calcification imaging, CAC scores, biochemical indices and bone mineral density were compared between two groups before and after the treatment. Besides, adverse reactions were observed during the treatment of STS. This study was approved by the Ethics Committee of Fuzhou General Hospital(2013No1). Results: Twenty-seven out of 38 patients (71.05%) had CAC, and the patients with CAC had significantly higher age, phosphate, the product of calcium and phosphate, intact parathyroid hormone (hPTH), hypersensitive C-reactive protein (hsCRP), and longer duration of dialysis (P=0.017, 0.038, 0.037, 0.012, 0.002, 0.037) and lower serum albumin (P=0.026) than patients without CAC.There was no significant statistical difference in the baseline characteristics. CAC score did not change significantly before and after treatment in the STS treatment group[1 045(47-12 734) vs 797(50-14 094), P=0.053], but increased significantly in the conventional treatment group[221(59-3 843) vs 174(50-3 369), P=0.021]. Difference of CAC score parameters before and after treatment showed statistically significant difference between the two groups[-67.5(-474-8) vs 52(-248-1 361) , P=0.004]. After STS treatment, level of hsCRP and HCO3- decreased (P=0.016 and P=0.020, respectively), and level of serum calcium increased (P=0.005). There was no significant statistical difference observed in iPTH, 25(OH)D, bone alkaline phosphatase (bALP), fibroblast growth factor 23 (FGF23) after STS treatment. Conclusion: STS treatment seems to be feasible, safe and may delay the rate of progression of vascular calcification, reduce inflammation in maintenance hemodialysis patients, but the adverse reactions needs further study.

Effects of zearalenone on calcium homeostasis of splenic lymphocytes of chickens in vitro.

Zearalenone (ZEA) is an estrogenic mycotoxin. It is produced by several Fusarium species and can contaminate food and feed. To investigate the role of calcium homeostasis in ZEA-induced toxicity of poultry and elucidate its cytotoxic mechanism, splenic lymphocytes isolated from chickens were exposed to ZEA (0-25 µg/mL) for 48 h. The intracellular calcium concentration ([Ca2+]i), pH, calmodulin (CaM) mRNA levels, and Na+/K+-ATPase activities and Ca2+-ATPase activities were detected by the fluorescent dyes Fluo-3/AM and BCECF/AM, quantitative real-time PCR, and chromatometry. Supernatant CaM concentrations were simultaneously detected by ELISA. As the ZEA exposure concentration increased, the [Ca2+]i and CaM mRNA levels gradually increased, while intracellular pH, CaM concentrations of supernatants, and intracellular Na+,K+-ATPase and Ca2+-ATPase activities gradually decreased in a dose-dependent manner. There were significant differences (P<0.05 or P<0.01) between the treatment groups and the control group. These results indicate that ZEA cytotoxicity arises by causing an imbalance in calcium homeostasis and intracellular acidification in lymphocytes.

Effects of zearalenone on IL-2, IL-6, and IFN-γ mRNA levels in the splenic lymphocytes of chickens.

Zearalenone (ZEN) is an estrogenic mycotoxin produced by several Fusarium species, which can contaminate food and feed. These compounds elicit a wide spectrum of toxic effects, including the capacity to alter normal immune function. In this study, the in vitro effects of the treatment of ConA-stimulated splenic lymphocytes with ZEN (0-25 µg/mL) were examined. ZEN modulates the expression of IL-2, IL-6, and IFN-γ. The IL-2 levels were up to fourfold higher (P < 0.05) compared with the levels in the control at toxin concentrations of 25 µg/mL after 48 h of treatment. The IL-6 levels were critically suppressed at this concentration; these changes were very statistically significant (P < 0.05). At lower ZEN concentrations (0.1, 0.4 and 1.6 µg/mL), the IFN-γ levels changed slightly; however at 6.25 and 25 µg/mL, the IFN-γ results reached statistical significance compared with the control levels (P < 0.05). These data suggest that ZEN has potent effects on the expression of chicken splenic lymphocytes cytokines at the mRNA level.

Effects of cold stress on nitric oxide in duodenum of chicks.

Animals may suffer from a variety of environmental stressors every day, among which is cold stress, which commonly exists in cold regions. The aim of this study was to investigate changes in antioxidative function and inducible nitric oxide synthase-nitric oxide (iNOS-NO) system activity in the duodenum of chicks as a reaction to cold stress. A total of 84 male chicks (15 d old) were randomly allocated to 12 groups (7 chickens/group). There were 1 control group and 5 treatment groups for acute cold stress and 3 control groups and 3 treatment groups for chronic cold stress. Antioxidative function was examined by superoxide dismutase (SOD), and oxidative damage was examined by malondialdehyde (MDA) detection. The iNOS-NO system activity was identified by NO content and NOS activity assay, and the transcription of iNOS mRNA was tested by fluorescence quantitative PCR. The results showed that under acute cold stress MDA level increased, the activity of NO in the duodenum fluctuated, and the activity of SOD and iNOS in the duodenum first increased and then decreased, whereas the expression of iNOS mRNA decreased. Under chronic cold stress the activity of SOD, NO, and NOS first decreased and then increased, whereas the MDA level and the expression of iNOS mRNA increased. The results indicated that both acute and chronic cold stress could cause duodenum oxidative stress and change in iNOS, which was related to the intestinal damage process.

Effects of cold stress on the messenger ribonucleic acid levels of peroxisome proliferator-activated receptor-{gamma} in spleen, thymus, and bursa of Fabricius of chickens.

This study was to investigate the expression trait of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) gene and the effect of cold stress on the mRNA levels of PPAR-gamma in spleen, thymus, and bursa of Fabricius of chickens. Eighty-four 1-d-old male chickens were randomly allocated to 12 groups (7 chickens per group). There was 1 control group and 5 treatment groups for acute cold stress and 3 control groups and 3 treatment groups for chronic cold stress. Chickens were maintained in our animal facility, kept under a 16L:8D cycle and temperature (30 +/- 2 degrees C), and given free access to standard chow and water. The cold stress was initiated when the birds were 15 d of age, with the duration of the acute cold stress being 1, 3, 6, 12, and 24 h, and the chronic cold stress was 5, 10, and 20 d, respectively. Cold stress temperature was 12 +/- 1 degrees C. Spleen, thymus, and bursa of Fabricius were collected for the assessment of the mRNA levels by real-time PCR after stress termination. The results showed that the PPAR-gamma gene is expressed in spleen, thymus, and bursa of Fabricius, and its expression level is different in different tissues and at different ages. Acute cold stress significantly decreased (P < 0.05) the mRNA levels of the PPAR-gamma gene of spleen and thymus in all treatment groups and significantly increased (P < 0.05) the mRNA levels of the PPAR-gamma gene of bursa of Fabricius in all treatment groups. Compared with the corresponding control groups, chronic cold stress resulted in a significant increase (P < 0.05) of the mRNA levels of the PPAR-gamma gene in spleen and a significant decrease (P < 0.05) of the mRNA levels of the PPAR-gamma gene in thymus and bursa of Fabricius. The results indicate that the PPAR-gamma gene is expressed in all 3 immune organs and has different expression traits. The magnitude and direction of change in PPAR-gamma gene expression differs with the type of cold stress applied and also varies by tissue.

Analysis of risk factors associated with outcomes in road traffic injury patients with acute lung injury.

Over 50% of road traffic injury (RTI) patients experience post-traumatic acute lung injury (ALI) and it is, therefore, extremely important to identify the risk factors related to the poor outcomes associated with ALI in RTI populations. This study evaluated 19 potential risk factors associated with the outcomes of ALI in 366 RTI patients. They were divided into two groups: a 'favourable outcomes group' and an 'unfavourable outcomes group'. The results indicated that the Acute Physiology and Chronic Health Evaluation II (APACHE II) score and the presence of gastrointestinal haemorrhage may help predict the outcomes of ALI in the early post-trauma phase of treatment. The duration of trauma and sepsis were shown to impact strongly on both the short- and long-term outcomes of ALI. Age (> 65 years) and disseminated intravascular coagulation in the early RTI phase were also independent risk factors for a poorer short- and long-term outcome in ALI.

Measurement of milk D-3-hydroxybutyrate with a simple UV spectrophotometer method: an alternative assay method.

The measurement of D-3-hydroxybutyrate (D-BHBA) in milk samples is an important tool for diagnosis of subclinical/clinical ketosis in dairy cows. We describe a simple UV spectrophotometric method for measuring the concentration of D-BHBA in milk of dairy cows. From two herds, 119 milk samples were taken from dairy cows. The standard-curve equation was y = 0.2582x + 0.0269 (R2 = 0.9967). The assay was highly specific with a minimum detection limit of 0.01 mmol/L and measuring range of up to 5 mmol/L. The recovery was between 99.35% and 100.22% and repeatability was 99.8%. The comparison between the spectrophotometric method and the fluorometric method revealed a close correlation (r = 0.9939). These results show that the spectrophotometric method can be successfully used as an alternative method to measure D-BHBA content in milk.

The impact of clinical risk factors in the conversion from acute lung injury to acute respiratory distress syndrome in severe multiple trauma patients.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are different stages of the same disease, the aggravated stage of ALI leading to ARDS. Patients with ARDS have higher hospital mortality rates and reduced long-term pulmonary function and quality of life. It is, therefore, important to prevent ALI converting to ARDS. This study evaluated 17 risk factors potentially associated with the conversion from ALI to ARDS in severe multiple trauma. The results indicate that the impact of pulmonary contusion, APACHE II score, gastrointestinal haemorrhage and disseminated intravascular coagulation may help to predict conversion from ALI to ARDS in the early phase after multiple-trauma injury. Trauma duration, in particular, strongly impacted the short- and long-term development of ALI. Being elderly (aged > or = 65 years) and undergoing multiple blood transfusions in the early phase were independent risk factors correlated with secondary sepsis, deterioration of pulmonary function and transfusion-related acute lung injury due to early multiple fluid resuscitation.

Effects of cold stress on the messenger ribonucleic acid levels of corticotrophin-releasing hormone and thyrotropin-releasing hormone in hypothalami of broilers.

In this study, seventy 1-d-old male broiler chicks were randomly allocated to 10 groups to investigate the effect of cold stress on the messenger RNA (mRNA) levels of corticotrophin-releasing hormone (CRH) and thyrotropin-releasing hormone (TRH) in hypothalami. The chicks were maintained at 30 +/- 2 degrees C temperature and given free access to standard chow and water. Until 15 d old, the 6 treatment groups were maintained at 12 +/- 1 degrees C. Hypothalami were collected for the assessment of the mRNA levels by semiquantitative reverse transcription-PCR after stress termination. Cold stress significantly decreased the mRNA levels of CRH in 6 and 12 h treatment groups and significantly increased the mRNA levels of TRH in 1, 6, and 12 h treatment groups during acute cold stress. There were no significant differences in the mRNA levels of CRH and TRH among different control groups during chronic cold stress. However, chronic cold stress resulted in a significant increase of the mRNA levels of CRH and a significant decrease of the mRNA levels of TRH compared with the corresponding control groups. The results indicate that the 2 genes show different response to cold stress at the mRNA levels, and on the other hand, the different degree of cold stress also produces different effects on the identical gene.