Endocrine dysfunction is not uncommon complication in patients with transfusion-dependent thalassemia and is thought to occur as a consequence of excessive iron overload. The primary objective of this study is to determine the frequency of adrenal insufficiency in patients with thalassemia major and thalassemia intermediate.This cross-sectional study was done at the Shiraz University of Medical Sciences, Shiraz, Southern Iran, in 2013. One hundred and ninety patients were divided into two groups; thalassemia major(TM) and thalassemia intermediate (TI) groups. We measured 8 AM serum cortisol, ACTH and ferritin concentrations in all patients.The mean age of the TM and TI group were 22.5±5.7 and 23.8±6 years, respectively. 90 patients (47.4%) were splenectomized, 34 (36.2%) with TM and 56 (58.2%) with TI (p :<0.001). The median and interquartile range of serum ferritin levels were 2184±3700 ng/ml and 437±443ng/ml in TM and TI respectively (p< 0.001). Three patients with TM (1.6%) had low basal cortisol and ACTH levels. However, their cortisol response to ACTH stimulation was normal.Low basal concentrations of cortisol and ACTH occurred in 1.6% of our adolescents young adult patients with TM suggesting a central defect in cortisol secretion at the basal state. However, cortisol response to standard – dose ACTH was normal in all patients with TM and TI. Three main forms have been described: thalassemia major(TM) thalassemia intermedia (TI) and thalassemia minor. Individuals with TM usually present within the first two years of life with severe anemia, requiring regular red blood cell (RBC) transfusions.1The life expectancy of children and adolescents with TM and TI has markedly increased, due to improvement of the quality and techniques of blood transfusion and new development of effective oral iron chelators.7Iron overload is toxic to parenchymal cells because it generates free radicals and induces oxidative stress causing damage to biomolecules, including lipids, proteins, and DNA.3Therefore, regular surveillance of these patients is essential for early detection and management of possible complications, such as: heart failure and arrhythmias, chronic liver diseases, endocrine problems , growth failure, osteoporosis and thrombophilia.We assessed basal and stimulated cortisol secretion in relation to the degree of iron overload in 190 consecutive adolescent patients with TM and TI.This study included all patients who were referred to the thalassemia clinic, Hospital and Outpatient Clinic, affiliated with the Shiraz University of Medical Sciences. The patients divided into two groups according to their diagnosis: TM and TI. All patients with TM were on regular blood transfusion every 2–4 weeks. TI patients were transfusion independent.The diagnosis of thalassemia was based on complete blood count (CBC), Hb electrophoresis and clinical history.Inclusion criteria were all TM and TI patients aged 10 years and above.Exclusion criteria included: history of any infection or stress such as surgery in the past four weeks, congestive heart failure, thyroid dysfunction, hepatic impairment, uncontrolled diabetes mellitus, and/or taking any medications that may have an effect on adrenal function. Four-hundred-ninety TM and 196 TI patients over 10 years old were eligible. From these patients, 190 consecutive patients were enrolled in our study.A venous blood sample was collected from all patients at 8 AM for measuring serum ferritin, cortisol, and ACTH concentrations.A 2.5 mL sample of fasting venous blood withdrawn from the participants was added to CBC tube contained EDTA anticoagulant. Plasma, centrifuged immediately, was preserved in −20 °C in the freezer, then carried by cold box to the laboratory to test ACTH level. Furthermore, 3 mL clot blood was also withdrawn from each patient. The serum was immediately separated without hemolysis, preserved in −20 °C in the freezer and then, carried by cold box to the laboratory to test cortisol level.The ACTH and cortisol levels were measured by use of an electrochemiluminescence immunoassay (ECLIA) method . Normal range of cortisol at 7–10 A.M. was 6.2–19.4 μg/dL and 2.3–11.9 μg/dL at afternoon, while that for corticotropin (ACTH) was 7.2–63.3 pg/mL.Analysis of the results was considered as follows:i.e. > 100 pg/mL) suggested primary AI.20A low plasma cortisol levels (< 6.2 μg/dL – 10th percentile), measured between 8.00–9.00 am, in the face of high ACTH levels , in the presence of low cortisol level (< 6.2 μg/dL – 10th percentile), suggested secondary AI.21Failure to increase cortisol levels above 18 μg/dL at 30–60 minutes post corticotropin intravenous (i.v.) stimulation test indicated adrenal insufficiency.22μg synthetic ACTH 1–24, cosyntropin, tetracosactin, Synacthen, as i.v. bolus followed by measurement of serum cortisol 30 and 60 min after the injection (CINACT ampoule).The ACTH stimulating test was performed with 250 Fasting serum ferritin was measured by Electro Fluorescent Assay (ELFA) method, Mini Vidas machine .Statistical analyses were performed with SPSS Software . Test of normality was performed by Shapiro-Wilk Test for serum ferritin, ACTH, and cortisol levels. Mann-Whitney test was used for comparison of serum ferritin, ACTH, and cortisol levels between TM and TI patients. Student t-test was used for comparison of age between the two groups. Comparison of qualitative data was done by Chi-square test. A p value <0 .05 was considered as statistically significant.The study was performed in accordance with provisions of the Declaration of Helsinki and Good Clinical Practice guidelines and was approved by Medical Ethics Committee of the Shiraz University of Medical Sciences. Written informed consent was obtained from the patients or their parents.Ninety-four TM and ninety-six TI patients were investigated. The mean age of the TM and TI group were 22.5±5.7 and 23.8±6 years, respectively. No significant age difference was found between the two groups (p:0.15).In the TM group, 55 subjects (58.5%) were females and 39 males (41.5%). In the TI group, 41 (42.7%) were women. All patients with TM have been on iron chelators with deferoxamine (DFO) (70%), combined DFO and deferiprone (DFP) (10%) or deferasirox (DFX) (20%).The compliance to DFO and DFX iron chelators were fair and good, respectively. Among the study subjects, 90 (47.4%) had splenectomy, 34 (36.2%) with TM and 56 (58.2%) with TI, respectively (p:0.001). The median and interquartile range of serum ferritin levels in TM and TI groups were 2184±3700 ng/ml and 437±443 ng/ml respectively (p< 0.001). In general, an early morning (8 am) plasma cortisol level lower than 6.2 μg/dL – (10th percentile) is suggestive for primary AI, whereas a value higher than 15 μg/dL makes the diagnosis highly unlikely. Therefore, we performed an ACTH stimulation test only in 3 patients  with low basal levels of cortisol .Patient 1 was on treatment with DFO and had dilated cardiomyopathy with normal left ventricular ejection fraction (LVEF). Patient 2 has short stature and pubertal delay with mild systolic and diastolic cardiac dysfunction. He has been on iron chelation therapy with DFO plus DFP and L-thyroxine therapy for primary hypothyroidism. Patient 3, while on treatment with DFX, presented with diabetes mellitus, dilated cardiomyopathy and normal LVEF. None of the 3 TM patients were on sex hormone replacement therapy or had received corticosteroids treatment.Patients 2 and 3 had poor compliance with iron chelation therapy.μg/dl in normal people.The plasma cortisol response at 60 minutes post ACTH injection should reach ≥ 18 All patients with TI had normal basal serum ACTH and cortisol concentrations.The median of ACTH concentrations did not differ significantly between patients with TM and TI ; but, this may result in a higher false-positive rate. In addition, the lack of a commercially available 1-μg dose may represent another potential error.Although 250 mcg ACTH test (owing to a massive dose of ACTH) is not sensitive for diagnosis of partial secondary adrenal insufficiency, the basal cortisol value during the standard-dose test has in most clinical situations a diagnostic accuracy close to that of a low-dose of ACTH test.38Our 3 TM patients with low basal secretion of cortisol and ACTH (secondary AI) were asymptomatic. It is well known that the generalized weakness; tiredness and fatigability are, in the early phase of the adrenocortical insufficiency, transient and appearing only after increased physical or psychical stress. They become gradually more intensive and in more advanced stages of chronic adrenocortical insufficiency.36A standard-dose of 250 μg cosyntropin is useful for excluding primary AI in those with low cortisol level during screening. A cortisol response higher than 18 μg/dL at 30 minutes after a standard-dose ACTH confirms adequate cortisol secretion. Most individuals with normal adrenal function achieve much higher cortisol levels at 60 minutes after cosyntropin injection.It should be also noted that a large variety of total cortisol assays is commercially available with considerable differences in specificity, sensitivity, accuracy, precision and reproducibility of many of these assays. Some of these assays appear to overestimate or underestimate actual cortisol levels and, as such, hamper the correct diagnosis of relative AI.39These findings raise several interesting, relevant issues. Hematologists need to be more vigilant about endocrine complications in thalassemia. Whenever possible, cases of AI should be interpreted and managed in consultation with a pediatric or adult endocrinologist, because adrenal insufficiency usually is not in thalassemia an acute event but results from gradual decline in pituitary function, due to iron overload.Deficiency of cortisol level (secondary AI) with intact renin-angiotensin-aldosterone system can cause adrenal crisis if it is severe or patients are in acute illness.Therefore, we recommend a systemic testing of the adrenal function prior infection, trauma, surgical intervention or other stress and at regular yearly interval in TM patients with iron overload especially in those with other endocrinopathies.For patients with proven AI, family education and stress steroids during times of illness, injury or surgery are imperative help reduce the morbidity and mortality associated with this serious complication. Recovery from other endocrinopathies may be possible in thalassemia by using intensive iron chelation therapy; however, this issue has not been studied in cases of AI.Our findings support the small prevalence of AI in adolescents and young adults with TM. All patients with TI have normal basal cortisol and ACTH secretion.An early morning (8 am) plasma cortisol level lower the 10° percentile is suggestive for adrenal insufficiency, whereas a value higher than 15 μg/dL makes the diagnosis highly unlikely. Cortisol levels of intermediate range may be seen in patients with primary, secondary or tertiary adrenal insufficiency. In those cases and in patients with low basal cortisol and standard response to ACTH a strict collaboration with the endocrinologist is needed.


Millettia macrophylla is used traditionally to treat menopause related symptoms. This plant was shown to exhibit estrogenic effects in vitro on human embryonic kidney cells and in vivo on ovariectomized rats. The present study aimed at elucidating underlying mechanisms by which M. macrophylla induced its estrogenic effects. To accomplish our goal, kidney Hek293T cells transiently transfected with estrogen alpha or beta receptor expression plasmids were cotreated with a pure antiestrogen ICI 182,780 and the dichloromethane or methanol soluble fractions of M. macrophylla. To follow up, we cotreated ovariectomized rats with both extracts and ICI 182,780 for 3 days in the classical uterotrophic assay. Animals were then sacrificed and the uterine wet weight, total protein levels in uteri, uterine, and vaginal epithelial heights, and mammary gland were assessed. In vitro, the results suggested that the induction of the estrogenic activity by M. macrophylla is due to the binding of its secondary metabolites to ERα and ERβ. In vivo, the cotreatment of extracts and ICI 182,780 significantly abrogated the biological responses induced by the extracts alone. Taken together, these results indicate that the active principles of M. macrophylla induce their beneficial effects on menopausal symptoms by activating the ERs. Millettia macrophylla, a Cameroonian medicinal plant used in the treatment of menopausal symptoms.Throughout the ages, plants have been the major source of medication for the treatment of diverse diseases and continue to be a major source of primary health care for today's populations in most areas of the world . The medMillettia macrophylla, also known as Millettia aboensis, is used to alleviate sexual transmitted diseases, as well as some symptoms related to menopausal complaints, like other species of Millettia genus such as M. conraui, M. drastica, M. griffoniana, M. zechiana, M. barteri, and M. dura -estra-1,3,5(10)-triene-3,17diol) and 17 Millettia macrophylla were harvested at the former reserve of Ediki  in January 2010. Our botanical sample was identified and authenticated by Mr. Victor Nana, botanist at the National Herbarium of Cameroon (HNC) in Yaounde (voucher specimen number 49654/HNC). The well-dried and pulverized stem bark of M. macrophylla (2500 g) was macerated in 10 liters of dichloromethane (DCM) for 72 h at room temperature, and 25 g of extract was obtained after filtration and evaporation of the solvent using a rotary evaporator in vacuum. The residue insoluble material was thereafter steeped in 10 liters of methanol for 72 h (MeOH), filtered through Whatman paper N°4, and concentrated to give 53.4 g of methanol extract of M. macrophylla. For phytochemical analysis of these extracts, the LC analyses were performed on a Thermo Finnigan HPLC system consisting of a vacuum degasser ), a quaternary pump, an autosampler, and a DAD (diode array detector)  as previously reported [The stem barks ofRattus norvegicus [ ab libitum. Animal housing and experiments were carried out following the guidelines of the institutional Ethics Committee of the Cameroon Ministry of Scientific Research and Technological Innovation, which has adopted the guidelines established by the European Union on animal care (CEE Council 86/609).Fifty female Wistar rats (rvegicus ) aged 3  M. macrophylla extracts are able to transactivate the estrogen receptors α and β, in human embryonic kidney 293T cell line (HEK293T) [α receptor expression plasmid (200 ng—HEK293T-ERα cells) or the estrogen β receptor expression plasmid (200 ng—HEK293T-ER β cells), together with the double estrogen response element (ERE) fused to a luciferase reporter (250 ng—(ERE)2-tk-Luc) plasmid and β-galactosidase reporter plasmid using Lipofectamine Reagent. They were then treated with different concentrations of M. macrophylla extracts alone or in combination with ICI 182,780 (500 nM) for 24 h. Cells treated with E2 (10 nM) served as positive control. Luciferase reporter gene assays were performed using a commercial kit  according to the manufacturer's instructions. Luciferase activity was measured and normalised against β-galactosidase activity determined by using the 2-nitrophenyl β-D-galactopyranoside (ONPG) method . Each experiment was performed in at least duplicate and repeated three times.In a previous study, we have found thatHEK293T) . In the  in vivo with M. macrophylla extracts is mediated through ERs. All substances  used in this experiment were dissolved in olive oil. The injection volume was 0.3 mL subcutaneously.This experiment aimed to elucidate whether the estrogenic activity observedμg/kg BW/d. Three groups received either DCM extract at the dose of 300 mg/kg or MeOH extract at the doses of 100 and 300 mg/kg BW/d. The last remaining five groups were treated with the same substances and at the same doses combined with ICI 182,780 at the dose of 300 μg/kg BW/d. After the 3-day treatment, animals were sacrificed; the uterine weight, total protein levels in uteri, uterine, and vaginal epithelial heights, and mammary gland were assessed.Fifty female Wistar rats were ovariectomized. Fourteen days after endogenous hormonal decline, animals were randomly distributed into 10 groups of 5 rats each. Five animal groups were treated as follows. The negative control received the vehicle (olive oil) and the positive control was treated with estradiol benzoate (E2B) at the dose of 0.75 Total uterine protein levels were determined in uteri using colorimetric methods described by Gornall et al. .The histomorphological parameters of mammary gland, uteri, and vagina were assessed as described previously . t-test was used for statistical comparison between different control and treated groups for in vivo and in vitro experiments, respectively. The significance of the difference was fixed at P < 0.05.The data from each experimental group were expressed as mean ± SEM. One-way analysis of the variance (ANOVA) followed by Dunnett's test for multiple comparisons and the Student'sP < 0.001) blocked the transactivation induced by E2 alone in both HEK-ERα and HEK-ERβ (α and ERβ showed a significant inhibition (P < 0.001) with M. macrophylla extracts and ICI 182,780  totally abrogated (P < 0.001) the increase of uterine wet weight induced by E2B (P < 0.01) decreased uterine total protein level (P < 0.01) decrease in the uterine wet weight. MeOH extract did not increase the uterine wet weight. However, it did significantly (P < 0.01) increase total protein levels and uterine epithelial height. ICI 182,780 significantly blocked the increase of these aforementioned parameters when cotreated with DCM and MeOH extracts  increased the vaginal epithelial height by 266.71% as compared with control (OVX). Treatment with M. macrophylla extracts increased the vaginal epithelial height (P < 0.01) at all tested doses. Cotreatment of M. macrophylla extracts with ICI 182,780 (Faslodex: 300 μg/kg) significantly (P < 0.01) abrogated the increase of vaginal epithelial height induced by E2B or M. macrophylla extracts treatment alone  which is the most potent estrogen receptor antagonist of the class of 7α-alkylamide that has been approved for treatment of postmenopausal breast cancer patients who fail to respond to tamoxifen therapy [ M. macrophylla extracts-induced transactivation of both α and β estrogen receptor subtypes in vitro. These results suggest that the induction of transactivation by M. macrophylla extracts is due to the binding of its secondary metabolites to ERs, thus corroborating the observation of many authors who also observed the inhibition of ERs (α and β) transactivation in cellular systems [f female , osteopof female , and malf female . We hypo therapy . It was  therapy . In our  systems –27. in vitro is not transferable in vivo because of the complexity of this later system, we also antagonized the plant extracts with ICI 182,780 in vivo in ovariectomized rats. The results showed that cotreatment of the extracts with ICI 182,780 significantly abrogated the increase of uterine wet weight, uterine, and vaginal epithelial height and uterine total protein levels after a 3-day treatment. Moreover, ICI 182,780 inhibits the effect of different treatments  on mammary gland. These results corroborate with known ICI 182,780 effects. In fact, ICI 182,780 blocks endometrial proliferation and uterine growth in rats and inhibits growth of breast tumor cells both in cell culture and in vivo [ M. Macrophylla extracts while cotreated with ICI lose their effects probably due to a low level of ERs available to exhibit the estrogen-like effects obtained in the absence of ICI. This confirms that the active principles of M. macrophylla bind directly to ERs or at least are part of the regulatory complex at the promoter region of ERs target genes.Based on the fact that any beneficial effect obtained in vivo , 28. ICI in vivo . Therefo M. macrophylla has exhibited estrogen agonistic effects in the range of some well-characterized phytoestrogens; and these estrogenic effects were lost in the presence of ICI 182,780. Therefore, Millettia macrophylla should be classified as source of phytoestrogen like a number of plants consumed by humans such as soy, flax, red-clover, hops, citrus, grape, block cohosh, dong quai, ginseng, licorice, and wild Mexican yam [Phytoestrogens have been found to bind the estrogen receptor with an affinity 10-fold to 1000-fold lower than estrogen . M. macrican yam . Our resican yam –34. M. macrophylla extracts may be due to one or more of its constituents that bind to ERs to induce their biological effects. More in vitro and in vivo studies will be needed to further characterise the molecular mechanism of action that underpins M. macrophylla extracts estrogenic activities. Nevertheless, M. macrophylla should be classified as a novel source of potent phytoestrogen.The estrogenic activities of


Fusarium oxysporum f. sp. cubense infection on banana is devastating banana plantations worldwide. Biological control has been proposed to suppress Panama disease, though the stability and survival of bio-control microorganisms in field setting is largely unknown. In order to develop a bio-control strategy for this disease, 16S rRNA gene sequencing was used to assess the microbial community of a disease-suppressive soil. Bacillus was identified as the dominant bacterial group in the suppressive soil. For this reason, B. amyloliquefaciens NJN-6 isolated from the suppressive soil was selected as a potential bio-control agent. A bioorganic fertilizer (BIO), formulated by combining this isolate with compost, was applied in nursery pots to assess the bio-control of Panama disease. Results showed that BIO significantly decreased disease incidence by 68.5%, resulting in a doubled yield. Moreover, bacterial community structure was significantly correlated to disease incidence and yield and Bacillus colonization was negatively correlated with pathogen abundance and disease incidence, but positively correlated to yield. In total, the application of BIO altered the rhizo-bacterial community by establishing beneficial strains that dominated the microbial community and decreased pathogen colonization in the banana rhizosphere, which plays an important role in the management of Panama disease.Panama disease caused by  Musa spp.) is a widely cultivated cash crop in tropical and subtropical climatic regions. This disease, caused by the infection of the soil-borne hyphomycete Fusarium oxysporum f. sp. cubense (FOC), leads to banana rot and death and is known as vascular fusarium-wilt or Panama disease5Banana  and fungicide application79Trichoderma, Actinobacteria, and Bacillus. These have been identified as dominant microbial groups in compost, serving to inhibit pathogen growth and confer plant protection from pathogen infection1922232425A disease suppressive soil is described as soil present in a severely disease infested field that has the potential to decrease pathogenesis1416Fusarium wilt disease in banana plantations. The following specific questions were addressed to further support our concept of manipulating the rhizosphere microbiome in order to suppress soil-borne disease: (i) Is the health status of the banana plant associated with the composition of the soil microbial community? (ii) What is the microbial community in disease-suppressive soil? (iii) Can BIO suppress Panama disease by manipulating the rhizosphere microbial community?In this study, we describe how a formulated bioorganic fertilizer specifically suppresses banana Panama disease. Comparisons of microbial community structure between disease-suppressive and diseased soils, beneficial strain isolation, and a field study implementing BIO application are investigated. We also demonstrate that BIO application establishes a beneficial taxa-dominated microbiome in the banana rhizosphere, which plays an important role in the management of Bacillus was the most dominant bacterial group in the disease suppressive soil followed by Rhizobium, Bhargavaea, Pseudolabrys, and Sinorhizobium. In comparison, the diseased soil harbored Acidovorax, Filimonas, Methylophilus, Oxalicibacterium, and Sphingomonas among the top 5 abundant genera . Clustering at 3% nucleotide dissimilarity resulted in 180 OTUs that were classified into 7 phylum, 16 classes, and 87 genera. t genera .Fusarium oxysporum f. sp. cubense (FOC) on potato dextrose agar plates. The isolate (NJN-6) with the highest suppression towards FOC was selected and identified as a member of the Bacillus amyloliquefaciens group , based on a maximum likelihood 16S rRNA based phylogenetic tree . This was applied to suppress Panama disease in a field with a 65% banana death rate during the last growing season. Overall, BIO succeeded in controlling Panama disease with a promotion of banana yield  decreased to 14%, resulting in a biological control percentage of 68.5% and a significantly increased unit yield (36 kg/plant)  and plot yield (927 kg/plot) ) (Liquid fermentations of strain na yield . For thena yield . After B< 0.05)) .F. oxysporum f. sp. cubense (FOC) colony forming units (CFU) in the banana rhizosphere was quantified by plating soil series dilutions on Komada’s selective medium K2 Petri plates for all samples collected in 2010. The FOC colonization was significantly lower in the disease-suppressive, compared to diseased soils  (p < 0.05). Pearson correlations revealed that FOC colonization was highly correlated to plant disease incidence , unit yield  and plot yield .The number of  < 0.05) . All disAfter quality trimming, the number of sequences obtained using pyrosequencing ranged from 7087 to 11727 per sample. Rarefaction curves were not saturated in all cases . After rp < 0.05), health status  and the treatment × health status interaction term . Overall sample dispersion was not significant . Multiple regression tree analyses resulted in a primary division of the bacterial community according to treatment, followed by health status  followed by Gemmatimonas, Ohtaekwangia, Acidobacteria-Gp1, Steroidobacter, Bacillus, Streptophyta, Flavobacterium, and Dongia , banana unit yield  and plot yield .Ordinations illustrated that all samples could be separated according to both health status and treatment . PERMANOh status . Acidobad Dongia . OverallBacillus (reads: CKH/BIOH = 1.19%/3.02%). Only one shared-OTU, which was classified as Bacillus, was observed in BIOH and CKD (reads: BIOH/CKD = 0.21%/0.21%), and in CKD and CKH (reads: CKD/CKH = 0.21%/2.13%). Pearson correlations revealed that Bacillus colonization was positively correlated to banana unit yield  and plot yield , but negatively correlated to disease incidence  and FOC abundance in banana rhizosphere .After BIO application (BIOH), healthy samples shared more OTUs with the disease suppressive soil (CKH) than diseased samples in the control treatment (CKD), and more shared OTUs were classified as Bacillus. In total, 48 OTUs were shared by BIOH and CKH samples (reads: BIOH/CKH = 7.67%/9.96%), 40 OTUs were shared by all the CKD and CKH samples (reads: CKD/CKH = 7.93%/8.85%), while 83 shared OTUs (reads: BIOH/CKD = 8.43%/11.89%) were observed in all the BIOH and CKD samples. The most abundant bacterial taxa of BIOH and CKH were Firmicutes and Actinobacteria were more abundant in CKH, while Verrucomicrobia, Bacteroidetes, Proteobacteria, and Gemmatimonadetes were more abundant in CKD . Bacillus was the top contributor  to the community differences between CKH and CKD, followed by Acidobacteria GP6, Gemmatimonas, Flavobacterium, Acidobacteria GP1, Rhodanobacter, Ohtaekwangia, Sphingobium, Steroidobacter, and Burkholderia  and diseased (CKD) soils within the control treatment was compared to elucidate the nature of the banana rhizosphere disease-suppressive microbiome. The microbial communities of CKH and CKD differed from the phylum level to genus level. At the phylum level holderia .Bacillus, while only one OTU (0.21% of reads) of 208 shared by all CKD samples was classified as Bacillus. This ‘core microbiome’ from the disease-suppressive soil was dominated by Bacillaceae, followed by Hyphomicrobiaceae, Gaiellaceae, Bradyrhizobiaceae, Sphingomonadaceae, Rhodospirillaceae, Paenibacillaceae, Nitrospiraceae, and Streptomycetaceae.In total, only 121 OTUs (21.44% of reads) were shared by all disease-suppressive samples (CKH), of which 56 OTUs (6.45% of reads) were classified as Fusarium oxysporus f. sp. cubense (FOC) infection resulted in larger populations of FOC colonizing the rhizosphere that, through direct agonistic/antagonistic interactions, may serve to alter the composition of the rhizosphere community. However, it should be noted that pathogenic or non-pathogenic FOC could not be separately quantifiedAn understanding of the composition of the rhizosphere microbiota in relation to plant health is essential for the prevention and treatment of plant diseases. In this context, we compared the rhizosphere bacterial community compositions of healthy plants with plants infected by Panama disease. Banana plant health status was associated with the composition of the rhizo-bacterial community in each treatment as revealed by beta-diversity comparisons and mantel tests. Two mechanisms may be responsible for these changes. First, plant health status may affect root exudates, which in turn have a selective effect on the rhizosphere bacterial community27Bacillus in the healthy soil and a negative correlation to FOC colonization in banana rhizosphere. Bacillus is a well-known plant growth promoting (PGPR) rhizo-bacterial group that has been shown to produce antifungal compounds and phytohormones in disease-suppressive soilsBacillus comprised one-third of the “core microbiome” in disease-suppressive samples. This is in contrast to a previous study on the microbial community of potato common scab-suppressive soil in which Bacillus was more abundant in disease-conducive soilB. amyloliquefaciens from other Bacillus species with up to only 54.4% of sequences classified at the genus level at 50% confidence.As a first step to disease management, the microbial community composition of a native disease-suppressive soil with Panama severe disease was assessed with clone libraries and pyrosequencing in order to identify key microbial taxa associated with suppression. Clone sequencing and pyrosequencing results agreed, with a high relative abundance of Burkholderia, Gaiellaceae, Paenibacillaceae, and Streptomycetaceae. Both Gaiellaceae and Streptomycetaceae belong to Actinobacteria, reported to be a dominant group in soils resistant to fusarium-wilt of bananaet al.Burkholderia sp. could colonize the surface of FOC hyphae and that the fungal macrospores resulted in mycelial deformation with terminal and intercalary swelling. Paenibacillaceae sp. has also been found to produce fusaricidins, an antifungal compounds group that suppresses F. oxysporum f. sp. neviumB. amyloliquefaciens NJN-6 as a potential biocontrol agent against Panama disease. Gram positive, spore-producing Bacillus strains are ideal for commercial production and mixing in organic fertilizers and soil, due to their survival characteristicsOther potentially beneficial groups were associated with disease suppression such as Fusarium wilt suppression and that pathogen abundances actually increasedB. amyloliquefaciens NJN-6 fermentation with an amino acid fertilizer and compost. In this study, BIO was expected to complement the establishment of a beneficial strain-dominated microbial community in the banana rhizosphere. Although there was a significant difference in overall microbial community structure between healthy and diseased samples, the rhizo-bacterial community was most affected by the addition of BIO. Multiple regression tree analyses confirmed that the BIO influence on the microbiome was able to overcome the effect of plant disease status. As a source of nutrition for bacteria involved in biocontrol, organic fertilizer may aid in antagonistic colonization in the banana rhizosphere by increasing soil microbial biomass and activity36Cucumis melo melonPrevious research has shown that compost application had no effect on B. amyloliquefaciens NJN-6 dominated BIO appeared to initiate microbial community shifts in the banana rhizosphere, leading to decreased FOC abundance, similar to results found in relation to watermelonet al.Fusarium oxysporum f. sp. niveum.The seedlings used in this study were generated through tissue culture and the germ-free seedling roots first came into contact with BIO during the nursery pot period, followed by the FOC infected field soil after transplantation. However, the field soil was also supplemented with BIO before the transfer of banana seedlings from the pot to the field. This likely resulted in an enhanced colonization of beneficial bacteria that contributed to the persistent beneficial effectsB. amyloliquefaciens colonization of the banana rhizosphere may be due to plant growth promotion. Specifically, strain NJN-6 has been found to produce indole 3-acetic acid (IAA) that enhances root system development, leading to increased root exudate production and a larger rhizosphere habitatBacillus dominated rhizosphere bacteria community was established that acted through direct and/or indirect antagonism against the pathogen, resulting in increased crop yields and decreased disease incidence.The suppression of Panama disease by Bacillus was the most abundant bacterial group in disease suppressive soils in the banana rhizosphere and that plant health status was correlated to soil microbial community composition. Field results demonstrated that the practical application of the BIO is a promising strategy for disease control and growth promotion in banana production. In addition, BIO application altered the rhizo-bacterial community by establishing beneficial strains that dominated the microbial community and decreased pathogen colonization pathogen in the banana rhizosphere, which plays an important role in the management of Fusarium wilt disease in banana plantations.In this study, we described the development of a specific Panama disease suppressive bioorganic fertilizer (BIO) to control plant disease and promote plant growth. In addition, comparisons of disease suppressive and diseased soil microbial communities, screening of biocontrol agents, and nursery pot applications of the bio-product were investigated. Both clone and pyrosequencing data showed that Musa AAA Cavendish cv. Brazil) seedlings used in this study were the same as previously planted in this field, supplied by Hainan Wan Zhong Co., Ltd., Hainan, China.The banana plantation under study is located in Ledong county , Hainan province, China, where Panama disease is widespread and has caused severe crop losses. The study site was a flat banana field that suffered severe Panama disease from 2007–09 and had been under continuous banana cropping for 5 years since 2005. In 2009, 65% of the banana plants died in this field. The germ-free banana  roots were collected in this field as well as the individual wilted banana (infected banana plants) roots. Soil remaining on the root segment after a strongly shaking was considered to be rhizosphere soil2+ free 10 × PCR buffer, 2 μl of Mg2+ (15 mM), 2 μl of mixed dNTPs (2.5 mM each), 10 pmol each forward and reverse primer, and 2 U of Taq polymerase . Amplification was performed with an initial denaturation of 4 min at 95 °C; followed by 30 cycles of 1 min at 94 °C, 1 min at 55 °C and 1.5 min at 72 °C, and a final extension at 72 °C for 10 min. The PCR products were purified using the Axygen PCR clean-up kit, ligated into a pMD19-T Vector (Takara), and transformed into E. coli DH5α. Transformants were randomly selected by plating onto Luria-Bertani /Ampicillin agar plates with IPTG and X-Gal and incubated at 37 °C overnight. White clones from each soil samples were randomly picked and sequenced using an ABI 3730 gene analyzer.The primer set 27f and 1492r was used to amplify the 16S rRNA genesF. oxysporum f. sp. cubense race 4  on potato dextrose agar plates to observe whether the strains inhibited pathogen growth. The isolate, which showed extremely strong suppressive efficacy on FOC growth, was selected and identified using 16S rRNA gene sequencing. A maximum composite likelihood phylogenetic tree of the 16S rRNA sequence was generated by MEGA 5.Disease suppressive banana roots with rhizosphere soil from a severe Panama disease field were collected and washed with sterilized water and a soil suspension dilution series was plated on 30% beef extract medium . Single colonies were obtained and cultured with 8 CFU g−1 fresh weight. The organic fertilizer was a mixture of amino acid fertilizer and pig manure compost  as described previouslyThe banana specific bioorganic fertilizer (BIO) used in this study was a mixture of organic fertilizer and the antagonistic isolates described above at approximately 1 × 102/plot). In the BIO treatment, banana seedlings were grown in nursery cups for 45 days and then pots for 60 days supplemented with BIO  that were then transplanted into the BIO supplemented holes (500 g /hole) in the field plots when the plants had 5 to 6 true leaves  and bioorganic fertilizer treatment (BIO) and assigned as CKD and BIOD, respectively. Healthy banana roots were collected in BIO and CK and assigned as BIOH and CKH, respectively. Five individual plants were randomly collected in each plot and mixed as one sample. Each sample had 4 replicates collected from the 4 parallel plots. Root soil was collected and DNA was isolated as described above. Replicated soil samples were not combined, but kept as 4 replicates.2, 0.2 μl BSA , 0.75 μl of mixed dNTPs , 10 pmol each forward and reverse primer, and 2.5 U of high fidelity Taq polymerase . Amplification was performed with an initial denaturation of 3 min at 95 °C followed by 30 cycles of 45 s at 94 °C, 45 s at 59 °C and 1 min at 72 °C, and a final extension of 7 min at 72 °C. The PCR products were purified using the Axygen DNA gel recovery kit . Sequencing was performed by Majorbio Biotech Co., Ltd. .The 454 pyrosequencing fusion primer set that included the 454 sequencing adaptor, barcode and the universal 16S rRNA gene primer set (27f/533r)F. oxysporum f. sp. cubense (FOC) colony forming units (CFU) colonizing the banana rhizosphere was quantified by plating soil series dilution solutions from rhizosphere soil suspensions on petri plates with modified Komada’s selective medium, as described previously2HPO4, 1 g, MgSO4•7H2O, 0.5 g, KCl, 0.5 g, Fe-Na-EDTA, 0.01 g, D-Galactose, 10 g , L-Asparagine, 2 g, and 16 g of agar. The basal medium was mixed with 100 ml of solution containing the following agents: 0.9 g PCNB , 0.5 g Na2B4O7•10H2O, 0.45 g oxgall, and 0.3 g streptomycin sulfate. The pH was adjusted to 3.8 ± 0.2 with 10% phosphoric acid. Plates were stored at 25 °C after plating for 10 days before colony counting. Triplicates were conducted for each sample.This soil solution obtained from root wash was used for pathogen quantification for all the samples collected in 2010. The number of de novo mode. Samples were randomly subsampled to 7018 reads per sample. For both the clone library and pyrosequencing data, sequences were aligned and clustered at 97% nucleotide identity and taxonomy was assigned using RDP Naive Bayesian classifierClone library sequences were analyzed with Mothurr and P values. To determine the significance of the differences, two-tailed, unpaired t–tests were performed on individual microbial groups in SIMPER analysis, disease incidence, unit yield, and plot yield. ANOVA followed by the LSD test in SAS (version 6.1)  was performed on FOC quantification data.For pyrosequencing data, non-metric multidimensional scaling (NMDS) was performed to illustrate beta-diversity between individual samples (Bray-Curtis distances between samples) after a hellinger transformation of the raw data. Similarity percentage (SIMPER) analysis was conducted to elucidate the indicator genera to the overall Bray-Curtis distances between samplesKJ600797 - KJ600996). The pyrosequencing sequences were deposited in the NCBI Sequence Read Archive (SRA) database (Accession number: SRR1185515- SRR1185517).All clone library sequences were deposited at GenBank .


Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes. Mutations were detected in QRDR of gyrA  and parE . Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL). Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes.The increased Salmonella is a human pathogen commonly found in developed and developing countries, causing clinical diseases ranging from mild gastroenteritis to septicaemia [ Salmonella infection. However, the worldwide emergence of quinolone-resistant bacterial strains raises public health concern. Increased incidence of quinolone-resistant Salmonella serovars has been documented in Southeast Asian region [ticaemia . Quinolon region –5. Salmonella: altered protein targets for quinolones, decreased uptake of quinolones by bacteria, and DNA gyrase protection via plasmid-derived qnr genes [Three major mechanisms contribute to quinolone-resistance amongnr genes . DNA gyrnr genes , 7. gyrA and gyrB genes, while topoisomerase IV is encoded by parC and parE genes. Mutation in quinolone-resistant determining region (QRDR) in these genes is associated with quinolone-resistance [ Salmonella [ gyrA gene represents the most frequently mutated amino acid in Salmonella [Both DNA gyrase and topoisomerase IV are tetrameric enzymes. DNA gyrase is encoded bysistance . Many stlmonella –12. Indelmonella , 14. Whilmonella , 12, 14,lmonella . Neverth parC and parE genes [ Salmonella strains can be discriminated based on their sequence, length, GC content, or strand composition.DNA sequencing is the gold standard for detecting genetic changes. However, it is relatively costly to sequence all four genes when examining a large sample pool. Furthermore, mutations are rare inrE genes ; thereforE genes –17. ThisrE genes . During rE genes . Hence,  Mycobacterium tuberculosis, Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Salmonella Typhi and Paratyphi A [ gyrA gene was examined. Although infrequent, mutations in gyrB, parC, and parE genes of Salmonella and their correlation with lower susceptibility to quinolones and fluoroquinolones have been documented too [ Salmonella gyrase and topoisomerase IV genes.HRM assay has been used for the identification of quinolone-resistance strains in pathogens such asatyphi A –23. In tnted too . The obj Salmonella enterica strains were obtained from the culture collection of Laboratory of Biomedical Sciences and Molecular Microbiology, Institute of Graduate Studies, University of Malaya. The susceptibility of the strains towards nalidixic acid and ciprofloxacin was examined and reported in previous studies [ Salmonella strains showing resistance or reduced susceptibility towards nalidixic acid were used to develop the HRM assay in this study . All selected strains were susceptible to ciprofloxacin. The minimum inhibitory concentration (MIC) of nalidixic acid for the strains was determined by using Etest strips . The test was performed according to manufacturer's instructions. Genomic DNA for each bacterial strain was extracted by using Wizard genomic DNA purification kit . The details of all Salmonella strains used in this study are listed in the Supplementary Table .A total of 195 studies , 25. Six gyrA, gyrB, parC, and parE QRDRs was performed using previously described primers [μL monoplex reaction mixture, containing 1x colourless GoTaq Flexi Buffer, 1.5 mmol/L magnesium chloride (MgCl2), 200 μmol/L deoxynucleoside triphosphate (dNTP) mix, 0.3 μmol/L of each primer pair, 1 U Taq DNA polymerase , and approximately 100 ng of bacterial genomic DNA. The PCR reaction mixtures were first incubated at 94°C for 2 min, followed by 25 cycles of 94°C for 30 s, 60–63°C  for 30 s, and 72°C for 45 s, with a final extension step of 72°C for 5 min. The appropriate annealing temperatures for gyrA, gyrB, parC, and parE are 60°C, 60°C, 63°C, and 62°C, respectively. Agarose gel electrophoresis was then carried out to confirm the presence of amplicons prior to DNA sequencing. Next, the PCR products were purified using Wizard SV gel and PCR clean-up system , and the purified PCR products were sent to First BASE Laboratories  for sequencing. Mutations in the target genes were determined by comparison with the wild-type sequence of the respective genes in Salmonella Typhimurium LT2 (GenBank accession number NC_003197).PCR amplification of primers . The sel gyrA, gyrB, parC, and parE gene sequences of Salmonella serovars from the NCBI GenBank database was performed. Sequence alignment was done using the molecular evolutionary genetics analysis software version 5 (MEGA5) [http://primer3plus.com/cgi-bin/dev/primer3plus.cgi), spanning the QRDR of each gene of interest, and were commercially synthesized . Approximately 20 ng of bacterial genomic DNA was added into 1x MeltDoctor HRM master mix , which was premixed with 0.3 μM of each primer pair. Sterile deionized distilled water was added to make up a final reaction volume of 20 μL. The reproducibility of the assay was determined by performing all reactions in triplicate. In each assay, the DNA sample from a wild-type reference strain was used as positive control. The selection of the wild-type reference strains for the different target genes was explained in the Results section  . Primers section . Negativ gyrA, gyrB, parC, and parE genes of 62 Salmonella strains were subjected to sequencing. Of these 62 strains, missense mutations were detected in QRDR of gyrA (n = 52) and parE (n = 1). Silent mutations within and beyond QRDR were observed in gyrB (n = 60), parC (n = 1), and parE (n = 50). In QRDR of gyrA, missense mutations occurred only in codons 83 , tyrosine (S83Y), and isoleucine (S83I)) and 87 (conversion of aspartic acid to glycine (D87G), tyrosine (D87Y), and asparagine (D87N)). In the parE gene, two missense mutations were detected, one within the QRDR (methionine to isoleucine (M438I)) and another outside of the QRDR ) . In the absence of mutations (STM057/05), the strain showed lowest MIC value (MIC 6 μg/mL). Detailed genetic analyses of the QRDRs of each Salmonella strain are listed in the Supplementary Table (Table S1).The(V521F)) . The MIC gyrA (Ala67-Gln106), gyrB (Asp426-Lys447), parC (Ala64-Gln103), and parE (Asp420-Lys441) genes. The primer pairs produce amplicons ranging from 150 to 250 bp . The QRDRs of gyrA, parC, and parE genes in the reference strain STM006/02 and the gyrB QRDR in the reference strain STM032/04 have matching sequences with the Salmonella Typhimurium reference genome LT2 (NC_003197). Detailed information for these two reference strains is listed in the Supplementary Table (Table S1).HRM primers were designed to span the QRDRs ofo 250 bp , with a  gyrA allele is 82.5–82.8°C, while that of the mutants  is at 82.0–82.4°C. Interestingly, Salmonella strains with gyrA D87G mutation produced a distinct melting curve compared to other mutants, at slightly higher melting temperature (82.9–83.5°C). The differences in the melting curves of the wild type versus mutants were distinguishable in both aligned and difference plot  with three mutations in gyrB has a slightly lower melting temperature at 86.0°C. The aligned plot did not show any difference when comparing the melt curves of wild-type versus mutant gyrB alleles. However, they can be distinguished in difference plot  and the mutant at 86.4°C. Although the differences in the melting temperature were small, the mutant produced a unique melting curve in the difference plot when compared to the wild type , which harbors a single mutation in parE QRDR, has a lower melting temperature at 83.9°C. The differences in wild-type versus mutant melt curves were apparent in both aligned and difference plot . The transition of melt curves in HRM analysis of all target regions successfully discriminated the wild types from mutants. Although the melting transitions were not very distinct in aligned melt curves, especially for gyrA, gyrB, parC, and parE. Consistent with the literature [ gyrA QRDR most commonly take place at codons 83 and 87, often resulting in amino acid change, and are associated with the nalidixic acid resistance in Salmonella strains. Gyrase A-DNA complex is the primary target for quinolones in Salmonella [ gyrA, since such mutations confer better resistance to quinolone compared to that of gyrB mutants [ gyrA successfully resolved the mutant strains into three distinct melting profiles. The melting profiles of the D87G mutants were highly divergent from those of the other mutants. The D87G variants consisted of a single base substitution from adenosine to guanine. This type of mutation represents class I SNP, which often results in a higher melting temperature shift compared to other classes of SNP.Point mutations were identified in QRDRs ofterature , 14, our mutants . HRM ana gyrB among Salmonella strains in this study. Furthermore, most studies did not identify mutations in gyrB of quinolone-resistant Salmonella [Mutation was not common in the QRDR oflmonella , 14, 28. parC and parE genes are uncommon among Salmonella [ parC genes and were identified in parE gene of only two Salmonella strains. The strains used in this study showed classical phenotype, that is, resistant to nalidixic acid but not to ciprofloxacin. These observations are in agreement with previous reports, which suggested that the mutations of these genes are most probably infrequently detected because they either are not important in quinolone-resistance or are only required for higher level of resistance, that is, resistance to fluoroquinolones [ parC and parE genes. However, these were mostly silent mutations. HRM assay successfully differentiated the mutants from the wild type. The melting profiles of the mutants were highly divergent from those of the wild type, as clearly shown in both aligned melt curves and difference plots.Topoisomerase is the secondary target of quinolones in Gram-negative bacteria. Mutations within QRDRs oflmonella , 14, 28.inolones . Multipl Salmonella.Despite the high efficiency of HRM analysis in mutation screening, this assay could only detect the presence of base substitutions. The melting profiles should not be used directly for variants classification without first validating with DNA sequencing. False positive may occur due to synonymous mutations, while false negative occurs due to mutations outside targeted regions or resistance caused by other mechanisms. To date, sequencing remains the ultimate proof for mutations that lead to amino acid changes and subsequently an alteration of enzyme structure . Neverth Salmonella gyrase and topoisomerase IV genes with sufficient sensitivity. The adoption of this assay for mutation screening prior to DNA sequencing is an attractive option to reduce the cost for mutational studies related to quinolone resistance. Furthermore, HRM analysis is time-saving, allows for high throughput screening, and is easy to perform since there is no post-PCR processing or purification steps. Nevertheless, sequencing shall be used for final validation and should not be completely replaced by HRM analysis.In conclusion, HRM analysis allows for detection of mutations inSalmonella strains used to develop the HRM assay is tabulated in the supplementary table (Table S1). Additional information include the susceptibility of each Salmonella strain to nalidixic acid and the corresponding minimum inhibitory concentration value, type of base substitution and the corresponding amino acid change in each strain, and the melting temperature of the HRM amplicons.The background of the


Anemia and red blood cell (RBC) transfusion occur frequently in hospitalized patients with cardiac disease. In this narrative review, we report the epidemiology of anemia and RBC transfusion in hospitalized adults and children (excluding premature neonates) with cardiac disease, and on the outcome of anemic and transfused cardiac patients. Both anemia and RBC transfusion are common in cardiac patients, and both are associated with mortality. RBC transfusion is the only way to rapidly treat severe anemia, but is not completely safe. In addition to hemoglobin (Hb) concentration, the determinant(s) that should drive a practitioner to prescribe a RBC transfusion to cardiac patients are currently unclear. In stable acyanotic cardiac patients, Hb level above 70 g/L in children and above 70 to 80 g/L in adults appears safe. In cyanotic children, Hb level above 90 g/L appears safe. The appropriate threshold Hb level for unstable cardiac patients and for children younger than 28 days is unknown. The optimal transfusion strategy in cardiac patients is not well characterized. The threshold at which the risk of anemia outweighs the risk of transfusion is not known. More studies are needed to determine when RBC transfusion is indicated in hospitalized patients with cardiac disease. Red blood cell (RBC) transfusion is common in critically ill adults and children -3. PatieThis narrative review critically appraises the available data on the relationship between anemia, RBC transfusion and outcome of critically ill adults and children with cardiac disease. We discuss the prevalence and risks of anemia in the ICU, the rationale for RBC transfusion, the evidence supporting restrictive transfusion strategies, and the growing interest in goal-directed transfusion therapy. Premature infants and intraoperative transfusions will not be addressed in this paper, nor will a detailed discussion of the relationship between length of storage of RBC units and outcomes, since the latter is discussed in other papers -7.2) to vital organs and tissues if DO2 drops below a critical DO2  and arterial content in:The prevalence of anemia in adults with heart failure ranges from 18 to 38% . Anemia 2 = Q’ × CaO2DO2 = {(Hb × SaO2 × 1.39) + (PaO2 × 0.0031)} [and CaO0.0031)} 2 and DO2 if the compensatory increase of Q’ is not high enough. Anemia may be poorly tolerated by patients with cardiac failure and/or coronary disease because their ability to increase cardiac output to compensate for anemia is limited [Any drop of the Hb level decreases CaO limited ,12.et al. [Carson et al.  studied et al. ,14 and set al. -20 on thet al. [A number of host characteristics specific to children, like growth, fetal Hb, different cardiac diseases and physiology, may impair their adaptive mechanisms to anemia. There are few data on the relationship between anemia and outcomes in cardiac children. Kammache et al.  reportedIn the United States, 7.8% to 92.8% of adults undergoing cardiac surgery are transfused -24. A tr2 , high lactate level, low SaO2/FiO2 ratio) would prompt them to prescribe RBC transfusions in this population [RBC transfusions are sometimes given to cardiac patients to rapidly replace blood volume in cases of postoperative blood losses . In the pulation . There ipulation -32. Howepulation , admissipulation ,33.RBC transfusion should be given only when the risk/benefit ratio is favorable (ratio < 1). The threshold at which the risks of anemia outweigh the risks of transfusion is currently unknown.P < 0.001) [There are little available hard data on the clinical benefits of RBC transfusion. In a prospective study that enrolled 303 Kenyan children with Hb below 50 g/L at hospitalization, 116 (38%) did not receive a transfusion, mostly because of blood unavailability, while 187 did; the mortality was significantly higher in non-transfused children  . This stThe available evidence suggests that RBC transfusion in patients with cardiac disease is an independent risk factor of mortality Table .et al. [et al. [et al. [Three studies reported some transfusion benefits if the Hb level is low (<80 g/L), but they also reported harm with high Hb level ,36,46. Aet al.  reported [et al.  reportedet al. [et al. [I2: 92% and 98% respectively). Garfinkle et al. [On the other hand, six studies of adults undergoing cardiac surgery, CABG or with myocardial infarction reported increased mortality in transfused cardiac patients ,42-44,47et al.  publishe [et al. , the rise et al.  also pubet al. [P < 0.01), but no association with mortality .There is less evidence in children. Three studies reported prolonged length of mechanical ventilation in children who received RBC transfusion after cardiac surgery ,49,54, tet al.  found anIs it justified to prescribe more RBC transfusions to cardiac than to non-cardiac ICU patients? The available data on this question are inconclusive: while some descriptive studies suggest a benefit to transfusion, most studies associate RBC transfusion to worse outcomes. Descriptive studies cannot prove that there is a cause-effect relationship between a risk factor and a given outcome: adjustment for confounders like volume of RBC transfusion and administration of other blood products can be done, but a possibility always remains that unknown confounders play a role. Moreover, severity of illness is associated with both transfusion and mortality ,24. A muThe safety of blood products with respect to transfusion-transmitted infectious diseases has improved greatly in recent decades. Presently, the greatest concern is non-infectious serious hazards of transfusion (NISHOT) -59.2 in small vessels and decrease coronary blood flow. Barr et al. [et al. [P = 0.007) and > 160 g/L  in patients with non-ST-elevation myocardial infarction.NISHOT may be non-immune or immune-mediated. Short-term non-immune NISHOT include transfusion-related circulatory overload (TACO) and overtransfusion. Overtransfusion is of concern because of the risk of TACO and/or hyperviscosity. Viscous blood flow may impair DOr et al.  reviewed [et al.  reportedImmune-mediated NISHOT include hemolytic and allergic reactions, transfusion-related immunomodulation (TRIM), transfusion-related acute lung injury (TRALI), nosocomial infections, transfusion-associated graft versus host disease, and alloimmunization to RBC and HLA antigens ,62,63.et al. [In critically ill cardiac patients, TRIM may represent a significant ‘second-hit’ when added to pre-existing organ dysfunction and/or a systemic inflammatory response syndrome (SIRS), which may result in TRALI and multiple organ dysfunction syndrome. Vlaar et al.  reportedet al. . TRALI met al. ,67.Although NISHOT are currently the most important causes of transfusion-related fatalities, their incidence rate in cardiac patients is not well characterized .2 and O2 consumption (VO2) remains horizontal as long as compensatory mechanisms (increasing cardiac output and O2 extraction) are still effective [2 drops with DO2 below a critical threshold. Anemia decreases DO2 and it is unmistakably deleterious below a certain threshold. The critical Hb level threshold below which DO2 becomes significantly impaired and transfusion becomes less harmful than persisting anemia is unknown and likely varies depending upon the underlying condition and clinical status of each patient. Anemia is well endured by healthy subjects: acute isovolemic reduction of Hb level down to 50 g/L was hemodynamically well tolerated by 11 resting healthy humans [2 than healthy adults and target Hb for transfusion might be different  in critically ill patients with cardiac disease Table .We found seven RCTs conducted in adults with cardiac illness ,52,73-76et al. [P = 1.00).In 1999, Hébert et al.  publisheet al. ; the 30-et al. [P = 0.93), cardiogenic shock , acute respiratory distress syndrome  and acute renal failure . The number of RBC units transfused was independently associated with 30-day all-cause mortality .Hajjar et al.  publisheet al. [Carson et al.  publisheIn summary, the available RCTs suggest that a restrictive transfusion strategy (threshold Hb level for transfusion: 70 or 80 g/L) appears as safe as a liberal strategy in stable adult ICU patients with cardiac disease.Three RCTs compared a restrictive and a liberal transfusion strategy after pediatric cardiac surgery.P = 0.36), PICU length of stay (7.0 ± 5.0 versus 7.4 ± 6.4 days) or 28-day mortality (3.2% versus 3.2%) [In the ‘Transfusion Requirements In PICU’ (TRIPICU) study , 637 staus 3.2%) .P = 0.063).An RCT compared the outcome of cardiac children older than 6 weeks randomized to receive a RBC transfusion if Hb level dropped below 80 g/L or 108 g/L . Patientet al. [Physicians target higher Hb values for critically ill children with cyanotic heart disease ,29. In aet al. , 60 chilet al. [2. Patients allocated to ScvO2 monitoring were also assigned to a bundle of treatment to maintain their ScvO2 over 70%; this bundle included, in sequential order, mechanical ventilation, fluid bolus, vasoactive drugs and RBC transfusion if the ScvO2 remained under 70% after all other interventions. Mortality was 30.5% with goal-directed therapy versus 46.5% in controls.Goal-directed RBC transfusion therapy targeting a physiological goal may be a more appropriate target than specific Hb levels. The concept of goal-directed therapy is well illustrated by the RCT conducted by Rivers et al. : 266 adu2 measured by a Swan-Ganz catheter or locally with near infrared spectroscopy (NIRS), global oxygen consumption (VO2), blood lactate, mixed venous O2 saturation (Sv’O2), ScvO2, tissue O2 saturation and O2 extraction rate [2 is another candidate. The results of the trial by Rivers et al. [There are currently no hard data on goal-directed transfusion therapy in cardiac patients. Goals that can be considered include physiologic parameters like DOion rate . Well-coion rate . Repeateion rate , but this et al.  suggestes et al. . Howevers et al. , 1,341 as et al. ,85. MoreCurrently, we do not know what physiologic parameters we must use to guide a goal-directed transfusion therapy in cardiac patients. Further clinical studies are required before a given goal that would help practitioners to precisely decide when to transfuse cardiac patients can be recommended in this population.Anemia is common in patients with cardiac disease and is associated with mortality and morbidity. RBC transfusion is the best way to rapidly increase the Hb level, but it is not risk-free: storage lesion and NISHOT, including prothrombotic and proinflammatory effects, may cause transfusion-related adverse events.The Hb level at which the risk of anemia outweighs the risk of transfusion is not well known. Three observational studies suggest that there might be some benefit to give a RBC transfusion to adults with acute coronary syndrome if their Hb level is < 80 g/L, but many more studies suggest that RBC transfusion might be harmful in this population if their Hb level is higher than 80 to 100 g/L. The question of RBC transfusion to adults with acute coronary syndrome is not addressed in the guidelines of the American College of Cardiology, the American Heart Association and the Canadian Cardiovascular Society . HoweverData in children are scarce. In stable acyanotic cardiac children, a Hb level above 70 or 80 g/L appears to be well tolerated without RBC transfusion. In children with cyanotic heart lesions, Hb level over 90 g/L appears safe. The threshold Hb level for unstable cardiac children and for neonates is unknown. Goal-directed transfusion therapy is a promising avenue for future research.Severity of illness, anemia and transfusions are interconnected with outcome in cardiac patients, and their individual contribution to outcome remains unresolved. Further RCTs are necessary to disentangle this relationship.2: O2 delivery; Hb: hemoglobin; Hct: hematocrit; ICU: intensive care unit; IE: ischemic events; HR: hazard ratio; MI: myocardial infarction; MODS: multiple organ dysfunction syndrome; MV: mechanical ventilation; NIRS: near infrared spectroscopy; NISHOT: non-infectious serious hazard of transfusion; OR: odds ratio; PCI: percutaneous coronary intervention; PICU: pediatric ICU; PRISM: pediatric risk of mortality; RBC: red blood cell; RCT: randomized controlled trial; RR: relative risk; ScvO2: central venous O2 saturation; Sv’O2: mixed venous O2 saturation; TACO: transfusion-associated circulatory overload; TRALI: transfusion-related acute lung injury; TRIM: transfusion-related immunomodulation; VO2: O2 consumption.ACS: acute coronary syndrome; aHR: adjusted HR; AMI: acute myocardial infarction; aOR: adjusted OR; APACHE: acute physiology and chronic health; CABG: coronary artery bypass graft; CI: confidence interval; DOThe authors declare that they have no competing interests.GD, KH and JL wrote, revised and approved the manuscript in its final form.


Background. Elevated urinary angiotensinogen (UA) was identified as novel prognostic biomarker capable of predicting chronic kidney disease, and in the present study, we will investigate the diagnostic value of UA in the patients of nephrolithiasis. Methods. Urine angiotensinogen levels and α1-microglobulin were measured by enzyme-linked immunosorbent assay (ELISA) in 60 patients presenting with nephrolithiasis and 50 sex- and age-matched healthy volunteers. Estimated glomerular filtration (eGFR) was calculated and, by simple regression analysis, the correlation of UA/α1-microglobulin levels and the decline of eGFR were analyzed as well. Results. Median UA levels was significantly increased in the nephrolithiasis patients compared with normal control . The mean serum creatinine levels in patients with higher UA levels (>1250 pg/mL) was significantly higher than those with lower UA levels (<1250 pg/mL) . According to the single variate analysis, UA levels were significantly and positively correlated with urinary α1-microglobulin , while they were significantly and negatively correlated with eGFR . Conclusion. Urinary UA is a novel biomarker for patients with nephrolithiasis, which indicates renal tubular injury. Further study on the molecular pathogenic mechanism of UA and larger scale of clinical trial is required. Nephrolithiasis is a condition involving the development of stones in the kidney; it is a common disease with a prevalence of 5–8% worldwide . The exaUrine is supersaturated with oxalate ions and calcium which promotes crystals formation, and then crystals grow into larger ones; aggregation and retention of crystals are important factors of nephrolithiasis formation. During the procedure, the role of renin-angiotensin system (RAS) in kidney tissue was identified recently . Moonen As already known, renin is made in the juxtaglomerular apparatus and released into the interstitial space, from where it may reach the circulation via diffusion across the peritubular capillaries. Proximal tubular fluid, however, also contains renin, suggesting that circulating renin is filtered in the kidney . CirculaRecent interest focuses on the occurrence of both renin and angiotensinogen in urine, as markers of renal renin-angiotensin system (RAS) activity, potentially reflecting the disease state . ElevateWe conducted a case control study between January 2012 and March 2013 at Tongji University tenth peoples' hospital. Patients who were diagnosed as having nephrolithiasis during the period were enrolled in this study. Briefly, they were diagnosed by ultrasonography and radiography. No case was found by X-ray to have radiolucent stones or by clinical evaluation to have cystine or uric acid stones. If stone specimens were removed by surgery or obtained after medical treatment or shock-wave lithotripsy, composition of the stones was confirmed by infrared spectroscopy  [Normal controls were randomly selected from subjects receiving general health examinations at the same hospital during the same period. The controls had no past history of nephrolithiasis and no clinical findings of stones, which were confirmed by plain abdominal X-ray and abdominal ultrasound. Both cases and controls were excluded if they had a history of chronic urinary tract infection, renal failure, chronic diarrhea, gout, renal tubular acidosis, autoimmune diseases, primary and secondary hyperparathyroidism, and cancer. All study subjects were living in Shanghai. The study protocol was approved by the Institutional Review Board of Tongji University School of Medicine. Each subject provided signed informed written consent.Since the literature reported that urinary angiotensinogen significantly correlated with decline of estimated glomerular filtration (eGFR), thus we measured fasting serum levels of creatinine (Cr) in each patient ; and the eGFR was calculated by the following equations :(1)eGFRα1-microglobulin and angiotensinogen were measured with enzyme-linked immunosorbent assay (ELISA) kits, LZ Test Eiken α1-M  and Human Total Angiotensinogen Assay Kit .Urinary excretions of α1-microglobulin, and other parameters were compared by the Mann Whitney U test. The concentrations of urinary angiotensinogen were compared by Student's paired t-test. SPSS software  was used for statistical analyses, and P < 0.05 was considered statistically significant.Data obtained by measurements were given as mean ± standard deviation. Urinary levels of angiotensinogen, P < 0.01); the median 24 h urinary angiotensinogen excretion was significantly higher in nephrolithiasis patients versus normal control ; urinary angiotensinogen-to-creatinine ratio was also significantly higher in nephrolithiasis patients versus normal control , as demonstrated in This study included 60 nephrolithiasis patients and 50 normal healthy people in the control group. The mean (±SD) of age of patients in the study and control groups was 45.5 ± 14.3 and 43.6 ± 12.7 years, respectively. The ratio of male/female was 37/23 in the study group and 35/15 in the control group. The demographic characteristics of both groups are summarized in μmol/L; however, serum creatinine levels were significantly higher (92.23 ± 18.13 μmol/L) in the nephrolithiasis patients with UA > 1250 pg/mL (P < 0.05), which was demonstrated in To compare the potential effect of UA on the renal function, we divided the patients into two groups according to their UA levels: UA < 1000 pg/mL and UA > 1000 pg/mL, and we compared their mean serum creatinine and found that the serum creatinine levels with UA < 1250 pg/mL were 70.07 ± 11.17 α1-microglobulin , while they were significantly and negatively correlated with eGFR   .α1-microglobulin and UA levels significantly and negatively correlated with eGFR, which suggests potential renal tubular injury in nephrolithiasis patients.Our study investigated the urinary angiotensinogen levels in nephrolithiasis patients and initially found that the UA concentrations in the patients with nephrolithiasis were significantly higher than those in normal subjects. In spite of normal renal function parameters in nephrolithiasis patients, the mean serum creatinine levels were significantly higher in patients versus normal control. In addition, we performed a simple regression analysis and demonstrated that UA levels significantly and positively correlated with urinary In the past studies, it has been suggested that urinary excretion of angiotensinogen reflects intrarenal angiotensin II levels , 15. AngIn Alge et al.'s study, urinary angiotensinogen could be useful as a prognostic acute kidney injury biomarker in the setting of the intensive care unit; in a recent study that used a definition of prerenal azotemia, which was very similar to Alge et al.'s, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), IL-18, and kidney injury molecule 1 (KIM)-1 were elevated in ICU patients with prerenal AKI compared to those without AKI but were lower than values for patients whose AKI did not resolve within 48 hrs [In humans, plasma angiotensinogen reaches urine via glomerular filtration, like albumin, and the normal urinary angiotensinogen levels in humans are ~0.2 pmol/mL versus ~1,200 pmol/mL in plasma. Thus, the urinary angiotensinogen levels range from 0.01% to 0.1% of the plasma levels in humans. In contrast, urinary angiotensinogen levels in rodents are much higher and range from 0.1 to 400 pmol/mL, implying that the urinary angiotensinogen levels in rodents are sometimes higher than their plasma levels . Less thα1-microglobulin is filtered freely through glomerular capillaries and reabsorbed by the proximal tubules [α1-microglobulin is a marker for proximal tubule dysfunction, and increased levels of urinary α1-microglobulin have been reported in patients with type 1/2 diabetes [α1-microglobulin allows the early diagnosis of diabetic nephropathy prior to the appearance of microalbuminuria and also predicts the progression of diabetic nephropathy. There is little literature about α1-microglobulin in nephrolithiasis patients, however [Urinary  tubules . Thus, udiabetes . The ass however . Plasma  however . Thus, p however . Another however . The preα1-microglobulin shows us the disease progression in nephrolithiasis cohort, which might require multicenter randomized study.In conclusion, we conclude in our study measurement of UA was a novel and useful marker for patients with nephrolithiasis. The investigation of UA suggests urologists to concern about previous neglected tubular injury in clinical kidney stone patients and greatly enhances our understanding of tubular injury in etiology of nephrolithiasis. Further validation of serum creatinine and urinary


Results showed that there were no discernible alterations in LC in rats. The serum NE concentrations, positive neurons, mean optical density (MOD), and protein levels of TH, DBH, and CRF in model group were significantly increased compared to the control group. But XYS-treated group displayed a significantly decreased in NE levels and expressions of TH, DBH, and CRF compared to the model group. In conclusion, CIS can activate LC-NE system to release NE and then result in a significant decrease in rats. XYS treatment can effectively improve depressive-like behaviors in rats through inhibition of LC-NE neurons activity.Most research focuses on the hypothalamic-pituitary-adrenal (HPA) axis, hypothalamus-pituitary-thyroid (HPT) axis, and hypothalamus-pituitary-gonadal (HPGA) axis systems of abnormalities of emotions and behaviors induced by stress, while no studies of Chinese herbal medicine such as Xiao Yao San (XYS) on the mechanisms of locus coeruleus-norepinephrine (LC-NE) system have been reported. Therefore, experiments were carried out to observe mechanism of LC-NE system in response to chronic immobilization stress (CIS) and explore the antidepressant effect of XYS. Rat model was established by CIS. LC morphology in rat was conducted. The serum norepinephrine (NE) concentrations and NE biosynthesis such as tyrosine hydroxylase (TH), dopamine- Stress response is a risk factor that can develop affective or mental disorders, including anxiety, posttraumatic stress disorder, depression, and other disorders  and is cThe NE contains axons that are widely distributed, indicating that this neurotransmitter plays a pivotal role in CNS function and behaviors , 5. The Many of studies have demonstrated that repeated or prolonged stressor can lead to complicated alterations in LC-NE neurotransmission. Although repeated or chronic stressors may not elicit NE to release, they can increase the capacity of this system to release NE, due to increased rates of NE synthesis . At pres Taiping Huiming Heji Jufang in the Song Dynasty (960–1127 A.D.) in ancient China. The XYS finished products including decoction, powder, and pill have been widely used to treat mental disorders for thousands of years in China [β production in hippocampus and paraventricular nucleus (PVN) induced by repeated stress and alter metabolic network abnormalities in rats with chronic unpredictable mild stress (CUMS) or CIS [XYS is a Chinese herbal formula that was initially recorded in the bookin China –17. The in China , 19. XYS) or CIS –22. In t) or CIS , 23, hyp) or CIS  . All animals were fed in standard animal room . Food and purified water were available studies . In this Angelica sinensis (Oliv.) Diels), Paeoniae Radix Alba , Bupleuri Radix (root of Bupleurum chinese DC), Atractylodis Macrocephalae Rhizoma , Glycyrrhizae Radix et Rhizoma , Poria (fungus nucleus of Poria cocos (Schw.) Wolf), Zingiberis Rhizoma Recens , and Menthae Haplocalycis Herba  (the ratio is 6 : 6 : 6 : 6 : 6 : 2 : 2 : 3). These herbal medicines were obtained from the Affiliated Nan Fang Hospital of Southern Medical University  and authenticated by Liu Q, Department of Pharmacognostical Identification in School of Chinese Medicine of Southern Medical University. All herbal medicines were immersed in 10 times volume of distilled water and boiled at 80°C for 1 h; the water extracts were then collected. This process was repeated again [The herbal formula of XYS comprises the following drugs: Angelicae sinensis Radix  was measured on days 7th, 14th, and 21st as previous described and made a minor modification . BrieflyThe experiment was terminated on the 21st day, and rats were anaesthetized with an intraperitoneal injection 10% chloral hydrate (0.35 to 0.40 mL/100 g bodyweight). Venous blood was drawn from abdominal aorta and serum was then separated to measure NE levels. Enzyme-linked immunosorbent assay (ELISA)  was measured using a microtiter plate reader  according to the protocol in the kit.μm). Preparation of tissue section and procedure of H&E staining were described in previous report [Six animals in each group were anaesthetized with 2% pentobarbital sodium (40 mg/kg) and perfused with cold 0.9% NaCl solution followed by perfusate solution . Brain stem contained locus coeruleus was cut out from brain tissues and fixed in 2.5% paraformaldehyde solution for 48 hours and was then cut into serial sections  for 10 min at room temperature in order to inhibit endogenous peroxidase. Antigen retrieval was conducted by heating for 15 min. Slices were incubated with primary antibodies  after blocking in the antisera. After incubation with the secondary antibody, sections were placed in DAB reagent  for 5–10 min at room temperature. After a further rinsing in PBS, sections were restained with hematoxylin and were mounted on gelatin-coated slides for observation under a light microscope.Protein levels of DBH, CRF, and TH were measured by Western blotting. The procedure was performed as previously described . BrieflyAfter washing three times for 5 min each in TBST, membranes were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibody which was diluted in enclosed liquid  for 1 h at room temperature. After three 5 min washes in TBST, membranes were developed using the enhanced chemiluminescence (ECL) detection reagent for 3 min and exposed to a CCD system . The intensity of protein band was measured using an Image J software.P < 0.05 was considered statistically significant difference.All data were presented as the mean ± SD and analyzed using an SPSS 17.0 statistical package. The multicomparison was performed. The mean values were conducted using one-way ANOVA as well as the homogenous variances test. The nonhomogeneous variances were compared by using Welch's test. P < 0.01). Body weights in rats were significantly increased after treating with XYS and fluoxetine . Three times measurements of sucrose preference in each group also showed significant differences (P < 0.01).In order to know depressive behaviors in rats during immobilization stress period, 1% sucrose preference in rats was recorded on days 7th, 14th, and 21st. A repeated measure ANOVA showed significant differences among four groups ; compared with the model group, XYS treatment group significantly increased the sucrose preference . Fluoxetine treatment group also significantly increased the sucrose preference compared with the model group (P < 0.05)  .P < 0.01). The serum levels of NE were 21.59 ± 1.28, 25.97 ± 4.17, 21.43 ± 0.912, and 21.31 ± 1.46 (ng/mL) in the control, model, XYS, and fluoxetine treatment group, respectively. The statistics analysis showed that serum NE levels were significantly increased in the model group compared to the control group (P < 0.01). After XYS or fluoxetine treatment, NE levels were significantly decreased compared to the model group (P < 0.01).In order to test whether chronic stress contributes to dysregulation of LC-NE system, peripheral NE concentrations were measured. In order to evaluate the impact of CIS on gross LC morphology in rats, H&E staining was conducted in this study. The results showed that LC adjacent located to the fourth ventricle in the brainstem . FurtherP = 0.036, 0.003, and 0.002, resp.) and there was a significant difference. Similarly, MOD in model group was significantly enhanced compared to the control group . After treatment of XYS and fluoxetine, the number of positive neurons of TH, DBH, and CRF were significantly decreased compared with model group; likewise, MOD in these three neurons in model group was also significantly decreased compared to control group.In order to determine whether CIS altered the function of LC neurons, TH, DBH, and CRF neurons were assessed by Immunohistochemistry. The results showed that LC expressed a number of TH, DBH, and CRF positive neurons in control group, 79.17 ± 13.56, 50.50 ± 6.09, and 46 ± 9.40, respectively. Quantity of the positive neurons of LC areas was increased and became dark brown, closely arranged, and round, shuttle or irregular shape of cell after modeling Figures . In modeP = 0.002, P < 0.01); protein levels of TH, CRF, and DBH in XYS and fluoxetine group were significantly decreased compared with the model group . XYS and fluoxetine group had no obvious difference compared with the control group (P > 0.05, In order to examine protein expressions of TH, DBH, and CRF in rats were measured using Western blotting. Compared with the control group, protein levels of TH, CRF, and DBH in model group were significantly increased (Chronic immobilization stress is usually used as a model to assess the underlying alterations in cellular and molecular relevant to some depressive diseases –31. FurtIn order to adapt to various stress, a body can timely adjust physiological status. Moderate stress is beneficial to both body and mind, while excessive stress is disadvantage to a body's mental and physical health. This can be explained that rats adapted to CIS in the beginning in this study and were in an agitated state and prone to irritate and struggle after modeling for seven days. Rats could not adapt to this stress resulting in passive manifestations such as fatigue, sluggish, loose stool, body weight loss, and others after CIS for 14 days. This indicated that rats could not adjust themselves under excessive stress. After treating XYS that function is soothe liver and tonifying spleen, a trend to reverse the weight loss induced by stress was displayed.Rodent is born with a preference for solutions or sweet foods. Decreased preference for sweet glucose solution in sucrose preference test means physical anhedonia, while therapies with chronic antidepressants can effectively reverse this reduction. SFT can detect the affective state or feelings of rodents . To evalLC-NE system firing can lead to differential NE levels to release. Specifically, NE system not only can be activated in response to acute stress across many preclinical studies, but also can be elicited by chronic stress paradigms. Varied outcomes in experiment studies depend on characteristics of stressor or individual. Findings from chronic psychosocial stress such as immobilization or cold restraint stressors have showed high NE reactivities and high plasma NE concentrations . Thus, iBoth DBH and TH were used to reveal the norepinephrine expression not only in locus coeruleus neurons, but also in the NE axons or fibers across the entire neuraxis , 35. SpeIn this study, no discernible changes in LC structure were found by H&E staining, suggesting that rats exposed to CIS for 21 days retained their brain structural integrity. Our results suggested increased positive neurons and MOD in CRF and synthesis enzyme of NE such as TH and DBH in the brain catecholaminergic system in response to 21-day chronic stress. It may contribute to a coordinated upregulation of these three genes expression. But these upregulation levels were decreased in response to XYS treatment as well as fluoxetine. We further confirmed these results in protein level by Western blotting in this experiment, and mRNA level had been confirmed in the former research . InteresTaking these together, our studies indicate that LC-NE system can be activated by acute and chronic stress. But the activity of LC-NE system is rapid, transient in response to an acute stress. Contrarily, this activity is persistent in response to a chronic immobilization stress. We will further explore the mechanism of activation of the LC-NE system in response to chronic stress at different time points such as 7 d stress, 14 d stress, and other and identify differentially expressed proteins using a proteomics approach.After exposure to an immobilization chronic stress, LC-NE system of rats can be activated resulting in a significant depression. Xiao Yao San can effectively improve depressive-like behaviors in rats. Further research is encouraged to detect the mechanism of activation of the LC-NE system in response to chronic stress in various time points and identify differentially expressed proteins or genes using an omics approach.