Indoxyl Sulfate Impairs Endothelial Progenitor Cells and Might Contribute to Vascular Dysfunction in Patients with Chronic Kidney Disease.

BACKGROUND/AIMS: Indoxyl sulfate (IS) is a protein-bound uremic toxin that accumulates in patients with chronic kidney disease (CKD). We explored the effect of IS on human early endothelial progenitor cells (EPCs) and analyzed the correlation between serum IS levels and parameters of vascular function, including endothelial function in a CKD-based cohort. METHODS: A cross-sectional study with 128 stable CKD patients was conducted. Flow-mediated dilation (FMD), pulse wave velocity (PWV), ankle brachial index, serum IS and other biochemical parameters were measured and analyzed. In parallel, the activity of early EPCs was also evaluated after exposure to IS. RESULTS: In human EPCs, a concentration-dependent inhibitory effect of IS on chemotactic motility and colony formation was observed. Additionally, serum IS levels were significantly correlated with CKD stages. The total IS (T-IS) and free IS (F-IS) were strongly associated with age, hypertension, cardiovascular disease, blood pressure, PWV, blood urea nitrogen, creatine and phosphate but negatively correlated with FMD, the estimated glomerular filtration rate (eGFR), hemoglobin, hematocrit, and calcium. A multivariate linear regression analysis also showed that FMD was significantly associated with IS after adjusting for other confounding factors. CONCLUSIONS: In humans, IS impairs early EPCs and was strongly correlated with vascular dysfunction. Thus, we speculate that this adverse effect of IS may partly result from the inhibition of early EPCs.

Indoxyl sulfate, not P-cresyl sulfate, is associated with advanced glycation end products in patients on long-term hemodialysis.

BACKGROUND/AIMS: Advanced glycation end products (AGEs) are pro-inflammatory and pro-oxidative compounds that play a critical role in endothelial dysfunction and atherosclerosis. Protein-bound uremic toxins, indoxyl sulfate (IS) and p-cresyl sulfate (PCS), inhibit endothelial function. We explored the association of IS and PCS with AGEs in a hemodialysis (HD) cohort. METHODS: This study was a cross-sectional study that recruited 129 stable patients on maintenance HD in a single medical center from July 1 to July 15, 2011. Serum levels of total and free IS, PCS and AGEs were measured concurrently. General laboratory results and patient background were also investigated. RESULTS: Serum levels of AGEs were associated with total IS (r = 2.7, p < 0.01) but not total PCS (r = 0.01, NS), free IS (r = 0.11, NS) or free PCS (r = 0.04, NS) using Pearson's analysis. Multiple linear regression analysis showed that total IS was significantly related to AGEs (ß = 0.296, p < 0.01), free IS (ß = 0.502, p < 0.01) and creatinine (ß = 0.294, p < 0.01). Serum AGEs levels were also independently correlated with diabetes status (ß = 0.250, p = 0.01) and total IS (ß = 0.341, p < 0.01) concentrations after adjusting for other confounding variables. Moreover, patients with diabetes had higher serum AGEs levels than patients without diabetes (p < 0.01). CONCLUSIONS: These findings suggest that serum levels of total IS were associated with AGEs levels, which may participate in the process of atherosclerosis.

CNS-targeted AAV5 gene transfer results in global dispersal of vector and prevention of morphological and function deterioration in CNS of globoid cell leukodystrophy mouse model.

Globoid cell leukodystrophy (GLD) is a devastating lysosomal storage disease caused by deficiency of the enzyme galactocerebrosidase (GALC). Currently, there is no definite cure for GLD. Several attempts with CNS-directed gene therapy in twitcher mice (a murine model of GLD) demonstrated restricted expression of GALC activity in CNS and failure of therapeutic efficacy in cerebellum and spinal cord, resulting in various degrees of correction of biochemical, pathological and clinical phenotype. More recently, twitcher mice receiving a combination of hematopoietic and viral vector gene transfer therapies were not protected from neurodegeneration and axonopathy in both cerebellum and spinal cord. This evidence indicates the requirement of sufficient and widespread GALC expression in CNS and rescue of cerebellum and spinal cord in the therapeutic intervention of murine model of GLD. In this study, we have optimized intracranial delivery of AAV2/5-GALC to the neocortex, hippocampus and cerebellum, instead of the thalamus as was previously conducted, of twitcher mice. The CNS-targeted AAV2/5 gene transfer effectively dispersed GALC transgene along the neuraxis of CNS as far as the lumbar spinal cord, and reduced the accumulation of psychosine in the CNS of twitcher mice. Most importantly, the treated twitcher mice were protected from loss of oligodendrocytes and Purkinje cells, axonopathy and marked gliosis, and had significantly improved neuromotor function and prolonged lifespan. These preclinical findings with our approach are encouraging, although a more robust response in the spinal cord would be desirable. Collectively, the information in this study validates the efficacy of this gene delivery approach to correct enzymatic deficiency, psychosine accumulation and neuropathy in CNS of GLD. Combining cell therapy such as bone marrow transplantation with treatment with the aim of reducing inflammation, replacing dead or dying oligodendrocytes and targeting PNS may provide a synergistic and more complete correction of this disease.

The discovery of potential acetylcholinesterase inhibitors: a combination of pharmacophore modeling, virtual screening, and molecular docking studies.

BACKGROUND: Alzheimer's disease (AD) is the most common cause of dementia characterized by progressive cognitive impairment in the elderly people. The most dramatic abnormalities are those of the cholinergic system. Acetylcholinesterase (AChE) plays a key role in the regulation of the cholinergic system, and hence, inhibition of AChE has emerged as one of the most promising strategies for the treatment of AD. METHODS: In this study, we suggest a workflow for the identification and prioritization of potential compounds targeted against AChE. In order to elucidate the essential structural features for AChE, three-dimensional pharmacophore models were constructed using Discovery Studio 2.5.5 (DS 2.5.5) program based on a set of known AChE inhibitors. RESULTS: The best five-features pharmacophore model, which includes one hydrogen bond donor and four hydrophobic features, was generated from a training set of 62 compounds that yielded a correlation coefficient of R = 0.851 and a high prediction of fit values for a set of 26 test molecules with a correlation of R² = 0.830. Our pharmacophore model also has a high Güner-Henry score and enrichment factor. Virtual screening performed on the NCI database obtained new inhibitors which have the potential to inhibit AChE and to protect neurons from Aß toxicity. The hit compounds were subsequently subjected to molecular docking and evaluated by consensus scoring function, which resulted in 9 compounds with high pharmacophore fit values and predicted biological activity scores. These compounds showed interactions with important residues at the active site. CONCLUSIONS: The information gained from this study may assist in the discovery of potential AChE inhibitors that are highly selective for its dual binding sites.

A novel mutation in PYCR1 causes an autosomal recessive cutis laxa with premature aging features in a family.

The autosomal recessive form of type II cutis laxa (ARCL II) is characterized by the appearance of redundant, inelastic skin with wrinkling, an aged look and additional variable systemic involvement including intrauterine growth retardation, failure to thrive, developmental delay, dysmorphism, osseous abnormality, and CNS manifestations. Several genetic defects have been found in patients and families with the clinical manifestations of ARCL II. Recently, mutations in PYCR1 have been linked to cutis laxa with progeroid features. We ascertained two siblings with of ARCL II born to non-consanguineous parents. Mutation analysis of PYCR1 revealed a novel single-base deletion (c.345delC) in exon 4 leading to frame-shift and premature stop of translation. The effect of this mutation results in a strong reduction of PYCR1 expression in skin fibroblasts from affected siblings. These two cases extend the genotypic spectrum of PYCR1-related ARCL II.

Compound heterozygous mutations in PYCR1 further expand the phenotypic spectrum of De Barsy syndrome.

De Barsy syndrome (DBS) is characterized by progeroid features, ophthalmological abnormalities, intrauterine growth retardation, and cutis laxa. Recently, PYCR1 mutations were identified in cutis laxa with progeroid features. Herein, we report on a DBS patient born to a nonconsanguineous Chinese family. The exceptional observation of congenital glaucoma, aortic root dilatation, and idiopathic hypertrophic pyloric stenosis in this patient widened the range of symptoms that have been noted in DBS. Mutation analysis of PYCR1 revealed compound heterozygous PYCR1 mutations, including a p.P115fsX7 null mutation allele and a second allele with two missense mutations in cis: p.G248E and p.G297R. The effect of mutation results in a reduction of PYCR1 mRNA expression and PYCR1 protein expression in skin fibroblasts from the patient. The findings presented here suggest a mutation screening of PYCR1 and cardiovascular survey in patients with DBS.

Transformation of 15-ene steviol by Aspergillus niger, Cunninghamella bainieri, and Mortierella isabellina.

Transformation of 15-ene steviol (ent-13-hydroxy-kaur-15-en-19-oic acid) by growth cultures of Aspergillus niger BCRC 32720, Cunninghamella bainieri ATCC 9244, and Mortierella isabellina ATCC 38063 was conducted to generate various derivatives for the development of bioactive compounds. Four previously undescribed compounds along with six known compounds were obtained. The newly identified isolates were characterized using 1D and 2D NMR, IR, and HRESIMS, and three compounds were further confirmed by X-ray crystallographic analyses. Subsequently, the effects of 15-ene steviol and its derivatives on lipopolysaccharide (LPS)-induced cytokine production by THP-1 cells were examined, with dexamethasone used as a positive control. Results indicated that most of the tested compounds showed lower inhibitory effects than those detected in the dexamethasone-treated group, except that 15-ene steviol showed better effects than dexamethasone on the reduction of LPS-induced monocyte chemoattractant protein (MCP)-1, -2, and -3 release. Three specialized products similarly showed better effects than dexamethasone on the inhibition of LPS-induced secretion of regulated on activation, normal T cell expressed and secreted (RANTES). Moreover, none of the tested compounds showed any cytotoxicity or triggered cell apoptosis, and none affected the protein integrity of toll-like receptor 4 (TLR4) or MyD88, suggesting that these compounds may exert the anti-inflammatory activity downstream of membrane-associated TLR4 and MyD88 molecules.

Molecular dynamics simulations to investigate the structural stability and aggregation behavior of the GGVVIA oligomers derived from amyloid beta peptide.

Several neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's diseases, are associated with amyloid fibrils formed by different polypeptides. Recently, the atomic structure of the amyloid-forming peptide GGVVIA from the C-terminal hydrophobic segment of amyloid-beta (Abeta) peptide has been determined and revealed a dry, tightly self-complementing structure between two beta-sheets, termed as "steric zipper". In this study, several all-atom molecular dynamics simulations with explicit water were conducted to investigate the structural stability and aggregation behavior of the GGVVIA oligomers with various sizes. The results of our single-layer models suggested that the structural stability of the GGVVIA oligomers increases remarkably with increasing the numbers of beta-strands. We further identified that SH2-ST2 may act as a stable seed in prompting amyloid fibril formations. Our results also demonstrated that hydrophobic interaction is the principle driving force to stabilize and associate the GGVVIA oligomers between beta-strands; while the hydrophobic steric zipper formed via the side chains of V3, V4, and I5 plays a critical role in holding the two neighboring beta-sheets together. Single glycine substitution at V3, V4, and I5 directly disrupted the hydrophobic steric zipper between these two beta-sheets, resulting in the destabilization of the oligomers. Our simulation results provided detailed insights into understanding the aggregation behavior of the GGVVIA oligomers in the atomic level. It may also be helpful for designing new inhibitors able to prevent the fibril formation of Abeta peptide.

Fibronectin modulates the morphology of osteoblast-like cells (MG-63) on nano-grooved substrates.

Cell interactions with biomaterials are affected by surface topographic and chemical cues. Although it is well-known that nanometrical grooves/ridges structure modulates cellular spreading, elongation, and alignment, the combinational influence of surface topographic and chemical cues is not well studied. In this study, nano-textured silicon substrata with parallel ridges of 90, 250, or 500 nm wide, separated by grooves with equal width, were fabricated by electron beam lithography and dry etching techniques. Osteoblast-like cells, MG-63, were cultured on the patterned substrata with or without pre-adsorption of fibronectin. The cell morphology was imaged by scanning electron microscopy, and analyzed by image software. We found that FN coating initially modulated cellular spreading, length, and orientation on all types of grooved surfaces. However, after 24 h of culture, the cell morphology was not affected by FN coating on the 250-nm and 500-nm surfaces, while FN decreased cell alignment on the 90-nm surfaces. Our results suggest that surface chemical cues influence the initial cell-substratum contact, while the long-term cellular morphology is dictated by surface topographic cues.

Dose-dependent effect of Lactobacillus rhamnosus on quantitative reduction of faecal rotavirus shedding in children.

Beneficial effects of probiotics in acute infectious diarrhoea in children are mainly seen in watery diarrhoea and viral gastroenteritis. Lactobacillus rhamnosus, one the most extensively studied probiotic strains, is effective in shortening courses of acute diarrhoea in children. However, the dose-dependent effect of Lactobacillus upon quantification of faecal rotavirus shedding in humans remains little known. Thus, an open-label randomized trial in 23 children with acute rotaviral gastroenteritis was undertaken by randomly allocating patients to receive one of the three regimens for 3 days: daily Lactobacillus rhamnosus 35 (Lcr35) with 0 CFU/day to six patients in the control group, 2 x 10(8) CFU/day to nine patients in the low-dose group, and 6 x 10(8) CFU/day to eight patients in the high-dose group. Faecal samples were collected before and after the 3-day regimen for measurements of rotavirus concentrations by ELISA. There was no statistically significant change in faecal rotavirus concentrations in either the control group (119.2 x 10(5) particles/ml vs. 23.7 x 10(5) particles/ml, p = 0.075) or the low-dose group (36.1 x 10(5) particles/ml vs. 73.5 x 10(5) particles/ml, p = 0.859). However, the high-dose group had a significant reduction of faecal rotavirus concentration (64.2 x 10(5) particles/ml vs. 9.0 x 10(5) particles/ml, p = 0.012). Without any exception, the faecal rotavirus concentrations of all eight patients in the high-dose Lcr35 group declined by 86% after 3 days when compared with those before Lcr35 administration. In conclusion, this is the first report to provide quantitative evidence of the dose-dependent effect of Lactobacillus rhamnosus, a minimal effective dose of 6 x 10(8) CFU for 3 days, upon the faecal rotavirus shedding in paediatric patients.

Correction: The role of indoxyl sulfate in renal anemia in patients with chronic kidney disease.

[This corrects the article DOI: 10.18632/oncotarget.18789.].

Resveratrol inhibits human leiomyoma cell proliferation via crosstalk between integrin αvß3 and IGF-1R.

Leiomyomas (myomas) are the most common benign smooth muscle cell tumor of the myometrium. Resveratrol, a stilbene, has been used as an anti-inflammatory and antitumor agent. In the current study, we investigated the inhibitory effect of resveratrol on the proliferation of primary human myoma cell cultures. Resveratrol arrested cell proliferation via integrin αvß3. It also inhibited integrin αvß3 expression and protein accumulation. Concurrently, constitutive AKT phosphorylation in myoma cells was inhibited by resveratrol. Expressions of proapoptotic genes, such as cyclooxygenase (COX)-2, p21 and CDKN2, were induced by resveratrol in myoma cells. On the other hand, expressions of proliferative (anti-apoptotic) genes were either inhibited, as in BCL2, or unchanged, as in cyclin D1 and proliferating cell nuclear antigen (PCNA). The accumulation of insulin-like growth factor (IGF)-1 receptor (IGF-1R) was inhibited by resveratrol in primary myoma cells. IGF-1-induced cell proliferation was inhibited by co-incubation with resveratrol. Therefore, growth modulation of myoma cells occurs via mechanisms dependent on cross-talk between integrin αvß3 and IGF-1R. Our findings suggest that resveratrol can be considered an alternative therapeutic agent for myomas.

Inhibitory Effect of Anoectochilus formosanus Extract on Hyperglycemia-Related PD-L1 Expression and Cancer Proliferation.

Traditional herb medicine, golden thread (Anoectochilus formosanus Hayata) has been used to treat various diseases. Hyperglycemia induces generation of reactive oxygen species (ROS) and enhancement of oxidative stress which are risk factors for cancer progression and metastasis. In this study, we evaluated hypoglycemic effect of A. formosanus extracts (AFEs) in an inducible hyperglycemia animal model and its capacity of free-radical scavenging to establish hyperglycemia-related carcinogenesis. AFE reduced blood glucose in hyperglycemic mice while there was no change in control group. The incremental area under blood glucose response curve was decreased significantly in hyperglycemic mice treated with AFE in a dose-dependent manner. AFE and metformin at the same administrated dose of 50 mg/kg showed similar effect on intraperitoneal glucose tolerance test in hyperglycemic mice. Free-radical scavenger capacity of AFE was concentration dependent and 200 µg/ml of AFE was able to reduce more than 41% of the free radical. Treatment of cancer cells with AFE inhibited constitutive PD-L1 expression and its protein accumulation. It also induced expression of pro-apoptotic genes but inhibited proliferative and metastatic genes. In addition, it induced anti-proliferation in cancer cells. The results suggested that AFE not only reduced blood glucose concentration as metformin but also showed its potential use in cancer immune chemoprevention/therapy via hypoglycemic effect, ROS scavenging and PD-L1 suppression.

Bioinformatics approaches for disulfide connectivity prediction.

Protein structure prediction with computational methods has gained much attention in the research fields of protein engineering and protein folding studies. Due to the vastness of conformational space, one of the major tasks is to restrain the flexibility of protein structure and reduce the search space. Many studies have revealed that, with the information of disulfide connectivity available, the search in conformational space can be dramatically reduced and lead to significant improvements in the prediction accuracy. As a result, predicting disulfide connectivity using bioinformatics approaches is of great interest nowadays. In this mini-review, the prediction of disulfide connectivity in proteins will be discussed in four aspects: (1) how the problem formulated and the computational techniques used in the literatures; (2) the effects of the features adopted to encode the information and the biological meanings implied; (3) the problems encountered and limitations of disulfide connectivity prediction; and (4) the practical usages of predicted disulfide bond information in molecular simulation and the prospects in the future.

Molecular dynamics simulations of human cystatin C and its L68Q varient to investigate the domain swapping mechanism.

Human cystatin C variant (L68Q), one of the amyloidgenic proteins, has been shown to form dimeric structure spontaneously via domain swapping and easily cause amyloid deposits in the brains of patients suffering from Alzheimer's disease or hereditary cystatin C amyloid angiopathy. The monomeric L68Q and wild-type (wt) HCCs share similar structural feature consisting of a core with a five-stranded anti-parallel beta-sheet (beta-region) wrapped around a central helix. In this study, various molecular dynamics simulations were conducted to investigate the conformational fluctuations of the monomeric L68Q and wt HCCs at various combinations of temperature (300 and 500K) and pH (2 and 7) to gain insights into the domain swapping mechanism. The results show that elevated temperature accelerates the disruption of the hydrophobic core and acidic condition promotes the destruction of three salt bridges between beta2 and beta3 in both HCCs. The results also indicate that the interior hydrophobic core of the L68Q variant is relatively unstable, leading to domain swapping more readily comparing to wt HCC under conditions favoring this process. However, these two monomeric HCCs adopt the same mechanism of domain swapping as follows: (i) first, the interior hydrophobic core is disrupted; (ii) subsequently, the central helix departs from the beta-region; (iii) then, the beta2-L1-beta3 hairpin structure unfolds following the so-called "zip-up" mechanism; and (iv) finally, the open form HCC is generated.

Molecular dynamics simulations to investigate the effects of zinc ions on the structural stability of the c-Cbl RING domain.

In eukaryotic cells, ubiquitylation of proteins plays a critical role in regulating diverse cell processes by the ubiquitin activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin protein ligase (E3). E3 is the key component that confers specificity to ubiquitylation and directs the conjugation of ubiquitin to a specific target protein. RING domains are small structured protein domains that require the coordination of zinc ions for a stable tertiary fold and some of them are involved in the E3 family. In this study, we reported the detailed relationships between the two zinc ions and the structural stability of the c-Cbl RING domain by molecular dynamics simulations. Our results show that these two zinc ions play an important role in maintaining both the secondary and tertiary structural stabilities of the c-Cbl RING domain. Our results also reveal that the secondary structural stability of the c-Cbl RING domain is mainly determined by the hydrogen-bonding networks in or near the two zinc ion binding sites. Our results further demonstrate that zinc ion binding site 2 is more structurally stable than site 1.

Molecular dynamics simulations to investigate the domain swapping mechanism of human cystatin C.

Human cystatin C (HCC), one of the amyloidgenic proteins, has been proved to form a dimeric structure via a domain swapping process and then cause amyloid deposits in the brains of patients suffering from Alzheimer's disease. HCC monomer consists of a core with a five-stranded antiparallel beta-sheet (beta region) wrapped around a central helix. The connectivity of these secondary structures is: (N)-beta1-alpha-beta2-L1-beta3-AS-beta4-L2-beta5-(C). In this study, various molecular dynamics simulations were conducted to investigate the conformational changes of the monomeric HCC at different temperatures (300 and 500 K) and pH levels (2, 4, and 7) to gain insight into the domain swapping mechanism. The results show that high temperature (500 K) and low pH (pH 2) will trigger the domain swapping process of HCC. We further proposed that the domain swapping mechanism of HCC follows four steps: (1) the alpha-helix moves away from the beta region; (2) the contacts between beta2 and beta3-AS disappear; (3) the beta2-L1-beta3 hairpin unfolds following the so-called "zip-up" mechanism; and finally (4) the HCC dimer is formed. Our study shows that high temperature can accelerate the unfolding of HCC and the departure of the alpha-helix from the beta-region, especially at low pH value. This is attributed to the fact that that low pH results in the protonation of the side chains of Asp, Glu, and His residues, which further disrupts the following four salt-bridge interactions stabilizing the alpha-beta interface of the native structure: Asp15-Arg53 (beta1-beta2), Glu21/20-Lys54 (helix-beta2), Asp40-Arg70 (helix-AS), and His43-Asp81 (beta2-AS).

Effects of Sevelamer Hydrochloride on Uremic Toxins Serum Indoxyl Sulfate and P-Cresyl Sulfate in Hemodialysis Patients.

BACKGROUND: Beside the phosphate binding effect, non-calcium non-aluminum phosphate binder, namely sevelamer hydrochloride (SH), has many other effects in dialysis patients. It can absorb many other compounds, decrease low-density lipoprotein cholesterol (LDL-C) level, and attenuate the progression of vascular calcification; it has been reported to have anti-inflammatory effect. However, it is not clear whether it has any effect on uremic toxins, i.e. serum indoxyl sulfate (IS) and p-cresyl sulfate, (PCS) in hemodialysis (HD) patients. This study was carried out to appraise the effect of sevelamer on serum IS and PCS in HD patients. METHODS: Five adult HD patients from a single medical center were enrolled in this study; these patients were treated with 800 mg of sevelamer thrice per day for 3 months; a series of biochemical parameters, serum IS and PCS were monitored concurrently. RESULTS: There was a significant reduction in the mean level of phosphate from 7.20 ± 0.70 mg/dL (mean ± SD) before treatment to 5.40 ± 0.50 mg/dL (mean ± SD) after treatment, total cholesterol from 151.00 ± 37.40 mg/dL (mean ± SD) before treatment to 119.20 ± 29.40 mg/dL (mean ± SD) after treatment, and PCS from 31.30 ± 10.60 mg/L (mean ± SD) before treatment to 19.70 ± 10.50 mg/L (mean ± SD) after treatment. On the contrary, this treatment had no effect on IS. CONCLUSION: A statistically significant reduction of serum phosphate and PCS in HD patients treated with SH suggests that beside the action of lowering serum phosphate, sevelamer may have an important role in the treatment of uremic syndrome by decreasing the uremic toxin.

Biological Mechanisms by Which Antiproliferative Actions of Resveratrol Are Minimized.

Preclinical and clinical studies have offered evidence for protective effects of various polyphenol-rich foods against cardiovascular diseases, neurodegenerative diseases, and cancers. Resveratrol is among the most widely studied polyphenols. However, the preventive and treatment effectiveness of resveratrol in cancer remain controversial because of certain limitations in existing studies. For example, studies of the activity of resveratrol against cancer cell lines in vitro have often been conducted at concentrations in the low µM to mM range, whereas dietary resveratrol or resveratrol-containing wine rarely achieve nM concentrations in the clinic. While the mechanisms underlying the failure of resveratrol to inhibit cancer growth in the intact organism are not fully understood, the interference by thyroid hormones with the anticancer activity of resveratrol have been well documented in both in vitro and xenograft studies. Thus, endogenous thyroid hormones may explain the failure of anticancer actions of resveratrol in intact animals, or in the clinic. In this review, mechanisms involved in resveratrol-induced antiproliferation and effects of thyroid hormones on these mechanisms are discussed.