",isbn:"978-1-80356-420-3",printIsbn:"978-1-80356-419-7",pdfIsbn:"978-1-80356-421-0",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,isSalesforceBook:!1,hash:"f188555eee4211fc24b6cca361983149",bookSignature:"Dr. Kim Ho Yeap",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/11509.jpg",keywords:"Inductive Coupling, Resonant Inductive Coupling, Magnetic Coupling, Magnetic Resonance, Transmitter, Receiver, Rectenna, Antenna, Induction Coil, Stationery Charging, Dynamic Charging, Rectifier",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"February 25th 2022",dateEndSecondStepPublish:"May 6th 2022",dateEndThirdStepPublish:"July 5th 2022",dateEndFourthStepPublish:"September 23rd 2022",dateEndFifthStepPublish:"November 22nd 2022",remainingDaysToSecondStep:"16 days",secondStepPassed:!0,currentStepOfPublishingProcess:3,editedByType:null,kuFlag:!1,biosketch:"Dr. Kim Ho Yeap is a senior member of the IEEE, a Chartered Engineer registered with the UK Engineering Council, a Professional Engineer (PEng) registered with the Board of Engineers Malaysia, and an ASEAN Chartered Professional Engineer. In 2008 and 2015 he underwent research attachment at the University of Oxford (UK) and the Nippon Institute of Technology (Japan). Dr. Yeap has been given the university teaching excellence award and 21 research grants. He has published more than 100 research articles.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"126825",title:"Dr.",name:"Kim Ho",middleName:null,surname:"Yeap",slug:"kim-ho-yeap",fullName:"Kim Ho Yeap",profilePictureURL:"https://mts.intechopen.com/storage/users/126825/images/system/126825.jpeg",biography:"Dr. Kim Ho Yeap is an associate professor at Universiti Tunku Abdul Rahman, Malaysia. He is a senior member of the IEEE, a Chartered Engineer (CEng) registered with the UK Engineering Council, a Professional Engineer (PEng) registered with the Board of Engineers Malaysia, and an ASEAN Chartered Professional Engineer. He received his BEng (Hons) in Electrical and Electronics Engineering from Universiti Teknologi Petronas in 2004, his MSc in Microelectronics from Universiti Kebangsaan Malaysia in 2005, and his PhD from Universiti Tunku Abdul Rahman in 2011. In 2008 and 2015, respectively, he underwent research attachment at the University of Oxford (UK) and the Nippon Institute of Technology (Japan). He is the external examiner and external course assessor of Wawasan Open University. He is also the editor-in-chief of the i-manager’s Journal on Digital Signal Processing. He has also been a guest editor for the Journal of Applied Environmental and Biological Sciences and Journal of Fundamental and Applied Sciences. In addition, he has been given the university teaching excellence award and 21 research grants. He has published more than 100 research articles (including refereed journal papers, conference proceedings, books, and book chapters), which are mostly related to electromagnetics. Among his notable publications—and those of which he is most proud—are the report on the design of the receiver optics used in the Atacama Large Millimeter/Submillimeter Array telescope and the formulations that rigorously describe wave propagation in lossy waveguides.",institutionString:"Universiti Tunku Abdul Rahman",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"3",totalChapterViews:"0",totalEditedBooks:"1",institution:{name:"Universiti Tunku Abdul Rahman",institutionURL:null,country:{name:"Malaysia"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"11",title:"Engineering",slug:"engineering"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"444315",firstName:"Karla",lastName:"Skuliber",middleName:null,title:"Mrs.",imageUrl:"https://mts.intechopen.com/storage/users/444315/images/20013_n.jpg",email:"karla@intechopen.com",biography:"As an Author Service Manager, my responsibilities include monitoring and facilitating all publishing activities for authors and editors. 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1. Introduction
Smith-Lemli-Opitz syndrome (SLOS), OMIM #270400, is one of the nine known disorders associated with altered post-squalene cholesterol biosynthesis [1, 2]. This autosomal recessive genetic disease was first described in 1964, as a syndrome of cognitive impairment and multiple malformations [3]. Thirty years were needed to further characterize the disorder as a metabolic disease and identify the underlying enzymatic defect. SLOS is caused by deficiency of 7-dehydrocholesterol redutase (7-DHCR, 3-hydroxysteroid redutase, EC.1.3.1.21) which catalyzes the conversion of 7-dehydrocholesterol (7DHC) to cholesterol, the terminal step of Kandutsch-Russell pathway [4, 5]. Consequently, SLOS patients typically show increased 7DHC and decreased serum and tissue cholesterol levels [5]. In 1998, mutations of the 3β-hydroxysterol Δ7-reductase gene (DHCR7) were shown to cause SLOS [6, 7, 8] and more than 154 DHCR7 mutations have been so far identified in SLOS patients [9]. The development of animal models has improved the understanding of SLOS physiopathology and provided material for in vivo and in vitro investigation of biological consequences of cholesterol deficiency. In 2001, two mouse models with null mutations in Dhcr7 gene have been created by homologous recombination [10, 11]. The malformations in the null mice are very mild compared to what would be seen in a null human infant (i.e., SLO type II). The mutant mice died within the first 24 h of extra-uterine life. Later, a mouse model with a milder phenotype was developed [12]. This hypomorphic mouse has a missense mutation equivalent to the human p.T93M, previously identified in SLOS patients often with Mediterranean heritage [13, 14, 15]. Like the majority of SLOS patients, the SLOS mouse models manifest a deficiency in cholesterol biosynthesis resulting in low levels of cholesterol in serum and tissues [11].
Effective cholesterol biosynthesis is especially critical at certain stages during development and continues to be important throughout life [16], since cholesterol is an essential lipid with multiple functions. Cholesterol is a major lipid component of membrane microdomains, which are crucial cell-surface dynamic structures responsible for many cellular signaling and communication events [17]. Membrane domains can form through a number of mechanisms involving lipid-lipid and protein-lipid interactions. One type of membrane domain is the cholesterol-dependent membrane raft [18]. Properties of these membrane domains have been primarily inferred from the study of detergent-resistant membranes (DRMs), composed by the non-ionic-detergent insoluble, low-buoyant density membranous fractions of cells [19]. Although it was initially thought that such microdomains enriched in cholesterol exist exclusively in the plasma membrane, increasing evidence suggests that similar lipid microdomains (sometimes referred as raft-like microdomains) are also present in internal organelles [20, 21, 22] with some of them being involved in the crosstalk between organelles [23].
Proteomics constitutes a powerful tool to study complex biological mechanisms and to identify alterations in protein expression induced by changes in the environment, drugs, or disease states. As such, proteomics is now widely employed to help understand pathological processes induced by the disease [24, 25, 26, 27]. The effect of an inborn error of cholesterol synthesis on skeletal muscle has not previously been reported. In this chapter, we report a comparative analysis of protein expression in skeletal muscle DRMs isolated from Dhcr7 T93M homozygous mutant mice (Dhcr7T93M/T93M) and wild type controls (Dhcr7+/+) controls. We analyzed sterols by GC–MS and we used amine-reactive isobaric tagging reagents (iTRAQ) for quantitative sub-cellular proteomics. We found the altered expression of key muscle proteins involved with bioenergetics, membrane transport and Ca2+ homeostasis.
2. Material and methods
2.1. Materials
Butylhydroxytoluene (BHT), N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA), triethylammonium bicarbonate (TEAB), trifluoroacetic acid (TFA), α-cyano-4-hydroxycinnamic acid (α-CHCA), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), protease inhibitor cocktail, formic acid and urea were purchased from Sigma (Karlsruhe, Germany). RC DC Protein Assay kit for protein quantification was from BioRad lab (Hercules, USA). The iTRAQ kit was purchased from Applied Biosystems (Foster City, CA). Sequencing grade modified trypsin (bovine) was from ABSciex (ABSciex, USA). HPLC-grade acetonitrile (ACN, Riedel, Seelze, Germany) and Milli-Q grade water were also used. Rabbit raised polyclonal anti-caveolin 1 (ab2910) and anti-annexin A2 antibodies were purchased from Abcom, Cambridge, UK. Analytical reagent grade chemicals were used unless stated otherwise.
2.2. Animals
The T93M mutation [12] was backcrossed into C57/BL6 for three generations. Homozygous (T93M/T93M) mice are viable and fertile [16]. Control C57/BL6 mice were obtained from IBMC Animal Centre from Oporto University.
All the animals were housed in plastic cages with free access to water and food (cholesterol free—chow -Mucedola, Ref: 4RF21). The animals were handled and maintained in controlled conditions according to international standards. Animals were euthanized using deep isoflurane anesthesia when they were 10 days old. Gastrocnemius and soleus muscles were dissected, submerged in ice-cold buffer (TRIS, HCl, pH 7.4, 10 mM mercaptoethanol, 0.28 M sucrose) and immediately frozen at −80°C.
2.3. DRMs extraction
Detergent-resistant membranes (DRMs) were isolated using cold Triton X-100 treatment followed by sucrose gradient centrifugation. In order to minimize individual variation, samples were analyzed as pools of several animals. The procedure was adapted from Kim et al. [28]. Briefly, the samples were allowed to thaw slowly on ice, and tissue from three mice (approximately 300 mg) was minced with scissors, mixed with 700 µL of cold (4°C) lysis buffer (25 nM HEPES-HCl, pH 6,5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and protease inhibitor cocktail) and homogenized 30 times with a Poter homogenizer. The sample was maintained in an ice bath during homogenization and then incubated for 30 min at 4°C. Aliquots of this homogenate were collected for protein quantification and sterol analysis.
The resulting extract was mixed with an equal volume of cold sucrose 80% (w/v) to give a final sucrose concentration of 40%, transferred to the bottom of a ultra-centrifuge tube (Ultra-clear 14 × 89 mm, Beckman Ref 344059) and overlaid carefully with 6.0 mL 30% and 3.0 mL 5% cold sucrose solutions, containing 25 nM HEPES-HCl, pH 6.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100. Discontinuous sucrose gradients were centrifuged for 20 h, at 187,000 g on a Sorvall Ultra Pro 80 UC, swinging bucket SW41 at 4°C. The DRMs fraction, which collects at the interface of the 5 and 30% sucrose layers, was isolated, washed with 9 mL of modified HEPES buffer and centrifuged 30 min at 49,500 g at 4°C. After centrifugation, the supernatant was discarded and the pellet containing purified DRMs was suspended in phosphate buffered saline (PBS) and stored frozen at −80°C until further proteomic or sterol analysis.
2.4. Sterols analysis
For extraction of neutral sterols, 50 µL of sample was mixed with 50 µL of BHT 0.05% in methanol (antioxidant), 50 µL of epicoprostanol 0.10 mmol/L (internal standard) and saponified at 60°C for 1 h by 16 adding 1 mL of 4% (w/v) KOH in 90% ethanol. The samples were then diluted with 1 mL of deionized water and the lipids were extracted twice with 2 mL of hexane. The pooled hexane extracts were dried under a gentle nitrogen stream at room temperature and derivatized with 50 µL BSTFA in 50 µL pyridine at 60°C for 1 h [29]. The trimethylsilylether derivatives of sterols were separated by GC–MS with a Supelco 28471_U SLB-5MS column (30 m × 0.32 mm i.d., 0.25 μm film thickness). The injector temperature was set at 270°C, and the splitless injection mode was used. The initial column temperature was 180°C for 1 min and programmed to increase at a rate of 20°C/min to 250°C and then increased again at 5°C/min rate till 300°C. The carrier gas was helium at a linear constant flow rate of 40 cm/s. The interface was programmed to 280°C, the quadrupole to 150°C and the ionization source to 230°C. After a solvent delay of 2.5 min, the eluted sterols were identified by their retention times (comparing with commercial standards) and respective mass spectra and quantified by selected ion monitoring (Table 1). The method was linear and reproducible for the range of amounts assayed.
Sterol
Retention time
m/z
Epicoprostanol
13.02
370
257
355
Cholesterol
13.72
329
353
368
7DHC
14.02
325
351
366
Desmosterol
14.10
343
327
Sitosterol
14.80
357
396
486
Lathosterol
15.80
255
443
458
Table 1.
Sterol parameters.
The m/z ions selected for sterol quantification are printed in bold.
2.5. Proteomic profile
2.5.1. Pellet solubilization and protein quantification
Each assay involved four sample pools: a wt-BL6 control and a Dhcr7T93M/T93M and their duplicates. For protein extraction, the DRMs pellets were treated with 10 volumes (w/v) of solubilization buffer (7 M urea, 2 M thiourea, 1% (w/v) CHAPS, 1% (w/v) Triton X-100, 1% (v/v) ampholytes (3–10) and 1 mM TCEP), sonicated briefly, then incubated for 10 min at room temperature under agitation. Clear cell lysates were obtained by centrifugation at 12,000×g for 30 min at 4°C and saved for analysis. Insoluble material was discarded.
The protein concentration was determined by RC DC Protein Assay kit, using BSA to generate the calibration curve.
2.5.2. Sample preparation for mass spectrometry analysis
Reduction, alkylation and digestion steps were performed according to the protocol provided by the manufacturer. Briefly, 100 mg of DRM protein was mixed with TEAB buffer (1 M, pH 8.5) and an enhancer of enzymatic digestion RapiGest (Waters) to give a final concentration of 0.5 M and 0.1%, respectively. Samples were then treated with: (1) a reducing agent—TCEP 5 mM—used to break disulfide bonds within and between proteins, for 1 h at 37°C and (2) a cysteine (sulfhydryl group) blocking reagent—S-methyl methanethiosulfonate (MMTS) 10 mM—for 10 min, at room temperature. Then, 2 mg of trypsin was added to each sample and the digestion was performed for 18 h at 37°C. The digested tryptic specimens were dried using a Speed-Vac. iTRAQ labeling was carried out according to the instructions provided by the manufacturer. Dhcr7T93M/T93M mice DRMs samples were marked with iTRAQ Tags 116 and wt-BL6 with114 and the duplicates with 115 and 113, respectively. The labeled samples were combined in pairs and dried in a Speed-Vac. The peptides were separated by reverse-phase liquid chromatography as previously published [30].
2.5.3. LC-MS/MS analysis
Peptide mass spectra were obtained in the mass range 700–4500 Da on a MALDI-TOF/TOF mass spectrometer (4800 Proteomics Analyzer, Applied Biosystems, Foster City, CA, USA) in the positive ion reflector mode. The obtained spectra were processed and analyzed by the ProteinPilot® software (v4.0 AB Sciex, USA), which uses an algorithm for protein/peptide identification based on the comparison of MS/MS data against the SwissProt protein database [31].
Protein clustering was performed according to biological and molecular functions derived from the PANTHER classification system.
2.6. Immunocytochemistry assay
Immunocytochemistry assays were performed in order to confirm the presence of (1) caveolin-1, a protein marker of a subtype of membrane microdomains which are rich in cholesterol, designated caveolae (anti-caveolin-1 antibody dilution 1:500) and (2) annexin 2 another protein associated with enriched cholesterol microdomains, that was found increased in the SLO mouse model (antibody dilution 1:2000). A sequential incubation with a secondary biotinylated anti-rabbit antibody was performed and diaminobenzidine (which stains brown) was employed as chromogen. Skeletal muscle samples were then counterstained with hematoxylin.
3. Results and discussion
3.1. Sterols
Sterols were extracted from two pools of biological samples: muscle homogenates and muscle DRMs, and then analyzed by GC-MS. Results are presented as the average ratios between sterols amounts. We observed a markedly elevated cholesterol/7DHC ratio in muscle homogenates from wild-type animals, reflecting the large amount of cholesterol and minimal levels of its precursor (7DHC), in controls. This ratio was also much greater than one in Dhcr7T93M/T93M mice, indicating that affected mice have significant residual 7-DHCR enzymatic activity and are thus capable of producing significant amounts of cholesterol (Figure 1A).
Figure 1.
Average ratios between the amounts of: cholesterol and 7DHC (A), 7DHC and desmosterol (B) and 7DHC and cholesterol (C), extracted from skeletal muscle homogenates (M) and DRMs of wt-BL6 controls and hypomorphic Dhcr7T93M/T93M mice pooled samples.
Desmosterol is a cholesterol precursor by an alternative biosynthetic route, and there are no significant differences in desmosterol levels between controls and affected animals. This is also reflected in the 7DHC/desmosterol ratio. This parameter showed an enrichment of Dhcr7T93M/T93M animals’ DRMs in 7DHC (Figure 1B). The ratio 7DHC/cholesterol also indicates that 7DHC is preferentially incorporated in membrane microdomains (Figure 1C). These findings corroborate the previous ones published by Rakheja and Boriack, based on liver analysis of SLOS patients, which showed that 7DHC accumulates in hepatic DRMs [32]. Furthermore, while wt-BL6 controls have essentially only cholesterol in DRMs, affected mice present a mixture of cholesterol and 7DHC (Figure 2).
Figure 2.
Comparative analysis of sterol composition of DRMs extracted from skeletal muscle of wt-BL6 controls and hypomorphic DHCR7T93M/T93M (SLOS) mice.
3.2. Proteomics
In order to explore the protein changes on sarcolemma, due to decreased 7-DHCR activity, we analyzed DRMs utilizing iTRAQ labeling and LC-MS/MS.
A total of 133 unique proteins were identified. Those identified based on a single peptide and those with a protein score less than 2.5 fold were excluded. Then a cut-off of 30% was applied to iTRAQ average ratios allowing us to select proteins with an important variation in SLOS mice relatively to controls. Differential protein expression was specific and not just a general finding. Caveolin-1, a protein known to be expressed in cholesterol-rich membrane microdomains did not show differential expression (Figure 3).
Figure 3.
Immunohistochemical stain for caveolin-1 (×10) shows no significant differences between Dhcr7 93M/93M (on the left) and wt-BL6 (on the right) skeletal muscle samples.
Of the 133 identified proteins, we observed an altered expression of 38 (29%) proteins. Increased and decreased expression was observed for 17 and 21 proteins, respectively (Table 2). The replicate samples demonstrated a strong positive correlation (r = 0.90) and indicated good reproducibility (Figure 4).
NADH dehydrogenase [ubiquinone] iron-sulfur protein 5
2
0.5
5.9
13
P68134
ACTS_MOUSE
Actin, alpha skeletal muscle
5
0.4
8.8
46
P19783
COX41_MOUSE
Cytochrome c oxidase subunit 4 isoform 1, mitochondrial
12
0.4
2.6
3
P68369
TBA1A_MOUSE
Tubulin alpha-1A chain
2
0.2
3.0
4
Q08857
CD36_MOUSE
Platelet glycoprotein 4
2
0.2
Table 2.
Proteins from the skeletal muscle DRMs with altered expression Dhcr7T93M/T93M mice.
Figure 4.
Comparison of the individual ratio values found for 38 proteins of DRMs extracted from skeletal muscle, which exhibited distinct levels of expression on hypomorphic Dhcr7T93M/T93M mice and wt-BL6 controls.
Most proteins showing an altered expression in DRMs preparations were found to participate in at least one of three main cellular processes: membrane trafficking, energy production and Ca2+ homeostasis.
Proteins with altered expression corresponded to a number of biological processes. We found alterations affecting several membrane transporters. Even though no results from skeletal muscle studies of SLOS are available, membrane trafficking abnormalities, in other cells harboring inborn errors of cholesterol biosynthesis, had already been reported. For example, in cultivated human skin fibroblasts from SLOS patients the membrane fluidity is altered, calcium permeability is augmented whereas folate uptake and membrane-bound Na+/K+ ATPase activity are markedly decreased [33]. In agreement with these data, we now report decreased expression of phospholemman, a small plasma transmembrane protein that acts as a channel or channel regulator and modulates Na+/K+ ATPase activity [34, 35]. Further, we detected significantly decreased expression of another integral membrane glycoprotein associated with DRMs, the fatty acid translocase (FAT also called Cd36 or platelet glycoprotein 4) on sarcolemma of SLOS mice, responsible for the uptake of long chain fatty acids [36, 37, 38, 39]. Upregulated transporters include mitochondrial phosphate carrier protein (which transports inorganic phosphate into the mitochondrial matrix, essential for the aerobic synthesis of ATP) and ADP/ATP translocases 1 and 2 (that catalyzes the exchange of cytoplasmic ADP with mitochondrial ATP across the mitochondrial inner membrane).
These abnormalities of mitochondrial transporters may be indicative of more generalized defect in mitochondrial function and ATP production. Decreased expression of subunits corresponding to complexes I, III and IV of the oxidative phosphorylation (OXPHOS) system was observed, while four ATP-synthase subunits showed increased expression (ATP synthase mitochondrial subunits alpha, beta, delta and b1). Despite their mitochondrial origin, these proteins should not be considered as contaminants of DRMs preparations. Several biochemistry and proteomic studies had already shown the presence of complex I and ATP-synthase subunits in DRMs, [40, 41] and Poston showed that Triton X-114-resistant DRMs are also present in mitochondria and contain proteins that facilitate ATP production and export from this organelle [23], compatible with the increasing evidence of the presence of raft-like microdomains in mitochondria [21, 42].
Ca2+ is one of the most important signaling compounds involved in various cellular processes being intracellular Ca2+ levels tightly regulated by specialized proteins in the plasma membrane, sarcoplasmic reticulum (SR) and mitochondria [43]. Recent studies suggest that membrane rafts are involved in coordinating the protein interactions required for proper Ca2+ exchange between the MAM and mitochondria [23]. In the present study, we found increased expression of four membrane proteins related with Ca2+ homeostasis namely: annexin A2, sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (SERCA1), calsequestrin-1 (CASQ1) and stromal interaction molecule 1 (STIM1).
Annexin A2 is a calcium-regulated membrane-binding protein, which holds two calcium ions. It has been proposed that it could play a key role in many processes including, (1) endocytosis and (2) exocytosis, (3) ion channel conductance, (4) link of F-actin cytoskeleton to the plasma membrane, (5) membrane organization, (6) formation of membrane cholesterol-rich microdomains and (7) regulation of cellular redox [44, 45, 46]. By LC-MS/MS, we found that annexin A2 was six times more abundant in DRMs obtained from Dhcr7T93M/T93M mice than in wt-BL6 and then confirmed such difference by immunocytochemistry (Figure 5).
Figure 5.
Immunohistochemical stain for annexin A2 (×4). Skeletal muscle samples of Dhcr793M/93M (left) and wt-BL6 (right). The Dhcr793M/93M (SLOS) sample shows a clear stronger coloration.
It is possible that the presence of 7DHC in SLOS mice membranes drives a higher annexin A2 incorporation in microdomains since 7DHC may promote microdomain formation [47]. An alternate hypothesis would be that increased annexin A2 expression is related to its role in the regulation of redox potential. In fact, several data suggest an increase of oxidative stress in SLOS: (1) over a dozen of oxysterols have been produced from 7DHC by free radical oxidation in solution [48], (2) the oxysterol mixture derived from 7DHC free radical oxidation is biologically active and leads to morphological changes in Neuro2a cells treated with these oxysterols [49], (3) the synthesis of 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), an oxysterol recently identified as a biomarker of 7DHC oxidation (in fibroblasts from SLOS patients and brain tissue), was found to be inhibited by an antioxidant compound in SLOS fibroblasts [47], (4) retinas from a SLOS rat model contain high levels of lipid hydroperoxides [50], (5) 7DHC peroxidation is a major source of oxysterols observed in cells [51] and (6) we previously identified a significant increase of the antioxidant enzyme superoxide dismutase mitochondrial in cultivated human SLOS fibroblasts. Considering these facts, we can hypothesize that there is an alteration of redox state of the cells in SLOS and annexin 2 is overexpressed as a part of cell strategy to compensate such a situation and protect cells’ biological compounds from peroxidation.
Another Ca2+ binding protein found upregulated in SLOS was SERCA1 an intracellular pump located in the SR of muscle cells, which catalyzes the hydrolysis of ATP coupled with the translocation of Ca2+ from the cytosol to the SR lumen, thus contributing to calcium sequestration involved in muscular excitation/contraction process. Another protein from this group is CASQ1, the major Ca2+-binding protein in the skeletal muscle SR. CASQ1 acts as an internal calcium store in muscle. The release of calcium bound to this protein through a calcium release channel triggers muscle contraction. Finally, stromal interaction molecule is a transmembrane protein essential for the activation of store-operated Ca2+ entry (SOCE), a major Ca2+ influx mechanism.
We also found integrin beta-1, overexpressed in affected mice. This microdomain-associated protein belongs to the integrin family which incorporates heterodimeric transmembrane proteins that function as major receptors for extracellular matrix proteins [52]. Integrin beta-1, plays a role in the maintenance of the cytoarchitecture of mature muscle as well as in the functional integrity of the muscle cells and is present at the neuromuscular junctions in skeletal muscle ones [53]. One wonders if its overexpression could be one of the factors which protect skeletal muscle from severe dysfunction in SLOS in opposition to other organs.
Ordered domains are formed when actin filaments attach to the plasma membrane [54]. Kwik and collaborators found changes in the organization and activity of actin and actin-modifying proteins after cholesterol depletion, and Gangully and Chattopadhyay demonstrated that cholesterol depletion mimics the effect of cytoskeletal destabilization [55, 56]. Differences in myofilaments and cytoskeleton proteins were also suspected in our study.
Furthermore, some of the proteins identified were not so far, according to the available bibliography, associated with lipid-rafts or MAMs; nevertheless, they are involved in intracellular physical connections like cytoskeleton-associated protein 4 (CKAP4) which is a transmembrane protein that further to its function as receptor also links endoplasmic reticulum (ER) to the cytoskeleton [57]. It is predictable that a membrane compact microdomain be involved in such function, in order to provide further support to the anchor. Such hypothesis is sustained by the fact that CKAP4 is a reversibly palmitoylated protein [58], and it is well described that palmitoylation of cytoplasmic proteins regulates the interaction of these soluble proteins with specific membranes or membrane domains. It is possible that palmitoylation controls the conformation of transmembrane segments, to modify the affinity of a membrane protein for specific membrane domains and to control protein–protein interactions [59].
Finally one should consider that both lipids and proteins for microdomains constructs are synthesized in the ER/Golgi before transport to the plasma membrane and, indeed, those proteins can be in a detergent-resistant, cholesterol-dependent state while residing there or in vesicle trafficking [60].
4. Conclusion
Our purpose was to contribute to the biological characterization of this Dhcr7T93M/T93M hypomorphic mouse model and identify differences that could help to go deeper in the understanding of SLOS physiopathology.
Proteomic analysis of DRMs of skeletal muscular samples clearly show differences between SLOS and wild-type mice, concerning proteins linked to membranes. The employed methodology allowed us to identify further alterations related with calcium homeostasis and membrane trafficking associated with SLOS and detected, for the first time, changes in mitochondrial energy metabolism in this mouse model.
To the best of our knowledge, this is the first research study focusing on the skeletal muscle of SLOS. The differential protein expression profile identified will open the way to comparative studies with more severally affected disease mice models as well as to similar approaches in human cells which may be helpful to uncover cellular mechanisms related to SLOS.
Acknowledgments
The authors are thankful to Prof Forbes D. Porter and Doctor Christopher A. Wassif from NIH who kindly provided the genetically modified mice model of SLOS used in this investigation. FDP and CAW are supported by the intramural research program of 30 the Eunice Kennedy Shriver National Institute of Child Health and Human Development.
Requimte received finnantial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2013.
This work received financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 (UID/MULTI/04378/2013 – POCI/01/0145/FERDER/007728).
Conflict of interest
The authors declare that there are no conflicts of interest.
Smith-Lemli-Opitz syndrome mouse, homozygous for T93M mutation
SR
sarcoplasmic reticulum
STIM1
stromal interaction molecule 1
TEAB
bicarbonate salt of triethylamine
TCEP
tris(2-carboxyethyl)phosphine hydrochloride
TFA
trifluoroacetic acid
UC
ultra-centrifuge
VDAC
voltage dependent anion-selective channels
wt-BL6
wild-type C57/BL6
\n',keywords:"skeletal muscle, detergent-resistant membranes (DRMs), Smith-Lemli-Opitz syndrome, SLO, comparative proteomics, mouse model, Dhcr7T93M/T93M",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/62552.pdf",chapterXML:"https://mts.intechopen.com/source/xml/62552.xml",downloadPdfUrl:"/chapter/pdf-download/62552",previewPdfUrl:"/chapter/pdf-preview/62552",totalDownloads:896,totalViews:138,totalCrossrefCites:0,totalDimensionsCites:0,totalAltmetricsMentions:0,impactScore:0,impactScorePercentile:46,impactScoreQuartile:2,hasAltmetrics:0,dateSubmitted:"December 12th 2017",dateReviewed:"April 27th 2018",datePrePublished:null,datePublished:"August 8th 2018",dateFinished:"July 10th 2018",readingETA:"0",abstract:"Smith-Lemli-Opitz syndrome (SLOS) is an inborn error of metabolism affecting the last step of cholesterol biosynthesis. It is characterized by a deficiency of the enzyme 7-dehydrocholesterol reductase and accumulation of 7-dehydrocholesterol (7DHC) in cells and body fluids. Given the similarities between 7DHC and cholesterol, 7DHC can be incorporated into cell membranes in lieu of cholesterol. Nevertheless, due to their structural differences and distinct affinity to other membrane components, this substitution alters membrane properties and one can expect to find abnormalities in membrane protein composition. In order to identify differences in membrane proteins that could facilitate our understanding of SLOS physiopathology, we isolated detergent-resistant membranes (DRMs) from the skeletal muscle of Dhcr7T93M/T93M mice and C57/BL6 controls and performed comparative proteomic analysis using iTRAQ for peptide quantification. A total of 133 proteins were identified in the DRM fraction: 17 (13%) proteins demonstrated increased expression in SLOS mice, whereas, 21 (16%) showed decreased expression. Characterization of functional point of view and bioenergetics pathway and transmembrane transport responded to the major differences between the two groups of animals.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/62552",risUrl:"/chapter/ris/62552",book:{id:"6752",slug:"cholesterol-good-bad-and-the-heart"},signatures:"Maria Luís Cardoso, Rui Vitorino, Henrique Reguengo, Susana Casal,\nRui Fernandes, Isabel Duarte, Sofia Lamas, Renato Alves, Francisco\nAmado and Franklim Marques",authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Material and methods",level:"1"},{id:"sec_2_2",title:"2.1. Materials",level:"2"},{id:"sec_3_2",title:"2.2. Animals",level:"2"},{id:"sec_4_2",title:"2.3. DRMs extraction",level:"2"},{id:"sec_5_2",title:"2.4. Sterols analysis",level:"2"},{id:"sec_6_2",title:"2.5. Proteomic profile",level:"2"},{id:"sec_6_3",title:"2.5.1. Pellet solubilization and protein quantification",level:"3"},{id:"sec_7_3",title:"2.5.2. Sample preparation for mass spectrometry analysis",level:"3"},{id:"sec_8_3",title:"2.5.3. LC-MS/MS analysis",level:"3"},{id:"sec_10_2",title:"2.6. Immunocytochemistry assay",level:"2"},{id:"sec_12",title:"3. Results and discussion",level:"1"},{id:"sec_12_2",title:"3.1. Sterols",level:"2"},{id:"sec_13_2",title:"3.2. Proteomics",level:"2"},{id:"sec_15",title:"4. Conclusion",level:"1"},{id:"sec_16",title:"Acknowledgments",level:"1"},{id:"sec_19",title:"Conflict of interest",level:"1"},{id:"sec_18",title:"Abbreviations",level:"1"}],chapterReferences:[{id:"B1",body:'Porter FD, Herman GE. Malformation syndromes caused by disorders of cholesterol synthesis. Journal of Lipid Research. 2011;52(1):6-34'},{id:"B2",body:'Cardoso M, Barbosa M, Serra D, Martins E, Fortuna A, Reis-Lima M, Bandeira A, Balreira A, Marques F. Living with inborn errors of cholesterol biosynthesis: Lessons from adult patients. Clinical Genetics. 2014;85(2):184-188'},{id:"B3",body:'Smith DW, Lemli L, Opitz JA. 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Neuro-Signals. 2009;17(1):100-108'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Maria Luís Cardoso",address:"m.luis.cardoso@insa.min-saude.pt",affiliation:'
Faculty of Pharmacy, University of Porto, Portugal
National Health Institute Doutor Ricardo Jorge (INSA), Portugal
BioISI-Biosystems and Integrative Sciences Institute, University of Lisbon, Portugal
UCIBIO-REQUIMTE, Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Portugal
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1. Introduction
A recurring theme in the statistical analysis is to separate the unstructured data into groups to detect the similarity or discrepancy within or between groups. This is especially true in the fields, e.g., discriminant analysis [1, 2, 3], pattern recognition [4, 5], gene expression [6, 7, 8], machine learning [9], and artificial intelligence [10]. In the literature, the clustering problem is often formulated within the cluster analysis framework, which is generally categorized into two classes: the non-probabilistic framework and the probabilistic framework. The non-probabilistic clustering method, including the K-means method [9, 11, 12] and the hierarchical/agglomerative clustering algorithms [13, 14, 15], is based on the distance between any two observations or groups. It clusters data by merging or removing observations according to the “closeness” specified by the distance. This method is more general since it does not impose any distributional assumptions on data, hence having greater flexibility in the real applications. Instead, the non-probabilistic clustering algorithm, also termed the model-based clustering, groups data by positing a probability model on data and then clustering data via configuration function related to the model. Compared with the non-probabilistic framework, the model-based methods enable us to assess the statistical properties of the solutions, e.g., how many clusters are there, how well the configuration function works, and how robust the method is against the model deviation and so on. There is rich literature on this issue. Among them, finite mixture model (FMM, [16, 17, 18]) perhaps is the most popular choice and has often been proposed and studied in the context of clustering (see a short review in Fraley and Raftery [2]). FMM assumes that each cluster is identified with a probability distribution indexed by the cluster-specific parameter(s), and each observation is related to clusters via configuration or membership function. The statistical task is the inference about the number of clusters, the estimation of the unknown parameters, and the allocation of observations.
In this chapter, we pursue a Bayesian model-based method to address the heterogeneity of fraction data. Fractional data are very common in the social and economical surveys. A distinguished feature of fractional responses is that its measurements are responded on a scale in the unity interval [0,1] but suffer from excessive zeros and unities on the boundaries. In understanding such type of data, the commonly used method is to separate the whole data into three parts: two corresponding to the zeros and unities respectively, and one corresponding to the continuously positive values. Two separative logistic models are suggested to model two discrete value parts respectively while single normal linear regression model is formulated for the continuous value part. This method, though more appealing, ignores the instinct association across different parts and readily leads to inconsistence of the occurrence probabilities on each part. Instead, we propose a three-category multinomial model for the occurrence variable, in which the usual separated models can be considered as the marginal models of our proposal. Such modeling always ensures the probabilities on each part to be proper, thus avoiding parameter constraints, see for example, [19]. To assess the heterogeneity underlying data, we formulate the problem into a finite mixture analysis of which each component is specified by two-part regression model. In view of the model complexity, we implement Markov Chains Monte Carlo sampling method to implement posterior analysis. Block Gibbs sampler is implemented to draw observations from the target distributions. The posterior inference including parameters estimates, model selection, and the configuration determination of observations are obtained based on the simulated observations.
The chapter is organized as follows. Section 2 introduces a general model-based clustering method to address the heterogeneity of regression model within the Bayesian framework. In Section 3, we apply the proposed method to the fractional data. Section 4 presents a cocaine use study. And Section 5 concludes the chapter.
2. Method description
2.1 General framework
Suppose that for i=1,2,⋯,n, yi is an observed response, each associated with an m dimensional fixed covariates xi=xi1⋯xim. In the context of regression analysis, the interest mainly focuses on exploring the pattern of the influence of xi on yi and predicting the mean of a future response y in terms of a new x. This is usually achieved by formulating xiyi as Eyixi=mxi for some mean function m⋅. In the parametric fitting framework, the function mx is assumed to be related to x via linking function as the form of
mx=hxTβE1
which induces the so-called generalized linear model [20] for xiyi, where β is the regression coefficients used to quantify the uncertainty about m, and h⋅ is the known linking function used to link the mean and the predictors.
More often, the single relationship such as Eq. (1) may not be sufficient when the patterns among the subjects take on the heterogeneity such as clustering. The heterogeneous data occur when the observations are generated from the different populations of which the number of populations and the membership of each observation to the population are unknown. The main objective is to separate data into different clusters to detect the possible similarity within clusters or the discrepancy between clusters. This is generally accomplished by defining a cluster’s membership/configuration function K:x1y1⋯xnyn↦1⋯K such that Ki=Kxiyi=k if xiyi belongs to the cluster k, where K is assumed to be less than n. The discrepancy between any two clusters is characterized by the cluster-specific parameters such as intercepters, regression coefficients, and/or disperse parameters.
The model-based clustering assumes that given the clusters membership Ki, xiyi within the cluster k has the following sampling density
yiKi=kxi∼ind.fkyixiTβkτkE2
while Ki is specified by
PKi=k=πkE3
where fk, maybe independent of k, is the probability density function, βk and τk are the cluster-specific regression coefficients and the disperse parameters, respectively, and πk is the mixing proportion identifying the proportion of the component k over the entire population. It is assumed that πk≥0 and ∑k=1Kπk=1.0.
Two important issues arise when formulating data clustering problem as Eqs. (2) and (3). One is related to the number of clusters, and the other is pertained to the determination of configurations. Within the Bayesian framework, several methods have been proposed for the first issue. One can, for example, follow [21] and treat K to be random and assign a prior to it. The reversible jump MCMC method (RJMCMC, [21, 22]) can be implemented to conduct the joint analysis of K with other random quantities. Another method is along the lines with the hypothesis test procedure and routinely to estimate K via model comparison/selection procedure. This perhaps is the most popular choice in the model-based clustering context, in which various measures such as the Akaike information criterion (AIC) [23], the corrected AIC (AICc) [24, 25], the Bayesian information criterion (BIC) [26], the integrated completed likelihood (ICL) [27], and Bayes factor (BF, [28, 29]) can be adopted to select a suitable model. It is worth pointing out that the deviance information criterion (DIC) [30] may not be appropriate for the mixture model comparison. The well-known software WinBUGS® [31] for Bayesian analysis does not provide DIC results for mixture analysis. In addition, many authors suggested modeling heterogeneous data into the mixture of Dirichlet process (MDP, [32, 33]). However, as discussed in Ishwaran and James [34], DP fitting often overestimates the number of clusters and readily leads to model over fitting.
For the second issue, the complexity of problem depends on the methods adopted in the analysis. In the frequency framework, for example, the configuration of observation i is often achieved by maximizing PKi=kYπ̂Ξ̂ over k=1,⋯,K, where π̂ and Ξ̂ are the maximum likelihood estimates (MLE) obtained via, e.g., the expectation-maximization algorithm (EM, [35]). In the next section, we will present a Bayesian procedure for determining K. Compared with the frequency approach, the nice feature of the Bayesian approach is its flexibility to utilize prior information for achieving better results. Also, the sampling-based Bayesian methods depend less on the asymptotic theory and hence have the potential to produce reliable results even with small sample size.
Let Y be the set of all observed responses and X be the set of fixed covariates; Write Ξ as the collection of βk and τk. Integrating over Ki produces a K-component mixture model for yi, which is given by
pyiπΞxi=∑k=1KπkfkyixiTβkτk.E4
The log-likelihood of the observed data conditional on K is given by
LπΞYX=∑k=1nlog∑k=1KπkfkyixiTβkτk.E5
As an illustration, Figure 1 presents a three-component normal linear mixture regression model with one covariate. It can be seen clearly that the density function illustrates strong heterogeneity. The regression line is obviously different from those of components, which indicates that single model is unappreciate in fitting such data. In what follows, we suppress X for notational simplicity.
Figure 1.
Plot of the three-component normal mixture model 0.3N−4−2x1+0.5N0.5+0.5x1+0.2N4.5+3x1. Left panel: Plot of the density functions of the mixture as well as their three weighted components ; right panel: plots of regression lines. Mixture model: solid line “−” component one: dotted lines “⋯” component two: dashed lines “−−” and component three: dotted-dashed lines “−⋅”
2.2 Bayesian model-based clustering via MCMC
Bayesian analysis for analyzing Eqs. (2) and (3) especially K requires the specification of a prior distribution pπΞ for the parameters of the mixture model. By model convention, it is naturally to assume that π and Ξ are independent, and the components among Ξ are also independent. In particular,
βk∼iid.Nmβ0Σ0,τk−1∼iid.Wρ0R0E6
in which Wρ0R0−1 is the Wishart distribution with the degrees of freedom ρ0 and the scale matrix R0, and reduces to the scaled Chi-square distribution when τk is a univariate; β0, Σ0, ρ0 and R0 are the hyper-parameters, which are treated to fixed and known. In the real applications, if no extra information can be available, the values of these hyper-parameters are often taken to ensure βk and τk to be dispersed enough. For example, one can set Σ0=λ0I with large λ0 (Throughout, we use I to signify an identify matrix). In this case, the values of β0 are not really important and can be set to any values, e.g., zeros. Note that for the mixture models, Diebolt and Robert [36] (see also, for example, [37]) showed that using fully non-informative prior distributions may lead to improper posterior distributions and hence is strictly prohibitive.
We assign a symmetric Dirichlet distribution to π as follows
π∣α∼DKα⋯αE7
in which α>0 is the hyper-parameter, which is treated to fixed and unknown. In the applications, we can take sensitive analysis by setting smaller and larger values for α. See section 4 for more details.
Let K=K1⋯Kn be the collection of all configurations. A Bayesian procedure for model-based clustering mainly focuses on exploring the behavior of the posterior of K given data, which is given by
pKY∝pYKpKE8
where pYK is the marginal distribution of pYπΞK with π and Ξ being integrated out. Generally, no closed form can be available for this target distribution. Markov Chain Monte Carlo [38, 39] sampling method can be used to conduct posterior analysis. In particular, one can follow the routine in Tanner and Wong [40] and treat the latent quantities πKΞ as the missing data and augment them with the observed data. Posterior analysis is carried out based on the joint distribution pπKΞY. In this case, block Gibbs sampler [41, 42] can be implemented to draw observations from such target distribution. The Gibbs sampler is iteratively implemented by drawing: (i) Ξ from pΞπKY; (ii) π from pπKΞY and K from pKπΞY till convergence. The convergence can be monitored by the “estimated potential scale reduction” (EPSR) values [43] or by plotting the traces of estimates against iterations under different starting values. Note that except for (i), all full conditionals involved in the Gibbs sampler are standard. However, drawing Ξ in (i) depends on the specific form of the density function fk and sometimes requires implementing Metropolis-Hastings algorithm (MH, [44, 45]) or rejection sampling [46].
2.3 Label switching
Formulating the model-based clustering problem into mixture model Eq. (2) faces the model identification. A statistical model is said to be identified if the observed likelihood is uniquely determined by unknown parameters. A less identified model may be problematic and will distort the estimates of unknown parameters. It is easily showed that the observed likelihood of data is only determined up to the permutation of the component labels. As a matter of fact, suppose that there are the pair π1Ξ1 and π2Ξ2 such that
pyπ1Ξ1=pyπ2Ξ2E9
then there exists a permutation ν:12⋯K↦12⋯K such that πk1=πνk2, βk1=βνk2 and τk1=τνk2. In this setting, we can not distinguish K and ν∘K in terms of data (“∘” denotes the operator of function composition). With this in mind, any exchangeable priors on π and Ξ like Eqs. (6) and (7) produces symmetric and multi-modal posterior distributions with up to K! copies of each “genuine” mode, which induces the so-called label switching problem on Bayesian estimate. Traditional approaches to eliminating such exchangeability is to impose identifiability constraints on the parameter space. However, as pointed out by Frühwirth-Schnatter [18], an unappropriate identifiability constraint may not be able to eliminate label switching. Many efforts have been devoted to coping with this issue, see Chapter 11 in Lee [47] for a review. Among them, the relabeling algorithm [48] is more appealing due to its simplicity and flexibility. The relabeling sampling procedure takes a decision-theoretical approach and requires specifying an appropriate loss function to measure the loss in terms of the classification probability. The model identification problem is addressed via postprocessing the MCMC output to minimize the posterior expected loss. Specifically, let θ be the collection of Ξ and π, and write Q={qikθ as the matrix of allocation probabilities of order n×K with qikθ=PKi=kYθ. In the context of clustering, the loss function can be defined on the cluster label K as follows
L0Kθ=−∑i=1nlogqiKiθ.E10
Given that θ1,⋯,θM are the sampled parameters and let ν1,⋯,νM be the permutation applied to them. The relabeling algorithm proceeds by selecting initial values for the νms, which are generally taken to be the identity permutations, then iterating the following steps until a fixed point is reached.
Choose K̂ to minimize ∑m=1ML0(K,νmθm;
For m=1,2,⋯,M, choose νm to minimize L0(K̂,vmθm.
2.4 Posterior inference
Once the label switching is taken care of, the MCMC samples can be used to draw posterior inference. For example, the joint Bayesian estimate of θ can be obtained easily via the corresponding sample means of the generated observations via ergodic average as follows:
The consistent estimates of the covariance matrix of estimates can be obtained via sample covariance matrix.
Given the observations Km:m=12⋯M drawn from the posterior pKY via MCMC sampling, serval methods can be available for arriving at a point estimate of the clustering using draws from the posterior clustering distribution. The simplest method, known as the maximum a posteriori (MAP) clustering, is to select the observed clustering that maximizes the density of the posterior clustering distribution, i.e.,
K̂:K̂i=argmaxk=1,⋯,KPKi=kYE12
in which PKi=kY can be approximated by
PKi=kY≈M−1∑m=1MIKim=k.E13
A more appreciate alternative to MAP is based on the pairwise probability matrix, an n×n association matrix δK with the ijth element formed by the indicator of whether the subject i is clustered with subject j. Element-wise averaging of these association matrices yields the pairwise probability matrix of clustering, denoted ψ̂. Medvedovic and Sivaganesan [49] and Medvedovic et al. [50] suggested a clustering estimate of K by using the pairwise probability matrix ψ̂ as a distance matrix in hierarchical/agglomerative clustering. However, as augured by Dahl [51], such routine seems counterintuitive to apply an ad hoc clustering method on top of a model which itself produces clusterings. In the context of Dirichlet process mixture-based clustering, Dahl [51] proposed a least-squares model-based clustering method by using draws from a posterior clustering distribution. Specifically, the least-squares clustering KLS is the observed clustering KLS, which minimizes the sum of squared deviations of its association matrix K from the pairwise probability matrix:
K̂LS=argminK∈K1⋯Km∑i=1n∑j=1nδijK−ψ̂ij2.E14
Dahl [51] showed that the least-squares clustering has the advantage over those in Medvedovic and Sivaganesan [49] since it utilizes the information from all the clusterings and is intuitively appealing for the “average” clustering instead of forming a clustering via an external, ad hoc clustering algorithm.
3. Assessing heterogeneity of two-part model
In this section, we first proposed a two-part regression model for the fractional data especially for the U shaped fractional data and then extend the method discussed above to the current situation to address the possible heterogeneity of the population underlying data.
3.1 Two-part model for U shaped fractional data
Suppose that for subject/individual i=1⋯n, yi is an univariate fractional response taking values in 01; xi is an m×1 fixed covariate vector denoting various explanatory factors under consideration. Usually, yi suffers from excess zeros and ones on the boundaries, and the whole data set takes on the U shape. In modeling such data, we introduce a three-category indicator variable di and a continuous intensity variable zi such that
where h⋅ is any monotone increasing function such that zi∈−∞+∞. That is, we break the data set into three parts: two parts corresponding to zeros and ones respectively and one part corresponding to the continuous values between 0 and 1. We formulate a two-part model for yi by first specifying a baseline-category logits model [52] for di and then a conditional continuous model for zi. The baseline-category logits model is assumed that conditional upon xi, dis are independent satisfying the following logits models simultaneously: for j=1,2,
logPdi=jxiPdi=3xi=xiTαjE16
where αj is an m×1 regression coefficients vector. We use category di=3 as the reference for the ease of parameters interpretation. For example, the magnitude of αjℓ in αj indicates that the increase of one unit in xiℓ will increase eαjℓ times chance of di=j over that of di=3.
The conditional continuous model for zi is given by
pzidi=3xi=pzzixiTγτE17
or equivalently
pyi0<yi<1xi=pzhyixiTγψ∣ḣyi∣E18
where ḣs=dh/ds, pzuaτ is the normal density with mean a and variance τ>0, and γ like that in Eq. (16), is the regression coefficient vector. Although the identical covariates are taken in Eqs. (16) and (17), this is not necessary in practice. Each equation can own their covariates. This can be achieved by imposing particular structure on the regression coefficients. For example, we can exclude xi1 from Eq. (17) by restricting γ1 in γ to be zero.
It follows from Eqs. (16) and (17) that marginal distribution of yi is given by
pyixiβτ=qi1δ0+qi2δ1+1−qi1−qi2pyi0<yi<1xiγτE19
where qij=Pdi=jxiαjj=12 is the response probability specified by Eq. (16) and β is the regression parameters constituted by α1,α2 and γ.
3.2 Assessing heterogeneity of two-part model
To detect the possible heterogeneity among yi, we extend the model Eq. (18) to the mixture case by assuming that conditional upon Ki=k, di and zi satisfy Eqs. (16) and (17) with αj replaced by αjk and γτ by γkτk respectively. This indicates that the mixture component fk in Eq. (1) in Section 2 is given by Eq. (19) with β=βk and τ=τk.
For the Bayesian analysis, the general forms of full conditionals involved in the model-based clustering have been given in Section 2. We here only focus on the technical details of the conditional distribution of Ξ in (i) in the Gibbs sampler.
We assume that the prior of τk is the same as that in Eq. (6), while the priors of βk are taken as pβk=pαk1pαk2pγk, in which
pαkℓ=DNmαℓ0Σαℓ0ℓ=12,pγk=DNmγ0Σγ0.E20
where αℓ0, γ0, Σαℓ0 and Σγ0 are the hyper-parameters treated to be known.
Gibbs sampling Ξ now becomes drawing αk, γk and τk alternatively from the full conditional distributions pαkKY, pγkτkKY and pτkγkKY respectively. By some algebras, it can be shown that
where d˜iℓ=Idi=ℓ and Cikℓ=log1.0+expxiTαk,−ℓ; αk,−ℓ denotes the set αk with αkℓ removed. Following the similar routine in Polson, Scott, and Windle [53], we recast the logistic function Eq. (25) as follows
in which κiℓ=d˜iℓ−1/2 and pPG is the well-known PG10 density function [53]. If one introduces n independent Pólya-Gamma variables ωiℓ into the current analysis, then,
with ηikℓ=κiℓ+Cikℓωiℓ. Consequently, drawing αkℓ is accomplished by first drawing ωiℓ from the Pólya gamma distribution and then drawing αkℓ from the normal distribution. The draw of ωiℓ is a little intractable since its density function involves the infinite sum. By taking advantage of series sampling method [54], Polson et al. [53] devised a rejection algorithm for generating observations from such type of distribution. Their method can be adapted to draw ωiℓ, see also [55].
4. A real example
In this section, a small portion of cocaine use data is analyzed to illustrate the practical value of the proposed methodology. The data are obtained from the 322 cocaine use patients who were admitted in 1988–89 to the West Los Angeles Veterans Affairs Medical Center. The original data set is made up of 68 measurements in which 17 items were assessed at four unequally spanned time points. In this study, we mainly focus on the measurements 1 year after treatment and ignore the initial effects at the baseline. The measurements cover the information on the cocaine use, treatment received, psychological problems, social status, employments, and so on. Among them, the measurement “cocaine use per month” (denoted by CC) plays a critical role since it identifies the severity of cocaine use of patients and therefore is treated as the dependent response. The CC is originally measured by 0–30 points but suffered from small portion of fractions. We identify CC/30 as the fraction response in [0,1]. In view of that the missing data are presented, we delete the subjects with missing values. The total sample size is 228. A primary analysis shows that CC/30 has excessive zeros and ones. Figure 2 gives the histograms of CC/30 and their fractional values in (0,1) via logistic transformation. It can be seen clearly that there is a large number of zeros and unities accumulated on the boundaries. The proportions of zeros and unities are about 15 and 4%, respectively. Moreover, panel (b) in Figure 2 indicates that single parametric model may be unappreciate for fitting the continuous valued variable.
Figure 2.
Plots of CC in cocaine use data: (a) Histograms of CC/30; and (b) histograms of CC/30 on logistic transformation conditional on CC/30 in (0,1).
To explore the effects of exogenous factors on the cocaine use, the following measurements are selected as the explanatory variables: the occupational status of a patient (x1). This is a binary indicator: 1 for employment and 0 for non-employment; the level of technical proficiency of patients engaged in work (x2): scaled on 0–4 points and the patient’s lifestyle (x3) with five-point scale. To unify the scales, all covariates are standardized. However, a preliminary analysis shows that there exists strong multiple collinearity among these covariates. The minimum eigenvalue of sample covariance matrix equals to 0.06284, which approaches zero. We remove such collinearity by implementing principle component analysis (PCA) and treat the scores of the first two components (still denoted by x1 and x2) as our explanatory variables. These two principle components can be interpreted as the levels related to the patients’ occupation and their live life.
To formulate a two-part model for the observed responses, we identity CCi/30 with di and zi, where di is the three-category indicator indicating the state of cocaine use after one year treatments: quitting cocaine successfully (state 1), insisting on cocaine use every day in a month (state 2) and taking the cocaine occasionally (state 3); zi is the intensity variable representing the numer of days of cocaine use in a month. We assess the effects of exogenous factors x1 and x2 on the cocaine use via Eqs. (16) and (17), respectively.
We proceed data analysis by first fitting data to the K-component mixture two-part models with K=1,2,⋯,6. The model fits are assessed via AIC, AICc, and BIC, which are defined as −2logpYθ̂K penalized by 2dK, 2ndK+1/n−dK−2, and dKlogn respectively, where θ̂K is the MLE of θK and dK is the dimension of unknown parameters under the model K. In view of that the Bayesian estimates and the ML estimates are close to each other, we replace the ML estimates by their Bayesian counterparts in evaluating AIC, AICc, and BIC. For computation, we take α=n−1, n0, n1, and n2 in Eq. (7), which represents our knowledge about πa prior. Note that for large value of α, the Dirichlet distribution places most of the mass on its center and the prior Eq. (7) tends to be informative. However, for small α, the Dirichlet distribution concentrates the mass on the boundaries of sampling space and the distribution tends to be degenerated and sparse. As a result, some components in π reduces to zeros. When α=1, DKα⋯α becomes an uniform distribution on the simplex SK. For the inputs of the hyper-parameters involved in the priors Eq. (20), we take α0ℓ=γ0=03, Σαℓ0=Σγ0=100I3, αγ0=2.0 and βγ0=2.0. These values ensure the priors Eq. (20) to be inflated enough and represent the weak information on the parameters.
The relabeling MCMC algorithm described in Section 2 is implemented to draw observations from the posterior. The convergence of algorithm is monitored by plotting the traces of estimates against iterations under three starting values. Figure 3 presents the values of EPSR of unknown parameters against the number of iterations under three different starting values with K=2. It shows that the convergence of the proposed algorithm is fast and the values of EPSR are less than 1.2 in less than 1000 iterations. Hence, 3000 observations, after removing the initial 2000 iterations, are collected for calculating AIC, AICc, and BIC. The resulting summary is given in Table 1.
Figure 3.
Plots of values of EPSR of estimates of unknown parameters against the number of iterations under three different staring values in the cocaine use example: K=2.
Model
α=1/n
α=n0
α=n
α=n2
AIC
K=1
921.3887
–
–
–
K=2
923.0580
907.4485
901.9474
901.4380
K=3
929.0698
926.5423
956.4945
994.5039
K=4
990.7506
949.5966
1014.5477
1006.4228
K=5
989.2483
971.4688
1069.1561
1037.5005
K=6
1097.6853
1007.4899
1091.8049
1086.8491
AICc
K=1
882.4025
–
–
–
K=2
885.5434
869.9339
864.4329
863.9234
K=3
875.9005
873.3730
903.3253
941.3347
K=4
925.3159
884.1618
949.1130
940.9880
K=5
915.5836
897.8041
995.4914
963.8357
K=6
1020.6483
930.4529
1014.7679
1009.8120
BIC
K=1
995.6821
–
–
–
K=2
995.0742
979.4647
973.9637
973.4542
K=3
1038.8088
1036.2814
1066.2335
1104.2429
K=4
1138.2125
1097.0585
1162.0096
1153.8846
K=5
1174.4330
1156.6534
1254.3408
1222.6851
K=6
1320.5928
1230.3973
1314.7124
1309.7565
Table 1.
Summary statistics of AIC, AICcc, and BIC for model selection in cocaine use data analysis.
Examination of Table 1 shows that all measures favor the model with K=2. This indicates that the proposed model with two groups seems to give a better fit to the data. It also indicates that large α favors the model fit. Furthermore, we calculate the posterior predictive density estimate of zi under the elected model. Results (not represented here for saving spaces) show that our method can be successful in capturing the skewness and modes of data. We also follow [56] to plot the estimated residuals δ̂i=zi−γ̂xiT and find that these plots lie within two parallel horizontal lines that are centered at zero, with nonlinear or quadratic trends detected. This roughly indicates that the proposed linear model Eq. (18) is adequate.
Table 2 presents the estimates of unknown parameters associated with corresponding standard deviation (SD) estimates under K=2. Based on Table 2, we can find the following facts: (i) for Part one, we observe that except for α̂23, the Bayesian estimates of unknown parameters within two clusters have the same signs but their magnitudes are more different. For example, the estimate of α11 within Cluster one is −1.540 with SD 0.587 while equals to −0.732 with SD 0.481 within Cluster two. This indicates that the baselines of logits Eq. (16) exist obvious difference. For α23, the estimates between two clusters have the opposite signs. Recall that α23 quantifies the magnitude of effects of live life on the probability Pdi=2 over Pdi=3 on log scale. This shows that increasing the level of live life will lead to an opposite effect among two clusters; (ii) for Part two, although all the estimates within two clusters have the same signs but the levels of effects among them are obviously different. The estimates of γ1 is −2.779 with SD 0.144 in the cluster one and attains −0.490 associated with SD 0.215 in the Cluster two. This indicates that the baseline of cocaine use in Cluster one is 50 times as much as that in Cluster two; and (iii) investigation of the estimate of τ also indicates that there exists the different amount of the fluctuation among two clusters.
Para.
Component I
Component II
Est.
SD.
Est.
SD
α11
−1.540
0.587
−0.732
0.481
α12
0.150
0.317
0.604
0.322
α13
0.261
0.703
0.188
0.601
α21
−1.337
0.480
−1.059
0.545
α22
−0.166
0.355
−0.229
0.418
α23
0.232
0.378
−0.184
0.411
γ1
−2.779
0.144
−0.490
0.215
γ2
−0.029
0.080
−0.011
0.154
γ3
0.087
0.144
0.179
0.240
τ
0.674
0.150
0.924
0.234
Table 2.
Summary statistics for the Bayesian estimates of unknown parameters in the cocaine use data.
5. Discussion
This chapter introduces a general Bayesian model-based clustering procedure for the regression model and proposed a Bayesian method for assessing the heterogeneity of fractional data within the mixture of two-part regression model framework. The heterogeneous fractional data arise mainly from two resources: one is that the excessive zeros and ones are accumulated upon the boundaries, and the other is that the underlying population may consist more than one components. For the first issue, we propose a novel two-part model, in which a three-category multinomial regression is suggested to model the occurrence probabilities of each part, and a conditional normal linear regression is used to fit the continuous positive values on logit scale. Such formulation is more appealing since it can ensure the probabilities on each part to be consistent and and at the same time maintains the coherent association across parts. For the second problem, we resort to the finite mixture model in which the cluster-specific components are specified via two-part model. MCMC sampling method is adopted to carry out the posterior analysis. The number of clusters and the configuration of observations are addressed based on the simulated observations from the posterior. We illustrate the proposed methodology in the analysis of cocaine use data.
When interest is concentrated upon the estimates, model identification is surely an important issue since it involves whether or not the estimates of component-specific quantities are meaningful. For a finite mixture model, model identification mainly stems from the label switching, in which the likelihood and the posterior are invariant under label permutation. Many efforts have devoted to alleviating such indeterminacy. Among them, parameters’ constraints may be the most popular choice. However, an unappreciated constraint fails to deal with the label switching. In this case, one can follow the routine in Frühwirth-Schnatter [18] and implement random permutation sampling to find the suitable identifiability constraints. The random permutation sampler is similar to the unconstrained MCMC sampling but only at each sweep, the labels 1⋯K are randomly permutated. The permutation aims to deliver a sample that explores the whole unconstrained parameter space and jumps between the various labeling subspaces in a balanced fashion. The output of such balanced sample can help us to find a suitable identifiability constraint. A more detailed discussion on model identification in the mixture context can be referred to, for example, [18, 57]. Instead, we resort to the relabeling algorithm for simplicity. Compared with the random permutation sampling, the relabeling method requires implementing MCMC samplng only once, thus saving the computation cost.
The methodology developed in this chapter can be extended to the case where latent factors are included to identify the unobserved heterogeneity due to some fixed convariates absent. Another possible extension is to establish a dynamic LVM, wherein model parameters vary across times. These issues may raise theoretical and computational challenges and therefore require further investigation.
Acknowledgments
The work presented here was fully supported by grant from the National Natural Science Foundation of China (NNSF 11471161). The authors are thankful to Professor Xin-Yuan Song, the Chinese University of Hong Kong, for using her cocaine use data in the real example.
Conflict of interest
The authors have no conflicts of interest to disclose.
\n',keywords:"model-based clustering, finite mixture model, two-part model, Markov Chain Monte Carlo sampling, cocaine use data",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/80937.pdf",chapterXML:"https://mts.intechopen.com/source/xml/80937.xml",downloadPdfUrl:"/chapter/pdf-download/80937",previewPdfUrl:"/chapter/pdf-preview/80937",totalDownloads:19,totalViews:0,totalCrossrefCites:0,dateSubmitted:null,dateReviewed:"February 7th 2022",datePrePublished:"March 23rd 2022",datePublished:null,dateFinished:"March 23rd 2022",readingETA:"0",abstract:"The purpose of this chapter is to provide an introduction to the model-based clustering within the Bayesian framework and apply it to asses the heterogeneity of fractional data via finite mixture two-part regression model. The problems related to the number of clusters and the configuration of observations are addressed via Markov Chains Monte Carlo (MCMC) sampling method. Gibbs sampler is implemented to draw observations from the related full conditionals. As a concrete example, the cocaine use data are analyzed to illustrate the merits of the proposed methodology.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/80937",risUrl:"/chapter/ris/80937",signatures:"Ye-Mao Xia, Qi-Hang Zhu and Jian-Wei Gou",book:{id:"10820",type:"book",title:"Data Clustering",subtitle:null,fullTitle:"Data Clustering",slug:null,publishedDate:null,bookSignature:"Prof. Niansheng Tang",coverURL:"https://cdn.intechopen.com/books/images_new/10820.jpg",licenceType:"CC BY 3.0",editedByType:null,isbn:"978-1-83969-888-0",printIsbn:"978-1-83969-887-3",pdfIsbn:"978-1-83969-889-7",isAvailableForWebshopOrdering:!0,editors:[{id:"221831",title:"Prof.",name:"Niansheng",middleName:null,surname:"Tang",slug:"niansheng-tang",fullName:"Niansheng Tang"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Method description",level:"1"},{id:"sec_2_2",title:"2.1 General framework",level:"2"},{id:"sec_3_2",title:"2.2 Bayesian model-based clustering via MCMC",level:"2"},{id:"sec_4_2",title:"2.3 Label switching",level:"2"},{id:"sec_5_2",title:"2.4 Posterior inference",level:"2"},{id:"sec_7",title:"3. Assessing heterogeneity of two-part model",level:"1"},{id:"sec_7_2",title:"3.1 Two-part model for U shaped fractional data",level:"2"},{id:"sec_8_2",title:"3.2 Assessing heterogeneity of two-part model",level:"2"},{id:"sec_10",title:"4. A real example",level:"1"},{id:"sec_11",title:"5. Discussion",level:"1"},{id:"sec_12",title:"Acknowledgments",level:"1"},{id:"sec_15",title:"Conflict of interest",level:"1"}],chapterReferences:[{id:"B1",body:'McLachlan GJ. Discriminant Analysis and Statistical Pattern Recognition. New York: John Wiley; 1992. DOI: 10.1002/0471725293.ch3'},{id:"B2",body:'Fraley C, Raftery AE. Model-based clustering, discriminant analysis, and density estimation. 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DOI: 10.1109/34.865189'},{id:"B28",body:'Berger JO. Statistical Decision Theory and Bayesian Analysis. New York: Springer-Verlag; 1985. DOI: 10.1007/978-1-4757-4286-2'},{id:"B29",body:'Kass RE, Raftery AE. Bayes factors. Journal of the American Statistical Association. 1995;90:773-795. DOI: 10.1080/01621459.1995.10476572'},{id:"B30",body:'Spiegelhalter DJ, Best N, Carlin B, van der Linde A. Bayesian measures of model complexity and fit. Journal of the Royal Statistical Society, Series B. 2002;64:583-640. DOI: 10.1111/1467-9868.00353'},{id:"B31",body:'Spiegelhalter DJ, Thomas A, Best NG, Lunn D. WinBUGS User Manual. Version 1.4. Cambridge, England: MRC Biostatistics Unit; 2003. DOI: 10.1001/jama.284.24.3187'},{id:"B32",body:'Ferguson TS. A Bayesian analysis of some nonparametric problems. The Annals of Statistics. 1973;1(2):209-230. DOI: 10.1214/aos/1176342360'},{id:"B33",body:'Antoniak CE. Mixtures of Dirichlet processes with applications to bayesian nonparametric problems. The Annals of Statistics. 1974;2:1152-1174. DOI: 10.1214/aos/1176342871'},{id:"B34",body:'Ishwaran H, James LF. Gibbs sampling methods for stickbreaking priors. Journal of the American Statistical Association. 2001;96:161-173. DOI: 10.1198/016214501750332758'},{id:"B35",body:'Dempster A, Laird N, Rubin D. Maximum likelihood from incomplete data via the EM algorithm (with discussion). Journal of the Royal Statistical Society, Series B. 1977;39:1-38'},{id:"B36",body:'Diebolt J, Robert CP. Estimation of finite mixture distributions through Bayesian sampling. Journal of the Royal Statistical Society, Series B. 1994;56:363-375. DOI: 10.1111/j.2517-6161.1994.tb01985.x'},{id:"B37",body:'Roeder K, Wasserman L. Practical Bayesian density estimation using mixtures of normals. Journal of the American Statistical Association. 1997;92:894-902. DOI: 10.1080/01621459.1997.10474044'},{id:"B38",body:'Geman S, Geman D. Stochastic relaxation, Gibbs distribution, and the Bayesian restoration of images. IEEE Transactions on Pattern Analysis and Machine Intelligence. 1984;6:721-741. DOI: 10.1109/TPAMI.1984.4767596'},{id:"B39",body:'Geyer CJ. Practical Markov chain Monte Carlo. Statistical Science. 1992;7:473-511. DOI: 10.1214/ss/1177011137'},{id:"B40",body:'Tanner MA, Wong WH. The calculation of posterior distributions by data augmentation(with discussion). Journal of the American statistical Association. 1987;82:528-550. DOI: 10.2307/2289463'},{id:"B41",body:'Gelfand AE, Smith AFM. Sampling-based approaches to calculating marginal densities. Journal of the American Statistical Association. 1990;85:398-409. DOI: 10.1080/01621459.1990.10476213'},{id:"B42",body:'Ishwaran H, Zarepour M. Markov chain Monte Carlo in approximation Dirichlet and beta-parameter process hierarchical models. Biometrika. 2000;87:371-390'},{id:"B43",body:'Gelman A, Rubin DB. Inference from iterative simulation using multiple sequences. Statistical Science. 1992;7:457-472. DOI: 10.2307/2246093'},{id:"B44",body:'Metropolis N, Rosenbluth AW, Rosenbluth MN, Teller AH, Teller E. Equations of state calculations by fast computing machines. Journal of Chemical Physics. 1953;21:1087-1092. DOI: 10.1063/1.1699114'},{id:"B45",body:'Hastings WK. Monte Carlo sampling methods using Markov chains and their applications. Biometrika. 1970;57(1):97-109. DOI: 10.1093/biomet/57.1.97'},{id:"B46",body:'Gilks WR, Wild P. Adaptive rejection sampling for gibbs sampling. Journal of the Royal Statistical Society. Series C (Applied Statistics). 1992;41(2):337-348. DOI: 10.2307/2347565'},{id:"B47",body:'Lee SY. Structural Equation Modeling: A Bayesian Approach. New York: John Wiley & Sons; 2007'},{id:"B48",body:'Stephens M. Dealing with label-switching in mixture models. Journal of the Royal Statistical Society, Series B. 2000;62:795-809. DOI: 10.1111/1467-9868.00265'},{id:"B49",body:'Medvedovic M, Sivaganesan S. Bayesian infinite mixture model based clustering of gene expression profiles. Bioinformatics. 2002;18(9):1194-1206. DOI: 10.1093/bioinformatics/18.9.1194'},{id:"B50",body:'Medvedovic M, Yeung KY, Bumgarner RE. Bayesian mixture model based clustering of replicated microarray data. Bioinformatics. 2004;20(8):1222-1232. DOI: 10.1093/bioinformatics/bth068'},{id:"B51",body:'Dahl DB. Model-based clustering for expression data via a Dirichlet process mixture model. In: Do KA, Müller P, Vannucci M, editors. Bayesian Inference for Gene Expression and Proteomics. Cambridge University Press; 2006. DOI: 10.1017/CBO9780511584589.011'},{id:"B52",body:'Agresti A. Categorical Data Analysis. 2nd ed. New York: John Wiley & Sons; 2003'},{id:"B53",body:'Polson NG, Scott JG, Windle J. Bayesian inference for logistic models using pólya Gamma latent variables. Journal of the American Statistical Association. 2013, 2013;108(504):1339-1349. DOI: 10.1080/01621459.2013.829001'},{id:"B54",body:'Devroye L. The series method in random variate generation and its application to the Kolmogorov-Smirnov distribution. American Journal of Mathematical and Management Sciences. 1981;1:359-379. DOI: 10.1080/01966324.1981.10737080'},{id:"B55",body:'Gou JW, Xia YM, Jiang DP. Bayesian analysis of two-part nonlinear latent variable model: Semiparametric method. Statistical Modeling. Published on line. 2021. DOI: 10.1177/1471082X211059233'},{id:"B56",body:'Xia YM, Tang NS, Gou JW. Generalized linear latent models for multivariate longitudinal measurements mixed with hidden Markov models. Journal of Multivariate Analysis. 2016;152:259-275. DOI: 10.1016/j.jmva.2016.09.001'},{id:"B57",body:'Jasra A, Holmes CC, Stephens DA. Markov Chain Monte Carlo methods and the label switching problem in Bayesian mixture modeling. Statistical Science. 2005;20(1):50-67. DOI: 10.1214/088342305000000016'}],footnotes:[],contributors:[{corresp:null,contributorFullName:"Ye-Mao Xia",address:null,affiliation:'
Department of Applied Mathematics, Nanjing Forestry University, China
Department of Applied Mathematics, Nanjing Forestry University, China
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It can improve soil fertility and plant growth. However, long-term application of treated sewage biosolids could result in heavy metal accumulation and some health problems. The objective of this study is to evaluate the effect of different fertilizers, especially Kala compost, on the soil fertility and plant productivity. An open field was divided into nine plots and received either treated municipal wastes (Kala compost) or inorganic fertilizer, or a mixture of both fertilizers. The field was irrigated by drip system, and commercial cucumber, tomato, cabbage, lettuce, carrot, and potato were grown in each plot. Soil and plant were monitored continuously and samples were taken at different stages of the study. No symptoms of physical or chemical problems were observed in the open field and measured soil samples. 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However, more monitoring is needed with treated biosolid application and good management could be the key to avoid any adverse effect of any contaminant.",signatures:"Ahmed Al-Busaidi and Mushtaque Ahmed",authors:[{id:"186164",title:"Dr.",name:"Ahmed",surname:"Al-Busaidi",fullName:"Ahmed Al-Busaidi",slug:"ahmed-al-busaidi",email:"ahmed99@squ.edu.om"},{id:"193988",title:"Dr.",name:"Mushtaque",surname:"Ahmed",fullName:"Mushtaque Ahmed",slug:"mushtaque-ahmed",email:"ahmedm@squ.edu.om"}],book:{id:"5358",title:"Soil Contamination",slug:"soil-contamination-current-consequences-and-further-solutions",productType:{id:"1",title:"Edited Volume"}}},{id:"52679",title:"Mitigating Environmental Risks of Wastewater Reuse for Agriculture",slug:"mitigating-environmental-risks-of-wastewater-reuse-for-agriculture",abstract:"The study was aimed to maximize and optimize treated wastewater reuse in conjunction with surface and ground waters resources. Moreover, environmental, agronomic and economic components were also considered. The project was funded by USAID and implemented in three countries (Oman, Tunisia and Jordan). In Oman, the study was done at Sultan Qaboos University experimental station field. Four types of waters (A: 50% of treated wastewater with 50% of groundwater, B: 100% of groundwater, C: 25% of groundwater with 75% of treated wastewater, and D: 100% of treated wastewater) were used to grow three different crops (okra, maize and sweet corn). Results showed no significant differences in soil physical and chemical properties with treatments irrigated with treated wastewater as compared to groundwater. On other hand, some chemical properties significantly increased (p<0.05) when treated wastewater was applied such as soil total carbon and some major elements (N, K, Mg). Crop physical analysis showed significant increases in plant productivity when plants were irrigated with treated wastewater and values of chemical properties were within the international standards. Crop biological analysis showed no effect on crop quality and all tested crops were free from any microbial contamination.",signatures:"Ahmed Al‐Busaidi and Mushtaque Ahmed",authors:[{id:"186164",title:"Dr.",name:"Ahmed",surname:"Al-Busaidi",fullName:"Ahmed Al-Busaidi",slug:"ahmed-al-busaidi",email:"ahmed99@squ.edu.om"},{id:"193988",title:"Dr.",name:"Mushtaque",surname:"Ahmed",fullName:"Mushtaque Ahmed",slug:"mushtaque-ahmed",email:"ahmedm@squ.edu.om"}],book:{id:"5417",title:"Biological Wastewater Treatment and Resource Recovery",slug:"biological-wastewater-treatment-and-resource-recovery",productType:{id:"1",title:"Edited Volume"}}}],collaborators:[{id:"185895",title:"Dr.",name:"Michael",surname:"Aide",slug:"michael-aide",fullName:"Michael Aide",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/185895/images/system/185895.jpg",biography:"Dr. Michael Aide received his Ph.D. in soil chemistry from Mississippi State University, US (1982) and a baccalaureate degree in chemistry and mathematics from the University of Wisconsin – Madison. He has been an educator and agronomic researcher at Southeast Missouri State University since 1982. His research interests involve the growth and development of rice in integrated systems involving soil fertility, water management, and integrated pest management. Rice research has permitted his travel to southeastern Asia, Central America and Egypt. Dr. Aide has also investigated the usage of rare earth elements in soils, particularly attempting to utilize them to indicate parent material uniformity and their fate/transport. Professional affiliations include the American Society of Agronomy and the Soil Science Society of America, and he is a certified professional soil scientist.",institutionString:"Southeast Missouri State University",institution:{name:"Southeast Missouri State University",institutionURL:null,country:{name:"United States of America"}}},{id:"185956",title:"Dr.",name:"Wilberth",surname:"Chan Cupul",slug:"wilberth-chan-cupul",fullName:"Wilberth Chan Cupul",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/185956/images/4765_n.jpg",biography:"I have a M. Sc. in Tropical Horticulture and a Ph. D. in Natural Resource Management by the Institute of Ecology (INECOL A. C.) from Xalapa, Mexico. My research area is the Applied Mycology. Principally, I am studying: 1) the biological control of insect-pest and plant diseases in horticultural crops; 2) the fungal interactions as a strategy to over production of fungal enzymes (laccase, LiP and MnP) and hidrogen peroxide to apply in contaminated soils with pesticides for its bioremediation; and 3) the solubilization of inorganic phosphates by soil borne micromycetes in stressed environments by heavy metals, pesticides and hydrocarbons.",institutionString:null,institution:{name:"University of Colima",institutionURL:null,country:{name:"Mexico"}}},{id:"186184",title:"Prof.",name:"Wlodzimierz",surname:"Bres",slug:"wlodzimierz-bres",fullName:"Wlodzimierz Bres",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Life Sciences in Poznań",institutionURL:null,country:{name:"Poland"}}},{id:"186789",title:"Dr.",name:"Yanzhao",surname:"Fu",slug:"yanzhao-fu",fullName:"Yanzhao Fu",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Dalian University of Technology",institutionURL:null,country:{name:"China"}}},{id:"186796",title:"Prof.",name:"Shiguo",surname:"Xu",slug:"shiguo-xu",fullName:"Shiguo Xu",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Dalian University of Technology",institutionURL:null,country:{name:"China"}}},{id:"186804",title:"Ph.D.",name:"Iryna",surname:"Loza",slug:"iryna-loza",fullName:"Iryna Loza",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Oles Honchar Dnipropetrovsk National University",institutionURL:null,country:{name:"Ukraine"}}},{id:"186838",title:"Dr.",name:"Yi",surname:"Xu",slug:"yi-xu",fullName:"Yi Xu",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"186839",title:"MSc.",name:"Qi",surname:"Wang",slug:"qi-wang",fullName:"Qi Wang",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"193279",title:"Prof.",name:"Barbara",surname:"Politycka",slug:"barbara-politycka",fullName:"Barbara Politycka",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"194101",title:"Dr.",name:"Refugio",surname:"Rodríguez Vázquez",slug:"refugio-rodriguez-vazquez",fullName:"Refugio Rodríguez Vázquez",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null}]},generic:{page:{slug:"edited-volumes",title:"Edited Volumes",intro:"
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Out of all of the publishing options available to researchers, why choose to contribute your research to an IntechOpen Edited Volume? The reasons are simple. IntechOpen has worked exceptionally hard over the past years to fine tune the Open Access book publishing process and we continue to work hard to deliver the best for all of our contributors. The quality of published content is of utmost importance to us, followed closely by speed, and of course, availability and accessibility. To view current Open Access book projects that are Open for Submissions visit us here.
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YOUR WORK, YOUR COPYRIGHT
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Out of all of the publishing options available to researchers, why choose to contribute your research to an IntechOpen Edited Volume? The reasons are simple. IntechOpen has worked exceptionally hard over the past years to fine tune the Open Access book publishing process and we continue to work hard to deliver the best for all of our contributors. The quality of published content is of utmost importance to us, followed closely by speed, and of course, availability and accessibility. To view current Open Access book projects that are Open for Submissions visit us here.
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Our books contain scientific content written by two Nobel Prize winners, two Breakthrough Prize winners and 73 authors who are in the top 1% Most Cited.
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ACCESS
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YOUR WORK, YOUR COPYRIGHT
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CURRENT PROJECTS
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To view current Open Access book projects that are Open for Submissions visit us here.
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\r\n\tTransforming our World: the 2030 Agenda for Sustainable Development endorsed by United Nations and 193 Member States, came into effect on Jan 1, 2016, to guide decision making and actions to the year 2030 and beyond. Central to this Agenda are 17 Goals, 169 associated targets and over 230 indicators that are reviewed annually. The vision envisaged in the implementation of the SDGs is centered on the five Ps: People, Planet, Prosperity, Peace and Partnership. This call for renewed focused efforts ensure we have a safe and healthy planet for current and future generations.
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\r\n\tThis Series focuses on covering research and applied research involving the five Ps through the following topics:
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\r\n\t1. Sustainable Economy and Fair Society that relates to SDG 1 on No Poverty, SDG 2 on Zero Hunger, SDG 8 on Decent Work and Economic Growth, SDG 10 on Reduced Inequalities, SDG 12 on Responsible Consumption and Production, and SDG 17 Partnership for the Goals
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\r\n\t2. Health and Wellbeing focusing on SDG 3 on Good Health and Wellbeing and SDG 6 on Clean Water and Sanitation
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\r\n\t3. Inclusivity and Social Equality involving SDG 4 on Quality Education, SDG 5 on Gender Equality, and SDG 16 on Peace, Justice and Strong Institutions
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\r\n\t4. Climate Change and Environmental Sustainability comprising SDG 13 on Climate Action, SDG 14 on Life Below Water, and SDG 15 on Life on Land
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\r\n\t5. Urban Planning and Environmental Management embracing SDG 7 on Affordable Clean Energy, SDG 9 on Industry, Innovation and Infrastructure, and SDG 11 on Sustainable Cities and Communities.
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\r\n
\r\n\tThe series also seeks to support the use of cross cutting SDGs, as many of the goals listed above, targets and indicators are all interconnected to impact our lives and the decisions we make on a daily basis, making them impossible to tie to a single topic.
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