**6. Chemotherapy for CRLM**

Systemic chemotherapy in CRLM is administered to attain surgical eligibility, for disease control, peri-operatively, or palliatively; since the treatment alone is rarely curative, with 5-year survival rates less than 10%, and historically less than 1% [25, 30, 117]. Polymetastatic liver disease faces treatment limitations with chemotherapy being the primary treatment. The survival rate is poor and a large demand exists for improved treatment options. As surgical resection offers the best long-term survival rates, the aim of systemic chemotherapy is often to downsize tumors to convert ineligible patients to surgical candidates, with systematic review showing a conversion rate for R0 resection in initially ineligible patients at 23% [118]. Chemotherapy regimens are administered neoadjuvantly prior to hepatectomy for cytoreduction, to reduce metastatic tumor size, allowing smaller resection volumes [119]. The regimens are also administered after resection to reduce recurrence [25, 120]. Hepatic intra-arterial infusion is often beneficial because the liver metastasis is supplied by the hepatic artery network, normal tissue is supplied by the portal vein, and locoregional treatment can be performed without exposing much healthy tissue [121–123]. The liver contains a capillary network of sinusoids that filter the blood as shown in **Figure 4**. Approximately 45% of metastatic tumor cells, predominately arriving from the hepatic arterial network [123], become embedded in the sinusoids [89]. Normal liver parenchyma receive about 80% of the blood supply from the portal vein and about 20% from the hepatic artery. In contrast, about 80% of the tumor blood supply arrives from the hepatic artery [116]. This allows locoregional embolization techniques, like radioembolization and chemoembolization, to both embolize the blood supply to specific tumor segments, and deliver locoregional radiotherapy or chemotherapy. These embolization techniques are suggested to be considered for metastatic CRC limited only to the liver, and after unsuccessful chemotherapy [1].

The chemotherapy regimen depends on a number of factors, including: aim of cytoreduction prior to surgery, aim of disease control, aim of palliation, type of somatic gene mutation, and wild-type or mutant phenotype. Somatic mutations of RAS proto-oncogenes have been found in up to 52% of CRLM hepatic resections, with up to 6–12% of resections expressing BRAF mutations, and co-occurring protooncogene RAS and TP53 tumor-suppressor mutations as common genetic events [1, 94]. According to ESMO guidelines, first-line chemotherapy for cytoreduction in RAS

#### **Figure 4.**

*Histological depiction of liver lobules. These units are microscale components of liver tissue. The liver sinusoids are small capillaries, with blood supplied by small branches of the hepatic artery and portal vein. Deesign/shutterstock.com.*

tumors should be recommended cytotoxic doublets (FOLFOX/CAPOX/FOLFIRI), in combination with VEGF antibody bevacizumab for RAS mutant-type tumors, and EGFR antibodies for wild-type tumors. FOLFOXIRI with bevacizumab are recommended as a first line treatment for cytoreduction in CRLM BRAF mutant tumors [1].

Chemotherapeutics can also exhibit many adverse side-effects on healthy liver tissue. Side-effects include sinusoidal obstruction syndrome and chemotherapyassociated steatohepatitis, that can lead to liver failure or increased mortality rates [119]. Additionally, the chemotherapy can cause missing metastases, making lesions unidentifiable on radiological imaging, complicating surgical decisions, and increasing the chance of recurrence [119]. Chemotherapy has difficulty supplying tumor cells with adequate drug dose. The maximum dose is limited by systemic toxicity effects and inadequate tumor penetration is common [8, 124]. Intrahepatic arterial delivery can exhibit acute side-effects of hepatocellular atrophy causing cirrhosis and necrosis [123, 125].

Doxorubicin is an anthracycline chemotherapeutic that can be administered during combination therapy. A liposomal form was created relatively early due to the need for better treatment in Kaposi sarcoma from autoimmune deficiency syndrome [126]. Clinical trials of FUS-mediated thermosensitive liposomal doxorubicin drug delivery to liver tumors [127, 128] have shown large increases in intratumoral doxorubicin concentration, and there are ongoing trials with MRgHIFU for pediatric tumors [129]. Similar ongoing trials are studying the enhanced ability for microbubbles to improve chemotherapy delivery to metastatic liver tumors [130].

### **7. Radiotherapy for CRLM**

Radiotherapy emits ionizing radiation at tumors, causing DNA damage, and apoptosis. The technique exhibits some similar drawbacks to focused ultrasound. Cumulative radiation exposure can occur in the beam's near and far field, resulting in unwanted tissue damage [8, 131, 132]. Also, systems require computed tomography

### *Magnetic Resonance-Guided Focused Ultrasound in the Treatment of Colorectal Cancer Liver… DOI: http://dx.doi.org/10.5772/intechopen.105906*

guidance and respiratory motion control [8, 133]. Local ablative techniques, including radiotherapy, are generally considered to be limited to patients with unresectable CRLM or oligometastatic disease [1]. CRLM radiotherapy has often been limited by liver parenchyma radio-sensitivity. External beam radiation doses of 70–90 Gy needed for CRLM and HCC tumor treatment exceeds tolerance limits of 35 Gy for radiationinduced liver disease (RILD) [57, 134] that can lead to liver failure and death [25]. The condition occurs two to sixteen weeks after treatment, is identified by ascites, high levels of alkaline phosphatases, and high levels of liver transaminases [135].

Stereotactic body radiation therapy (SBRT) with linear accelerators has recently gained much interest for surgical ineligibility, particularly in oligometastatic disease. With SBRT, fiducial markers are percutaneously placed near the tumor site to allow precise tumor targeting [57]. Though MRI guidance reduces invasiveness, without the need for fiducial markers [116]. SBRT is recommended by ESMO to be considered for patients with oligometastatic disease who are ineligible for surgery and ablative therapy [1]. One major advantage of SBRT compared to ablative therapies is that the treatment is non-thermal, mitigating some of the common side-effects seen in local ablative techniques, such as fluid perfusion effects [116]. Studies have shown that liver failure is infrequent when only a portion of the liver is irradiated [135]. The liver toxicity is mild to moderate, with liver failure in less than 1% of patients [136, 137]. Treatment of oligometastatic CRC in the liver with SBRT, suggests one and two year overall survivability at about 67.1% and 56.5%, respectively [137]. Many early phase clinical trials are recruiting, active, or recently completed, for treatment of primary or secondary hepatic tumors with magnetic resonance guided linear accelerators [138, 139] and magnetic resonance guided SBRT [140–143]. Recent phase I trial results with magnetic resonance guided SBRT, showed improved toxicity, with estimated 2-year overall survival of 51%, and median overall survival of 29 months [144].

### **8. Focused ultrasound clinical studies for liver cancer**

A substantial number of clinical studies, cohorts, and randomized control trials for non-liver MRgHIFU and MRgFUS have been reported, including: treatment with bone osteomas or palliative bone metastasis [145, 146], uterine fibroids [147–151], gynaecological tumor recurrence [152], prostate cancer [153, 154], essential tremor [155, 156], and breast cancer [157]. Many clinical studies have been reported for USgFUS ablation for liver tumors [158–165], with most studies reporting on HCC ablation [166]. Similar to USgFUS, new histotripsy devices using cavitation rather than thermal ablation, are currently being studied for the treatment of primary and secondary tumors, with an active prospective clinical trial [45, 46, 167, 168]. No Phase III trials for USgFUS or MRgHIFU ablation of CRLM have been published [116]. Early USgFUS studies in liver malignancies, not distinguishing between metastatic liver tumors and primary liver tumors, showed a median survival time of 13.4 months, 6-month survival times of 82.6%, and 12-month survival time of 53.4% [159]. More recent systematic reviews of FUS for liver malignancies have given 1 year, 2 year, and 5-year survivability of 81%, 60%, and 39%, respectively [166]. Most studies have been conducted using the Chongqing Haifu JC system, capable of up to 300 W acoustic power and peak intensity up to 20,000 W cm2 [166]. The system has received the mark Conformite´ Europeenne´ (CE), being the most reported system for clinical liver tumor ablation [2, 3]. The permission is granted to individual commercial models rather than general treatment procedures. The magnetic resonance guided

systems that have received regulatory approval for alternative treatments include the ArcBlate (Episonica, Hsinchu, Taiwan), Exablate (Insightec, Tirat Carmel, Israel), and Sonalleve (Profound Medical, Mississauga, Canada) systems.

Local ablative techniques, including focused ultrasound ablation, are generally recommended only in cases of unresectable liver metastases or oligometastatic disease [1]. Most FUS ablation therapy studies for liver tumors are USgFUS for HCC, with less reports of metastatic liver tumor treatment [166]. Particularly advantageous in FUS is the improved side effect profile and reduced morbidity compared to standard treatment options. The treatment can occur multiple times with no cumulative radiation-like side effects. In relation to chemotherapy, it is much more focused, with less toxicity to healthy tissues [8, 169]. Additionally, extracorporeal FUS liver ablation is completely non-invasive and offers very fast recovery times [170]. Benefits of MRgHIFU compared to USgFUS include near real-time temperature mapping, integration into existing imaging systems, less propensity for radiofrequency interference in the imaging system, and capability of assessing treatment response during the procedure. Though ultrasound-guided devices do not provide real-time temperature map-ping, assessment of grey-scale change are indicative of coagulative necrosis [166]. Treatment plans with FUS generally depend on the cancer staging. Curative ablation of early stage tumors often include a 1.5–2.0 cm peripheral tissue margin. The treatment is administered palliatively for late-stage tumors to slow progression or alleviate symptoms [8, 160].

Drawbacks to hepatobiliary focused ultrasound studies have been the need for general anesthesia, long treatment times, scattering by the thoracic cage, high power requirements, respiratory motion, skin burns, osteonecrosis, skin pain, skin edema, rib resection, fever, the need for intrapleural effusion, and reduced thermal dose from fluid perfusion of surrounding vessels [2, 39, 165, 166, 170–177]. A systematic review of USgFUS for the treatment of malignant hepatobiliary tumors indicated the primary complications were skin burns in 15% of cases, followed by localized pain in 5%, then fever at 2% [166]. Major post-treatment complications include fluid and/or air accumulation in the lungs, biliary obstruction, and fistula occurrence [177].

Some studies have reported focused ultrasound ablation in primary and secondary liver tumors in difficult locations, including near major hepatic veins and arteries, and near surrounding organs of the heart, gallbladder, stomach, and intestine [162, 165, 178]. Tumors located near surrounding organs are high-risk. Particularly sensitive are the bowel and gallbladder due to the thin walls and risks of peritonitis [162].

Skin and rib burns have been addressed in a variety of manners. Skin burns have been reported to occur with tumors located near the subcapsular area, resulting from possible rib reflection or reflections from internal gas pockets in the bowel or lung parenchyma [166]. The right lobe is more susceptible as it is predominately located behind the ribs [162]. Intrapleural effusion can distance the tumor site from the subcapsular area, or rib resection can be performed [162, 179]. Particularly troublesome are tumors of the liver dome in Couinaud segments 7 and 8, due to the close proximity to the lungs, the close proximity to the ribs, and that this region tends to remain behind the rib cage under general anesthesia due to reduced respiration [162]. A small cohort for USgFUS reported that proper intraoperative assessment of the soft tissue prevented skin burns in all patients [161].

A variety of techniques have been tested to overcome respiratory motion and rib interaction. Respiratory motion creates complications requiring organ image

### *Magnetic Resonance-Guided Focused Ultrasound in the Treatment of Colorectal Cancer Liver… DOI: http://dx.doi.org/10.5772/intechopen.105906*

registration techniques [180] and MRI motion artifact compensation [174, 181]. Numerous preclinical studies have undertaken new technologies to address respiratory motion and rib interactions [39, 172, 174–176, 180, 182–186]. Previous USgFUS human studies have generally been successful at performing ablation through the ribs; though additional measures have included left lung ventilation with endotracheal intubation and general anesthesia to reduce liver movement, intrapleural effusion, and rib resection [160, 162, 179]. MRgHIFU pilot studies used intermittent sonications, and limited to the treatment to the left liver lobe, in tumor sites not blocked by the ribs [170, 187–189].

Handheld intraoperative HIFU devices under ultrasound-guidance are in development, and being tested in early phase clinical trials for CRLM tumor abla-tion. The technique is similar to intraoperative radiofrequency and microwave ablation, but prevents the need for an intraparenchymal probe. Results have been reported using the device for ablating tissue near tumors in segments prior to surgical resection, to assess accuracy and safety. Applications include reduction of hemorrhaging during surgery and potentially bridging more patients for surgical resection [190–193]. The device was shown capable of in vivo hepatic vessel occlusion for diameters of 2 mm [194], and studies have reported diameters of left hepatic arteries and right hepatic veins between 3 and 4 mm [195].

Several small clinical studies have been reported for MRgHIFU ablation for HCC [170, 187–189, 196, 197]. There is currently an ongoing Phase I clinical trial with MRgHIFU for a variety of pediatric solid tumors, in which hepatic tumors are eligible [198].

In the study from Okada et al. [187], MRgHIFU liver tumor ablation was performed on a single patient. The MRI system utilized respiratory gating and ablation was performed on a 15 mm HCC lesion. The procedure required about two hours to ensure complete coagulation by repeated coverage. Gadolinium contrast agent was administered post-treatment and no increased signal intensity was observed at the tumor site, indicating expected ablation contrast. The authors noted the need for better technology for avoiding bowel loops, ribs, and respiratory liver motion. Though, the patient only complained of slight skin heating discomfort during treatment and was released from the hospital the following day.

Anzidei et al. treated a single HCC patient more comprehensively with MR-FUS [188]. The patient refused surgery and percutaneous ablation, then opted for MR-FUS. The individual had no distant metastases, was treated successfully, and later underwent total liver transplant. Excised liver histopathology showed complete coagulative necrosis with only slight recurrence at the ablation periphery. The investigators noted the procedure can be improved with better respiratory motion control and expected that future applications would use automated feedback algorithms.

Gedroyc conducted a series of pilot studies for MRgHIFU liver tumor ablation [170, 189]. It was reported that the absorption from the ribs was problematic and the treatments were limited to patients with exposed tumor sites, such as below the rib line or in the left lobe of the liver. One case was a female with HCC arising from Hepatitis-B infection. She was previously treated with hepatic arterial chemoembolization and laser ablation. Recurrence occurred with a 1.5 cm HCC lesion in the left lobe within Couinaud segment 3, a position that was not covered by the ribs. The site was ablated with MRgHIFU. In another case, the patient was a male with HCC, Hepatitis-C, extensive cirrhosis, and elevated alpha-fetoprotein levels. He was treated for a 3 cm HCC in the anterior portion of the left liver lobe.


*Sustainable Management of Phosphorus in Agriculture for Environmental Conservation DOI: http://dx.doi.org/10.5772/intechopen.113086*

Therefore, agricultural management practices should focus on reducing P-transport caused by excessive consumption of inorganic fertilizers and organic manures [67]. Both soil and hydrological factors are responsible for the movements of P [68]. The former decides the initial transport, while the latter is associated with transport as well as the pathways. There are three major pathways by which P is transported to surface water bodies. Those are surface runoff, subsurface flow, and vertical flow [67]. Minimizing soluble P concentrations or increasing water infiltration in soil for P-adsorption will help in decreasing the dissolved P-transport [69]. The subsurface flow of P is a concern only for soils having high P-saturation or preferential flow. At high rainfall intensity (hydrological factor), fertilizers containing a higher concentration of water-soluble P can increase P-transport in runoff [70]. Manures containing high organic P (like poultry manure) may contribute to dissolved P-losses via leaching and displacement of loosely bound inorganic P [71, 72]. The possible transport of P in the environment has been highlighted in **Table 1**.

Long-term application of P-fertilizers (like triple superphosphate) may increase the concentrations of some trace elements or heavy metals (arsenic, cadmium, copper, chromium, nickel, vanadium, and zinc) in soil [73]. Mining and processing of rock phosphate cause significant contamination (from radioactive to heavy metal pollutants) in the environment [74, 75]. Phosphogypsum, a by-product obtained during the processing of rock phosphate, marks a serious potential hazard for human health and soils, water, and atmospheric pollution [75]. The use of organic wastes (pig slurry and cattle slurry) above crop P demands saturates soil adsorption sites, leading to P-migration in the soil profile and subsurface water contamination [76]. Therefore, instead of the bulk application of the bioresources, technologies should be framed to recover P from human and animal wastes including sewage, sludge, manure, incineration ashes, etc., by developing contaminant-free fertilizers, e.g., struvite (ammonium magnesium phosphate) and/or other soluble products for efficient P-cycling [77].

#### **Table 1.**

*Transport of phosphorus in the environment.*


*2.1.2 Chemical preparation method*

The chemical preparation process involves a bottom-up technique and is the most frequently employed method for preparing silver nanoparticles in water or organic solvents as a stable, colloidal dispersion. Various chemical preparation methods are available, such as sol system, chemical preparations: CdS base, CdS/ HgS/CdS composite, sol-gel process, hydrothermal, pyrolysis and chemical vapor deposition (CVD) process [3] (metallic) silver salt to produce nanoparticles. Here, commonly used reductants are borohydride, citrate, ascorbate and elemental hydrogen that reduce (metallic) silver salt to produce nanoparticles. Chemically synthesized nanoparticles are made of Cu, Ag, Au, Gold, etc., although the use of toxic chemicals in the synthesis protocol cannot be avoided. Alternative methods for nanoparticle synthesis are therefore explored in order to minimize the use of chemicals, and several physical methods were developed and implemented for synthesis of nanoparticles to resolve the chemical preparation.

#### *2.1.3 Biological preparation method*

Physical methods are expensive and require strong material, toughness, strength, etc. for nanoparticle synthesis, which may not be suitable for large-scale

**97**

**Table 1.**

and the respective nanoparticle size.

**3. Nanofabrication using plant sample**

*Phytonanofabrication: Methodology and Factors Affecting Biosynthesis of Nanoparticles*

**Sr. no. Plant species Plant part NP size References** *Aloe vera* Leaves 50–350 nm [5] *Argyreia nervosa* Seeds 20–50 nm [6] *Acorus calamus* Rhizome 31.83 [7] *Allium sativum* Sucrose and fructose 4–22 nm, 4 4 ± 1.5 nm [8] *Boerhaavia diffusa* Whole plant 25 nm [9] *Citrus sinensis* Peel 10–35 nm [10] Cocos nucifera Inflorescence 22 nm [11] *Calotropis procera* Plant 19–45 nm [12] Olive extract 1 ml 30 nm [13] *Passiflora foetida* Leaf disc 14 nm [14] *Terminalia chebula* Fruit extract 100 nm [15] *Thevetia peruviana* Latex 10–60 nm [16]

production of nanoparticles. Although chemical methods are used in the mass production of nanoparticles, the key product generated in the synthesis of nanoparticles tends to cause pollution that ultimately poses a risk to life in terrestrial and aquatic life, leading to environmental accumulation. Physical and chemical methods therefore constitute invaluable processes, while the problem can be overcome by using a biological solution that uses living organisms, bacteria, plants, etc., which is sometimes called the green method. These green strategies include plants, microorganisms and enzymes that are considered to be nonhazardous to humans through the use of natural biological agents that are ecofriendly, biodegradable as reducing, capping and stabilizing agents for nanoparticle synthesis. These biological preparations require a living origin, such as insects, parts of plants, microorganism such as *F. oxysporum*, enzymes, amino acid, vitamin polysaccharides, polyphenols, amino acid, tea, coffee, wine, winery waste, banana, red grape, table sugar and glucose are used for capping and stabilizing. Plant components for the manufacture of nanoparticles have significant advantages over other methods such as easy preparation, processing, cost-effective, rapid development, reproducible, stable components, environmentally friendly, avoiding the use of harsh and toxic chemicals and zero contamination of the environment [4]. Nevertheless, plant-based preparation has become common due to its simple, comprehensive availability and extensive range of samples, including plant part extracts and natural products. In addition, plant, leaf core, root, latex, seed and stem contain polyphenols and have been successfully used for metal particle synthesis [4]. Moreover, plant extract, more specific leaf extract, seed extract, stem extract, fruit extract, root extract and flower extract are used for nanoparticle synthesis. **Table 1** displays many studies of plants mediated silver nanoparticles synthesis reports with their reducing biomolecules

*Reports of silver nanoparticle synthesis using plant species, biomolecules, nanoparticle sizes and references [17].*

The biological method that uses plant samples is the easiest and cheapest way to use a bottom-up approach to nanoparticle synthesis. In broad aspect, there are large

*DOI: http://dx.doi.org/10.5772/intechopen.90918*

*Phytonanofabrication: Methodology and Factors Affecting Biosynthesis of Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.90918*

#### **Table 1.**

*Smart Nanosystems for Biomedicine, Optoelectronics and Catalysis*

**2.1 Types of nanoparticle preparation**

*2.1.1 Physical preparation method*

*2.1.2 Chemical preparation method*

*2.1.3 Biological preparation method*

In nanosynthesis, the reactant A (metallic salts) interacts with the reactant B (capping agent) where the nucleation reaction occurs. The entire phenomenon occurs at a specific temperature, and the nanoparticle synthesis is significantly improved by the stabilizing (agent) to form metallic nanoparticles and by-products. In several cases, the by-product or larger reaction structures may lead to a mass agglomeration, which suggests aggregation by chemical bonding of larger particles. However, the reacting metallic salts and the capping agent form nanoparticles that have been stabilized by a capping agent that directly prevents mass synthesis.

The physical method uses a "top-down" approach in which the bulk material is pulverized into fine particulate matter by means of force applied, such as crushing, impact, disruption, degradation, cutting, cryo-grinding, grinding, processing and homogenization. Physical methods use the milling process in micro-particle fracturing, and few examples involve ball milling, high-energy ball milling (HEBM), grinding, cryo-grinding, refining and homogenization: high process homogenization (HPH) and medium-pressure homogenization (UHPH). These physical productions are versatile methods that manufacture nanoparticles of larger size, diameter and volume, which are still controlled, generate surface defects, contamination, costly and time-consuming. It may also be possible to produce nanoparticles of the same size, but instrument assembly is very expensive and maintenance is too costly. This imperfection in the form of efficient and expensive maintenance culminated in the space travel of biological sources and methods for the nanoparticle manufacturing process [2]. The synthesis of nanoparticles using biological methods includes the synthesis of nanoparticles using living things or matter. There are several other ways to synthesize nanoparticles, such as chemical and biological

The chemical preparation process involves a bottom-up technique and is the most frequently employed method for preparing silver nanoparticles in water or organic solvents as a stable, colloidal dispersion. Various chemical preparation methods are available, such as sol system, chemical preparations: CdS base, CdS/ HgS/CdS composite, sol-gel process, hydrothermal, pyrolysis and chemical vapor deposition (CVD) process [3] (metallic) silver salt to produce nanoparticles. Here, commonly used reductants are borohydride, citrate, ascorbate and elemental hydrogen that reduce (metallic) silver salt to produce nanoparticles. Chemically synthesized nanoparticles are made of Cu, Ag, Au, Gold, etc., although the use of toxic chemicals in the synthesis protocol cannot be avoided. Alternative methods for nanoparticle synthesis are therefore explored in order to minimize the use of chemicals, and several physical methods were developed and implemented for

synthesis of nanoparticles to resolve the chemical preparation.

Physical methods are expensive and require strong material, toughness, strength, etc. for nanoparticle synthesis, which may not be suitable for large-scale

**2. Nanosynthesis**

**96**

methods.

*Reports of silver nanoparticle synthesis using plant species, biomolecules, nanoparticle sizes and references [17].*

production of nanoparticles. Although chemical methods are used in the mass production of nanoparticles, the key product generated in the synthesis of nanoparticles tends to cause pollution that ultimately poses a risk to life in terrestrial and aquatic life, leading to environmental accumulation. Physical and chemical methods therefore constitute invaluable processes, while the problem can be overcome by using a biological solution that uses living organisms, bacteria, plants, etc., which is sometimes called the green method. These green strategies include plants, microorganisms and enzymes that are considered to be nonhazardous to humans through the use of natural biological agents that are ecofriendly, biodegradable as reducing, capping and stabilizing agents for nanoparticle synthesis. These biological preparations require a living origin, such as insects, parts of plants, microorganism such as *F. oxysporum*, enzymes, amino acid, vitamin polysaccharides, polyphenols, amino acid, tea, coffee, wine, winery waste, banana, red grape, table sugar and glucose are used for capping and stabilizing. Plant components for the manufacture of nanoparticles have significant advantages over other methods such as easy preparation, processing, cost-effective, rapid development, reproducible, stable components, environmentally friendly, avoiding the use of harsh and toxic chemicals and zero contamination of the environment [4]. Nevertheless, plant-based preparation has become common due to its simple, comprehensive availability and extensive range of samples, including plant part extracts and natural products. In addition, plant, leaf core, root, latex, seed and stem contain polyphenols and have been successfully used for metal particle synthesis [4]. Moreover, plant extract, more specific leaf extract, seed extract, stem extract, fruit extract, root extract and flower extract are used for nanoparticle synthesis. **Table 1** displays many studies of plants mediated silver nanoparticles synthesis reports with their reducing biomolecules and the respective nanoparticle size.


*2.4 Exam under anesthesia (EUA) considerations*

If EUA is warranted, general anesthesia will be needed to conduct detailed examination and ancillary testing at one time. Preparation of the room for EUA should consist of examination materials including: external photography, portable slit lamp, tonometry, indirect ophthalmoscope with condensing lens and scleral

#### **Figure 1.**

*10 mHz ultrasound in A and B scan mode showing a large intraocular retinoblastoma with irregular mass and intrinsic calcification with acoustic shadowing.*

**31**

*Retinoblastoma: Presentation, Evaluation, and Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.85744*

pinealoblastoma in trilateral disease.

successfully without long term sequelae [16].

hypoplastic thumbs, cleft palate, or microcephaly [17].

buphthalmos and increased corneal diameter.

**2.6 Examination under anesthesia**

*2.6.1 External examination*

*2.6.2 Anterior examination*

disease [18].

**2.5 Anesthesia**

depressor, wide-angle hand-held fundus camera, intravenous fluorescein dye, high resolution ophthalmic ultrasound, ultrasound biomicroscopy, electroretinography. When retinoblastoma is confirmed, an MRI of the brain and orbits with and without contrast should be ordered at the time of the EUA to assess for extrascleral extension, orbital anatomy, posterior portion of the optic nerve, and presence of

The type of general anesthetic and airway support varies depending on institution and available resources. Safe anesthesia methods range from mask anesthesia, to laryngeal mask airway (LMA), to complete endotracheal intubation. Both inhaled anesthetics and intravenous anesthetics, or a combination of the two, are suitable for examination. General guidelines recommend that heavy fatty meals be discontinued 8 h prior to procedure, light meals, formula, and nonhuman milk 6 h prior to surgery, human milk 4 h prior to anesthesia, and clear liquids 2 h prior to anesthesia [15]. These recommendations will vary by location and anesthesiologist, and type of anesthesia administered. Recently there has been literature support for use of LMA without placement of intravenous lines, which reduces total time under anesthesia without increased anesthesia complications, all of which were managed

An orderly approach to the exam should be taken to efficiently access and treat the patient to avoid prolonged anesthesia exposure. The overall features of the child should be assessed, evaluating for any abnormalities that are consistent with retinoblastoma and a 13Q deletion syndrome which is strongly associated with retinoblastoma risk (**Figure 2**). Patient's with 13Q deletion syndrome may have hypotelorism, micrognathia, hypoplasia of midface, absent nasal bones, large ears,

Intraocular pressures should be measured using Schiotz tonometer, tonopen, Perkins tonometer, or pneumotonometer. Elevated intraocular pressure in patients with retinoblastoma can be secondary to iris neovascularization or angle closure, and has been associated with increased risk of optic nerve invasion and metastatic

After intraocular pressure is checked, corneal diameter should be measured with a caliper both in horizontal and vertical directions. Conditions such as persistent fetal vasculature (PFV) is associated with asymmetric corneal diameter size as well as eyes with congenitally elevated intraocular pressures can be associated with

A portable slit lamp should be used to assess the anterior segment. The clinician should evaluate anterior depth, clarity of the lens, neovascularization of the iris or atrophy, retrolental membrane, anterior chamber cells, nodules of the iris, or anterior vitreous seeds seen behind the lens. Presence of a pseudohypopyon should raise suspicion for endophytic tumor and anterior chamber seeing of cancer. It may be possible to visualize retinal vascular, retrolental masses, or retrolental membranes. Retrolental retinal tissue should reveal blood vessel branching patterns depressor, wide-angle hand-held fundus camera, intravenous fluorescein dye, high resolution ophthalmic ultrasound, ultrasound biomicroscopy, electroretinography. When retinoblastoma is confirmed, an MRI of the brain and orbits with and without contrast should be ordered at the time of the EUA to assess for extrascleral extension, orbital anatomy, posterior portion of the optic nerve, and presence of pinealoblastoma in trilateral disease.


**3. The benefits of healthcare supply chain management digitization**

The connected store network, or inventory network 4.0, is solid and versatile, able to do quickly answering changing circumstances and recuperating from surprising interferences. Medical services organizations are carefully modernizing medical care production network the board by embracing the most current medical services store network innovation, simplifying it for themselves as well as their exchange accomplices to streamline and improve the production network. They are using this innovation to acquire continuous, definite access into their stock chains from starting to complete the process of, permitting them to recognize and address potential hardships and obstacles early and turn quickly on a case by case basis.

They might use medical services store network investigation to all the more accurately gauge interest, advance stock preparation and the executives, and answer all the more successfully to changing economic situations by joining inventory network and clinical information. Computerized inventory network the executives innovations can help raise efficiency and lessen time to advertise by advancing expanded correspondence and joint effort all through the medical services production network. Besides, by upgrading trust and straightforwardness all through the stock organization, they

further develop store network flexibility, aiding the production of a medical services esteem chain that can more readily answer and recuperate from future pandemics and other general wellbeing disasters.

Work the executives for provider networks across a few ventures. Resolve provider episodes up to 80% faster.

### **3.1 Healthcare supply chain management in the future**

A computerized production network organization, intended to deal with the intricacy of medical services production network the board, gives associations in this quickly developing area with the straightforwardness and start to finish perceivability they expect to deal with a convoluted and extending organization of exchanging accomplices by means of constant data streams, specialized devices, and cooperative work processes. It can help them in distinguishing and settling organic market concerns right off the bat, before patients and their primary concern endures. Since it is cloud-based, network individuals benefit from the versatility and adaptability expected to incorporate and utilize mechanical cycle computerization, man-made consciousness and AI, prescient investigation, and other state of the art store network 4.0 advancements.

Work the board programming likewise helps firms in producing cooperative advancement and creating patient-driven inventory network organizations. Supply accomplices might team up more successfully to organize patient results in original ways and make new advanced working models for the fate of medical care. Medical organizations might utilize work the board instruments and a computerized supply organization to ensure that all patients get the treatments they require, when and when they require them. Individuals from the organization are engaged to team up for everyone's best interests, and worldwide populaces benefit from expanded admittance to life-saving drugs and better treatment made conceivable by computerized medical services store network the board.

## **3.2 Blockchain technology could enhance the caliber of clinical research**

Since it empowers information recording, sharing, and care, blockchain can possibly impact clinical examination. Without a doubt, it involves a decentralized secure global positioning framework for any information connections that might happen with regards to clinical preliminaries, as well as a distributed comprehensive organization that empowers information sharing on the examination side while likewise guaranteeing all vital straightforwardness and protection worries on the patient local area side.

Therefore, this technique might encourage more confidence in clinical exploration, whose standing has been seriously hurt by ongoing scandals [9, 10]. Blockchain innovation might be seen as an establishment for improved clinical examination strategies as well as a push toward more transparency to construct certainty inside research networks and among exploration and patient networks.

### **3.3 Building reliable clinical studies: At each step, keep track and timestamp**

Information sacredness and trustworthiness are two crucial qualities of information at the utilitarian level, "the information level." as far as information sacredness and accuracy, Blockchain guarantees that occasions are kept in their exact sequential control, significantly forestalling deduced reproduction study.


!531A2!DB353AC4!AB!C25D!

with the corresponding vector of weights:

$$(1, 1, 1, 2, 2, 2, 1, 1, 1, 1, 1, 2, 1, 2, 1, 1, 1, 1, 1, 1, 1, 2, 3)$$

#### *2.3. VSST Data Analysis Method*

We are going to formulate the VSST problem in terms of a pairwise sequence alignment, where both the target scan-path and the obtained scan-path are strings.

Let *T* = *t*1 ... *tn* be any string of length *n* over the alphabet A = {1, 2, 3, 4, 5, *A*, *B*, *C*, *D*, *E*, !}, and let *P* = 1*A*2*B*3*C*4*D*5*E*. Given *T* and *P*, we look for the matches of *P* in *T*, that is the occurrences of symbols of *P* in *T*. Regions of identity (matches) can be visualized by the so-called dot-plot. A dot-plot is a 10 × *n* binary matrix *M* such that the entry *mij* = 1 if and only if *pi* = *tj*, otherwise *mij* = 0. Some toy examples are shown in Figure 3 where the identity is visualized by a dot.

**Figure 3.** Dot-plots for the toy-sequences: (**a**) T = 1A2B3C4D5E; (**b**) T = E1!A2B3CA4D54E and (**c**) T = 4C1BA2!3C4E5DA.

It is easy to see that "diagonals" of dots correspond to consecutive matches of *P* in *T*. This can be formalized as follows. A substring of *T* is a finite sequence of consecutive symbols of *T*, while in a subsequence symbols are not necessarily consecutive. Thus, *P* is a subsequence of *T* if there exist indices *i*1 < ... < *im* such that *p*1 = *ti*1 , *p*2 = *ti*2 , *pm* = *tim* and *T* = *ti*1*ti*1+1 ... *tim* is the substring of *T* containing *P*.

Let us define the VSST problem as an approximate string matching problem. The approximate string matching problem looks for those substrings of the text *T* that can be transformed into pattern *P* with at most *h* edit operations: a deletion of a symbol *x* of *T* changes the substring *uxv* into *uv*; an insertion of a symbol *x* changes the substring *uv* of *T* into *uxv*; a substitution of a symbol *x* of *T* with a symbol *y* changes the substring *uxv* into *uyv*. When deletion is the only edit operation allowed and we choose *h* = *k* − *m*, the problem is equivalent to finding all substrings of T of length at most *k* that contain *P* of length *m* as a subsequence.

In the VSST problem we search for the first occurrence of *P* in *T*, i.e., we find the substring of *T* starting in the leftmost symbol in *T* containing *P* as a subsequence. This can be done in linear time in the size *n* of *T* with the naïve algorithm.

#### *2.4. The Score Scheme*

Let *T* = *t*1 ... *tk* be the substring of *T* containing *P*. Next step consists of scoring the approximate matching between *T* and *P*. Actually, *h* = *k* − 10 provides a first evaluation of the distance between *T* and *P* since they differ by *h* symbols. Note that this corresponds to defining a scoring system that assigns value 1 to each deletion and sums up each value. However, this measure is oversimple to provide a meaningful evaluation, and moreover we prefer to measure the complementary information, to calculate a "similarity score" between *T* and *P*. Indeed our goal is to assign a final score assessing the performance of the patient in the VSS test. The first step in the definition of the scoring function is to assign a positive value (a reward) to each match, i.e., to each occurrence of a symbol of *P* in *T*. On the contrary, each deletion of symbols of *T* must be assigned a negative value (a penalty). We decided to weakly penalize a deletion of the symbol ! with respect to the deletion of any other symbol, since we consider a fixation of the background as an intermediate pause in the process, but not a true selection of an ROI. We refer to these three values as *penalty scale constants*.

In addition, in the latter case (deletion of a symbol not ! in *T*), we compute the distance of the centroid of the ROI corresponding to the deleted symbol to the centroid of the ROI of the next expected symbol of *P*, to take the spatial relation between the two ROIs. The set of the distances for each pair, normalized by the maximum distance, is then collected in a *distance matrix* (Figure 4).

**Figure 4.** Distance matrix.

Another factor included in the score is the duration of the fixation. We store the information in a parallel array as explained in Section 2.2.2. We assume that the fixation duration is associated to hesitation in the VSST. Since duration corresponds to consecutive repetitions of any symbol, we define a function decreasing in the number of repetitions for scoring the match and increasing in the number of repetitions for scoring the deletion. We refer to it as the *duration function*.

Finally, since the fixations outside the ROIs may be part of the exploration strategy, we compute the frequency of each symbol in the prefix ending there, to amplify the penalty: the frequency corresponds to the number of times that the symbol has been already fixed in the exploration so that it reflects the number of times needed to learn its position.

To summarize, the final score of *T* is the sum of the contributions to the score for each symbol in *T* where each score is obtained by the product of the following factors: the penalty scale constant *v*, the duration function *f* , and, in case of deletion of a symbol non !, an item of the distance matrix, *dist*, and the frequency *f req* of the symbol. The computation of the score is sketched in Algorithm 1.

#### **Algorithm 1** Similarity score evaluation

**Require:** *T*, *w*, *align*, *v*, *P*, *f*(*w*) **Ensure:** *score j* ← 0 index for P *i* ← 0 index for T' *score* ← 0 *f req*(*k*) ← 0 ∀*k in P* **while** *j* = *length*(*P*) *AND i* = *length*(*T*) **do if** *i* = *align*(*j*) **then** match *p*\_*score* ← *v*(0) · *f*(*w*(*i*)) *f req*(*P*(*j*)) ← *f req*(*P*(*j*)) + 1 *j* ← *j* + 1 **else if** *T*(*i*) =! **then** deletion *p*\_*score* ← −*v*(1) · [1.1 − *f*(*w*(*i*))] **else** *f req*(*T*(*i*)) ← *f req*(*T*(*i*)) + 1 *p*\_*score* ← −*v*(2) · *f req*(*T*(*i*)) · *dist*(*T*(*i*), *P*(*j*)) · [1.1 − *f*(*w*(*i*))] **end if** *score* ← *score* + *p*\_*score i* ← *i* + 1 **endwhile**

We remark that this algorithm uses three vectors: the substring *T*, the vector *w* of the weights of size *k* and a vector *align* of size *m* = 10, which stores the indices of the items of *P* such that *align*(*j*) = *i* iff *ti* = *pj*, else *align*(*j*) = −1. The algorithm scans *T* based on the index *i* and *P* based on *j*. Initially *i* = *j* = 0. Then, it checks if *i* is equal to *align*(*j*): if true, it scores the match (*ti* is equal to *pj*) and both indices are increased, otherwise it scores the deletion of *ti* and then increases *i*. In case of deletion, it checks if *ti* is equal to ! and, consequently, computes the appropriate score. Each access to the vectors takes *O*(1) and the algorithm scans the whole vector *T* so that it runs in *O*(*k*) time.

#### **3. Experimental Results**

After the pre-processing phase described in Section 2.2.2, the data consist of strings with their weights divided into three classes, depending on the individuals performing the test: 46 strings from patients with extrapyramidal syndrome, 284 from patients affected by chronic pain and 46 healthy participants. From now on, we refer to them as the Extrapyramidal (E), the Chronic (C) and the Healthy (H) classes.

For each member of the classes, we computed the score using the algorithm described in Section 2.4. In particular we used *v* = [1, 0.25, 0.5] for the penalty constant vector, and the inverse of the weight of the symbol for the duration function *f* .

Figures 5 and 6 illustrate the dot-plots and the scores computed for a member of each class, respectively. We are going to show that these members are good "representatives" of their classes. At a glance, the dot-plots suggest that the first image corresponds to a performance better than the second, which in turn, looks better than the third.

**Figure 5.** A dot-plot of the strings: (**a**) !5511DAAB3223BBD!53DECCE44ADD55E, member of the Healthy class; (**b**) !!!2CEB1!52!AA55!!24EBBB!!!334!ECC4!B, member of the Chronic class; (**c**) !!!C!3354CCAA!B!A!!E5!!!2!!C!2!5A23122!2!EC!25D!EEE!1353131ACCA!5A!525!2!, member of the Extrapyramidal class.

In the images of Figure 6, we illustrate the score as the bar graph obtained by visualizing each value assigned to each symbol of the sequences as a bar. Let us notice that bars of positive height correspond to the score of matches, whereas bars of negative height correspond to the score of deletions. Matches can be scored with values lower than 1, when repeated; deletions are scored differently depending on repetition, frequency, and distance from the next symbol objective.

**Figure 6.** A bar graph of the scores of the strings: (**a**) !5511DAAB3223BBD!53DECCE44ADD55E, member of the Healthy class; (**b**) !!!2CEB1!52!AA55!!24EBBB!!!334!ECC4!B, member of the Chronic class; (**c**) !!!C!3354CCAA!B!A!!E5!!!2!!C!2!5A23122!2!EC!25D!EEE!1353131ACCA!5A!525!2!, member of the Extrapyramidal class.

Before the analysis, we dropped some outliers for each class, according to the Chebischev Theorem. Setting *γ* = 2, we were sure to retain at least 75% for each class; such a dropping resulted in retaining 43 sequences out of 46 for the Healthy class, 265 out of 284 for the Chronic class and 44 out of 46 for the Extrapyramidal class.

Therefore, we analysed the results using the R language for computing the basic statistics and graphics. A summary divided by the groups is shown in Figure 7, while in Figure 8 we report the box plots.

**Figure 7.** Main statistics for the three classes of patients.

**Figure 8.** Box plots of the score distribution for each class: Healthy (**left**), Chronic (**center**), and Extrapyramidal (**right**).

Based on the obtained results, we can notice that the data seems not to follow a normal distribution, as we can see from Figure 9, at least for two of the three classes (the Healthy class and the Chronic one). Indeed we run the Kolgomorov–Smirnov test for comparison with the normal distribution on each class, obtaining *p*-values, respectively, equal to 6.56388017310154 · 10−30, 9.711223032427313 · 10−105, 1.5674138951676932 · 10−12.

**Figure 9.** Q-Q plots for each patients' class: (**a**) Healthy, (**b**) Chronic, and (**c**) Extrapyramidal.

Thus, we used the non-parametric Kruskal–Wallis test by rank which extends the two-sample Wilcoxon test in the situation where there are more than two groups. It turns out that at 0.05 significance level, the medians of the data of the three groups are different. In particular, the *p*-value for the Kruskal–Wallis test is *p*-value = 6.553 · 10−8. In order to know which pairs of groups are significantly different we used the function pairwise.wilcox.test() to calculate pairwise comparisons between group levels with corrections for multiple testing and Bonferroni correction. The results confirm that the pair exhibiting the most significant difference is the Healthy–Extrapyramidal as expected (see Table 1). Indeed, patients with extrapyramidal disease have well known difficulties in visual spatial exploration and executive functions that result in difficulties from the subject to maintain a top-down (human intention) internal representation of the visual scene during task execution. This is reflected in a bad performance in the VSST. Differently, patients in the Chronic class are affected by several kinds of chronic pain syndromes so that they may have different behaviours in performing the task.

Nevertheless, note that, actually, all the pairs have *p*-values less than 0.05 so that they are significantly different.

## **4. Discussion**

In this study, we propose a method for the analysis of gaze in a top-down visual search task and find a score for the VSST performance. The whole pipeline for the process is illustrated in Figure 1. The considered method and score have been validated by comparing the performance of three different subjects' groups. The first group includes 46 patients with extrapyramidal disease, who have well known difficulties in visual spatial exploration and executive functions. The second group is composed of 238 patients suffering from chronic pain syndrome and the third, collecting 46 patients, is a control group.

The identification of a robust method of analysis and the detection of a reliable indicator for the VSST performance, would allow to give a measure of executive functions in a clinical setting for diagnostic and prognostic purposes and eventually in clinical trials. Moreover, scoring the performance of such a VSST may have implications in the rehabilitation of cognitive functions and in general may be used for upgrading mental activity by exercise.

Indeed, cognitive rehabilitation is an effective non–pharmacological treatment that consists of learning compensatory strategies and exploiting residual skills in order to counteract, for instance, cognitive impairments and degenerative diseases. In fact, as for dementia, unfortunately, there is no specific pharmacological treatment, being existing drugs able to counteract the symptoms of the disease, but do not change its course. Consequently, the disease progresses: there is a continuous and constant progressive decline of cognitive functions for the patient, which negatively affects the various daily skills. Instead, changing the course of the disease, "pushing forward" the degenerative progression allows the patient to maintain their autonomy for a longer time and reduces the disinterest, anxiety and depression that degenerative diseases entail. Finally, cognitive rehabilitation is also fundamental for maintaining cognitive functions in efficiency and to combat the consequences of normal aging. Similarly, it is possible to implement intellectual stimulation with a preventive purpose.

The main characteristic of VSST is that it forces the subject to perform a default and logic path using high level cognitive resources. In this task the target of the next fixation changes continuously and, thus, in order to perform an adequate eye movement, each fixation must contain the information on the current target position and the next target location [25]. Previously, Veneri et al. [26] suggested that the re-sampling of the spatial element in such a visual search task requires a ranking of each element of the sequence during fixations. To be effective, this process requires a maximization of the discrimination abilities of the peripheral vision. The comparison of the *expected scan-path* with *the observed scan-path* provides a valuable method to investigate how a task forces the subject to maintain a top-down (human intention) internal representation of the visual scene during task execution. The proposed method has proved to be really effective in distinguishing between healthy people and patients affected by extrapyramidal pathologies, and less sensitive to the differences among the other cohort combinations. Actually, patients with chronic pain syndrome may be affected by very different pathologies—from severe neoplasms to chronic migraines—not all equally disabling from a neurocognitive point of view, which makes this group of patients extremely heterogeneous and difficult to distinguish, for example, from healthy people. Anyway, the main advantage of the proposed VSS test, equipped with the automatic procedure to score its outcomes, lies in the possibility of standardizing the test—making the obtained results repeatable—as well as memorizing them permanently. In this way, for each patient, a historical series of their performance can reliably be collected and analysed, a suitable procedure for evaluating both the course of a disease or the recovery based on a cognitive rehabilitation process.

Some issues concerning the proposed method naturally arise:

designing a more appropriate weight function, which could take into account cognitive features associated with the repetitive behaviour.

## **5. Conclusions**

In this manuscript, we have described a new method for the analysis of the Visual Sequential Search Test, a neurocognitive task commonly used in clinical settings as a diagnostic tool for the evaluation of frontal functions. The VSS test is an eye-tracking version of the Trail Making Test to discover how selection (fixations) guides next exploration (saccades), and how human top-down factors interact with bottom-up saliency. The problem of analysing the VSST outcomes is faced as an episode–matching problem, where an event corresponds to a fixation, and an episode to a scan–path. In this way, a score can be devised able to quantify how much a particular outcome diverges from the expected one. Based on this score, we are able to predict, with a high statistical confidence, if a particular scan-path corresponds to a patient with an extrapyramidal disease or suffering from the chronic pain syndrome or if it describes a "normal" cognitive behaviour. Having a standardized way to evaluate the VSST can help for monitoring the evolution of a disease, for neurological rehabilitation and for intellectual stimulation with a preventive purpose. In particular, the preventive aspect is taking on an increasingly important role, both in terms of the physical and intellectual well–being of the population, and in a more general process of optimizing economic resources for healthcare. We are perfectly aware of the small size of the used dataset. This is a common problem with medical data. Apart from collecting new data, another possible way to overcome this problem could consist in applying data augmentation techniques in order to both balance and enlarge its size. This will be an issue to discuss in future investigations.

**Author Contributions:** Conceptualization, G.A.D., S.B. and M.L.S.; Data curation, S.B.; Formal analysis, G.A.D., S.B. and M.L.S.; Investigation, G.A.D., S.B., M.L.S., A.R. and M.B.; Methodology, G.A.D., S.B., A.R. and M.B.; Project administration, M.B.; Resources, D.F.M.; Software, G.A.D., S.B. and M.L.S.; Supervision, M.L.S., A.R. and M.B.; Validation, A.R.; Visualization, G.A.D., S.B. and M.L.S.; Writing — original draft, G.A.D., S.B., M.L.S., A.R. and M.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Patient consent was waived due to the anonymous nature of analyzed data.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors wish to thank RoNeuro Institute, part of the Romanian Foundation for the Study of Nanoneurosciences and Neuroregeneration, Cluj-Napoca, Romania, represented by Dafin Fior Muresanu, for providing the datasets used here for the experiments.

**Conflicts of Interest:** The authors declare no conflict of interest.


**3.1. Antitumor activities**

The seeds extract of *Cerbera manghas* were found possessing obvious cytotoxic activity against some human cancer cell lines by MTT methods. Feng and coworkers obtained two cardiac glycosides named GHSC-73 and GHSC-74. Further study suggested GHSC-73 and GHSC-74 can significantly inhibited growth and proliferation of HepG2 cells in dose-dependent manner. GHSC-73 inhibited the growth and proliferation of HepG2 cells through blocking S phase and inducing apoptosis, while GHSC-74 through blocking S and G2 phases and inducing apopto‐ sis.Wang and coworkers tested 24 compounds isolated from *Pluchea indic* and found that valenc-l (10)-ene-8α, 13-diol showed inhibiting activity against Bel-7402 and A2780 cells. Lanceolatin B purified from *Pongamia pinnata* can prevent the development of cancer [20]. The cytotoxic activities of mansonone D, mansonone H, thespesone, and thespone were tested against MCF-7 cells by Johnson using the MTT methods. The results indicated that they all showed certain cytotoxic activities [21]. Ethnomedical survey has shown that the seeds of *Barringtonia racemosa* are traditionally used in certain remote villages of Kerala (India) to treat cancer like diseases. Thomas [22] tested the seed extracts for their anti-tumor activity and toxicity. Intraperitoneal (i.p.) daily administration of 50% methanol extract of this seed to mice challenged with 1 million Dalton's Lymphoma Ascitic (DLA) cells resulted in remarkable dose dependent anti-DLA activity in mice. The optimum dose was found to be 6 mg/kg. This dose protected all the animals challenged with the tumor cells. The efficacy of the drug was found to be better than that of a standard drug, vincristine in this tumor model. However, the oral administration showed only marginal activity compared to i.p. administration. The extract was found to be devoid of conspicuous acute and short-term toxicity to mice, when adminis‐ tered daily, (i.p.) for 14 days up to a dose of 12 mg/kg (which was double the concentration of optimum therapeutic dose). The treated mice showed conspicuous toxic symptoms only at 24 mg/kg. The LD (50) to male mice for a single i.p. dose was found to be 36 mg/kg. Consequently, they found that the seed extract is an attractive material for further studies leading to drug development. Anbu and coworkers [23] evaluated anti-tumor activity of the roots of *Hibiscus tiliaceus* against Dalton's Ascitic Lymphoma (DAL) in Swiss albino mice. A significant enhancement of mean survival time (MST) of *H. tiliaceus* treated tumor bearing mice was found with respect to control group. *H. tiliaceus* treatment was found to enhance peritoneal cell counts. When these *H. tiliaceus* treated animals under-went intraperitoneal (i.p.) inoculation with DAL cells, tumor cell growth was found to be inhibited. The results indicated that, *H. tiliaceus* treated group were able to reverse the haematological parameters, protein and Packed Cell Volume (PCV) consequent to tumor inoculation with in fourteen days after the trans‐ plantation.

**3.4. Others**

**4. Conclusions**

**Acknowledgements**

**Author details**

Xiaopo Zhang

**References**

Chakraborty reported the bark, seeds of *Barringtonia acutangula* could be used as a fish poison. Pongamia pinnata was evaluated by Elanchezhiya [31] for antiviral properties against herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) by *in vitro* studies in Vero cells. A crude aqueous seed extract of *P. pinnata* completely inhibited the growth of HSV-1 and HSV-2 at

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Among the 12 mangroves, *Pongamia pinnata*, *Cerbera manghas*, *Barringtonia racemosa* have been carried on deep researches concerning their chemical constituents, biological activities. Some compounds isolated from the semi-mangroves were proved to have obvious activities as the two cardenolides of GHSC-73 and GHSC-74. However, as proposed by Shao Changlun "Some semi-mangrove plants have not yet been analyzed and interpreted on their chemical constit‐ uents and pharmacological effects", the further researches of the semi-mangrove plants will

We would like to express our sincerest gratitude to the financial support of initial funding by Hainan Medical University (Project No. HY2010-012). We also thank Dr. Yinfeng Tan for

[1] Lin, P, & Fu, Q. Environmental ecology and economic utilization of mangroves. Bei‐

concentrations of 1 and 20 mg/ml (w/v), respectively.

make an important contribution to for the finding of new drugs.

helpful discussions regarding the pharmacological analysis.

jing: higher education press, (1995).

School of Pharmaceutical Science, Hainan Medical University, Haikou, PRC

[2] Lin, P. Zoology of China's mangroves. Beijing: Science press, (1997).

[3] Wang, W. Q, & Wang, M. Mangroves in China. Beijing: Science press, (2007).


**3. Conclusions**

In this short review paper, I have discussed some of the most unusual quantum transport effects that have been recently observed in silicon CMOS compatible devices. If from one side valley-orbital effects can be linked to the novel observation of effects such as for example the SU(4) Kondo [8] and the Fano-Kondo ones [10], from another side single-atom transistors and other silicon devices [4,12–15,46,48], because of their immunity to thermal and non-adiabatic detrimental effects, have demonstrated the ability to provide a unique environment for the ultra-fast control of electrons [12–15,46,48]. It is also very interesting to observe that even in the quantum pumping regime, the special valley-orbit energetic structure of silicon nano-materials plays a fundamental role in opening the way to high-performant devices. This is a special outcome as the origin of these powerful effects, i.e., the indirect band gap properties observed for silicon, was in the past considered one of main limitation of these materials [1–4].

**Conflicts of Interest:** The funders had no role in the design of the review; in the collection, in the analyses, nor in the interpretation of data; in the writing of the manuscript, nor in the decision to publish the results.


**4. Social and economic development**

The project assigns importance to the diffusion of ethical behaviors among companies, but what characterizes it is the widening of this vision through the dimension of social interventions, called social commitment, which has the aim of promoting the real participation of companies in supporting national and local welfare system according to a modern logic of public-private integration [4]. In particular, the role of the government is to identify areas of intervention, that, at a national and local level, require specific action and direct funding on priority projects [4]. Companies can join on a voluntary basis and contribute to the financing of welfare policies, while social intervention is managed directly by the associations and by voluntary work.

Government's objective is to develop the ethical supplementary funds. The standard proposed by the ministry envisages a system articulated on two levels [5]: the first level, called CSR level, easy to access and articulated on the basis of already known schemes (e.g., the International Organization for Standardization approach), has the main purpose of promoting the culture of social responsibility; the second level (social commitment level) requires the company to cofinance social projects in order to benefit from tax bonuses and facilitates access to the financial market through ethical funds. For companies wishing to participate in the reward system, an easily usable, simple and innovative tool was developed: the social statement. This is a selfassessment device for social responsibility activities and a support in the drafting of a report whose objective is to guarantee transparency of performance in the field of social responsibility. Lastly, the Ministry of Labor and Social Policies established within the framework of the commitments undertaken in the context of the Third European Conference on Corporate Social Responsibility, the Italian multi-stakeholder forum for corporate social responsibility [6]. The initiative was to encourage the dissemination of CSR among Italian companies and organizations by increasing the level of awareness of the importance of relations between CSR and sustainable development; facilitate the exchange of experiences and best practices; promote transparency and convergence of CSR practices and tools, with particular attention to the needs of small- and medium-sized enterprises, and, lastly, to promote the sharing of partner-

ship experiences between institutions, companies and organizations and civil society.

With socioeconomic progress, there has been an increase in household incomes followed by demand increase and differentiation that have stimulated the transition from a craft production to an industrial production, stimulating in turn a profound innovation of products and processes [7]. Environmental protection, seen as a capital belonging to all humanity, has made it possible to develop consumers' sensitivity toward their assets, particularly toward food products: sensitivity especially forced by scandals that have interested the agri-food sector,

Nowadays, the subjective component of the demand attributable to the preference of the consumer is subordinated to cultural and natural factors, level of education, age, and social context that takes a leading role in the market [8]. The consumer, with his/her preference and his/ her taste, is able to modulate the demand and, consequently, the market itself, structured on

**3. Consumer social responsibility**

particularly in recent years.

102 Social Responsibility

In this context, the sociodemographic changes connected to economic development, product and process innovations [12] have pushed consumers toward an increasing sensitivity in terms of the safety of food. This sensitivity has been sharpened by food scandals and health emergencies that have hit the agricultural and agri-food sector on a global scale in the years between the old and the new millennium [13].

However, not only the food risks are important, but also the tendencies and sensitivities spread toward issues closely related to food and its processing (that includes the treatment of raw materials, the phases of preparation and the packaging of the product), from ecosystem pollution to biodiversity loss, from the conditions of animal breeding to genetic manipulation and from the new forms of labor exploitation to the increase in social inequalities [14]. It follows that the subjective component of the demand attributable to personal consumer's taste, considered enduring in the past, now is highly influenced by factors of a cultural and sociodemographic nature (education, age, social context), taking a leading role in the market. This component allows a better interpretation of the demand (not just the food one) and its transformation, since the traditional explanatory variables (price, relative prices and income) are no longer sufficient [15].

For at least three decades, markets have been structured according to the different variables that consumer can converge each time: affordability in terms of price, opportunity of purchase, ease of use, durability, packaging, quality, ecology, ethics, respect for workers' rights, pleasure, health, diet and gluttony. In Italy, a scenario is evolving in which the citizen-consumer is driven to look for food products to satisfy a multiplicity of needs [16]. These needs range from food safety, in terms of hygienic and nutritional characteristics, to environmental sustainability, in terms of prevalent use of local and renewable resources, of the use of natural products and processes and of the respect of living conditions of farmed animals, up to a whole series of ethical components, from workplace safety to workers' protection, from social cohesion of the community to the enhancement of rural production areas and local food and wine traditions.

**5.1. Consumer survey: objective and methodology**

of responsible consumption.

Hp1: Attitude is positively related to Behavior;

**5.2. Features and results of the survey**

skewed toward the better educated one.

**5.3. Social responsibility and consumer**

wives and 28% are students.

Hp2: Knowledge is positively related to Behavior.

The research has set itself the objective of investigating in detail the role of social responsibility of the company in relation to the impact that it can have on consumption of agri-food products. We take into consideration the knowledge and opinion that buyers have about the product. The analysis is also aimed at highlighting the importance that consumers give to corporate social responsibility when purchasing agri-food and the receptivity of consumers toward the

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The degree of knowledge of particular categories of food products other than conventional (organic, fair-trade, genetically modified, etc.) was also measured and, lastly, the readiness of consumers to buy products of ethically oriented companies was measured, even when involving a higher payment. The research was carried out through the administration of a survey aimed at investigating preferences, behavior and participation regarding the theme

In this context, in order to formalize the research, three factors that play an important role in determining consumer behaviors were identified: knowledge, attitude and behavior that could influence the consumers' behavior toward CSR. The important meaning of the variables also stems from their role in other studies [18] that have focused on knowledge, attitude and practices or perception or training needs. The formulated research hypotheses are as follows:

The analyzed sample is composed of 46% for men and 54% for women. The considered sample appears to be fairly well distributed also with respect to age. As a matter of fact, 29% of respondents are aged between 20 and 30 years; 28% of respondents are aged between 30 and 45 years; 26% of respondents are aged between 45 and 60 years and 17% are over the age of 60. With regard to social and age variables, the role attributed to the company can be influenced by level of education obtained and by professional status. Among the respondents, 3% have obtained an elementary school diploma; 11% have obtained a middle school diploma; 50% have obtained a high school diploma and 36% have obtained a complete university degree. Therefore, from this point of view, the criterion was balanced but the participant sample was

This situation is similar when the professional status of the sample is assessed. Namely, 16% are self-employed; 21% are employees; 11% are retirees; 6% are unemployed; 19% are house-

The results of the survey highlight the different degrees of confidence that consumers show toward the companies that control the production, the management and the distribution of agri-food products. The analysis showed that the majority of respondents, 97%, do not have

sustainability strategies implemented above all by the main companies of the sector.

The evolution of Italians' eating habits and consumption in the last 40 years has enucleated the characteristics and the main economic indicators subdividing them into five stages, each corresponding to just as many periods (source: Istat data—the Italian National Institute of Statistics) [17]. From the post II World War period until the 1970s, it is possible to observe: progressive satisfaction of basic needs; consumption grows by about 300% in real terms during 15 years. Then, income increases; consumption and savings grow; food consumption at home dominates; almost 50% of the households have two income earners; food expenditure is one-third of the total expenditure. During the 1980s, income increases; the new acquisition consumption starts; food consumption experimentation shows new trends, signs of excesses as income rises further. Later, as financial incomes take off, consumption continues to grow, including food consumption, but at a slower pace; the organized large-scale distribution develops (supermarkets, ipermarkets, etc.); about 50% of marketing takes place through the organized large-scale distribution. In the end, since 2000, the race to high levels of consumption stops; attention to quality, safety, eco-social impact grow; eating out counts like eating at home; the organized large-scale distribution is the main sales channel; there is a reduction in the per-capita spending. The latter can be related to the global economic crisis.


*Edited by Alicia Esther Ares*

Almost all processing of technologically important materials includes a process where liquid material is cooled to form a solid, called "solidification." In order to form a solid from an undercooled melt, the formation of crystalline nuclei and growth of these nuclei to form a solid are necessary. The process of an atom jumping from the liquid to the solid is a diffusive jump with a driving force. The book Solidification is logically developed through a careful presentation of the relevant theories and models of solidification occurring in a variety of materials. Mathematicians, chemists, physicists, and engineers concerned with melting/freezing phenomena will also find this book to be valuable.

ISBN 978-953-51-3905-8

Solidification

Photo by NaokiKim / iStock

## Solidification

*Edited by Alicia Esther Ares*

**SOLIDIFICATION**

Edited by **Alicia Esther Ares**


*2.1. Physicochemical Characterization*

The color parameters (L\*, a\*, b\*) were measured using a LabScan XE System (Hunter Associates Laboratory, Inc., Reston, VA, USA) colorimeter by triplicate using a CIELAB system. Instrumental color was expressed as the Hue angle (Equation (1)), Chroma (Equation (2)), and Yellowness Index (YI, Equation (3); [28]). Evaluation of viscosity was determined in triplicates using a rheometer (MCR 302, Anton Paar Canada Inc, Quebec, Canada) at 25 ◦C. All samples (30–40 mL) were measured for pH at 25 ◦C in triplicates using a Fisherbrand Accumet® AB15 pH meter (Fischer Scientific, Hampton, NH, USA) calibrated against standard buffers (3-point calibration: pH 4.00–0.1 M Potassium hydrogen phthalate buffer, pH 7.00—Potassium Phosphate Monobasic/Sodium Hydroxide buffer, pH 10.00–Potassium Carbonate/Potassium Tetraborate/Potassium Hydroxide/Disodium EDTA Dihydrate buffer). Titratable acidity (TA, as % acetic acid) was measured in duplicates, with the method described by Okafor et al. [29], and the following modifications. A total of 5 mL was titrated with 0.1 M NaOH until pH = 7.0 was achieved using bromothymol blue as an indicator.

$$\text{Hue angle} = \arctan\left(\frac{\mathbf{b}^\*}{\mathbf{a}^\*}\right) \times \frac{180}{3.14} \tag{1}$$

$$\text{Chroma} = \left(\mathbf{a}^{\ast 2} + \mathbf{b}^{\ast 2}\right)^{0.5} \tag{2}$$

$$\text{YI} = 142.86 \times \frac{\text{b} \ast}{\text{L} \ast} \tag{3}$$

The density of samples was assessed based on weight and volume (50 mL). Total dissolved solids (TDS) were measured in triplicates using a Yuelong YL-TDS2-A digital water quality tester (Zhengzhou Yuelong Electronic Technology Co., Ltd., Zhengzhou City, Henan Province, China). Salt concentration was obtained using two drops of the sample in triplicates added to a digital salt-meter (PAL-SALT Mohr, Atago Co., Ltd. Saitama, Japan). On the other hand, alcohol content was assessed using 18 mL of the sample at room temperature (20 ◦C) injected to an Alcolyzer Wine M alcohol meter (Anton Paar GmbH, GRAZ, Austria) with the wine extension method found in the equipment settings; the instrument has a maximum error of 0.1% vv−1 .


**2. Dyslipidemia induced by stress: Physiological mechanisms**

Many studies have shown the effect of stress on lipid metabolism. Stress associated with a major disaster, such as an earthquake or loss of job and income is associated with increased total cholesterol, LDL, and triglycerides in the bloodstream (Stoney, 2007). The perception of increased stress during a period of high workload is associated with elevated cholesterol in the bloodstream and ingestion of foods that increase cholesterol (McCann et al., 1990). Acute psychological stress in healthy men and women reduces the clearance rate of exogenous fat (Stoney et al., 2002). Chronic psychological stress increased the plasma cholesterol level in medical students (O'Donnell et al., 1987). In a more recent study, Yoo et al., 2011, showed high prevalence of hypercholesterolemia in stressed female law enforcement officers in comparison with the general female population. Moreover, elevated basal cortisol concentrations and lower circadian cortisol variability can induce dyslipidemia in patients with depressive and anxiety disorders (Venn et al., 2009; Vogelzangs et al., 2007). These patients presented hypercortisolism, increased serum levels of total cholesterol, LDL, and triglycerides and decreased serum levels of HDL (Venn et al., 2009).

In animal studies, it has been shown that electric shock stress increases plasma cholesterol concentrations (Berger et al., 1980), and unpredictable immobilization stress decreases HDL, increases blood LDL, and very-low-density lipoprotein (VLDL) concentrations in rats (Bryant et al., 1988). Chronic mild unpredictable stress increases triglycerides, total cholesterol, VLDL, and LDL concentrations in the bloodstream of stressed rats compared with control rats and this effect was observed 15 days after the stress protocol had ended (Neves et al., 2009).

The stressful modern lifestyle exerts a strong influence on lipid metabolism (Black, 2002) and may transform adaptative responses to pathophysiological changes. Acute increases in blood lipids are necessary for the individual to survive and adapt to the stressor. However prolonged changes in lipid metabolism induced by chronic stress can result in cardiovascular diseases such as atherosclerosis, coronary heart disease, and stroke (Brindley et al., 1993).

The negative effects of sustained stress-induced dyslipidemia are related to a bidirectional relationship between stress hormones and insulin. Catecholamines directly stimulate free fatty acid and glycerol secretion in the bloodstream from fat depots, a process that may result from increased blood flow through adipose tissue or from adipose-β2 adrenoceptor stimulation (Stoney, 2007). Stress-induced high glucocorticoid concentration exerts a permissive effect on these lipolytic actions of catecholamines (Brindley et al., 1993). Since insulin regulates triglyceride synthesis and hepatic VLDL production, insulin resistance results in unregulated triglyceride synthesis and VLDL production (Stoney, 2007) and

with the greatest demand during the stress reaction, and support the fight or flight reaction

During acute stress, there is a rapid and transient increase in blood concentrations of total cholesterol, low-density lipoprotein (LDL), apoprotein B, triglycerides, and free fatty acids (Stoney, 2007). This increase persists as along as the stressor is maintained (Black, 2002), and disappears in stress-free periods (Stoney et al., 1999). In chronic stress situations, it has been shown that dyslipidemia is maintained and may persist even after the stressor is no longer

Many studies have shown the effect of stress on lipid metabolism. Stress associated with a major disaster, such as an earthquake or loss of job and income is associated with increased total cholesterol, LDL, and triglycerides in the bloodstream (Stoney, 2007). The perception of increased stress during a period of high workload is associated with elevated cholesterol in the bloodstream and ingestion of foods that increase cholesterol (McCann et al., 1990). Acute psychological stress in healthy men and women reduces the clearance rate of exogenous fat (Stoney et al., 2002). Chronic psychological stress increased the plasma cholesterol level in medical students (O'Donnell et al., 1987). In a more recent study, Yoo et al., 2011, showed high prevalence of hypercholesterolemia in stressed female law enforcement officers in comparison with the general female population. Moreover, elevated basal cortisol concentrations and lower circadian cortisol variability can induce dyslipidemia in patients with depressive and anxiety disorders (Venn et al., 2009; Vogelzangs et al., 2007). These patients presented hypercortisolism, increased serum levels of total cholesterol, LDL, and

In animal studies, it has been shown that electric shock stress increases plasma cholesterol concentrations (Berger et al., 1980), and unpredictable immobilization stress decreases HDL, increases blood LDL, and very-low-density lipoprotein (VLDL) concentrations in rats (Bryant et al., 1988). Chronic mild unpredictable stress increases triglycerides, total cholesterol, VLDL, and LDL concentrations in the bloodstream of stressed rats compared with control rats and this effect was observed 15 days after the stress protocol had ended

The stressful modern lifestyle exerts a strong influence on lipid metabolism (Black, 2002) and may transform adaptative responses to pathophysiological changes. Acute increases in blood lipids are necessary for the individual to survive and adapt to the stressor. However prolonged changes in lipid metabolism induced by chronic stress can result in cardiovascular diseases such as atherosclerosis, coronary heart disease, and stroke (Brindley

The negative effects of sustained stress-induced dyslipidemia are related to a bidirectional relationship between stress hormones and insulin. Catecholamines directly stimulate free fatty acid and glycerol secretion in the bloodstream from fat depots, a process that may result from increased blood flow through adipose tissue or from adipose-β2 adrenoceptor stimulation (Stoney, 2007). Stress-induced high glucocorticoid concentration exerts a permissive effect on these lipolytic actions of catecholamines (Brindley et al., 1993). Since insulin regulates triglyceride synthesis and hepatic VLDL production, insulin resistance results in unregulated triglyceride synthesis and VLDL production (Stoney, 2007) and

**2. Dyslipidemia induced by stress: Physiological mechanisms**

triglycerides and decreased serum levels of HDL (Venn et al., 2009).

to a stressor.

present (Neves et al., 2009).

(Neves et al., 2009).

et al., 1993).

triglycerides are secreted by the liver in large quantities within the VLDL particles (Black, 2003). Therefore both catecholamines and glucocorticoids antagonize the actions of insulin, contributing to insulin resistance (Kyrou & Tsigos, 2009; Lafontan & Langin, 2009).

Moreover, hyperinsulinemia acts centrally to stimulate sympathetic nervous system activity, resulting in increased secretion of catecholamines (Black, 2003), and the absence of satisfactory insulin action facilitates the actions of cortisol and glucagon, which in turn stimulate phosphatidate phosphohydrolase activity to synthesize hepatic triglyceride (Brindley et al., 1993).

The cortisol also induces apoprotein B (apo B) secretion from the liver in the proportion of one apo B molecule per VLDL particle (Brindley et al., 1993), consequently increasing the VLDL concentrations in the bloodstream. As each VLDL particle is metabolized to intermediate-density lipoprotein (IDL) or LDL, the action of the cortisol that stimulates apo B secretion also results in increased LDL particles in the blood. Furthermore, in the presence of stress-induced insulin resistance, high levels of glucocorticoids suppress the hepatic LDL receptors, which delay LDL clearance (Stoney, 2007).

Contributing to all these processes, it has been shown that perilipin, which coats the surface of lipid droplets to restrict lipase access to the triglyceride core within the droplet, may suffer phosphorylation and/or down-regulation by glucocorticoid action, thereby facilitating the lipolysis of triglycerides in fatty acids and glycerol (Xu et al., 2001). This sets off a vicious cycle, leading to more and more triglycerides being produced by the liver and secreted in VLDL particles, as a result of the stimulation of glucocorticoids and fatty acids.

In addition, norepinephrine and cortisol inhibit lipoprotein lipase activity, leading to diminished triglyceride clearance, decrease in HDL concentration, and increase in VLDL, IDL, and LDL concentrations in the bloodstream (Stoney, 2007). Norepinephrine also diminishes hepatic triglyceride lipase activity, which in turn promotes high concentrations of lipoproteins rich in triglycerides in the blood (Stoney, 2007).

In the context of stress-induced dyslipidemia, changes in food ingestion must also be considered. During acute stress, transient dyslipidemia and food intake inhibition are mediated by β-adrenergic activation and increased hypothalmic corticotrophin releasing hormone (CRH) levels which act as catabolic signals. On the other hand, chronic activation of the hypothalamic-pituitary-adrenal axis has been associated with overeating and obesity (Dallman et al., 2004; Nishitani & Sakakibara, 2006). Many studies have supported this relationship. Lemieux & Coe, 1995, related that approximately 50% of women with posttraumatic stress disorder as a result of childhood sexual abuse were overweight, and also showed high concentrations of norepinephrine, epinephrine, and dopamine in urine. Changes in sleep-wake cycles associated with stress, resulting in sleep loss, induce decreased leptin levels, increased ghrelin levels, and increased hunger and appetite (Pejovic et al., 2010; Spiegel et al., 2004). In addition, the parent's lifestyle can influence metabolism, and individuals exposed to maternal stress during intrauterine life can exhibit deregulation of body weight control mechanisms and blood lipid profile (De Moura, 2008). The relationship between excessive glucocorticoids and visceral fat accumulation has also been discussed by Björntorp & Rosmond, 1999.

Thus, the typical response to chronic stress is not by way of avoiding food but by increasing the intake of sugar- and fat-rich comfort foods, which make people feel better

Dyslipidemia Induced by Stress 371

HPA axis Sympathetic nervous system

**STRESSOR**

Central Nervous System

> Insulin resistance

**DYSLIPIDEMIA**

Fig. 1. Schematic representation of physiological mechanisms of dyslipidemia induced by stress. Hypothalamic-pituitary-adrenal axis (HPA), low-density lipoprotein (LDL), verylow-density lipoprotein (VLDL), free fatty acids (FFA), neuropeptide Y (NPY), tumor necrosis factor (TNF-α), interleukin 6 (IL-6). Solid arrows show stimulatory effects; dashed

Atherogenic dyslipidemia is a major underlying cause of the development of atherosclerosis, which is an inflammatory disease (Mullick et al., 2006; Sheril et al., 2009). Since the stress-induced atherogenic lipid profile potentiates the effects of dietary and genetic factors in atherogenesis (Brindley et al., 1993), stress has been recognized as a risk factor for atherosclerosis (Kyrou & Tsigos, 2009; Shively et al., 2009). However, despite the association between dyslipidemia and atherosclerosis, many individuals develop severe atherosclerotic lesions associated with low serum lipid concentration, and others develop far more severe atherosclerosis than would be expected on the basis of a modest elevation of serum lipids (Kaplan et al., 1983). In this context, other effects of stress, not related specifically to dyslipidemia, are also involved in atherogenesis (Bierhaus et al., 2003; Gu et al., 2009) and approximately 40% of cases without known causal factor, have been attributed

**3. Stress, dyslipidemia and atherosclerosis: Putative mechanisms**

Catecholamines

Lipoprotein lipase

Abdominal obesity

NPY

Hyperinsulinemia

Comfort food ingestion

NPY

Peripheral effects via sympathetic nerves

Fat depots (lipolysis)

LEPTIN TNF-α IL-6

**LDL receptor**

arrows indicate inhibitory effects.

to stressful situations (Black, 2002).

LDL

FFA Glycerol

Liver

VLDL Triglycerides

Glucocorticoids

(Stoney, 2007; Torres & Nowson, 2007). Dallman et al., 2003, suggested that people or animals eat comfort food in an attempt to reduce activity in the 'chronic stress-response network' with its attendant anxiety. They suggested the following mechanism: first, in the periphery, glucocorticoids stimulate accretion of mesenteric energy stores; second, as the abdominal energy-generated (unidentified) signal increases, the negative input to catecholaminergic cells in the nucleus tractus solitarius reduces the synthesis of enzymes required for norepinephrine synthesis; third, the decreased noradrenergic signal to the hypothalamic paraventricular nucleus (PVN), in turn, decreases CRH synthesis and secretion. Thus, there is a powerful metabolic feedback control of CRH in the PVN, which may indirectly decrease glucocorticoid-action in the central nucleus of the amygdala; and thereby control anxiety (Korte et al., 2005). Consequently, all these mechanisms can lead to obesity and dyslipidemia due to overeating. In addition, it has been proposed that when chronic stress, to which animals and humans cannot easily adapt, is combined with high-fat high-sugar diets, it stimulates the sympathetic nerves to upregulate the expression of neuropeptide Y, an adrenergic cotransmitter and stress mediator. Stress and hypercaloric diets also increase glucocorticoid concentration in visceral fat, which in turn upregulates the expression of neuropeptide Y and its receptor Y2R, resulting in fat growth, hyperinsulinemia and hyperlipidemia (Bartolomucci et al., 2009; Kuo et al., 2008).

Some studies have also shown that glucocorticoid actions in the target tissues depend not only on circulating hormone levels, but also on intracellular glucocorticoid receptors and activities of both 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) and 2 (Bose et al., 2009). The effects of glucocorticoid are enhanced by the enzyme 11β-HSD1 in the stromal cells of visceral fat, since this enzyme catalyzes the conversion of inactive cortisone to active glucocorticoid in local tissue. It has been shown that transgenic knockout mice, which overexpress 11β-HSD1 in adipose tissue, present accumulation of visceral adipose tissue, hypertension, dyslipidemia and glucose intolerance (Masuzaki et al., 2001; Masuzaki & Flier, 2003). Therefore 11β-HSD1 plays an important role in the development of metabolic disease associated with stress (Bose et al., 2009; Walker & Stewart, 2003).

In addition, cytokines such as interleukin 6 (IL-6), tumor necrosis factor (TNF)-α, and leptin released from fatty cells also contribute to dyslipidemia induced by stress. IL-6 increases the activity of 11β-HSD1 with consequent expansion of visceral fat. TNF-α induces lipolysis in adipose tissue. Both IL-6 and TNF-α decrease lipoprotein lipase activity, contributing to the increase in triglyceride levels induced by stress (Black, 2003). Moreover, TNF-α induces insulin resistance because it depresses insulin receptor activity (Yudkin et al., 2000). TNF-α also induces IL-6 synthesis, and stimulates leptin synthesis, which acts centrally to decrease appetite and increase thermogenesis to decrease fat storage (Black, 2003). Leptin increases the activity of sympathetic nervous system centrally (Mohamed-Ali et al., 1998), which in turn stimulates increased release of TNF-α and IL-6 from adipocytes (Black, 2003). This sympathetic nervous system hyperactivity induced by high levels of leptin in the bloodstream would provide an additional effect of catecholamines on the genesis of insulin resistance and dyslipidemia associated with stress in obese individuals.

Therefore, dyslipidemia induced by stress involves complex interactions among stress hormones, insulin, adipose tissue metabolism and cytokines. Figure 1 indicates the physiological mechanisms of dyslipidemia induced by stress.

(Stoney, 2007; Torres & Nowson, 2007). Dallman et al., 2003, suggested that people or animals eat comfort food in an attempt to reduce activity in the 'chronic stress-response network' with its attendant anxiety. They suggested the following mechanism: first, in the periphery, glucocorticoids stimulate accretion of mesenteric energy stores; second, as the abdominal energy-generated (unidentified) signal increases, the negative input to catecholaminergic cells in the nucleus tractus solitarius reduces the synthesis of enzymes required for norepinephrine synthesis; third, the decreased noradrenergic signal to the hypothalamic paraventricular nucleus (PVN), in turn, decreases CRH synthesis and secretion. Thus, there is a powerful metabolic feedback control of CRH in the PVN, which may indirectly decrease glucocorticoid-action in the central nucleus of the amygdala; and thereby control anxiety (Korte et al., 2005). Consequently, all these mechanisms can lead to obesity and dyslipidemia due to overeating. In addition, it has been proposed that when chronic stress, to which animals and humans cannot easily adapt, is combined with high-fat high-sugar diets, it stimulates the sympathetic nerves to upregulate the expression of neuropeptide Y, an adrenergic cotransmitter and stress mediator. Stress and hypercaloric diets also increase glucocorticoid concentration in visceral fat, which in turn upregulates the expression of neuropeptide Y and its receptor Y2R, resulting in fat growth, hyperinsulinemia and hyperlipidemia (Bartolomucci et al., 2009; Kuo et al.,

Some studies have also shown that glucocorticoid actions in the target tissues depend not only on circulating hormone levels, but also on intracellular glucocorticoid receptors and activities of both 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) and 2 (Bose et al., 2009). The effects of glucocorticoid are enhanced by the enzyme 11β-HSD1 in the stromal cells of visceral fat, since this enzyme catalyzes the conversion of inactive cortisone to active glucocorticoid in local tissue. It has been shown that transgenic knockout mice, which overexpress 11β-HSD1 in adipose tissue, present accumulation of visceral adipose tissue, hypertension, dyslipidemia and glucose intolerance (Masuzaki et al., 2001; Masuzaki & Flier, 2003). Therefore 11β-HSD1 plays an important role in the development of metabolic

In addition, cytokines such as interleukin 6 (IL-6), tumor necrosis factor (TNF)-α, and leptin released from fatty cells also contribute to dyslipidemia induced by stress. IL-6 increases the activity of 11β-HSD1 with consequent expansion of visceral fat. TNF-α induces lipolysis in adipose tissue. Both IL-6 and TNF-α decrease lipoprotein lipase activity, contributing to the increase in triglyceride levels induced by stress (Black, 2003). Moreover, TNF-α induces insulin resistance because it depresses insulin receptor activity (Yudkin et al., 2000). TNF-α also induces IL-6 synthesis, and stimulates leptin synthesis, which acts centrally to decrease appetite and increase thermogenesis to decrease fat storage (Black, 2003). Leptin increases the activity of sympathetic nervous system centrally (Mohamed-Ali et al., 1998), which in turn stimulates increased release of TNF-α and IL-6 from adipocytes (Black, 2003). This sympathetic nervous system hyperactivity induced by high levels of leptin in the bloodstream would provide an additional effect of catecholamines on the genesis of insulin resistance and dyslipidemia associated with

Therefore, dyslipidemia induced by stress involves complex interactions among stress hormones, insulin, adipose tissue metabolism and cytokines. Figure 1 indicates the

disease associated with stress (Bose et al., 2009; Walker & Stewart, 2003).

physiological mechanisms of dyslipidemia induced by stress.

2008).

stress in obese individuals.

Fig. 1. Schematic representation of physiological mechanisms of dyslipidemia induced by stress. Hypothalamic-pituitary-adrenal axis (HPA), low-density lipoprotein (LDL), verylow-density lipoprotein (VLDL), free fatty acids (FFA), neuropeptide Y (NPY), tumor necrosis factor (TNF-α), interleukin 6 (IL-6). Solid arrows show stimulatory effects; dashed arrows indicate inhibitory effects.