**5. SL versus KAR signaling**

there is no evidence that MAX2/D3 can interact with SLs, there are reasons to assume that

For a long time, it was not known which proteins are recognized by the SCFMAX2/D3 complex, but recently the SL repressors degraded during SL signal transduction were identified in rice (D53) [51, 52] and in *A. thaliana* (Suppressor of MAX2‐Like 6 to 8, SMXL 6 to 8) [53–55]. Gain‐of‐function mutation in D53 resulted in semi‐dwarf plants with increased tillering, a phenotype which is characteristic for SL mutants. Similar effects were observed after over‐ expression of *D53*, whereas reduced expression of *D53* in a *d53*‐mutant background inhib‐ ited tiller formation [52]. D53 shows the nuclear localization and it was confirmed that in the presence of SL molecules degradation of D53 occurs. This degradation proceeds through the proteasome‐dependent pathway and requires the presence of D14 and D3 proteins [51, 52]. There are evidences that D53 may also interact with D3 in the absence of D14, although this interaction is less efficient. In contrast to rice, where only one SL repressor has been identi‐ fied, *A. thaliana* contains three proteins—SMXL 6 to 8—that may act redundantly. First reports indicated that only a triple mutant *smxl6/7/8* will result in a phenotype with reduced number of tillers [54]. Later on, it was found that the phenotype characteristic for SL mutants could be produced by the expression of the non‐degradable form of SMXL 7 under a native promoter [55]. All three SMXLs interact with D14 and are degraded in an SL‐dependent manner in the presence of MAX2 and D14 [53, 54]. It still remains an open question whether SMXLs 6 to 8 do

The SL repressors of both *A. thaliana* and rice contain the conserved amino acid sequences (F/L‐D‐L‐N‐L) which is known as an ethylene‐responsive element‐binding factor‐associated amphiphilic repression (EAR) motif. This motif plays a key role in interactions with transcrip‐ tional corepressors from Topless (TPL) and Topless‐Related Proteins (TPR) families [52, 53]. TPL and TPR regulate the expression of genes in response to different classes of hormones, such as auxin or jasmonates [56]. The presence of an EAR motif in SL repressors suggests that D53/SMXLs may bind TPL/TPR corepressors. An ensuing degradation of these corepressors may then result in the expression of transcriptional factors, previously suppressed by TPL/ TRP. Interactions between SMXL6 to 8 and proteins from the TPR family were already con‐

Phytohormones induce a change in gene expression. This response is usually mediated by transcription factors. Until now, only one family of transcription factors has been identified as a downstream component in SL signaling, namely the TEOSINTE BRANCHED1/CYCLOIDEA/ PROLIFERATING CELL FACTOR1 family (TCP). In different species, single transcription fac‐ tors from this family, related to SL signal, were already characterized: Branched1 (BRC1) in *A. thaliana* [57], Fine Culm1/Teosinte Branched1 (FC1/OsTB1) in rice [58], TB1 in *Zea mays* L. [59] and PsBRC1 in *Pisum sativum* L. [60]. Expression of these genes is particularly strong in axillary buds, and mutations in these genes lead to an increased branched phenotype, which

MAX2/D3 may act as a receptor for other signaling molecules.

108 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

act redundantly or they are involved in different responses to SLs.

firmed using a yeast‐two hybrid and co‐immunoprecipitation assays [54].

**4. Transcription response to SL signal**

The unique features of SLs signaling have been discussed elsewhere [7, 63]. Here, the simi‐ larities and differences between SLs and KARs will be summarized. Though SLs and KARs play different roles in plant development [64], there are some striking similarities in the signal transduction mechanisms of these two classes of plant growth regulators [65] what might be crucial in understanding the mechanisms of their actions in plants. As already mentioned, the signal transduction of SL and KAR is mediated by the same F‐box protein MAX2. However, since the signals generated by SLs and KARs are not interchangeable these phytohormones must be recognized by different receptors. Indeed, the D14 receptor has found to be specific for SLs, whereas the KAI2 receptor is specific for KARs and based on the differences in the size of active pocket site they cannot recognize the signal from second group of plant growth regulators [66, 67]. Both D14 and KA12 display a conserved catalytic triad, but only in case of D14 its catalytic activity was confirmed [33, 37]. Not only has a catalytic function of KAI2 never been proven, modeling studies of the KAR‐KAI2 complex indicate that the distance between the KAR molecule and the catalytic Ser from KAI2 prohibits nucleophilic attack [67, 68]. Nevertheless, since mutation in the catalytic triad of KA12 can abolish the function of this receptor [69], the catalytic triad of the KA12 receptor may be essential for ligand binding. Similar observations have been made for D14 [38]. The second similarity between both recep‐ tors is their degradation during perception, though in the case of KAI2 the presence of MAX2 is not required for its degradation [70].

MAX2 is a component of SCF complexes which are involved in the conversion of SL and KAR signals. Therefore, the phenotypic effects caused by a mutation of MAX2 are due to an insen‐ sitivity to both plant growth regulators.

Due to the presence of different receptors, the respective SCF complexes guide the degrada‐ tion of different suppressors: SMXL6 to 8 in the case of D14‐SL‐Max2 and Supressor of Max2 1 (SMAX1) in the case of KAI2‐KAR‐MAX2 [71]. *SMAX1* and *SMXL6/7/8* show similar patterns of expression in *A. thaliana* seedlings and all four proteins may interact with TPL corepres‐ sors *via the* EAR motif [53] which indicates that ultimately SLs and KARs may regulate gene expression by a similar mechanism.


**Factors affecting gas sensing mechanism:**

Several factors should be taken into account when assessing the effectiveness of gas sensing techniques (**Figure 1** depicts the classification of gas sensor):

The operating stability of gas sensors that are intended for the market must also be guaranteed; or they must display a highly stable as well as repeatable signal over time.

**Figure 1.** *Chart representing the classification of gas sensors.*

#### *Optical Gas Sensors DOI: http://dx.doi.org/10.5772/intechopen.108971*

But, the instability of the gas sensor is caused by a number of reasons such as design mistakes, structural modifications (such as changes in grain size or grain network), phase transitions (which typically refers to separation of the additives doped with sensing materials), poisoning brought on by the chemical processes as well as change in the environment.

The use of optical techniques for gas sensing is typically simple and can produce results with more sensitivity, stability as well as selectivity in respect to other non-optical techniques while having a significantly longer lifetime. Their quick response time makes on-line real-time detection possible. The changes in the atmosphere or poisoning of the catalyst brought on by particular gases, etc. will not affect performance. The majority of optical techniques utilized for sensing gas are based on spectroscopy.

### **2.1 Basic principles**

There has been an extensive discussion about the principles of molecular absorption spectroscopy [12, 13]. Optical gas sensing techniques have been commercialized and while the majority are absorption-based, several other methods also play a significant role. Myriad of chemical substances show high absorption mainly in the region dominated over ultraviolet–visible range, near-infrared (NIR) as well as mid-infrared range of electromagnetic spectrum. Each substance has its unique set of absorption lines or bands that serve as the foundation for its detection and quantification. The parameters of the absorption spectra show variation in the various spectral bands displayed in **Table 2**.

Optical gas sensors work on the principle of the Beer and Lambert law and relation for the same is [14].

$$I(\mathcal{X}) = I\_\circ(\mathcal{X})e^{-ad} \tag{1}$$

were, (λ ) *o I and I* are the detected as well as emitted optical intensity at a particular wavelength ( λ) , (respectively)

α λ( )[*L g* / ] = Absorption coefficient of the gas.

*cg L* [ / ] = Concentration of gas.

l[*m*] = beam-gas interaction path length.

**Figure 2** depicts a sensor comprised of different parts, namely

The spectroscopic investigation relied mainly on the spectrometry absorption as well as emission. Absorption analysis is completely based on the Beer and Lambert

#### **Table 2.**

*Origin of absorption spectra in the various regions of the electromagnetic spectrum.*

law and is basically concentration-dependent absorption of the photons at a particular wavelength of the gas. Other than this plethora of improvised techniques of absorption spectrometry, namely Differential Optical Absorption Spectroscopy, Raman Light Detection, Tunable Diode Laser Absorption Spectroscopy, Differential Absorption LIDAR and Intra-Cavity Absorption Spectrometry (ICAS), etc. [15–17]. Spectrometry based on emission is observed when the atoms sitting in the excited levels on their transition to the ground state emits photons. Emission-based techniques are namely Laser-Induced Breakdown Spectroscopy, Fourier Transform Infrared Analysis (FT-IR) can be utilized for both the spectroscopy, namely absorption and emission. Furthermore, the photo-acoustic and correlation spectroscopy techniques also come in the category of spectroscopic analysis. These spectroscopic techniques are generally employed to gas detectors. This can contribute to the designing of the more complicated system at a higher cost level and can incorporate exceptional sensitivity, selectivity and reliability as compared to the other available gas sensors. Extensively utilized optical-based sensors utilize infrared source. The infrared source-based gas sensors work on the principle of absorption spectrometry. That implies that each and every gas is having a specific absorption property to infrared radiation of varying wavelength and results in unique fingerprint of infrared absorption. This implies that each and every gas has some unique property of absorption to infrared radiation range with varying wavelengths and thus shows a rare fingerprint of infrared absorption. Gas sensors based on infrared source consist of mainly three major parts, namely (depicted in **Figure 2**),

#### iii.Infrared Detector

At the primary state, the infrared source emits broad-band of radiation wavelength containing the wavelength range corresponding to the absorption of target gas. After that the gas sample present in the gas cell absorbs the incoming radiation beam of their specific wavelength. Later on the optical filters are utilized to screen out the entire wavelength of the radiation except the radiation wavelength absorbed by the target gas. Thus, the gas of interest can be easily detected as well as measured just by employing infrared detector. The entire setup is named as Non-Dispersive Infrared gas sensor.

Non-dispersive sensing technique is utilized, wherein the beam (unfiltered) is utilized for the interaction with the gas. This sensing technique encourages the selective detection of beam (λ ) , just by filtering the beam detected by characteristic absorption spectra of the molecular species. Non-dispersive infrared (NDIR) sensors conventionally configured with infrared emitters and infrared detectors [18]. Sensors

#### **Figure 2.**

*Beer and Lambert law–based optical gas sensor; (A) off state (no any signal being detected, (B) on state maximum detected signal, (C) decreased detection with gas concentration.*

work on the same principle of Beer–Lambert's Law, if utilized for different spectral regions or configured for acoustic detectors.

A different gas cell consisting of the reference gas other than the single detector mode can be incorporated to uplift the overall accuracy of the infrared sensing method just by truncating the ambient environmental factors without inducing much more complexity. **Figure 2** depicts the layout of two detectors. Just to keep the radiation parameters of infrared source identical, gas cell utilizes the reflected infrared beams from the single source just utilizing the mirror property. The assortment of the range of wavelength of the infrared beam utilized as source impacts a lot on the concluding detection result. Spectral region of the mid-infrared gains more attention as it encourages stronger molecular absorption as compared to spectral region of near-infrared. The conventional mid-infrared laser sources suffer major drawbacks, namely wavelength tunability, lower power of output, complexity, requirement of coolants. Plethora of lasers have been developed and investigated in order to overrule the above mentioned disadvantages, e.g. Quantum-Cascade Lasers blessed with tunability, highly narrow line-width as well as lower average power for the operation at the room temperature.

Myriad of topologies have been employed for the fabrication of optical gas sensors. The commonly utilized sensors are based on gas cells formed between face-to-face configured emitters and optical detectors. Several approaches followed for the miniaturization of the gas cell include utilization of enhancement layers namely, Utilization of photonic crystals [19], optical cavities [20], multi-pass cells [21], gas enrichment layers [22–24], in order to enhance beam-gas interaction, the planar configurations of the emitters as well as detectors, utilization of waveguides for the interaction of evanescent field. Optical detectors, namely photodiode, thermopile or pyroelectric, acoustic detector are mainly implemented in order to detect the beam being absorbed ( *I*(λ) ) [25–28]. The mechanism of lock-in detection is employed to

extract the sensor response signal ( *I*(λ ) ) and further known frequency is utilized to modulate the emitter. The reference detector is frequently utilized in order to compensate for the variation in emitted beam. Further, additional sensors can be utilized in order to compensate for the several parameters of environment, namely temperature, pressure, etc.


**9.4.4 Biologics**

An increasing number of "biologics" have been developed in the last decade. They counteract extremely potent immunological targets such as tumor necrosis factor-α (TNF-α), T cells, B cells, various cytokines, some immunoglobulins and key enzymes. A variety of them has been used in psoriasis, often with astonishing success (Lawry, 2007). Also nail psoriasis was the object of some studies. This chapter is, however, not suited to discuss all as this will be discussed in the psoriasis treatment chapters.

#### **<IP_ADDRESS> TNF- inhibitors**

TNF- promotes cytokine secretion, such as interleukin (IL)-1, IL-6, IL-8, by macrophages and other inflammatory cells, activates T cells and induces adhesion molecule expression by vascular endothelial cells which in turn promote angiogenesis and keratinocyte proliferation, both key events in the pathogenesis of psoriasis (Smolen and Emery, 2011).

Table 5. Application, dosage and indications of the commonly used TNF- antagonists

**Infliximab** (Remicade®) is an inhibitor of TNF-, which is a proinflammatory cytokine in psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's disease and several more chronic inflammatory diseases. Given as an intravenous infusion of 5 mg/kg at weeks 0, 2, 6, and then every 8 weeks, infliximab, a chimeric monoclonal antibody, is effective in psoriasis, psoriatic nail lesions (Reich et al, 2005), psoriatic arthritis and Reiter's disease including its nail changes (Gaylis, 2003). Many reports have described the dramatic infliximab-induced improvement of both psoriatic skin and nail lesions (Antoni et al, 2005a, 2005b, Rich et al, 2008, Hussain et al, 2008, Reich, 2009). Another study showed a reduction of the mean NAPSI of 55.8 at baseline to 29.8 at week 14 and 3.3 at 38 (Rigopoulos et al, 2008). In the evaluation mentioned above, NAPSI improvement after 12,

Nimesulide is a non-steroidal anti-inflammatory agent. After first positive experience (Piraccini e al 1994) it was given to 13 patients with pustular nail psoriasis of whom 4 responded well to twice daily 100 mg. All these 4 responders relapsed after withdrawal of

An increasing number of "biologics" have been developed in the last decade. They counteract extremely potent immunological targets such as tumor necrosis factor-α (TNF-α), T cells, B cells, various cytokines, some immunoglobulins and key enzymes. A variety of them has been used in psoriasis, often with astonishing success (Lawry, 2007). Also nail psoriasis was the object of some studies. This chapter is, however, not suited to discuss all

TNF- promotes cytokine secretion, such as interleukin (IL)-1, IL-6, IL-8, by macrophages and other inflammatory cells, activates T cells and induces adhesion molecule expression by vascular endothelial cells which in turn promote angiogenesis and keratinocyte proliferation,

> **Adalimumab**  Humira®

Loading dose: week 0 2x40 mg, week 1 40 mg, then 40 mg every

Psoriasis, psoriatic

antibody

2 weeks

arthritis

**Infliximab** (Remicade®) is an inhibitor of TNF-, which is a proinflammatory cytokine in psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's disease and several more chronic inflammatory diseases. Given as an intravenous infusion of 5 mg/kg at weeks 0, 2, 6, and then every 8 weeks, infliximab, a chimeric monoclonal antibody, is effective in psoriasis, psoriatic nail lesions (Reich et al, 2005), psoriatic arthritis and Reiter's disease including its nail changes (Gaylis, 2003). Many reports have described the dramatic infliximab-induced improvement of both psoriatic skin and nail lesions (Antoni et al, 2005a, 2005b, Rich et al, 2008, Hussain et al, 2008, Reich, 2009). Another study showed a reduction of the mean NAPSI of 55.8 at baseline to 29.8 at week 14 and 3.3 at 38 (Rigopoulos et al, 2008). In the evaluation mentioned above, NAPSI improvement after 12,

Table 5. Application, dosage and indications of the commonly used TNF- antagonists

Application Intravenous infusion Subcutaneous Subcutaneous

**Etanercept**  Enbrel®

Human fusion protein

50 mg/week, or 2x50mg/week for 12

Psoriasis, psoriatic

weeks

arthritis

the drug (Piraccini et al 2001). It was judged as not being superior to other drugs.

as this will be discussed in the psoriasis treatment chapters.

Structure Chimaeric antibody Human monoclonal

both key events in the pathogenesis of psoriasis (Smolen and Emery, 2011).

**9.4.3 Nimesulide**

**9.4.4 Biologics**

**<IP_ADDRESS> TNF- inhibitors**

**Infliximab**  Remicade®

Dosage 5 mg/kg at weeks 0,

weeks

Indications Psoriasis, psoriatic arthritis

2, 6, then every 8

24 and 48 weeks was 50%, 81%, and 92%, respectively (Sánchez-Regaña et al 2011). There appears to be general agreement that infliximab is the most potent antipsoriatic biologic (Noiles and Vender, 2009).

**Adalimumab** (Humira®) is a human antibody. In an open study, significant NAPSI reductions were obtained for finger and toe nails both in patients with cutaneous psoriasis as well as with psoriatic arthritis (Rigopoulos et al, 2010). In a large cohort of 442 patients with psoriatic arthritis, the mean NAPSI was reduced by 44% (Van den Bosch et al, 2010). Nail psoriasis response may be rapid (Irla and Yawalkar, 2009) although some authors found skin lesions to respond less than articular inflammation (Otten et al, 2011). In a group of ankylosing spondylitis and psoriatic arthritis patients, the NAPSI score was demonstrated to be reduced by 6 points (Rudwaleit et al, 2010). Adalimumab-induced improvement in nail psoriasis correlated with a good response in palmar plantar psoriasis (Langley et al, 2011). In the Spanish study, NAPSI improvement after 12, 24 and 48 weeks was 37%, 73%, 84%, respectively. Adalimumab was also beneficial for nail psoriasis after etanercept treatment (Puig et al, 2010).

**Etanercept**, a fully human TNF-α receptor fusion protein, binds TNF-α with greater affinity than natural receptors. The bound TNF-α is biologically inactive and many of the proinflammatory pathways responsible for initiation, maintenance, and recurrence of skin lesions in psoriasis are inhibited (Weinberg, 2003). The starting dose is twice weekly 50 mg subcutaneously, which may be reduced to once weekly 50 mg or twice weekly 25 mg. In a comparison of systemic nail psoriasis treatments, NAPSI improvement after 12, 24 and 48 weeks was 24%, 68%, and 87%, respectively (Sánchez-Regaña et al, 2011). The commonest adverse effect is an irritation reaction at the injection site. Infections and reactivations may occur as in infliximab treatment though probably less commonly. It should not be combined with systemic corticosteroids (Sanchez et al, 2006, Scheinfeld, 2004). There are some otherwise rare skin diseases that have been observed during etanercept treatment, such as lupus erythematosus, vasculitis, eosinophilic cellulitis like inflammation and interstitial granulomatous dermatitis (Scheinfeld, 2004, Winfield et al, 2006, Deng et al, 2006).

**Golimumab** (Simponi®) is a new human monoclonal antibody against TNF- binding with high affinity and specificity to soluble and transmembrane TNF-. It was studied once in psoriasis and nail psoriasis and showed an improvement in the NAPSI score of 25% and 43% after 14 weeks and 33% and 54% after 24 weeks in a dose of 50mg or 100 mg subcutaneously, respectively, at weeks 0, 4, 8, 12, 16, and 20 (Kavanaugh et al, 2009).

**Certulizumab** (Cimzia®) has not been used in nail psoriasis (Gartlehner et al, 2009).

All TNF- inhibitors were reported to have induced psoriasis or psoriasiform skin and nail lesions (Sfikakis et al, 2005, Wollina et al, 2010). The spectrum of conditions induced by TNF- is very wide and it apparently does not depend on the specific disease treated nor on the anti-TNF- agent used (Pine et al, 2010, Conrad et al, 2011; Lee et al 2011). In more than half of the cases, the TNF- induced skin lesions were successfully suppressed despite continuation of the drug. It is speculated that as TNF- blockade is one of the strongest inducers of interferon- production an unabated IFN- production by plasmocytoid dendritic cells might result in these paradoxical psoriasis flares under anti-TNF- treatment (Conrad et al, 2011).

Nail Psoriasis 177

marked improvement of his nail signs was noted after 4 weeks.  No reports on nail psoriasis treatment

**Abatacept** (Orencia®) is a soluble chimeric protein consisting of the extracellular domain of human CD152 linked to the modified Fc portion of human IgG1. By binding to B7-1 (CD80) and B7-2 (CD86) molecules on antigen presenting cells, CTLA4Ig blocks the CD28-mediated costimulatory signal for T cell activation. Success with abatacept has been noted in psoriasis. Abatacept was administered to two patients with refractory psoriasis and psoriatic arthritis after the patients had failed all conventional treatment methods. Both patients experienced

**Rituximab,** a B cell depleting chimeric antibody has no place in the treatment of nail

In clinical routine, different treatments are often combined as one is either insufficient or too slow. The combination should always consist of drugs with different mechanisms of action. In contrast to skin psoriasis, there are almost no studies on the effect of combination therapy on nail psoriasis (Jiaravuthisan et al 2007). A single-blinded study on 54 patients with nail psoriasis examined the effects of cyclosporine monotherapy versus cyclosporine systemically plus calcipotiriol cream topically (Feliciani et al 2004). The cyclosporine dose was 3.5 to 4.5 mg/d, calcipotriol was applied twice daily. After 3 months, the combined treatment showed significant improvement of pitting, subungual hyperkeratosis and onycholysis in 79%, whereas the cyclosporine monotherapy group showed 48% marked improvement. Six months after treatment, the cyclosporine monotherapy group showed a relapse rate of 52.9% (9/17), whereas only 37% (10/27) of patients in the combined therapy

Nail psoriasis is frequent in psoriatic subjects with about 50% of psoriasis patients presenting with nail changes at any time and a life-time prevalence of up to 90%. Nail psoriasis has a strong genetic background and a frequent association with psoriatic arthritis. The most frequent signs of nail matrix involvement are pitting, leukonychia, crumbling and red spots in the lunula, whereas salmon or oil spots, subungual hyperkeratosis, onycholysis and splinter haemorrages represent changes of nail bed involvement. Understanding the mechanism of psoriatic nail sign development requires some basic knowledge of the nail organ, its specific reaction patterns and of nail histopathology. Nail psoriasis has a serious impact on the quality of life interfering particularly with manual work but also being cosmetically embarrassing. Treatment of nail psoriasis is difficult as the matrix pathology is hidden by the proximal nail fold and the nail bed changes are protected against treatment by the overlying nail plate and nail bed hyperkeratosis. Progress has been made with the new biologic drugs, which are

however, usually only administered for skin plus nail involvement.

achieved 4 weeks later after the second injection (Rallis et al, 2010).

very brief improvement in disease (Altmeyer et al, 2011).

have been published hitherto.

**9.5 Combined treatments**

group had any signs of recurrence.

**10. Conclusion**

**T cell inhibitors**

psoriasis.

Serious adverse events of all TNF- inhibitors include the development of viral, bacterial, mycobacterial, and fungal infections (Lowther et al, 2007), reactivation of tuberculosis, hepatitis B and C, allergic infusion reactions, malignancies, autoantibody formation with lupus erythematosus, pancytopenia and aplastic anaemia, neurological disorders and worsening of congestive heart failure (Smolen and Emery, 2011). Experience in pregnancy is lacking. Further, infliximab comes with an information what to look for before starting a treatment. Paradoxical sarcoidosis while on anti-TNF- treatment was also reported (Pine et al, 2010)


**5. Changes in the serum protein electrophoretic pattern**

[213]. It also plays a role in the destruction of the infectious agent through the interaction with specific receptors on phagocytes, which may help in the reduction of tissue damage, and

There are considerable species differences in the magnitude and duration of changes in CRP concentrations during health disorders. In humans, dogs, and pigs, CRP is the major acutephase protein with approximately 1000-fold increase in serum concentrations during acute inflammatory states [214]. In cattle, CRP has been reported to be a constitutive protein with only a minor increase during disease processes [7]. Despite this disadvantage, Schrodl et al. [215] evaluated the CRP concentrations in cows with mastitis, and found approximately 10-fold higher values in these cows (1083 ± 93 ng/ml) compared with healthy ones (82 ± 66 ng/ml). The data recorded by Lee et al. [216] showed a correlation between serum CRP concentrations and

Fibrinogen (Fbg), a precursor of fibrin, is also an acute-phase protein, which in coagulation cascade is the final substrate in the formation of a clot being converted to its insoluble fibrin form [217]. Fibrinogen belongs to the group of β-globulins and is present in the plasma. It is composed of three polypeptide chains linked by disulfide bridges and a glycoprotein [218]. Fibrinogen plays an important role in homeostatic processes, providing a substrate for fibrin formation. It is also involved in tissue reparation, and provides a matrix for the migration of inflammatory-related cells [219]. During an inflammatory reaction, fibrinogen can increase 2–3 folds, which may significantly increase blood viscosity and cause red cell aggregation, as well as may contribute to the growth of atherosclerotic plaques [220]. In human, studies showed an association between fibrinogen concentrations and subsequent cardiovascular disease risk, atherosclerosis, and acute thrombosis [221]. In cattle, fibrinogen has been used for many years to evaluate inflammatory and traumatic diseases, and is characterized by mark-

The γ-globulin fraction is predominantly composed of immunoglobulins (Ig) of various classes (IgG, IgA, IgM, IgD, and IgE). While in some animal species (cattle and goats) the γ-globulins constitute one overall fraction, in sheep, they may be visualized as two subpeaks: the γ1

 subfractions [104]. According to Kaneko [22], immunoglobulins from the γ fraction may migrate as fast or slow, which may be seen in these two subfractions. On the other hand, Vavricka et al. [76] indicated that some classes of immunoglobulins may migrate into the β-γ zone or β-region. Immunoglobulins (or antibodies) have major roles in the immune responses of the body, especially in response to foreign molecules, the so-called antigens. Their primary function is the protection of the host due to specific binding of one or a few closely related antigens in order to mediate their neutralization and elimination [223]. Immunoglobulins are produced by cells of the adaptive immune system, activated B cells and plasma cells, in response

and

contribute to the tissue repair and regeneration [162].

120 Ruminants - The Husbandry, Economic and Health Aspects

edly increased synthesis in response to infection [222].

the health condition of dairy cattle.

*<IP_ADDRESS>. Fibrinogen (in plasma)*

*4.3.3. The γ-globulins*

to the exposure to antigens [8].

γ2

Several factors, including non-pathological and pathological conditions, may influence the concentrations of proteins in the serum, thus the entire profile of serum proteins [226]. Many disease processes are associated with abnormal serum protein profiles. Changes in the protein profile commonly occur as secondary symptoms in numerous diseases, but may be also the primary symptom of some specific disease conditions [227]. Thus, the results of the electrophoretic analyses of serum or plasma proteins may provide a basis for the establishment of further specific diagnostic procedures and may be helpful by the differential diagnosis of several disease processes. However, abnormalities in the serum protein profile must be interpreted with regards to many influences that are not associated with pathological processes.


**Section 2: "Carbon Fiber Reinforced"**

Chapter 3: "Unidirectional Carbon Fiber Reinforced Thermoplastic Tape in Automated Tape Placement Process" by Svetlana Risteska.

Chapter 4: "Mechanically Improved and Multifunctional CFRP Enabled by Resins with High Concentrations Epoxy-Functionalized Fluorographene Fillers" by Junhua Wei


**4. Conclusion**

The advantage of metagenomic method is that it allows us to study the genome of the bacterial community directly from the natural environment in which the function and role of certain bacteria on the environment can be determined. Studies have shown that metagenomics are a very useful technology in finding genes that can be applied in industrial, biotechnology, pharmaceutical and medical fields such as esterase, lipase, agarase, polymerase, polyketide synthases, chitinase and so on. Some potentially uncultured microbes and new genes were discovered through this study. In this study, metagenomic libraries using fosmid vectors were constructed from the bacterial community associated with A. tamiyavanichii. A total of 1501 fosmid clones ranging from 30 to 40 Kbp have been obtained and this is equivalent to 13 bacterial genomes. Finally, to our knowledge, the metagenomic library in this study was the first being constructed from the bacterial communities associated with toxic marine dinoflagellate. This collection of libraries can be used as a major source for finding new genes or pathways for biosynthesis and to study the interactions of dinoflagelates more profoundly, especially in the production of the saxitoxins as there is a hypothesis that the toxins produced by dinoflagelates are derived from bacteria.

## **Acknowledgements**

We thank all the people involved directly or indirectly in this study especially staffs from the School of Fisheries and Aquaculture Sciences, Universiti Malaysia Terengganu and Bachok Marine Research Station, University of Malaya.

**45**

**Author details**

and Gires Usup3

Muhd Danish-Daniel1,2\*, Mohd Ezhar Mohd Noor1

1 School of Fisheries and Aquaculture Sciences, Terengganu, Malaysia

3 University College Sabah Foundation, Kota Kinabalu, Sabah, Malaysia

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Institute of Marine Biotechnology, Terengganu, Malaysia

\*Address all correspondence to<EMAIL_ADDRESS>
provided the original work is properly cited.

, Yik Sung Yeong2

, Tan Min Pau1,2

*Construction and Analysis of Metagenome Library from Bacterial Community Associated…*

*DOI: http://dx.doi.org/10.5772/intechopen.88751*

### **Conflict of interest**

The authors declare no conflict of interest.

*Construction and Analysis of Metagenome Library from Bacterial Community Associated… DOI: http://dx.doi.org/10.5772/intechopen.88751*


**HMF oxidation route under base-free conditions**

**Scheme 5.** Reaction mechanism with inclusion of 18O (blue). Main focusing units are boxed in dashed lines (reproduced from [64] with permission from Elsevier, copyright [2014]).


1.4.2. Novel Variants of Cas Protein and Applications

In commonly used CRISPR-Cas9 systems, the *Streptococcus pyogenes* (SpCas9)-gRNA complex generally recognises the region 20 nt upstream of the PAM sequence (5 -NGG-3 ). To broaden the Cas9 protein recognition sequence and reduce the off-target editing rate, several approaches have been used to broaden PAM compatibility and enhance specificity. These approaches are based on the structural characteristics of SpCas9 binding to gRNA and target DNA. For example, the Cas9 variants SpCas9-VQR (NGA-PAM), SpCas9-EQR (NGAG-PAM), and SpCas9-VRER (NGCG-PAM) functioned, but their cleavage activity levels were lower compared to that of the wild-type SpCas9 in *Arabidopsis* and rice [99–102]. SpCas9-NG has a broader recognition sequence with enhanced compatibility, recognising NG-PAM, and has successfully generated targeted mutations in rice and *Arabidopsis* [103–105].

The variants SpCas9-HF1, eSpCas9, and HypaCas9 have been developed to enhance Cas9 protein-cleavage specificity. They show reduced off-target editing activities, indicating high specificity in plant cells [106–108]. Cas9 protein-directed evolution has been developed for Cas9 engineering, conferring high-specificity engineered SpCas9 proteins such as xCas9 [109], evoCas9 [110], and Sniper-Cas9 [111]. xCas9 recognises the NG, GAA, and GAT PAM sequences. Although the gRNA containing these PAM sequences can be mutated in plants, the mutation efficiencies and the preferences for different corresponding PAM sequences differ between cells. For example, the cleavage activity of xCas9 is lower in rice callus than in mammalian cells [112], and xCas9 does not recognise the NG-PAM sequence in tomatoes [32]. Cas9-NG has a stronger cleavage activity than xCas9, especially at CGG, AGC, TGA, and CGT sequence recognition sites [98], making Cas9-NG more suitable for genome editing at the NG-PAM site in plants. The single-base editing system developed based on Cas9 variants (SpCas9-NG and SpCas9-VQR) has been applied to precise base-editing of plant genomes [103,113].

At present, the most commonly used Cas9 protein comes from *Streptococcus pyogenes*, in order to broaden the Cas9 protein recognition sequence, orthologous Cas9 proteins have been isolated from other bacteria; for instance, NmCas9 has been isolated from *Neisseria meningitidis* [114], SaCas9 from *Staphylococcus aureus* [115], StCas9 from *Streptococcus thermophilus* [116], FnCas9 from *Francisella novicida* [117], and CjCas9 from *Campylobacter jejuni* [118]. These proteins are smaller than SpCas9, which is an advantage in cassette delivery. In *Arabidopsis*, the SaCas9 and SpCas9 systems do not interfere with each other [119], so they can fully utilise Cas9 orthologues that recognise different PAM sequences. Such simultaneous targeting by Cas9 orthologues with different PAM sequences would enable multiplex genome engineering by simultaneously targeting more than one site.

With the continued discovery and investigation of the functions of CRISPR protein family members, new types of Cas proteins have been discovered including the type VI CRISPR-Cas system Cas13 (C2c2) protein, which recognises RNA sequences and exhibits RNA editing activity without altering the genome sequence [120,121]. This system has been successfully applied to knockout gene function in rice and tobacco and promote resistance to RNA viruses in *Arabidopsis* [121,122]. Likewise, the CRISPR-Cas13 system created RNA-guided immunity against RNA viruses in plants. Type V CRISPR-Cas systems, such as Cas12c, Cas12g, Cas12h, Cas12i, and Cas14, are distinguished according to the type of their target template (ssRNA, ssDNA, dsDNA, or ssDNA) and cleavage activity strength. Their functions range from dsDNA nicking and cleavage, and can have collateral cleavage activity on ssRNA, ssDNA, dsDNA, and ssDNA [123,124]. In short, the functional differentiation of these Cas protein variants can be used to target mutations in different nucleic acid types, induce a small number of 100 kb sequence deletions, and expand the repertoire of plant genome-editing tools.


**4.1 Prenatal factors**

The association between prenatal insults and the pathogenesis of autism has been reported recent decades. Early in 2005, Beversdorf *et al*. conducted surveys regarding incidence and timing of prenatal stressors. They found a higher incidence of prenatal stressors in autism at 21-32 weeks gestation, which peaks at 25-28 weeks. Their finding supported the hypothesis of prenatal stressors as a potential contributor to autism, and the timing was consistent with the embryological age suggested by neuroanatomical findings seen in the cerebellum in autism [231]. More specifically, Meyer *et al* demonstrate that the effects of maternal immune challenge between middle and late gestation periods in mice are dissociable in terms of several neuropsychiatric disorders including autism [232]. However, this conclusion was challenged by another group of scientists. Ploeger *et al*. proposed pleiotropic effects during a very early and specic stage of embryonic development, namely early organogenesis (day 20 to day 40 after fertilization) in order to explain the effect of uterine disturbances to the development of autism [233]. They provided ample evidence from literature for the association between autism and many different kinds of physical anomalies such as limb deformities, craniofacial malformations, brain pathology, and anomalies in other organs, which agrees with the hypothesis that pleiotropic effects are involved in the development of autism.

Drugs are the most important prenatal factors affecting embryo and fetal development. Cumulating data support the relationships between maternal medication and fetogeneous diseases including autism. The obnoxious drug thalidomide turned out not only to relate to fetal abnormality but also to autism. Stromland group retrospectively investigated 100 Swedish thalidomide embryopathy cases and found possible association of thalidomide embryopathy with autism [234]. Another example of drug relating to autism is valproate. Williams *et al* reported six cases whose clinical phenotype was compatible with both fetal valproate syndrome (FVS) and autism. Although the sample size is small, the authors claimed the association between this known teratogen and autism had both clinical and research implications [235]. Similarly, Rasalam group provided another line of evidence that prenatal exposure to sodium valproate is a risk factor for the development of an ASD [236].

Another prenatal factor is intrauterine inflammation. Kannan *et al* conducted an animal study to demonstrate intrauterine inflammation results in alterations in cortical serotonin and disruption of serotonin-regulated thalamocortical development in the newborn brain therefore resulting in impairment of the somatosensory system, such as autism [237]. More persuasive evidence comes from Girard's report. According to their results, end of gestation exposure of pregnant rats to systemic microbial product such as lipopolysacharide (LPS) is an independent risk factor for neurodevelopmental diseases such as cerebral palsy, mental deficiency, and autism. And coadministration of IL-1 receptor antagonist with LPS alleviated the detrimental effects caused by LPS [238].

Autoimmune Disorder and Autism 531

the previous results are hardly conclusive. Further research is needed to explore the possible

Mouse models provide a powerful strategy to explore experimentally candidate genes for autism susceptibility, and to use environmental challenges to induce gene mutations and cell pathology early in development. Mouse models have also been used to investigate the effects of alterations in signaling pathways on neuronal migration, neurotransmission and brain anatomy, which are relevant to findings in autistic subjects [250]. These models have

There are currently several mouse models for autism research, most of which are primarily developed by knocking out different candidate genes for other neuropsychiatric diseases such as fragile X syndrome [250, 251], Rett syndrome [252], but now are used as autistic models because of their autistic-like behaviors. Other examples include *Engrailed 1&2* and *PTEN* genetic mice [253, 254]. In addition, there is another group of models constructed by surgical or toxic treatments of candidate regions in the brain, in general during development [255]. Some other reports regarding autistic-like behaviors in BALB/c and A/J mice have

Here the author would like to stress an inbred mouse strain for autistic research. BTBR T(+)*tf/J* mouse, also named as BTBR mouse, is an inbred strain with black top coat and blond undercoat. Anatomically BTBR mice get total absence of the corpus callosum, and severely reduced hippocampal commissure, which are also attributed to their phenotypes [259-262]. Although primarily used as type 2 diabetes model [263-268] and phenylketonuria (PKU) model [269-274], BTBR mice were recently found to be a promising mice model for autism research because they exhibited the three core symptoms for diagnosing autism [275-282]. Using this strain, several groups have begun to explore the pathogenesis of autism. It was well documented that circulating corticosterone is higher in the BTBR than in B6. And higher basal glucocorticoid receptor mRNA and higher oxytocin peptide levels were detected in the brains of BTBR as compared to B6, although their relationship to autism remain disputable [283, 284]. In the meanwhile, potential treatments for autism have been proposed based on the experimental results using BTBR mice. Two independent groups confirm the efficacy of the SERT blocker, fluoxetine for enhancement of social interactions [285, 286]. Another experiment reported repetitive self-grooming behavior in the BTBR mouse model of autism was blocked by the mGluR5 antagonist Methyl-6-phenylethynylpyridine (MPEP) [287]. Behavioral therapies offer another option for autism treatment, Young group reported social peers rescued autism-relevant sociability deficits in adolescent

However, the tools to analyze these animals are not yet standardized, and an important effort needs to be made. Crawley *et al* proposed three standards to evaluate animal model, namely face validity (i.e. resemblance to the human symptoms), construct validity (i.e. similarity to the underlying causes of the disease) and predictive validity (i.e. expected responses to treatments that are effective in the human disease) [290]. Using these standards,

Autism spectrum disorders (ASD) is a common neurodevelopment disorder. Diagnosed before three years old, autistic children present significant language delays, social and

newly developed tests are used to screen more animal models for autism research.

elucidated neuropathology that might underlie the autism phenotype.

mechanisms underlying these results.

also been seen [250, 256-258].

BTBR mice, but not cross-fostering [288, 289].

**6. Summary and conclusions**

**5. Mouse models for autism research**

In addition, maternal complications of pregnancies are proved to be associated with autism. One group performed a discriminant analysis to explore perinatal complications as predictors for autism. They found three maternal medical conditions including urinary infection, high temperatures, and depression to be highly significant and contribute to the separation between the autistic and normal subjects [239].

#### **4.2 Postnatal factors**

Heavy metals have also been generally considered to contribute to the pathogenesis of autism. Mercury is one of the most widely studied heavy metals. Palmer *et al* studied the association between environmentally released mercury, special education and autism rates in Texas using data from the Texas Education Department and the United States Environmental Protection Agency, and found there was a significant increase in the rates of special education students and autism rates associated with increases in environmentally released mercury. They reported a 43% increase in the rate of special education services and a 61% increase in the rate of autism [240]. Windham group included 284 children with ASD and 657 controls from the San Francisco Bay area in order to explore possible associations between autism spectrum disorders (ASD) and environmental exposures. Their results suggested a potential association between autism and estimated metal concentrations including mercury, cadmium, nickel [241]. Consistent with previous results, Geier *et al* conducted a prospective study which provided biochemical/genomic evidence for mercury susceptibility/toxicity in ASDs indicating a causal role for mercury [242, 243], and they further explored the threshold effect of mercury in a recent publication [244]. In spite of these different pieces of evidence, disagreement exists. IP *et al* performed a cross-sectional cohort study to compare the hair and blood mercury levels of autistic children and a group of normal children. There was no difference in the mean mercury levels. Thus, they concluded that there is no causal relationship between mercury as an environmental neurotoxin and autism [245].

In addition of mercury, lead is also associated with autism. Very early evidence came from a case report, which explored the interaction and possible casual relationship of an elevated blood-lead and autism, as well as treatment of the behavioral symptoms [246]. Later, Canfield *et al* concluded that blood lead concentrations, even those below 10 microgram per deciliter, were inversely associated with children's IQ scores at three and five years of age, and associated declines in IQ were greater at these concentrations than at higher concentrations [247]. Supporting these results, Yorbik group reported that autism could be associated with significant decrease in excretion rate of lead [248].

Hazardous air pollutants have long been related to the development of autism and more evidences have begun to emerge in recent years. Kalkbrenner *et al* conducted a case-control study to screen perinatal exposure to 35 hazardous air pollutants using 383 children with autism spectrum disorders and, as controls, 2,829 children with speech and language impairment. Although the results were biased by exposure misclassification of air pollutants and the use of an alternate developmental disorder as the control group, they provided evidence based on their analysis that methylene chloride, quinoline, and styrene were the plausible candidate exposures for autism spectrum disorders [249]. In another study conducted by Windham group, trichloroethylene, and vinyl chloride have also been related to autism [241].

However, one should notice that the currently available data are mainly derived from epidemiological studies. Considering the limited sample sizes and the different populations,

In addition, maternal complications of pregnancies are proved to be associated with autism. One group performed a discriminant analysis to explore perinatal complications as predictors for autism. They found three maternal medical conditions including urinary infection, high temperatures, and depression to be highly significant and contribute to the

Heavy metals have also been generally considered to contribute to the pathogenesis of autism. Mercury is one of the most widely studied heavy metals. Palmer *et al* studied the association between environmentally released mercury, special education and autism rates in Texas using data from the Texas Education Department and the United States Environmental Protection Agency, and found there was a significant increase in the rates of special education students and autism rates associated with increases in environmentally released mercury. They reported a 43% increase in the rate of special education services and a 61% increase in the rate of autism [240]. Windham group included 284 children with ASD and 657 controls from the San Francisco Bay area in order to explore possible associations between autism spectrum disorders (ASD) and environmental exposures. Their results suggested a potential association between autism and estimated metal concentrations including mercury, cadmium, nickel [241]. Consistent with previous results, Geier *et al* conducted a prospective study which provided biochemical/genomic evidence for mercury susceptibility/toxicity in ASDs indicating a causal role for mercury [242, 243], and they further explored the threshold effect of mercury in a recent publication [244]. In spite of these different pieces of evidence, disagreement exists. IP *et al* performed a cross-sectional cohort study to compare the hair and blood mercury levels of autistic children and a group of normal children. There was no difference in the mean mercury levels. Thus, they concluded that there is no causal relationship between mercury as an environmental

In addition of mercury, lead is also associated with autism. Very early evidence came from a case report, which explored the interaction and possible casual relationship of an elevated blood-lead and autism, as well as treatment of the behavioral symptoms [246]. Later, Canfield *et al* concluded that blood lead concentrations, even those below 10 microgram per deciliter, were inversely associated with children's IQ scores at three and five years of age, and associated declines in IQ were greater at these concentrations than at higher concentrations [247]. Supporting these results, Yorbik group reported that autism could be

Hazardous air pollutants have long been related to the development of autism and more evidences have begun to emerge in recent years. Kalkbrenner *et al* conducted a case-control study to screen perinatal exposure to 35 hazardous air pollutants using 383 children with autism spectrum disorders and, as controls, 2,829 children with speech and language impairment. Although the results were biased by exposure misclassification of air pollutants and the use of an alternate developmental disorder as the control group, they provided evidence based on their analysis that methylene chloride, quinoline, and styrene were the plausible candidate exposures for autism spectrum disorders [249]. In another study conducted by Windham group, trichloroethylene, and vinyl chloride have also been related

However, one should notice that the currently available data are mainly derived from epidemiological studies. Considering the limited sample sizes and the different populations,

associated with significant decrease in excretion rate of lead [248].

separation between the autistic and normal subjects [239].

**4.2 Postnatal factors**

neurotoxin and autism [245].

to autism [241].

the previous results are hardly conclusive. Further research is needed to explore the possible mechanisms underlying these results.


5.2. History and intuition of neural networks

In the twentieth century scientists were able to definitively identify the primary unit of the brain – the neuron. One theory of the time was that information is not pre-loaded in the brain of a newly born child, only the basic structure and connections between the neurons in its brain exist, the brain learns to function by strengthening/weakening various neural pathways.

1 Machine Learning, Tom Mitchell, McGraw Hill, 1997.

Therefore it was theorized that the neuron, which either fires or does not, can be modeled as a function that has multiple inputs, a single output (which may be connected to several other neurons) and only 'fires' when the sum of inputs exceeds a certain threshold. The connections between neurons have weights, which strengthen or weaken a connection between two neurons.

"Neurons that wire together, fire together"

The above quotation may be familiar. It is based on Donald Hebb's theory to explain neural adaptation and learning and this forms the basis of learning in modern-day artificial neural networks.


*2.5. Determination of PAHs in Soil, Forage, and Milk by Gas Chromatography–Tandem Mass Spectrometry (GC-MS/MS)*

Soil samples (10 g) were extracted with dichloromethane:acetone [1:1 (*v*/*v*)] in a Soxtherm system (Gerhardt, Bonn, Germany). The extracts were cleaned with silica gel, then concentrated by rotary evaporation (Heidolph, Schwabach, Germany). PAH concentrations were determined after injection into a 7890A GC System coupled to a 5975C Inert XL MSD with a Triple-Axis Detector (Agilent Tech., Santa Clara, CA, USA), following EPA Method 8272, with modifications. The samples were run on a capillary column DB-5ms with a length of 30 m, an inner diameter of 0.25 mm, and a film thickness of 0.25 μm (Agilent Tech. Santa Clara, CA, USA), with He as the carrier gas at 1 mL min−1. The initial oven temperature of 70 ◦C was held for 2 min; ramped up to 220 ◦C at 20 ◦C min−1, then to 270 ◦C at 10 ◦C min−1, where it was held for 1 min; ramped up to 290 ◦C at 10 ◦C min−1, where it was held for 1 min; and finally ramped up to 300 ◦C at 10 ◦C min−1, where it was held for 7 min. The total run time of GC separation was 30 min The gas chromatography injector was operated in splitless mode for 2 min, and its temperature was maintained at 260 ◦C. The mass spectrometer was operated in electron ionization mode (EI) at 70 eV and calibrated daily by auto-tuning with perfluorotributylamine (PFTBA). PAH calibration standards (AccuStandard, New Haven, CT, USA) were used. Blanks (one for every five samples), duplicate samples, and cross correlation were used for quality assurance and quality control (QA/QC) purposes. RSD for individual PAHs was below 10% in all cases. The following species (*m*/*z*) were quantified: 128 (naphthalene), 152 (acenaphthylene), 153 and 154 (acenapthene), 165 and 166 (fluorene), 178 (anthracene/phenanthrene), 202 (fluoranthene/pyrene), 228 (benzo(a)anthracene/chrysene), 252 (benzo(b)fluoranthene/benzo(k)fluoranthene), 276 (indene(1,2,3-c,d)pyrene/benzo(g,h,i)perylene), and 278 (benzo(a,h)anthracene). In Appendix A, the most representative chromatogram of soil samples has been included.

Forage and milk samples were treated according to the "QuEChERS" extraction method, with some modifications [26]. Briefly, samples (10 g) were extracted with 30 mL acetonitrile and vortexed at 3000 rpm for 1 min. A total of 4 g anhydrous MgSO4 and 1 g NaCl were added and immediately vortexed for 1 min, then 50 μL of an internal standard solution were added and the mixture was vortexed for another 30 s. The mixture was centrifuged at 2800× *g* for 5 min at room temperature and the supernatant was purified by a dispersive solid-phase extraction method [26]. An aliquot of supernatant (5 mL) was transferred to a flat-bottomed flask, concentrated in a 40 ◦C water bath until neardrying, and dissolved in 5 mL of cyclohexane [26]. An aliquot of 1 μL was injected into a 7890B gas chromatograph (Agilent Tech.) equipped with a Select PAH CP7462 capillary column with a length of 30 m, an inner diameter of 0.25 mm, and a film thickness of 0.15 μm. He was used as carrier gas at 2 mL min−1. The initial oven temperature of 70 ◦C was held for 0.7 min, ramped up to 180 ◦C at 85 ◦C min−1 and then to 230 ◦C at 3 ◦C min−1, where it was held for 7 min; ramped up to 280 ◦C at 28 ◦C min−1, where it was held for 10 min; and, finally, ramped up to 350 ◦C at 14 ◦C min−1, where it was held for 3 min. The total run time of GC separation was 60 min. The GC injector was operated in splitless mode for 1 min and its temperature was maintained at 300 ◦C. The compounds were detected using a 7000D mass spectrometer (Agilent Tech.), which was operated in electron ionization mode (EI) at 70 eV. The following *m*/*z* ratios were monitored: 178 (anthracene/phenanthrene), 202 (fluoranthene/pyrene), 228 (benzo(a)anthracene/chrysene), 252 (benzo(b)fluoranthene/benzo(k)fluoranthene/benzo(a)pyrene), 276 (benzo(g,h,i) perylene/Indene(1,2,3-c,d)pyrene), and 278 (benzo(a,h)anthracene). Each sample was analyzed per duplicate. The method was validated using five internal standards (AccuStandard, New Haven, CT, USA), prepared by adding isotopically labelled PAHs to sample extracts. Concentrations of PAHs in the samples were determined by comparing their peak areas to those of the internal standards.


**4. Very small embryonic like stem cells (VSELs)**

VSELs were identified by Ratajczak and group in 2006 by multi parameter sorting in adult murine BM. They express several morphological (e.g., relatively large nuclei containing euchromatin) and molecular (e.g. expression of SSEA-1, Oct4, Nanog, Rex1) markers

characteristic for embryonic stem cells (ESCs) [33]. The morphology of the cells was investigated using transmission electron microscopy which showed their distinctive morphology and size differentiating VSELs from HSC in particular in terms of size (3–6 μm vs. 6–8 μm for HSC), chromatin structure and nucleus/cytoplasm ratio. Based on their small size, presence of PSC markers, distinct morphology (open-type chromatin, large nucleus, narrow rim of cytoplasm with multiple mitochondria) and ability to differentiate into all three germ layers, including mesoderm-derived cardiomyocytes, these cells were named very small embryoniclike stem cells. The true expression of Oct-4 and Nanog in BM-derived VSELs (BM-VSELs) was recently confirmed by demonstrating transcriptionally active chromatin structures of Oct4 and Nanog promoters. A mechanism based on parent-of-origin-specific reprogramming of genomic imprinting that keeps VSELs quiescent in a dormant state in tissues has been described. VSELs highly express Gbx2, Fgf5, and Nodal, but express less Rex1/Zfp42 transcript as compared to ESC-D3s what suggests that VSELs are more differentiated than ICM-derived ESCs and share several markers with more differentiated EpiSCs. VSELs also highly express Dppa2, Dppa4, and Mvh, which characterize late migratory PGCs. The expression of germ line markers (Oct4 and SSEA-1) and modulation of somatic imprints suggest a potential developmental similarity between VSELs and germ line-derived primordial germ cells (PGCs) [39, 40].

VSELs in Bone Marrow and Cord Blood 77

, CD133+ SSEA-4+. They also

have also reported that VSELs are mobilized into peripheral blood in response to injury/ stress in animal models [27,54-56] as well as in humans [28-30,57] – thus suggesting a role in

We studied the VSELs in UCB and discarded fraction of BM [46]. Usually the 'buffy coat' obtained after Ficoll-Hypaque centrifugation is considered to be rich in stem cells and used for various studies over several decades. **However, we reported that VSELs settle along with the RBCs rather than getting enriched in the 'buffy coat (Figure:3). Similarly we found that the 'discarded' RBC pellet obtained during initial processing of bone marrow was also rich in VSELs. These results were explained on the basis of buoyancy. The adult stem cells have abundant cytoplasm, are relatively larger and thus observed in the buffy layer whereas the VSELs are the pluripotent stem cells, with high nucleo-cytoplasmic ratio, minimal cytoplasm and thus sink to the bottom of the tube along with the RBCs.**

exhibit other primordial germ cell markers like Stella and Fragillis, thus supporting their origin from the epiblast stage embryo at the same time when PGCs migrate via the dorsal

**Figure 3. Isolation and characterization of VSELs from Cord Blood: A-Separation** of cord blood into

These studies have several implications e.g. the stem cell biologists should ask themselves what is getting banked in the cord blood banks. VSELs unknowingly get discarded and only adult stem cells (and progenitors) including HSCs and MSCs get banked. Similarly autologus stem cell therapy for various indications other than blood related diseases have resulted in

four layers on Ficoll-Hypaque; B-Description of cells observed in each layer separated; C-Immunolocalization studies on MNC (A) and VSEL (B) using polyclonal Oct-4 (40X); D-Markers

characterized on VSELs using Quantitative PCR and immunofluorescence

**5. Our studies on VSELs in cord blood and bone marrow**

These VSELs exhibited various pluripotent markers, like CD45-

mesentry to the gonadal ridges to become a source of germ cells.

regeneration and homeostasis.

*Developmental Origin of VSELs*: VSELs are epiblast-derived PSCs deposited early during embryonic development in developing organs as a potential reserve pool of precursors for TCSCs and thus this population has an important role in tissue rejuvenation and regeneration. VSELs originate from or are closely related to a population of proximal epiblast migratory Stem Cells (EpiSCs) that approximately at embryonic day (E)7.25 in mice, become specified to PGCs, and egress from the epiblast into extra-embryonic tissues (extraembryonic mesoderm) [41]. VSELs follow developmental route of HSCs colonizing together with HSCs first fetal liver and subsequently BM [37].

Thus PGCs, HSCs, and VSELs form all together a unique highly migratory population of interrelated Stem Cells (SCs) that could be envisioned to be a kind of ''fourth highly migratory germ layer.'' [37]

*Self-renewal and in vitro differentiation of VSELs*: VSELs exist in various mouse organs [42], have been well-characterized and are capable of differentiating into all three lineages, supporting their true pluripotent character. Murine VSELs form embryoid body-like structures in co-cultures over C2C12 supportive cell line [24] and could become specified into HSCs after co-culture over OP-9 stroma cells. VSELs-derived HSCs harvested from these co-cultures reconstitute murine bone marrow after total body irradiation [43]. The Umbilical Cord Blood (UCB)-purified VSELs have also been reported to differentiate into neural cells [44] and after co-culture over OP-9 stroma cells were specified into HSCs similar to murine BM-derived VSELs [45]. Apart from umbilical cord blood and bone marrow, VSELs have also been reported in Wharton's jelly and gonadal tissue [46- 51]. Their presence amongst the MSCs in the Wharton's jelly is in agreement with observations made by other groups that MSCs contain a sub-population of more primitive stem cells [52] or even as postulated by Taichman and group [53] that VSELs are precursors of MSCs. Various studies have also reported that VSELs are mobilized into peripheral blood in response to injury/ stress in animal models [27,54-56] as well as in humans [28-30,57] – thus suggesting a role in regeneration and homeostasis.


**6. References**

Numerical Modeling of Solidification Process 541

Mochnacki, B. & Szopa, R. (2008). Generalization of temporary temperature field correction

Mochnacki, B. & Majchrzak, E. (2010). Numerical modeling of casting solidification using

Mochnacki, B. & Szopa, R. (1998). Application of the boundary element method in numerical

Mochnacki, B. & Szopa, R. (2002). Numerical modelling of heat transfer in spherical domains

Kleiber, M. (1997). *Parameter Sensitivity in Nonlinear Mechanics*, J. Wiley & Sons Ltd.,

Szopa, R. (2005). The parametric sensitivity analysis of solidification process, *Archives of*

Szopa, R. & Wojciechowska, W. (2003). Sensitivity analysis of solidification process with respect to grains geometry, *Archives of Foundry*, Vol. 3, No 10, pp. 255-260. Szopa, R., Siedlecki, J. & Wojciechowska, W. (2004). The influence of initial conditions on the

Mochnacki, B. & Szopa, R. (2009). Application of sensitivity analysis in numerical simulation of

Majchrzak, E., Dziewonski, M. & Kaluza, G. (2007). Identification of cast steel latent heat by

Majchrzak, E., Mochnacki, B. & Suchy, J.S. (2008a). Identification of substitute thermal

Majchrzak, E. & Mendakiewicz, J. (2009). Identification of cast steel latent heat on a basis of

Mochnacki, B. & Majchrzak, E. (2006). The methods of inverse problems solution in the

Mendakiewicz, J. (2008). Application of identification methods in solidification process modelling, *Archives of Foundry Engineering*, Vol. 8, No. 1, pp. 203-210. Majchrzak, E. & Mendakiewicz, J. (2007). Gradient method of cast iron latent heat identification, *Archives of Foundry Engineering*, Vol. 7, No. 4, pp. 121-126.

*Theoretical and Applied Mechanics*, Vol. 2, No. 36, pp. 457-468.

Dems, K. & Rousselet, B. (1999). *Structural Optimization*, Vol. 17, pp. 36-45.

*Mechanics*, Vol. 2, No. 40, pp. 357-370.

*Foundry*, Vol. 5, No. 15, pp. 395-404.

*Computer Science*, 1(3), pp. 201-208.

*Engineering*, Vol. 1, No. 5, pp. 555-570.

Polish Academy of Sciences, pp. 239-254.

8, No. 1, pp. 219-222.

Chichester, England.

pp. 271-286.

2, pp. 257-268.

pp. 195-199.

2681.

method in numerical modelling of solidification, *Archives of Foundry Engineering*, Vol.

generalized finite difference method, *Materials Science Forum*, Vols. 638-642, pp. 2676-

modelling of solidification Part I. The One Domain Approach, *Journal of*

by mean of the BEM using discretization on time, *Journal of Theoretical and Applied*

course of solidification process, *Scientific Research of the Institute of Mathematics and*

solidification process, In: *Progress of Foundry Theory and Practice*, Pol. Ac. of Sciences,

means of gradient method, *Int. J. Computational Materials Science and Surface*

capacity of solidifying alloy, *Journal of Theoretical and Applied Mechanics*, Vol. 46, No.

thermal and difference analysis, *Computer Methods in Material Science*, Vol. 9, No. 2,

thermal theory of foundry processes, In: *Research in Polish Metallurgy at the beginning of XXI century*, Ed. K. Świątkowski, Committee of Metallurgy of the

Majchrzak, E. & Mendakiewicz, J. (1993). Application of the generalized alternating phase

Majchrzak, E., Suchy, J. S. & Szopa, R. (2006). Linear model of crystallization. Identification

Mochnacki, B. & Szopa, R. (2007) Model of pure metal solidification using the power-type

Fraś, E., Kapturkiewicz, W. & Lopez, H.F. (1993). Macro and micro modelling of the solidification kinetics of casting, *AFS Transactions*, Vol. 48-92, pp. 583-591. Lupa, M., Szopa, R. & Wojciechowska, W. (2004). Sensitivity analysis of crystallization with

Mochnacki, B. & Szopa, R. (2010). Generalized micro/macro model of crystallization and its

Majchrzak, E. & Piasecka, A. (1997). Numerical micro/macro model of solidification process, *Journal of Materials Processing Technology*, Vol. 64, pp. 267-276. Domański, Z., Ciesielski, M. & Mochnacki, B. (2009a). Voronoi tessellations description of cast

Domański, Z., Ciesielski, M. & Mochnacki, B. (2009b). Applicatiom of CVM using the Voronoi

Mochnacki, B. & Ciesielski, M. (2007). Micro/macro model of solidification. Numerical

Brebbia, C.A. & Dominguez, J. (1992). *Boundary elements, an introductory course*, Computational Mechanics Publications, McGraw-Hill Book Company, London. Majchrzak, E. (2001). *Boundary element method in heat transfer*, Publication of the Czestochowa

Majchrzak, E. & Mochnacki, B. (1996). The BEM application for numerical solution of non-

Majchrzak, E. & Szopa, R. (2001). Analysis of thermal processes in solidifying casting using

Mochnacki, B. & Majchrzak, E. (2007b). Application of the shape sensitivity analysis in

steady and non-linear thermal diffusion problems, *Computer Assisted Mechanics and*

the combined variant of the BEM, *Journal of Materials Processing Technology*,

numerical modelling of solidification process, *Materials Science Forum*, Vols. 539-

University of Technology, Czestochowa, Poland (in Polish).

*Engineering Sciences*, Vol. 3, No. 4, pp. 327-346.

*Engineering and Computer Science*, Vol. 2, pp. 1197-1201.

*of Metals and Alloys*, Vol. 18, pp. 103-112.

*Computer Science*, 1(3), pp. 91-98.

New York, pp. 17-27.

Vol. 109, pp. 126-132.

543, pp. 2524-2529.

Verlag.

pp. 65-71.

266.

truncation method for numerical modelling of cast iron solidification, *Solidification*

of nuclei density, *Giessereiforschung, International Foundry Research*, Vol. 2, pp. 29-32.

function, *Journal of Achievements in Materials and Manufacturing Engineering*, Vol. 22,

respect internal parameters, *Scientific Research of the Institute of Mathematics and*

numerical realization, *Journal for Multiscale Computational Engineering*, 8(3), pp. 259-

composite solidification process, *World Congress on Engineering 2009*, *Lecture Notes in*

tesselation in numerical modelling of solidification process, *Current Themes in Engineering Science 2009*. Ed. M. Korsunsky, American Institute of Physics, Melville,

simulation using the control volume method, *17th International Conference on Computer Methods in Mechanics CMM-2007*, CD ROM Proceedings, ISBN-978-83-923982-3-3. Brebbia, C.A., Telles, J.C.F. & Wrobel, L.C. (1984). *Boundary Element Techniques*, Springer-

**0**

**25**

*Italy*

**Field Simulations**

*Università Politecnica delle Marche*

**Performance Evaluation of Adaptive Algorithms**

**for Wave Field Analysis/Synthesis Using Sound**

The main objective of a multi-channel sound reproduction system is to give an optimal acoustical sensation to the listener. These systems are designed to produce sounds that are as natural as possible, so that the listener does not realize that they are generated by a loudspeakers system. For this purpose, the knowledge of only audio temporal information is not sufficient: also spatial information is needed. Furthermore, since the invention of stereophony, it is well known that at least two loudspeakers are needed in order to generate a virtual source that is not spatially localized at the speaker position. However, the stereo reproduction is very limited because the optimal source localization is focused on one point, called sweet spot. Starting from this assumption, in the recent literature, research efforts have focused on reproduction techniques that use an extremely high number of loudspeakers in order to reproduce not only a simple audio source but a complete sound field. Various techniques, able to record and to reproduce the entire sound field, have been proposed in the

One of the most studied techniques is Wave Field Synthesis (WFS) that is directly based on the Huygens' principle. It has been introduced in the late '80s by Berkhout, who showed that audio reproduction can be linked to audio holography concepts (Berkhout, 1988). WFS is based on Kirchhoff-Helmholtz integral which permits the calculation of the pressure field inside a volume by knowing pressure and normal particle velocity on the enclosing surface. The underlying idea of WFS is to generate a sound field inside a volume bounded by an array of loudspeakers. Actually, the surface is reduced to a 2D curve positioned on the ear plane. The number of loudspeakers on this curve depends on the desired localization quality. Similarly, Wave Field Analysis (WFA) implements a sound field recording technique based on microphone arrays (Hulsebos et al., 2002). Therefore, this approach allows to record the entire sound field in the recording room (WFA) and subsequently to reproduce it in the listening room (WFS) more or less accurately depending on loudspeakers/microphones number. As it happens in traditional reproduction systems, digital algorithms based on adaptation processing have to be applied inside the WFS/WFA in order for these techniques to be used in real applications (music, cinema, theatre, etc.). Examples of adaptive algorithms that are very useful in real applications are Acoustic Echo Cancellation (AEC), Active Noise Control (ANC), room compensation, etc. (Haykin, 1996). A straightforward implementation of these algorithms in WFA/WFS systems is not feasible due to the dramatically high

literature (Berkhout et al., 1993; Daniel et al., 2003; Fazi et al., 2008).

**1. Introduction**

Paolo Peretti, Stefania Cecchi, Laura Romoli and Francesco Piazza