In addition to p21Cip1*/*WAF mRNA, which has been demonstrated to be posttranscriptionally stabilized after DNA damage, Fornace and colleagues identified Gadd45α mRNA as posttranscriptionally stabilized in response to genotoxic stress (Jackman et al., 1994) (**Figure 1**). Gadd45α is part of a family of genes consisting of Gadd45α, Gadd45β, and Gadd45γ that is widely expressed in mammalian cells following different stress stimuli. Gadd45α is induced following hypoxia, IR, oxidants, UV, and growth factor withdrawal (Zhan, 2005). Gadd45α has been mechanistically linked to numerous cellular processes, including apoptosis, cell cycle arrest, nucleotide excision repair and repair-mediated DNA demethylation, maintenance of genomic stability and signaling through the p38MAPK, and JNK kinase pathways (Hollander et al., 1999; Wang et al., 1999; Smith et al., 2000; Amundson et al., 2002; Hildesheim et al., 2002; Barreto et al., 2007). Gadd45α expression is rapidly induced after genotoxic stress. This transcriptional activation has initially been thought to be primarily induced by p53 (Kastan et al., 1992). In fact, p53 was the first transcription factor reported to induce Gadd45α transcription and, at least in response to IR, Gadd45α transcription strictly depends on p53 (Kastan et al., 1992). However, it is now clear that additional transcription factors, including WT1, Oct1, NF-YA, FoxO3a, Egr-1, and C/EBPα are also capable of inducing Gadd45α transcription, even in the absence of p53 (Constance et al., 1996; Zhan et al., 1998; Jin et al., 2001; Takahashi et al., 2001; Tran et al., 2002; Hirose et al., 2003; Thyss et al., 2005). For example, we recently showed that Gadd45α was induced in p53-deficient murine embryonic fibroblasts (MEFs) following treatment with doxorubicin (Jiang et al., 2009). In resting cells, Gadd45α transcription appears to be repressed through c-Myc and a repressive complex consisting of ZBRK1 and BRCA1 (Marhin et al., 1997; Amundson et al., 1998; Bush et al., 1998; Zheng et al., 2000; Tan et al., 2004). Interestingly, c-Myc itself is translationally repressed through miR-34c via a highly conserved target-site within the 3 UTR in response to etoposide-induced DNA damage. While miR-34c can be induced by p53 following genotoxic stress, Cannell et al.(2010) showed that miR-34c expression in p53-deficient cells depends on the p38MAPK/MK2 signaling complex (Cannell et al., 2010). In addition to this elaborate network of transcriptional control, Fornace and colleagues reported as early as 1994 that Gadd45α mRNA is posstranscriptionally stabilized in response to UV or MMS exposure (Jackman et al., 1994). However, the molecular details of this posttranscriptional regulation remained largely obscure. These posttranscriptional regulatory mechanisms might impact on Gadd45α mRNA molecules at different steps of their maturation, from their *de novo* synthesis as pre-mRNA until the eventual degradation or translation. These steps include premRNA splicing and maturation (3 polyadenylation, 5 capping), followed by mRNA export to the cytoplasm, sub-cytoplasmic transport, escape from ribonucleolytic cleavage and translation (Mitchell and Tollervey, 2000; Orphanides and Reinberg, 2002; Moore, 2005). Recent studies from Gorospe and co-workers have identified the RBPs AUF1 and TIAR as critical posttranscriptional regulators of Gadd45α mRNA (Lal et al., 2006). Both proteins were found to form RNP complexes through a direct interaction with the 3 UTR of the Gadd45α mRNA in resting cells. However, when cells were exposed to UV or MMS these RNP complexes rapidly dissociated, which correlated with a substantial increase in Gadd45α mRNA stability an enhanced association of Gadd45α mRNA with actively translating ribosomes and increased Gadd45α protein accumulation (Lal et al., 2006). When Lal et al. examined the molecular mechanisms of Gadd45α repression in resting cells, they found AUF1 to render Gadd45α mRNA unstable while TIAR prevented the association of Gadd45α mRNA with translating polyribosomes. Thus, the combined effect of AUF1 and TIAR is a potent repression of Gadd45α biosynthesis through AUF1-mediated mRNA destabilization and TIAR-dependent translational suppression at resting state. The genotoxic stress-induced dissociation of AUF1 and TIAR from the Gadd45α mRNA represents a mechanism of posttranscriptional derepression resulting in mRNA stabilization and enhanced translation in response to DNA damage. Both of these posttranscriptional regulatory steps were found to be essential for proper induction of Gadd45α protein levels following DNA damage (Lal et al., 2006).

The report by Lal et al. implicated AUF1 and TIAR as RBPs that are critical for the posttranscriptional de-repression of Gadd45α mRNA. However, it remained unclear which molecular mechanisms underlie the DNA damage-induced dissociation of these RBPs from the Gadd45α mRNA. A plausible explanation might be DNA damage-dependent phosphorylation events. Indeed, AUF1 was reported to be a phospho-protein and GSK3β and PKA were subsequently identified as kinases capable of AUF1

phosphorylation *in vivo* (Zhang et al., 1993; Wilson et al., 2003). Nonetheless, whether these phosphorylations occur *in vivo* following genotoxic stress persists to be elusive.

TTP. In addition, MK2 phosphorylates hnRNP A0, promoting its binding to and stabilization of Gadd45α mRNA. In the absence of DNA damage, Gadd45α

We have recently identified the p38MAPK/MK2 pathway as a critical regulator of RBPs that mediate posttranscriptional stabilization of Gadd45α mRNA in response to genotoxic stress (Reinhardt et al., 2010). In analyzing the molecular details of MK2 function in response to DNA damage, we found that MK2 knockdown prevented the accumulation of Gadd45α mRNA and protein in response to adriamycin. We identified the known MK2 substrate hnRNP A0 as a novel Gadd45α mRNA-binding protein (Reinhardt et al., 2010). MK2-mediated phosphorylation of hnRNP A0 on Ser-84 following DNA damage was required for the formation of hnRNP A0:Gadd45α mRNA RNP complexes and overexpression of a non-phosphorylatable hnRNP A0 on Ser-84 to Ala mutant prevented Gadd45α mRNA and protein accumulation in response to adriamycin (Reinhardt et al., 2010). These data suggest that MK2-dependent phosphorylation of hnRNP A0 is critical for the formation of hnRNP A0:Gadd45α mRNA RNP complexes, which in turn appears to be essential for the posttranscriptional stabilization of Gadd45α mRNA. In addition, we found that MK2 phosphorylates *P*oly-(*A*) *r*ibo*n*uclease (PARN) on Ser-557 in response to adriamycin (Reinhardt et al., 2010). Two major pathways of mRNA degradation exist in eukaryotes. In both cases, shortening of the poly(A) tail is the first, timelimiting, step. Three distinct protein complexes—Pan2/Pan3, or PAN complex; PARN; and the Ccr4/Pop2 complex—govern this deadenylation. After deadenylation, degradation occurs in 3 –5

MK2. Green circles indicate DNA damage-activated kinases, red circles

indicate RNA-binding and metabolizing proteins.

direction through the RNase-containing exosome complex. In an independent pathway, deadenylation is followed by removal of the 7-methyl-guanosine cap of mRNAs and then proceeds in the 5 –3 direction. The mechanisms of mRNA turnover have been reviewed recently (Meyer et al., 2004). We found PARN phosphorylation on Ser-557 to be critical for prolonged Gadd45α mRNA and protein expression after adriamycin (Reinhardt et al., 2010). However, the molecular details of this apparent inhibition of Gadd45α mRNA degradation remain somewhat unclear. Despite our best efforts, we failed to observe any changes in PARN activity or RNA binding affinity following MK2-mediated phosphorylation on Ser-557 (Schmedding, Reinhardt, Yaffe unpublished). In addition to these MK2-mediated posttranscriptional mechanisms of Gadd45α mRNA stabilization, we confirmed that TIAR dissociates from the Gadd45α mRNA in response to genotoxic stress (Reinhardt et al., 2010). Furthermore, we could show that p38MAPK directly phosphorylates TIAR after adriamycin exposure, both *in vitro* and *in vivo* [(Reinhardt et al., 2010) and Morandell, Reinhardt, Yaffe unpublished]. Pretreatment of cells with the p38α*/*β-specific inhibitor SB203580 completely prevented the adriamycin-mediated dissociation of TIAR:Gadd45α mRNA RNP complexes. Thus, we have identified three novel mechanisms of posttranscriptional Gadd45α mRNA control. We identified hnRNP A0 as a critical MK2-dependent posttranscriptional inducer of Gadd45α mRNA. In addition to AUF and TIAR, which have been described as posttranscriptional repressors of Gadd45α mRNA, we have identified PARN as a further molecule that appears to be involved in Gadd45α mRNA repression at resting state. Lastly, we could show that the DNA damage-induced dissociation of the TIAR:Gadd45α mRNA RNP complex depends on p38MAPK-mediated TIAR phosphorylation.

In additional experiments we could confirm data provided by Bulavin et al. showing that Gadd45α interacts with p38MAPK (Bulavin et al., 2003). Bulavin et al. further showed that Gadd45α is critical for H-rasV12-induced activation of p38MAPK. We made a similar observation in response to adriamycin-invoked genotoxic stress. RNAi-mediated knockdown of Gadd45α prevented the prolonged phosphorylation and activation of MK2, likely through a lack of p38MAPK activity. MK2 remained active in control cells for at least 30 h. However, MK2 activity dropped precipitously after ∼24h in Gadd45α-depleted cells. These data suggest that the initial activation of MK2 after genotoxic stress does not depend on Gadd45α, but subsequent p38MAPK/MK2 dependent stabilization of Gadd45α, through phosphorylation of TIAR, PARN, and hnRNP A0, becomes essential for maintaining MK2 activity at late times. Further experiments showed that particularly this late MK2 activity was critical to maintain checkpoint control after genotoxic stress invoked by doxorubicin through a mechanism involving Cdc25B/C inactivation. Members of the Cdc25 family of dual-specificity phosphatases are phosphorylated by the checkpoint effector kinases Chk1 and MK2 in response to DNA damage. We and others previously showed that the cell cycle arresting checkpoint function of MK2 is mediated through MK2-dependent Cdc25B/C phosphorylation and subsequent cytoplasmic sequestration (Lopez-Aviles et al., 2005; Manke et al., 2005; Reinhardt et al., 2007).

We note that MK2 and its activating kinase p38MAPK form a tight nuclear complex in resting cells (Ben-Levy et al., 1995, 1998; ter Haar et al., 2007). MK2 contains a nuclear localization signal (NLS) and a nuclear export signal (NES) located at the C-terminus. At resting state, the NES is masked by a direct intramolecular interaction (ter Haar et al., 2007). Following p38 mediated activating phosphorylation of MK2 on Thr-334, this interaction is relieved and the NES becomes exposed, resulting in cytoplasmic translocation of the p38MAPK/MK2 complex (Ben-Levy et al., 1995, 1998; ter Haar et al., 2007). We could show that MK2 rapidly leaves the nucleus in response to DNA damage via a Crm1-dependent nuclear export mechanism (Reinhardt et al., 2010). Thus, we hypothesized that late cytoplasmic MK2 activity might be required to maintain Cdc25B/C sequestered in the cytoplasm in the context of active cell cycle checkpoints. We have hence used live cell imaging to follow the subcellular distribution of Cdc25B and C after genotoxic stress in control cells or cells that were depleted of either Chk1 or MK2. Cytoplasmic accumulation of GFP-tagged Cdc25B/C was used as a readout for active checkpoint signaling. These experiments revealed that adriamycin exposure induces a robust cell cycle checkpoint in control cells that is relieved after ∼30 h and is followed by a cytologically normal mitotic cell division. Cdc25B/C was maintained in the cytoplasm until cells entered mitosis. In contrast, Chk1 depletion resulted in premature nuclear re-entry of Cdc25B/C after ∼15 h, followed by catastrophic mitotic cell division resulting in apoptosis. We observed a similar phenotype in MK2-depleted cells. However, Cdc25B/C nuclear re-entry did not occur until ∼23 h following doxorubicin. Intriguingly in this regard is the observation that this time corresponds perfectly to the time when MK2 activity returned to baseline levels in Gadd45α-depleted cells that were treated with doxorubicin. These data strongly suggest that the positive feedback loop involving MK2-dependent stabilization of Gadd45α, and Gadd45α-dependent maintenance of MK2 activity, are essential for prolonged cell cycle arrest through cytoplasmic Cdc25B/C sequestration in response to adriamycin. Together, these data suggest that a feed forward loop consisting of p38, MK2, and Gadd45α is critical to provide time to recover from adriamycin-induced genotoxic insults before entering the next mitotic cell division.

# **CONCLUDING REMARKS**

Posttranscriptional control of gene expression has recently moved into the focus of scientists working in various areas of life sciences. This is owed to the discovery of miRNA-mediated gene silencing mechanisms and the uncovering and characterization of a number of RBPs that are involved in the stabilization and translatability of mRNAs. The DDR network has classically been regarded as consisting of a fast-acting kinase signaling branch, leading to the rapid inactivation of Cdk-cyclin complexes and a delayed transcriptional response, resulting in the transactivation of genes encoding for Cdk inhibitors, such a p21. As a consequence of numerous recent discoveries, a clearer picture is emerging stressing the molecular mechanisms involved in posttranscriptional control of gene expression and expanding the complex DDR signaling network with a third layer. These recent reports strongly suggest that cells employ complex regulatory circuits impacting on transcript stability and translatability in response to genotoxic stress. The major challenges in this emerging area of research in the field of DNA damage signaling are the identification of transcripts that are posttranscriptionally regulated and the identification and functional characterization of proteins that mediate this posttranscriptional control. New technologies, such as, genome-wide RNAi screening and next generation sequencing of cell lines and primary tumor material will promote the identification and functional characterization of non-coding RNAs, RBP, and regulatory RNA sequences involved in the initiation, maintenance and termination of DDR signaling in human tissue.

# **REFERENCES**

stress-activated protein kinase p38 mediated by its substrate MAPKAP

# **ACKNOWLEDGMENTS**

We apologize to our colleagues for the omission of many important contributions to the field, and their references, due to space limitations. We thank the members of the Reinhardt laboratory for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft (RE2246/1-1, RE2246/2-1, SFB-829, and SFB-832 to H. Christian Reinhardt), the Deutsche Nierenstiftung (to Katja Höpker) and the Köln Fortune Program (to Grit S. Herter-Sprie and Katja Höpker).

T., Negrini, M., Bullrich, F., and Croce, C. M. (2002). Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. *Proc. Natl. Acad. Sci. U.S.A.* 99, 15524–15529.

a novel human protein phosphatase that is induced in response to ionizing radiation in a p53-dependent manner. *Proc. Natl. Acad. Sci. U.S.A.* 94, 6048–6053.

Zhan, Q., Fernandez-Salguero, P. M., Morgan, W. F., Deng, C. X., and Fornace, A. J. Jr. (1999). Genomic instability in Gadd45a-deficient mice. *Nat. Genet.* 23, 176–184.

translatability. *Biochem. Soc. Trans.* 30, 963–969.

BRF1 is regulated by mitogenactivated protein kinase-activated protein kinase 2. *RNA* 14, 950–959.

p38 in response to DNA damage. *EMBO J.* 26, 2005–2014.

Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1. *Mol. Cell. Biol.* 13, 7652–7665.

and Lee, W. H. (2000). Sequencespecific transcriptional corepressor function for BRCA1 through a novel zinc finger protein, ZBRK1. *Mol. Cell* 6, 757–768.

Zhou, B. B., and Elledge, S. J. (2000). The DNA damage response: putting checkpoints in perspective. *Nature* 408, 433–439.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 26 July 2012; accepted: 06 August 2012; published online: 2 August 2012. 5*

*Citation: Boucas J, Riabinska A, Jokic M, Herter-Sprie GS, Chen S, Höpker K and Reinhardt HC (2012) Posttranscriptional regulation of gene expression—adding another layer of complexity to the DNA damage response. Front. Gene. 3:159. doi: 10.3389/fgene. 2012.00159*

*This article was submitted to Frontiers in Cancer Genetics, a specialty of Frontiers in Genetics.*

*Copyright © 2012 Boucas, Riabinska, Jokic, Herter-Sprie, Chen, Höpker and Reinhardt. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

# Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling

#### *Elisa Ferrando-May1 \*, Martin Tomas 2, Philipp Blumhardt 2, Martin Stöckl 1, Matthias Fuchs 3 and Alfred Leitenstorfer 2*

*1 Department of Biology, Bioimaging Center and Center for Applied Photonics, University of Konstanz, Konstanz, Germany*

*2 Department of Physics, Chair for Ultrafast Phenomena and Photonics and Center for Applied Photonics, University of Konstanz, Konstanz, Germany*

*3 Soft Matter Theory Group, Department of Physics, University of Konstanz, Konstanz, Germany*

#### *Edited by:*

*Antonio Porro, Ecole Polytechnique Federale de Lausanne, Switzerland*

#### *Reviewed by:*

*Jurek W. Dobrucki, Jagiellonian University, Poland Christoffel Dinant, Danish Cancer Society, Denmark*

#### *\*Correspondence:*

*Elisa Ferrando-May, Department of Biology, Bioimaging Center and Center for Applied Photonics, University of Konstanz, Universitätstrasse 10, 78464 Konstanz, Germany e-mail<EMAIL_ADDRESS>
Our understanding of the mechanisms governing the response to DNA damage in higher eucaryotes crucially depends on our ability to dissect the temporal and spatial organization of the cellular machinery responsible for maintaining genomic integrity. To achieve this goal, we need experimental tools to inflict DNA lesions with high spatial precision at pre-defined locations, and to visualize the ensuing reactions with adequate temporal resolution. Near-infrared femtosecond laser pulses focused through high-aperture objective lenses of advanced scanning microscopes offer the advantage of inducing DNA damage in a 3D-confined volume of subnuclear dimensions. This high spatial resolution results from the highly non-linear nature of the excitation process. Here we review recent progress based on the increasing availability of widely tunable and user-friendly technology of ultrafast lasers in the near infrared. We present a critical evaluation of this approach for DNA microdamage as compared to the currently prevalent use of UV or VIS laser irradiation, the latter in combination with photosensitizers. Current and future applications in the field of DNA repair and DNA-damage dependent chromatin dynamics are outlined. Finally, we discuss the requirement for proper simulation and quantitative modeling. We focus in particular on approaches to measure the effect of DNA damage on the mobility of nuclear proteins and consider the pros and cons of frequently used analysis models for FRAP and photoactivation and their applicability to non-linear photoperturbation experiments.

#### **Keywords: non-linear optics, fluorescence, microirradiation, DNA strand break**

# **INTRODUCTION**

The DNA damage response plays a crucial role in oncogenesis (Bartek et al., 2007, 2012). Consequently, cancer biologists have a strong interest in experimental methods that address the spatiotemporal dynamics of the complex chain of cellular events triggered by DNA lesions. In recent years, microirradiation with focused laser beams has emerged as a useful tool to introduce DNA damage in live cells and to study the ensuing cellular responses via fluorescence imaging. In the first part of this Review, we will recapitulate the pros and cons of the most widespread microirradiation methods and point at new developments involving the use of pulsed near infrared lasers.

In eucaryotes, all reactions to DNA damage occur within chromatin. The local chromatin environment affects the susceptibility to genotoxic agents, influences the choice of DNA repair pathway and the efficiency of the repair reaction (Ziv et al., 2006; Fernandez-Capetillo and Murga, 2008; Jakob et al., 2011; Xu and Price, 2011). Despite successful restoration of the DNA sequence, long-lasting and heritable marks that persist at the level of chromatin structure may contribute to cellular transformation and cancer at a later stage (Lukas et al., 2011). To understand the biological consequences of genotoxic stress it is therefore essential to integrate the role of chromatin into our picture of the DNA damage response. Methods for visualizing chromatin rearrangements induced by DNA damage in living cells at high spatial and temporal resolution are of fundamental importance to achieve this goal. We will present imaging techniques suited to this purpose in the second part of this article focusing on combinations of laser microirradiation and fluorescence photoperturbation. Finally, to achieve an accurate quantitative description of how chromatin dynamics is affected by DNA lesions kinetic data need to be correctly interpreted. We will conclude this Review discussing current modeling approaches and their appropriateness for quantifying alterations of protein mobilities in microirradiated nuclei.

# **LASER MICROIRRADIATION METHODS FOR THE INDUCTION OF LOCALIZED DNA DAMAGE**

Among the available methods to expose cells to DNA damage like treatment with genotoxic chemicals or diffused irradiation, focused laser beams offer the fundamental advantage of free choice of the target area. With respect to irradiation with heavy ions or alpha particles which can also be employed for this purpose (Heiss et al., 2006), laser sources are easier to handle and to integrate into confocal microscopes for sample observation. Using high aperture objective lenses, the microbeam can be directed to any subcellular or subnuclear region of interest. UV lasers have been used for this purpose since the late 60's and were applied to the study of chromosome structure and of the repair of UV-photolesions in the nucleus (Berns et al., 1969; Berns, 1978; Cremer et al., 1981). In this wavelength region damage to DNA occurs mainly via direct linear absorption. Selective induction of UV-photoproducts, i.e., cyclobutane pyrimidine dimers (CPDs) was reported at 266 nm, a wavelength well-matching the absorption maximum of DNA bases at 260 nm (Voet et al., 1963). At longer wavelengths (340–500 nm), side reactions are observed due to the production of reactive oxygen species in the aqueous cellular environment which also contains endogenous sensitizers. The outcome are (unwanted) oxidative base modifications such as 8-oxo-guanine and DNA strand breaks (Kielbassa et al., 1997). The main microirradiation studies exploiting linear absorption in the UV/VIS region to induce localized DNA damage are summarized in **Table 1**.

In the presence of exogenous photosensitizers such as the base analogue bromodeoxyuridine (BrdU) or the intercalating dye Hoechst, exposure of cells to UVA illumination leads to single and double strand breaks (Limoli and Ward, 1993). Rougakou et al. exploited this finding to introduce DNA strand breaks in nuclei irradiated with a UVA laser at a wavelength of 390 nm (Rogakou et al., 1999). Since then, the combination of photosensitization and UVA has become very popular because it can be easily performed using a GaN-based UV laser diode at a wavelength of 405 nm which is frequently provided with commercial confocal microscopes. However, at this longer wavelength and only in presence of Hoechst 33342 a residual production of CPD has been reported (see **Table 2**). The combination of this sensitizer with irradiation at 405 nm seems to trigger an unusual and yet poorly characterized response (Dinant et al., 2007). In general, photosensitizers may have undesired effects on chromatin structure and cellular metabolism, not to mention the fact that they will mediate additional damage throughout the imaging procedure performed with visible light (Solarczyk et al., 2012).

In sum, methods based on linear absorption achieve optimal damage specificity with high efficiency only in the UVC region around 260 nm, leading to almost exclusive induction of UVphotoproducts. This technique requires specialized optics with high UV transmittance. In addition, Solarczyk et al. demonstrated selective induction of DNA strand breaks at 488 nm at power levels normally used in confocal imaging (Solarczyk et al., 2012). Independently of the wavelength used, linear absorption occurs throughout the entire irradiation path. Although the photon flux above and below the focal plane is much lower than in the focus of the objective lens, this may lead to substantial damage and, in flat cultured cells, cause harm to the nuclear membrane.

To achieve confinement of the photomanipulated volume in three dimensions, multiphoton (non-linear) excitation is the tool of choice. First described in theory by Maria Göppert-Mayer in 1931 and demonstrated experimentally by Kaiser and Garrett

**Table 1 | Laser microirradiation methods to induce DNA damage predominantly via linear absorption.**

*The induced lesions were characterized via immunofluorescence (IF), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and recruitment of DNA repair factors, as indicated. CPD, cyclobutane pyrimidine dimers; 6-4PP, 6-4 photoproduct; DSB, DNA strand break; 8-oxoG, 8-oxo-guanine;* γ*H2AX, phosphorylated histone H2AX.*

*The induced lesions were characterized via immunofluorescence (IF), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and recruitment of DNA repair factors, as indicated. CPD, cyclobutane pyrimidine dimer; DSB, DNA strand break; 8-oxoG, 8-oxo-guanine;* γ*H2AX, phosphorylated histone H2AX.*

in 1961, this process relies on the simultaneous absorption of multiple photons at very high photon densities, as they are present within the focus of the objective lens (Göppert-Mayer, 1931; Kaiser and Garrett, 1961). At high numerical apertures the affected volume is restricted to a few femtoliters. The high intensities (GW/cm2) required for these transitions are delivered via ultrashort pulses (ps to fs) thus limiting the average laser power to levels compatible with cell viability. For non-linear excitation, the sum of the energy of the incoming photons has to match the definite energy gap between two electronic states. For DNA bases, the maximum of linear absorption lies at 260 nm and excitation at this wavelength leads to the formation of UV-photoproducts, as mentioned above. Hence, the same type of lesion can be generated by irradiating cell nuclei with femtosecond pulses at a wavelength of 780 nm, corresponding to the simultaneous absorption of three near-infrared photons (Meldrum et al., 2003; Trautlein et al., 2008), or in the visible range (∼500 nm) via a two-photon process (Daddysman and Fecko, 2011). The relative probabilities of linear and non-linear absorption processes depend strongly on the intensities of the light field applied. Generation of UVphotoproducts via two-photon absorption at the relative moderate intensities used for imaging with continuous wave lasers in the visible range is very inefficient and can thus be neglected.

An additional mechanism that has been proposed to contribute to DNA damage by ultrashort NIR pulses is low-density plasma formation (Kong et al., 2009; Botchway et al., 2010). The high photon density and the extremely strong peak electric field of the laser pulse within the small focal volume may lead to photoionization events. In the presence of water and reactive biomolecules solvated electrons and radical intermediates are formed. This low-density plasma has been observed in pure water after exposure to femtosecond laser pulses over a broad range of conditions (Vogel et al., 2005). Its effects on DNA compare to that of ionizing radiation and include the generation of single and double strand breaks and chemical base modifications.

Thermal heating arising from laser irradiation is expected to play only a minor role as a source of DNA damage. According to calculations of Schönle and Hell irradiation of water for 1 s at a wavelength of 850 nm with 100 mW average power (*NA* = 1.2) causes a temperature rise of 0.2 K due to linear absorption (Schonle and Hell, 1998). Commonly used average powers range far below these conditions. In addition to linear absorption, the high peak power of the ultrashort laser pulses leads to collisions between free electrons and atoms and therefore to a local rise of the temperature. Although this heating can denature biomolecules, the chemical reactions triggered by free electrons strongly dominate the process of DNA damage (Vogel et al., 2005).

So far, most studies have observed a complex mixture of products after non-linear excitation of nuclear DNA (see **Table 3**). A direct comparison of the results is difficult because the exact irradiation parameters (wavelength, pulse duration, peak power and diameter of the focal spot) are not always fully specified. Furthermore, damage detection methods may vary. Not all types of lesions can be detected directly at the level of the DNA structure. So far, specific and robust antibodies are available for UV-photoproducts only. Methods for the direct detection of base modifications in cells *in situ* are not as well-established. DNA strand breaks have been monitored via TUNEL and Comet assays (Dinant et al., 2007; Harper et al., 2008), but more frequently via antibodies recognizing the phosphorylated form of the histone H2AX (Rogakou et al., 1999), although the latter may also occur subsequent to UV-damage (Halicka et al., 2005). The presence of a defined type of lesion is often inferred from the binding of a fluorescently labeled repair factor in live-cell microscopy experiments. Given the high degree of cross-talk between DNA

**Table 3 | Laser microirradiation methods to induce DNA damage via non-linear absorption.**

*The induced lesions were characterized via immunofluorescence (IF), and recruitment of DNA repair factors, as indicated. CPD, cyclobutane pyrimidine dimer; 6-4PP, 6-4 photoproduct; DSB, DNA strand break; 8-oxoG, 8-oxo-guanine;* γ*H2AX, phosphorylated histone H2AX.*

repair pathways the concomitant occurrence of multiple lesions cannot be excluded by this approach. More extensive reviews comparing the outcome of laser-induced DNA microdamaging methods, including NIR irradiation, are provided in (Dinant et al., 2007; Inagaki et al., 2009; Kong et al., 2009; Nagy and Soutoglou, 2009; Botchway et al., 2010).

Mode locked Ti:sapphire laser oscillators are frequently used to induce DNA damage with NIR irradiation because these systems are well-established as excitation sources for commercial two-photon confocal microscopes. For multiphoton imaging they are usually operated at a wavelength of ∼800 nm delivering pulses of a duration of 100–200 fs. From the studies mentioned above it has become clear that these pulses produce a mixture of lesions with strand breaks and photoproducts being detectable at similar levels. What is the perspective for improving the lesion specificity of pulsed NIR irradiation in order to fully exploit the advantages of three-dimensional confinement of the damage and low collateral damage? As we have recently shown, one way to achieve this goal is to increase the center wavelength of the pulse: at λ = 1050 nm the efficiency of formation of UV photoproducts drops drastically while that of DNA strand breaks remains unaltered. This observation let us propose an irradiation method that leads to the preferential induction of DNA strand breaks vs. UV photoproducts without the need of exogenous photosensitizers (Trautlein et al., 2009). As this work suggests, the efficiency of the different damage mechanisms may depend distinctly not only on the wavelength, but also on other pulse parameters. Along this line, Kong et al. have reported an impact of repetition rate and pulse duration on the threshold for phosphorylation of H2AX (Kong et al., 2009) while another study has shown a dependence of cell viability from pulse length at constant pulse energy (Konig et al., 1999).

In sum, non-linear excitation with ultrashort near infrared laser pulses enables to introduce highly localized DNA lesions on a sub-μm scale in the nuclei of living cells with minimum collateral photodamage. As future perspective we envision that these pulses may be tailored to become lesion-specific through a careful analysis of the influence of parameters such as laser central wavelength, pulse duration, repetition rate and peak irradiation on the type of damage.

# **IMAGING APPROACHES FOR VISUALIZING CHROMATIN DYNAMICS AT SITES OF LOCAL DNA DAMAGE**

The ability to target only a selected small area of the nucleus via laser microirradiation is one important prerequisite for addressing the role of nuclear organization in the chromatin response to DNA damage. A reliable indicator of chromatin remodeling is histone mobility as can be measured via fluorescence photoperturbation methods. Fluorescence recovery after photobleaching (FRAP), and, to a lesser extent, photoactivation, have yielded important insights into the dynamics and binding properties of histones in intact, native chromatin (Kimura, 2005; Beaudouin et al., 2006; Wiesmeijer et al., 2008; Martin and Cardoso, 2010; Raghuram et al., 2010; Stasevich et al., 2010). Mobility changes triggered by DNA damage can be investigated easily on a global scale, by treating cells with genotoxic agents such as chemicals or ionizing radiation prior to a FRAP or photoactivation experiment. These approaches disregard local differences in chromatin architecture, and they detect mobility changes only on the time-scale of several minutes to hours depending on the type and duration of the genotoxic treatment. To study the effect of local DNA damage on chromatin dynamics one can perform a FRAP experiment within a subnuclear region previously exposed to the DNA damaging laser. This strategy is straightforward if the fluorescently labeled protein of interest visibly binds to the damage and accrues along the laser trajectory. The photobleaching laser is then targeted to this area when the signal increase, i.e., the binding reaction in the irradiated region, has reached steady-state to avoid superimposition of the recruitment and the bleaching recovery kinetics. Using this approach, Mortusevicz et al have shown distinct mobilities of XRCC1 and PCNA at DNA damage induced at 405 nm (Mortusewicz and Leonhardt, 2007), while two other studies have investigated the biphasic dynamics of HP1-β and compared the mobilities of Cdt1, Cdt2 and PCNA at laserirradiated sites, respectively (Ayoub et al., 2008; Roukos et al., 2011). Further reports compared the recovery kinetics of PARP1 and a catalytic mutant at DNA damage (Mortusewicz et al., 2007). The behavior of members of the MRN complex and of 53BP1 at DNA strand breaks was also investigated (Lukas et al., 2004; Bekker-Jensen et al., 2005). For proteins that do not recruit to DNA damage including most chromatin components it is possible to measure FRAP curves within a pre-irradiated, damaged area via its known coordinates. However, unintentional bleaching due to the damaging laser—in particular if wavelengths in the UV/VIS are used—will interfere with the subsequent FRAP experiment. The latter can be performed only after the signal has recovered from the first irradiation step, thus severely limiting the temporal resolution of this approach.

Fluorescence photoactivation has also been combined with laser microirradiation to study local changes of chromatin structure due to DNA strand breaks (Kruhlak et al., 2006). In this study, cells expressing a photoactivatable fusion of histone H2B (PAGFP-H2B) were irradiated with a UV laser (364 nm) either in the absence or presence of the DNA intercalator Hoechst 33342. DNA strand breaks were produced only under the latter condition, as demonstrated by the appearance of phosphorylated H2A along the laser track. Under both conditions the DNA damaging laser simultaneously activated PAGFP-H2B. Significantly, a local broadening of the fluorescence signal could be observed only in the presence of the sensitizer indicating an expansion of chromatin in the direct proximity of DNA strand breaks.

These examples highlight one basic requirement for combining DNA damage via laser microirradiation with fluorescence photoperturbation for mobility measurements: the two irradiation procedures must not influence each other, i.e., they have to either induce DNA damage or photobleach/photoactivate, respectively. In the study by Kruhlak et al., this distinction was reached by adding a photosensitizer. Recently, we have presented an alternative strategy based on the use of femtosecond laser pulses of different wavelengths (Tomas et al., 2012). In a first step, DNA strand breaks are introduced via irradiation at λ = 1050 nm in a defined subnuclear volume, as described above. Subsequently the protein to be studied, which is expressed as a photoactivatable fusion is spot-activated within the lesioned zone via two-photon absorption at λ = 775 nm. We demonstrate that the two irradiation procedures can be performed in a single cell nucleus without mutual interference if the pulse parameters are chosen properly. Under these conditions it is possible to vary the time delay between the infliction of damage at 1050 nm and the photoactivation step at 775 nm arbitrarily. Hence, alterations in protein mobilities can be probed at different stages of the DNA damage response. The spatial precision of non-linear excitation also enables to vary the position of the photoactivation spot with respect to the damaged region. Using this method, we could show that DNA strand breaks lead to an increase in the mobility of histone H1.2 in the time range of 2 min after infliction of damage and that this change is spatially confined because distant chromatin is not affected (Tomas et al., 2012). The spatial and temporal flexibility of this assay will enable to visualize how the chromatin response to DNA damage emanates within the cell nucleus thus contributing to dissect the spatiotemporal complexity of this fundamental biological process.

# **MODELING CHANGES IN CHROMATIN DYNAMICS IN RESPONSE TO LOCAL DNA DAMAGE**

To extract information from the complex data sets on the kinetics generated by the combination of laser microdamage and fluorescence photoperturbation it is necessary to elaborate proper modeling approaches. Their aim is to describe quantitatively how structural damage affects the interaction of DNA with chromatin proteins.

Numerous studies have undertaken quantitative analysis of FRAP and photoactivation data of nuclear proteins under undisturbed conditions (i.e., in the absence of intentional DNA damage). In general, a reaction-diffusion model is assumed to be the best mathematical description of the dynamic behavior of these proteins. According to our current understanding, nuclear proteins diffuse stochastically in all three dimensions within the nucleoplasm until they collide with binding partners with whom they undergo transient interactions. This model assigns to each protein the following characteristic parameters: the binding and release constants *k*on and *k*off describing binding events, and the diffusion coefficient D accounting for phases of unrestrained movement. Solving the system of differential equations required for a full reaction-diffusion model is a complex task. Therefore, different simplifying assumptions have been made in different studies, depending on the experimental setting used to generate the mobility data (FRAP, or photoactivation, size of the bleached/photoactivated area etc.). These simplifications have enabled analytical solutions of the reaction-diffusion model, exactly describing the redistribution of the fluorescence signal to equilibrium as a function of time. The basic assumptions that are employed for this type of approach are the following:

1. The system's properties remain close to unperturbed, i.e., the number and the distribution of binding sites do not change during the observation period. In addition, binding sites are assumed to be homogenously distributed within the nucleus. The latter does not hold true for chromatin proteins because chromatin density varies between subnuclear regions (e.g., in hetero- vs. euchromatin.).

Otherwise, the solution of the reaction-diffusion model has to be found via numerical modeling where changes in fluorescence are approximated consecutively for each time point for a given set of parameters. For both the analytical and the numerical procedure, the obtained solutions are optimized by testing combinations of different parameters iteratively until the simulated behavior fits the experimental data.

The power of kinetic modeling consists in its ability to make quantitative predictions about the reaction of the biological system under study. For FRAP models, this ability has been questioned, because different approaches have yielded very different results for the same or similar proteins. Therefore, a crossvalidation strategy that compares different models as well as different experimental methods to generate the primary data is highly recommended (Mazza et al., 2008, 2012; Mueller et al., 2010).

For modeling changes of nuclear protein dynamics triggered by localized DNA damage yet other issues have to be considered. The main difference with respect to the undamaged situation is that at the time point of the mobility measurement the system itself may not be in an unperturbed state (see point 1 in the above list). Exposure to short laser pulses to induce DNA damage initiates a signal chain that may develop on different time scales for different types of lesions/binding sites. This amplification process is inherent to the biological response to DNA damage and leads to a fast and massive propagation of the damage signal, as best exemplified by the spreading of the phosphorylation of histone H2AX from the initial strand break to chromatin regions of the size of a few megabases (Rogakou et al., 1999; Iacovoni et al., 2010). Docking sites for proteins are thus generated in reactions secondary to proper DNA damage, as has been described in detail for many DNA repair factors, but also holds true for chromatin constituents (see e.g., Li et al., 2010; Lukas et al., 2011; Xu et al., 2012). To verify if the steady-state criterium is fulfilled it is thus very important to assess qualitatively on which time scale the mobility of the protein under investigation is influenced by DNA damage. The equilibrium approximation can be made only for observation times that are much shorter than the chromatin remodeling processes themselves.

Concerning the number and distribution of binding sites, a spatial discontinuity is implicit in the localized nature of the damage inflicted by microirradiation. Obviously, a different number of binding sites is present within the damaged region as compared to the rest of the nucleoplasm. The approximation of a uniform distribution can be made only if the area in which fluorescence redistribution is measured is much smaller than the DNA damaged one, and only for short observation times during which the protein does not move outside of this region. In our assay, this condition is fulfilled since the protein of interest is photoactivated in a small spot (diameter 1μm) within a significantly larger zone containing the damage (6μm2) (Tomas et al., 2012). These caveats concerning the temporal and spatial equilibrium criteria apply in general to all methods combining microirradiation with fluorescence photoperturbation.

An aspect that needs to be specifically considered when attempting to model data from non-linear photoactivation experiments concerns the shape of the photoactivated volume. Due to the three-dimensional confinement of non-linear excitation the signal intensity is not uniform along the optical axis. Thus, the conditions for a two-dimensional approximation are not fulfilled strictly (point 2). On the other hand, the small spot size minimizes the influence of nuclear geometry which may therefore be neglected without inducing significant errors (point 3).

An overview of recently proposed approaches for the analysis of FRAP and photoactivation experiments specifying their most important features is given in **Table 4**. The study of Mazza et al. addresses the point of the three-dimensionally confined nonlinear excitation volume and proposes an analytical solution of the diffusion equation for a two-photon photoactivated/bleached circular area of variable size. The method enables to determine diffusion coefficients over a wide range of values, but binding, i.e., a reaction dominated protein dynamics, is only taken into account as an immobile fraction. A solution for the reactiondiffusion model is not given (Mazza et al., 2008). Sprague et al. develop an analytical solution of the reaction-diffusion model which applies to a spatially localized cluster of binding sites and use it to simulate binding of the glucocorticoid receptor to an array of promoter sites in live cells. This approach has the advantage of taking into account a non-homogenous distribution of binding sites as it occurs in microirradiated cell nuclei. However, it is only applicable if the size of the cluster (the damaged area) is smaller than the bleached/photoactivated region. This model has been recently applied to describe the behavior of Nbs1 and Mdc1 at sites of DNA damage introduced by charged particle irradiation (Sprague et al., 2006; Tobias et al., 2013).

Numerical solutions of the reaction-diffusion model provide a more accurate approach to describe protein dynamics. In their study, Beaudouin et al. present a method that includes both the real geometry of the nucleus and an inhomogeneous distribution of binding sites and is independent of the shape of the bleached/photoactivated region. The differential equations are solved numerically using a finite difference method. The approach was validated for the photoactivation of five different chromatin proteins (Beaudouin et al., 2006). It is interesting to note that in the case of histone H1, these authors do not detect

subpopulations with higher and lower mobility, contrarily to what was reported previously in FRAP studies (Raghuram et al., 2010; Stasevich et al., 2010). Since the bleaching laser may induce phototoxic effects including DNA damage (Dobrucki et al., 2007; Solarczyk et al., 2012) and the mobility of the protein is increased in the presence of such lesions (Tomas et al., 2012), the partial fluorescence recovery observed in FRAP experiments can be due to a change of the protein's mobility within the bleached area leading to a local decrease in concentration rather than to the presence of

# **REFERENCES**

3D from diffraction-limited sources of PAGFP or sinks of EGFP created by multiphoton photoconversion. *J. Microsc.* 225, 49–71. doi: 10.1111/j.1365-2818.2007.01715.x

an immobile fraction. These effects have to be considered when choosing experimental conditions for photoperturbation studies.

None of the currently available models addresses the issue of the temporal non-equilibrium of the system that is characteristic for mobility measurements performed subsequently to DNA microdamage, either via FRAP or photoactivation. Addressing this issue is a promising avenue for future work and a prerequisite for the proper quantitative description of the response of nuclear proteins to DNA damage.

apoptosis. *Cell Cycle* 4, 339–345. doi: 10.4161/cc.4.2.1486

required for rapid response to DNA damage in living cells. *Nucleic Acids Res.* 35, 7665–7675. doi: 10.1093/nar/gkm933

light to study chromatin repair. *DNA Repair* 11, 996–1002. doi: 10.1016/j.dnarep.2012.09.008

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 15 April 2013; paper pending published: 03 May 2013; accepted: 25 June 2013; published online: 16 July 2013.*

*Citation: Ferrando-May E, Tomas M, Blumhardt P, Stöckl M, Fuchs M and Leitenstorfer A (2013) Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling. Front. Genet. 4:135. doi: 10.3389/fgene.2013.00135*

*This article was submitted to Frontiers in Cancer Genetics, a specialty of Frontiers in Genetics.*

*Copyright © 2013 Ferrando-May, Tomas, Blumhardt, Stöckl, Fuchs and Leitenstorfer. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*


**7.3 Weight variation**

The weight variation is usually calculated to guarantee that each film has the same quantity of a drug every time it is used. It's computed by weighing individual films and averaging the weights of a group of films. The individual weight of patches is deducted from the average weight of the film.


**Theorem 6.**

$$\mathcal{H}^{i}(\Delta^{2k+1}\mathbf{x}\_{1}) = \begin{cases} \mathbb{Z} \oplus \mathbb{Z} & i = 0 \\ 0 & i = 1 \\ 0 & i = 6k + 1 \end{cases}$$

,

**Proof.** The proof is similar to the proof of Theorem 5: Obtain all states, organized them in columns, assign a graded vector space to each state, form chain groups as a direct sum of all vector spaces along a column, and form the chain complex. Then, write the differential maps in terms of matrices using the ordered bases of the chain groups, and compute their kernels and images. Finally, find the Khovanov homology groups using the relation H*r*(*L*) = ker *dr* im*dr*+1 .


**Author details**

Oumayma Arbani1 , Mariette Ducatez2 , Mohammed El Houadfi1 and Siham Fellahi1 \*

1 Avian Pathology Unit, Department of Veterinary Pathology and Public Health, Agronomy and Veterinary Institute Hassan II, Rabat, Morocco

2 IHAP, Toulouse University, INRAE, ENVT, Toulouse, France

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 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, provided the original work is properly cited.

*Low Pathogenic Avian Influenza: A Permanent Threat to Poultry Farming in Africa DOI: http://dx.doi.org/10.5772/intechopen.104480*


Preface

**Chapter 9 155**

Effective Parameters on Increasing Efficiency of Microscale Heat Sinks

*by Yousef Alihosseini, Amir Rezazad Bari and Mehdi Mohammadi*

and Application of Liquid Cooling in Real Life

**II**

Both microfluidics and nanofluids are rapidly growing technologies that have emerged as multidisciplinary research fields. The impact and importance of these two technologies can be manifested from their tremendous potentials and diverse applications such as virus detection, cell manipulation and separation, 3D printing, anticancer drug screening, advanced thermal management, and energy harvesting and storage. This book covers eclectic areas of these emerging technologies starting from their fundamentals to development to applications and it is composed of nine chapters that are organized into two sections: one related to microfluidics and the other on nanofluids and cooling.

The first chapter presents an overview of advances in both microfluidic and nanofluid technologies. This chapter provides the overall information and discusses the progress of these technologies. In addition to reviewing their key features, their applications and challenges are also highlighted.

In the second chapter, we discuss the applications of microfluidic devices specifically developed for the investigation of time-resolved analysis of growth kinetics and the structural evolution of nanoparticles and nanofibers. Focus is placed on the design considerations required for spectrometry and SAXS analysis. This chapter also discusses the use of these devices for time-resolved research.

The third chapter demonstrates commonly used manufacturing technologies and the process chain for prototyping and mass production of microfluidic chips. It details various types of rapid prototyping technologies besides presenting some important research findings. It also provides good guidance from processing materials and method selection for chip production to end-users.

A review of microfluidic flow sensing approaches is presented in Chapter 4. It covers numerous aspects including currently available products, microfluidic flow sensing technologies, major factors impacting flow metrology, and prospective sensing approaches for future microfluidic flow sensing.

Chapter 5 provides a comprehensive review of the fabrication of circular and rectangular cross-section channels of microfluidic devices using micromilling process. Various process and machining parameters are also discussed in this chapter.

The application of micromixers for wastewater treatment and an assessment of their life cycle are presented in Chapter 6. Six different micromixer designs and an evaluation of the performance of each in the treatment of wastewater are discussed. Their performance in terms of environmental impact was assessed through the life cycle assessment (LCA) methodology.

In the second section of this book, three contributions to nanofluids and cooling are included. The first one (Chapter 7) is on the fundamentals and applications of solar thermal conversion of plasmonic nanofluids. It summarizes the preparation

methods of plasmonic nanofluids and reviews the solar absorption performance of these nanofluids based on the theoretical and experimental design.

The recent advancements of nanocomposite and nanofluids toward sustainable carbon capture, utilization, and storage are presented in Chapter 8. This chapter focuses primarily on nanomaterial applications for both fossil and renewable energies.

The last chapter deals with the effective parameters for increasing the efficiency of heat sinks and the application of liquid cooling in real life. Recent advances in developing an efficient heat sink including different parameters like geometry and flow parameters are reviewed. It also highlights the current gap between academic research and industry.

The book is expected to be a source of information for different communities including students, researchers, manufacturers, academicians, and professionals of these fields.

I would like to thank all the authors for their high-quality contributions and the publishing team for their cooperation and support.

I dedicate this book to my parents and siblings for their sacrifices and support. I owe my success to them.

> **S. M. Sohel Murshed** Instituto Superior Técnico, University of Lisbon, Lisbon, Portugal

**Chapter 1**

Technologies

*S.M. Sohel Murshed*

**1. Introduction**

devices and systems.

Introductory Chapter: An

Overview of Advances in

Microfluidics and Nanofluids

Both microfluidic and nanofluids are rapidly growing and hugely potential technologies which emerged as multidisciplinary research fields. While microfluidic technology began with the development of the first lab-on-a chip in 1979 microfluidic research had its first step forward only when Manz introduced the idea of μ-TAS (Micro total analysis systems) in 1990 [1]. Other main timelines of the development of microfluidic technology include introduction of microfluidics in cell biology and biochemistry in 1994, employing PDMS (polydimethylsiloxane) in microchips production in 1998, introduction of digital microfluidics in 2000, investigations on microfluidics cell culture systems in 2004, development of organon-chip technology between 2005 and 2010, emergence of paper-based microfluidics in 2007, application of 3D printing in microfluidic technology in 2010 and the latest development of microfluidics for theranostics between 2012 and 2015 [2, 3]. Microfluidic technology deals with small amounts (i.e., microliter or nanoliter) of fluids (liquids and gases) in micron or sub-micron size devices/systems/geometries. Microfluidics is a very popular research field which can be evidenced from the rapid growth of numbers of publications on this topic (**Figure 1**). Year-wise publication data on microfluidics for the past two decades (data obtained by searching topic: "microfluidic" in Web of Sciences on 10th May 2021) are presented in **Figure 1**. The impact and importance of microfluidic technology can be manifested from its diverse real-world applications ranging from virus detection and bioanalytical, cell manipulation and separation, 3D printing, paper microfluidics to anticancer drug screening as highlighted in the following section. The advantages of microfluidics include very small quantity (e.g., microliter) of sample or reagent usage, contamination risk reduction, low cost (e.g., for analysis/diagnosis), automation, enhanced sensitivity, accuracy and reliability. Although different materials have been used, PDMS is currently the most widely used material for the fabrication of microfluidic

On the other hand, nanofluids, which is a new class of heat transfer fluids coined in early 1990's [4, 5] have also attracted tremendous attention from the researchers due to their enhanced thermophysical properties, potential benefits, and numerous applications [6–9]. **Figure 1** also shows publication records on nanofluids during the last two decades and the number of publications on nanofluids (search by the topic: Nanofluids) increases exponentialy. It is to note that nanofluid is a much smaller


**6. Effects of humic+fulvic acid on symbiotic nitrogen fixation**

Organic matter is one of the most important issues of agriculture and it contains three very important components: humic acids, fulvic acids and humin. Plants and microorganisms in soil benefit from applications of humic acid in several ways. Humic acid stimulate root growth, increase carbohydrate production, have a hormone-like affect within the plant, and increase soil microorganisms (Lawn Care Academy, 2010). The incorporation of humic acid fractions in media designed for the enumeration of soil micro-organisms belonging to specific physiological groups was found to result for some groups in appreciably higher counts. It is suggested that by influencing the enzyme systems of certain micro-organisms, humic compounds may affect the range of substrates which they can utilize. The effect could have implications on the activity of organisms in environments in which humic substances are normally present, such as soils and natural waters (Visser, 1984).

Coskan et al. (2010) carried out a pot experiment to represent effects of humic + fulvic acid (HFA) applications on biological nitrogen fixation under soybean vegetation. Humic + fulvic acid application realized by either incorporate to soil or admixing by irrigation water. Seeds are inoculated by appropriate Bradyrhizobium japonicum strain, before sowing. In flowering stage, roots are removed from soil and the number of nodule determined (Fig 5).

Fig. 5. Effects of humic + fulvic acid application on nodule number of soybean (-Inoc: noninoculated)

Due to the fields where soil was taken is not previously introduced with the rhizobia that match with soybean; no nodule occurrence was observed in the pot which non-inoculated control variant. A few nodules observed in non-HFA applied pots however HFA application increased nodule occurrence considerably. Both "incorporation to soil" and "admixing by irrigation" applications were effective on formed nodule number; however because of the dilution effect in incorporation to soil application, admixing by irrigation application seems to be more effective than incorporation to soil. (dilution effect). Increasing doses of HFA increased the number of nodule, thus findings expressing the considerable positive effects of HFA on biological nitrogen fixation.


**6. Conclusion**

We want to finish with the conclsuive statement made by ESGE [35]; *"In conclusion, urgent ERCP with sphincterotomy did not reduce the composite endpoint of major complications or mortality in patients with predicted severe gallstone pancreatitis, compared with conservative treatment. These findings support a conservative strategy with an ERCP indicated only in patients with cholangitis or persistent cholestasis. With this conservative strategy, about two-thirds of patients did not need to undergo ERCP".* In the presence of cholangitis, ERCP as E-ERCP should be done only after hemodynamic stabilisation and relieved organ hipoperfusion with aggressive fluid replacement and antibiotic treatment within the first 24 hours, Elective ERCP to prevent ABP attacks is suggested only for patients unsuitable for an choecystectomy procedure.

*Endoscopic Retrograde Cholangiopancreatography in Acute Biliary Pancreatitis DOI: http://dx.doi.org/10.5772/intechopen.96545*


**2.2 Early irrigation system in Punjab**

The Punjab province is located downstream to the Kashmir, and early developments in irrigation system of this region occurred during the thirteenth to sixteenth century. The first canal in Punjab was constructed about five centuries ago by the Mughal Emperors. In the beginning, only inundation channels were designed to deliver river water to the cropland during the high flow season. Because of some technical problems, viz., unpredicted high flow season, siltation, and breaching, these inundation canals could not deliver the water effectively to the cropland. Later on, some primitive types of headworks on rivers were constructed to get more control over the water. These headworks either did not extend across the entire stream or allowed the floods to pass over their crests. Mughal Emperor Jahangir constructed an 80 km perennial canal on Ravi River to deliver the water to the Gardens of Sheikhupura [21]. In 1643, Shah Nahr developed to provide water to the Shalamar gardens and other irrigational lands around Lahore city [22]. Later on, *Hajiwah* and *Tiwana* Canals were developed in Punjab with support of British rulers to irrigate croplands in the *Mailsi* and *Sargodha* regions. Furthermore, 11 canals were built on the Indus River to irrigate the agricultural lands of D.G. Khan Region during 1875 [23].


**2.2.2 Materials and methods**

The determination of the rate of potassium chloride clearance was done by the same methods as in study 1. A 1-mol/L solution of potassium chloride was mixed with sufficient agarose (Electrophoresis Purity Reagent; BioRad Laboratories, Richmond, CA) to give a 1.0% solution which was heated until the agarose dissolved. The acrylic chambers(Fig. 3) to hold the gel were rectangular (16 mm in length, 8 mm wide, and 1.5 mm thick) with a cylindrical central depression (6 mm diameter and 1.5 mm depth). The potassium concentration in agarose was analyzed by absorption spectrophotometry.

The subjects were 4 boys and 8 girls, 5 years of age, who were all in good health and with complete primary dentitions. Six subjects had primary spaces (located mesial to the maxillary and distal to the mandibular canines) and developmental spaces (present between the remaining teeth) (Fig. 7), and the other 6 subjects had no spaces in their arches (Fig. 8). The mean values of right and left primary spaces and total developmental spaces for 6 subjects who have spacing arch were 1.1 ± 0.4 mm, 1.2 ± 0.4, and 4.2 ± 1.9 mm for the upper dentition and 0.8 ± 0.3 mm, 0.7 ± 0.3, and 3.8 ± 1.4 mm for the lower dentition, respectively.

Fig. 7. Spacing arch at 5 years old.

56 Macro to Nano Spectroscopy

Nursing bottle caries (Fig.6) is a specific form of rampant decay on the buccal surface of the upper anterior primary teeth. Some etiological factors, such as the types of microorganisms, tooth structure, and diet, have been reported, but there is little information about the

Very little research has been carried out on the salivary flow rate or salivary clearance in children. Although the average thickness of the salivary film covering teeth and oral mucosa in children is essentially identical with values reported for adults, marked differences were found between children and adults for such parameters as unstimulated and stimulated whole-salivary flow rates, the volume of saliva in the mouth before and after swallowing,

The aim of this study was to evaluate the rates of salivary clearance at different locations in the mouths of children and the effect of the spaces in the primary dentitions to determine whether prolonged clearance would occur in sites particularly susceptible to nursing bottle caries.

The determination of the rate of potassium chloride clearance was done by the same methods as in study 1. A 1-mol/L solution of potassium chloride was mixed with sufficient agarose (Electrophoresis Purity Reagent; BioRad Laboratories, Richmond, CA) to give a 1.0% solution which was heated until the agarose dissolved. The acrylic chambers(Fig. 3) to hold the gel were rectangular (16 mm in length, 8 mm wide, and 1.5 mm thick) with a cylindrical central depression (6 mm diameter and 1.5 mm depth). The potassium

concentration in agarose was analyzed by absorption spectrophotometry.

**2.2 Salivary clearance in children with complete primary dentitions**

**2.2.1 Aim**

influence of the salivary flow rate.

Fig. 6. Nursing bottle caries

and the surface area of the mouth.

**2.2.2 Materials and methods**

Fig. 8. No spacing arch at 5 years old.

Seven different sites in the mouth were chosen for measurements. These were the lower anterior lingual (LALi) and buccal (LAB), lower posterior buccal (LPB) and lingual (LPLi), upper posterior lingual (UPLi) and buccal (UPB), and upper anterior buccal (UAB). The flow rate of unstimulated whole saliva was also measured on each occasion for 5 min by being allowed to drip off the lower lip into a weighed container.

Estimation of the Velocity of the Salivary Film at the Different Regions in the Mouth

**2.2.4 Discussion**

in different regions.

**2.3.1 Aim**

British children aged 30-36 months.

– Measurement of Potassium Chloride in the Agar Using Atomic Absorption Spectrophotometry 59

The present study showed that the rate of clearance of substances from agarose gels into saliva varies markedly in different regions of the primary dentition. The location closest to the submandibular and sublingual ducts (LALi) showed the lowest half-time, whereas the UAB site had a clearance half-time 6.5 times longer than that for clearance into a large volume in vitro. As the opening of the parotid duct is situated on the rearward of the upper second primary molar in the children's mouth, there was a relatively long half-time in the UPB site. Since the mean salivary film thickness in 5-year-old children has been estimated to have almost the same value (0.06-0.09 mm) (Watanabe and Dawes, 1990) as in adults (Collins and Dawes, 1987), these results suggest that the velocity of the salivary film varies

The relative order of the half-times at the different sites in the no-spacing arches was identical with that found in a study on adult subjects when saliva flow was unstimulated. Although in the spacing arch, the LAB site had a shorter clearance half-time than the UPB site. These results may be due to the fact that in the spacing arches, the tongue pushes out a portion of saliva from the lingual to the buccal side during swallowing, and this is in accordance with clinical findings that these sites are not susceptible to caries. The ideal arch in the primary dentition has spacing between the teeth (Pinkham et al, 1988), but Foster and Hamilton (1969) reported that only 33% had spacing between all the incisors in the upper and lower arches and that only 12% had spacing between all teeth in both arches in 100

Although it is known that nursing bottle caries depends on the feeding pattern in infancy, the present results suggest that the upper anterior buccal site in a no-spacing arch will be the most cariogenic site in a child's mouth because it has the lowest rate of salivary clearance.

The rate of oral clearance was shown to vary markedly at different locations in the mouth. Oral clearance is slower for teeth in the maxilla than for those in the mandible and slower for the buccal surfaces of the teeth than for the lingual. Oral clearance on the labial surface of the upper anterior region is the slowest, while that for the lingual surface of the lower anterior region is the fastest. The lingual surface of the lower anterior region is near the openings of the ducts of the submandibular and sublingual glands, which probably accounts for the fastest rate of oral clearance being there. The effect of unstimulated parotid saliva on clearance around the maxillary first molar is not very striking, perhaps because the volume ratio of parotid saliva to total saliva is only about 15% at rest for each side. However, with stimulation, the proportion of parotid saliva increases, increasing clearance over the maxillary first molar, which is closest to the parotid duct. Few studies have examined positional relationships between the parotid duct orifice and the maxillary molars or individual differences in this positional relationship (Suzuki et al. 2009). The present study sought to ascertain the location of the parotid duct orifice in relation to the maxillary molars and whether oral clearance at locations 1 cm mesial and distal to the duct opening

**2.3 Influence of the location of the parotid duct orifice on oral clearance**

would be as rapid as that directly opposite the opening of the duct.

The rate of potassium chloride clearance from the gels into a large volume (1 liter) of 100 ppm NaCl fluid at 37℃ stirred by a magnetic stirrer was determined for the estimation of the half-time.

#### **2.2.3 Result**

The mean half-times for in vitro clearance into the large volume of stirred 100 ppm NaCl at 37°C was 3.9 ± 0.5 min.

The half-times in the mouth varied with location as shown in Table 1. The half-times of all sites for the spacing arch and of the LALi site for the no-spacing arch were reduced (p<0.05) as compared with the values when the flow rate was unstimulated.

Statistical analyses werw carried out between UAB site and the other sites in each group: \*p<0.05, \*\*p<0.01, \*\*\*p<0.001.

Mean unstimulated salivary flow rates were 0.47±0.2 ml/min.

Table 1. Half-times (mean ± SD) and salivary flow rates when salivary flow was unstimulated

When the saliva flow rate was stimulated (Table 2), the shortest halftimes occurred in the LALi site and the longest in the UAB site. The clearance from the LAB site in the spacing arch showed almost the same value as those from the LALi sites in both groups.

Halftimes(mean±SD)and salivary flow rates when salivary flow was stimulated

Halftime in large volume at 37°C=3.9 ± 0.5min.Statistical analyses were carried out detween UAB site and the other sites in each group :\*p<0.05 \*\*p<0.01

Table 2. Halftimes (mean ± SD) and salivary flow rates when salivary flow was stimulated


**3.3. Perspective for future of CTC technology: combinations of several methods**

formed according to the manufacturer's instructions. Briefly, the RAM M280 Dynabeads were incubated with either BerEP4, 9189, or MOC31 mAbs at a concentration of 1/u.g/107 beads for 30 min at 4°C under gentle rotation, followed by three magnet washes in PBS/0.1% HSA and then stored at 4°C. Before use, the Dynabeads were washed once with separation medium.

separation medium to a concentration of 2 ×107 MNC/ml (0.5 ml volume) and incubated with RAM IgGl M280 Dynabeads coated with either BerEP4 317G5, or MOC31 mAb. The bead

bead/cell suspension was incubated under gentle rotation for 30 min at 4°C. The sample was then diluted to 3 ml and placed against a magnet for 7 min to recover the rosetted cells, followed by two additional washes as follows. The supernatant was removed, and the rosetted cells were resuspended in 3 ml separation medium, followed by treatment with the magnet (7 min). To facilitate ICC TC detection, the positive LMS product was finally resuspended to contain 5–7 × 106 beads/ml, and 0.5 ml aliquots were centrifuged onto each cytospin slide for further

separation medium to a concentration of 2 × 107 MNC/ml (0.5 ml volume) and incubated with anti‐CD45‐conjugated M450 Dynabeads at a bead/cell ratio of 2.5:1, 5:1, or 10:1. The bead/cell suspension was incubated under gentle rotation for 45 min. The solution was then diluted to about 30 ml, and the magnet was applied for 5 min, with initial rotation of the tubes onto the magnet to reduce trapping of tumor cells. The supernatant was collected and centrifuged at 450*g* for 10 min, counted, and resuspended in 10% FBS in PBS to 1 × 106 cells/ml. Then, cytospins containing 5 × 105 cells were prepared. All the slides were air‐dried overnight and stained by

In novel era of huge advances of microfluidic devices as mentioned in the section of "Label‐ free isolation strategy" [45, 46, 56, 60, 173–186]. In fact, the vast majority of microfluidic devices were designed based on EpCAM‐ or CK‐identifying mechanism, which is positive selection method. The CTC‐Chip [25, 243], and the herringbone chip [244, 245] have been proven effective to isolate CTCs with both high CTC purity (50–62%) [25, 245] and high recovery rate (90–95%) [244, 245]. There are several microfluidic devices designed to use positive selection strategy for proof‐of‐concept purpose [34, 57, 59, 124, 184, 210, 246–269] and for specific cancer in clinical trials, (e.g., breast [270, 271], pancreas [272, 273], ovarian [274], prostate [275], esophageal cancer [270], gastric [271], colorectal cancer [276], cancer of unknown primary [277]) and for mutational analysis [278]. Moreover, combined preparation using positive, negative, or label‐free selection methods with microfluidic devices for better performance is

peripheral blood mononuclear cells (PBMNC) were resuspended in cold

peripheral blood mononuclear cells (PBMNC) were resuspended in cold

b/ml, as described in individual experiments. The

*3.1.2. Positive immunomagnetic separation technique*

*3.1.3. Negative immunomagnetic separation technique*

**3.2. Microfluidic‐based methods for the high purity CTC isolation**

concentration varied from 2.5 to 40 × 106

immunocytochemical analysis.

A total of 1 × 107

158 Tumor Metastasis

A total of 1 × 107

immunocytochemistry.

also feasible and have been reported [93].

Our perspectives for future CTC isolation is mainly combined methodologies instead of conventional ones based on a single isolation strategy.

Yamamoto et al. [49] displayed a combination of size‐based filtration plus a magnetic column method for CTC isolation. The combined use of the column and filter decreased the required time for the spiked cancer cell capture, and the recovery rate of the spiked cancer cells from blood was significantly higher using the combination process (80.7%) than that using the filter alone (64.7%). Moreover, the recovered CTCs are more abundant by the combination process. Another combination was ISET and CellSearch™ systems [76, 93] and the combination had better performance in CTC detection in non‐small cell lung cancer (NCSCL) patients than ISET or CellSearch™ alone [93]. Furthermore, density separation plus flow cytometry or cell sorting systems have been postulated to be potential tools of CTC isolation and identification consid‐ ering their high sensitivity and purity since 1998 [27]. This method could be seen as a combi‐ nation of negative selection strategy and a positive confirmation with surface markers, such as EpCAM or cytokeratins. Later in 2011, leukapheresis plus fluorescence‐activated cell sorting (FACS) elutriation were also reported to be effective for large volume blood process for CTC isolation with molecular analysis [211]. These studies illustrated the possibility and better efficacy the combination can achieve, therefore, in our opinion, to find a suitable combination of CTC isolation protocols considering the balance of efficiency, time, sample and costs is very important in the future CTC field.

Intriguingly, several liquid biopsies, as aforementioned, could be combined to be tested in a single sample and at the same time. To realize the goals and minimize the blood sample required, Chudziak et al. [248] reported a novel device, Parsortix system, could negatively select CTCs and perform cfDNA analysis simultaneously. The system recovered more CTCs than CellSearch™ system in the comparison.


**15. Conclusions**

Topical 0.2% nifedipine with lidocaine appears today as the most efficient mode of treatment for anal fissures in children, with a significant healing rate and no side effects. It is safe and effective, prevents the evolution of acute anal fissures in children to chronicity, avoids surgical procedures in the great majority of cases, avoids complications and does not require hospitalization.

Nifedipine Gel with Lidocaine in the Treatment

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**7. Measuring saline cell permittivity with mis**

122 Advanced Aspects of Spectroscopy

perform these measurements with electrodes.

small values, reaching values used to 1 kΩ .

with various degrees of salinity.

**Figure 20.** Measurement of Z12 of four saline cells

The values obtained for both measurement and calculation as shown in Table 2 allows us to make the cell preparation of constant section for measuring conductivity. For this preparation had to consider the mechanical error sources such as physical dimensions of the saline cell, the effects of temperature on the cell, and error handling and positioning of it. It also ponder the discrepancy between the measurements of a real physical element such as a capacitor in parallel with a resistor, and the resistivity and permittivity expected of an electrolytic cell, it was therefore necessary to have a truer reference of known salt solution to evaluate deviation of our measurements and bring the measuring physical model to a virtual model and simulate their behavior in this way to "induce" their performance, come to

In Figure 19 shows a sample obtained from a series of measurements made under the scheme of Figure 18, changing the values of the capacitances in parallel with resistors first

**Figure 19.** Comparisons between reference measurements with physical components and estimated values

Although performing multiple measurements and their subsequent acquisition of information due to the speed and flexibility of use of equipment to automate this process, include only the most representative of the behavior of a saline cell, as seen in Figure 20,

> The resistivity of an aqueous sodium chloride dissolution (NaCl) [21] is obtained from considering the current density (J), determined this by the ions types (Na + and Cl – are the most abundant ions) of the solution, this is directly proportional to the factor "α" of dissociation of molecules, approximately equal to 1 if the electrolyte is strong, with a concentration "c "gram-equivalent, and a mobility" μ "of the ions, also the electric field" E "as shown in the following relationship: J = F α c μ E.

> A solution consisting of 9 grams of sodium chloride dissolved in one liter of water in medicine2 is called Normal Saline, since the concentration of 9 grams per liter divided by 58 grams per mole (approximate molecular weight of sodium chloride ) provides 0.154 moles per liter, that is, contains 154 mEq / L of Na + and Cl -. The fact contain more solute per liter,

2 In chemistry, the normal concentration of sodium chloride is 0.5 mol of NaCl assuming complete dissociation. Physiological dissociation is approximately 1.7 ions per mole, so that a normal NaCl is 1/1.7 = 0.588 molar. This is approximately 4 times more concentrated than the medical term "normal saline" of 0.154 mol

makes this solution with a slightly higher osmolarity3 than blood, on an average day the natremia4 can range between 130-150 mEq / L (normonatremia) . However, the osmolarity of normal saline is very close to the osmolarity of the NaCl in the blood[22].

From Figure 22 and considering the dimensions of our saline cells, presents three values of resistivity, ρ = 2.65Ωm, ρ = 0.55Ωm and ρ = 1.3Ωm, respectively.

**Figure 22.** Measured frequency behavior of three electrolytic cells

**Figure 23.** Saline solutions response at resonance frequency of the coil system

3 Measuring solute concentration

4 Concentration of sodium in the blood

Figure 23 presents a summary of values acquired with the network analyzer, Figure 24 represents the values obtained with the HP4192A impedance analyzer.

**Figure 24.** Frequency responses of Saline Solutions.

#### **8. Biological suspensions**

124 Advanced Aspects of Spectroscopy

makes this solution with a slightly higher osmolarity3 than blood, on an average day the natremia4 can range between 130-150 mEq / L (normonatremia) . However, the osmolarity of

From Figure 22 and considering the dimensions of our saline cells, presents three values of

normal saline is very close to the osmolarity of the NaCl in the blood[22].

resistivity, ρ = 2.65Ωm, ρ = 0.55Ωm and ρ = 1.3Ωm, respectively.

**Figure 22.** Measured frequency behavior of three electrolytic cells

**Figure 23.** Saline solutions response at resonance frequency of the coil system

3 Measuring solute concentration 4 Concentration of sodium in the blood Typically, in vitro measures, is considered a sample of uniform section (A) and length (L), in the majority of cases it is more practical to consider the volume (V) in aqueous solutions. In this case the ratio between the conductivity and the permittivity of the sample with the resistance and reactance are:

$$\mathcal{L} = \frac{\varepsilon \varepsilon\_0 \mathcal{V}}{\mathcal{L}^2} \text{ [ $F$ ]} \tag{22}$$

$$R = \frac{L^2}{\sigma V} \text{ [ $\Omega$ ]} \tag{23}$$

$$Z = \frac{L^2}{\left(\sigma + \int \omega \,\varepsilon \,\varepsilon\_0\right) V} \tag{24}$$

Following experiments with saline solutions, was considered to have the information needed to proceed to carry out experiments with biological solutions, hence the first use of coil system as a reference and according to the above expressions, and also with a 500 mL volume, and a tank length of 85 mm was obtained with a suspension of high concentration of biomass (80 g / l of yeast), a 8.305 dB attenuation, comparing this with a 13.71 dB attenuation of coil system, this means a representation of a 65% increase in impedance in the suspension.

 It should be mentioned that the embodiment of the vast majority of measurements for suspension was made with the HP4192A impedance analyzer, considering the structure and existing coil system, the utilization ratio of the impedance analyzer is because the probes used to interconnect arrangement coils are constructed on purpose, greatly decreasing the amount of error attributable to the length of the coaxial cables.

**Figure 25.** Impedance Representation of Biological Suspension, comparatively to the coil system.

After 35 minutes, the yeast was deposited at the bottom of the container; we proceeded to extract 350ml of water and recovered the same amount of water (350ml), but now with a salinity 8.78mS/cm.

The interesting thing in this experiment as shown in Figure 25, having increased conductivity, as a result of water replaced without electrolytes, with water with electrolytes (8.78mS/cm) decreased by 6% the impedance of the suspension by the lower proportion of yeast and major salinity, Zlev = 384.37Ω, Zlev + NaCl = 361.87Ω, ΔΩ = 22.5


2. Golden ratio

Golden Ratio has many perspectives: geometrically, mathematically, and arithmetically. A full understanding of all perspectives as well as the origin of the term is essential, in order to grasp the magnitude from the microscale to the macroscale. In this section, Golden Ratio will be explained term and perspective wise.

The origin of the term Golden Ratio was termed by Phidias. Phidias is a Greek painter, sculptor, and architect. Phidias sculpted the famous Zeus at temple of Olympia, which was considered one of the Seven Wonders of the World. The golden ratio was termed mean of Phidias [6, 8, 15]. There are many terms that reflect the term Golden Ratio: golden mean [2, 7, 10] and extreme/ mean ratio, (Euclid) medial section [3], divine proportion, divine section (Latin: sectio divina), golden proportion, golden cut, [11] golden number [6, 15, 8]. In current history, Golden Ratio was termed as Golden Section (goldene Schnitt) by a German mathematician named Martin Ohm who lived from 1792 till 1872. Mark Barr coined the term (phi) to reflect the idea of Golden ratio, in commemoration of Phidias, because many historians [7, 13] claim that Phidias used the Golden Ratio in his sculptures.

Simply, Golden Ratio is a proportion of two unequal segments: the proportion of the long segment to the short segment is equal to the total of the two segments to the long segment, both equal 1:1.61803398874989. In Figure 1, the longer segment a and the short segment b, whereby the proportion of a:b is equal to the total of a and b to a. The number 1.61803398874989 is the value of phi. In this context, the ensuing question is how to derive b from a while keeping the Golden Proportion, phi?

To derive the b segment from the a segment, the following must be conducted: First, draw a square with length a, as articulated in Figure 2, and construct a unit square. Then, at midpoint on the side of the square, draw a line to the opposite side. Next, connect the midpoint to an opposite corner. When using that line as the radius to draw an arc, accordingly, the long dimension of the rectangle is defined.

The simpler picture is reflected in Figure 3 where the "a" and the "b" are shown and the both hold the value of phi, the Golden proportion.

Concluding the section where the origin of the term Golden Ratio was not only clarified but thorough explanation of the geometrical aspect of the ratio was also put forward, in order to comprehend the magnitude of this proportion.

Figure 1. The Golden Ratio represented in line segments [3, 4].

Figure 2. Construction of a golden rectangle.

Vincicirca both based on the golden proportion notion. First, the research gives an introduction about golden proportion known also as golden section. Then, in the second section, the research explains the three dimensions of golden proportion: mathematically, geometrically, and arithmetically. The third section of the research presents the work of FIBONACCI and his series in the golden proportion. Followed by a discussion of the geometry of Golden ratio and the essential proportions in Vitruvian Man, in the fourth section, where 15 essential proportions are explained based on the Vitruvian Man. In fifth section, BAUENTWURFSLEHRE by ERNST NEUFERT [20] is further explained with circle geometry and 29 golden proportions are reflected in the work. In the sixth section, the face and hand proportions in relation to the golden proportion are further explained. Based on the findings of the previous sections, the seventh section suggests two algorithms to be used when building a human body model. Thereby, presenting more than 35 proportions and measurements used to model human body model along with a simple algorithm that calculates exact measurements. As such, the research facilitates and explains for the human modeler the process of human modeling in multimedia arena. Hence, this research will be a spring board for researchers, practitioners in human

Dubbed by the Greeks, "Golden Ratio" is a mathematical relation and proportion, where the length to width of a rectangle proportion is 1:1.61803398874989484820, such proportion is most suited for human eye and is used by architects, artists, sculptures in their work. While the Golden Ration does not embody every structure or pattern in the universe, yet, historically, this ratio emerged in Great Pyramid of Giza/Khufu/Cheops (2560 BC) [21], and in Vitruvian Man by Leonardo da Vinci and Neufert. More notably, in today's multimedia, Golden Ratio is used in many applications: plastic surgery simulation software, animation software, art, architecture, sculpture, anatomy. A close exploration of such divine proportion and the related applications, offers opportunities to connect an understanding the conceptions of ratio and proportion to the geometry, proportions, and ratio related to the Vitruvian Man and its relation to Bauentwurfslehre by Ernst Neufert (1936) [20]. Throughout the next sections, a detailed explanation will be given to golden ratio: geometrically, mathematically, and arithmetically.

Golden Ratio has many perspectives: geometrically, mathematically, and arithmetically. A full understanding of all perspectives as well as the origin of the term is essential, in order to grasp the magnitude from the microscale to the macroscale. In this section, Golden Ratio will be

The origin of the term Golden Ratio was termed by Phidias. Phidias is a Greek painter, sculptor, and architect. Phidias sculpted the famous Zeus at temple of Olympia, which was considered one of the Seven Wonders of the World. The golden ratio was termed mean of Phidias [6, 8, 15]. There are many terms that reflect the term Golden Ratio: golden mean [2, 7, 10] and extreme/ mean ratio, (Euclid) medial section [3], divine proportion, divine section (Latin: sectio divina), golden proportion, golden cut, [11] golden number [6, 15, 8]. In current history, Golden Ratio was termed as

modeling in multimedia field.

114 Machine Learning and Biometrics

2. Golden ratio

explained term and perspective wise.

Figure 3. A golden rectangle with longer side a and shorter segment b.