**3.2 Laser-induced generation of micro-bubbles in a glass capillary**

Liquid flows are more clearly observed in the microscopic measurements of the laserinduced hydrodynamic effects in the vicinity of the fiber tip surface of the laser fiber that is placed in the glass capillary filled with water (model of the laser channel).

As it follows from Fig. 8, the attached vapor-gas bubbles at a laser power of 1–2 W emerge at the tip surface and the convective motion is observed in the liquid. A qualitatively different scenario corresponds to a power of 3 W: the microscopic bubbles ejected from the fiber tip move along arc shaped trajectories and entrain liquid flows (Fig. 8a). The intensity of the resulting vortices rapidly increases with increasing radiation power (Fig. 8b). In accordance with the estimations based on the frame-to-frame analysis of the video records, the period of the typical circulating liquid flows at laser powers of 3– 5 W ranges from 0.2 to 1 s. Note that the above effects can be observed in the experiments with the blackened fiber tip at both laser wavelengths (0.97 µm and 1.55 μm). In the absence of the preliminary blackening, the effects are observed only for a radiation wavelength of 1.55 μm. Such a difference is caused by the fact that the radiation with a wavelength of 1.55 μm (unlike the short wavelength

Fig. 8. Water flows that actively circulate inside the glass capillary (with a diameter of 1 mm) in the vicinity of the blackened tip surface I heated by the laser radiation with a wavelength of 0.97 µm and a power of 3 W (a) and 5 W (b)

Laser-Induced Hydrodynamics in Water and Biotissues Nearby Optical Fiber Tip 105

Knowing the level of the meniscus in a capillary it is possible to determine easily the total volume of vapor-gas bubbles. Fig.10 shows change in the volume of generated bubbles at different laser powers and different laser wavelengths. Our experiments show that the total volume of bubbles rises gradually with time by a logarithmic low after the laser radiation switching on. The total volume at 1 W of laser power rises with time monotonically for both wavelengths, while at higher laser power quite strong fluctuations take place, with the growing in time amplitude. As this takes place, at laser power of 3 W the strong eruption of liquid from the capillary was observed after 4.7 s of laser irradiation (curve 3 at Fig. 10a). At that moment the curve 3 interrupts, since the meniscus went out of visualization zone

The total volume of generated bubbles increases with laser power. Comparison of curves 1 and 2 at Fig.10b shows that twofold increase of laser power (from 1 to 2 W) causes about the fourfold rise of the generated bubbles volume. After the laser radiation switching off, the total volume of bubbles first rapidly decreases (vapor condensation inside bubbles), ant next decreases more slowly. It should be noted that quite a strong low-frequency oscillations are

because of the abrupt decrease of meniscus level.

Arrows show the moments of laser on and laser off. Digits at curves shows laser power in Watts.

(Sandler et al., 2004; Chudnovskii & Yusupov, 2008).

and 1.56 m (b) wavelengths of radiation.

observed, caused by variation of total bubbles volume in a capillary.

In the case of 0.97m wavelength the fiber tip surface was covered by a thin carbon layer.

hydrodynamic processes in water-saturated biotissues at medium laser powers.

Fig. 10. Change of the total bubbles volume at different powers of lasers with 0.97 m (a)

Thus, the hydrodynamic processes related to the explosive boiling in the vicinity of the hot tip surface are observed in the liquid even at medium laser powers. Note that the intracapillary liquid exhibits effective mechanical oscillations with a frequency of 1– 5 Hz and appears saturated with microbubbles. We expect the development of such laser-induced

On the one hand, such processes provide the saturation of cavities and fractures in a spinal disc or bone with the water solution containing vapor-gas bubbles. On the other hand, they give rise to high-power acoustic oscillations and vibrations in the organ containing the connective tissue. Apparently, the filling of hernia with vapor-gas bubbles provides the reproducible decrease in the density of herniation immediately after the laser treatment

radiation) is capable of heating a thin water layer in the vicinity of the tip surface to the boiling point, since the absorbance at a wavelength of 1.56 µm is higher than the that at a wavelength of 0.97 µm by a factor of about 20 (Hale & Querry, 1973).

It is possible to visualize the hydrodynamic flows occurring in capillary and caused by laser-induced bubbles generation by microscope visual observing the meniscus. To accomplish this, the silica optical fiber with a 400 m diameter was introduced into a thin water-filled capillary with a 500 m internal diameter. The volume of liquid in a capillary was about 20 mm3, and meniscus was located at a 25 mm distance from the fiber tip surface.

Fig. 9 demonstrates the observed variations of a meniscus shape in a glass capillary at a power of laser radiation of 1 W and at laser wavelength of 1.56 m. Switching of laser radiation on has resulted in growing the distance between optical fiber tip surface, which is caused by the fact that vapor-gas bubbles are formed in a liquid in the course of laser irradiation nearby a fiber tip. Simultaneously with a gradual rise of average volume of liquid in a capillary, quite a strong variations of meniscus shape takes place in this case, which are caused by hydrodynamic processes observing in a capillary water cell. At a certain period of laser irradiation time even water flows occur (Fig. 9b and 9c) caused, presumably, by the appearance and fast motion of quite large vapor-gas bubbles in a water capillary cell. Decrease of laser power causes increase of water streams, and in some cases the eruption of some portion of liquid from a capillary takes place.

Fig. 9. Variation of a meniscus shape in a capillary caused by laser induce hydrodynamics and bubbles formation. Laser wavelength is 1.56 µm, laser power – 1W, internal diameter of capillary - 500 m.

radiation) is capable of heating a thin water layer in the vicinity of the tip surface to the boiling point, since the absorbance at a wavelength of 1.56 µm is higher than the that at a

It is possible to visualize the hydrodynamic flows occurring in capillary and caused by laser-induced bubbles generation by microscope visual observing the meniscus. To accomplish this, the silica optical fiber with a 400 m diameter was introduced into a thin water-filled capillary with a 500 m internal diameter. The volume of liquid in a capillary was about 20 mm3, and meniscus was located at a 25 mm distance from the fiber tip

Fig. 9 demonstrates the observed variations of a meniscus shape in a glass capillary at a power of laser radiation of 1 W and at laser wavelength of 1.56 m. Switching of laser radiation on has resulted in growing the distance between optical fiber tip surface, which is caused by the fact that vapor-gas bubbles are formed in a liquid in the course of laser irradiation nearby a fiber tip. Simultaneously with a gradual rise of average volume of liquid in a capillary, quite a strong variations of meniscus shape takes place in this case, which are caused by hydrodynamic processes observing in a capillary water cell. At a certain period of laser irradiation time even water flows occur (Fig. 9b and 9c) caused, presumably, by the appearance and fast motion of quite large vapor-gas bubbles in a water capillary cell. Decrease of laser power causes increase of water streams, and in some cases

Fig. 9. Variation of a meniscus shape in a capillary caused by laser induce hydrodynamics and bubbles formation. Laser wavelength is 1.56 µm, laser power – 1W, internal diameter of

wavelength of 0.97 µm by a factor of about 20 (Hale & Querry, 1973).

the eruption of some portion of liquid from a capillary takes place.

surface.

capillary - 500 m.

Knowing the level of the meniscus in a capillary it is possible to determine easily the total volume of vapor-gas bubbles. Fig.10 shows change in the volume of generated bubbles at different laser powers and different laser wavelengths. Our experiments show that the total volume of bubbles rises gradually with time by a logarithmic low after the laser radiation switching on. The total volume at 1 W of laser power rises with time monotonically for both wavelengths, while at higher laser power quite strong fluctuations take place, with the growing in time amplitude. As this takes place, at laser power of 3 W the strong eruption of liquid from the capillary was observed after 4.7 s of laser irradiation (curve 3 at Fig. 10a). At that moment the curve 3 interrupts, since the meniscus went out of visualization zone because of the abrupt decrease of meniscus level.

The total volume of generated bubbles increases with laser power. Comparison of curves 1 and 2 at Fig.10b shows that twofold increase of laser power (from 1 to 2 W) causes about the fourfold rise of the generated bubbles volume. After the laser radiation switching off, the total volume of bubbles first rapidly decreases (vapor condensation inside bubbles), ant next decreases more slowly. It should be noted that quite a strong low-frequency oscillations are observed, caused by variation of total bubbles volume in a capillary.

In the case of 0.97m wavelength the fiber tip surface was covered by a thin carbon layer. Arrows show the moments of laser on and laser off. Digits at curves shows laser power in Watts.

Fig. 10. Change of the total bubbles volume at different powers of lasers with 0.97 m (a) and 1.56 m (b) wavelengths of radiation.

Thus, the hydrodynamic processes related to the explosive boiling in the vicinity of the hot tip surface are observed in the liquid even at medium laser powers. Note that the intracapillary liquid exhibits effective mechanical oscillations with a frequency of 1– 5 Hz and appears saturated with microbubbles. We expect the development of such laser-induced hydrodynamic processes in water-saturated biotissues at medium laser powers.

On the one hand, such processes provide the saturation of cavities and fractures in a spinal disc or bone with the water solution containing vapor-gas bubbles. On the other hand, they give rise to high-power acoustic oscillations and vibrations in the organ containing the connective tissue. Apparently, the filling of hernia with vapor-gas bubbles provides the reproducible decrease in the density of herniation immediately after the laser treatment (Sandler et al., 2004; Chudnovskii & Yusupov, 2008).

Laser-Induced Hydrodynamics in Water and Biotissues Nearby Optical Fiber Tip 107

fiber tip surface occurs in the regime of channel formation when the fiber is shifted inside the wooden bar that mimics the biotissue. In this case, we observe substantial modifications and distortion of tip surface. The comparison of the sequential photographs (Fig. 12) shows a significant increase in the volume of the fiber fragment (swelling) in the vicinity of fiber tip.

Fig. 12. Modifications of the profile of the blackened fiber tip surface (side view) for regime of channel formation (the channel is formed by the fiber that moves inside the wooden bar with water and the radiation power is 5 W). The left-hand panel shows the original fiber just

SEM images (Fig. 13) show that the laser action in the regime of the channel formation in the presence of water causes substantial modifications of the working surface: the sharp edge is rounded and surface irregularities (craters) emerge on the tip surface. The image shows that a thin shell (film) with circular holes is formed at the tip surface of the optical fiber. Multiple cracks pass through some of the holes. In addition, we observe elongated crystal-like structures on the surface (Fig. 13b). Looking through the largest hole in the film on the tip surface (at the center of the lower part of the fragment at Fig. 13a), whose dimension in any

direction is greater than 10 µm, we observe the inner micron-scale porous structure.

Fig. 13. The microstructure of the fiber tip surface after laser action. **a -** SEM image of a fragment of the fiber end surface; **b -** magnified SEM image of a fragment of the end surface

with the crystal-like structures on the surface (Yusupov et al., 2011a).

after its blackening (Yusupov et al., 2011a).

It is known from (Bagratashvili et al., 2006) that the mechanical action on cartilages in the hertz frequency range actively stimulates the synthesis of collagen and proteoglycans even at relatively small amplitudes. The above estimations show that the pressure on biotissue provided by the vapor-gas bubbles can reach tens of kilopascals. In accordance with (Buschmann et al., 1995; Millward-Sadler & Salter, 2004), such pressures in the hertz frequency range can lead to regenerative processes in cartilage owing to the activation of the interaction of the extracellular matrix with the mechanoreceptors of chondrocytes (integrins).


*Edited by Erik Froyen*

*Fatty Acids - From Biosynthesis to Human Health* discusses how fatty acids are associated with various diseases and health conditions, such as cardiovascular disease, cancer, inflammation, autoimmune diseases, and metabolic disorders. Nutrition, fatty acid, and therapeutic recommendations are provided for individuals with certain diseases and health conditions and for those interested in decreasing the risk factors for chronic diseases. This book also covers biosynthesis and processing of fatty acids, which address human health and environmental concerns.

*Miroslav Blumenberg, Biochemistry Series Editor*

ISBN 978-1-80356-440-1

Fatty Acids - From Biosynthesis to Human Health

ISSN 2632-0983

Published in London, UK © 2023 IntechOpen © monsitj / iStock

## IntechOpen Series Biochemistry, Volume 42


*3.3. Di*ff*erentially Expressed Genes Associated with Leaf Senescence*

Comparative RNA-seq analysis was conducted to identify differentially-expressed transcripts, using 30-day-old leaves from Hwaseong and CR2002 to gain a better understanding of the mechanism involved in delayed senescence. A total of 10,339 and 9885 DEGs were identified in Hwaseong and CR2002 under dark treatment compared with the control, respectively (Figure 8a). To examine the difference between Hwaseong and CR2002, Hwaseong- and CR2002-specific DEGs were extracted. In CR2002-specific DEGs, 33 GO terms were enriched, and these included iron ion transport, response to hormone stimulus, cell cycle, response to endogenous stimulus, and chloroplast. Transporter genes are closely associated with leaf senescence process. During senescence, remobilization of nutrients from senescing cells to developing tissues is mediated by transporters, and transporter genes show notable changes in expression [13,29]. Chloroplast GO term contains 15 CR2002-specific DEGs, including Os07g0462000 (*OsSG1*), Os07g0558500 (*NYC4*), Os02g0152400 (*RbcS1*) [28,30]. Os07g0462000 encodes glutamate-cysteine ligase and possibly controls chlorophyll content and stay-green phenotype [30]. Os07g0558500 (*NYC4*) is an ortholog of *THF1* in *Arabidopsis.* The *AtTHF1* is known as a multi-function protein involved in acclimation to high light, sugar sensing, and disease resistance, while *nyc4-1* mutant shows the stay-green phenotype in rice [28]. Os02g0152400 encodes the rubisco small subunit 1, which reflects involvement in the regulation of rubisco catalytic activity.

Transcription factors (TFs) play an important role in regulating leaf senescence. NAC (NAM, ATAF1, and CUC2) transcription factor family is one of the large TF families, and many NAC TFs are up-regulated during leaf senescence [31]. In addition, several NAC TFs regulating leaf senescence have been characterized in various plant species [13,32–36]. Transcript levels of NAC transcription factors (*OsNAP, ONAC120*, and *ONAC122*) were specifically up-regulated in Hwaseong (Table 3). *OsNAP* is a positive regulator of leaf senescence, and its orthologs *TtNAM-B1* and *AtNAP* have shown similar function in wheat and *Arabidopsis* [13,32,33]. *OsNAC122* (or *OsNAC10*, Os11g0126900) over-expression and root-specific expression transgenic have shown significantly improved drought, high salinity, and low-temperature tolerance in rice [37].

Phytohormone signaling and biosynthesis-related genes were identified in Hwaseong- and CR2002-specific DEGs. During dark treatment, ethylene (*OsERF5*), ABA (*OsABI1*), and jasmonic acid (*OsJAZ1*) signaling genes were significantly down-regulated, while auxin synthesis (*OsNIT2*), brassinosteroid biosynthesis (*OsDWF1*), and cytokinin signaling (*OsRR3*) genes were up-regulated in CR2002 (Table 3). In contrast, ethylene biosynthesis and signaling (*OsACS2* and *OsERF2*) and auxin signaling (*OsARF2*) genes were up-regulated in Hwaseong, whereas cytokinin signaling (*OsRR6*, *OsRR9*, and *OsRR10*), brassinosteroid signaling (*OsBRI1*), and *gibberellin 20-oxidase* (*OsGA20ox1*) were down-regulated (Table 3). These results indicated that delayed leaf senescence in CR2002 (*gw2-ko*) was associated with phytohormone signaling or biosynthesis pathways in rice. The rice stay-green mutant *oself3.1* has shown down-regulated transcript levels of ABA-, ethylene-, JA-associated genes in microarray analysis [7]. The results from the present study were consistent with the report that *OsELF3.1* promoted leaf senescence by modulating signaling pathways [7]. To further investigate this relationship with phytohormones, gene expression of *GW2* in response to plant hormones was examined using the RiceXPro database. *GW2* did not show large changes in response to plant hormones (data not shown) [38]. In durum wheat, a *GW2* knock-down mutant has shown enhanced *cytokinin dehydrogenase 1* (*CKX1*) transcript level and down-regulation of *cytokinin dehydrogenase 2* (*CKX2*) and *gibberellin oxidase 3* (*GA3-ox*) [39]. In addition, a wheat near-isogenic line (NIL) harboring the *TaGW2-6A* allelic variant has shown an increase in not only grain size but also endogenous cytokinin content (Z+ZR) [40]. Compared to the control (Chinese Spring), the expression level of cytokinin biosynthesis genes (*TaIPT2*, *TaIPT3*, *TaIPT5*, and *TaIPT8*) are found to be up-regulated, while cytokinin degradation genes (*TaCKX1*, *TaCKX2*, and *TaCKX6*) are found to be down-regulated in the NIL during the endosperm development [40]. These results supported the possibility that delayed senescence in CR2002 and *gw2-ko* was mediated by phytohormone-related pathways.

#### *3.4. GW2 as an E3 Ubiquitin Ligase*

The ubiquitination process is a post-translational modification. E3 ligases function in the regulation of polyubiquitination and are involved in target protein degradation. Several studies have reported interacting partners of GW2. Target proteins of GW2 have been revealed through yeast two-hybrid screening [24]. Yeast two-hybrid and pull-down assays have shown that expansin-like 1 (EXPLA1) directly interacts with *GW2*. In addition, in vitro assays have identified the ubiquitination of EXPLA1 by *GW2* at lysine 279 (K279). Proteomic analysis has shown that chitinase 14 (CHT14) and phosphoglycerate kinase (PGK) directly interact with GW2 [23]. However, genes that are directly associated with leaf senescence have not been reported. Identification of GW2 substrate protein(s) responsible for leaf senescence would be key to understanding the post-translational molecular mechanism of leaf senescence in rice.

### **4. Materials and Methods**