**3.2 Anesthesia options**

• The type of anesthesia can be decided based on the preference of the patient and the surgeon, the amount of fat and the necessity of ancillary surgical procedures to be applied. If it is a limited volume liposuction and no additional surgical procedures is planned then, local and regional anesthesia may be the

type of anesthesia chosen. If it is going to be a large volume liposuction and h additional surgical procedures will be added then, general anesthesia may be preferred. The importance of fluid replacement should be kept in mind throughout surgery. And it is crucial especially for mega volume liposuction.


**2.5. Ammonium sulfate precipitation of proteins**

The Crude (supernatant) was saturated to 45% (277g/l) by slowly adding ammonium sulfate salt at 40C for precipitation, kept it for overnight [5,6]. After precipitation the solution was centrifuged at 20,000g for 25 minutes [6,]. Collected the yellow supernatant saturated again to 95% by adding (NH4)2SO4 (372g/l) slowly and kept at 40C for overnight. The overnight precipitated solution was centrifuged at 23000g for 45 minutes. Pale supernatant was discarded. Precipitate (contains azurin) were collected and resuspended in 0.02M Potassium Phosphate buffer pH 7[11, 12].

## **2.6. Dialysis of the supernatant**

Azurin suspended in 0.02M potassium phosphate buffer pH 7 was dialysed by standard dialyses bag purchased from Sigma-Aldrich, (Kolkata, India) having 3 kDa molecular weight cut off at 40C for 20 hours on the same buffer for overnight with continuous gentle stirring. Dialysis was done until the solution attains its buffer pH. The solutions were kept at 40C after dialysis for further purification [11, 12].

## **2.6.1. Purification of Azurin on Ion – Exchange chromatography**

## *<IP_ADDRESS>. DEAE cellulose treatment*

Dialysate (contains azurin) were initially treated with DEAE. 100 ml slurry of DEAE cellulose equilibrated in 0.02M potassium phosphate buffer pH 7 were treated with the

dialysate and stirred for 20-30 min at 40C.The suspension was centrifuged at 10,000g for 15 min. Azurin does not adsorb in the gel remains in supernatant but most of the unwanted proteins like yellow flavo proteins are removed. Supernatant was collected. DEAE cellulose precipitate was resuspended in the same buffer and again centrifuged at 10,000g for 15 minutes to remove all unattached proteins [11-13].

Purification of Azurin from *Pseudomonas Aeuroginosa* 315

*2.6.4. Characterization of Azurin (purified from P. aeruginosa MTCC2453)*

systems Illinois, USA) for analysis by using nitrogen laser at 337 nm.

*(MALDI-ToF)*

*SDS-PAGE*

purification process.

*2.6.6. FTIR analysis*

transilluminator (Biorad, PA, USA) [16].

*<IP_ADDRESS>. Molecular weight determination by matrix-assisted laser desorption/ionization time of flight*

The most successful method to analyze biopolymers such as, proteins, peptides, sugars and large organic molecules which are tend to be fragile and fragment when ionized by more conventional ionization methods [15]. The Fractions collected from G-25, G-75 and CM cellulose were performed MALDI for molecular weight determination. Two micro liter from each fraction of the chromatography was added with 20µl of 3, 5-Dimethoxy, 4-Hydroxy cinnamic acid otherwise called as sinnapinic acid (Sigma-Aldrich. Kolkata, India). Tiny spots were made on silver plate and kept for drying for 4-6 hours to drain the water molecules. Further spots were dried with a vacuum drier to make a crystalline molecule. After drying the samples were placed in the MALDI-ToF chamber (Voyager De pro, applied

*2.6.5. Purification profile of Azurin ((synthesized from P. aeruginosa MTCC2453) by*

Five ml of 12% resolving gel contains 1ml distilled water, 30% acryl amide, 1.5M Tris (pH 8.8), 10% SDS, 10% APS and 0.002µl TEMED for polymerization was casted in the glass slab without any bubbles and kept it for 10-15 minutes. After polymerization of the resolving gel, 3ml of stacking gel (4%) were loaded over the resolving gel which contains 0.68 ml distilled water, 30% acryl amide, 1M Tris (pH 6.8) 10% SDS,10% APS, and 0.001ml TEMED. After casting the gel, proteins purified from different chromatography were loaded with bromophenol (molecular weight marker dye) at different lanes for profiling the protein

Glass slab gel were kept in the electrophoresis tank with tank buffer (196 mM glycine, 0.1%SDS, 50mM Tris-Hcl pH 8.3 made by diluting a 10x stock solution). This setup was connected with power pack initially in 80mV to 100 mV. After running the gel up to its anode end, was removed and stained with 0.2% coomassie brilliant blue for overnight. Destained with destaining solution (45: 45: 10 – methanol: water: acetic acid) which destains the comassie blue until it reveals the bands. The bands (figure 4) were observed under UV

Infrared spectroscopy experiments were performed using a Nexus 870 (Thermo Nicolet Corporation, Madison, USA) spectrometer equipped with a potassium bromide (KBr) beam splitter and DTGS (deuterated triglycine sulfate) detector in the range of 3,000-4000 cm−1. We recorded 32 scans per spectrum at a 2 cm−1 resolution for 100 µl of azurin liquid samples in 0.02 M PBS buffer (pH 7.0). We kept the same buffer as a background medium and performed all measurements at room temperature. We corrected spectra for the moisture

The supernatant after DEAE treatment was saturated to 100% (766g/l) with (NH4)2SO4 at 40C for overnight for precipitation. After saturation, precipitates are mixed gently and kept for centrifugation at 10000g for 10 min. supernatant was collected for dialysis at 40C for overnight with gentle stirring with the same before. Dialysis was continued till the solution pH attains its buffer pH [11-13].


[27] Martin JE, Hurd AJ. Scattering from fractals. Journal of Applied Crystallography. 1987;20:61-

[28] Schmidt PW. Small-angle scattering studies of disordered, porous and fractal systems. Journal of Applied Crystallography. 1991;24:414-435. DOI: 10.1107/S0021889891003400

[29] Cherny AY, Anitas EM, Osipov VA, Kuklin AI. Deterministic fractals: Extracting additional information from small-angle scattering data. Physical Review E. 2011;83:036203.

[30] Anitas EM. Small-angle scattering from fat fractals. European Physical Journal B.

[31] Cherny AY, Anitas EM, Osipov VA, Kuklin AI. Small-angle scattering from multiphase fractals. Journal of Applied Crystallography. 2014;47:198-206. DOI: 10.1107/S16005767130

[32] Anitas EM. Microscale fragmentation and small-angle scattering from mass fractals. Advances in Condensed Matter Physics. 2015;2015:5. ID 501281. DOI: 10.1155/2015/501281

[33] Anitas EM. A structural model for scattering intensities with multiple fractal regions of variable length. Journal of Optoelectronics and Advanced Materials. 2015;17:1122-1127

[34] Anitas EM. Scattering structure factor from fat fractals. Romanian Journal of Physiology.

[35] Cherny AY, Anitas EM, Osipov VA, Kuklin AI. The structure of deterministic mass, surface and multi-phase fractals from small-angle scattering data. Romanian Journal of

[36] Anitas EM, Osipov VA, Kuklin AI, Cherny AY. Influence of randomness on small-angle scattering from deterministic mass fractals. Romanian Journal of Physiology. 2015;61:457-463

[37] Cherny AY, Anitas EM, Osipov VA, Kuklin AI. Scattering from surface fractals in terms of composing mass fractals. Journal of Applied Crystallography. 2017;50:919-931. DOI:

[38] Cherny AY, Anitas EM, Osipov VA, Kuklin AI. Small-angle scattering from the cantor surface fractal on the plane and the Koch snowflake. Physical Chemistry Chemical Phys-

Biophysical Journal. 2014;107:185-196. DOI: 10.1016/j.bpj.2014.05.025

Condensed Matter. The Netherlands: North Holland; 2002. 552 p

DOI: 10.1107/S0907444910017658

190 Complexity in Biological and Physical Systems - Bifurcations, Solitons and Fractals

78. DOI: 10.1107/S0021889887087107

DOI: 10.1103/PhysRevE.84.036203

29956

2015;60:647-652

Physiology. 2015;60:658-663

10.1107/S1600576717005696

ics. 2017;19:2261-2268. DOI: 10.1039/C6CP07496K

2014;87:139. DOI: 10.1140/epjb/e2014-41066-9

## *Edited by Ricardo López-Ruiz*

Modeling and simulating biological and physical systems are nowadays active branches of science. The diversity and complexity of behaviors and patterns present in the natural world have their reciprocity in life systems. Bifurcations, solitons and fractals are some of these ubiquitous structures that can be indistinctively identified in many models with the most diverse applications, from microtubules with an essential role in the maintenance and the shaping of cells, to the nano/microscale structure in disordered systems determined with small-angle scattering techniques. This book collects several works in this direction, giving an overview of some models and theories, which are useful for the study and analysis of complex biological and physical systems. It can provide a good guidance for physicists with interest in biology, applied research scientists and postgraduate students.

Published in London, UK © 2018 IntechOpen © Cappan / iStock

Complexity in Biological and Physical Systems - Bifurcations, Solitons and Fractals

Complexity in Biological

and Physical Systems

Bifurcations, Solitons and Fractals

*Edited by Ricardo López-Ruiz*


**4. Nailfold capillaroscopy in autoimmune connective tissue diseases**

AICTD complex pathogenesis usually includes microvascular changes, with occurrence of progressive structural and functional damage of the capillaries. Therefore, NFC became an important diagnostic and prognostic tool to use while managing these disorders.

### **4.1 Systemic sclerosis (SSc)**

Systemic sclerosis has probably been the most studied disease with NFC. It is a severe AICTD in which the main pathological events are endothelial dysfunction,

fibrosis, and inflammation, which usually results in skin and vascular changes [24]. These include an important damage of microvascular network, with enlarged and giant capillaries, capillary loss with disarrangement of capillaries' architecture, and neoangiogenesis. As mentioned above, the scleroderma patterns are divided into three different patterns: "early," revealing only few enlarged capillaries, few hemorrhages, and preserved capillary density; "active," showing frequent giant capillaries and hemorrhages, mild capillary loss, and disorganization of the microvascular network; and "late," where irregular and giant capillaries can be found, as with few hemorrhages, neoangiogenesis, and avascular areas [25] (**Figure 5**).

A large multinational study based on EULAR Scleroderma Trials and Research (EUSTAR) registry disclosed a scleroderma pattern in more than 86% of SSc patients [24]. Subjects without this pattern did not have organ involvement and RP and some had negative autoantibodies. These results suggest that although these patients were classified as having an SSc, they did not have an overt disease and, then, nonspecific NFC changes may precede an evolving scleroderma pattern. In patients with overt disease, capillaroscopic findings mirror somehow internal organ involvement evolution. Following disease progression, dynamic transition of microvascular abnormalities through different NFC patterns can be found in up to 50% of SSc patients [18, 26]. On the other hand, capillaroscopy patterns can improve after up to 4 years of combined treatment, revealing a progressive significant recovery in structure and function of microcirculation, associated to ameliorated outcomes, independently of disease severity [27–29].

Capillaroscopy abnormalities became one of the diagnostic criteria for SSc in the 2013 classification [6]. NFC should not only be used for a diagnostic purpose but also for monitoring the disease process and determining its prognosis, because, as explained above, its dynamic changes occur, and its severity directly relates to the extent of organ involvement [8].

#### **4.2 Systemic lupus erythematosus (SLE)**

A systematic review was recently performed by a EULAR study group, in order to establish capillaroscopic parameters in SLE patients and its correlations with clinical and laboratory characteristics [30]. According to this study, SLE patients present more tortuous and abnormal capillaries than healthy controls, as well as more hemorrhages (**Figure 11**). An NFC score was created by these authors to set the microangiopathy severity, SLE patients being those who had the higher scores. "Nonspecific patterns" and "scleroderma-like patterns" were also described. A correlation between NFC abnormalities and clinical and laboratory parameters was established, since a relationship between NFC score and SLE activity was disclosed. Further, it is an important note to highlight that, once SLE is a heterogeneous disease, with altered vascular involvement, probably capillaroscopic changes will only be seen in the active phase of the disease [31]. Also, as the microvasculopathy profile in SLE is quite different from the SSc's, which is typically obliterative, changes as neoangiogenesis are less common in SLE patients [32, 33].

#### **4.3 Inflammatory idiopathic myositis**

In inflammatory idiopathic myopathies (IIM), it is frequent to find tortuosities, capillary loss, enlarged and giant capillaries, microhemorrhages, and neoangiogenesis, as well as a disorganization of the vascular network and avascular areas [1, 34]. In dermatomyositis (DM), patients present more severe NFC findings, compared with those with polymyositis (PM). Ramified and bushy capillaries represent a hallmark of microvascular damage in DM (**Figure 10**). In these patients,

**179**

*Cabral.*

**Figure 12.**

**Figure 11.**

*The Impact of Nailfold Capillaroscopy in the Approach of Microcirculation*

capillaroscopic abnormalities seem to be related with disease duration: in the first 6 months of disease duration, capillary density is usually reduced and giant capillaries are frequent; after that period, scleroderma pattern becomes more common. In PM patients, NFC findings do not significantly differ from healthy controls. It has also been demonstrated that there is a potential relationship between capillary changes and organ involvement, especially in patients with lung disease [35]. A recent multicenter study in antisynthetase syndrome revealed that NFC changes are usually independent from the presence of RP [27]. In these patients, the scleroderma pattern is associated to positivity for anti-Jo1 antibodies and a longer disease duration. An interesting finding was that significant correlation was established between ILD and ramified capillaries, but not with SSc-like pattern. Together, these studies suggest that NFC can become an important indicator of interstitial lung disease in patients with IIM, disclosing early this potential life-

In MCTD, different abnormalities can be found: minor changes, hemorrhages, dilated and giant capillaries, reduced density, and neoangiogenesis [19] (**Figure 12**). The patients can present with a scleroderma-like pattern, but less severe when compared to systemic sclerosis. The dilated loops are usually more dystrophic, and the dilated loops are long. The neoangiogenesis features are normally present in patients who progress to SSc. The avascular areas are more frequent in patients with

*Systemic lupus erythematosus. Images from three different patients, revealing crossing capillaries (x), dilated loops (square), meandering capillaries (black circle), tortuosities (arrow), ingurgitated venous plexus (plus sign), and loop aneurysm (\*). Images were taken using Videocap biomicroscope, version 3.0, magnification*

*Images from 3 different patients with mixed connective tissue disease. Capillaroscopic findings include crossing (x), meandering (black circle), tortuosity (arrow), dilated loops (black square), giant capillaries (black dot), neoangiogenesis (black star), and clear avascular areas on the right image. Images were taken using Videocap biomicroscope, version 3.0, magnification ×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry*

*DOI: http://dx.doi.org/10.5772/intechopen.90525*

threatening manifestation.

**4.4 Mixed connective tissue disease (MCTD)**

pulmonary involvement or under immunosuppressants [36]**.**

*×200. Courtesy of Nailfold Capillaroscopy Clinic of Hospital Curry Cabral.*

#### *The Impact of Nailfold Capillaroscopy in the Approach of Microcirculation DOI: http://dx.doi.org/10.5772/intechopen.90525*

capillaroscopic abnormalities seem to be related with disease duration: in the first 6 months of disease duration, capillary density is usually reduced and giant capillaries are frequent; after that period, scleroderma pattern becomes more common. In PM patients, NFC findings do not significantly differ from healthy controls. It has also been demonstrated that there is a potential relationship between capillary changes and organ involvement, especially in patients with lung disease [35].

A recent multicenter study in antisynthetase syndrome revealed that NFC changes are usually independent from the presence of RP [27]. In these patients, the scleroderma pattern is associated to positivity for anti-Jo1 antibodies and a longer disease duration. An interesting finding was that significant correlation was established between ILD and ramified capillaries, but not with SSc-like pattern.

Together, these studies suggest that NFC can become an important indicator of interstitial lung disease in patients with IIM, disclosing early this potential lifethreatening manifestation.