## *Edited by Gulden Kokturk and Tutku Didem Akyol Altun*

People have been finding inspiration in nature in solving their problems, from the very beginning of their existence. In the most general sense, biomimicry, defined as "inspire from the nature," has brought together the engineers and designers nowadays. This collaboration creates innovative and creative outcomes that encourage people with their interdisciplinary relationships. Accordingly, the aim of this book is to bring together different works or developments on biomimetics in interdisciplinary relationship between different areas, especially biomimicry, engineering, and design. The twentyfirst century has conceived many new and amazing designs. The book in your hands will surely be an important guide to take a quick look at the future possibilities.

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Interdisciplinary Expansions in Engineering and Design With the Power of Biomimicry

Interdisciplinary Expansions

in Engineering and

Design With the Power of

Biomimicry

*Edited by Gulden Kokturk*

*and Tutku Didem Akyol Altun*


*2.8. Statistical Analysis*

Statistical analysis was performed using GraphPad Prism version 7. The Shapiro–Wilk test was used to determine the normality of data. *T*-tests and Pearson's test were used to compare and correlate histology and immunohistochemistry scores, respectively. A two-way ANOVA with multiple comparisons was used to analyse the growth kinetics of the cells treated with heparanase and a paired, one-sample *t*-test for the fold change in gene expression. A *p*-value ≤ 0.05 was considered statistically significant.


**5. Sampling procedure and data collection**

Most of that data used for analyses in the study were primary data obtained from sampling and laboratory analyses. Data of benthic macroinvertebrate assemblage and water quality assessment were obtained from *in situ* analyses and further analyses in the laboratory. River habitat and morphology data were measured on‐site in field surveys. On the other hand, secondary data such as land use, rainfall data, and stream catchment maps were obtained from several agencies such as the Department of Environment, Department of Drainage and Irrigation, Department of Survey and Mapping and local authorities.

### **5.1. Benthic macroinvertebrate**

The multihabitat approach of USEPA's Rapid Bioassessment Protocol (RBP) was adopted for this study as it was suitable for sampling a wide variety of stream types [22]. In this study, a rectangular dipnet with 500‐µm mesh attached to a 0.5 m × 0.3 m frame and a long pole were used for this purpose. For multiple habitats, the habitat types were sampled in proportion to their relative surface area within the sampling reach. A total of 20 sample units were collected from all major habitat types by kicking the substrates or jabbing with a dipnet within a sampling station to obtain a composite of 60 sample units in total. The samples were washed and any detritus present was removed on‐site as it would be impractical to wash large samples in the laboratory. Following this, benthic materials were sieved and rinsed before preservation in 70% ethanol. The sample containers were labeled to show all the essential information, including date and sampling location. Preprinted labels were preferably used using marker pens as ethanol would remove writing. Moreover, labeling on container lids was avoided in case they were interchanged. In laboratory, benthic macroinvertebrates were rinsed thoroughly in 500 µm‐mesh sieves to remove preservative and sediment, while remaining debris was visually inspected and discarded. During the separation process, the samples were soaked for about 15 min in tap water to hydrate the preserved organisms and prevent them from floating on the water surface during sorting. The samples were then spread over an enamel tray and sorted out into major taxa. All organisms were identified to the lowest practical level using a dissecting microscope and taxonomic Key from Yule and Yong [23].

### **5.2. Water quality**

However, there is active logging in the area planted with *Acacia mangium*, a tree species of the pea family, Fabaceae. *Acacia mangium* is suitable as raw material for sawn timber, and wood‐ chips in the pulp and paper industry, and reconstituted wood for the furniture industry. In the Asia–Pacific region, Japan is among the larger importers of wood chips, while the pulp and paper industry finds markets in Taiwan and South Korea. Based on the macro‐EIA for forest management units (FMU) in Johor, Sg Berasau supports a variety of fish species such as Terbul,

Sg Mengkibol is a second‐order river located within the Endau watershed. This river receives flows from Sg Melantai before joining Sg Semberong. This river basin is approximately 185

Most of that data used for analyses in the study were primary data obtained from sampling and laboratory analyses. Data of benthic macroinvertebrate assemblage and water quality assessment were obtained from *in situ* analyses and further analyses in the laboratory. River habitat and morphology data were measured on‐site in field surveys. On the other hand, secondary data such as land use, rainfall data, and stream catchment maps were obtained from several agencies such as the Department of Environment, Department of Drainage and

The multihabitat approach of USEPA's Rapid Bioassessment Protocol (RBP) was adopted for this study as it was suitable for sampling a wide variety of stream types [22]. In this study, a rectangular dipnet with 500‐µm mesh attached to a 0.5 m × 0.3 m frame and a long pole were used for this purpose. For multiple habitats, the habitat types were sampled in proportion to their relative surface area within the sampling reach. A total of 20 sample units were collected from all major habitat types by kicking the substrates or jabbing with a dipnet within a sampling station to obtain a composite of 60 sample units in total. The samples were washed and any detritus present was removed on‐site as it would be impractical to wash large samples in the laboratory. Following this, benthic materials were sieved and rinsed before preservation

 width and 20 km long. The study area is located in the middle section of Sg Mengkibol, starting from the Sg Mengkibol Riverine Park until the wet market. From December 2006 until January 2007, Malaysia experienced large‐scale flood events that affected most of the state of Johor. This phenomenon was caused by extremely high rainfall attributed to Typhoon Utor that made landfall in the Philippines and Vietnam. A series of massive floods hit the states of Malacca, Pahang, and Negeri Sembilan, with Johor as the worst hit state. Among the major towns affected by the floods were Batu Pahat, Johor Bahru, Kluang, Kota Tinggi, Mersing, Muar, Pontian, and Segamat. Consequently, the Department of Irrigation and Drainage (JPS) allocated RM2 million to deepen Sg Mengkibol at its banks for flood mitigation in low‐lying

Sebarau, Baung, Seluang, and Tapah.

**4.3. Sg Mengkibol**

residential areas [21].

**5.1. Benthic macroinvertebrate**

**5. Sampling procedure and data collection**

Irrigation, Department of Survey and Mapping and local authorities.

km2

320 Water Quality

Water quality assessment data were obtained by two methods, namely *in situ* and laboratory analyses. *In situ* measurements were made on temperature, pH, conductivity, and dissolved oxygen (DO) by using a YSI Professional Plus handheld multiparameter instrument. Meanwhile, other parameters such as biochemical oxygen demand (BOD5), total suspended solid (TSS), ammoniacal nitrogen (NH3N), and chemical oxygen demand (COD) were measured in the laboratory based on the Standard Methods for the Examination of Water and Wastewater [24]. Both *in situ* readings and water samples were collected from the same location. Water samples were collected in 1‐L polyethylene bottles and chilled in a cold box filled with ice cubes (4°C) to minimize the metabolism of organisms contained in the water. The water samples were labeled in a manner similar to that used for the benthic samples.

The water quality index (WQI) was calculated to indicate the level of pollution and the corresponding suitability for use according to the National Water Quality Standards for Malaysia (NWQS). Water quality class was determined based on the water quality index (WQI), ascertained by the six parameters, *viz*. pH, DO, BOD5, COD, TSS, and NH3N, according to the DOE formula (1):

$$\begin{aligned} \text{WQI} &= (0.22 \text{\*SIDO}) + (0.19 \text{\*SIDO}) + (0.16 \text{\*SICO}) + (0.15 \text{\*SINN})\\ &+ (0.16 \text{\*SISS}) + (0.12 \text{\*SlpH}) \end{aligned} \tag{1}$$

where SI is the subindex of the respective water quality parameters which is used to calculate the WQI (**Table 1**). The WQI classification based on water use is shown in **Table 2**.

**Table 1.** Best fit equations for the estimation of various subindex values.

**Table 2.** Water classes and uses.

### **5.3. Characterization of river habitat**

A visual‐based habitat assessment based on the USEPA habitat assessment survey was carried out simultaneously with the biological sampling. Several features in habitat assessment include a general description of the site, a physical characterization and water quality assess‐ ment, and also a visual assessment of instream and riparian habitat quality. Physical charac‐ terization comprised documentation of general land use, description of the stream origin and type, and a summary of the riparian vegetation features that included measurements of instream parameters such as width, depth, flow, and substrates. The observed channel dimensions were carried out in the survey stretch, located either in the presence of riffle or at a suitable shallow section of the river. Channel dimension measurements were taken according to the river habitat survey (RHS) method.

### **5.4. Streamflow gauging**

**Subindex for DO (in % saturation)**

*Subindex for COD*

322 Water Quality

*Subindex for NH3-N*

*Subindex for SS*

*Subindex for pH*

**Class Uses**

Class IV Irrigation

Class V None of the above

**Table 2.** Water classes and uses.

SIBOD = 108 × exp(−0.055x) − 0.1x for x > 5

SICOD = −1.33x + 99.1 for x ≤ 20 SICOD = 103 × exp(−0.0157x) − 0.04x for x > 20

SIAN = 100.5 − 105x for x ≤ 0.3 SIAN = 94 × exp(−0.573x) − 5 × |x − 2| for 0.3 < x < 4 SIAN = 0 for x ≥ 4

SISS = 97.5 × exp(−0.00676x) + 0.05x for x ≤ 100 SISS = 71 × exp(−0.0061x) − 0.015x for 100 < x < 1000 SISS = 0 for x ≥ 1000

SIpH = 17.2 − 17.2x + 5.02 × 2 for x < 5.5 SIpH = −242 + 95.5x − 6.67 × 2 for 5.5 ≤ x < 7 SIpH = −181 + 82.4x − 6.05 × 2 for 7 ≤ x < 8.75 SIpH = 536 − 77.0x + 2.76 × 2 for x ≥ 8.75

Fishery III—common, of economic value and tolerant species; livestock drinking

A visual‐based habitat assessment based on the USEPA habitat assessment survey was carried out simultaneously with the biological sampling. Several features in habitat assessment include a general description of the site, a physical characterization and water quality assess‐ ment, and also a visual assessment of instream and riparian habitat quality. Physical charac‐ terization comprised documentation of general land use, description of the stream origin and

**Table 1.** Best fit equations for the estimation of various subindex values.

Water supply I—practically no treatment necessary

Fishery I—very sensitive aquatic species Class IIA Water supply II—conventional treatment required Fishery II—sensitive aquatic species

Class III Water supply III—extensive treatment required

Class I Conservation of natural environment

Class IIB Recreational use with body contact

**5.3. Characterization of river habitat**

Streamflow gauging was conducted to measure the flow rate of the study area. The equipments used in this study were flow meter, measuring tape, staff level, hammer, and ropes/cables. The Cole Parmer Model BS 11000 flow meter used in this study was equipped with a propeller to allow it to rotate according to the velocity of the water. The mean section method was used to measure the river discharge using the flow meter and other tools, whereby cross‐sectional area of the river was divided into several subsections. Stream velocity was measured at depths of 0.6d, 0.2d, and 0.8d depending on the need, where d represents the variable of depth from the water surface. This method was used to obtain the average velocity of the represented river. The general hydraulic formula for river discharge is as follows (2)

$$\mathbf{Q} = \mathbf{A}\mathbf{V} \tag{2}$$

where Q is discharge (volume/unit time‐e.g. m3 /s, also called cumecs), A is the cross‐sectional area of the stream (e.g. m2 ), and V is the average velocity (e.g. m/s).


**3. Fuel characteristics calculator (FCCAL)**

The main thrust of FCCAL [2] is to analyze the plenum gas conductivity with fission gas accumulation and its analytical evaluation. With irradiation of the fuel inside the reactor core, fission noble gases Xenon and Krypton accumulate in the plenum gap which changes the gap conductivity from the initial fuel behavior that is for the Helium-filled during manufacturing. The analytical model is a better approximation over the use of correlations to estimate the effect of noble gases in the plenum gap. The change in conductivity is observable in the fuel in CANDU reactors where on-power refueling is practiced and the old fuel bundles move to higher power generating regions of the core. The fresh fuel along with the old bundles leads to a higher fission rate and hence the release of trapped noble gases towards the plenum. This results in a higher temperature of the old bundles even without an appreciable change in the power. We will discuss these phenomena in the sections below.

#### **3.1 Fission gas release**

Fission gases are considered to be released from the fuel when they reach any space that is connected to the free volume within the fuel pin. The released gases accumulate in the fuel-cladding gap, the central void, and porosity within the fuel which communicates directly with the fuel-pin gas space [1]. Cracks or interlinked gas bubbles or pores are an important type of open porosity. The fission gas that has been released from the fuel is responsible for the change in plenum gap conductivity and is assumed to have the following properties. (a) Once the gas is released, the probability of its re-entering the solid from the free volume is negligible (b) the gas pressure in open porosity is equal to that in the free volume of the pin. Because of the insolubility of Xenon and Krypton in solids, there is no effect of plenum fission gas pressure on the rate of gas escape from the fuel (c) while the fission gas contained by the fuel tends to cause swelling, fission gas that has been released promotes shrinkage in the fuel by pressurizing the solid pellets leading to collapse of the internal porosity and bubbles. FCCAL carries out an explicit calculation for changes in gap gas conductivity due to a binary mixture of Helium and Xenon. As the heat generated in the pellet is transferred across the fuel to the coolant, the heat transfer across discontinuities is calculated in the following steps. (i) Heat transfer from the meat of the pellet to the pellet surface. It is estimated by the heat transfer coefficient of the natural uranium oxide pellet (hPÞ. (ii) Heat transfer across the plenum gap and the Zircalloy clad hGg þ hGs þ hs . hGg is the heat transfer coefficient due to plenum gas,hGs is the heat transfer coefficient of the solid-solid contact points between the pellet and the sheath, andhs is the heat transfer of the Zircalloy sheath. (iii) Heat transfer from the clad outer surface to the coolant is estimated by the heat transfer coefficient of the coolant film near the fuel surface hð Þ cf . Total heat transfer coefficient hð ÞT is the sum of terms in (i), (ii), and (iii) i.e.

*Plenum Gas Effect on Fuel Temperature DOI: http://dx.doi.org/10.5772/intechopen.101098*

$$\mathbf{h}\_{\rm T} = \mathbf{h}\_{\rm P} + \mathbf{h}\_{\rm Gg} + \mathbf{h}\_{\rm Gs} + \mathbf{h}\_{\rm s} + \mathbf{h}\_{\rm cf} \tag{1}$$


*Aspergillus flavus* **as a Model System to Test the Biological Activity of Botanicals: An Example on** *Citrullus colocynthis* **L. Schrad. Organic Extracts**

**Francesca Degola 1,\*, Belsem Marzouk 2, Antonella Gori 3, Cecilia Brunetti 3,4, Lucia Dramis 1, Stefania Gelati 5, Annamaria Buschini 1,6 and Francesco M. Restivo 1**

Received: 15 April 2019; Accepted: 17 May 2019; Published: 22 May 2019

**Abstract:** *Citrullus colocynthis* L. Schrader is an annual plant belonging to the Cucurbitaceae family, widely distributed in the desert areas of the Mediterranean basin. Many pharmacological properties (anti-inflammatory, anti-diabetic, analgesic, anti-epileptic) are ascribed to different organs of this plant; extracts and derivatives of *C. colocynthis* are used in folk Berber medicine for the treatment of numerous diseases—such as rheumatism arthritis, hypertension bronchitis, mastitis, and even cancer. Clinical studies aimed at confirming the chemical and biological bases of pharmacological activity assigned to many plant/herb extracts used in folk medicine often rely on results obtained from laboratory preliminary tests. We investigated the biological activity of some *C. colocynthis* stem, leaf, and root extracts on the mycotoxigenic and phytopathogenic fungus *Aspergillus flavus*, testing a possible correlation between the inhibitory effect on aflatoxin biosynthesis, the phytochemical composition of extracts, and their in vitro antioxidant capacities.

**Keywords:** antimycotoxigenic activity; *Citrullus colocynthis*; *Aspergillus flavus*; model system; HPLC-MS/MS

#### **1. Introduction**

Oxidation is considered an underlying mechanism in the incidence of chronic diseases: Reactive oxygen species (ROS) such as superoxide anions, hydroxyl radicals, and hydrogen peroxide are cytotoxic, leading to tissue injuries. As in a "domino effect", oxidative stress resulting from the imbalance between the generation of reactive oxygen species and endogenous antioxidant systems induces inadvertent enzyme activation and consequent oxidative damage to cellular systems [1]. It is widely reported that cellular oxidative damage is responsible for numerous disorders, such as cardiovascular [2], Alzheimer's [3], and Parkinson's disease [4]—as well as ulcerative colitis [5], atherosclerosis [6], and cancer [7]. A key defense mechanism against radical mediated toxicity is represented by antioxidants, which protect cells from the damage caused by free radicals [8]. Consequently, during the last thirty years, several antioxidant-based formulations for the prevention and treatment of complex diseases have been developed [9–11]. Among these antioxidant formulations, a high number of plant-derived drugs, widely used for ethnopharmaceutical preparations, have been applied as "natural" principles included in modern medical applications [12,13]. Accordingly, interest in botanicals as a source of bioactive compounds has increased worldwide, and the finding of new biologically remarkable natural compounds affects not only the pharmaceutical field, but the nutraceutical and cosmeceutical fields too [14–17]. Clinical studies aimed at confirming the scientific bases (chemical and biological) of pharmacological activity of many plants used in folk medicine often rely on the results obtained from preliminary laboratory tests. For example, the antioxidant properties of plant extracts are typically evaluated through in vitro analyses, such as DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) and ABTS [2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonate)] assays [18], and to date, the number of in vitro studies far exceeds the number of in vivo studies, remaining the most cost effective and predominant type of research investigations performed. In fact, factors including costs, interspecific differences that preclude the adequate predictive value of the experiments, feasibility of testing procedures, and ethical concerns generally limit the utilization of animal models and human subjects for this kind of research [19]. The possibility of using high-throughput small-scale in vivo or ex vivo model systems to predict a possible antioxidant biological activity of plant extracts before their medical application is therefore desirable. Eukaryotic microorganisms, though few, have sometimes been selected for this purpose: For example, the yeast *Saccharomyces cerevisiae* has been employed to determine the antioxidant activity of different berry juices, which reportedly contain high amounts of phenolics [20].

*Aspergillus flavus*, a saprophytic plant pathogen, is the predominant species producing aflatoxins (AFs). Among multiple events that contribute to aflatoxin production, those involving ROS accumulation—such as during morphological and metabolic transitions—and the establishment of an oxidative intracellular environment seem to possess a key role in triggering AF biosynthesis. This correlation has been supported by the identification of at least one transcription factor, which responds to the cellular oxidative stress by activating a series of enzymes responsible for the scavenging of cytoplasmic ROS excess [21–23]. Conversely, many compounds with antioxidant properties (such as ascorbate, eugenol, ethylene, methyl jasmonate, and α-lipoic acid) showed to exert an inhibition/containment effect on AF biosynthesis [24–27], mainly through the stimulation of catalase, superoxide dismutase, and glutathione peroxidase activity [28]. An inhibitory effect on AF biosynthetic pathway and *A. flavus* growth has been recently reported for a wide number of botanicals and essential oils [29–31]. Since the response of aflatoxin metabolism to redox balance alterations is well known, as they are considered a sort of "defense molecule" synthesized to cope with an excess of ROS in the late phase of growth [22,23], we proposed this airborne microorganism as a model system to screen the antioxidant potential of plant extracts.

Here we analyzed the effect of organic extracts of *Citrullus colocynthis* L. Schrader, an annual plant belonging to the Cucurbitaceae family which grows in arid and semi-arid regions, on AF biosynthesis and *A. flavus* growth. Native to tropical Asia and Africa, *C. colocynthis* is is now widely distributed in the desert areas of the Mediterranean basin (in Italy the only known population is located in the Aeolian island of Vulcano). Many pharmacological properties (anti-inflammatory, anti-diabetic, analgesic, anti-epileptic) are ascribed to different organs of this plant [32–35]: Extracts and derivatives of *C. colocynthis* are used in folk Berber medicine for the treatment of numerous diseases; the root is used for arthritic pain, breast inflammation, ophthalmia, and uterine pain; and the leaves are used for treatment of cough, many tumors, and as a cholagogue [36]. Recently, antifungal and antibacterial activities of organic extracts from leaves and seeds were reported [37–39]. However, despite various studies on the medical use of *C. colocynthis* derivatives, information about its antimycotoxigenic potential are still scarce. The aim of this work is to evaluate the antiaflatoxigenic effect of organic

extracts of *C. colocynthis* stem, leaf, and root through the use of *A. flavus* as a model system, comparing their effect on the basis of phytochemical composition.

#### **2. Results and Discussion**

#### *2.1. Phytochemical Characterization of C. colocynthis Extracts*

Several studies reported a high in vitro antioxidant potential of organic *C. colocynthis* extracts obtained from various tissues, due to their polyphenolic composition [40,41]. The antioxidant capacity of root, stem, and leaf extracts and phenolic content were then measured according to the DPPH and Folin-Ciocalteu's methods, respectively. As a general consideration, it should be noted that stem and leaf extracts showed a wider range of antioxidant activity, depending on the extraction solvent, than root extracts (Figure 1). The highest antioxidant capacity was determined in the methanol (MET) leaf extract and ethyl acetate (EA) root extracts, followed by chloroform (CHL) and methanol root extracts. On the contrary, MET stem extracts showed the lowest activity (Figure 1).

**Figure 1.** DPPH scavenging activity of *Citrullus colocynthis* root, leaf, and stem extracts. Data are means of three replicates ± S.D. Same letters indicate absence of statistically significant differences (*p* < 0.05).

A detailed phytochemical characterization of the leaves, stems, and roots of *C. colocynthis* revealed the presence of different classes of metabolites—such as coumarins, hydroxycinnamic acid derivatives, flavan-3-ols glycosides, flavone glycosides, and tetracyclic triterpenes (Table 1)—as according to previous investigations [42–44]. These compounds, which have been indicated as responsible for antifungal activity against *Aspergillus* strains [38], were abundant in the analyzed extracts (Table 1). Esculetin (1), p-coumaric acid derivatives (2, 5), orientin (3), vitexin (6), apigenin derivatives (4, 7, 8, 9, 12, 17), and epicatechingallate (18) were identified according to their MS fragmentation pattern and absorption spectra (Table 1). The remaining identified peaks corresponded to different flavone derivatives (10, 11), and cucurbitacin derivatives (13, 14, 15, 16) were elucidated by their molecular weight obtained by MS analysis, their UV spectra, and by comparing experimental data with respective literature data (Table 1) [45–47]. In particular, cucurbitacin derivatives and colocynthoside B did not furnish any MS fragment as previously reported by Chawech et al. [44]. In order to exclude that these compounds were artefacts due to solvent extraction, they were also compared with MS and UV spectra of authentic standards of cucurbitacin E and I. Compound **19** (Table 1) was tentatively identified as colocynthoside B on the basis of its molecular weight, its UV spectrum, and the comparison with literature data [48]. Marked differences were observed among the tissues. However, in all three extracts, the leaf contained the highest variety of phenolics, as well as the higher content of secondary metabolite, except for compound **12** (Table 1), which was more abundant in the ethyl acetate extract of the stem. Overall, our results showed that ethyl acetate was the most efficient solvent to extract

phenolic constituents in all tissues, apart from orientin, coumaric flavone derivative, two apigenin hexosides (compounds **7** and **17**), epicatechin gallate, vitexin, and compound **5**—which were more concentrated in methanol extracts. On the contrary, chloroform was efficient to extract cucurbitacin I derivatives (compounds **13**, **14**, **16**) and colocynthoside B. The most abundant cucurbitacin derivative (compound **13**) resulted in extraction to the same extent as in ethylacetate and chloroform. The presence of cucurbitacins is relevant to the bitterness and toxicity of the plant, but it also has some biological effects—such as anti-inflammatory, purgative, and anti-cancer activities [49].

#### *2.2. Antifungal and Anti-Aflatoxigenic Activity*

The prediction of the biological activity of natural extracts may often be difficult due to the variation in their chemical constituents, that in turn depend on the growth stages of the plants, and/or their geographic origin [50]. On the other hand, screening plant crude extracts can simplify the discovery of new and promising bioactives, and allows a further, more specific identification of the chemical compounds responsible for the observed activity. We performed a preliminary assay to evaluate the effect of the different *C. colocynthis* extracts on *A. flavus* growth, intended as daily radial increase of fungal colonies diameter. Concentration of 500 μg/mL was tested for each extract. As reported in Table 2, none of them resulted in a significant reduction of radial mycelium growth, suggesting that the composition of extracts did not possess appreciable antimicrobial activity against the fungus.

These preliminary results led to the exclusion of any antifungal or fungistatic potential of extracts on the mycelium long-term growth. However, various studies have reported that several compounds, both synthetic and natural, are effective in lowering AF production without apparently interfering with the fungal development [51,52]. At present, these compounds are a promising tool for uncovering the regulatory mechanisms triggering the mycotoxigenic metabolism, one of the main targets for mycotoxin diffusion/contamination control strategies [53,54].

To assess and compare the efficacy of different organic extracts of *C. colocynthis* tissues on total AF biosynthesis, conidia of *A. flavus* were inoculated in clarified coconut medium (CCM) and the fluorescence-based microplate procedure was used [27]. Leaf, stem, and root organic extracts were tested at increasing concentrations (data not shown); the lower and the higher concentrations (100 and 500 μg/mL, respectively) are reported in Figure 2. Chloroform (CHL), ethyl acetate (EA), and methanol (MET) extracts were administrated to aflatoxigenic *A. flavus* cultures. After six days of incubation, aflatoxin accumulation showed a dose-dependent alteration in response to extract exposure: The lowest dose (100 μg/mL) was less effective in limiting the amount of toxins in the culture, while the effect increased when extracts were added at the concentration of 500 μg/mL. Among tissues, leaf and root extracts had the highest levels of aflatoxin inhibition (Figure 2B,C), exceeding 80% inhibition in both CHL extracts. In addition, root extracts were able to lower the aflatoxin concentration by up to 12% in the most effective (CHL), and around 45% in the case of the least effective (MET; Figure 2B).

**Table 2.** Effect of 500 μg/mL *C. colocynthis* extracts on *A. flavus* radial growth. Radial increment is expressed as the mean of daily radial increase of colonies radius (cm/d) ± S.D. Same letters indicate absence of statistically significant differences (*p* < 0.05).

**Figure 2.** Activity on toxin accumulation and mycelium growth. Aflatoxin accumulation (reported as fluorescence arbitrary units) in *A. flavus* six-days after CCM cultures treated with stem (**A**), leaf (**B**), and root (**C**) extracts. (**D**) Early mycelium growth inhibition of *A. flavus* conidia treated with 500 μg/mL extracts, measured 48 h after inoculum by optical density increasing, and expressed as percentage in respect to control. Error bars refer to mean values of four replicates ± S.D.

A similar correlation between solvent and aflatoxin inhibition rate was observed for the highest concentration of EA leaf and root extracts in eculetin, p-coumaric acid, apigenin, and cucurbitacin derivatives (Table 1). Interestingly, the high activity of the CHL extract could be related to cucurbitacin derivatives and colocynthoside B. In particular, colocynthoside B was detected only in the chloroform extracts and may be responsible of the observed aflatoxin inhibition (Figure 2). Prevention of AF accumulation has, for a long time now, been associated with the action of molecules and/or conditions that interfere with fungal growth [24,29,30]; however, during the last decades, several other substances have been shown to be effective in completely blocking the biosynthesis of mycotoxins without affecting mycelium development [52,55]. Thus, the correlation between aflatoxin metabolism and *A. flavus* growth should be considered under different perspectives of fungal development: Colonies may have the same radius, but vary significantly in hyphal density and, therefore, biomass. In fact, hyphae branching, which is necessary for an efficient colonization and utilization of the substrate, responds to nutrient gradients, growing away from areas staled by metabolic by-products of existing hyphae. However, colony radial growth is not influenced by the concentration of nutrients, since existing hyphal tips at the colony margin, which determine the colony diameter, have priority over all other hyphal tips (i.e., the branches). For this reason, the evaluation of colony radial growth as a unique parameter for the assessment of any antifungal effect could be misleading about possible fungistatic

activities of tested compound/mixture, or might disguise an early stadium effect. As reported in Figure 2D, when *C. colocynthis* extracts were administrated to *A. flavus* conidia in YES liquid cultures at the higher concentration (500 μg/mL), early mycelium development was delayed by the majority of extracts. The exerted effect depended either on the tissue or solvent: For example, while CHL and EA extracts from the stem and leaf did not significantly differ in their inhibitory effect (50 vs. 60% and 45 vs. 50%, respectively), EA root extract proved to be more highly effective against the initial development of mycelium than CHL extract from the same tissue (70 vs. 25%).

#### *2.3. Aflatoxin-Modulating Activity*

Time-dependent aflatoxin production was analyzed by time-course experiments, where the kinetic of toxin accumulation was "real time", determined by starting from 65 to 146 h after inoculum in CCM medium. As previously reported [27] the AF concentration in the control cultures progressively increased for up to 85–90 h, maintaining, from here on, a 'plateau' value. AF production in the *A. flavus* cultures treated with a 500 μg/mL extract concentration showed a similar time course, but the maximum quantity of toxins produced varied consistently, with the extract-dependent inhibition rate observed in the end-point cultures. Stem extracts did not significantly differ from each other in terms of global inhibition level, blocking AF accumulation to 50% of the control value (Figure 3A). On the contrary, leaf and root extracts resulted in a variable range of toxin containment: The AF accumulation course, in cultures treated with leaf extracts, split from control at 72 h after inoculum, reaching a peak at 89 h (Figure 3B). A similar pattern was observed in root MET treated cultures, whereas root CHL and root EA avoided AF accumulation already before 65 h (Figure 3C).

**Figure 3.** Aflatoxin time-course accumulation. Effect of stem (**A**), leaf (**B**), and root (**C**) extracts on AF time-course accumulation in *A. flavus*. (**D**) Comparison between 500 μg/mL stem, leaf, and root CHL extract; α-lipoic acid 1 mM is used as a reference. Error bars refer to mean values of four replicates ± SD.

Due to the variety of synergistic phenomena occurring in the cell, the antioxidant activity (and resulting biological effect) may rarely be calculated on the basis of chemical in vitro assays, mainly in the case of botanicals and phytocomplexes as those reported here. Therefore, the effect of different extracts on the redox balance could only in part be predicted. Additionally, the use of high concentrations of single constituents (when, actually, a plant extract is a complex mixture) may result in the in vitro system being exposed to an overstated and unrealistic concentration. However, a comparison with a standard molecule, owning well-documented antioxidant properties consistent with the biological effect in the object of this study, should be done. Lipoic acid is a well-known ROS scavenger, and its efficacy in preventing aflatoxin production in *A. flavus* was already reported [27]. At the highest dose considered here (500 μg/mL), CHL root extracts resulted in AF accumulation containment, which was comparable to that observed for 1 mM α-lipoic acid. Stem and leaf CHL extracts were less efficient inhibitors of AF biosynthesis, as compared to α-lipoic acid and root CHL extracts. It thus appears that CHL root extracts are a promising source of anti-aflatoxigenic molecules. On the other hand, reinforcing evidence shows that *A. flavus* aflatoxin-producing strains may be used as an in vivo model to test the antioxidant activity of new mixture/compounds.

#### *2.4. Time Course of Extract Administration on Aflatoxin Accumulation*

According to various authors, many plant extracts showing an inhibitory effect against aflatoxin accumulation at the early stage seemed to become almost ineffective after protracted incubation [56,57], suggesting that their biological activity might depend not only on phytochemical composition, but also on the chemical structure and related properties of single components. Additionally, evidence has been provided showing that the delivery time of an anti-oxidant compound during fungal growth may affect its inhibitory efficacy on aflatoxin biosynthesis and/or accumulation [40]. In Figure 4, the time course of *C. colocynthis* root extracts (CHL and EA) administration is reported. As a general observation, early administration (time 0; at the germination stage) of the relevant extract in the medium resulted in the highest inhibition of AF accumulation, whereas delaying the time of administration (from 65 h onwards) did not block mycotoxin biosynthesis but, in the best case (65 h), retarded aflatoxin accumulation.

**Figure 4.** Effect of 500 μg/mL CHL (**A**) and EA (**B**) root extract over time on aflatoxin production. Extracts were added to conidia of *A. flavus* inoculated in CCM after 65, 72, and 86 h of incubation. Error bars refer to mean values of four replicates ± SD.

As previously reported for lipoic acid [27], the efficacy of CHL and EA root extracts in preventing AF accumulation is limited to a short time interval (0–65 h) that precedes the burst of AF biosynthesis. It would be worth analyzing the metabolic and regulatory networks operating during this time window to uncover possible molecular targets for designing new and specific anti-aflatoxigenic compounds.

#### *2.5. Conidia Production and Conidiophores Morphology*

In Aspergilla, vegetative reproduction and subsequent colonization of the surrounding environment rely on the differentiation of specialized structures (conidiophores) bearing vegetative spores (conidia), whose formation process characterizes the late phase of mycelium growth. We tested the effect of *C. colocynthis* CHL and MET stem, leaf, and root extracts on the production of conidia; as reported in Figure 5, a tissue and solvent (CHL and MET) dependent efficacy of the various extracts on lowering the number of conidia accumulated by treated mycelia was observed.

The overall pattern of tissue/solvent efficacy on conidia production was quite different with that observed for mycelium growth (Table 2) or hyphae elongation, and for AF accumulation (Figure 2). Interestingly, the scanning emission microscopy analysis (SEM) conducted to evaluate the conidiophore organization showed that no significant alteration of either the morphology or the general aspect of these reproductive structures occurred (an example is reported in Figure 5B), providing evidence that the relevant treatment affected the number of conidia or conidiophores.

**Figure 5.** Effect of 500 μg/mL root, stem, leaf (R, S, L) MET and CHL extracts on conidia production (**A**) and conidiophores morphology (**B**) (left: Control; right: R-CHL treated cultures) in *A. flavus* 96-wells CCM cultures. Values are reported as inhibition percentages in respect to control; Error bars refer to mean values of four replicates ± S.D. Different letters over the bars indicate the differences that were statistically significant (*p* < 0.05).

#### **3. Conclusions**

Plants with significant pharmacological properties have often been found to be rich in polyphenols and other secondary metabolites that have been proven to possess high antioxidant potentials due to their activity as reducing agents, metal chelators, and free radical quenchers. In this sense, every bioactive able to interfere with the oxidative status of fungal cells, on which the mycotoxin metabolism relies, should be validated as a biocontrol agent in organic strategies aimed at reducing aflatoxin contamination in food and feed commodities. On the other hand, a search for new antifungal and antimycotoxigenic substances is of increasing interest, with the perspective of improving antifungal resistance and understanding the underlying mechanisms of these new drugs with a wide range of applications—from medical mycology to agricultural and food safety.

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

#### *4.1. Plant Materials*

*Citrullus colocynthis* L. Schrader plants were collected near Medenine (Tunisia), in the municipality of Sidi Makhlouf. The identification was performed according to the flora of Tunisia [58] and a voucher specimen (C.C-01.01) deposited in the biological laboratory of the Faculty of Pharmacy of Monastir.

#### *4.2. Extraction Protocol*

Fresh tissues (roots, stems, and leaves) were dried and powdered using a tissue blender. Different solvents, in ascending polarity (petroleum ether, chloroform, ethyl acetate, and methanol) were used for Soxhlet extraction to fractionate the soluble compounds from the plant material. The extraction was performed with dried powder (100 g) placed inside a thimble made by thick filter paper, loaded into the main chamber of the Soxhlet extractor, which consisted of an extracting tube, a glass balloon, and a condenser. The total extracting time was 6 h for each solvent, continuously refluxing over the sample at a temperature not exceeding the boiling point. The resulting extracts were evaporated at reduced pressure to obtain the crude extracts. The organic solvents used were 99% pure. Extracts were all ethanol resuspended for further analysis. All the chemicals were obtained from Sigma (St. Louis, MO, USA).

#### *4.3. Determination of the Total Phenolic Contents*

Phenolic compound concentration in the different extracts was determined by using the Folin–Ciocalteu's phenol reagent, according to Singleton and Rossi [59], with some modifications. Briefly, 100 μL of the extract solution was mixed with 100 μL of Folin–Ciocalteu's phenol reagent. After 3 min, 100 μL of saturated sodium carbonate solution was added to the mixture and adjusted to 1 mL with distilled water. The reaction was kept in the dark for 90 min, after which the absorbance was recorded at 720 nm. Gallic acid was used to design the standard curve. The contents of total phenolic are expressed as mg of gallic acid equivalents (GAE)/g of extract. Data were reported as means of three replicates ± S.D.

#### *4.4. Determination of DPPH Radical Scavenging Activity*

The ability to scavenge the DPPH-free radical was monitored according to a method first introduced by Blois (1958) and developed by Brand-Williams et al. [60]. Various concentrations of sample extracts (0.5 mL) were mixed with 0.5 mL of methanolic solution containing DPPH radicals (6 × 10−5 M). The mixture was shaken vigorously and left to stand in the dark until stable absorption values were obtained. The reduction of the DPPH radical was measured by continuously monitoring the decrease of absorption at 517 nm. The DPPH scavenging effect was calculated as a percentage of DPPH discoloration using the following equation: % scavenging effect = [(ADPPH × AS)/ADPPH] × 100, where AS is the absorbance of the solution when the sample extract has been added at a particular level, and ADPPH is the absorbance of the DPPH solution. Three experiments were performed in triplicate. The antiradical activity was expressed in terms of the amount of antioxidant necessary to decrease the initial DPPH absorbance by 50% (IC50). The IC50 value for each extract was determined graphically by plotting the percentage of DPPH scavenging as a function of extract concentration.

#### *4.5. UPLC-DAD-ESI-MS*/*MS Analysis*

For the chemical characterization, 60 mg of each extract were re-dissolved in methanol-distilled water (1:1 *v*/*v*) and filtered with a PTFE membrane. 5 μL were injected in a LC–DAD-MS/MS system, consisting of a Shimadzu Nexera UPLC system (Kyoto, Japan) coupled with a diode array detector (DAD), and a Shimadzu LCMS-8030 quadrupole mass spectrometer (Kyoto, Japan) equipped with a electrospray ionization source (ESI). Analytical separation was performed on a reversed-phase Waters

Nova-Pak C18 column (4.9 × 250 mm, 4 μm) (Water Milford, MA, USA), operating at 30 ◦C. The mobile phase consisted of 1% aqueous formic acid (solvent A) and 1% formic acid in acetonitrile (solvent B). The elution gradient consisted of 3% B isocratic for 5 min, from 5 to 100% B linear for 30 min, 100% B isocratic for 7 min. The flow rate was 0.5 mL/min. The mass spectrometer operated in Negative Ion Scan and in Product Ion Scan mode, acquiring over a mass-range from *m*/*z* 50 to 1100 and using Argon as Collision Induced Dissociation (CID) gas at a pressure of 230 kPa. The interface voltage was set to −3.5 kV; desolvation line (DL) temperature was 250 ◦C and the heat block temperature was 400 ◦C.

Identification of the major secondary metabolites in the different extracts was carried out using their retention times, and both UV-VIS, MS and MS/MS spectra. Quantification of single compounds was performed by UPLC-DAD in triplicates through an external standard method, using stock solutions of the following compounds: Esculetin, p-coumaric acid, orientin, catechin, vitexin, (all from Sigma-Aldrich, Milan, Italy) and cucurbitacin I, cucurbitacin E, and apigenin-7-O-glucoside from Extrasynthese (Lyon, France). All solvents used for the analyses were purchased from Sigma-Aldrich (Milan, Italy).

#### *4.6. Fungal Strains, Media and Culture Condition*

*Aspergillus flavus* toxigenic strain Fri2 and atoxigenic strain TOφ used were previously isolated from corn fields of the Po Valley [61]. Conidia suspensions were obtained from 10-day YES-agar [2% (*w*/*v*) yeast extract (Difco, Detroit, MI, USA), 5% (*w*/*v*) sucrose (Sigma, St Louis, MO, USA), 2% (*w*/*v*) agar (Difco)] cultures incubated at 28 ◦C; conidia concentration (quantified by OD600) and viability (>90%) were determined according to Degola et al. [42]. Coconut milk-derived medium (CCM) used for microplate assays was obtained as described in Degola et al. [27]: Briefly, 400 mL of commercial coconut cream was diluted to the final volume of 1.2 L with bidistilled water, sterilized by autoclaving (10 min, 120 ◦C), cooled at 4 ◦C overnight, and clarified by centrifugation (15 min at 3200× *g*). The residual floating material and the pellet were discarded, and the intermediate phase was then recovered and used as culture medium in the aflatoxin inhibition assays.

#### *4.7. Aflatoxin Production Assay*

The extracts' effects on aflatoxin biosynthesis were assessed by the microplate fluorescence-based procedure described in Degola et al. [27]. Standard flat-bottom 96-well microplates (Sarstedt, Newton, NC, USA) were used. Suspensions of conidia were diluted to the appropriate concentrations and brought to the final concentration of 5 × 102 conidia/well; cultures were set in a final volume of 200 μL/well of CCM medium. *C. colocynthis* organic extracts were ethanol resuspended and added to the culture medium. The plates were incubated in the dark under stationary conditions for up to 6 days at 25 ◦C; visual inspection of mycelium development and conidiation served as an indicator of the culture growth. Total aflatoxin accumulation was monitored by fluorescence emission determination; readings were performed directly from the wells bottom of the culture plate with a microplate reader (TECAN SpectraFluor Plus, Männedorf, Switzerland) using the following parameters: λex = 360 nm; λem = 465 nm; manual gain = 83; lag time = 0 μs; number of flashes = 3; integration time = 200 μs). Inocula were performed in quadruplicate.

#### *4.8. Aspergillus Flavus Growth*

*A. flavus* radial growth was performed in YES-agar added with *C. colocynthis* extracts at 500 μg/mL final concentration: Three equidistant single spots (5 μL of a 107 conidia/mL suspension each) of aflatoxigenic strain Fri2 were inoculated in Petri dishes (9 cm Ø), plates were incubated for 4 days at 25 ◦C, and the mycelium growth was evaluated daily by measuring colonies reverse along two orthogonal diameters. YES-agar plates supplemented with 0.5% EtOH (*v*/*v*) were used as control. Early mycelium development was assessed, recording changes in optical density of liquid cultures over time: In a 96 wells microtiter plate (Sarstedt, Newton, NC, USA), 1 × 104 conidia were inoculated in a final volume of 200 μL of YES 5% liquid medium, added with 500 μg/mL organic extracts, and incubated at

28 ◦C. The optical density at 620 nm (OD620) was recorded for each well, using a microplate reader (RosysAnthos ht3; AnthosLabtec Instruments GmbH, Salzburg) without shaking. Experiments were performed in quadruplicate.

#### *4.9. Conidiation Rate Assessment and Reproductive Structures Analysis*

Conidia production was estimated for CHL and MET leaf, stem, and root extract treated cultures (500 μg/mL). From the eight replicates of each condition, four mycelia were collected from the CCM microplates used in the AF accumulation assay, and individually washed three times in a 0.1% (*v*/*v*) Tween20® aqueous solution by vortexing 1 min. The spore suspensions were then washed three times with a 80% (*v*/*v*) ethanol solution and conidia concentration was then determined with a Burker chamber. The remaining four CHL and MET leaf, stem, and root extract treated cultures from AF accumulation plates were observed using a Scanning Electron Microscope (SEM) JEOL IT 300, in high vacuum mode. Samples were fixed for 4 h in 3% (*v*/*v*) glutaraldehyde, acetone dehydrated (from 30% to 100% water/acetone solutions), critical point dried, and coated with a thin layer of gold by means of a Sputter Coater "Agar". Observations were conducted at an acceleration voltage of 10.0 kV and at a 450× magnification.

#### *4.10. Statistical Analysis*

The data were analyzed using the statistical and graphical function of PASW Statistics (SPSS Inc., Chicago, IL, USA). Differences were assessed using analysis of variance (ANOVA), followed by Dunnet-t post hoc test.

**Author Contributions:** Conceptualization, F.D., F.M.R., A.B., and B.M.; Investigation and Visualization, F.D.; Chemical Analyses, B.M., C.B., and A.G.; Electron Scanning Microscopy Analysis, L.D., F.D., and S.G.; Resources, B.M., F.M.R., C.B., and S.G.; Writing—Original Draft Preparation, F.D. and B.M.; Writing—Review & Editing, F.M.R., A.B., C.B., and A.G.

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

**Acknowledgments:** We are indebted to the Laboratoire de biologie végétale, Unité de Pharmaco-économie et développement des médicaments, Faculté de Pharmacie, Monastir (Tunisia) for providing the *C. colocynthis* organic extracts. We are also indebted to Massimo Cigarini for the technical help. We would like to thank Prof. Justice Johannson for the English language editing of proof.

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

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*


**5. Putting brain monitored propofol ketamine ("Goldilocks") anesthesia into practice**

*Teaching the technique is simple. Teaching cooperation is not.*

*Daniel HS Lin, DO, Cosmetic surgeon*

Along with the patient, many egos enter the operating room, not the least of which is that of the surgeon's. The surgeon's cooperation with local anesthesia is essential for success. The surgeon must also understand the need for *adequate* local anesthesia. **Surgeon perfection injecting local analgesia is not a requirement, only persistence.** Surgeons typically equate a blanched surgical field with adequate analgesia. Only with brain monitoring can the anesthesiologist hope to convince the surgeon that a blanched field only equals adequate vasoconstriction.

that it is resolved when the ointment is wiped from their eyes. One patient in the author's 40-year career became panic-stricken upon emergence thinking something bad had happened to her eyes. Full disclosure to avoid repetition of this unfortunate patient's experience

Brain Monitored Propofol Ketamine for Elective Cosmetic Surgery

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Ketamine increases salivation. Glycopyrrolate causes less tachycardia and, for that reason, is chosen as an antisialagogue in preference to atropine. Because propofol can sometimes cause patient discomfort, lidocaine is added to the glycopyrrolate. The patient is advised to tell the anesthesiologist if there is additional aching during induction so that more lidocaine can be

Between 1992 and 1997, a 50-ml IV bag with 400 mg (40 ml) propofol was connected to a 60 drops/ml IV set (mini-drip, pedi-drip, micro-drip). The initial rate was set to the patient's heart rate and adjusted until **loss of lid reflex** and **loss of verbal response** were observed. This was a **qualitative** approach that was far more subjective and less reproducible. After the demand for the propofol numbers for publication, the author began using an infusion pump. A completely programmable, albeit more expensive, infusion pump is better than less expensive, commonly found pumps that only permit settings of 25, 50, 75 or 100 mcg/kg/min. The ability to deliver a separate bolus in addition to maintaining an infusion rate is also important. This **quantitative** approach along with BIS/EMG monitoring makes "Goldilocks" anesthesia

Set the pump to deliver propofol for 50 mcg/kg minibolus. Induce with miniboluses given in *one after another* until a decrease in the real-time frontalis muscle electromyogram (EMG) is observed (see YouTube Going under with Goldilocks anesthesia). The BIS value and trend line will display behind the EMG as it is based on algorithm-derived information that is delayed

The induction goal is to observe whether or not 25 mcg/kg/min basal infusion rate is sufficient to maintain 60 < BIS < 75 with baseline EMG. Bolus with 50–100 mcg/kg to maintain BIS <75 and upwardly adjust the basal propofol rate to 35–50 mcg/kg/min to maintain that level PRIOR to administering ketamine. Most patients have been maintained at 60 < BIS < 75 with baseline EMG with propofol 25–50 mcg/kg/min. However, as little as 2.5 mcg/kg/min and as much as 200 mcg/kg/min propofol have also been observed by the author in his 20-year, >4000

Avoid the textbook advice of 1–2 mg/kg propofol bolus for induction. A bolus will produce peak propofol levels in the brain quickly and just as quickly begin to redistribute, decreasing a protective level just as the ketamine arrives to the brain. The brain may then not be protected from ketamine side effects when that drug is administered. The value of the brain monitored, incremental induction is providing a **numerically reproducible, stable propofol brain level to prevent negative ketamine side effects** [13]. The very sensitive as well as the very resistant patient can be identified from the outset of the case rather than trying to understand a body weight–derived dose effect. Lastly, the incremental propofol induction preserves baseline blood pressure as well as the muscles that maintain a patent airway*, i.e. the temporalis, masseter, orbicularis* and *genioglossus*. Nevertheless, one must always be prepared with available bag mask ventilation and suction for the exceptional patient, no matter how often it will be unnecessary.

administered. Propofol ache is more common with distally placed IV.

is strongly recommended.

numerically reproducible.

from real time by 15–30 s.

patient clinical experience.

Prompt essentially PONV free and minimal pain recovery are the hallmarks of "Goldilocks" anesthesia. Once the surgeon observes how much better his patients do postoperatively, the need for his/her cooperation should become apparent. The office-based setting has a higher incentive for surgeon cooperation. Recovery space is limited and personnel are often multitasked compared with free-standing or hospital-attached day surgery center facilities. As superior and **numerically reproducible** as the "Goldilocks" anesthesia outcomes are, there are still surgeons reluctant to cooperate. Just as disappointing is the numbers of anesthesiologists who remain shackled to the outdated teachings that ketamine causes hallucinations, hypertension and tachycardia or are unwilling to reconsider using real-time BIS/EMG to titrate propofol.

The clinical pathway for brain monitored propofol ketamine ("Goldilocks") anesthesia is a simple straightforward three-drug technique: glycopyrrolate, propofol and ketamine. Induce hypnosis first, next dissociate, then inject local analgesia (**Table 4**). (Atropine may be substituted when glycopyrrolate is unavailable. Tachycardia will more commonly result than administering glycopyrrolate). Unexplained tachycardia, not temperature elevation, is the cardinal recognition sign for MH. Once the brain is protected with propofol incrementally titrated to loss of lid reflex/loss of verbal response or 60<BIS<75 with baseline EMG, the 50mg ketaamine by itself does not cause tachycardia or hypertension!

Prior to placing the BIS sensor on the forehead, the patient is advised **the number from their forehead allows them to "control" their anesthetic dose**. This control means that neither too much nor too little can be given. Then the patient is encouraged to touch the sensor pad to assure them their skin cannot be scarred even though the sensor application will be a little uncomfortable. A small discomfort for a great relief about anesthetic dosing is greatly appreciated by patients. Patients are additionally advised that 5% or one in 20 may have pleasant, colorful dreams.

The preoperative consultation includes disclosure of the dry mouth and the need to maintain eye lubrication with ointment. Patients are told to expect blurry vision upon awakening and

Propofol infusion rate start 25 mcg/kg−1/min−1

Adjust infusion rate as needed to maintain 60 < BIS < 75 with baseline EMG

Ketamine 50 mg IV 3 minutes prior to local analgesia injection

Lidocaine 1 mg/lb−1 or 2 mg/kg−1 IV STAT for laryngospasm

Labetalol 10 mg iv for HR > 100–110. Avoid if patient asthmatic

**Table 4.** Clinical pathway.

Glycopyrrolate 0.2 mg with 30 mg lidocaine IV

Propofol 50–100 mcg/kg−1 minibolus repeated to 60 < BIS < 75 with baseline EMG

that it is resolved when the ointment is wiped from their eyes. One patient in the author's 40-year career became panic-stricken upon emergence thinking something bad had happened to her eyes. Full disclosure to avoid repetition of this unfortunate patient's experience is strongly recommended.

equate a blanched surgical field with adequate analgesia. Only with brain monitoring can the anesthesiologist hope to convince the surgeon that a blanched field only equals adequate

Prompt essentially PONV free and minimal pain recovery are the hallmarks of "Goldilocks" anesthesia. Once the surgeon observes how much better his patients do postoperatively, the need for his/her cooperation should become apparent. The office-based setting has a higher incentive for surgeon cooperation. Recovery space is limited and personnel are often multitasked compared with free-standing or hospital-attached day surgery center facilities. As superior and **numerically reproducible** as the "Goldilocks" anesthesia outcomes are, there are still surgeons reluctant to cooperate. Just as disappointing is the numbers of anesthesiologists who remain shackled to the outdated teachings that ketamine causes hallucinations, hypertension and tachycardia or are unwilling to reconsider using real-time BIS/EMG

The clinical pathway for brain monitored propofol ketamine ("Goldilocks") anesthesia is a simple straightforward three-drug technique: glycopyrrolate, propofol and ketamine. Induce hypnosis first, next dissociate, then inject local analgesia (**Table 4**). (Atropine may be substituted when glycopyrrolate is unavailable. Tachycardia will more commonly result than administering glycopyrrolate). Unexplained tachycardia, not temperature elevation, is the cardinal recognition sign for MH. Once the brain is protected with propofol incrementally titrated to loss of lid reflex/loss of verbal response or 60<BIS<75 with baseline EMG, the 50mg

Prior to placing the BIS sensor on the forehead, the patient is advised **the number from their forehead allows them to "control" their anesthetic dose**. This control means that neither too much nor too little can be given. Then the patient is encouraged to touch the sensor pad to assure them their skin cannot be scarred even though the sensor application will be a little uncomfortable. A small discomfort for a great relief about anesthetic dosing is greatly appreciated by patients. Patients are additionally advised that 5% or one in 20 may have pleasant,

The preoperative consultation includes disclosure of the dry mouth and the need to maintain eye lubrication with ointment. Patients are told to expect blurry vision upon awakening and

ketaamine by itself does not cause tachycardia or hypertension!

Propofol 50–100 mcg/kg−1 minibolus repeated to 60 < BIS < 75 with baseline EMG

Adjust infusion rate as needed to maintain 60 < BIS < 75 with baseline EMG

Ketamine 50 mg IV 3 minutes prior to local analgesia injection Lidocaine 1 mg/lb−1 or 2 mg/kg−1 IV STAT for laryngospasm Labetalol 10 mg iv for HR > 100–110. Avoid if patient asthmatic

vasoconstriction.

48 Anesthesia Topics for Plastic and Reconstructive Surgery

to titrate propofol.

colorful dreams.

**Table 4.** Clinical pathway.

Glycopyrrolate 0.2 mg with 30 mg lidocaine IV

Propofol infusion rate start 25 mcg/kg−1/min−1

Ketamine increases salivation. Glycopyrrolate causes less tachycardia and, for that reason, is chosen as an antisialagogue in preference to atropine. Because propofol can sometimes cause patient discomfort, lidocaine is added to the glycopyrrolate. The patient is advised to tell the anesthesiologist if there is additional aching during induction so that more lidocaine can be administered. Propofol ache is more common with distally placed IV.

Between 1992 and 1997, a 50-ml IV bag with 400 mg (40 ml) propofol was connected to a 60 drops/ml IV set (mini-drip, pedi-drip, micro-drip). The initial rate was set to the patient's heart rate and adjusted until **loss of lid reflex** and **loss of verbal response** were observed. This was a **qualitative** approach that was far more subjective and less reproducible. After the demand for the propofol numbers for publication, the author began using an infusion pump. A completely programmable, albeit more expensive, infusion pump is better than less expensive, commonly found pumps that only permit settings of 25, 50, 75 or 100 mcg/kg/min. The ability to deliver a separate bolus in addition to maintaining an infusion rate is also important. This **quantitative** approach along with BIS/EMG monitoring makes "Goldilocks" anesthesia numerically reproducible.

Set the pump to deliver propofol for 50 mcg/kg minibolus. Induce with miniboluses given in *one after another* until a decrease in the real-time frontalis muscle electromyogram (EMG) is observed (see YouTube Going under with Goldilocks anesthesia). The BIS value and trend line will display behind the EMG as it is based on algorithm-derived information that is delayed from real time by 15–30 s.

The induction goal is to observe whether or not 25 mcg/kg/min basal infusion rate is sufficient to maintain 60 < BIS < 75 with baseline EMG. Bolus with 50–100 mcg/kg to maintain BIS <75 and upwardly adjust the basal propofol rate to 35–50 mcg/kg/min to maintain that level PRIOR to administering ketamine. Most patients have been maintained at 60 < BIS < 75 with baseline EMG with propofol 25–50 mcg/kg/min. However, as little as 2.5 mcg/kg/min and as much as 200 mcg/kg/min propofol have also been observed by the author in his 20-year, >4000 patient clinical experience.

Avoid the textbook advice of 1–2 mg/kg propofol bolus for induction. A bolus will produce peak propofol levels in the brain quickly and just as quickly begin to redistribute, decreasing a protective level just as the ketamine arrives to the brain. The brain may then not be protected from ketamine side effects when that drug is administered. The value of the brain monitored, incremental induction is providing a **numerically reproducible, stable propofol brain level to prevent negative ketamine side effects** [13]. The very sensitive as well as the very resistant patient can be identified from the outset of the case rather than trying to understand a body weight–derived dose effect. Lastly, the incremental propofol induction preserves baseline blood pressure as well as the muscles that maintain a patent airway*, i.e. the temporalis, masseter, orbicularis* and *genioglossus*. Nevertheless, one must always be prepared with available bag mask ventilation and suction for the exceptional patient, no matter how often it will be unnecessary.

Only after the patient's propofol level has stabilized should the 50 mg ketamine, **independent of body weight**, be given, **3 minutes prior** to local analgesia injection. Any patient movement accompanied with an EMG spike during injection must be given more propofol to drive the EMG spike back to the baseline. As little as repeated 100 mcg/kg propofol boluses or as much as 200–400 mcg/kg propofol boluses may be required. An upward basal rate should be considered to maintain the patient's propofol level at 60 < BIS < 75 *with* baseline EMG.

saturation occurs. "Goldilocks" anesthesia provides numerically reproducible propofol levels to define adequate hypnosis as well as the absence of EMG spikes with local anesthesia injec-

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Propofol ketamine is like the martini cocktail wherein the propofol is the 'vodka' and the ketamine is the 'olive.' Ketofol is when the propofol in mixed with the ketamine. These are two very different approaches that should never be confused with one another. **Do not mix**

Propofol hypnosis is protective of ketamine hallucinations [13]. **Do not give ketamine before propofol hypnosis.** Bolus propofol induction will not provide a stable CNS propofol level to protect against ketamine hallucinations. Do not induce with 1–2 mg/kg propofol

Propofol at BIS < 75 with baseline EMG is a numerically reproducible protection against ket-

**Adequate local analgesia is critical** to minimizing or eliminating the need for postoperative opioid pain rescue. Do not fail to have the surgeon reinject the immediate area of dissection when patient movement occurs *without* EMG spike and BIS < 75. More propofol or ketamine is an inadequate response for patient movement at BIS < 75 with baseline EMG. Adding opioids instead of more local analgesia is not only inadequate to correctly deal with insufficient local

Aggregate ketamine doses >200 mg is associated with prolonged emergence. **Do not exceed 200 mg ketamine or give ketamine in the last 20 minutes** of the case or use ketamine in cases less than 20 minutes. Spontaneous ventilation is a prized safety feature of "Goldilocks" anesthesia. **Do not introduce an MH trigger like SCH to treat laryngospasm.** Because of the

Do not mix propofol and ketamine. Titrate separately to define goals of adequate hypnosis and midbrain NMDA

amine hallucinations. **Do not give ketamine if BIS > 75 or EMG is not at baseline.**

tion to define midbrain NMDA receptor saturation (**Table 5**).

analgesia but also increases the probability of PONV.

**propofol with ketamine.**

boluses.

saturation.

Do not give ketamine before propofol.

Do not fail to provide adequate local analgesia.

Do not give opioids in lieu of adequate local analgesia. Do not give ketamine in lieu of adequate local analgesia. Do not exceed an aggregate ketamine dose >200 mg.

Do not paralyze instead of using lidocaine to break laryngospasm. Do not use succinylcholine instead of lidocaine to treat laryngospasm.

Do not give ketamine in the last 20 minutes of a case or for cases <20 minutes duration.

Do not give ketamine at BIS > 75.

**Table 5.** Pitfalls to avoid.

Do not bolus propofol for induction. Incrementally induce.

The surgeon should inject as much of the proposed surgical field(s) with the initial ketamine dose whenever possible. Review of 1000 patient records demonstrated 80% were performed with one or two 50 mg ketamine doses. **Aggregate ketamine doses greater than 200 mg were associated with prolonged emergence**. Many surgeons are concerned that the lidocaine or epinephrine effect will not last if a lengthy procedure is contemplated. Long experience with Klein's ultradilute solution [21] has shown that concern is unwarranted even in 6-hour cases. Ketamine is to facilitate a painless injection of **'virgin' surgical fields** and is not necessary for reinjections when needed.


**4. Knowledge versus experience**

**2. Curriculum ideologies**

40 Contemporary Pedagogies in Teacher Education and Development

into existence a better society [20, 21].

**3. Research site and methods**

in daily school curriculum.

*centered* (child study), and the *social reconstruction* ideology.

play an important role in the success of any reform movement [25].

According to Schiro [20, 21], a curriculum ideology is a practical philosophy that influences a teacher's day-to-day behaviors toward curriculum issues. However, other scholars use the phrase curriculum orientation (e.g., [14, 19, 22, 23]). In the current study, I draw on Schiro's [20, 21] framework, in which the beliefs about the purpose of curriculum (education) are divided into four distinctive ideologies: the *scholar academic*, the *social efficiency*, the *learner-*

The scholar academic ideology is concerned with maintaining "cultural literacy" by having students study the content and modes of inquiry of traditional academic subjects. The social efficiency ideology aims at efficiently carrying out a task for society, providing knowledge and skills that give students the ability to function in society. The learner-centered ideology places the student in the center of the educational endeavor and is concerned with helping each student grow into a unique individual who has a healthy self-concept. The social reconstruction ideology attempts to help students understand the crises facing society, develop a value stance toward those crises, and learn how to act to relieve the crises, thereby bringing

According to Schiro [20, 21], these ideologies are roughly parallel to the academic, vocational, personal, and social goals for education. Each curriculum ideology is more akin to a pedagogical subculture than to a goal; each has an impact on teachers' thinking about the nature of knowledge, the curriculum intent (aims, goals, and learning objectives), teaching strategies, learning, students, and evaluation methods (e.g., [19–21, 25, 26]). They also affect students because the underlying values and beliefs of each ideology not only influence what is taught but also how and why it is taught [20, 27]. It has also been shown that curriculum ideologies

In connection with two undergraduate courses entitled "Inclusive Education" and "Teaching Practicum," the current study was conducted at a multidisciplinary research university within the Primary School Teacher Education (PSTE) program in Finland. All participants were enrolled in both courses. The participants were 115 pre-service teachers who were completing their teacher qualification for primary school. The "Inclusive Education" course focused on discussing the theoretical underpinnings of student diversity (such as language, abilities, background, religion, and gender) and the current instructional support system as a means of enhancing inclusive school culture. The students were encouraged to work in small teams to construct a shared understanding and build new ideas of how to enhance inclusion

In addition, the course included a week-long field experiment that asked students to visit one school community and find out how the inclusion principle was implemented in accordance with the NCC [9]. They were encouraged to observe the school community and classroom practices, conduct interviews with the in-service teachers, special needs teachers, school Although the basis of scholarly academic ideology rests on the belief that acquiring an understanding of academic knowledge involves learning the content knowledge and ways of thinking [20], the learner-centered ideology focus is solely on the student. The student's educational needs and interests are central to learning and must be incorporated in the learning experience. In the following, two pre-service teachers make meaning and reflect on their experiences of the teaching practicum. The former episode refers to a scholarly academic ideology and the latter one to a learner-centered ideology:

*It is not enough that you have understood and your pupils have understood the subject matter; rather everyone should even understand the same matter by the same time and in addition together. (…) I have been wondering, indeed, how I know my subject matter of school so badly. I must concentrate on using the right terms. I fail even with my well-practiced demonstrations. My authority feels insufficient. If the students take the upper hand, it all ends up in chaos. The problem with questions is that you prepare a new topic with questions, that you ask children questions about things they cannot have enough information about. (…) My personal opinion is that it is the teacher's task to teach.* (PRAC 89).

*There was a one student labeled as receiving intensified support in the Finnish class. This student sat in front of the classroom, in the middle of two other students. The lesson proceeded through a common conversation into the writing task. The task was made independently. Most of the teacher's time was spent in assisting that one 'intensified support' receiving student. Writing was not easy for him, and he needed the teacher's help frequently. So, the teacher had to go through the text word by word with him. When assisting that student all the time, the teacher did not have any time to go around the class during the performance of the assignment. She shouted the instructions to other students while sitting beside the demanding student, and the instructions were essentially to ensure that the class was disciplined, and all behaved in an appropriate manner. The demanding student suffered from the situation as well as the class that remained ignored by the teacher* (PRE 26).

Curriculum Ideologies Reflecting Pre-Service Teachers' Stances toward Inclusive Education

http://dx.doi.org/10.5772/intechopen.76326

43

*Some of us have obstacles that require more than normal enterprise and assistance, but despite that diversity is not an obstacle to moving forward or working together. This observation raised strongly during the field work. I looked at that so-called tomboy in the class, and I admired his way to see the world, in that case, the class, differently. I wondered, what kind of things he saw and felt. He was not cynical at all. I noticed that shared values were more important for him than the values of the individual. He gave his attention with a smile to the student sitting next to him, although he himself was in turbulent. He was empathic! I understood that the way how we usually differentiate learning and try to slick down these students' learning paths by taken them away from the mainstream classes, is not the way I believe in.* (PRE 101). The above episode refers to the preference of viewing the subject matter as the most valued property, which must be delivered to the students; as Schiro [20] stated, the curriculum is intended to acculturate children into a discipline. This inclination made pre-service teachers concerned about how they should accomplish goals regardless of the imbalance between mastering the subject matter and managing the classroom (cf. [32]). In turn, the latter episode illustrates that such a learner-centered ideology has the learning process as an end in itself. The view differs from an educational policy-driven paradigm of student centrality that emphasizes that curriculum should be characterized by "learning outcomes" describing the measurable skills or abilities that students should be able to do or demonstrate as a result of

This returns to a question about the focus of learning: should we pay attention to generic skills capacities instead of a detailed specification of content knowledge? Wheelahan [34] raised the view about the "crisis of curriculum," arguing that the worldwide education policy drives toward stripping knowledge out of the curriculum due to the shift in learning outcomes (cf. [34, 35]). But then, the current knowledge society and information overload within the explosion of digital technologies raise a critical question about the changing nature of knowledge and children's life worlds, meaning making, and learning as a whole. Accordingly, this leads to the question of which one is more valuable: the subject matter or generic skills. For example, there is literacy, which guarantees equal access to the most powerful forms of knowledge; literacy is crucial because it allows learners to gain these basic skills and builds students' discipline-specific understanding of how texts represent both the knowledge and the ways of knowing, doing, and believing in different disciplinary communities (cf. [36, 37]). Hence, the subject matter and generic skills are two sides of the same coin and should not be separated

learning (e.g., [33]).

from each other.

*A successful moment is when students are eager to learn new things and feel encouraged to use their new skills (…) when we experience a moment of flow together, when we experience joy and freedom. There was a kind of positive atmosphere in the classroom. I listened and gave them opportunities, and then I felt that I created security in a way that nobody laughed at anybody no matter what they said. It's important to stay positive.*

*All learning seems to be based on interaction (…) only in interaction first with another person and later with other persons will students get a mirror to look in and a frame in which he or she can develop.* (PRAC 08).

The episodes above illustrate the tension between knowledge and experience, which occurs between the traditional curriculum, developed to guide students on how to acquire knowledge and solve problems, and the learner-centered curriculum, advocated by constructivism as a way to emphasize an individual's activity and learning experience.

According to scholarly academic ideology, the meaning of knowledge is its ability to contribute to the extension of the academic discipline (cf. [20]). Thus, the subject matter is determined and selected from the knowledge bases of disciplines, such as biology or physics, by "specialist scholars" who are expected to bring down this knowledge to teachers and students. The impact on teachers is that they should also be specialists in the disciplines being taught, keeping the hierarchy between scholars at the top and students at the base.

In turn, the main idea of the learner-centered ideology is that learning is seen as an active process driven by the interest of the individual student. Schiro [20] exemplified the distinction to the previous ideology as "I have experienced" is more important than "I know," that is, the firsthand experience of reality is more important than the second-hand experience gained through a textbook. Curriculum is designed in line with Piagetian ideas about the stages of development, assuming that the construction of the learning process should be in harmony with students' cognitive stages (cf. [30, 31]). The impact on the teachers is that they are seen as facilitators of the discussion, debate, and interaction between students and their learning environment.

Although scholarly academic ideology prefers teaching methods such as Socratic questioning, didactic discussion and supervised practice, for the learner-centered ideologist, teaching is to be adapted to the individual student, and the student's authentic activity is seen as a prerequisite for education [20]. These distinctions between the stances on teaching are crucial in terms of inclusive education. The following episodes demonstrate two contrasting ways pre-service teachers observed and formed an attitude regarding the activities in the inclusive classroom. The first episode echoes scholarly academic ideology, even though the participant attempted to recognize the individual students, while the latter one refers to more learnercentered ideology:

*There was a one student labeled as receiving intensified support in the Finnish class. This student sat in front of the classroom, in the middle of two other students. The lesson proceeded through a common conversation into the writing task. The task was made independently. Most of the teacher's time was spent in assisting that one 'intensified support' receiving student. Writing was not easy for him, and he needed the teacher's help frequently. So, the teacher had to go through the text word by word with him. When assisting that student all the time, the teacher did not have any time to go around the class during the performance of the assignment. She shouted the instructions to other students while sitting beside the demanding student, and the instructions were essentially to ensure that the class was disciplined, and all behaved in an appropriate manner. The demanding student suffered from the situation as well as the class that remained ignored by the teacher* (PRE 26).

*It is not enough that you have understood and your pupils have understood the subject matter; rather everyone should even understand the same matter by the same time and in addition together. (…) I have been wondering, indeed, how I know my subject matter of school so badly. I must concentrate on using the right terms. I fail even with my well-practiced demonstrations. My authority feels insufficient. If the students take the upper hand, it all ends up in chaos. The problem with questions is that you prepare a new topic with questions, that you ask children questions about things they cannot have enough information about.*

*A successful moment is when students are eager to learn new things and feel encouraged to use their new skills (…) when we experience a moment of flow together, when we experience joy and freedom. There was a kind of positive atmosphere in the classroom. I listened and gave them opportunities, and then I felt that I created security in a way that nobody laughed*

*All learning seems to be based on interaction (…) only in interaction first with another person and later with other persons will students get a mirror to look in and a frame in which he or she*

The episodes above illustrate the tension between knowledge and experience, which occurs between the traditional curriculum, developed to guide students on how to acquire knowledge and solve problems, and the learner-centered curriculum, advocated by constructivism

According to scholarly academic ideology, the meaning of knowledge is its ability to contribute to the extension of the academic discipline (cf. [20]). Thus, the subject matter is determined and selected from the knowledge bases of disciplines, such as biology or physics, by "specialist scholars" who are expected to bring down this knowledge to teachers and students. The impact on teachers is that they should also be specialists in the disciplines being taught, keep-

In turn, the main idea of the learner-centered ideology is that learning is seen as an active process driven by the interest of the individual student. Schiro [20] exemplified the distinction to the previous ideology as "I have experienced" is more important than "I know," that is, the firsthand experience of reality is more important than the second-hand experience gained through a textbook. Curriculum is designed in line with Piagetian ideas about the stages of development, assuming that the construction of the learning process should be in harmony with students' cognitive stages (cf. [30, 31]). The impact on the teachers is that they are seen as facilitators of the

Although scholarly academic ideology prefers teaching methods such as Socratic questioning, didactic discussion and supervised practice, for the learner-centered ideologist, teaching is to be adapted to the individual student, and the student's authentic activity is seen as a prerequisite for education [20]. These distinctions between the stances on teaching are crucial in terms of inclusive education. The following episodes demonstrate two contrasting ways pre-service teachers observed and formed an attitude regarding the activities in the inclusive classroom. The first episode echoes scholarly academic ideology, even though the participant attempted to recognize the individual students, while the latter one refers to more learner-

discussion, debate, and interaction between students and their learning environment.

*(…) My personal opinion is that it is the teacher's task to teach.* (PRAC 89).

*at anybody no matter what they said. It's important to stay positive.*

as a way to emphasize an individual's activity and learning experience.

ing the hierarchy between scholars at the top and students at the base.

*can develop.* (PRAC 08).

42 Contemporary Pedagogies in Teacher Education and Development

centered ideology:

*Some of us have obstacles that require more than normal enterprise and assistance, but despite that diversity is not an obstacle to moving forward or working together. This observation raised strongly during the field work. I looked at that so-called tomboy in the class, and I admired his way to see the world, in that case, the class, differently. I wondered, what kind of things he saw and felt. He was not cynical at all. I noticed that shared values were more important for him than the values of the individual. He gave his attention with a smile to the student sitting next to him, although he himself was in turbulent. He was empathic! I understood that the way how we usually differentiate learning and try to slick down these students' learning paths by taken them away from the mainstream classes, is not the way I believe in.* (PRE 101).

The above episode refers to the preference of viewing the subject matter as the most valued property, which must be delivered to the students; as Schiro [20] stated, the curriculum is intended to acculturate children into a discipline. This inclination made pre-service teachers concerned about how they should accomplish goals regardless of the imbalance between mastering the subject matter and managing the classroom (cf. [32]). In turn, the latter episode illustrates that such a learner-centered ideology has the learning process as an end in itself. The view differs from an educational policy-driven paradigm of student centrality that emphasizes that curriculum should be characterized by "learning outcomes" describing the measurable skills or abilities that students should be able to do or demonstrate as a result of learning (e.g., [33]).

This returns to a question about the focus of learning: should we pay attention to generic skills capacities instead of a detailed specification of content knowledge? Wheelahan [34] raised the view about the "crisis of curriculum," arguing that the worldwide education policy drives toward stripping knowledge out of the curriculum due to the shift in learning outcomes (cf. [34, 35]). But then, the current knowledge society and information overload within the explosion of digital technologies raise a critical question about the changing nature of knowledge and children's life worlds, meaning making, and learning as a whole. Accordingly, this leads to the question of which one is more valuable: the subject matter or generic skills. For example, there is literacy, which guarantees equal access to the most powerful forms of knowledge; literacy is crucial because it allows learners to gain these basic skills and builds students' discipline-specific understanding of how texts represent both the knowledge and the ways of knowing, doing, and believing in different disciplinary communities (cf. [36, 37]). Hence, the subject matter and generic skills are two sides of the same coin and should not be separated from each other.


**4. Microbiological therapy**

#### **4.1 Pathogens of DFO**

Beside possible surgical intervention there is need for an antibacterial treatment. Choosing an active agent is always first empirical, and subsequently targeted to the microbiological culture results. Knowledge of the possible microorganisms is a precondition to an empirical therapy [35]. Dependent on the country, most isolates of DFO are *Staphylococcus aureus*, β-hemolytic streptococci, coagulasenegative staphylococci and Gram-negative pathogens such as *Pseudomonas aeruginosa* [36, 37]. Interestingly, the location of the infection is critical as well, so are calcaneal infections associated with *P. aeruginosa* in diabetic patients [19]. Unfortunately, despite the advocated greenish color of superficial *Pseudomonas* infections and a presumed characteristic smell of the infected wound, even longstanding clinicians cannot predict the presence of *P. aeruginosa* by visual and olfactory means alone. The microbiological laboratory assessment is still necessary [38]. Multi-resistant pathogens in DFO are increasing in frequency worldwide [39] such as extended-spectrum beta-lactamase-producing Gram-negative rods (ESBL). Compared to DFI without involvement of the bone a meta-analysis found a three times higher chance in DFO for isolating a multi-resistant pathogen [40]. Fungi are rarity. Enterococci are equally rare but relatively more prevalent in the infected diabetic foot compared to other osteoarticular infections in the body such as steptococci and staphylococci [41, 42]. It is important to recognize that in the DFO patient, we may retrieve any bacteria, including avirulent coagulase-negative staphylococci and corynebacteria. Unlike to other infections such as pneumonia or endocarditis, the causative pathogens can also change during the current therapy of DFO, by selection of new (more resistant) pathogens by the therapeutic antibiotics and iterative surgeries during treatment. Therefore, if ever there is surgery during ongoing systemic antibiotic treatment, we recommend to re-sample again. The incidence of such a new microbiological finding can be as high as 10% [43].

#### **4.2 Antibiotic therapy**

The systemic antibiotic therapy is – next to a possible surgery, iterative professional debridement, (podiatric) wound care, enhancement of the patient's compliance, and off-loading – always required, if the goal is the healing of DFO. A clear recommendation for a specific antimicrobial agent, or the general administration route, cannot be made. A lot of studies and meta-analyses failed to show a superiority of one specific drug against the others [44]. During the initial empirical treatment, we recommend to cover *S. aureus*. If the therapy fails to achieve a proper reduction of local inflammatory symptoms, then the therapy should be broadened to include (aerobic and anaerobic) Gram-negative bacteria [45]. In severe infections, (sub)tropical regions, or sepsis, a relatively broad empirical coverage targeting the local epidemiology of Gram-negative pathogens could be chosen [46] from the start. Further there is few data supporting parenteral therapy [47]. Of note, the microbiological culture results can lead to necessity of parenteral agents due to resistant pathogens.

Ideally, the DFO therapy is accompanied by professional debridement, or the resection of necrotic and infected bone (total amputation). A study of 50 patients with chronic toe DFO showed that patients with surgical resections had a significantly lower relapse rate. This was also witnessed in single-center survey with partial amputations [48]. In well-selected patients and neuropathic DFO cases without progressive ischemia, other studies report successful treatment

without surgery, with selected remission rates of 60–70% [49, 50]. When surgery is not necessary for various reasons, a strictly conservative antibiotic therapy is very reasonable. Of note, the proportion of antibiotic-related side effects in randomized-controlled DFO trials may compromise up to 20–30% of all systemic antibiotic DFO regimens [49–55].