**2. Overview of recent results**

#### **2.1. Graphene: basic properties**

for instance in natural photosynthesis [2] for efficient capturing and transport of the sun‐ light energy, and has been recently implemented in artificial light-harvesting assemblies [3]. The efficiency of ET depends on the spectral properties of a donor and an acceptor,their mutual orientation, as well as separation between them [4]. In particular, the distance dependence of the ET efficiency, which for localized dipoles scales with d−6, has been exploited as a useful tool to measure lengths at the nanoscale, both statically and dynamically. In particular, the energy transfer has been considered an attractive way to control light harvesting and bio(sensing) [5–7]. As a result, various strategies had to be devised to fabricate hybrid nanostructures with well-defined morphology required for controlling the energy transfer efficiency. Among the most feasible, one can find robust nanolayers, either polymer or dielectric, deposited on a surface of nanoparticles or a substrate [8, 9], and more flexible linkers

Another critical parameter that influences the interaction between two dipole moments in the context of the energy transfer is the relation of their spectral properties. Namely, as shown in **Figure 1**, the absorption of one of the molecules (acceptor) has to overlap with the emission of the second molecule (donor). The larger the overlap, the higher the efficiency of the energy transfer. The final parameter that has to be considered in energy transfer geometry is the mutual orientation of the dipole moments of a donor and an acceptor. All these factors are included in the equation shown in **Figure 1**. It is important to note that the equation de‐

**Figure 1.** Schematic representation of the energy transfer between two dipole moments of a donor (green) and acceptor (red). The energy transfer is possible only when the emission of the donor overlaps with the absorption of the acceptor.

Optical spectroscopy, and in particular fluorescence spectroscopy, provides a variety of tools to probe the energy transfer in hybrid nanostructures. It stems from the fact that the emer‐ gence energy transferresults in the decrease of the emission intensity of a donor at the expense emission intensity of an acceptor. This is in fact the most straightforward consequence energy transfer between two nanostructures. In addition to the intensity flow between donors and acceptors, another signature of the energy transfer is a shortening of the fluorescence decay time of the donor. Indeed, the energy transfer can be considered as a new channel for nonradiative recombination from the point of view of the donor, and as such it should result in

shortening of the lifetime.

152 Recent Advances in Graphene Research

based for instance on DNA strands or biotin-streptavidin conjugation [10, 11].

scribes the case where both donors and acceptors are classical dipole moments.

Graphene is nowadays one of the most intensively studied materials. Since 2004, when it was for the first time obtained by mechanical exfoliation, many research groups worldwide have focused on understanding and proving uniqueness of this one-atom thin material [12, 13]. Part of these efforts were inspired by the combination of properties rarely met in any other material, such as exceptionally high electronic and thermal conductivity, mechanical strength, unusu‐ al electronic structure and opticaltransmittance, impermeability to gases and many others [14].

The key property of graphene, which impacts its electronic and optical character, and is particularly important in the context of light-matter interactions, is an unusual zero bandg‐ ap structure and linear dispersion near the Brillouin zone corners. Indeed, in the case of graphene, the conduction and valence bands meet at Dirac points and in their vicinity the energy depends linearly on the wavevector [15]. Consequently, both electrons and holes mimic massless relativistic particles with effective velocity of *c* \* ≈ 1/300 the speed of light.

Fully occupied valence band combined with an empty conduction band, and no energy gap between them, leads to unique electronic absorption of graphene. Remarkably, it is rather high, and for a suspended graphene monolayer is defined solely by the fine-structure constant, which translates to 2.3% absorption of incident light (**Figure 2**) [16]. In addition, the absorp‐ tion of graphene shows no wavelength dependence from ultraviolet to near-infrared. Therefore, graphene sheets can be visualized using optical microscopy, as shown in **Figure 2**, and the absorption of a few-layer graphene can be roughly described as a sum of noninteracting single layers with each layer contributing 2.3% of opacity. From the point of view of hybrid assemblies where the energy transferis exploited, graphene can be utilized as energy acceptor due to uniform absorption, but at the same time, it features no fluorescence emis‐ sion. Consequently, any effects attributable to the energy transferfrom any emitterto graphene will have to be probed and quantified based solely on the behaviour of energy donors.

**Figure 2.** (A) Photograph of a 50-mm aperture partially covered by monolayer and bilayer graphene. The line-scan profile shows the intensity of transmitted white light along the yellow line. (Inset) Sample design. (B) Transmittance spectrum of single-layer graphene (open circles). The red line is the transmittance expected for two-dimensional Dirac fermions, whereas the green curve takes into account a non-linearity and triangular warping of graphene's electronic spectrum. (Inset) Transmittance of white light as a function of the number of graphene layers (squares). From Nair et al. [16]. Reprinted with permission from AAAS.

#### **2.2. Graphene: fabrication methods**

There are several ways of obtaining graphene and its derivatives, with each method holding specific advantages depending on particular application [17], but also facing limitations from the point of view of scalability, cost, reproducibility and alike.

First experiments on graphene have been carried out for the highest-quality pristine gra‐ phene obtained by mechanical exfoliation. This method, which is still arguably the leading one, allows preparing pure hexagonal carbon lattice without defects or dopants, thus exfoli‐ ated graphene has been at the centre of fundamental studies of its properties [16, 18]. It suffers, however, from small and irregular sample sizes and shapes, low throughput and high prices.

Growing demand for larger and reproducible graphene pieces has resulted in the develop‐ ment of other methods used for the production of two-dimensional (2D) carbon materials. Epitaxial techniques, by both sublimation and chemical vapour deposition (CVD), are the most promising due to the comparably high quality of fabricated graphene combined with high reproducibility and scalability. The first approach involves decomposition of SiC in low pressures (or ultra-high vacuum) and high temperatures. Addition of the annealing in argon atmosphere significantly improves homogeneity of graphene [19]. Upon sublimation of silicon from SiC surface, the remaining carbon atoms form graphene layers. Although the price of SiC substrate is relatively high, it could be compensated by excellent electronic parameters of graphene and performance of fabricated devices [13].

An alternative approach to produce graphene is CVD, which involves deposition of hydro‐ carbons onto a transition metal surface, usually copper or nickel, which works as a catalyst [20, 21]. Due to the differences of solubility of carbon in Ni and Cu, the processes also differ from each other. As the solubility of carbon in Cu is rather low, the formation of graphene layers weakly depends on the actual conditions of the growth process. This allows for a better control over graphene growth, and when the monolayer is formed the deposition process stops. For this reason, large domains of single graphene layers are formed (>95% of the surface) [20]. Although graphene growth on Cu displays some disadvantages, such as wrinkles and grain boundaries, it is a relatively simple and inexpensive approach, suitable for mass production of high-quality graphene. Importantly, graphene grown on metallic surfaces can be transferred via polymer-assisted method on any arbitrary substrate, what is important for constructing graphene-based devices (photovoltaic cells, transistor, etc.) [22].

For uniform graphene layers obtained using exfoliation or CVD, hybrid assemblies for studying the energy transfer have to be constructed in a layer-by-layer geometry, where emitters are deposited onto the graphene layer (or on a polymer/oxide layer that is sup‐ posed to separate emitters from graphene).

In addition to methods that allow for growing large-area uniform graphene with wellcontrolled number of layers, other approaches have also been developed, based on the concept of liquid phase exfoliation. The approach of synthesizing two-dimensional carbon flakes in liquid relies on the reduction of graphene oxide (GO) [23] and key advantages thereof include solubility in aqueous and organic solvents, easy processing and surface functionalization, cheap synthesis for scalable production and relatively mild conditions of synthesis [24].

**Figure 2.** (A) Photograph of a 50-mm aperture partially covered by monolayer and bilayer graphene. The line-scan profile shows the intensity of transmitted white light along the yellow line. (Inset) Sample design. (B) Transmittance spectrum of single-layer graphene (open circles). The red line is the transmittance expected for two-dimensional Dirac fermions, whereas the green curve takes into account a non-linearity and triangular warping of graphene's electronic spectrum. (Inset) Transmittance of white light as a function of the number of graphene layers (squares). From Nair et

There are several ways of obtaining graphene and its derivatives, with each method holding specific advantages depending on particular application [17], but also facing limitations from

First experiments on graphene have been carried out for the highest-quality pristine gra‐ phene obtained by mechanical exfoliation. This method, which is still arguably the leading one, allows preparing pure hexagonal carbon lattice without defects or dopants, thus exfoli‐ ated graphene has been at the centre of fundamental studies of its properties [16, 18]. It suffers, however, from small and irregular sample sizes and shapes, low throughput and high prices. Growing demand for larger and reproducible graphene pieces has resulted in the develop‐ ment of other methods used for the production of two-dimensional (2D) carbon materials. Epitaxial techniques, by both sublimation and chemical vapour deposition (CVD), are the most promising due to the comparably high quality of fabricated graphene combined with high reproducibility and scalability. The first approach involves decomposition of SiC in low pressures (or ultra-high vacuum) and high temperatures. Addition of the annealing in argon atmosphere significantly improves homogeneity of graphene [19]. Upon sublimation of silicon from SiC surface, the remaining carbon atoms form graphene layers. Although the price of SiC substrate is relatively high, it could be compensated by excellent electronic parameters of

An alternative approach to produce graphene is CVD, which involves deposition of hydro‐ carbons onto a transition metal surface, usually copper or nickel, which works as a catalyst

al. [16]. Reprinted with permission from AAAS.

154 Recent Advances in Graphene Research

**2.2. Graphene: fabrication methods**

the point of view of scalability, cost, reproducibility and alike.

graphene and performance of fabricated devices [13].

Briefly, the process usually starts with oxidizing graphite using one of the many popular oxidation methods: Hummers, Brodie or Staudenmaier [25, 26]. In the next step, due to the presence of oxygen-containing functional groups (hydroxyls, carbonyls, carboxyls or oxygen epoxides), graphite oxide can be easily exfoliated into GO via ultrasonication or mechanical stirring.  However, it is an ideal precursor for the synthesis of reduced GO (rGO), also called chemical graphene. Important‐ ly, by reducing GO, and thus restoring the sp2 -carbon network without additional compo‐ nents and residues [50, 51], it is possible to not only diminish fluorescence but also retrieve electrical and thermal conductivity. In contrast to graphene, rGO can be dispersed in water, and preparation of rGO flakes in solution makes it feasible to incorporate various functional groups on the surface, as required for many applications.

Among the key challenges is the establishment of reproducible methods of fabricating largearea rGO flakes and assuring the control of the number of layers, although these two factors frequently compete against each other: it is difficult to fabricate large single-layer rGO. The most commonly applied strategies are mild oxidation conditions, which promote the forma‐ tion of larger flakes simply by reducing cracking of graphite flakes [27], adding a pretreat‐ ment step (an incubation in sulphuric acid with gentle stirring) before oxidation [28], or even skipping sonication to avoid breakage of flakes [29]. An important step is a separation of large flakes from aggregates and smaller sheets, and this can be performed with a high-speed centrifugation [27, 30, 31]. Also, GO might be deposited on a substrate using LangmuirBlodgett technique or self-assembling, before subsequent reduction, either chemically or thermally, to prevent re-aggregation of rGO flakes [29, 32].

From the point of view of the energy transfer, rGO offers important degree of freedom as compared to epitaxial graphene, namely it is possible to prepare mixtures in solution, which is often technically simpler and more suitable for—for instance—fluorescence-sensor design.

#### **2.3. Graphene as energy acceptor**

Constant absorption covering ultraviolet, visible and near infrared spectral regions (300–2500 nm), together with unusual electronic structure, renders graphene as an exceptional energy acceptor. It is possible to obtain not only energy transferfrom any emitterto graphene but also the zero-energy gap of graphene implies that any interactions in graphene-based hybrid nanostructures can be investigated exclusively by studying optical properties of a donor. These properties result in a unique potential of graphene as a component of devices designed for photonics, optoelectronics, as well as photodetectors and biosensors. In particular, in recent years several studies emerged, where the energy transfer from various emitters to graphene has been investigated.

**Figure 3.** Images of the same area of the sample containing a monolayer graphene sheet (darker field on the left image) obtained with optical microscope, fluorescence microscope, where the emission intensity of Rhodamine dyes was measured, and fluorescence lifetime imaging microscope, where decay times of fluorescence were measured. Signifi‐ cant decrease of the intensity of Rhodamine on graphene is accompanied with drastic reduction of the decay time. From Gaudreau et al. [33]. Reprinted with permission from ACS.

One of the first hybrid structures where the energy transfer to graphene was investigated comprised a layer of Rhodamine molecules on a graphene flake [33], as shown in **Figure 3**. The position and shape of the graphene flake can be determined by optical microscopy and next by collecting a fluorescence image of the same area, any influence of the presence of gra‐ phene on the fluorescence properties of Rhodamine can be extracted. In this work [33], the authors included also time-resolved fluorescence lifetime-imaging microscopy. In order to study the dependence of the energy transfer on the distance between the emitters and graphene, a layer of PMMA polymer with a thickness from 5 to 20 nm was deposited on graphene. As can be seen in **Figure 3**, for molecules placed on graphene the fluorescence intensity is significantly less as compared to the reference. This decrease of emission intensi‐

ty is accompanied with strong reduction of fluorescence lifetime. Both these spectral signa‐ tures are indication of the energy transfer from Rhodamine to graphene. Estimated maximum efficiency of the energy transfer was equal to 99% and it decreased with the spacer thickness. It was experimentally proved that the energy transfer to graphene scales with the distance as d-4, which is different from the classic case of two interacting dipole moments. This differ‐ ence is indicative of the universal value of the optical conductivity (assigned to gapless and 2D lossy media) and is in agreement with theoretical results obtained by Sebastian and Swathi [34]. There are two major consequences of these results: high efficiencies of the energy transfer indicate that in order to study energy transfer from a dye to graphene it might be critical to use a spacer, as otherwise fluorescence of the emitters can be totally quenched. There are, however, examples, where particular orientation of the molecules on a graphene surface partially inhibited the energy transfer [35]. Furthermore, weaker relation between the decay rate and distance can make it possible to investigate donor-acceptor interactions for distan‐ ces unavailable for pairs of two classical dipoles.

Blodgett technique or self-assembling, before subsequent reduction, either chemically or

From the point of view of the energy transfer, rGO offers important degree of freedom as compared to epitaxial graphene, namely it is possible to prepare mixtures in solution, which is often technically simpler and more suitable for—for instance—fluorescence-sensor design.

Constant absorption covering ultraviolet, visible and near infrared spectral regions (300–2500 nm), together with unusual electronic structure, renders graphene as an exceptional energy acceptor. It is possible to obtain not only energy transferfrom any emitterto graphene but also the zero-energy gap of graphene implies that any interactions in graphene-based hybrid nanostructures can be investigated exclusively by studying optical properties of a donor. These properties result in a unique potential of graphene as a component of devices designed for photonics, optoelectronics, as well as photodetectors and biosensors. In particular, in recent years several studies emerged, where the energy transfer from various emitters to graphene

**Figure 3.** Images of the same area of the sample containing a monolayer graphene sheet (darker field on the left image) obtained with optical microscope, fluorescence microscope, where the emission intensity of Rhodamine dyes was measured, and fluorescence lifetime imaging microscope, where decay times of fluorescence were measured. Signifi‐ cant decrease of the intensity of Rhodamine on graphene is accompanied with drastic reduction of the decay time.

One of the first hybrid structures where the energy transfer to graphene was investigated comprised a layer of Rhodamine molecules on a graphene flake [33], as shown in **Figure 3**. The position and shape of the graphene flake can be determined by optical microscopy and next by collecting a fluorescence image of the same area, any influence of the presence of gra‐ phene on the fluorescence properties of Rhodamine can be extracted. In this work [33], the authors included also time-resolved fluorescence lifetime-imaging microscopy. In order to study the dependence of the energy transfer on the distance between the emitters and graphene, a layer of PMMA polymer with a thickness from 5 to 20 nm was deposited on graphene. As can be seen in **Figure 3**, for molecules placed on graphene the fluorescence intensity is significantly less as compared to the reference. This decrease of emission intensi‐

From Gaudreau et al. [33]. Reprinted with permission from ACS.

thermally, to prevent re-aggregation of rGO flakes [29, 32].

**2.3. Graphene as energy acceptor**

156 Recent Advances in Graphene Research

has been investigated.

**Figure 4.** Optical and fluorescence images of individual nanocrystals on single-layer graphene and on the quartz sub‐ strate; (b) optical reflectivity image in the emission range of nanocrystals; (d) wide-field fluorescence image of individ‐ ual CdSe-ZnS nanocrystals in the region shown in panel (b). The colour scale bar indicates the number of emitted photons (in arbitrary units) integrated over 30 s. From Chen et al. [36]. Reprinted with permission from ACS.

An important advancement into elucidating the energy transfer to graphene was experimen‐ tal observation of fluorescence quenching of individual semiconductor nanocrystals [36]. For this experiment, micrometer-size sheets of graphene monolayer were used, on which highly diluted solution of CdSe/ZnS nanocrystals was deposited (**Figure 4**). By combining optical and fluorescence imaging, it was possible to correlate the differences in fluorescence intensity of individual nanocrystals with the locations where graphene was present. The result was dramatic (70-fold) quenching of fluorescence intensity for nanocrystals placed on graphene. Moreover, the quenching efficiency was found to increase with a number of graphene layers. This observation was explained using a simple model of a few-layer graphene, in which weak interactions between layers can be neglected, so the quenching factor is calculated for *n* layers of non-interacting single graphene planes. The significance of these results is in removing any

ensemble averaging that is always present in experiments performed on layers, as the one described above. In addition, when layers of emitters are studied, there is always a certain thickness of this layer, across which the strength of the interaction, in this case the energy transfer, varies, sometimes considerably. As a result, an average effect is measured in fluorescence microscopy experiment, similarly as in the case of plasmonic interactions associated with metallic nanoparticles [37]. In this context, the value of single nanocrystal experiment manifests itself in the observation of suppression of fluctuations of fluorescence intensity of single nanocrystals upon deposition on graphene. This indicates the effect of graphene-induced fluorescence quenching also on the photophysics of the nanocrystals.

Very recently, both concepts were combined in a single experiment, where distance depend‐ ence of the energy transfer rate to a monolayer graphene was studied for individual emitters, both zero-dimensional CdSe/CdS nanocrystals and two-dimensional CdSe/CdS/ZnS nanopla‐ telets [38]. Both types of energy donors were separated from graphene with ultrasmooth dielectric film of magnesium oxide with a thickness varied from just a few Å up to several tens of nanometers. In terms ofradiative energy transfer efficiency (>95%), both structures exhibited similar behaviour upon direct coupling to graphene. Important differences appear when emitters are separated from graphene with a spacer. While for zero-dimensional nanocrys‐ tals the energy transfer rate scales with a distance according to d-4 law, the energy transfer rate of the two-dimensional platelet decays is affected to a lesser degree. This is explained by a theoretical model, which includes the contribution of thermal distribution of free excitons in a two-dimensional quantum well at finite temperatures. The results confirm that graphenenanocrystal hybrid structure, governed by both charge transfer and Förster-type resonant energy transfer, is a suitable system to explore the influence of exciton dimensionality and localization, as well as distance, on the energy transfer rate (**Figure 5**).

**Figure 5.** (a) Selected luminescence decays of individual CdSe/CdS nanocrystals separated from graphene by an MgO spacer with increasing thickness. The thin black lines are fits based on biexponential decays convoluted with the in‐ strument response function. (b) Statistically averaged measured decay rate *γ* as a function of the thickness of the MgO spacer. (c) Statistically averaged product of the number of emitted photons per exciting laser pulse *N*em and the decay rate *γ*. From Federspiel et al. [38]. Reprinted with permission from ACS.

Very efficient energy transfer from organic dyes to graphene-based materials has also been used to visualize the structure and determine the morphology of graphene-dye hybrids.

Fluorescence quenching microscopy is a practical tool for detecting and mapping of gra‐ phene flakes [39]. Contrary to Raman spectroscopy or optical techniques, in this case an additional component of energy donor is necessary. This is compensated by significantly increased contrast, as well as faster and more sensitive data recording of large areas com‐ pared to optical imaging (**Figure 6**) and Raman spectroscopy.

**Figure 6.** Images of mechanically exfoliated graphene on a SiO2/Si substrate taken by (a) AFM, (b) optical microscopy and (c) FQM using PVP/fluorescein. All scale bars = 10 μm. From Kim et al. [39]. Reprinted with permission from ACS.

Similarities between graphene and rGO are also reflected in their role as energy acceptors. However, in contrast to graphene, rGO is used most frequently as a fluorescence quencher in solutions, instead of in layer-by-layer geometries. This method, although less ordered and less controllable, may be advantageous for increasing energy/charge transfer efficiency in rGObased assemblies. Reduced graphene oxide has been studied as an efficient fluorescence quencher of polymers [40, 41], quantum dots [42], dye-labelled aptamer [43] and also in hybrid nanostructures involving photosynthetic complexes [44]. While graphene-based hybrid structures are applied primarily for fundamental studies and to define parameters that determine energy/charge transfer, in the case of rGO composites the main focus is on poten‐ tial devices and applications and optimization of their performance. Such hybrid nanostruc‐ tures are considered promising for easy and relatively cheap scalable mass production of biosensors, as well as light-harvesting and optoelectronic platforms.


**1. Introduction**

The antibiotic crisis is now well acknowledged worldwide as a serious health problem. Antibiotics which enabled saving millions of lives in the world since their discovery are now facing a rapid decrease in efficiency due to the bacterial resistance crisis. The reasons usually reported to explain such a situation include the overuse and misuse of conventional antibiotics, and their inappropriate prescription. Moreover, possibilities to refill the antibiotic pipeline will be very limited in the near future due to reduced economic

**Citation:** Madi-Moussa, D.; Deracinois, B.; Teiar, R.; Li, Y.; Mihasan, M.; Flahaut, C.; Rebuffat, S.; Coucheney, F.; Drider, D. Structure of Lacticaseicin 30 and Its Engineered Variants Revealed an Interplay between the N-Terminal and C-Terminal Regions in the Activity against Gram-Negative Bacteria. *Pharmaceutics* **2022**, *14*, 1921. https://doi.org/10.3390/ pharmaceutics14091921

Academic Editors: Scavello Francesco, Jean-Eric Ghia and Amiche Mohamed

Received: 3 July 2022 Accepted: 7 September 2022 Published: 12 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

41

incentives expected by the major pharmaceutical companies [1,2]. In 2014, antimicrobial resistance (AMR) was estimated to cause 10 million deaths per year by 2050 [3]. To face this overwhelming situation, numerous alternatives to antibiotics have been explored, among which bacteriophage therapy [4,5], predatory bacteria [6], competitive exclusion of pathogens [7] and bacteriocins [8–10] are included. These approaches offer clear advantages, such as their specificity and low detrimental impact on beneficial microbial communities, unlike antibiotics which generally have collateral damage on commensal bacteria [11].

Bacteriocins are ribosomally synthesized antimicrobial peptides (AMPs) produced by Gram-positive and Gram-negative bacteria as well as Archaea [12,13]. They exhibit extensive variations in their molecular masses, inhibitory spectra, modes of action, and mechanisms of biosynthesis, export and self-protection of the producing strains [14]. These AMPs are considered to be significant actors of microbial competitions because of their role in colonizing niches, killing competing strains and their use of cross-talk or quorum-sensing networks within bacterial communities [15–17]. Bacteriocins from Gram-positive bacteria are predominantly produced by lactic acid bacteria (LAB). These bacteriocins, referred to here as LAB-bacteriocins, are safe for cells from the Eukarya domain [18,19]. They show most often a narrow spectra of activity, acting therefore selectively on members of species identical or closely related to the producer, and in rarer cases they exhibit broad spectra, thus targeting other species [20,21]. During the last few years, several classifications of LAB-bacteriocins have been proposed [17,22,23]. Among those, the classification proposed by Alvarez-Sieiro et al. [22], dividing LAB-bacteriocins into three main classes, is largely used. According to this classification, the class I of bacteriocins includes peptides that have undergone extensive post-translational modifications during their biosynthesis, resulting in the introduction of rare amino acids, such as lanthionines that are present in lanthipeptides. Class II includes unmodified bacteriocins having molecular masses below 10 kDa, while class III contains thermo-labile unmodified bacteriocins of more than 10 kDa with a bacteriolytic or non-lytic mechanism of action [23]. Depending on their structural and functional characteristics, many LAB-bacteriocins act on the cytoplasmic membrane of target bacteria by forming pores, leading to the leakage of ions and small essential molecules, or by degrading the cell walls. With continuing research, LAB-bacteriocins have been allocated with further functions such as antiviral activity or inhibition of proliferation of unscheduled and unregulated tumor cell lines [17,24]. Gram-negative bacteria are generally resistant to LAB-bacteriocins due to their outer membrane, which confers upon them supplementary protection against antimicrobial agents. Nonetheless, a limited number of LAB-bacteriocins possessing activity against Gram-negative bacteria, including *Escherichia coli,* have been reported in the literature [25–27]. However, if the mode of action of LAB-bacteriocins against Gram-positive bacteria is globally well documented [8,28], while their action against Gram-negative bacteria remains to be understood.

Here, we focused on *Lacticaseibacillus paracasei* CNCM I-5369, a strain isolated from an Algerian dairy product recently shown to produce five class II bacteriocins endowed with activity at pH 5 against Gram-negative bacteria including *E. coli* strains resistant to colistin [29]. Moreover, we have heterologously produced each of these bacteriocins, which are encoded by *orf010*, *orf012*, *orf023*, *orf030* and *orf038*. The bacteriocin encoded by *orf030*, here referred to as lacticaseicin 30, was obtained in large quantities in the soluble fraction, contrarily to the other produced peptides [30], and was shown to exhibit potent activity against *E. coli*.

In this study, the structure-activity relationship of lacticaseicin 30 was investigated. For this purpose, the predicted secondary structure of this bacteriocin, which includes five helices distributed over the 111 amino acid sequence, was used to design a series of variants, exhibiting either truncated sequences including one helix, two helices or three helices, or specific point mutations. All lacticaseicin 30 variants were assessed for their anti-Gram-negative activity.


**4.3 Colon cancer**

Ng et al. from Singapore found 58 studies that provided incidences of colorectal adenoma and cancer and cancer survival outcomes and found that metformin significantly lowered the risk of colorectal adenoma (RR 0.77, CI 0.67–0.88, *P* < 0.001), advanced adenoma (0.61, CI 0.42–0.88, *P* = 0.008) and colorectal cancer (RR 0.76, CI 0.69–0.84, *P* < 0.001) respectively. Overall cancer survival (HR 0.6, CI 0.53–0.67, *P* < 0.001), even among metastatic cases was also higher among metformin users (HR 0.77, CI 0.68–0.87, *P* < 0.001), and it was concluded that metformin significantly reduces colorectal adenoma and cancer incidence as well as enhanced colorectal cancer survival at all stages [78].

#### **4.4 Endometrial cancer**

In 19 studies reviewed in 2017, metformin used reversed atypical endometrial hyperplasia to normal, and decreased cell proliferation from 51.94% (CI = 36.23% to 67.46%) to 34.47% (CI = 18.55% to 52.43%) [79], while separately, a review of seven studies showed that metformin could significantly improve overall survival of in endometrial cancer (HR = 0.61, 95% CI 0.48–0.77, P < 0.05) and reduce their recurrence risk (OR = 0.50, 95% CI 0.28–0.92, *P* < 0.05) [80], whereas another review of six retrospective cohorts if 4723 endometrial cancer cases demonstrated that metformin use was associated with a significant reduction in overall mortality in comparison with not using metformin (adjusted HR 0.64, 95% CI 0.45–0.89, *P* = 0.009) irrespective of diabetic status [81], and these results corroborated the improved overall (HR, 0.58; 95% CI, 0.45–0.76; *P* = 0.207) as well as progression free survival (HR, 0.61; 95% CI, 0.49–0.76; *P* = 0.768) found in another review of 6242 patients from fourteen studies [82].

#### **4.5 Lung cancer**

An analysis of 13 observational studies found lung cancer incidence to be reduced in diabetic patients on metformin vs. no metformin (RR = 0.89; 95% CI, 0.83–0.96; *P* = 0.002) [83]. A separate meta-analysis found six studies comparing metformin usage and non-metformin usage significantly improved overall survival in diabetic patients with NSCLC [pooled HR =0.87 (0.77–0.99), *P* = 0.04] [84]. Especially noteworthy was an ambitious prospective clinical trial conducted by Marrone et al. which studied non-diabetics with advanced or metastatic NSCLC receiving platinum-based doublet chemotherapy and bevacizumab with or without metformin 1000 mg twice daily followed by maintenance therapy with bevacizumab and metformin combined or bevacizumab alone and showed a significant clinical benefit in PFS (9.6 vs. 6.7 months) with the addition of metformin [63].

#### **4.6 Pancreas cancer**

A review of seventeen studies involving 36791 participants study has evidenced a significant association of metformin adjuvant treatment in pancreas cancer with overall survival benefit (HR = 0.88, 95% CI = 0.80–0.97) especially in Asians, those with early stage disease and those undertaking surgery [85]. In terms of overall survival with metformin use in pancreas cancer, a study of 8 retrospective cohort studies and 2 randomized clinical trials representing 3,042 patients revealed overall survival to be improved with metformin (meta-HR = 0.79; 95% CI: 0.70, 0.92, *P* < 0.001) [86].

#### **4.7 Prostate cancer**

In a systematic review involving eleven studies with 877,058 patients, the odds ratio of metformin use for reducing prostate cancer was estimated at 0.89 (95%CI: 0.67–1.17) and it was concluded that metformin consumption reduced the risk of prostate cancer, although the result was not statistically significant [87]. Separately, a review of eight studies on diabetic patients with prostate cancer found no metformin use was associated with an increased risk of cancer recurrence (RR, 1.20; 95% CI, 1.00–1.44) [88], which concurs with another review of eight retrospective cohort studies and one nested-case–control study, metformin was found to be associated with a reduced risk of biochemical recurrence (pHR: 0.82, 95% CI 0.67, 1.01, *P* = 0.06) [89]. Finally, a large review of 30 cohort studies, including 1,660,795 prostate cancer patients revealed that metformin treatment compared with no treatment improved overall, prostate cancer specific, and recurrence free survival (HR = 0.72, 95% CI: 0.59–0.88, *P* = 0.001; HR = 0.78, 95% CI: 0.64–0.94, *P* = 0.009; and HR = 0.60, 95% CI: 0.42–0.87 *P* = 0.006, respectively) [90].

#### **4.8 Ovarian cancer**

*One review of 13 studies involving ovarian cancer incidence and prognosis revealed metformin use to be associated with* a lower incidence (pooled OR 0.76, 95% CI 0.62 to 0.93, *P* = 0.008) as well as improved prognosis (pooled OR 0.55, 95% CI 0.36 to 0.84, *P* = 0.006) [91].

#### **4.9 Other cancers**

Metformin is also increasingly studied or planned in less common cancers, such as glioblastoma, thyroid cancer, and non-Hodgkin's lymphoma. The recent study on newly diagnosed glioblastoma showed that temozolomide plus memantine, mefloquine, and metformin are feasible as an adjuvant therapy [92]. One planned phase 1b/2 clinical trial of metformin and chloroquine was recruiting patients with IDH1-mutated or IDH2-mutated solid tumors, including glioma [93]. In another recent retrospective study from Korea, cancer preventative effects of metformin on thyroid cancer were observed in individuals with T2DM on long duration or higher doses of the drug [94]. Separately, a trial in head and neck squamous cell cancer patients revealed metformin to inhibit cancer by enhancing apoptosis, and increasing cellular immune infiltration of the cancer [95]. In non-Hodgkin's lymphoma,

a retrospective analysis of looking at T2DM patients treated with standard therapy found improved progression-free survival and overall survival compared to control not taking metformin [96].


**Abstract**

Abdominal trauma is difficult to identify, especially in a patient with multiple injuries. Mechanism of injury can guide us to the likely organs injured, but the extent and location cannot be accurately pinpointed in most cases. Owing to the multitude of structures located in the abdomen, timely identification and appropriate intervention are crucial to ensure the good patient outcomes. Focused assessment with sonography in trauma (FAST) and its extended version (eFAST) has become the standard care as per ATLS guidelines in patient evaluation. The main goal is to identify hemoperitoneum, hemothorax, and/or pneumothorax. However, sonography can be applied to detect varying injuries to abdominal viscera, beyond the elementary eFAST examination. This includes assessment of solid organs, hollow viscus, vascular structures, and even soft tissues. Sonography, when wielded with necessary knowledge and practice, can be an incredible asset at the bedside. This chapter aims to explore these possible applications of point of care ultrasonography (POCUS) in abdominal trauma.

**Keywords:** POCUS, solid organ injury, bedside sonography, vascular injury, blunt trauma abdomen


**Abstract**

This chapter focuses on olive tree fertilization in Morocco: Describe the practices used by olive growers, diagnose the nutritional status of olive orchards and synthesize the different results and the recommendations of research carried out in Morocco around this theme. Before that, a general overview of the olive tree nutrition and its needs in mineral elements as well as the role of each of these nutrients in the olive tree growth and development will be presented. An introduction to the importance given to the olive tree in national agricultural strategies is necessary. The surveys carried out in the Sais region have shown that farmers do not control olive tree fertilization. This affected negatively the soil fertility level and the olive tree's nutritional status, which were determined through soil and leaf analyzes. From the results of three field trials, carried out in the Fez-Meknes region, it can be concluded that nitrogen and potassium are the two most important elements for the olive tree nutrition and which can affect both its productivity and its quality. The impact of phosphorus on the crop has not been significant, whereas our farmers provide it in high doses compared to the crop's need.

**Keywords:** Olea Europea, Morocco, fertilization, macronutrients, surveys, experimentations


*2.3. Enzyme Activity and the Ratio of Transfructosylation Activity to Hydrolytic Activity (T/H) Assay*

The reaction systems of Psor-LS were set as 1 mL containing 30% (*w*/*v*) sucrose and 10 μg enzyme. The activity of Psor-LS was determined at 65 ◦C for 15 min. The reaction was stopped in boiling water for 10 min. One unit of total and hydrolytic activity was designated as the amount of enzyme catalyzing the release of 1 μmol glucose and fructose per minute, respectively. The transfructosylation activity was the subtraction between total and hydrolysis activity.

The ratio of transfructosylation activity to hydrolytic activity (T/H) of Psor-LS was determined at different concentrations of substrate (from 10% to 60%), different pH (4.0–9.0), and different temperatures (30–80 ◦C). The glucose and fructose contents of the reaction mixture were analyzed by high-performance liquid chromatography (HPLC). The Waters e2695 system (Waters Corporation, MA, USA) has a Waters 2414 RI detector and a Sugar-Pak I column (6.5 mm × 300 mm, Waters, USA). The column temperature and the mobile phase velocity were set as 85 ◦C and 0.4 mL/min.

## *2.4. Biochemical Characterization*

Three types of buffers with different pH ranges were used to determine the influence of pH on the activity of Psor-LS: acetate buffer (pH 4.0–6.0, 50 mM), sodium phosphate buffer (pH 6.0–7.5, 50 mM), and Tris-HCl (pH 7.5–9.0, 50 mM). The interval of each pH was set as 0.5. The reaction conditions were the same as those for the enzymatic activity assay.

The reactions were performed at various temperatures within 35–80 ◦C at intervals of 5 ◦C to measure the optimal temperature of Psor-LS. The other reaction conditions were the same as the enzymatic activity determination except for the temperature. The enzyme was incubated at 45, 55, and 65 ◦C for different time intervals to determine the thermostability of Psor-LS. The initial activity without pre-incubation treatment was taken as 100%. The formula calculated the half-life (*t*1/2) value, t1/2 = ln2/*k*d, and the inactivation rate constant (*k*d) was determined by the linear regression with the equation: ln (At/A0) = *k*d × t (At: residual activity; A0: initial activity; t: incubation time).

Nano DSC III (TA Instrument, New Castle, DE, USA) was introduced to detect the denaturation temperature of this recombinant Psor-LS. The Nano-DSC was equipped with flow-through capillary cells loaded with a pipette mounted on both the inlet and outlet. The sodium phosphate (pH 7.0) buffer was used as the dialysis fluid overnight for the sample, and it was injected into the reference cell for buffer baseline determination. All samples were placed horizontally in a rack on an orbital shaker in a temperature-controlled

incubator with gentle agitation. All samples must be degassed under a vacuum and loaded into the DSC. Scans were conducted by elevating temperatures from 20 ◦C to 100 ◦C, with a scan rate of 1 ◦C min−1. To measure molar heat capacities (Cp), sample scans were obtained from the subtraction of the buffer scans to measure molar heat capacities (Cp), and the experimental thermograms with protein concentration and the volume of the calorimeter cell were also normalized. The apparent denaturation temperature Tm values of Psor-LS were determined, and experiment data from Nano DSC III were analyzed using the Nano Analyze software package.

The kinetic parameters for Psor-LS against sucrose were determined at 65 ◦C. The reactions were prepared in potassium phosphate buffer (pH 6.5) containing sucrose 10–900 mM. The apparent Michaelis−Menten constant (Km) and the turnover number (kcat) were obtained through the nonlinear least square regression method (nonlinear regression Michaelis and Menten (http://biomodel.uah.es/en/metab/enzimas/MM-regresion.htm).

#### *2.5. Optimization of Levan Production*

A reaction containing 10–60% sucrose (*w*/*v*) was carried out to determine the optimal sucrose concentration. Under optimized conditions, different enzyme dosages, ranging from 5 to 100 μg/mL, were added to the reaction mixture to investigate the optimal enzyme dosages. The highest enzyme activity determined the optimal sucrose concentration. Differently, the optimal enzyme dosage was decided upon the benefit ratio, which is the ratio of the product formed in 15 min (including FOS and levan) to enzyme dosage. The biological production of Psor-LS was studied by measuring different saccharides at specific time intervals (0.5, 1, 2, 3, and 6 h).

#### *2.6. Purification of Polysaccharide and FOSs*

#### 2.6.1. Purification of Polysaccharides

Sevag reagent (N-butanol: chloroform = 1:4, *v*/*v*) was used to remove protein from the reaction mixture to purify fructans. The polysaccharides in the system were separated by ethanol (final concentration 60%, *v*/*v*) precipitation. Subsequent alcohol precipitation was used to increase the purity of polysaccharides (until HPLC could detect no monosaccharide and sucrose). The samples obtained were then dried via lyophilization using a LABCONCO FreeZone (LABCONCO Co., Kansas City, MO, USA).


**4. Polyimide films' characterization**

**Figure 6** shows the main intrinsic electrical properties of spin-coated polyimide films measured at wafer level. On the one hand, the DC bulk conductivity of polyimide shows very low values around 10−16 S/m over an applied electric field range up to 40 V/μm (i.e., ohmic range), but remaining quite low at least up to 150 V/μm. On

### **Figure 6.**

*Main intrinsic electrical properties of spin-coated polyimide films measured at wafer level: (a) DC conductivity versus electric field and (b) AC breakdown field distribution.*

**Figure 7.** *Surge performance for isolators with polyimide films 30 μm thick.*

the other hand, the AC breakdown field of the polyimide films exhibits a minimum value of 450 Vrms/μm at 60 Hz. All these make spin-coated polyimide films very good insulating materials for reliable digital isolator application.

**Figure 7** shows the surge performance for isolators with 30-μm thick polyimide films. As it can be seen, these isolators will pass surge tests up to 18 kV, and the first failure voltage is 19 kV for negative pulse and the first failure voltage is 20 kV for positive pulse.