*3.1. Synthesis and Characterization of Cationic CNCs*

CNCs with cationic polymer brushes were synthesized by grafting poly (METAC) or poly(AEM) via SI-SET-LRP from the surface of pristine CNCs (Scheme 1). The first step required the covalent attachment of an initiator on the surface of CNCs using a well-known esterification method of the hydroxyl groups of CNCs with an acyl bromide [23,26,27]. CNC-initiator (CNC-BriB) were prepared at two different initiator loading capacity with respect to anhydroglucose units (AGU) in CNCs ([Br]/[AGU]), 5:3 for CNC-BriB-1 and 5:12 for CNC-BriB-2). The next key step involved a grafting from approach to introduce cationic poly(METAC) or poly(AEM) brushes by SI-SET-LRP on CNCs under mild reaction conditions (Scheme 1B). SET-LRP is a robust and versatile polymerization technique for materials synthesis [28] and had been previously used to graft thermoresponsive and methacrylamide-based polymers from the surface of CNCs [23,26,27,29]. A series of cationic CNCs was synthesized by varying the ratio between the immobilized initiator and the monomers (Table 1). After the polymerization, the resulting cationic CNCs were extensively purified by several centrifugations and dialysis over a week to ensure the removal of unreacted materials or any trace impurities.

**Scheme 1.** (**A**) chemical structure and schematic representation of pristine CNCs; (**B**) synthetic strategy for the fabrication of a series of cationic CNCs.

The success of the grafting of cationic brushes on the surface of CNCs was confirmed by FTIR spectroscopy, DLS, zeta potential and elemental analysis. Pristine CNCs displayed typical IR peaks characteristic of cellulosic functional groups at 3000–3600 cm−1, 1645 cm−1 and 900–1150 cm−1 corresponding to the -OH, -OH bending of water and -C-O-C vibrations, respectively [30,31] (Figure 1, spectrum A; Supplementary Figure S2). The spectra of CNCs after the polymerization showed a notable change with the appearance of new IR peaks at 1730 cm−1 and 1728 cm−1 for CNC-METAC-1A (Figure 1, spectrum B) and CNC-AEM-2A (Figure 1, spectrum C), respectively. These peaks were attributed to the typical carbonyl stretching vibration (C = O) of ester moieties confirming the presence of poly (METAC) and poly (AEM) on the surface of CNCs [27].

**Figure 1.** FTIR spectra of cationic CNCs: (A) pristine CNC, (B) CNC-METAC-1A, (C) CNC-AEM-2A.

Dynamic light scattering (DLS) and zeta potential measurements were conducted to explore the surface properties and stability of CNC suspensions before and after polymer grafting. Although the DLS technique is used for spherical particles, the measurements have been commonly employed to compare the relative sizes of rod-like CNCs before and after a chemical modification as well as the state of dispersion of the particles [16,21]. However, particle size measurements by DLS are not absolute and should not be considered as the exact dimensions of pristine and modified CNCs. The apparent hydrodynamic diameter of CNC particles was obtained by DLS in water at a 0.05 wt% concentration. Pristine CNCs showed an apparent particle size of ~101 nm which is in close agreement with the data obtained from AFM analysis (Figure 2D). An increase in apparent particle size was observed for all cationic CNCs (Table 2, Supplementary Figure S3) which indicated the presence of the polymer brushes on the surface of CNCs and as expected, the cationic CNCs would be highly hydrated. Furthermore, DLS analysis indicated no apparent aggregation of CNC particles at low concentrations. The nature of the surface charges on pristine and cationic CNCs was examined by measuring the zeta potential of the particles in water at 0.25 wt% concentration. Pristine CNCs had a negative zeta potential (-34.8 mV) due to the presence of anionic sulfate half-ester groups. On the other hand, cationic CNCs displayed positive zeta potential values in the range of +31.8 to +45.0 mV (Table 2). This further confirmed a successful polymerization reaction and the grafted cationic polymer brushes shielded the anionic sulfate half-ester groups. An increase in zeta potential (becomes more positive) was observed with an increase in monomer concentration ([monomer/AGU]: 50:3 v/s 60:3, Table 1). For instance, CNC-METAC-1B which had a higher monomer concentration compared to CNC-METAC-1A showed a more positive zeta potential value (+44.9 mV v/s +31.8 mV). The same trend was observed for CNC-METAC-2B (+38.2 mV) compared to CNC-METAC-2A (+32.0 mV). Overall, the cationic nature and colloidal stability of CNC-METAC and CNC-AEM materials are crucial for consistent results in biological studies.

The atomic composition of CNC samples before and after polymer grafting was measured by elemental analysis (Table 3). As expected, a small percentage of sulfur was detected for pristine CNCs due to the presence of the sulfate half-ester groups. While pristine CNCs displayed the absence of nitrogen, all the prepared cationic CNCs indicated the appearance of nitrogen deriving from the cationic monomers (METAC or AEM). This further supported the success of the polymerization reaction. The cationic CNC with a fixed initiator content and increased monomer concentration (CNC-METAC-1B v/s CNC-METAC-1A, Table 1) had a higher nitrogen content (~7.68%).

**Figure 2.** Morphological analyses of pristine and cationic CNCs by TEM and AFM: Representative TEM images of (**A**) pristine CNCs; (**B**) CNC-METAC-1A; (**C**) CNC-METAC-1B and AFM phase images of (**D**) pristine CNCs, (**E**) CNC- METAC-2A; (**F**) CNC-METAC-2B. TEM and AFM images of other cationic CNCs are depicted in Figures S4 and S5.

**Table 3.** Elemental analysis of pristine and cationic CNCs.

The morphological features of the cationic CNCs were analyzed by both transmission electron microscopy (TEM) and atomic force microscopy (AFM) to verify whether the characteristic rod-like crystalline structure of pristine CNCs (Figure 2A,D) was maintained after the surface modification reactions. Figure 2B–F, Figures S4 and S5 indicated that the rod-like morphology of the cationic CNCs was retained and the samples did not degrade into more simple carbohydrates [32]. Accurate size measurement of the rod-like particles by TEM was difficult as the edges of the grafted CNCs were less defined as well as a tendency to aggregate during air-drying or staining [33]. AFM analysis of pristine CNCs depicted a good distribution of the nanorods possessing an average length of 104 ± 68 nm and diameter of 4.0 ± 0.5 nm with a resultant aspect ratio of 26 (Figure 2D). The size of the cationic CNCs was found to be in the range of 112–164 nm in average length and 4.9–5.3 nm in cross-section. Moreover, AFM phase images of the cationic CNCs (Figure 2D,E) showed a halo that was indicative of the presence of grafted polymer brushes [26]. Aggregation of nanoparticles was observed for CNC-METAC-2B (Figure 2F) as it was prepared with a high monomer concentration (compared to CNC-METAC-2A (Figure 2E)), and hence a high degree of polymerization was expected [34].


**1. Introduction**

Due to the increase of aged patients with atherosclerosis, more attention should be paid to the endothelial damage of great vessels. The damaged endothelium is more susceptible to bacterial infections. The reports or papers of infected aneurysms (mycotic aneurysms) are increasing in Pubmed search (Figure 1), which is consistent with personal experience in dai‐ ly practice. The reasons for the increase were due to several causes: 1) the increase of aged patients with more risk of atherosclerosis than before, 2) an improvement of CT imaging and MR imaging, 3) more awareness of the disease. We focused the diagnosis and manage‐ ments of infected aneurysms, and the diseases needed to differentiate from infected aneur‐ ysms, such as collagen vascular diseases and periaortitis.


**1. Introduction**

The world is living in an era of information outburst thanks to rapid speed of technological developments in computational domain. Many factors contribute to the immense amount of data available online: Mobile devices are becoming more accessible to everyone with their decreasing cost, underlying network technologies are facilitating data transfer by providing faster and more reliable communication, data processing methods (such as editing, compressing) are helping customize data format, and increasing data storage capacities are turning bulk data storage more effective.

With all technology and convenience so easily reachable, an ordinary user is allured further to handle even more information by either transferring, processing, or storing them. Yet, the issues of security and privacy are often taken for granted, and unfortunately this ignorance may cause a destructive consequence by totally violating the security and privacy of information, as well as its owner.

In this chapter, three fundamental states of information, i.e., information in transit, information in process, and information in storage, are defined first. Then the chapter addresses security and privacy of information in each state by giving examples. It should be noted that as information is the processed form of data, these two terms, i.e., information and data, are used interchangeably throughout the text. The chapter then provides algorithms to achieve privacy and security of information in these three states and how they apply to particular states of information for ensuring security.

The rest of the chapter is organized as follows: In Section 2, three states of information are defined together with examples for each. In Section 3, security mechanisms, i.e., privacy and security algorithms that apply to each state of information, are discussed in detail, and examples are provided. Finally, the chapter is concluded in Section 4.


**5. Seasonal variation at onset and birth**

The first report of seasonal variation in the manifestation of T1DM was presented by Franklin Adams in 1926[32] although a consistent picture on the real seasonality of the disease has not been established. The increased incidence of T1DM diagnosis during

Altering Trends in the Epidemiology of Type 1 Diabetes Mellitus in Children and Adolescents 329

A seasonal variation of birth has been also observed in children who developed T1DM later in life in many countries, which suggests that environmental factors during pregnancy, in the neonatal period or very early in life play a role in its development. Several studies from Europe [35-37] and Israel [38] showed higher rates of T1DM among youth born in spring and lower rates among youth born in winter. Although McKinney maintained that there is no relationship between T1DM diagnosis and date of birth[39]. It has been suggested that seasonability pattern may be explained by reduced vitamin D production [40] during the

The genetics of T1DM cannot be classified according to a specific model of inheritance. Susceptibility to autoimmune T1DM is determined by multiple genes with HLA genes having the strongest known association. HLA antigens are present on the surface of the leucocytes and participate in some immune reactions. The genes coding for these antigens are located on chromosome 6. The class II sub region of HLA consists of the DR, DQ, and DP loci. These class II molecules are involved to the immune destruction of the pancreatic beta cells because they participate in the presentation of the antigen to the helper T cell,

Inheritance of HLA-DR3 and HLA-DR4 appears to confer a 2 to 3 fold increased risk for the development of T1DM. When both HLA-DR3 and HLA-DR4 are inherited the relative risk for the development of T1DM is increased by 7-10 folds. It is estimated that 48 percent of the familial aggregation can now be ascribed to known loci, and the Major Histocompatibility Complex (MHC) contributes 41 percent [41]. As an example, siblings with the highest risk HLA DR and DQ alleles, who inherit both HLA regions identical by descent to their diabetic sibling, may have a risk of developing anti-islet autoimmunity as high as 80 percent and a similar long-term risk of diabetes[42]. Moreover HLA DR2 and HLA DR5 are both protective in most studies. Furthermore, stronger associations of DM1 have been reported

In our effort to detect the genetic susceptibility of Greek Cypriot population to DM1, we studied 101 DM1 patients with age of onset less than 15 years through HLA serological typing for the DR and DQ1 alleles and compared them to 209 healthy controls. Our findings support the strong association of HLA-DR4 and DR3 with DM1. The most frequent allelic combination was that of HLA-DR3/DR4 (27%) followed by that of DR2/DR4 (21.6%). The percentage of HLA antigens in patients with DM1 and controls are shown in figure 4. The protective role of HLA-DR5 was shown, whereas the presence of HLA-DR2 is neutral, in contrast with most findings among Caucasian population where DR2 is protective. In addition, high resolution testing of the DR4 and DR3 alleles revealed the predominant presence of the DRB1\*0403 (0% vs 36%), similar frequency of the DRB1\*0402 in both groups (19% vs 14%) and that the DRB1\*301 was the only DR3 allele detected. The DQB1 alleles present in our T1DM patients as shown in figure 5 were nearly exclusively DQB1\*0201 and DQB1\*0302 [44]. The relative risk of developing T1DM in children carrying the DQB1\*0201 and the DQB1\*0302 alleles is 5.05 and 2.56 respectively whereas the protective role of

Furthermore, although most T1D cases occur in individuals without a family history of the disease, T1D is strongly influenced by genetic factors. The lifelong risk of T1DM is markedly

critical intrauterine and neonatal periods of life.

which initiates the immune reaction.

DQB1\*0301 is documented.

with other MHC loci: HLA-DQA1 and DQB1 antigens[43].

**6. Genotype**

Fig. 3. Ratio of males and females in relation to year of diagnosis (at 2-yr intervals).

Autumn and Winter months could support the hypothesis that infections may act as participating factors in the clinical onset of the disease, possibly accelerating an autoimmune process that may have been initiated months or years before [33]. Based on the average temperature record in our island, the newly diagnosed cased were grouped according to the month of diagnosis as follow: November, December, January, and February were defined as cold months, October, March, April and May as neutral months and, June, July , August and September, as warm months. More children were significantly diagnosed with T1DM during the cold months compared to those who manifested the disease during the warm months (p<0.001), whereas no difference was observed in the incidence between neutral and cold months ((p>0.05) throughout the study period (1990-2009) as depicted in Table 2. A recent study on seasonal variation in DM in 53 countries has suggested that seasonality in the diagnosis of T1DM occurs and that the pattern of seasonality appears to be related to the geographical position, at least as far as the northern/southern hemisphere dichotomy is concerned [34].

Table 2. Percentage of children diagnosed with T1DM at cold months, neutral months and warm months. Binomial test performed to compare proportions compared to "Cold Months" group: \* NS (p>0.05), \*\* p<0.05, \*\*\* p<0.01, \*\*\*\*p<0.001

A seasonal variation of birth has been also observed in children who developed T1DM later in life in many countries, which suggests that environmental factors during pregnancy, in the neonatal period or very early in life play a role in its development. Several studies from Europe [35-37] and Israel [38] showed higher rates of T1DM among youth born in spring and lower rates among youth born in winter. Although McKinney maintained that there is no relationship between T1DM diagnosis and date of birth[39]. It has been suggested that seasonability pattern may be explained by reduced vitamin D production [40] during the critical intrauterine and neonatal periods of life.


**2. Origin and distribution**

Garlic is native to India (Central Asia), where was considered a spice with mystical implications due to its medicinal attributes. Egyptian hieroglyphs and Roman texts refer to garlic as a source for health and strength required for physical work. During the Middle Ages was used to prevent cholera. Nowadays, it is known for its antiseptic, diuretic, vermifuge and vasodilator activities. It also stimulates bile and stomach secretions, and acts against atherosclerosis and thrombosis.

Spaniard conquerors carried with them garlic; first to Cuba and, later, to the rest of the American colonies. Early reports of garlic fields in Mexico appeared at the beginning of the twentieth century, and fifty years later, the central region of Mexico (Bajío) was the main area for garlic production. The time of harvest in that region made possible to start exporting surplus, since at that time of the year the world production is low.

Garlic species are widely distributed on boreal areas having temperate climates and mountainous areas from tropical regions. Most of the species diversity is found from Mediterranean countries to Central Asia. USA is considered as diversification center for *Allium* (Lagunes, 2009).


**1. Introduction**

328 Thermoplastic Elastomers

Yong-Hing, K., J. Reilly, et al. (1980). "Prevention of nerve root adhesions after

Nowadays thermoplastics are widely used in industries; many products used different specifications of the thermoplastics that are adapted to their requirement. The importance of consideration to the environmental protection leads the mankind to thinking about producing environment-friendly material, and also controlling the waste material by recycling or reducing them. In the meantime the natural materials such as cellulose based material are one of the major resources that can be used to replace with many manufactured materials. However, cellulosed based material such as natural fibres, because of their degradability, need protection from any circumferential agents. The protection may require a special condition in order to utilise in soil, due to water absorption, soil organisms, and minerals. Natural fibres are amenable to modifications as they bear hydroxyl groups from cellulose and lignin. In addition coating the fibres with any chemical materials reduce their water absorptions and protect them from any bacteria and fungi attack. The hydroxyl groups may be involved in the hydrogen bonding within the cellulose molecules. This weakness of the natural material and good characteristics of natural fibres is the basis of biocomposites invention.

\*OPEFB : Oil Palm Empty Fruit Bunch

Table 1. Chemical and mechanical properties of some important natural fibres

Application of Thermoplastics in Protection of Natural Fibres 331

OPEFB fibre is obtained after the subtraction of oil seeds from fruit bunch for oil extraction. OPEFB fibre is extracted by the retting process of the EFB. Average yield of OPEFB fibre is about 400 g per bunch. Previous studies report the mechanical properties of OPEFB fibres. Table 2 and Table 3 show the summary of oil palm fibre properties (Jacob et al., 2004;

Thermoplastics as a coating can lead to improving the natural fibre performance in two ways: 1. the thermoplastics cover the fibres and keep the fibres from any fungi or bacteria attacks by decreasing the water absorption and contact of the fibres to the soil and any organism inside it, 2. The physical performance of the fibre such as tensile strength and elongation can be affected by modification and coating with any kind of thermoplastics. Therefore, a method was developed to coat the fibres with the thermoplastics. The solvent was used to prepare soluble thermoplastic since the natural fibres cannot reside in high temperature. Different density of the thermoplastic solution was used to evaluate the coated fibres to reach the best strength and resistance. Two types of the fibre were used as a reinforcement of composites such as soil, first the discrete fibres where it needs to be coated one by one, and second is the sheet fibres that were made by compaction of bulk fibres. The fibre was coated by acrylonitrile butadiene styrene (ABS) solution and the characterisation test results for both single and sheet fibres are

ABS is an important engineering copolymer widely used in industry due to superior mechanical properties, chemical resistance, ease of processing and recyclability (Yang et al., 2004). ABS is a common thermoplastic used to make polymeric wood composites, has good physical properties in comparison with other commodity plastics and is cheap in

ABS is derived from acrylonitrile, butadiene, and styrene. The chemical structure of the ABS is shown in Figure 1. Acrylonitrile is a synthetic monomer produced from propylene and ammonia. Butadiene is a petroleum hydrocarbon obtained from butane. Styrene monomers, derived from coal, are commercially obtained from benzene and ethylene from coal. The advantage of ABS is that this material combines the strength and rigidity of the acrylonitrile and styrene polymers with the toughness of the polybutadiene rubber. The most amazing

comparison with other engineering plastics (Huang & Mo, 2002).

mechanical properties of ABS are resistance and toughness.

C

H

C

C

H

H

*l m n*

C C

H

H

C

H

H H

*Acrylonitrile Butadeine Styrene*

H

Sreekala et al., 2001; Sreekala et al., 1997)

**3. Thermoplastic coat**

described in the following sections.

**3.1 Acrylonitrile butadiene styrene**

C

C

C N

H

H

Fig. 1. Chemical structure of ABS

H


**Influence of the Phenological State of in the Antioxidant Potential and Chemical Composition of** *Ageratina havanensis***. Effects on the P-Glycoprotein Function**

**Trina H. García 1, Claudia Quintino da Rocha 2, Livan Delgado-Roche 3, Idania Rodeiro 3, Yaiser Ávila 4, Ivones Hernández 3, Cindel Cuellar 3, Miriam Teresa Paz Lopes 5, Wagner Vilegas 6, Giulia Auriemma 7, Iraida Spengler 1,\* and Luca Rastrelli 7,\***

Academic Editors: Ra ffaele Pezzani and Carmen Formisano Received: 31 March 2020; Accepted: 30 April 2020; Published: 2 May 2020

**Abstract:** *Ageratina havanensis* (Kunth) R. M. King & H. Robinson is a species of flowering shrub in the family Asteraceae, native to the Caribbean and Texas. The aim of this work was to compare the quantitative chemical composition of extracts obtained from *Ageratina havanensis* in its flowering and vegetative stages with the antioxidant potential and to determine the e ffects on P-glycoprotein (P-gp) function. The quantitative chemical composition of the extracts was determined quantifying their major flavonoids by UPLC-ESI-MS/MS and by PCA analysis. The e ffects of the extracts on P-gp activity was evaluated by Rhodamine 123 assay; antioxidant properties were determined by DPPH, FRAP and inhibition of lipid peroxidation methods. The obtained results show that major flavonoids were present in higher concentrations in vegetative stage than flowering stage. In particular, the extracts obtained in the flowering season showed a significantly higher ability to sequester free radicals compared to those of the vegetative season, meanwhile, the extracts obtained during the vegetative stage showed a significant inhibitory e ffect against brain lipid peroxidation and a strong reductive capacity. This study also showed the inhibitory e ffects of all ethanolic extracts on P-gp function in 4T1 cell line; these e ffects were unrelated to the phenological stage. This work shows, therefore, the first evidence on: the inhibition of P-gp function, the antioxidant e ffects and the content of major flavonoids of *Ageratina havanensis*. According to the obtained results, the species *Ageratina havanensis* (Kunth) R. M. King & H. Robinson could be a source of new potential inhibitors of drug e fflux mediated by P-gp. A special focus on all these aspects must be taking into account for future studies.

**Keywords:** *Ageratina havanensis*; flavonoids; UPLC-ESI-MS/MS; P-glycoprotein; antioxidant potential


**2.1.1 Not only bacteria**

62 Malaria Parasites

response. The effectors of this transmission-blocking immunity are antibody molecules directed against particular protein epitopes (Spano et al., 1996). Anti-Pbs 21 may be used in paratransgenic experiment in the murine system to prove the feasibility of the approach.

Some studies have already shown the possibility to use mosquito guts bacteria to deliver anti-*Plasmodium* effector molecules within the mosquito vector (Riehle and Jacobs-Lorena

In these studies are reported that *E. coli* bacteria expressing SM1 and PLA2, are able to partially inhibit the parasite development. Very recently, Bisi & Lampe (2011) described *Pantoea agglomerans* engineered to express and secrete anti-*Plasmodium* effector proteins. To this purpose, plasmids that included the pelB or hlyA secretion signals from the genes of related species were constructed and tested for their efficacy in secreting SM1, anti-Pbs21,

Very interestingly, *P. agglomerans* was able to secrete HlyA fusions of anti-Pbs21 and PLA2, and now ongoing experiments are evaluating these strains for anti-*Plasmodium* activity in

It is worth to remind that parasites tend to have a heterogeneous genome that favours selection of individuals able to overcome barriers such as drugs or possibly effector gene products favouring the insurgence of resistant strains. Thus the possibility to release paratrasgenic mosquito carrying microbial symbionts modified to express several different

The results cited above, represent a clear "proof of principle" about the applicability of

In this context, the symbiotic bacterium *Asaia* previously described*,* may be very

We engineered *Asaia* strains that were transformed to produce fluorescent proteins; they showed a remarkable capacity to colonize the midgut, reproductive organs ad salivary glands of recipient mosquitoes (Favia et al., 2007; Damiani et al., 2008, 2010). These studies underline the possibility of using this bacterium to express anti-parasite molecules within

Furthermore, *Asaia* can be transmitted, vertically to the progeny (by maternal, paternal and trans-stadial routes) and horizontally between mosquito individuals by mating and co-

As stated above, vertical transmission is important because it offers the chance of introducing engineered bacteria into mosquito populations in the field. In *An. gambiae* it has been proven that vertical transmission occurs by an egg-smearing mechanism, where the extracellular *Asaia* symbionts are smeared onto the egg surface, thus is quite likely that larvae, after emergence, are infected by the bacteria by feeding on them and on egg

Larvae infection by bacteria in the breeding water, offers a way to spread recombinant bacteria in the field, moreover the vertical transmission of *Asaia* symbiont would allow the

molecules would circumvent the selection of resistant malaria strains.

the mosquito body, to inhibit the transmission of the parasite.

feeding (Favia et al., 2007, 2008; Damiani et al., 2008, 2010).

"passage" of recombinant symbionts through the generations.

2005; Riehle et al., 2007).

infected mosquitoes.

successfully implied.

and PLA2 in *P. agglomerans* and *E. coli*.

paratransgenesis in malaria control.

remnants (Damiani et al., 2010).

It is worth to mention that not only bacteria symbiont may find application in paratransgenesis but several evidences indicate that virus, yeast and fungi may also be successfully use.

Ren and collaborators (2008) discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in *An. gambiae*. By the development of an AgDNV-based expression vector to express gene(s) of interest in *An. gambiae*, an exogenous gene (enhanced green fluorescent protein; EGFP) was expressed in *An. gambiae* mosquitoes. Wild-type and EGFP-transducing AgDNV virions were able to consistently infect *An. gambiae* larvae, expressed EGFP was detected in adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations.

This work produced clear evidence that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-*Plasmodium* effectors or insect-specific toxins in *Anopheles* mosquitoes.

In the last few months we have identified the yeast *Wickerhamomyces anomalus* as stably associated to some malaria vector species localising at the level of midgut and reproductive organs of the host (Ricci et al., 2011b, 2011c). This localization, the possibility to easily manipulate yeast and the chance to express effector molecules in a eukaryotic organism render *W. anomalus* a potential good candidate for paratransgenesis. Interestingly, very often we found in the midgut and reproductive organs of the mosquito a large number of *Asaia* bacteria in association with a relevant number of *W. anomalus.* This finding may support the idea to use synergistically these two symbionts to release different effector molecules in the mosquito.

We are now proceeding to achieve rapidly a stable transformation system to produce strains of *W. anomalus* able to express and delivery anti-*Plasmodium* peptides.

Very recently, Fang et al (2011) develop transgenic fungi that kill malaria parasite in the mosquito.

They used the fungus *Metarhizium anisopliae* to infect mosquitoes through the cuticle. Recombinant strains of *M. anisopliae* expressing three effector molecules (SM1, a single chain antibody called PfNPNA-1 and an antimicrobial peptide called scorpine) targeting sporozoites during their travel through the hemolymph to the salivary glands were used to infect mosquitoes that had a *Plasmodium*-infected blood meal eleven days before.

The reduction in sporozoite counts was relevant and even more when using *M. anisopliae* strain expressing scorpine and an [SM1](8): scorpine fusion protein (with the reduction in

Facing Malaria Parasite with Mosquito Symbionts 65

From this point of view, the "*Ae. aegypti* lesson", can be particularly important. Similar to *Anopheles*, *Ae. aegypti* has never been detected as naturally infected by *Wolbachia*, transinfection of selected strains of *Wolbachia* in *Ae. aegypti* can block the development of dengue

Very recently the wMel *Wolbachia* strain was introduced into *Ae. aegypti* from *Drosophila melanogaster* showing a successfully invasion two natural *Ae. aegypti* populations in Australia, reaching almost the fixation in a few months after the releases of wMel-infected

These findings demonstrate that *Wolbachia*-based strategies can be a successful approach to eliminate dengue infection and a similar approach may be developed to control malaria

The concept that manipulation of microbial symbionts may represent an important tool to

Recent studies have also pointed out that is possible to overcome many of the limitations that since some time ago represented strong limitation to this approach, including the difficulty to culture and transform many symbionts. As discussed in the previous paragraph, to date several symbionts of insect pests and vector borne diseases can be cultivated outside the host, manipulated to express specific factors and reintroduced within the host to produce *in situ* the effector molecules. A very informative example of advanced studies in genetic manipulation of insect symbionts is *Rhodococcus rhodnii*, a bacterial symbiont of the reduviid bug *Rhodnius prolixus.* This bacterium, if manipulated appropriately, can be reintroduced to the vector and inhibit the transmission of *Trypanosoma cruzi* that causes Chagas disease (Beard et al., 2001). Another example regards the bacterium *Alcaligenes*, a gut symbiont of the sharpshooter *Homalodisca coagulata*, the vector of Pierce's

Concerning malaria control, we have described some symbionts that can be cultured outside the mosquito and can be genetically modified to produce specific molecules that have proven to have an inhibitory effect on *Plasmodium* development. In the last 5 years our group has been focused in the study of symbiosis in mosquito vector. Our group as well as others research units, have indicated few microorganisms that possess a strong potential in the paratransgenic control of mosquito-borne diseases and in the very few last years, the number of parasite of medical and veterinary for which the paratransgenic approach has been proposed as one of the element of an integrated control strategy is increasing as demonstrated by study aimed to develop control method of *Leishmania* parasite (Hurwitz et al., 2011). However, even if the genetic modification of insect symbionts to inhibit parasite development, is clearly feasible and achievable in laboratories, many concerns need to be properly addressed before this approach can be applicable in the field. Even though the release of paratransgenic mosquito poses much less ethical and safety concerns that the release of genetically modified mosquitoes, for example the release of large-scale transgenic mosquitoes, would cause not only an increase of the nuisance but also the health risk related to other mosquito borne diseases (it is worth to remind that malaria vector mosquitoes may

contrast insect pests and insect vectors of infectious diseases is now widely accepted.

infection (Walker et al., 2011).

infection.

*An. aegypti* adults (Hoffmann et al., 2011).

**3. Future development and conclusion**

crop disease (Bextine et al., 2004).

sporozoite counts of 98%). These experiments prove that *Metarhizium*-mediated inhibition of *Plasmodium* development could be an additional tool to contrast malaria development within the vector.


**2.4 Preparation of blood sample for zeta potential measurement**

About 0.1 ml of blood sample was transferred into 50 ml of freshly prepared 5% w/w dextrose solution which is isotonic with the red blood cells [12].