Functionalized Gold Nanoparticles for Inhibition of Vascular Endothelial Growth Factor in Arthritic Patients

Nanotechnology has recent biological and medical applications especially in bioanti-VEGFA/GNPs conjugate research. The antiVEGFA/GNPs conjugate based on nanoparticles are introducing materials with new characteristics because sensing elements are biocompatible so it may be cells, proteins, or nucleic acids also the sensors are used to evaluate biological processes changes. Among the nanomaterials used as component in anti-VEGFA/ GNPs conjugate, gold nanoparticles (GNPs) have received greatest interests because they have various unique properties [1-6]. GNPs have been used in various medical applications due to its stability, controlled geometrical, optical, and surface chemical properties [7]. GNPs have high surface-to-volume ratio and high surface energy that provide a stable immobilization of a large amount of biomolecules retaining their bioactivity [8,9]. Nanoparticles have a unique characteristics as it not detected by immune system. Nanoparticles play an important role in treatment of some diseases for example, caner, Osteoarthritis (OA) and Rheumatoid Arthritis (RA) diseases through active targeting of angiogenic cells. Osteoarthritis (OA) or degenerative joint disease is the most common type of arthritis. It’s usually prevalent among the elderly people; recently it is becoming more common among younger people. The joints most commonly affected are hands, knees, ankles and spines. Often the cause of osteoarthritis is unknown but it may be due to a combination of the following risk factors a family tendency, obesity, a joint injury, repetitive destruction from activities and problems with bones in a joint not lining up properly [10]. Over many years these factors can wear away the articular cartilage, expose bony surfaces rub together. This along with the growth of bony projections causing swelling, pain and limited movement of the joint. Damage starts at the molecular scale and progressively spreads to the higher structural architecture of the articular cartilage that cushions the joints [11].

Introduction
Nanotechnology has recent biological and medical applications especially in bioanti-VEGFA/GNPs conjugate research. The anti-VEGFA/GNPs conjugate based on nanoparticles are introducing materials with new characteristics because sensing elements are biocompatible so it may be cells, proteins, or nucleic acids also the sensors are used to evaluate biological processes changes. Among the nanomaterials used as component in anti-VEGFA/ GNPs conjugate, gold nanoparticles (GNPs) have received greatest interests because they have various unique properties [1][2][3][4][5][6]. GNPs have been used in various medical applications due to its stability, controlled geometrical, optical, and surface chemical properties [7]. GNPs have high surface-to-volume ratio and high surface energy that provide a stable immobilization of a large amount of biomolecules retaining their bioactivity [8,9]. Nanoparticles have a unique characteristics as it not detected by immune system. Nanoparticles play an important role in treatment of some diseases for example, caner, Osteoarthritis (OA) and Rheumatoid Arthritis (RA) diseases through active targeting of angiogenic cells. Osteoarthritis (OA) or degenerative joint disease is the most common type of arthritis. It's usually prevalent among the elderly people; recently it is becoming more common among younger people. The joints most commonly affected are hands, knees, ankles and spines. Often the cause of osteoarthritis is unknown but it may be due to a combination of the following risk factors a family tendency, obesity, a joint injury, repetitive destruction from activities and problems with bones in a joint not lining up properly [10]. Over many years these factors can wear away the articular cartilage, expose bony surfaces rub together. This along with the growth of bony projections causing swelling, pain and limited movement of the joint. Damage starts at the molecular scale and progressively spreads to the higher structural architecture of the articular cartilage that cushions the joints [11].
Rheumatoid Arthritis (RA) is the second common type of arthritis and known as an autoimmune disease affecting approximately 1% of the worldwide population, characterized by the infiltration and accumulation of activated immune cells in the synovial joints. In RA the immune system attacks the joints and organ tissue. Subsequent chronic production of pro-inflammatory cytokines leads to destruction of the joint architecture and severe disability [12,13]. One of the earliest histopathology responses in RA is the generation of new blood vessels as it is known as Angiogenesis and inflammatory cell recruitment. This event is accompanied by the transudation of fluid and the transmigration of both lymphocytes into the synovium and polymorph nuclear leukocytes into the synovial fluid [14,15]. In the mature RA synovium, the mass of tissue is too much for the multiple new capillaries to nourish leading to local tissue ischemia. Relative synovial hypoxia is associated with an increased production of the transcription factor hypoxia inducible factor 1 (HIF-1) which activates transcription of genes that are of fundamental importance for angiogenesis, including those for Vascular Endothelial Growth Factor (VEGFA) and the VEGFA receptor [5,16].
The most known rote for treatment of OA and RA are the use of angiogenesis inhibitors which interfere with various steps in the role of VEGFA binding to its receptor process such as bevacizumab(Avastin ® ), is a monoclonal antibody that binds to VEGFA antigen [17]. When VEGFA is attached to bevacizumab, it is unable to activate the VEGF receptor. Also sorafenib and sunitinib, bind to VEGF receptors and blocking their activities [18]. Recent progresses of the GNPs-labeled antibody or antigen for immunoassay researchers generally accept the theory that mechanisms related to the residues of the particular amino acids play an important role in binding proteins to gold particles. Appropriate active targeting ligand should be selected for designing RA-targeted particles, For example, there are certain biologic drugs, such as anti-Tumor necrosis factor agents or B-cell depleting agents, which target cells other than macrophages [19,20]. The objective of the present in vitro study is to introduce bioconjugation technique (anti-VEGFA/GNPs conjugate) as novel modality for inhibition of serum VEGFA level for treatment of OA and RA angiogenic diseases.

Methods
Preparation of Anti-VEGFA/GNPs conjugate Synthesis of GNPs: Synthesis of gold nanoparticles (GNPs) was carried out by the reduction of Gold chloride hydrate by trisodium citrate according to Frens method as follow [21,22]. In brief; 50 mL aqueous solution of 0.01 mM HAuCl 4 in flask was heated until boiling with stirring. After that 1 mL of 1% trisodium citrate solution was added. The solution color turned from yellow to intrinsic bright red. The solution has been left to cool until reach room temperature with gentle stirring. GNPs were characterized by UV-Vis spectroscopy, particle size analyzer, High Resolution Transmission Electron Microscopy (HR-TEM), Fourier Transform Infrared Resonance (FTIR).

Conjugation of Anti-VEGFA and GNPs:
The determination of optimum anti-VEGFA-Pab concentration was carried out according to the method described by Ye et al. [22,23] with modification as follows: Serial dilutions of anti-VEGFA antibody solutions have been prepared (10,50,100,150,200,250, 300, and 500ng/mL) in 20mM tris buffer. Then 10 µL from each anti-VEGFA concentration have been added to an eppendorf containing 100 μL colloidal gold adjusted to pH 7.3 using 2M sodium bicarbonate. The anti-VEGFA/ GNPs conjugate were allowed to interact for an additional 2 hr at room temperature with gentle shaking. The residual surface of the GNPs was blocked and stabilized by adding 10 µL of 1% BSA in 20mM tris buffer with stirring for 30 min. The optimum anti-VEGFA concentration estimated using UV-Vis spectroscopy [24] and particle size analysis. After determination of antibody optimum concentration for conjugation and functionalization of GNPs, the anti-VEGFA/GNPs conjugate has been prepared at optimum condition as follows; 10 mL of GNPs adjusted to pH 7.3 was mixed with 1 mL of anti-VEGFA (100 ng/mL) and allowed to react for 2 hr.
The residual surface of the GNPs was blocked and stabilized by adding 1 mL of 1% BSA in 20mM Tris buffer with stirring for 30 min, and then stored at 4 • C for 2 hr. After that the anti-VEGFA/GNPs conjugate has been centrifuged (SORVALL -MX-150 centrifuge, Hitachi, Japan) at 10,000 rpm at 4 ºC for 40 min after that the Supernatant is discarded. The pellet was dispersed in 1 mL of 20 mM Tris buffer containing 1% BSA and 0.05 % NaN 3 . GNPs and Anti-VEGFA/GNPs conjugate have been characterized by an UV-Vis-NIR spectrophotometer (Cary5000 UV-Vis-NIR, Varian Instrument, U.K), Fourier transform infrared spectroscopy (FTIR) NICOLET IS 10 spectrophotometer (Thermo Fisher Scientific, USA), Zetasizer Nanoseries (Malvern Instruments, U.K) and High resolution transmission electron microscopy Tecnai G20 (FEI, Netherlands) operating at 200 kV. The characterization procedures and experiments were done at Nanotechnology and Advanced Materials Central Lab, Agriculture Research Center, Egypt.

Participant
Forty three serum samples have been collected and divided into three groups according to the American College of Rheumatology /The European League Against Rheumatism (ACR/EULAR 2010) [25], classification criteria as follow. First group, 22 patients with RA disease. Second group,11 patients with primary knee OA upon clinical and radiological criteria of primary OA. Third group,10 healthy volunteers subjected as control group. All Patients had been selected from the outpatient's clinics and inpatient of Rheumatology department at Ain Shams University Hospital.

Inclusion criteria
The patients of OA had symptoms and signs consistent with knee OA and fulfilled the American College of Rheumatology clinical radiological criteria of idiopathic knee OA. The patients after signing a consistent have been included and subjected to the following procedures: Complete medical history, physical examination including body mass index and X-rays of the knee joints.

Excluding criteria
Patients have been excluded if one at least of the following history were present: knee trauma, Intra-articular steroid of hyaluronate injection within 3 months prior to enrolment, Knee surgery and Arthroscopy and evidence of secondary knee OA.

Ethical consideration
The nature of the study was explained to all participants. The laboratories represent standard care and pose no ethical conflicts. All patients gave written informed consent to participate in the study, which was approved by the Ain Shams medical ethics committee.

Laboratory Investigations.
Seven mL of venous blood were withdrawn from all groups and has been divided into three parts; 3mL has been putted in a tube with EDTA for complete blood count (CBC) using (BECKMAN COULTER HMXAL) automated differential cell counter, 2 mL has been putted in sodium citrate tube for estimation of 1sthour erythrocyte sedimentation rate (ESR) using Westergren method [26], and 2 mL has been putted in a plain tube and left to be clotted then centrifuged at 9000 rpm for 5 min for separation of serum after that serum has been stored at -80 ºC until use in estimation of VEGFA antigen level. VEGFA antigen assay was performed by sandwich ELISA kit. All practical details were operated strictly in accordance with the instructions in the manual of the ELISAkit [27].

Treatment procedure
In vitro application of anti-VEGFA/GNPsconjugate to both OA and RA patient serum samples for inhibition of VEGFA antigen, then estimation of human serum samples with and without addition of anti-VEGFA/GNPs conjugate was carried as follow; 10 µL of anti-VEGFA/GNPs conjugate has been incubated with 100µL of serum, without any dilution of serum, with gentle shaking at room temperature for 2 h.Estimation of the conjugation efficacy evaluated by ELISA kit [27]. At the same time 10 µL of anti-VEGFA antibody (100 ng/mL) has been incubated with 100µL of patient's serum, without any dilution of serum, with gentle shaking at room temperature for 2 hr. Also, 10 µL of GNPs has been incubated with 100µL of patient's serum with gentle shaking at room temperature for 2 hr. these procedures have been done to compare the effect of conjugate, anti-VEGFA antibody and GNPs alone on the VEGFA antigen concentration in patient's serum.

Statistical analysis
Statistical presentation and analysis of the study has been conducted, using the mean, median standard deviation, unpaired student t-test, paired student t-test and linear correlation coefficient tests. Statistical data analyses were performed using GraphPad Prism 5 software.

Characterization of GNPs and Anti-VEGFA/ GNPsconjugate
New approach to arthritis inhibition include anti-VEGFA/ GNPs conjugate has been designed for inhibition of VEGFA growth factor to serve as a starting point for inhibition of VEGFA-mediated blood vessel proliferation. The conjugation process between GNPs and anti-VEFGA antibody is concentration dependent therefore inadequate conditions will lead to precipitation and flocculation of GNPs. VEGFA antibody concentration has a great effect on the conjugation process, where the optimal antibody concentration was found to be 100 ng/ml as shown in Figure 1A, as these affect the absorbance intensity differences between 520 nm and 580 nm of GNPsanti-VEGFA/GNPs conjugate (A 520 -A 580 ). In addition to that, at VEGFA antibody concentration of 100ng/ml, the lowest optimum particle size with no aggregation and better stability of the anti-VEGFA /GNPsanti-VEGFA/GNPs conjugate was achieved as observed in Figure 1B. The present study found that 100 ng/ ml VEGFA antibody is the optimum minimum concentration of anti-VEGFA for a stable anti-VEGFA/GNPs conjugate with about 45 nm average size. Spectrophotometer measurements of gold nanoparticles GNPs showed sharp characteristic absorption peak at 520 nm which corresponding to Surface Plasmon Resonance (SPR) absorbance peak, while anti-VEGFA/GNPs conjugate showed slight red shift at 532 nm as shown in Figure 2A.  The measurements of particle size distribution based on dynamic light scattering technique. Figure 2B shows that average particle size of GNPs is 18.3±6.3 nm with narrow peak, indicating monodispersity and no aggregation. While, the average particle size of anti-VEGFA/GNPs conjugate is 43±8.3 nm with narrow peak, indicating no aggregation formed upon conjugation which indicate particle size growth that support the formation of Anti-VEGFA antibody cap around the gold nanoparticles.
Zeta potential measurements of GNPs and anti-VEGFA/ GNPs conjugate represent a decrease in the net negative charge surround the GNPs upon conjugation with anti-VEGFA according to replace of citrate anions from the dielectric layer surround GNPs with anti-VEGFA and keeping the anti-VEGFA/GNPs conjugate stable due to the repulsion between neighbor GNPs. The GNPs surface net charge has been-38.9 mV and the zeta potential for anti-VEGFA/GNPs conjugate showed a noticeable change in the surface net charge which decreased to the -25 mV as shown in Figure 2C which also confirm the formation of anti-VEGFA/ GNPs conjugate .HR-TEM imaging showed that the formation of spherical gold nanoparticles with average size around 20 nm, as shown in Figure 2D. A more specific characterization tool that confirm the conjugation formation was FTIR spectrum. FTIR analysis of GNPs showed a broad absorption bands appearing at 623 cm −1 and 1635 cm −1 due to CH 2 -O deformation and C-O stretching of citrate ligand in GNPs spectra, FTIR of anti-VEGFA/GNPs conjugate shows presence of peak specific for C=C stretching of aromatic amine group which appear at1116cm −1 and 1384cm −1 involve changing in both the band positions and redistribution of intensities, from FTIR spectra of anti-VEGFA alone which appear at 1118cm −1 and 1384cm −1 upon their adsorption onto GNPs ( Figure 2E).

Significance of clinical data
Laboratory investigations: Serum samples were obtained from 43 patients, First group, 22 patients with RA disease. Second group, 11 patients with primary knee OA upon clinical and radiological criteria of primary OA. Third group, 10 healthy volunteers subjected as control group. The mean age at the time of the study was 35-55 years and the mean duration of the disease was not more than one year. For complete blood count (CBC) parameters, it shows that there is no differences than normal values in OA. In RA patients, the mean value of Hb (9±0.3 g/dL) were lower than the control patients (12.5±0.5 g/dL), the mean value of WBCs (7.825±0.6 103/mL) were higher than those of the control subjects (4.1±0.23 103/mL). In addition to that, the mean value of the Erythrocyte Sedimentation Rate, ESR. In OA patients was (34.5 ± 3 mm/h) while that of RA patients was (54.5± 5 mm/h) higher than those of the control (7±0.15 mm/h), as shown in Table 1.

Correlations of VEGFA with clinical indicators
In osteoarthritis patient (OA), there was non-significant correlation in the level of VEGFA with complete blood count ( Table 2). While in RA patients there was significant positive correlations in the level of VEGFA with WBCs, High-grade systemic inflammation observed in active RA and significant positive correlations in the level of VEGFA with ESR, useful for monitoring inflammatory diseases especially Rheumatoid Arthritis ( Table 3). Table 4 & 5 represent the individual concentrations of VEGFA in OA and RA patient's serum, respectively, before and after treatment with anti-VEGFA/GNPs anti-VEGFA/GNPs conjugate, Anti-VEGFA and GNPs after incubation for 2 hr the results revealed that the treatment with anti-VEGFA/GNPs conjugate has the highest inhibitory effect on the serum VEGFA concentrations compared to treatment with Anti-VEGFA and GNPs. The inhibitory effect of the anti-VEGFA/GNPs conjugate was directly proportional to the concentration of VEGFA in patient serum irrespective to OA or RA patients. Where for OA patient with highest VEGFA level, 815 pg/ml, before treatment, was inhibited to be 299 pg/ml, 600 pg/ml and 800 pg/ml after treatment with anti-VEGFA/GNPs anti-VEGFA/GNPs conjugate, Anti VEGFA and GNPs, respectively. While for OA patient with lowest VEGFA level, 68 pg/ml, before treatment, was inhibited to be 55 pg/ml, 50 pg/ml and 60 pg/ ml after treatment with anti-VEGFA/GNPs anti-VEGFA/GNPs conjugate, Anti-VEGFA and GNPs, respectively. For RA patient with highest VEGFA level, 2500 pg/ml, before treatment, was inhibited to be 952 pg/ml, 1500 pg/ml and 2000 pg/ml after treatment with anti-VEGFA/GNPs conjugate, Anti VEGFA and GNPs, respectively. While for OA patient with lowest VEGFA level, 55 pg/ ml, before treatment, was inhibited to be 27 pg/ml, 45 pg/ml and 50 pg/ml after treatment with anti-VEGFA/GNPs conjugate, Anti-VEGFA and GNPs, respectively. It was found that the mean VEGFA concentration in reference group (control) was (30-100) pg/ml. Table 6 summarizes the mean inhibitory effect anti-VEGFA/ GNPs conjugate on the level of VEGFA in both OA and RA patient serum. Results indicate that, significant decrease in VEGFA level after incubation with anti-VEGFA/GNPs anti-VEGFA/GNPs conjugate. The mean concentration of VEGFA in OA patient's serum was (407±77 pg/ml) and decreased after incubation of serum with anti-VEGFA/GNPs conjugate to be (161±26 pg/ml). Similar results were obtained for RA patient's serum, where the mean concentration of VEGFA was (424±115 pg/ml) and decreased after incubation of serum with anti-VEGFA/GNPs anti-VEGFA/GNPs conjugate to be (155±44 pg/ml). There is a significant decrease in level of VEGFA after incubation of serum with anti-VEGFA/GNPs conjugate for 2 hr in both OA and RA patient's serum at p value =0.001 and 0.002, respectively, with mean percent of cure reach to be more than 59 % and 62 % for OA and RA patient's serum, respectively.

Discussion
The mean idea behind the present work is to find a new hope to relief the pain of arthritic patients through decreasing the level of VEGFA in serum. The affinity and sensitivity of anti-VEGFA/GNPs conjugate is affected by antibody concentration so that insufficient amount of antibody can lead to aggregation and increased amount will lead to flocculation that have been confirmed by subtraction of absorbance intensity of anti-VEGFA/GNPs conjugate at (A 520 -A 580 ) as shown in Figure 1A [28]. Where A 520 represent SPR absorbance peak of GNPs with no aggregation and A 580 represent beginning of aggregation so as the difference between them be great as the optimum anti-VEGFA/GNPs conjugate was obtained without flocculation [29]. Variation of GNPs particle size distribution, ( Figure 1B) with antibody concentrations shows that the anti-VEGFA concentration increases the stability of the GNPs and no aggregation affect the anti-VEGFA/GNPs conjugate until it reaches the minimum antibody concentration that stabilize GNPs, and then excess of the antibody will lead to increase of the size that reflects presence of aggregation and flocculation in the anti-VEGFA/GNPs conjugate leading to loss of affinity and sensitivity to antigen [30][31][32][33][34].
The functionalization of GNPs with anti-VEGFA antibody lead to mild red shift in the absorbance peak of GNPS from 520 nm to 532 nm as a function particles size growth due to antibody conjugation, (Figure 2A). Appearance of the characteristic antibody peak exhibited at 280 nm corresponding to tryptophan group, assure the formation of the anti-VEGFA/GNPs conjugate [35].
Zeta potential measurements of GNPs and anti-VEGFA/ GNPs conjugate, ( Figure 2C) the antibody/gold nanoparticles conjugation process. Where the zeta potential of GNPs was -38.9 mV due to encompassing surface of GNPs with citrate carboxylic groups. Zeta potential of anti-VEGFA/GNPs conjugate was -25 mV indicates the replacement of the negative charges of citrate ions with the positive charged VEGFA antibody. The type of interaction between anti-VEGFA and GNPs was not studied under this study but it may be electrostatic interaction rather than covalent interaction.
FTIR, particle size distribution and HRTEM characterization techniques confirm functionalization and formation of anti-VEGFA/GNPs conjugate at optimum conditions without aggregation or loss of affinity and sensitivity.
It is known that VEGFA antigen is produced during angiogenesis by rheumatoid synoviocytes and acts specifically on endothelial cells [36,37]. The angiogenesis is important in the development of new cartilage and mineralization in OA and the neoangiogenesis process contributes to synovitis, pannus formation and articular cartilage destruction in RA [38]. In RA and OA patients, the mean levels of VEGFA have been significantly higher than in healthy one.
Elevated Serum VEGFA was non-significant positively correlated with high WBCs in RA indicating high-grade systemic inflammation in active RA and OA has indeed been associated with the perpetua tion of synovial angiogenesis including VEGFA-mediated neovascularization [14]. Normochromic and normocytic anemia is an extra-articular symptoms of RA. Anemia in RA is multi-factorial-disease activity, drug-induced, nutritional, bone marrow suppression, and ineffective erythropoiesis. Anemia correlates with the disease activity, particularly the degree of articular inflammation [39].
The nanoconjugation strategy of anti-VEGFA to GNPs surface to which targeting VEGFA specifically over expressed in inflamed cells or in angiogenesis could reduce the side-effects due high level of VEGFA widespread in injured tissues in addition to healthy tissues and organs [40,41].
The comparison between OA and RA as regarding the effect of anti-VEGFA/GNPs conjugate on VEGFA level antigen shows that a significant decrease in level of VEGFA compared to the anti-VEGFA and GNPs alones as shown in Tables 4&5. The mean results are summarized in Table 6, where the mean value of VEGFA antigen have been significantly decreased in both RA and OA patients after incubation of serums with anti-VEGFA/GNPs conjugate with cure efficiency more than 60 %. The obtained results support the idea of this study which postulate that, anti-VEGFA/GNPs conjugate is highly effective for targeting certain angiogenic factor which may be used for treatment of arthritis disease which confirmed that the targeting of VEGFA is a fea sible anti-inflammatory and antiangiogenic therapeutic strategy in arthritis, as well as in malignancies [14, 41,42].
As the nanoconjugation improve the targeting and the efficacy of antibody in the serum make it more feasible to immune system. It was remarked that treatment efficacy was concentration dependent, for patients with high level of VEGFA, the efficacy of anti-VEGFA/GNPs conjugate was very high reach to more than 60% in contrast to that for patients with low level of VEGFA concentration the efficacy was not more than 20% that may be due to the loading capacity effect. The introduced anti-VEGFA/ GNPs conjugate can be used with patients complain of arthritis and will not affect the normal function of VEGFA antigen in blood vessel proliferation in normal situation as a growth factor, that makes the anti-VEGFA/GNPs conjugate verifies the requirements of ideal drugs which works only at high antigen levels as in case of severe arthritis [42,43].

Conclusion
Targeting of VEGFA is a fea sible anti-inflammatory and antiangiogenic therapeutic strategy in arthritis, as well as in malignancies and could reduce their side-effects. Anti-VEGFA/ GNPs conjugate was highly effective for scavenging VEGFAantigen in serum suppressing its effect and inhibit its interaction with the VEGFA receptors on macrophages and blood vessels. The Anti-VEGFA/GNPs conjugate, represented by this work, reduced the level of VEGFAantigen in both OA and RA patients serum (in vitro) at percent 60%which is considered to be promising approach in arthritis treatment as the traditional treatments is just a combination of drugs for inflammations and relief of pain symptoms.
The work done in this study was on in vitro scale for standardization of the technique. The group welling to apply this product (Anti-VEGFA/GNPs conjugate) in future for in vivo on animal models as a new modality for treatment of OA and RA patients through decrease level of VEGFA antigen in circulating blood.


s2fieldsofstudy: Biology Medicine

text:
Comparison of ankle plantar flexor activity between double-leg heel raise and walking

[Purpose] We aimed to evaluate the difference in the muscle activity between the double-leg heel raise (DHR) and treadmill walking. [Subjects] Thirty healthy males aged 21.5 ± 1.6 years (body mass 63.6 ± 9.3 kg, height 171.0 ± 4.5 cm) participated in the study. [Methods] Electromyograms were simultaneously recorded from both heads of the gastrocnemius and the soleus of the right side during the DHR and treadmill walking. The DHR conditions were maximum plantar flexion (MPF), 3/4 MPF, 2/4 MPF, and 1/4 MPF, and the walking speeds were 20, 40, 60, 80, and 100 m/min. [Results] The muscle activity during the DHR and walking significantly increased with increments in the height of the heel raise and walking speed, respectively. Comparison of the muscle activity at MPF with that at each walking speed revealed that the muscle activity in the soleus and gastrocnemius medial head during walking exceeded that during the DHR in less than 3.3% of cases. [Conclusion] The DHR test is useful for evaluating the ankle plantar flexor activity necessary for walking.

INTRODUCTION
The heel raise test is often used in a clinical setting to evaluate the function of the calf muscle. Muscle activities are increased during activities such as walking, running, and jumping. The standing heel raise, called the "calf raise", is a commonly prescribed exercise for improving the strength and power of the ankle plantar flexors. In physical therapy, the strength of the ankle plantar flexors is also measured during one-footed standing [1][2][3][4][5][6][7] . However, the single-leg heel raise can be difficult for patients with ankle disorder and elderly people because of decreased balance function. Therefore, the double-leg heel raise (DHR) is potentially useful for the clinical evaluation of ankle plantar flexors 8) .
There have been few studies on the maximum plantar flexion (MPF) moment during the DHR. Flanagan 9) reported that the MPF moment for older adults was 0.85 ± 0.18 Nm/ kg during the DHR and 1.50 ± 0.23 Nm/kg during the singleleg heel raise. The strength of the plantar flexor muscles is particularly important for walking. The activity of the plantar flexor muscles increases during the single stance phase: from the mid-stance to terminal stance phase. Ishikawa 10) indicated that tendinous tissues of both the soleus and gas-trocnemius muscles slowly stretched during the single support phase and dramatically recoiled during the pre-swing phase, contributing to the storage of elastic energy. Akizuki et al. 11) reported that the plantar flexor activity was 30% of the maximum voluntary contraction (MVC) during normal speed walking. However, little attention has been paid to the plantar flexor activity during walking in comparison with that during the calf raise. Therefore, in the present study, we aimed to evaluate the difference in the muscle activity between the DHR and walking.

SUBJECTS AND METHODS
Thirty healthy young males participated in this study (age 21.5 ± 1.6 years, weight 63.6 ± 9.3 kg, height 171.0 ± 4.5 cm). All the subjects provided written informed consent prior to participation, and the Human Subjects Ethics Committee of Tohoku Bunka Gakuen University approved the study.
The passive ROM of plantar flexion at the right ankle joint was measured with subjects in a supine position using a goniometer (TTM-KJ; Sakai Medical Co., Ltd., Tokyo, Japan). The active ROM of plantar flexion was measured in a standing position under weight-bearing conditions. EMG data were simultaneously acquired from both heads of the gastrocnemius and the soleus muscle of the right leg during the DHR and treadmill walking. After shaving and scrubbing the skin with alcohol, disposable Ag/AgCl surface electrode discs with a diameter of 10 mm (Blue-Sensor N-00-S, Ambu, Copenhagen, Denmark) were attached to the skin at locations recommended by Perotto and Delagi 12) .

Original Article
For each muscle, two electrodes were placed approximately 20 mm apart, in the direction of the muscle fiber. A reference electrode, shared by the four measurement channels, was placed on the bony part of the malleolus lateralis. EMG was amplified by bipolar leads with an input impedance >10 MΩ and a gain was 80dB (MT-11; GEHC-J, Tokyo, Japan).
The foot-switch sensors (P002; DKH Co., Ltd., Tokyo, Japan) were attached to the planta pedis, at the center of a line between the malleolaris lateralis and the malleolaris medialis. The pressure data from the foot-switch sensors were amplified (PH-462; DKH Co., Ltd., Tokyo, Japan), and downloaded onto a personal computer through an A/D converter at a sampling frequency of 1 kHz.
After EMG and the foot-switch sensors were set up, each subject performed the DHR on a flat structure consisting of a load cell (SB-200K12; Kyowa, Japan) and hard wooden boards. The right foot was placed on the load cell and loaded as much as the leg on the other side. The subjects received feedback information from the load cell via a digital amplifier and indicator (AD-4532; A&D, Tokyo, Japan), and they controlled the load placed on the right foot so that it was half of body weight.
The heel raise was performed under four conditions: MPF, 3/4 MPF, 2/4 MPF, and 1/4 MPF. MPF was based on the active ROM in a standing position under weight-bearing conditions. The subjects followed a rhythm of 1 Hz produced with an electronic metronome, raising their heels for 1 s and lowering them until they touched the ground in the next second; therefore, the complete movement cycle was over 2 s. For each heel-raise condition, the DHR was performed five times in succession. The height of the heel raise was controlled using the bar of a device that was adjusted to touch the dorsum pedis at the selected heel-raise height.
After the DHR test, the subjects walked on a treadmill (Evolve JP; Jonson Health Tech Japan, Tokyo, Japan). The walking speed was set at 20, 40, 60, 80, or 100 m/min, and the subjects walked for 1 min at each speed. The EMG and foot-switch data were recorded during the last 30 s at each speed.
The analog EMG signals and foot-switch data were passed through a 16-bit A/D board (PowerLab; ADInstruments, Nagoya, Japan), and all signals were sampled at 1 kHz. The foot-switch data were filtered with an 89-ms moving average window. The onset of right heel contact was determined during the DHR and treadmill walking. The EMG data were filtered with a 10-Hz high-pass IIR digital filter and zero-phase filtering. After the signal was full-wave rectified, the moving average was calculated for an 89-ms window. For the EMG data during the DHR, the peak values were detected for each heel-raise movement. We calculated the average of five peak values and adopted it as the indicator of the muscle activity during the DHR. On the other hand, during treadmill walking, EMG data of each gait cycle were converted into 100 data using a spline; the data for each gait cycle was interpolated to 100% of gait cycle at 1% intervals. After calculating the addition average values for all gait cycles, the peak value was determined for each subject. We adopted the peak value as the indicator of the muscle activity during treadmill walking.
A within-subject one-way analysis of variance (ANOVA) was used to analyze differences in the activity of the three muscles. The independent variables were the height of the heel raise and the walking speed, and the dependent variable was muscle activity. Significant individual differences were evaluated using the Scheffe test. Differences were assessed using two-sided tests, and an alpha level of 0.05.

RESULTS
The passive MPF in the supine position was 53 ± 6 degrees, and the active MPF in the standing position was 50 ± 6 degrees. There was no significant difference between the passive MPF and the active MPF. There were significant main effects of both the height of the heel raise (p < 0.001) and walking speed (p < 0.001). The muscle activity during the DHR significantly increased with the increase in the height of the heel raise (Table 1). In addition, there were significant differences in muscle activity between walking speeds for each muscle (p < 0.001). During treadmill walking, muscle activity significantly increased with the increase in the walking speed (Table 2). There were significant differences in the muscle activity of the gastrocnemius lateral head between all walking speeds; however, there were no significant differences in the gastrocnemius medial head activity between 40 and 60 m/min and between 60 and 80 m/ min, and there were no significant differences in the soleus activity between 20 and 40 m/min and between 40 and 60 m/min.
In the comparison of the muscle activity at 1/4 MPF with that at each walking speed, the activity of the soleus There were significant differences between all conditions in each muscle (mV). in 26.7% of the subjects at a walking speed of 100 m/min was higher than that during the DHR, while the activity in the gastrocnemius lateral head in 80.0% of the subjects was higher than that during the DHR. However, in the comparison of the muscle activity at MPF with that at a walking speed of 100 m/min, this value decreased to 23.3% for the gastrocnemius lateral head. Furthermore, in the comparison of the muscle activity at MPF with that at each walking speed, the activities of the soleus and gastrocnemius medial head during walking exceeded those during the DHR in only 3.3% of subjects (Table 3).

DISCUSSION
The heel-raise test is often used in a clinical setting to evaluate the function of the calf muscles. However, the single-leg heel raise can be difficult for the elderly and other people with decreased balance function. Therefore, we proposed that the DHR test could be a useful alternative to the single-leg heel raise test for the clinical assessment of ankle plantar flexors. The results of this study indicated that the DHR test is useful for evaluation of the ankle plantar flexor activity necessary for walking.
There have been many studies on the plantar flexion torque of the ankle joint during walking; however, the findings have been variable, and many gait characteristics are also dependent on gait velocity 13,14) . DeVita and Hortobagyi 15) showed that the peak torque of the ankle joint at a comfortable walking velocity of 89 m/min was 136 ± 27 Nm in young subjects and 102 ± 29 Nm in elderly subjects. Browning and Kram 13) studied plantar flexion torque during walking at different velocities and found that the peak plantar flexion torque was 63.9 ± 4.85 Nm at 30 m/min and 85.8 ± 8.03 Nm at 90 m/min. Flanagan et al. 9) reported that the peak torque was 0.85 Nm/kg during the DHR. Therefore, if the body mass of the subjects ranged from 60 to 80 kg, the peak torque would range from 51 to 68 Nm. In that study, the plantar flexion peak torque for the DHR was lower than that for walking. However, analysis of the muscle activity of the main plantar flexors in the present study provided different results.
The heel-raise test involves eccentric-concentric muscle contraction of the plantar flexors. Eccentric pre-activation renders an increase in performance during the concentric phase, partly through the utilization of the elastic energy stored during the pre-activation phase. Mueller et al. 16) reported that the plantar flexion peak torque, measured using an isokinetic device, accounted for 40% and 53% of the variance in peak ankle moment and power during walking, respectively, and that there was a strong correlation between plantar flexion peak torque and dorsiflexion ROM. The authors explained that an increase in the dorsiflexion ROM allows for an increase in the requirement for the external dorsiflexion moment and necessarily increases the requirement for internal (muscle generated) control of the plantar flexion moment during walking. However, ankle dorsiflexion during the mid-stance phase may be important for other reasons. Ishikawa et al. 10) noted that the tendinous tissues of both the soleus and gastrocnemius muscles were slowly stretched during the single support phase and dramatically recoiled during the pre-swing phase, contributing to the storage elastic energy. In this respect, the DHR is similar to walking. However, during walking, the kinetics change from the closed kinetic chain of the stance phase to the open kinetic chain of the swing phase. On the other hand, there is no change during the DHR. This suggests that humans use muscle activity more efficiently during walking. In the present study, the activities of the soleus and gastrocnemius muscles during walking were less than those of these muscles during MPF.
Suzuki et al. 17) reported that the isometric strength of the plantar flexors was an important independent predictor for habitual gait velocity in community-dwelling older women. The plantar flexor muscles of the ankle joint play an important role in the generation of torque during walking. In addition, low torque of the plantar flexor muscles has been linked to an increased risk of falling in older individuals. Therefore, a simple method for evaluation of plantar flexor function is important for clinical assessment, and the DHR test fulfils this role.
In the present study, measurement of the plantar flexor muscle activity was limited to two muscles. However, the gastrocnemius and soleus muscles can produce a large amount of torque compared with other plantar flexor muscles. Although the maximum force of the triceps surae is 1620 N, the maximum force of the tibialis posterior is only 267 N 18) . If a moment arm is considered, the plantar flexion torque of the tibialis posterior is much smaller than that of the triceps surae. In addition, the double-leg heel raise does not allow us to evaluate the strength of the right and left calf muscle independently. However, the present study, which examined the DHR with half the body weight bearing by each lower extremity, indicates that it may be used as a criterion of muscle weakness if the center of gravity shifts into the left or right.


extfieldsofstudy: Biology Medicine

text:
Insect Pathogenic Bacteria in Integrated Pest Management

The scientific community working in the field of insect pathology is experiencing an increasing academic and industrial interest in the discovery and development of new bioinsecticides as environmentally friendly pest control tools to be integrated, in combination or rotation, with chemicals in pest management programs. In this scientific context, market data report a significant growth of the biopesticide segment. Acquisition of new technologies by multinational Ag-tech companies is the center of the present industrial environment. This trend is in line with the requirements of new regulations on Integrated Pest Management. After a few decades of research on microbial pest management dominated by Bacillus thuringiensis (Bt), novel bacterial species with innovative modes of action are being discovered and developed into new products. Significant cases include the entomopathogenic nematode symbionts Photorhabdus spp. and Xenorhabdus spp., Serratia species, Yersinia entomophaga, Pseudomonas entomophila, and the recently discovered Betaproteobacteria species Burkholderia spp. and Chromobacterium spp. Lastly, Actinobacteria species like Streptomyces spp. and Saccharopolyspora spp. have gained high commercial interest for the production of a variety of metabolites acting as potent insecticides. With the aim to give a timely picture of the cutting-edge advancements in this renewed research field, different representative cases are reported and discussed.

Introduction
Bacteria are widespread in the environment and they have evolved a variety of interactions with insects including essential symbiosis [1]. While many bacterial species inhabit bodies of insects establishing different levels of mutualistic relationships, only a limited number of them behave as insect pathogens. The latter have evolved a multiplicity of strategies to invade the host, to overcome its immune responses, to infect and to kill it. The mechanisms leading to these kinds of interactions are presumed to have ancient origin and to have developed throughout a long co-evolution process [2]. In line with this concept, a variety of insecticidal toxins produced by certain spore forming entomopathogenic bacteria, have a similar structure and mode of action. This is the case for protein toxins produced by Bacillus thuringiensis Berliner (Bt) and localized in parasporal bodies [3]. These toxins are normally very specific to a limited range of targets, while in other cases bacteria produce metabolites that show a broader insecticidal spectrum. Important information to understand the molecular mechanisms involved in diverse pathogen-host interactions are being produced as a result of modern "omic" studies. However, many aspects are still unrevealed and after few decades of microbial pest management dominated by B. thuringiensis, novel bacterial species with innovative modes of action have been discovered and formulated as new biopesticidal products [4].
The entomopathogenic bacteria domain has traditionally been well represented by members of the Bacillaceae family, such as B. thuringiensis, Lysinibacillus sphaericus (Meyer & Neide) Ahmed et al., Paenibacillus spp. and Brevibacillus laterosporus Laubach. The entomopathogens belonging to the Gammaproteobacteria class, which includes the entomopathogenic nematode symbionts Photorhabdus spp. and Xenorhabdus spp., Serratia species, Yersinia entomophaga Hurst and Pseudomonas entomophila (Mulet et al.), are also important.
More recent is the discovery of Betaproteobacteria species that show broad-spectrum insecticidal properties. This group includes specific strains of Burkholderia spp. and Chromobacterium spp. Lastly, certain Actinobacteria species have gained high scientific and commercial interest in relation to the production of a variety of metabolites acting as potent insecticides. This is the case for Streptomyces and Saccharopolyspora species.
As a result of continuous industrial and academic screening activities, the discovery of new bacterial species and insecticidal metabolites is expected in the near future [5,6]. This trend is also the result of modern legislative frameworks fostering the use of bioinsecticides in Integrated Pest Management (IPM) programs.
The purpose of the present review is to give an overall and concise picture of the knowledge advancements of insect pathogenic bacteria and of their use as bio-insecticidal products for IPM.

Bacillus thuringiensis
Bacillus thuringiensis (Bt) is the most studied entomopathogenic species and some of its crystal producing strains have certainly represented the main active substances used for the microbial pest management during the last decades [7]. The pathogenic action of this bacterium normally occurs after ingestion of spores and crystalline inclusions containing insecticidal δ-endotoxins that specifically interact with receptors in the insect midgut epithelial cells [8]. It is largely demonstrated that these toxins, mostly represented by Cry proteins, after being solubilized and activated in the insect midgut, act through a pore-forming mechanism determining the disruption of natural cell membrane permeability, with consequent cell lysis followed by gut paralysis and death [9].
Most commercially available formulations are based on spore-crystal mixtures with effectiveness against different pest species. B. thuringiensis subsp. kurstaki (Btk) is generally used against young Lepidopteran larvae and includes different strains with significant commercial interest like HD-1, SA-11, SA-12, PB 54, ABTS-351 and EG2348. Strains of B. thuringiensis subsp. aizawai (Bta) (i.e., ABTS-1857) are also used against armyworms and diamondback moth larvae. Besides, strains belonging to the subsp. israelensis (Bti) and tenebrionis (Btt) have been employed for the management of mosquitoes and simulids, and against coleoptera, respectively [10]. Continuous research activities have led to the isolation of many strains and to the discovery and characterization of various Bt insecticidal toxins produced in different bacterial stages (i.e., Cyt, VIP) [3]. The insecticidal potential of bacterial strains closely related to B. thuringiensis has also been demonstrated [11].
Additionally, the integration of cry genes into genetically modified plants has been successfully implemented to confer resistance to specific crop pests.

Lysinibacillus sphaericus
Entomopathogenic strains belonging to the L. sphaericus (formerly Bacillus sphaericus) species group are featured by the production of spherical endospores closely associated with parasporal crystals containing an equimolar ratio of binary protein toxins (BinA and BinB) [12]. The insecticidal mode of action includes damages to the microvillar epithelial cells in the midgut comparable to the ones known for B. thuringiensis [13]. In addition, vegetative cells of certain strains produce mosquitocidal toxins (Mtx proteins).
The main targets of commercial formulations based on L. sphaericus strains are mosquitoes, blackflies and non-biting midges.

Paenibacillus spp.
The genus Paenibacillus includes different species showing pathogenicity against insects like the causative agent of the honeybee disease American Foulbrood (AFB), P. larvae subsp. larvae [14].
On the other hand, the spore-formers P. popilliae (Dutky) Pettersson et al. and P. lentimorbus (Dutky) Pettersson et al. are the causal agents of milky disease in phytophagous coleopteran larvae. The production of parasporal inclusions within the sporangial cells has been observed in P. popilliae, even if they are not directly responsible for the insecticidal action. However, homology between a 80 kDa parasporal protein this species produces and Bt Cry toxins has been demonstrated [15]. After spores are ingested by the host, they germinate in the midgut. The following pathogenicity seems to be in relation to the septicemia caused by vegetative cells.

Brevibacillus laterosporus
Brevibacillus (former Bacillus) laterosporus is a pathogen of invertebrates and a broad spectrum antimicrobial species [16]. During sporulation it produces a typical canoe-shaped parasporal body (CSPB) firmly associated with the spore coat, which gives this species a unique morphological feature. The insecticidal action of different B. laterosporus strains has been reported against insects in different orders, including Coleoptera, Lepidoptera and Diptera, and against mollusks, nematodes, phytopathogenic bacteria and fungi. In relation to its antifungal and antibacterial properties, due to the production of antibiotics, it has also found use in medicine.
The whole genome of B. laterosporus has recently been published [17,18], which reveals the potential to produce different toxins.
Certain strains showing toxicity against the corn rootworms (Diabrotrica spp.) and other coleopteran larvae, produce insecticidal secreted proteins (ISPs) that act as binary toxins in the insect midgut and have high homology with B. thuringiensis vegetative insecticidal proteins (VIPs) [19].
Specific strains toxic to mosquitoes produce parasporal inclusion bodies reminiscent of those produced by B. thuringiensis. These bodies contain proteins and their implication in the mosquitocidal action has been reported [20]. Spores of a strain lacking parasporal crystals are highly toxic to the house fly Musca domestica L., and the mode of action implies histopathological changes in the midgut with disruption of the microvillar epithelium [21,22].

Clostridium bifermentans
A strain of C. bifermentans (Weinberg and Séguin) Bergey et al. serovar malaysia (C.b.m.), isolated in Malaysia, shows high toxicity against mosquitoes and black flies. During sporulation, this bacterium produces three major proteins involved in the insecticidal action [23]. These include the mosquitocidal protein Cbm71 showing homology to B. thuringiensis delta endotoxins [24].

Photorhabdus spp. and Xenorhabdus spp.
The entomopathogenic members of the genera Photorhabdus and Xenorhabdus are represented by endosymbionts of insecticidal nematodes. The first are typically associated with entomopathogenic nematodes in the genus Heterorhabditis, while the second to Steinernema species. The pathogenic action usually involves the release of symbiotic bacteria in the insect hemocoel once the nematodes have actively entered the insect body. Here the bacteria proliferate producing various antimicrobial compounds to contrast the growth of other microorganisms. They also release different enzymes that contribute to the degradation processes in the hemocoel, thus creating an ideal environment for the development of the nematode population.
A variety of bacterial virulence factors are involved in the interaction with the susceptible host. Different Photorhabdus and Xenorhabdus species producing an insecticidal toxins complex (Tc) have high potential for pest management [25]. Generally, the Tcs are high-molecular weight and multi-subunit proteins that include three components, A, B and C, orally active against different insects [26]. While the mode of action is not completely understood, all these components are normally needed to achieve full toxicity [27].
Another example of insecticidal proteins produced by these bacterial species is represented by the Photorhabdus insect related (Pir) proteins, produced by P. luminescens (Thomas and Poinar), that show similarity to B. thuringiensis delta-endotoxins and have been proposed to be mimics of the juvenile hormone esterases (JHEs) interfering with insect development regulation [28].
In addition to insecticidal toxins and various metabolites, these bacterial endosymbionts have evolved different mechanisms to face the insect immune response. For instance, it has been shown that certain Photorhabdus species exploit lipopolysaccharide modifications to resist the action of insect antimicrobial peptides (AMPs) [29], while X. nematophila (Poinar and Thomas) interferes with the expression mechanisms of host AMPs [30].
So far, the commercial use of these bacterial endosymbionts is related to the employment of the nematode species with which they are associated.

Serratia spp.
The association of Serratia spp. with insects or with entomopathogenic nematodes is well documented [32][33][34]. Different species in this genus produce a variety of virulence factors [35]. Common is the production of toxin complexes analogous to those produced by Xenorhabdus spp. and Photorhabdus spp. S. entomophila Grimont et al., a pathogen of the grass grub, Costelytra zealandica (White) (Coleoptera: Scarabaeidae) [36], produces Sep proteins (SepA, SepB, SepC), a group of insecticidal toxins showing similarities to the insecticidal toxins of P. luminescens [37].
On the other hand, the recent genome sequencing of S. nematodiphila Zhang et al. also highlighted other pathogenic factors of Serratia species. Among these was a variety of secreted extracellular enzymes such as proteases, lipases, and chitinases [38].
It has recently been demonstrated that the pathogenicity of S. marcescens Bizio is increased by the action of a serralysin metalloprotease it secretes and that this bacterium is able to suppress cellular immunity by decreasing the adhesive properties of immunosurveillance cells of the insect host [39].

Yersinia entomophaga
Isolated from the New Zealand grass grub, C. zealandica, Y. entomophaga is a non-spore-forming entomopathogenic bacterium characterized by the production of an insecticidal toxin complex (Yen-Tc) showing similarity to those produced by Photorabdus spp. [40]. These complexes include three Yen protein families, A, B and C, and two chitinases (Chi1 and Chi2) [41].
The broad insecticidal range of these toxins and their post-ingestion histopathological action in the insect midgut epithelium have been reported [42].
Promising are the studies conducted in field conditions with insecticidal formulations containing Y. entomophaga against the pasture pest porina (Wiseana spp. larvae) [43].

Pseudomonas entomophila
P. entomophila is a ubiquitous bacterium that orally infects larvae of insects in different orders determining extensive gut cell damages. Host-pathogen interactions have been studied in experiments with Drosophila melanogaster Meigen (Diptera: Drosophilidae), which highlighted a specific post-ingestion immune response [44]. Recent complete genome sequencing of P. entomophila revealed a specific secretion system and the associated toxins probably responsible for its entompathogenic properties [45].

Burkholderia spp.
Different insect species harbor symbiotic bacteria of the genus Burkholderia, mostly in association with specific gut regions [46,47]. In addition to these mutualistic relationships with insects, Burkholderia sp. has recently been reported to affect ovipositon and fecundity of the bean bug Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) [48]. The pontential of Burkholderia species as biocontrol agents against different plant pathogens has also been reported [49,50]. More recently, the insecticidal properties of a new strain isolated in soil from Japan and identified as B. rinojensis sp. nov., were discovered [51]. Whole cell broth cultures of this bacterial strain, named A396, show oral toxicity and contact effects against the beet armyworm Spodoptera exigua Hübner (Lepidoptera: Noctuidae) and the two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae). Insecticidal and miticidal properties are maintained after heat-treatment, hence commercial formulations against a variety of chewing and sucking insects and mites are based on heat-killed cells and spent fermentation media. Different bacterial metabolites might be involved in the insecticidal action. Interestingly, a recent study highlighted the biochemical properties of natural compounds produced by fermentation of a Burkholderia sp. strain showing high homology to B. rinojensis [52].

Chromobacterium spp.
A strain of Chromobacterium subtsugae Martin et al., isolated from a soil sample in Maryland (USA) and named PRAA4-1T, was discovered to show high insecticidal activity against insect species in different orders, including the Diamondback moth Plutella xylostella L. (Lepidoptera: Plutellidae), the Sweet potato whitefly Bemisia tabaci Gennadium (Rhynchota: Aleyrodidae), the Southern green stink bug Nezara viridula L. (Rhynchota: Pentatomidae), the Southern corn rootworm Diabrotica undecimpunctata Mannerheim (Coleoptera:Chrysomelidae spectrum), the Western corn rootworm Diabrotica virgifera Le Conte (Coleoptera: Chrysomelidae), the Colorado potato beetle Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), and the Small hive beetle Aethina tumida Murray (Coleoptera: Nitidulidae) [53,54]. The broad spectrum activity of this strain is related to multiple modes of action probably involving different chemical compounds produced by the bacterium. Among the bacterial metabolites, C. subtsugae synthesizes the tryptophan derivative violacein, which confers a typical violet color to its colonies. In addition to this, various molecules produced by this species have been characterized and associated with the insecticidal action [55]. Bioactive compounds were reported to be associated to the stationary growth phase [56], and the heat-stability of insecticidal toxins was also demonstrated [57]. The active ingredient of available commercial formulations is represented by C. subtsugae strain PRAA4-1T and spent fermentation media.
More recently, a new strain identified as Chromobacterium (Csp_P), isolated from the midgut of Aedes aegypti L. (Diptera: Culicidae) was shown to be able to colonize the insect midgut and to display entomopathogenic and anti-pathogen properties [58].

Streptomyces spp.
Different Streptomyces spp. are associated with herbivorous insects that take advantage of their cellulolytic properties [59]. Other species and strains in this genus produce a variety of metabolites acting as potent toxins against either phytopathogenic microbials or insect pests [60]. Among the first discovered insecticidal substances produced by Streptomyces species are flavensomycin [61], antimycin A [62], piericidins [63], macrotetralides [64] and prasinons [65]. Later on, the insecticidal and anthelmintic activities of avermectins produced by the soil Actinomycete Streptomyces avermitilis MS & Dwas, were discovered [66]. These macrocyclic lactone derivatives target the gamma-aminobutyric acid (GABA) receptor in the insect peripheral nervous system. The enhancement of GABA binding generates a cascade of events resulting in the inhibition of neurotransmission and paralysis of the neuromuscular systems [67].
Insecticides based on avermectins include a mixture of avermectin B1a and avermectin B1b, known as abamectin, that act by contact and ingestion and have a limited plant translaminar activity. Analogous substances produced by Streptomyces species include Emamectin, especially toxic to Lepidoptera, and Milbemectin, isolated from S. hygroscopicus Jensen. A variety of other secondary metabolites produced by diverse Streptomyces species, have been isolated and characterized so far. Commercialized products have been very successful against ectoparasites and endoparasites with medical and veterinary importance.

Saccharopolyspora spinosa
Saccharopolyspora spinosa Mertz and Yao was discovered during a screening program where the insecticidal activity of the isolate A83543 emerged [68]. Subsequent assays highlighted the broad toxicity of specific compounds isolated from the fermentation broth that were given the generic name of spynosins. The major component is spinosyn A, whose structure comprises a tetracyclic polyketide aglycone to which a neutral saccharide substituent is attached [69]. A variety of spinosyn analogs have been isolated and many studies have been conducted to investigate the pathway of spinosyn biosynthesis, which led to the characterization of specific gene clusters [70].
Since the first experimentations, spinosyns exhibited broad-spectrum activity against insect species in different orders, especially Lepidoptera and Diptera [71]. The natural S. spinosa fermentation-derived mixtures were named "spinosad" and contain spinosyn A and spinosyn D, as major and minor component, respectively. The biological activity of numerous semisynthetic derivatives has been studied [72].
The insecticidal mode of action of spinosyns is not completely understood, but is considered unique in comparison with other insecticides. It has been demonstrated their interaction with g-aminobutyric acid receptors and nicotinic acetylcholine receptors, eventually leading to the disruption of neuronal activity and consequent insect paralysis and death [73]. Despite their broad spectrum of activity against insects, spinosyns are associated with a low risk toward non-target species, including mammals and various aquatic organisms, in comparison with other insecticides [74].
The continuous research and industrial interest in this field has recently led to the discovery of a variety of spinosyn-related compounds produced by another Saccharopolyspora species, S. pogona [75].

Use of Entomopathogenic Bacteria in Integrated Pest Management
Insect pathogenic bacteria and their derived products represent the active substances of various "biopesticides". There is a range of definitions for this term, but it essentially includes mass reared living organisms (natural insect predators and parasitoids), nematodes and micro-organisms (bacteria, fungi, microsporidia, virus), natural compounds from plant extracts, and semiochemicals (e.g., insect pheromones). Besides, in countries where their use is permitted, biopesticides comprise genetically modified plants that express genes conferring resistance against pests or diseases (plant incorporated products).
Biological control agents, like bacterial entomopathogens, are generally recognized as lower risk substances than conventional chemical pesticides, and various benefits are associated with their use. For instance, their mode of action is normally more complex than conventional chemicals, targeting a diversity of action sites, which makes the development of resistant pests less likely. Although entomopathogenic bacteria can be used as stand-alone products for pest management in organic farming, their use in rotation or combination with chemicals is strongly encouraged to achieve full efficacy and eco-sustainability. Many studies have highlighted compatibility and synergistic effects of entomopathogenic bacteria and chemical substances [76][77][78]. Among the other advantages of including biopesticides in pest management programs are their safety for workers, the reduction of residues on crop and the flexibility on harvests, due to minimal or no pre-harvest interval.
The effectiveness of entomopathogenic bacteria is often associated with a proper application in the field. For instance, in the case of products that act by ingestion (i.e., Bt based products), timing is critical to ensure that the bacterial toxins remain stable in the environment until they are ingested by their target insect stage [7]. Another aspect is to ensure a proper coverage of substrates (e.g., foliage) frequented or eaten by insects. This has led to the development of special processing and formulation of bacteria-based bioinsecticides, with the aim of maximizing shelf-life, improving dispersion and adhesion, reducing spray drift and above all enhancing efficacy. A variety of adjuvants and additives for microbial formulations have been developed by the industry. These include dispersants, surfactants, wetters, spreaders, drift control agents, pH buffers, antifoam agents, carriers, phagostimulants and attractants [79]. Depending on the application target and on the adverse environment conditions, a choice of solid and liquid formulations is available. The first include dusts, granules, briquettes and wettable powders (WPs), while liquid suspensions may consist of suspension concentrates (SCs), emulsions and encapsulations. Advanced technologies aiming at increasing residual effects comprise microencapsulations and microgranules.
The variety of entomopathogenic bacteria based active substances authorized for commercialization and of their relative commercial products is significantly increasing worldwide. Consequently, their market is expanding into new segments, thus broadening their use for pest management, traditionally relegated to niche contexts (forest, public health, protected crops) [6].
Besides optimizing efficacy, modern pest management strategies tend to minimize the impact on the environment and on non-target organisms. This is also in line with the need to meet current regulations on the maximum residue levels (MRLs) for synthetic pesticides. The management of pest counter-adaptation (resistance) to pesticidal products is another concern in the agro-ecosystem. The integration of bio-based pesticides in pest management programs in many cases represents an important resource to face these challenges. During the last years, the number of microbial products available has grown significantly and many efforts are dedicated to increasing the awareness and to fostering the adoption of biopesticides in integrated pest management programs. This includes the implementation of worldwide industrial understandings like the Biopesticide Industry Alliance (BPIA) and the International Biocontrol Manufacturers' Association (IBMA). According to this trend, a database of available biopesticides is maintained within the context of the biopesticide program of the Interregional Research Project Number 4 (IR-4) at Rutgers University (U.S.).
Many biopesticides have recently gained interest within the legislative framework of most important regions like USA and Europe, fostering the use of low risk active substances in agriculture. In the United States, for the pre-market approval (registration) of any pesticide, the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) requires a specific evaluation by the Environment Protection Agency (EPA).

Market Overview
Based on a recent report published by BCC Research LLC (CHM029E, June 2014), the global market for pesticides, including both the synthetic pesticides and the biopesticides segments, is estimated to grow at a compound annual growth rate (CAGR) of 6.3% and to reach more than $80 billion by 2019.
The fastest growing segment of this market is represented by biopesticides (around 4%) that are expected to increase at more than twice the rate of synthetic pesticides. This segment comprises both biochemical (i.e., semiochemicals, plant extracts) and microbial biopesticides. Among the latter are included products based on bacteria, fungi, virus and nematodes, targeting different kinds of plant pests and parasites. A different category is represented by biocides, including products for public health like microbials targeting mosquitoes and flies, whose commercialization is differently regulated. This market is also expected to experience substantial growth in future (Research and Markets, ID: 2692355, October 2013).
The increased industrial interest in biopesticides is shaping a changing global scenario featured by strategic partnerships, mergers and acquisitions of companies. Significant examples of this trend are the recent acquisitions of the US-based biological company AgraQuest and of the German company Prophyta GmbH by Bayer CropScience. Similarly, BASF acquired Becker Underwood Inc., while Syngenta agreed to acquire the US-based biotechnology company Pasteuria Bioscience Inc. and the Swiss Devgen. On the other side, Monsanto and Novozymes have established an historical BioAg Alliance committed to the discovery and development of new microbial solutions for agriculture.

Conclusions
The need to feed a growing human population requires continuous advancements in pest management systems limiting production losses in major crops [80]. On the other side, the land available for cultivation and farming on Earth is limited, which requires the development of new technologies supporting improvements in productivity. Safeguarding the environment and human health, and the need to manage the development of insect resistance to pesticides, are additional concerns. For all these reasons, the integration of bio-based insecticides in combination or rotation with synthetic formulations is strongly recommended. This is in line with the expectations of fruit and vegetable consumers and with the requirements of a modern legislative framework on the use of IPM in agriculture.
Following this trend, an increasing academic and industrial interest in the discovery and development of new bioinsecticides is being experienced by the scientific community working in the field of insect pathology. The relative market segments and the industrial interest in this field are also growing at a significant rate. As a result, researches on entomopathogenic bacteria are gaining momentum and new discoveries are expected in the near future. Due to the regulatory issues related to the pre-market authorization of new active substances, the availability of bio-based products, including entomopathogenic bacteria, is still limited to certain crop pests. However, both research funding bodies and industry are progressing in the direction of increasing investments in this field, which will result in a continuous expansion of the repertoire of insect pathogenic bacteria-based formulations available for integrated pest management.