Strategies in Protein Engineering to Evolve Proteins and Mimicking Evolution in the Laboratory Scale-Juniper Publishers

Trends in Technical & Scientific Research

Abstract

Nature has its own way of synthesizing molecules and materials with an array of bio-catalysts known as enzymes. Enzymes comprise of a combination of proteins which in turn are the building blocks of life. With just a backbone combination of Carbon, Hydrogen, Oxygen and Nitrogen, proteins can be perceived as delightfully intelligent and adaptive. Over billions of years, nature have provided a wide range of survival questions in front of these molecules and somehow, they have evolved and survived, as proved by Darwin. This adaptive nature of proteins has been exploited and successful evolution of enzymes is possible towards a desired interest on a laboratory scale using nature’s machinery. Thus, mimicking evolution on a much shorter time-scale. In this article, we address the strategies involving the idea of evolution in a laboratory directed to fulfil our chemical needs. Also, how with time, the strategies have evolved covering up the loop-holes of previous generations. The advent of polymerase chain reactions made combinatorial libraries creation easy and then screen towards a fitness test. With time we learnt, a combination of evolution with rational designing creates shorter and smarter libraries that have a strong ‘impact factor’ upon the desired goal and reduce false-positives. However, while controlling biological systems, scientists are manipulating the factories that actually make the molecules and materials into newer forms that are not natural. Nature, out-does scientists via evolution. Through survival of the fittest, evolution collects all the beneficial mutations through multiple iterated genetic diversification and screening.

Keywords: Directed evolution; Protein engineering; Combinatorial libraries; Mutagenesis; Enzymes; Promiscuous ancestral proteins; Escherichia coli metabolism; Chemical origins; Thermoanaerobacter brockii alcohol dehydrogenase; X-ray crystallographic structures

Introduction

Enzymes are thought to be highly specific biological catalysts having a very discreet range of target molecules or substrates [1, 2]. The evolution of enzymes has been occurring under different selection pressures of nature from promiscuous ancestral proteins. A genomic analysis in Escherichia coli metabolism reveals that 37% of its enzymes have a wide variety of substrate range. However, they are found to catalyze 65% of the known metabolic reactions [3]. The evolution of enzymes in response to environmental demands is the key to the overall organism’s fitness in the nature. This is manifested in the inherent promiscuity of many enzymes. Although, we are thankful to the ‘specialist’ nature of the enzyme molecule, we also do question, how much further can the ‘generalist’ nature be pushed in-vitro or in-vivo so that an enzyme can be evolved more. In the 3-D structure analysis, the active site of an enzyme is the engine, that performs the turn-over of a substrate into the product (catalytic machinery) and more specifically amino-acid residues have been identified from the X-ray crystallographic structures that are key players in the process. Textbook definition of enzymatic activity reveals that the work of the enzyme is broadly related to the binding of the substrate into the active site, followed by a possible conformation change, then the catalytic activity happening in the core of the enzyme and finally, the product leaving the enzyme-product complex. To explain the evolution of enzymatic catalytic activities, scientists have used models of chemical origins to propose the evolutionary initiation of the diversity. One such model demonstrates via chemical models and assumes key mutations in the active site that can lead towards enhanced specificities or sometimes enhanced specificities but with compromised efficiency. Then it proposes that designing a realistic trade-off constraint is possible with mutation combinations where the evolution can be directed towards both enhanced specificity and efficiency [4].

To improve the characteristics of an enzyme structural bioinformatics has been extensively used and with de-novo approaches enzymes have designed for enhanced specificity and functionality. Evolution has been a brute force that pushes the enzymes to evolve and then select the survivors based on their functionality. From Figure 1 it is evident that the evolution not only results in various changes in chemistry and substrates but are also supplemented with rarer gain or loss in the function in the form of moonlighting and pseudoenzymes respectively [5]. Due to the innate promiscuity, many enzymes do show off-target activity even when the natural selection pressure ceases to exist over evolutionary time. At that point of saturation, further catalytic/specificity changes do not improve the fitness in its highly conserved active site. The phenomenon was demonstrated by -lactam antibiotic resistance in bacterial populations over many generations. The promiscuity can also vary between orthologous enzymes which originate from the same family [5-7]. An approach of rational design relying on somewhat of a thorough knowledge of the protein structure that exists for many years is called directed evolution [8,9]. It is a popular technique that has been used to evolve a particular enzyme towards a user-defined goal. The ‘user defined’ goals narrows down the chances to produce the false-positives in comparison to natural selections. Also, this gives an opportunity to mimic natural evolution in a laboratory pilot scale operation. The limitations of this technique however is, screening may be needed for enormous random mutagenesis libraries which in-turn needs a robust high-throughput assay. A combinatorial approach has also been implied to create ‘focused’ libraries that will concentrate on regions of the enzyme active site that has been predicted by rational design to produce beneficial mutations and reduce the number of mutants to screen by a significant number. However, it must also be mentioned that our understanding of the link between the genetic sequence and function lags well behind our desire for designing new enzymes. Which makes predictions of performance-enhancing mutations, extremely challenging.

In the early 90s the most user-defined properties that were targeted for evolution included thermostability, nonnative substrates or activity in harsh organic solvents and after rounds of screening it was sometimes revealed that beneficial mutations could be nowhere near the active site of the enzyme [5,6]. Hence, it would be a hitting a deadended loop to just look for the key in the active site of the enzyme. The power of a combinatorial library is enormous. With a stable high-throughput assay it is possible to analyze millions of mutant proteins in one day. In some other cases however, the number can be less than a few hundred per day. So, development of smarter libraries is very important in this aspect. Using site-directed mutagenesis and site-directed saturation mutagenesis techniques, identification and construction of a ‘mutable landscape’ has been done in case of cytochrome P450 evolution [10-12]. This has been proved to be a very effective technique recently to tackle the very important challenge of the sequence-function relationship in case of directed evolution. The libraries thus generated on mutability landscapes can be used to engineer any fitness trait of user-defined interest [6,7,13]. We are entering into an era where the ‘user-defined’ interest has changed a lot. The demand for synthetic compounds has been growing and ‘green synthesis’ has been one of the most valued tools in the 21st century that uses enzymes as biocatalysts and minimizes the production of non-degradable bye-products and pollutants. So, directed evolution of stereo- or regio-selective enzymes that can behave as biocatalysts in asymmetric transformations is of particular interests to bio-organic chemists. Towards this goal, raised the need to develop the small and smart libraries. And hence forth different strategies including triple-code saturation mutagenesis (TCSM) at multiresidue sites of the Thermoanaerobacter brockii alcohol dehydrogenase by using distinct reduced amino acid alphabets and with requirement minimal screening have been recently reported [13-15]. So not only the physical labor has been greatly reduced, but also chances of false-postive results and codon-degeneracy can be minimized under this protocol [13].

Discussion

To push the promiscuous nature inherent in enzymes has been what scientists been brain-storming over the decades. The further challenges are to create enzymes for reactions that will let them solve problems that neither synthetic organic chemistry nor biological chemistry can address. Being macromolecular catalysts, it would be easier for enzymes to stabilize transition-states which otherwise would be inaccessible to chemical small molecule catalysts as they would likely compete with other lower-energy reaction pathways. Such was reported from cytochrome P450 from Labrenzia arregata [16]. Strategies and efforts are currently being invested to design enzymes with artificial cofactors as well. However, designed enzymes are yet to have the sophistication of nature’s products and struggles remain with other co-factors and prosthetic groups that are currently being addressed.

Conclusion

Over time, nature has been the best template to mankind. In our case, it has been the best chemistry teacher, solving the very challenges of existence of life over billions of years through a range of astonishing conditions. This has been possible only at the molecular level because of the enzymes which act as the fundamental bio-molecules. These systems are good models for a sustainable chemical industry or energy industry that uses renewable natural resources and recycles almost all of it without causing significant damage to the environment. It is not far where we stand today that DNA programmable organisms can produce many of our desired chemical needs.

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Heavy Metal Bioaccumulation by Cestode Parasites of Mustelus Schmitti (Chondrichthyes: Carcharhiniformes), from the Bahía Blanca Estuary, Argentina-Juniper Publishers

Journal of Dairy & Veterinary sciences

The environment of the Bahía Blanca estuary is considered a hot spot in terms of pollution. Bioindicators should have the ability to react relatively fast to certain pollutants and environmental disturbances. Therefore, an exploratory study was carried out determining and quantifying the concentrations of cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb) and zinc (Zn) in the muscle and liver of Mustelus schmitti narrownose smooth-hound and were compared with the values obtained from their respective helminth assemblies. In most of the fishes analyzed, the concentration of heavy metals was higher in the infra communities of cestodes compared to the host. Our results position the cestodes as efficient sentinel species of pollution by bioaccumulating higher concentrations of heavy metals than the host tissues, thus behaving in excellent early warnings of environmental pollution, more real than quantifications in sediments, in water and fish

Keywords: Heavy metals; Bioaccumulation; Sentinels parasites

Introduction

The estuary of Bahía Blanca (39° 03′44 ″ S 62° 04′00 ″ W) is an adequate environment to develop pollution studies, considering that it is an area that includes urban centers, several industrial parks and deep-water ports. All the effluents are discharged with different degrees of pretreatment, so they generate different impacts on the ecosystem. In environmental monitoring to detect heavy metals, organisms are often used as bioindicators, which have the ability to react relatively fast to certain toxic products and environmental disturbances. Some of these organisms, such as parasites, may be highly sensitive to brief exposures, poorly detected in water, sediment or fish [1-5]. Our previous studies in the estuary have focused on evaluating the parasitism of fish in the time scale to be able to compare and analyze them as effect indicators altering some parasites population parameter such as prevalence and abundance or causing symptoms in their hosts in response to environmental disturbances [6-10]. The narrownose smooth-hound Mustelus schmitti Springer, 1939 is a resident fish of the estuary of Bahía Blanca and third in importance as a fishing resource. Based on the fact that some parasites, such as cestodes, have the ability to absorb and accumulate more chemicals than their host tissues [11].

The objective of the present study was to analyze whether a higher concentration of metals in the parasites respect to their host was applicable in the cestodes- narrownose smooth-hound model and to evaluate if these helminths possess ecotoxicological value and could be used in the study area as early bioindicators of anthropic impact. Therefore, an exploratory study was carried out in order to determine and quantify the cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb) and zinc (Zn) concentrations in the muscle and liver of the narrownose smooth-hound and compared with the values obtained from their respective cestode assemblages (Dollfusiella sp., Orygmatobothrium schmittii, Calliobothrium australis and Symcallio sp.) The samples were analyzed by Inductive Coupling Plasma Atomic Emission Spectrometer (ICP-AES, LANAQUI-CERZOS-CONICET-UNS). The values were compared with the limit values allowed by the European Union for fish meat.

Results and Discussion

In most of the fishes analyzed, the concentration of heavy metals was higher in the infra communities of cestodes compared to each host. The parasites concentrated 270 times more Cadmium than the fish muscle. For this metal, the standards established in the liver and those of the parasites were exceeding the limits established by the European Union for muscle or liver. Chromium was bioconcentrated in the cestodes two times more than the muscle and six times more than the liver. Copper was accumulated with values 65 times more than muscle and up to four times more than in the liver. Lead had values 48 times more in helminths than both muscle and liver of fishes. For this metal in most dosages, the concentration measured in parasites exceeded the limit value established by the European Union. Zinc bioaccumulated in parasites seven times more than muscle and four times more than liver. Only in one case the Zinc concentration was three times higher in the liver than in parasites.

The environment of the Bahía Blanca estuary is considered a hot spot in terms of pollution and is included among the most eutrophic coastal ecosystems known [12]. Also, since many years it have been reported high concentrations of heavy metals and pesticides in water and sediments [13,14]. The combined effect of these pollutants, plus the sewage discharge, the industrial effluents of petrochemical origins, and the overheated water from a thermoelectric power station (620MW) all of them represent a growing threat to the environment. These increase in anthropogenic activity around estuaries, coupled with the persistence of heavy metals, their high toxicity, strong tendency to bioaccumulate, and non-degradability [15], usually affect water. As negative effects it could change the trofic web in the aquatic fauna, eliminate the spawning and larval recruitment sites and a potential decrease in diversity, affecting all the ecosystem [16,17]. That is why the need to choose of efficient bioindicators in the evaluation of the quality of the environment [18].

Conclusion

The estuary of Bahía Blanca is one of the most important in Argentina, having the main deep-water port system in the country. Although fish species can be used as efficient and useful bioindicators, our results position cestodes parasites as sentinel species of contamination for the fact that reported higher concentrations of heavy metals than their hosts. This would avoid possible underestimations in pollution levels by being quantified only in sediments, in water and in fish. 

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Pulmonary Hydatid Cysts Causing Massive Hemoptysis and Hydatoptysis Treated Surgically: A Case Report and Review of Literature-Juniper Publishers

International Journal of Pulmonary & Respiratory Sciences

A 52 years old male nonsmoker, presented with a history of shortness of breath, cough, hemoptysis and upper right upper quadrant abdominal pain for the last one month. No history of loss of weight or appetite. Patient was diagnosed six months ago as hydatid disease of liver and lung and he was commenced on oral scolicidial agent Abendazole by referring hospital. Computed tomographic scan of thorax and abdomen Demonstrated large bilateral pulmonary and hepatic cysts with typical radiological findings of hydatid cyst. Pulmonary cysts were excised by staged procedure and later on hepatic cyst were drained by interventional radiologist.

Keywords: Echinococcus granuloses liver; Lung; Hemoptysis; Hypertonic saline; Surgery

Background

Parasitic infestation by Echinococcus Granuloses was described by Rudolphy in 1808 as a hydatid cyst also this is known as echinococcosis and hydtasois [1]. There are four other species of Echinococcus which can affect human but among all Echinococcus Granulosus is the most common cause of zoonotic parasitic infection (Table 1). Epidemiologically, this disease prevails almost all over the world but there are endemic areas as Australia, New Zealand, South and Central America, Middle East, Sub Saharan Africa, Russia, China, Turkey [2,3].

Case

A 52 years old male smoker presented to our tertiary care hospital with history of cough and hemoptysis. He was diagnosed six months ago in the referral hospital as case of hydatid disease of lung and was commenced on Albendazole 800 mg daily. He had scattered hemoptysis for 3-4 times in a month but prior to admission he had massive hemoptysis. On clinical examination he had dyspnea with the respiratory rate of 24/min, no cyanosis or jaundice, breath sounds were decreased over right hemi thorax. Laboratory tests revealed Hemoglobin (8gm/dl), liver enzymes Alanine Transaminase (ALT) 28U/lit Aspartate Transaminase (AST) 16U/lit, Alkaline phosphatase 300 U/lit. Chest radiograph and CT scan of thorax and upper abdomen showed large bilateral pulmonary and hepatic cysts. Immunoglobulin G for echinococcus was positive. As the patient had massive hemoptysis and was symptomatic urgent surgery was planned. Surgical approach was right thoracotomy and we found a very large cyst densely attached and compressing the lung parenchyma. A purse string suture was placed and in the center of cyst and surrounding lung tissues were covered with gauze soaked in hypertonic 14% saline. A 14 F catheter was inserted in the cyst and tightly snugged to avoid any spillage then 200 ml of 14% hypertonic saline was installed in the cyst cavity and left for five minutes after that fluid was sucked, catheter was removed and purse suture snugged. With meticulous dissection cyst was removed in total sparing the lung parenchymal tissues (Figures 1 & 2). The left side cysts were removed as staged procedure after four weeks in similar fashion. Histopathology report showed dead daughter cysts and no live parasite (Figure 3).

Discussion

Echinococcosis also known as hydatisosis is the most common parasitic disease caused by larval cestodes. This parasite harbors in two hosts: primary and intermediate to complete its life cycle. Livestock animals like sheep, goat, horse, pigs, camels as well as humans are intermediate host while the Carnivorous animals like wolves, dogs, are definite host (Figure 4A-4F). Humans are infected accidently by ingesting the vegetables, fruits and water contaminated by soil with dog stool containing eggs. Echinococcus eggs can survive for a year outside in the atmosphere and are the main source of contamination [4-6]. On ingestion eggs hatch to oncosphere larva in the small intestine thereafter, enter in to the blood stream and is carried to the liver by portal circulation or can by pass to pulmonary system and then develop to metacestodes. These fluid filled cysts are composed of hundreds of protoscolices, which are the source leading to formation of daughter cysts or mature worm (2-3 mm long). This life cycle is usually completed in 2-7 weeks. Human to human spread is not possible as carnivores’ (definite host) is required to complete the life cycle. Hydatid cysts are tri layers, (pericyst, laminated and germinal) filled with nutritious fluid that promote larval growth [7,8] (Figure 5).

The best diagnostic modality is imaging chest x-ray and CT scan of thorax and abdomen [9,10]. Serological methods, Weinberg complement fixation and Casoni tests were used in the past and their yield is very poor. ELISA and IHA are most widely used serological tests [11]. Patients with pulmonary hydatid disease are initially asymptomatic. Most of the symptoms are either due to mass effect of cyst on neighboring structures or rupture of cyst leading to anaphylactic reaction, hemoptysis, hydatoptysis, infection, pneumothorax and empyema (Table 2). Clinically patient can present with chest pain, fever, tachycardia, hemoptysis, and hydatoptysis. Pulmonary hydatid cysts increase in size faster than in other parts of body probably due to elastic nature of lungs and negative intrathoracic pressure [12-14].

Treatment is chemotherapy and surgery. The medical treatment is very beneficial in patients with multisystem (disseminated) disease and can be given as neodjuvant therapy or adjuvant therapy in patients at risk of spillage during the surgery. The commonly used chemotherapeutic drugs are Mebendazole, Albendazole, Benz imidazole and Praziquantal. Albendazole is preferred because of its better bioavailability and the dose is 15mg/kg body weight per day in two daily doses approximately 800mg daily dose for minimum 3-6 months. It is not recommended in pregnancy particularly in first trimester due to its teratogenicity [15-18]. Horton et al. [19] treated 500 patients of Echinococcosis giving those 800 mg of Albendazole daily two and half cycles with 14 days interval. After evaluation of 250 patients only 47 required surgery and resected specimen demonstrated that only five patients have viable parasite. Recurrence rate in patients who received preoperative Albendazole as compared to those who did not receive Albendazole was 18.75& 4.16 % respectively. In medical literature Little et al. [20] reported recurrence rate of 22%, Mottaghlan and sadi observed 11.3%. Medical treatment alone is not enough to eradicate the disease once there are cystic lesions in lung and liver [19,20].

Radical Surgical resection of host tissues and entire cyst is mandatory if patient is symptomatic or any signs of infection or invasion in to surrounding structures. Postoperative chemotherapy for 1-2 years is recommended. Surgical approach is designed according to the location of the cysts. Most common surgical approaches are thoracotomy, median sternotomy and video assisted thoracoscopic resection. The basic surgical principal is that spillage of cyst contents should be avoided and adjacent tissues should be packed with gauze soaked in hypertonic saline solution (15%,20%) to avoid contamination. In case of cyst rupture during surgery or if cyst is infected, after the removal of germinal layer its recommended to wash the cyst cavity with hypertonic saline solution [21]. Dekak et al. [22] reported 202 patients who underwent surgery out of 422 cases. Enucleation, capitonage, segmentectomy, and lobectomy were the procedures performed [22]. Biswas & Burhan et al. [23] reported 26 and 24 cases respectively which were treated surgically. Ashok et al. [24] reported 33 cases out of 72 who required surgery [24].

Liver is the most common site of hydatid cyst formation, right lobe and left lobes are affected 60-75% and 20% respectively. Hepatic cysts generally remain asymptomatic for long time. The most common complication is intrabillary rupture 3-17% or rarely can rupture in to pericardial, pleural or peritoneal cavity, and neighboring organs in 20-50% of cases. Sometimes cysts develop secondary bacterial or fungal infection. The best diagnostic modality is Ultrasound and Spiral CT scan. ERCP can be helpful for diagnosis of biliary rupture or communication. Liver hydatid cyst rupture can be categorized as contained, communicating or direct. Reported incidence of communicating rupture in to biliary system is 44-64% [25]. The patient with intrabillary rupture present with right upper quadrant pain, obstructive jaundice, fever, cholangitis and sepsis (Table 2). There are multiple treatment modalities for the management of liver hydatid disease and surgery is reserved for complicated cysts. Rupture of the cyst into adjacent organs, complicated with biliary fistula, compression of adjacent vital structures and cysts with infection or hemorrhage require surgical intervention. Surgery is also recommended for cysts with many daughter vesicles that are not amenable to percutaneous treatment (WHO stage CE2 and CE3b) [26] (Table 3). If the cyst diameter is more than 10cm and or percutaneous facilities are not available then surgery is advisable. The other management options recommended by WHO are drug therapy (Albendazole) for stage CE1 and CE3 a provided the cysts are less than 3cm. If because of any reason medical management is not feasible then percutaneous treatment with puncture, aspiration, injection and respiration (PAIR) is the alternative. Combination treatment with PAIR and Albendazole is recommended for stage CE1 and CE3 that are more than 5cm in size [27-29].

Conclusion

In conclusion we treated a patient who had simultaneous hepatic and bilateral pulmonary hydatid cysts who presented with hemoptysis and right abdominal upper quadrant pain. The pulmonary hydatid cysts were resected as staged procedure and liver cysts were treated with PAIR and Albendazole therapy for six months. Our improvised technique of installation of hypertonic saline in the cyst prior to surgical dissection is effective and safe.

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Smart Stiction-Juniper Publishers

Soft robotics could loosely be described as the engineering science of expanded dexterity through controllable flexibility. The exploitation of controllable compliance through the judicious choice of soft flexible members, as opposed to a finite number of rigid kinematic joints, can result in greater dexterity without compromising simplicity. One example is the replacement of segmented mechanical legs with simple compliant material eruciform prolegs. To achieve this in robotics, without introducing additional mechanical joints, mobile surfaces with switchable coefficients of friction is essential. This paper explains how, using silicone based smart materials, the rapid alternation between kinematic and static friction (stiction) may be achieved.

Introduction

Stiction (sometimes referred to as “stick-slip”) is a word construction derived from friction and sticking. It represents the forces due to static friction which manifests itself as a cohesion force threshold which must be overcome to enable relative motion between otherwise stationary objects in contact [1]. Following the onset of movement, resistance to motion is provided exclusively by dynamic friction. In engineering, stiction is usually considered to be a problem [2]. However, in certain applications, stiction can be deliberately implemented to advantage, as in the case of soft robotics where movement is to be achieved through controlled sliding rather than rolling or crawling. It is not easy to make a clear distinction between adhesion and stiction. Both are related to inter molecular viscoelastic effects and both are influenced by surface roughness. Adhesion (for example magneto adhesion and electro adhesion) concerns the application of an astrictive force which causes prehension whereas magnetostriction and electrostriction pertain to the control of friction though a magnetic or electric field respectively. Should it not be possible to sustain post contact retention on lifting, then adhesion properties are absent. If, despite this, the frictional coefficient is still controllable by means of some external influence, then stiction is present.

The coefficient of static friction μ_s denotes the necessary frictional force between two surfaces in order to maintain two objects relative to one another immobile (1).

Once the applied force exceeds the maximum static frictional force, sliding will commence. The force due to kinetic friction (2) always opposes the direction of motion.

In expressions (1) and (2) F_N is the force (usually due to gravity, F_N = m g) normal to the direction of motion. The surfaces of highly polished planar objects tend to adhere to one another. This is often attributed to electrostatic and/or Van der Waals forces. After overcoming the initial stiction forces, relative motion occurs, and the resistance shifts into the kinetic region as shown in Figure 1. Should the movement direction be reversed, then a degree of positional hysteresis will be experienced.

F_N is a function of gravity and is otherwise only dependent on mass. However, F_N can be increased by the application of additional force, though this is not necessarily advantageous. What is important is the relationship between μ_s and μ_k.

In order to increase the relationship in (3), the mechanical characteristics of one or both contacting material surfaces must be precisely controlled. There are several relevant factors here.

Frictional Materials

Before introducing magnetically and electrically controllable friction, it pays to consider the frictional coefficients of general engineering materials. These are commonly available in almost any mechanical engineering textbook [3]. The values given are usually in the range 0 (no friction) to 1 (the frictional force is equal to the normal force F_N). Mechanically compliant materials such as rubbers demonstrate some of the highest frictional coefficients. In fact, silicone rubbers can have frictional coefficients higher than unity [4]. What is required is a manner in which these coefficients can be changed by means of an external influence.

Compliance is the inverse of stiffness or the potential of a surface to elastically change its geometry to comply with a different topology. The ability to switch between two elastic states is the basis of shape memory materials [5]. In the form of a practical device, what is ideally needed is a switchable mating surface, where in one condition the surface has a high elastic modulus and a very low coefficient of friction (similar to PTFE) and in the other condition is extremely compliant and has a high coefficient of friction (like silicone rubber).

In a real situation, the entire area of a compliant surface is not in contact with the rigid surface over which it is intended to propagate. The compliant mating surface contains a plurality of approximately semi-spherical asperities, which may or may not be in contact with the lower surface. This is illustrated in Figure 2.

Given a nominal height between the two surfaces h, as shown in Figure 2a, then only a small amount of asperity will be in direct contact with the lower planar surface. Figure 2b illustrates the situation for a single asperity with curvature radius R and displacement z from the reference plane. At the point of contact, the elastic asperity will deform with displacement [6].

Given a random variation in height h following a Gaussian probability distribution, then the probability of contact is given by (4).

The number of contacts n can be calculated from the amount of asperity N (5).

Using Hertz’s theory, the area of contact between the surface and an asperity with displacement z is given by expression [7] (6).

The area of contact (7) and the contact force {8} may be calculated accordingly [6].

For the two mating surface materials, E₁ and E₂ are the elastic moduli and υ1 and υ2 are the respective Poisson’s ratios.

For an individual contact point, dividing (8) by (7), gives the load pressure (9).

The greater the effective elastic modulus, the higher the applicable load pressure. A lower elastic modulus results in a correspondingly lower pressure for a given deformationcompliance. When one of the surfaces is highly compliant, then R also becomes a function of the applied pressure. Transposing for R in (9) and differentiating gives (10).

The function (10) tends to zero for large values of pressure, thus making the effect stable. Furthermore, for a given surface, either E₁ or E₂ will be rigid and constant. The other must be controllable in accordance with (3). Halling [3] also provides a similar analysis for conical asperities but for the sake of brevity this will not be included here [3].

Clearly, given a composite material with two sets of controllable asperity, one with a high and one with a low frictional coefficient, the criteria for a practical device laid out earlier, is achievable. This principle already exists in nature. Eruciform lifeforms employ soft prolegs with hard crochets on the ends to allow switching between sliding and prehension. Although the Gecko foot enjoys the ultimate natural prehension, it does not adhere to a PTFE surface [8]. Furthermore, the objective in soft robotics is to achieve this without resorting to direct methods of mechanical alternation.

Magnetostiction

Here it must be distinguished between magneto adhesion and magnetostiction. The former concerns simple magnetic force produced when a magnetic field is applied against a ferrous surface, while the latter relates to frictional changes in the properties of a material caused by the application of a magnetic field. When the coefficient of static friction s is a function of an applied magnetic field, then the resulting force which resists motion is the product of this frictional coefficient m and the normal force FN. Magnetostiction should also not be confused with magnetostriction. The latter refers to a second order magneto-mechanical effect which leads to reversible dimensional changes in specific magnetic materials [9].

Magnetoactive polymers (MAP) are polymer matrices containing a dispersion of ferromagnetic or paramagnetic particles distributed within a soft, nonmagnetic elastomer [10]. Their mechanical [11] and electrical [12,13] properties have been extensively investigated. Under the influence of a magnetic field gradient, both magnetodeformation and an increase in elastic modulus results [14]. Magnetodeformation can result in extremely large displacements as is exploited in the design of origami actuators [15].

If E₁ or E₂ in (9) is a function of the applied magnetic field, as in the case of a magnetoactive polymer [14], then the criterion outlined in (3) can be, at least partly, fulfilled. However, the bulk permeability of a magnetoactive polymer changes with mechanical deformation. This, in turn, changes the magnetic flux density in the medium and with it the degree of deformation [16]. Magnetizations measurements carried out on 1mm³ samples from the same production batch using a SQUID magnetometer (Quantum Design MPMS XL) including MPMS RSO Controller and digital R/G Bridge can be seen from Figure 3. Although the maximum carbonyl iron powder (CIP) concentration does not coincide with the maximum magnetization (which occurs between 12%wt and 27%wt CIP), a compression of the MAP will inevitably result in a localized increase in CIP.

The inclusion of hard magnetic particles allows the MAP to be permanently magnetized [17]. For example, Nd-Pr-Fe-Co-Zr- Ti-Bm, SmCo or NdFeB are materials commonly used in modern permanent magnets. Because of potential oxidation, the finished magnets are usually chrome or epoxy resin coated. As well as providing the elastic matrix, the polymer in a MAP serves the same purpose. Unfortunately, this introduces a slight problem. Profiling of a MAP surface alone rarely suffices as it is impossible to use the magnetic particles themselves as asperities [18]. Inevitably in a MAP each magnetic particle is surrounded by a thin layer of polymer, so the friction coefficient remains the same with or without magnetic field. Magnetic particles not completely surrounded by the polymer matrix rapidly detach themselves from the matrix thus playing no further role in the dynamics of the system. In addition, although immediately after magnetization, each of the particles are magnetized in the same direction, this situation rapidity changes as the magnetized particles attempt to rotate within the polymer matrix in order to reach the level of lowest energy [19]. This effect can be clearly seen using a magnetic field camera (Matesy MagView) in Figure 4a which shows the magnetic flux density over the surface of a hard-magnetic MAP immediately following magnetisation and Figure 4b the same MAP several hours later where a substantial portion of the particle orientation has changed.

In order to create an effective switchable stiction device with two frictional states, two discrete areas of the same substrate having different frictional coefficients must be presented. A number of potential physical implementations exist, the most effective of which are bistable devices [15] similar to the previously mentioned insect prolegs. One portion of the device comprises highly compliant addition-curing RTV-2 silicone (Wacker Silicones) polydimethylsiloxane (PDMS) elastomer sans magnetic content which exhibits a high coefficient of friction between its under-surface and any surface in which it comes into contact with. The other portion is magnetically active and hardens under the influence of the magnetic field thus providing a lower frictional coefficient and less compliance. This may also be augmented by a metallic or PTFE layer to further reduce friction.

The traditional means of ascertaining frictional coefficients is the tilted plane method, where an object is placed on a sloped surface and the slope angle gradually increased. Static friction resistance develops when the object starts sliding [20]. At a given time, the ratio between the load applied at right angles to the sloped surface (W) and the friction resistance (F) is called the coefficient of friction (f) and is determined according to the following equation:

Much more accurate results may be obtained by measuring the force threshold at the point of movement. Replacing FN with mg in (1) and transposing for s gives (12).

Preliminary experiments were conducted on such a device comprising two interspaced surface structures effectively selectable by means of a magnetic field.

Experimental

A 6 axis Staübli RX60 [21] industrial robot equipped with an ATI FTN-Mini40 6 d.o.f. force/torque sensor provided linear movement of such a bistable element in both active and inactive states [22]. Figure 5 shows the measured force in the Y-axis (direction of movement) and the Z-axis (normal to the direction of movement) for a linear displacement in the Y-direction. Because compressive stress is by convention negative, the values of force in the Z-axis of Figure 5 have been inverted.

The slight decrease in Fy at the beginning of movement is the initial changeover from static to dynamic friction of the sliding condition. The deliberate change from low to high friction occurred at increment 4 along the abscissa of Figure 5.

Conclusion

Controllable stiction, as described in this paper, can be implemented in many possible ways. The manipulation of mechanical asperities on a surface can give rise to controllable friction. However, this can only be achieved by the application of some external influence such as an applied magnetic or electric field. This paper has demonstrated how magnetostiction can be implemented through magnetic field induced changes in the mechanical properties, and in particular the elastic modulus, of magnetoactive polymer substrates in order to control the friction between two surfaces. The application of magnetostiction and its relevance to controllable motion in soft robotics has been demonstrated as a practical example.

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Airway Microbiota and Allergic Diseases: Clinical Implications-Juniper Publishers

INTERNATIONAL JOURNAL OF PULMONARY & RESPIRATORY SCIENCES

Introduction

Bronchial Asthma is an airway disease with variable degrees of bronchial mucosal inflammation and intermittent episodes of airway obstruction and bronchial hyperesponsivness. That asthma is a syndrome consisting of different phenotypes has been recognized for a long time by clinicians [1]. New evidence indicates that the composition of airway microbiota differs in states of health and disease. Different chronic airway diseases had been related to changes in microbiota due to various factors which could affect severity of symptoms and even response to treatment [2]. Micro biome may be one of the protective factors against asthma in early life [3].

What is Airway Microbiota

It a complex variety of microbes present intrachea and different generations of the bronchi either on the mucus layer or the epithelial surfaces or even both. These microbes include bacteria, yeasts, viruses and bacteriophages. The bacterial part of microbiomeis the most prevalent component with various genera: Prevotella, Sphingomonas, Pseudomonas, Acinetobacter, Fusobacterium, Megasphaera, Veillonella, Staphylococcus, and Streptococcus. The bronchial tree for instance contains a mean of 2000 bacterial genomes per cm2 surface [4]. The mucosal surfaces in the human body are the home of 10-100 trillion microbes with a diversity of greater than 1,000 species [5]. The highest concentration of microbes is found in the GI tract, compared to those found in the lower airways. Healthy human lungs are not sterile, as previously believed, but it is unknown whether the microbes in the lungs form a stable community or are a series of transient colonizers [6].

However, various theories about the origin of lower airway microbiota in healthy individuals had been suggested. As it may represent true colonization of the lower generations of bronchi, or it is the result of turnover of the microbial community or it is just contamination of oropharynx during lower airway sampling or even linked potentially to those who are incorrectly categorized as truly healthy [7].

Importance of microbiota

The commensal bacteria are nonpathogenic and defend our airways against the pathogens. There are several possible mechanisms:

1. Commensals are the native competitors of pathogenic bacteria, because they occupy the same niche inside the human airways.
2. They are able to produce antibacterial substances called bacteriocins which inhibit the growth of pathogens. Genera Bacillus, Lactobacillus, Lactococcus, Staphylococcus, Streptococcus, and Streptomyces are the main producers of bacteriocins in respiratory tract.
3. Commensals are good inducers of anti allergic Th1 cascade with anti-inflammatory interleukin (IL)-10, FOXP3, and secretory immunoglobulin A (sIgA) production [7].

Airway epithelial cell and microbiota interaction

The airway epithelium together with alveolar macrophages and dendritic cells collectively can recognize of bacterial products trapped into the lower airways with the inhaled air. Some of these products are can potentiate pro inflammatory stimuli. So it is a challenging issue to distinguish between pathogens and commensals to avoid development of constant or persistent inflammation and help to develop tolerance against harmless microbiota [8].

Once pathogenic bacterium (e.g., S. pneumoniae, P.aeruginosa) has been attached to activated pattern recognition receptors located on/in bronchial epithelial cells, the proinflammatory cytokines pathways are predominant via release of IL-1, IL-6 and IL-8 which induce neutrophils, dendritic cells and macrophages chemotaxis to target cells (e.g., neutrophils, dendritic cells and macrophages. Standard microbiota fail to induce strong signaling, thus aborting inflammation. (Figure 1) [9].

This process becomes much more intriguing when taking into account that commensals often share their surface molecules with pathogens. Epithelial cells are equipped with very sensitive recognition tools - toll like receptors (TLRs), NOD like receptors (NLRs) and retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) which determine presence of non commensal bacteria which activate cellular components of the adaptive and innate immunity and recruit them to the infection site [7].

NF-κB is the principal regulators of different response to harmful microbiota as it is become activated by a number of stimuli as bacterial cell walls or inflammatory cytokines. This results in its translocation from the cytoplasm into the nucleus to activate epithelial cells pro-inflammatory genes. These specific genes can recognize a particular nucleotide sequence (5’-GGG ACT TTC T-3’) in upstream region of response genes. [10]. Inspite of expressing express the same microbe-associated molecular patterns (MAMPs), harmless bacteria fails to translocate NF-κB into the nucleus thus preventing the inflammation. The balance between pathogens and commensals is extremely important in the maintenance of homeostasis in the respiratory tract [9].

Pediatric acterial airway microbiota in early life

A neonatal mouse exposed to a broad-spectrum antibiotic has been shown to increase allergen-induced airway inflammation susceptibility [4]. Germ-free mice also exhibit enhanced airway inflammation upon allergen exposure [3], while colonizing OF germ free mice with microbiota from conventional mice decreased accumulation of natural killer T (NKT) cells in their airways .This was only observed in neonates not in adult mice. This highlights the importance of early life as a critical period for intervention [11].

Absence of airway colonization during this critical neonatal window resulted in sustained susceptibility to allergicinflammation through adulthood. This ensure long-term control of allergic airway inflammation via controlling commensal bacteria communities early in early life [12].

Microbiota and climax community

Climax community is defined as a microbial community that has reached a final or “climax” steady state best adapted for growth at that specific niche along the mucosa. However, this climax community is dynamic and still exhibits both resistance and resilience [13]. Evidence is now accumulating that longterm dietary pressures , repeated antibiotic use, GI illnesses or medications such as antacids, proton pump inhibitors, and nonsteroidal anti-inflammatory drugs can break both the resistance and resilience of a community and result in it re-assembling into another climax community, although this may be accompanied by detrimental changes in host mucosal immuno biology and physiology. One mechanism underlying the activity of probiotic microbes and prebiotic nutrients may be the ability to restructure a climax community to improve host mucosal immuno biology and physiology [14].

Microbiota (microflora) hypothesis

Several theories had been suggested to explain the increase in the incidence of asthma and other allergic diseases over the past 30 years and the discrepancy between the higher rates of allergic disease among industrialized relative to developing countries. One rising assumption is a lack of early microbial stimulation which results in aberrant immune responses to innocuous antigens later in life “hygiene hypothesis” [15]. Life style modifications and over use of broad spectrum antibiotics raise the concept of disturbance of mechanisms of mucosal immunologic tolerance due to changing diversity of gastrointestinal (GI) microbiota composition in westernized areas [16].

Epidemiologic and clinical data supporting this interpretation include

1. a positive correlation between increasing risk for asthma/allergies and increasing use antibiotics in industrialized countries,
2. Altered fecal microbiota composition had been correlated to different atopic diseases
3. Oral probiotics orsignificant dietary changes lead to some successful prevention/reduction of severity of allergic diseases.

Experimental data in mice compared that immune response generation and normal ones which showed numerous defects in immune response [17]. Altogether, these experimental, epidemiologic, and clinical observations support the hypothesis that even minor changes in the quality or quantity of airway microbiota can be one of the predisposing factors for allergic disease [10].

Cross-talk between the gut and the lung

The existence of the gut–lung axis and its implications for airway disease provide a portal for potential therapeutic intervention in prevention or management of asthma [18]. Oral supplementation with probiotic strain of Bifidobacterium and prebiotic non-digestible oligosaccharides reduced airway IL6 and IL4 levels and protected against HDM-induced airway inflammation. This suggest that some intestinal bacteria have the capacity to suppress inflammation at a distal mucosal site [19].

Oral tolerance and airway tolerance

Oral tolerance is defined as the propensity of ingested antigens to abort subsequent systemic immune responses. Gastrointestinal tract may be also involved in tolerance to inhaled and ingested antigensvia CD4+ regulatory T cells (Tregs) that produce immunosuppressive cytokines, IL-10 and TGFβ, in what is termed “bystander suppression.” [19,20]. Mucosal signals, such as those from the microbiota, keep resident dendritic cells in an immature or non-inflammatory state [15].

Airway microbiata diversity in asthma

In asthmatic patients, certain airway microbial composition was associated with airway eosinophilia and AHR to mannitol but not airway neutrophilia. Comparing eosinophilic and noneosinophilic asthmaas regards airway microbiome revealed that Asthmatic patients with the lowest levels of eosinophils had an altered bacterial microbial profile, with more Neisseria, Bacteroides, and Rothia species and less Sphingomonas, Halomonas, and Aeribacillus species compared with asthmatic patients with high eosinophilia. This may invite furtherresearch on effect of modulating diversity of microbiota to modulate various asthma phenotypes [21].

Airway microbiota dysbiosis in asthma

Airway dysbiosis in patients with severe asthma appears to differ from that observed in those with milder asthma. Specific Bacterial communities as Proteobacteria were associated with worsening ACQ scores and sputum total leukocyte values in severe and poorly controlled asthma. Actinobacteria had been associated with stable or even improving ACQ scores and can predict steroid responsiveness [22].

Airway microbiota and asthma heterogeneity

Dissecting the role of the microbiome in asthma is challenged by the heterogeneity of the disease at multiple levels (Figure 2). These levels include asthma’s clinical and inflammatory heterogeneity, genetic factors that contribute to asthma risk, and the multiplicity of immune pathways involved in asthma. The potential effects of environmental exposures on gene function, immune responses, as well as microbiota composition add further complexity. As with genetics, mechanistic consequences of the altered microbiome may explain certain aspects or phenotypes of asthma as the development of allergic or non-allergic asthma,and treatment-resistant asthma) [22] Components of the depicted system-host genetics and immunology, microbiota, environmental exposures, and the disease of asthma- are themselves heterogeneous entities, presenting challenges to more precisely dissect the role(s) of the microbiome in asthma.

Upper airway microbiota and asthma

Bisgaard et al. [23] demonstrated that the nasopharyngeal microbiome composition was influenced by the early life exposures, including attending day care, having siblings, and taking antibiotics. Haemophilus, Streptococcus, Moraxella had been previously associated with airway disease and increased risk for asthma exacerbations. Early colonization with either Moraxella, or Streptococcus was strongly associated with acute lower respiratory viral infections. This colonization can be predictor for asthma development later in life.

Thus, probiotic intervention studies of animals provide encouraging evidence for intentional manipulation of the intestinal microbiota as a strategy for asthma prevention and management. A meta-analysis of a large number of randomized trials of probiotic supplementation, on atopic sensitization and asthma in children, however, shows that the success of these interventions in mice does not translate easily to disease prevention in humans. At a minimum, this highlights that different probiotics may have distinct interactions with the host microbiome and that some strains might be more specific for modulating atopic inflammation but many other considerations, such as diet, age of intervention, coincident environmental exposures, length of supplementation period, and other as yet unknown factors, are likely important [24].

Airway microbiota and severity of asthma

Relationships between the airway microbiome and disease features have also been examined in patients with in severe asthma. Different clinical phenotypes of severe asthma have been described, suggesting the possible involvement of alternate mechanistic pathways, as has been surmised for asthma in general. A preliminary analysis of the bronchial microbiome in these subjects, poorly controlled despite high-dose ICS therapy, noted significant relationships between different bacterial community profiles and features such as body-mass index and measures of asthma control [25]. A similar study of sputum bacterial composition in 28 treatment-resistant asthmatics found that the relative abundance of M. catarrhalis, Haemophilus, or Streptococcus spp. correlated with worse lung function and higher sputum neutrophil counts and IL-8 concentrations [19].

Microbiota and therapy of allergic disease

The composition of the microbiota can be manipulated by combinations of antibiotics, probiotics, and dietary components which may have direct growth promoting or inhibiting activity for specific microbes. [26]. Certain types of fatty acids, phenolic compounds, and carbohydrates may modulate these microbiota. However, a single type of probiotic or dietary component will not be efficacious in all individuals. This likely due to differences in the types of microbial communities in different individuals. The objective of the international Human Microbiome Project is to characterize and define the human microbiome in states of health and disease [10]. The challenge for future research is to use this information to optimize probiotic/dietary therapy to improve human health and prevent microbiota-associated diseases, such as allergies .They are likely to include short chain fatty acids and ionic polysaccharides [27] .

Microbiota and prevention of allergic disease

Probiotic intervention studies of animals provide encouraging evidence for intentional manipulation of the intestinal microbiota as a strategy for asthma prevention and management. However, A large number of randomized trials on the value of probiotic supplementation, on asthma incidence and severity in children, could not show the same success of these interventions as in mice [28-30]. This may be due to many other considerations, such as diet, age of intervention, coincident environmental exposures, length of supplementation period, and other as yet unknown factors, are likely important [24,31- 34].

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