Ultrasonic guided wave (UGW) is one of the most commonly used technologies for non-destructive evaluation (NDE) and structural health monitoring (SHM) of structural components. Because of its excellent long-range diagnostic capability, this method is effective in detecting cracks, material loss, and fatigue-based defects in isotropic and anisotropic structures. The shape and orientation of structural defects are critical parameters during the investigation of crack propagation, assessment of damage severity, and prediction of remaining useful life (RUL) of structures. These parameters become even more important in cases where the crack intensity is associated with the safety of men, environment, and material, such as ship’s hull, aero-structures, rail tracks and subsea pipelines. This paper reviews the research literature on UGWs and their application in defect diagnosis and health monitoring of metallic structures. It has been observed that no significant research work has been convened to identify the shape and orientation of defects in plate-like structures. We also propose an experimental research work assisted by numerical simulations to investigate the response of UGWs upon interaction with cracks in different shapes and orientations. A framework for an empirical model may be considered to determine these structural flaws. Mechanical structures and their reliability deteriorate continuously under the influence of operating and ambient environmental conditions. The structural degradation processes may occur in different forms of corrosion, fatigue cracks, erosion, and strength reduction . The extThe SHM approach involves collecting data from various sensors installed on structures and interpreting the findings to make reliable and cost-effective and CBM decisions for structures under different operating conditions. Numerous health assessment techniques such as bulk wave ultrasonic, X-rays, infrared thermography, and eddy current are used effectively for non-destructive evaluation (NDE) of structures. However, majority of these techniques tend to be slow and cumbersome, especially in case of large structures . The guided waves ultrasonic testing (GWUT) potentially offers a smart alternative solution to conventional approach towards NDE and SHM . The guiThe SHM is generally performed by two methods: the passive and active SHM . The pasThe UGW is a rapidly emerging SHM technique for inspection of metallic/composite structures ,12. The This paper aims to review research publications on structural defect detection with GW technique, particularly in metallic structures. It signifies the achievements of this technique to detect various features of defects (crack/notches) on metal surfaces. We identify certain research gaps with respect to the classification of some crucial facets (shape and orientation of cracks/notches) of structural flaws, detectable by UGW techniques. Furthermore, to the best of our knowledge, the determination of these characteristics of defects (using UGW method) in plate type structures has not been addressed in any research study to date. To overcome the research gap, this paper also proposes an experimental research assisted by numerical simulation on thin metal plates . It aims to determine the signal variations in excited Lamb waves (short tone burst) after interacting with the surface cracks  in several shapes and orientations. Based on the theoretical knowledge and previously held research work on GWs, it seems quite possible to sense the projected changes in an output signal  upon interaction with these aspects of structural discontinuities.The rest of the paper is organized as follows: p) and group (Cg) velocity in metals. In here, the displacement of medium occurs both in the direction of wave propagation as well as in a perpendicular direction [The GWs are elastic waves transmitting in form of wave packets, and confined along the structures with distinct boundaries. The propagation of GWs depends on the material properties and slight variation may take place with variation in environmental conditions. These waves are highly dispersive, and almost infinite modes are generated by the superposition of basic longitudinal and transverse waves. The Lamb wave is one of the main types of GWs used in SHM. They are elastic perturbations normally traveling with a different phase  are a non-contact technique based on the principles of Lorentz force or magnetostriction which have been used enormously in defect diagnosis studies. The EMATs are ideal to directly excite and detected Sals only ,32,33. Tetection . These aetection ,10,35,36etection . PWAS caetection . A new terospace  E is Young’s modulus, T and CL represent transverse and longitudinal wave velocities, respectively. Readers can also refer to [The propagation of GWs can be divided into two broad categories: longitudinal and transverse waves. When these waves propagate in a traction-free metal plate, the longitudinal wave particles tend to travel along the direction of wave propagation (in from of compressions and rarefactions) whereas the transverse wave particles oscillate perpendicular to the direction of wave propagation. The velocity of acoustic waves depends on various properties of the medium of propagation, as shown in the following equations:refer to ,39,40,41The Lamb ultrasonic guide waves, also known as plate waves, are guided between two parallel traction free surfaces, such as the lower and upper surface of a plate. The Lamb wave speed is also dependent on the frequency of propagating waves . The Lam0 and A0) exist [Similarly, for smaller f-d product, only fundamental Lamb wave modes of symmetric and antisymmetric  exist ,36,43. T0) exist ,44.Use of Lamb waves on structural defect detection was first started in early 1960. Since then, rigorous research studies have been conducted for application of these waves in health prognostics of metallic/composite structures ,46,47,48H) and the Shear vertical (SV) waves. The particle vibration of SH waves is polarized parallel to the plate surface, as illustrated in H waves exist in both symmetric and antisymmetric modes and both are dispersive in nature, with the exception of very fundamental SH0 mode [H waves has increased in recent years by the inculcation of new techniques to overcome difficulties associated with SH wave generation. Therefore, superior transducers have been introduced for using shear waves in SHM, such as magnetostrictive transducers and EMATs.The Shear waves are also called transverse waves in which most of the movement of the medium is perpendicular to the direction of wave propagation. They exist in two forms: the Shear horizontal  and normally found in solids having free surfaces. As Sed waves . The Rayed waves . Figure These waves travel over the surface of a semi-infinite solid or a very thick plate, where the wavelength is very small as compared to the thickness of the plate. These waves have also been used in NDE of the structural defects, confined to surfaces . They cap) of an acoustic wave is the rate at which the phase of the wave propagates in spatial dimensions. The individual harmonics travel with different Cp, but the superimposed packet travels with the group velocity (Cg). In dispersive elastic wave propagation, the packets of waves travel with a different velocity (Cg) than individual wave velocity Cp [The phase velocity  signal from any structural discontinuities or boundaries . Figure ® Xi series) for ease of signal processing, analysis, and interpretation.The output signal received on the sensor is the exclusive source of information about the nature of the defect. It may encompass several changes in the characteristics of the induced signal after interaction with structural discontinuities. The error signal is obtained by taking the difference between undamaged and damaged signal. The highly advanced oscilloscopes , laser vibrometry, and UGWs have been used immensely for SHM and NDE of metallic and composite structures ,65,66,67The GWs scatter at various angles after interaction with structural defects and this scattering phenomena is proportional to the severity/size of discontinuity. It is difficult to collect most of the dispersed signals by limited number of sensors. Therefore, the use of multiple detached transducers in various array configurations has become instrumental to investigate large structural areas with better accuracy . When a The GWUs have shown enormous potential in SHM of aviation based metallic and composite structures. These structures are prone to abrupt variation in ambient conditions of humidity level, ambient temperature, stress loading, and pressure that can instigate the corrosion and fatigue based structural cracks . The traThe ice deposition on structures may compromise the efficacy of NDE and remote monitoring systems by warning for a false damage or missing the detection of actual defect failure. More recently, some selective modes of guided waves have been used successfully for detection of ice on various metallic/composite structures such as wind turbine blades, aero-structures, helicopter rotors and ship’s hull. The icing phenomena is a critical issue in the lube oil/fuel circuits of aircrafts and their aerodynamic efficiency and maneuverability. Some researchers used GW modes to differentiate water and ice content as well as deicing on structures in cold climates. The guided waves produced by a piezoelectric transducer (bonded/embedded on a surface) can be used to induce shear stresses to delaminate/fracture ice deposits on various structural surfaces ,79. Disp0 mode of Lamb wave to differentiate between the water and ice layers on aluminium plate (1.6-mm thick). A circular array of 16 sensors were used with an exciting frequency of 350 KHz. The S0 mode having dominant in-plane displacement on metal surface was found to be insensitive to the water layer. However, it remained very sensitive towards the ice loading. Later, the probability-based reconstruction (PRA) algorithm was used to construct the ice images in other types of material such as titanium and carbon-fiber-reinforced titanium plates. In case, ships maneuvering in the polar region, the amalgamated ice layers and water disturbances may cause variation in the signal response of GWs and the ice detection may be misleading the damage detection process. Memmolo et al. [®) for ice detection on ship structures using both pitch-catch and pulse-echo methods. It was revealed that the back-scattering in the pulse-echo signal and forward scattering in the pitch-catch configuration are highly dependent on the length of the ice layer. This study proposed that it is quite much possible to quantify the dimensions of the ice layer.In an ice sensing experiment, Zhao and Rose  deliberao et al.  conducte0 wave mode were found to be more sensitive to the variations in the thickness of the ice loading than the temperature factor, whereas the group velocity of S0 mode was deeply influenced by the changes in both the temperature and the thickness of ice layer. Complexities in the estimation of ice formation and temperature variation were addressed effectively using a principal component analysis (PCA) based ice monitoring method.In the cold countryside, icing of wind turbine structures  is a common problem, which may hinder its operation. The ice layers on the surface of the blades can change the propagation characteristics of the acoustic waves used for condition monitoring. Wang et al.  investigDuring a research study on the use of antisymmetric  cylindrical GWs in the underwater pipelines, Na and Kundu  used a nApart from use of GWUT in structures, Tse and Rostami  demonstrTime-shift average algorithms were used by Michaels and Michaels  in diffeThe advancement of sensor equipment over the years has benefitted SHM/NDE techniques and assisted in the optimization of maintenance/RUL prediction for structures . The imp0 mode exhibited strong out of phase motion than S0 mode. It was noticed that the transmission index was found stronger than the reflection index in half-through-thickness cracks than full through-thickness cracks. This effect was attributed to the fact that the metal portion underneath the half-thickness crack allows transmission of forwarding wave. The material impedance is another factor for deciding transmission and reflection of the acoustic signal. The effect of defect orientation  on reflection and transmission coefficients of GWs has been described in [Yu et al.  used UGWribed in . It has ribed in  conducteThe GWUT has been able to diagnose cracks or CUIs in the bends/elbows of pipelines. Only a limited portion of the wrapped material is required to be removed for placement of sensors and defect diagnosis process is conducted without removal of entire material ,96,97. TIn the oil and gas industry, the underground and subsea pipelines are more prone to general corrosion, fatigue, stress cracking and pitting . Most ofIn a non-contact approach (laser-based), a pulsed laser is used to generate elastic waves and a laser Doppler vibrometer or interferometers are used for collection and detection of structural discontinuities . The FabGuided wave excitation and sensing is limited (by contact method) to some discrete points, therefore it is hard to achieve a high resolution to sense minor emerging defects;High cost for transducer, assemblies, installation, and labour cost. The vulnerability for equipment damage is high, leading to high maintenance budgets;Limitation for use of contact transducers under harsh environmental conditions and application to convoluted/high speed rotating surfaces such as high temperature/pressure, radioactive surfaces, rail wheels.In comparison with non-contact methods, some researchers believe that the contact based techniques have some limitations as follows : Guided 0 wave mode was observed, whereas the attenuation of A0 mode found to be around 57 dB/m. In a similar research, An et al. [The laser ultrasonic excitation can be performed by using pulse lasers, continuous lasers, or laser interferometry. The non-contact method has been preferably used in crack/corrosion detection, quantification, and localization of structural defects in various areas ,110,111.n et al.  used lasRao et al.  reportedThe pulse-echo technique was used by Djili et al.  for dete0) signal is greater than that of symmetric (S0) signal [0 mode of GWs  they have been used as a preventive technique for bio-fouling on ship structures [A predictive numerical based study was conducted in a steel plate using acoustic emission (AE) method. It was revealed that peak amplitudes of antisymmetric (A) signal . They alructures . Carvalhructures  used theructures . 0 wave mode than the S0 mode on the higher frequency. Fromme and Sayir [0 wave mode around rivet holes, notch cut and fatigue cracks in an aluminium plate. The piezo transducers were used for excitation of antisymmetric Lamb GWs and its scatter measure was taken with the help of laser interferometer. The field results depicted significant changes with the presence of a notch or a crack, which were much smaller than the wavelength of excited GWs.Recently, Martinez et al.  presentend Sayir  observedH and Rayleigh waves are also used extensively in structural diagnostic studies of plate type and tubular shaped metallic/composite structures [H wave is polarized parallel to a surface, so they reflect without mode conversion. Additionally, during this process the amplitude changes are insignificant, therefore, SH wave is considered to be superlative for crack detection in welds [H wave with EMAT was found effective in defect identification in a 7 mm thick carbon steel sample. It was found to be highly reliable in defect detection, axial sizing and approximation of circumferential positioning of a defect for both artificial cracks and corrosion defect, even with a thick paint coating [H waves. The defect location and quantification for corrosion loss was clearly established from high amplitude reduction and delayed arrival timings of received signal.In addition to the uses of Lamb guided waves, the review of the literature revealed that the Sructures . As duriin welds ,121,122.in welds . The SH  coating . The red coating  using SH0 and S0) were simultaneously excited using wedge transducer (at a center frequency of 450 KHz) and the reflected signal was collected with a laser interferometer. The change in the response signal due to the wave mode interference was measured and the wall thinning was monitored. A good agreement was found between experimental results and theoretical estimation. In partial non-contact method, the contact less mechanism can be used for either excitation or sensing. This experimental setup is shown in In ships and offshore marine platforms, accelerated rates of corrosion based deterioration has been reported, primarily due to the harsh seawater climatic conditions. The general corrosion and pitting occur more frequently. Apart from these, crevice, galvanic, fretting, groove, fatigue, edge and stress corrosion are also encountered in various loaded and dynamic loaded structures ,125,126.Some researchers have reported that the minor temperature change up to 40 °C) does not affect the performance of GWs therefore special compensation techniques may not be required, except in cases where the structural damage to be sensed is very small in size  °C does  revealedDuring a research study on SHM of rail tracks, Moustakidis et al.  presentePerformance of transducers is crucial in the GWUT especially under variant operating conditions. This performance is highly influenced by several external factors such as environmental dynamics, high operating temperatures in particular. As the general purpose piezoelectric sensors used in UGWS have low curie temperatures, therefore their signal reception performance deteriorates significantly in higher temperatures. The intrinsic E/M impedance, admittance signatures of PWAS degrades when exposed to higher temperatures . In thesIn some cases, non-contact GWs methods have also been preferred for SHM and NDE at elevated temperatures . SeveralThe role of health monitoring in operational availability, upkeep, and maintenance of equipment and structures has increased exponentially in recent years. Conventional maintenance procedures such as corrective and preventive are transforming rapidly towards technology-driven upkeep methods  due to an ever-increasing reliance on progressive technological tools. The role of these technologies is more vital in equipment health prognostic studies, RUL assessment and through-life engineering support (TES) of structures, especially in cases when they are operating away from base maintenance facilities, e.g., aircraft, ships, subsea pipelines, and offshore energy structures. The GWUT is a powerful and rapidly evolving technology with enormous potentials to be used as NDT and SHM of various platforms. Its long-range defect diagnostic capability with minimal preparatory costs  for inspection is the biggest competitive edge over contemporary diagnostic techniques. Contrary to the limitations of other types of defect diagnostic technologies, guided waves have a lower level of operational barriers for usage in any type of material and environmental conditions. They have been used enormously in magnetic/non-magnetic, metals/composites isotropic/anisotropic structures of various shapes. The flexibility of use with contact or non-contact methods has enhanced its viability and future scope. The latter is relatively a more sophisticated technique, which has frequently been used in recent days for investigation of defects in complex, sensitive, dynamically loaded structures, especially in high temperatures where the use of simple contact type sensors may not be appropriate for use. Nevertheless, the contact transducer technique is still the most widely used method in UGWs because of its low initial cost, the flexibility of use, and portability.In the reviewed literature, the efficacy of GWUT in different materials, operating and environmental conditions has been studied through experimental and numerical methods. This technique has been successfully used to locate and quantify the defects in various types of structures by classification of variations in numerous parameters of output signals, like frequency, amplitude, reflected/transmitted coefficient, velocity, TOF, and impedance. Certain limitations of UGWs have also been deliberated in the literature especially in bends/joints, complex geometries, submerge and thickly painted structures. These performance degradations have been attributed to the highly dispersion nature, scattering effects and attenuation of some acoustic guided waves under specific conditions. In the real world situations, the performance of UGWs can be affected by intermingling/interference of external acoustic signatures of running machinery (in surrounding) with the of GWs signals. More improvements in the use of GWs methodology is still needed to overcome the highlighted impediments, increase reliability, effectiveness, and diagnostic ability for identification of various defect features. The Lamb wave interaction with damaged surface influences their propagation characteristics significantly, tempting various distinctive scattering phenomena ; dependent on locations and severity of damaged area. The omnidirectional scattering (around the hole) of GWs has been reported in literature, when interacted with a through-thickness hole in the composite surface. The positioning of the sensor with respect to crack location becomes crucial in cases where the cracks are oriented in specific dimension/directions, shapes and angles. Therefore, the development of meticulous damage identification approach with an appropriate number of sensors would be required to capture the scattered waves optimally.The literature has reported that the angle of crack orientation and length of crack has an important relationship with the reflection and transmission coefficients of incident elastic waves in plate-like metal surface. It has been revealed by some researchers that the reflection coefficient decreased whereas the transmission coefficient increased with the crack angle. This relationship was found to be even more complex in case of change of length of crack and angle. However, it is suggested that the reflection coefficient alone may not be able to define the length of the crack/notch exclusively. Moreover, the notch width can also influence the reflection and transmission ratio of GWs. Similarly, some research studies have reported that the transmission coefficient is higher in half through-thickness crack than in the full through-thickness, because of the fact that the GWs travel in the remaining half through-thickness part of the metallic surface.The estimation of a crack depth has a great significance in determining the severity of crack and pit to crack transition. In absence of near-surface boundaries in metallic plates, the reduction in transmitted signal between actuator and sensor can be attributed to the quantitative measure of the defect. Nevertheless, in real-world situations the performance of active SHM with GWs can be adversely influenced by external excitation signatures; hence, defect investigation process can be further complicated. In the applications where external excitations are significant, passive SHM methods (such as acoustic emission) have been used by some researchers with contact/contactless methods. For example, the contact between wheels of train and rail track generates high excitation acoustic emissions which have been used for health inspection of rail tracks using non-contact methods.Marine structures are often subjected to corrosive seawater conditions and enormous stress levels due to internal and external loading conditions. Therefore, the rate of various degradation mechanisms and failure probability are quite phenomenal. In research literature, the crack identification on water submerged metal structures has been deliberated at length. It has been reported that the defects were detected successfully with minor variations in the location of actual defects. This variation may be accredited to refraction phenomena in the denser medium. Behaviour of various modes of guided waves have been investigated to control antifouling process, detection of ice layers and de-icing in the ship’s underwater hull, aerospace, and wind turbine structures. Many experiment/numerical based research studies have presented that the GWs can be used effectively to differentiate between the ice layer, water content and structural defects on external surfaces of engineering structures that are operating under dynamic climatic conditions.The GWUT can be extremely useful for SHM, fracture diagnostics in obscure locations and subsequent CBM planning of dynamically loaded marine structures. It has been reported that presently the structural integrity inspection of ships and associated harbour installations (floating docks) is carried out using either conventional NDT methods like bulk ultrasonic waves, which require complete removal of surface protective paints or destructive methods like sand or grit blasting. These inspections can only be performed in dedicated graving/floating docks, which make inspection tasks expensive, labour intensive and time-consuming. The use of autonomous vehicles to facilitate video imaging of underwater structures and inspection of difficult to access areas  has already been reported in the literature ,137,138.The literature review disclosed that the decision-making for the sensor(s) placement methods  is extremely important. The precise identification/quantification of defect features  is very much reliant on the selection of crack detection approach and the positioning of the sensors. The experimental results on metallic and composite plates presented in ,95 wouldVarious breakthroughs have been reported on defect characterization in metallic structures. However, to the best of knowledge of the authors of this paper, the research publications for determination of shape and orientation of structural defects in plate type metal structures are still obscure. The prominence of these two parameters (shape and orientation) is extremely vital in the purview of fatigue and fracture mechanisms, especially for the structures working under extreme static/dynamic loading conditions. The fatigue growth propagation and pit to crack transition processes are highly dependent on shape and orientation of the developed cracks in metallic structures, specifically those operating under complex loading and corrosive conditions. The identification of these prominent features of structural defects can be even more interesting than just detection or location of the defect. Advanced working methodologies, statistical/optimization tools and experimental equipment would be desirable for interpretation and empirical formulation of output (reflected/transmitted) signals. Therefore, the use of advanced and high-resolution equipment (Lecory wave-runner oscilloscope-Xi series), actuators/sensors  would be instrumental to minimize the equipment driven uncertainties.This paper proposed an experimental-based research study to determine the shape and orientation of structural defect. The cracks/notches in several shapes and orientations can be artificially crafted on thin plate type aluminum plates. Formulation of an empirical model is possible based on variations in output signal parameters on various sensors placed in different configurations, as illustrated in The UGWs have immense potential in various types of structures for CM, SHM, and NDE. They have the propensity to be used without disrupting the ongoing operations in the structure under investigation, be it a pipeline embedded in soil/insulation applied exhaust/air conditioning ducts, rail track, aircraft, or a marine structure. The use of various modes of guided waves have been reported for ice detection, de-icing and differentiation between actual defect, ice layer and pure liquid phases in structural applications. The flexibility of using GWs technology with several methods  provide greater opportunities to extend its scope and applications in various other fields. To overcome the limitation of GWs in variant environments, various methodologies  have been adopted as compensatory strategies to optimize the performance. The potential of Lamb guided wave modes has been accepted in health monitoring of marine structures, aircraft, wind turbine blades, pigging of pipelines and antifouling on ship structures. Researchers have recognized the potential of UGWs to localized defects in various geometries and complex structures; hence, it provides the opportunity to analyze the severity of the defect, optimize inspection/maintenance interval and predict failure probability and RUL of valuable assets. During this review, it was established that no research work has been published yet suggesting a methodology or an empirical method to determine the shape, widths, depths, and orientation of defects in thin plate structures. In the proposed research methodology, cracks/notches on thin metal plates will be produced artificially in various geometries (shapes and orientation). In the proposed method, the defects may be investigated at various angles with respect to the position of transducers. Upon successful identification of defect geometries, an empirical model would be designed to predict the defect features and quantification. The numerical study can be conducted for validation and optimization of designed experiment and its methodology.The outcome of experimental results based on this proposed plan can be of highly valuable to bring further improvement in GWUT based SHM/NDE in various field applications, particularly for identification of damage tolerance of structural defects and subsequent prediction of RUL and life extension (LE). It can be made as a part of future structural maintenance/inspection strategy of offshore installations and ships, which normally operates with limited man-power and away from base maintenance facilities. The big-data of SHM acquired from large marine structures (using ROV/AUV integrated GWs methods) can be continuously provided to the remote maintenance experts, through wireless transmission or internet of things (IOT). In presence of ground maintenance teams, this data can be interpreted and the defect (if any) can be investigated through comparison of data . Subsequently, this priori information on structural health can be used to update/optimize the forthcoming maintenance/inspection schedules, using probabilistic inference methods such as Bayesian inference. Determination of characteristics and directional properties of defects  through GW technologies can be extremely helpful to predict the future growth rate and defect intensity. Notwithstanding, further research studies are essential to minimize the glitches with the use of guided wave technology for health monitoring of assets in practical field as well as to increase its reliability and effectiveness, under vibrant operating conditions Although some researchers have developed the experimental-based compensatory methods to uplift the performance of GWUT affected by the environmental (mostly temperature) variations. However, implementation of these benign methods in the field experiments is not free from numerous complications due to the abrupt transient behavior of a wide range of climatic factors which ca be triggered simultaneously. The ship’s hull is consistently under static/dynamic loading conditions from internally (machinery) or external factors (momentum/wave action) which aggravate the challenges for effective SHM, exponentially. Contrary to the lab experiments, the GWUT based health inspection of these giant structures involves several factors at the same time, which have the tendency to adversely affect the defect diagnostic capability. A few prominent factors are the non-uniform thermal gradients, wetting/icing of external surfaces, exterior acoustic signatures, and the surface protective measures. In the future research studies, field-oriented experimentation under marine weather conditions is necessitated to explore the capability of various GWs  on large and valuable marine assets. The FEM approaches can also be used for the validation process, which is a cost-effectiveness way to evaluate the proficiency of GWs for characterization of salient defect features.


Such a database would enable us to apply the SbD approach more efficiently.The physicochemical properties of nanobiomaterials, such as their small size and high surface area ratio, make them attractive, novel drug-carriers, with increased cellular interaction and increased permeation through several biological barriers. However, these same properties hinder any extrapolation of knowledge from the toxicity of their raw material. Though, as suggested by the Safe-by-Design (SbD) concept, the hazard assessment should be the starting point for the formulation development. This may enable us to select the most promising candidates of polymeric nanobiomaterials for safe drug-delivery in an early phase of innovation. Nowadays the majority of reports on polymeric nanomaterials are focused in optimizing the nanocarrier features, such as size, physical stability and drug loading efficacy, and in performing preliminary cytocompatibility testing and proving effectiveness of the drug loaded formulation, using the most diverse cell lines. Toxicological studies exploring the biological effects of the polymeric nanomaterials, particularly regarding immune system interaction are often disregarded. The objective of this review is to illustrate what is known about the biological effects of polymeric nanomaterials and to see if trends in toxicity and general links between physicochemical properties of nanobiomaterials and their effects may be derived. For that, data on chitosan, polylactic acid (PLA), polyhydroxyalkanoate (PHA), poly(lactic-co-glycolic acid) (PLGA) and policaprolactone (PCL) nanomaterials will be evaluated regarding acute and repeated dose toxicity, inflammation, oxidative stress, genotoxicity, toxicity on reproduction and hemocompatibility. We further intend to identify the analytical and biological tests described in the literature used to assess polymeric nanomaterials toxicity, to evaluate and interpret the available results and to expose the obstacles and challenges related to the nanomaterial testing. At the present time, considering all the information collected, the hazard assessment and thus also the SbD of polymeric nanomaterials is still dependent on a case-by-case evaluation. The identified obstacles prevent the identification of toxicity trends and the generation of an assertive toxicity database. In the future,  Over the last decades, several nanomaterials (NMs) have been developed and studied as promisor drug delivery vehicles and medical devices, including magnetic, metallic, ceramic and polymeric nanomaterials. At present, there is fragile consensus regarding the “nano” definition among different regulatory organizations. In detail, considering medical regulatory authorities, such as the European Medicines Agency (EMA) or the United States Food and Drug Administration (FDA) some considerations can be made. In a reflection paper about nanotechnology-based medicinal products for human use published in 2006, EMA defined nanotechnology as “the production and application of structures, devices and systems by controlling the shape and size of materials at nanometer scale,” considering that “the nanometer scale ranges from the atomic level at around 0.2 nm (2 Å) up to around 100 nm” , dendrimers, polymeric micelles and drug conjugates Bhatia, . On its All considered, most of these advantages are frequently attributed to their distinctive size which contributes to their high surface area to mass ratio, and is also responsible for the different toxicokinetic fate of the NMs , (2) oxidative stress, (3) inflammation, (4) genotoxicity, (5) toxicity on reproduction and (6) hemolysis. Importantly, articles were carefully examined regarding minimal characterization parameters, such as chemical composition, particle size, surface charge and endotoxin contamination (when relevant).We started this review by discussing the pillars of human health risk assessment: exposure assessment and hazard assessment. Next, in order to analyze the state of the art about the toxic effects of polymeric NMs, peer reviewed original research articles from the last 10 years were analyzed and discussed, addressing the following endpoints: (1) To perform human health risk assessment of any material is necessary to integrate the exposure assessment with hazard assessment. The first intends to determine routes of exposure and estimate exposure dosages  while the second intends to characterize the possible hazards (toxic effects) of polymeric NMs when in contact with the human body.Human exposure to polymeric NMs should be considered in the context of intentional nanomedicine applications, and in the context of occupational exposures of workers during the manufacturing processes, testing methods, distribution and handling/administration of polymeric NMs. Moreover, it cannot be disregarded situations where misuse and overuse are easily attained , each route presenting its own biodistribution pattern, resulting in different effects on human health. Indeed, the same composition, size and surface charge of the polymeric NM, might produce a different effect only by changing the exposure route  reaching cells and tissues that are generally protected from bulk size materials  the difficulty to have complex in vitro systems, based on human cells or primary cell lines, that mimic the physiological complexity of the human body and its interaction with the materials  the difficulty on transposing accurately human effective doses to in vitro assays recommended for bulk materials  of the different NMs , incubation time, cell culture media or cell culture supplementation , which aims to promote international cooperation on the human health and environmental safety of manufactured NMs, and involves the safety testing and risk assessment of manufactured NMs. Over the years they have published numerous reports and some test guidelines which are published in the OECD Series on the Safety of Manufactured Nanomaterials to provide up-to-date information on the OECD activities in this area (OECDin vitro MTS assay for measuring the cytotoxic effect of nanoparticles), discussed below . In parallel, the European Nanomedicine Characterization Laboratory (EUNCL) is also developing standard operating procedures (SOPs) to allow the physical, chemical, in vitro and in vivo testing of nanobiomaterials (http://www.euncl.eu/).An important contribution to this field is being given by the US National Cancer Institute Nanotechnology Characterization Laboratory, whose main objective is to facilitate the development and translation of nanoscale particles and devices for clinical applications. In fact, they have described several protocols for in vivo and in vitro assays considering its effect in the host physiological and immunological integrity , while in vivo assays, particularly acute and repeated dose toxicity assays assess the effects on vital organ functions .NMs toxicity should be evaluated by in vivo tests in animals (most time non-primates) to evaluate acute and repeated-dose  toxicity. These studies, although highly valuable to understand the adsorption, distribution, metabolism and excretion (ADME) of the NMs as well as the immune system interactions, should be limited to a minimum according to the 3Rs strategy   used in the assays, the number of days (for the repeated-dose toxicity studies) and even for the endpoints that are analyzed. Some of the most reported endpoints are the clinical appearance of the animal, clinical signs of infection, hematological parameters, serum hemoglobin levels and albumin/globulin ratio, organ weights, and enhanced histopathology evaluation different organs , the animal models  and the dosing schedules (7–19 days), all revealed no On its turn, PLGA NPs also exhibited no toxicity on repeated oral administration studies , superoxide anion (O2−•) and hydroxyl (•OH) radicals  are produced during cellular metabolism in the forms of hydrogen peroxide , which diffuses freely through the cell membrane and is hydrolyzed inside the cells into H2DCF carboxylate anion form, which is in its turn non-permeable , which is excited at 495 nm and emits at 520 nm  assay for evaluating nanoparticle-induced intracellular reactive oxygen species (ROS) production in RAW 264.7 macrophage cell line. Nonetheless, according to this ISO, other cell lines similar to RAW 264.7  can be used with due validations. In this technical specification, the protocol was validated for conducting the assay in 24 well-plates, for 6 and 24 h incubation with the NPs and controls, and 30 min incubation with the probe before flow cytometry analysis. To note, the recommendation is the use of Sin-1 as positive control (maximum ROS production due to cell death) and polystyrene NPs as negative control.Free radical production is the highest in macrophages , superoxide anion  is widely applied as a simple mean to perform a qualitative and quantitative analysis of cytokines, chemokines, growth factors and immunoglobulins, with a spectrophotometric readout  or the intracellular levels of the cytokines can be measured by flow cytometry analysis using specific antibodies fluorescently labeled   is the most commonly used initial screening performed. Also, the Allium cepa model, allows for a simple and cost-effective assay where DNA damage is assessed after the roots of the plant grow in direct contact with the substance of interest  due to their molecular and physiological similarities with humans, therefore giving a high-throughput for genotoxicity , the results were similar. Another interesting fact, is the heterogeneity of results that may be achieved with different cell lines. For instance, Platel et al. used three different cell lines, and three different PLGA NPs and evaluated genotoxicity using the comet assay and the micronucleus test  or use ex vivo placentae or sperm from healthy donors  and proving effectiveness of the drug loaded formulation, using the most diverse cell lines  toxicity on reproduction, and hemolysis. Importantly, one of the first conclusions to retain is that among different research groups, the methodologies, the animal or cellular model, the dose or concentration, the assay duration and notably, the polymeric NM properties, are not the same, making it difficult to compare and establish trends. This issue derives in part from the absence of regulatory binding and standardized methodologies and guidelines which hardens the comparison of safety/toxicity assessments in different reports  on chitosan and other polymeric NMs is still scarce, which compromises some of the results found in the literature regarding their bioactivity and toxicity. In addition, despite testing the presence of endotoxins is a common procedure in laboratory and several commercial tests are available, they need to be validated for use with NMs, since most are based on optical assays and may be affected by the optical density of NPs  and positive controls with NMs, can reveal whether these NMs might be generating false positive or negative results.in vitro and in vivo harmonized toxicity studies using unloaded polymeric NMs, extensively characterized regarding their intrinsic and extrinsic properties and by performing all necessary controls it is possible to generate such database. At the present time, taking everything into account, the human health risk assessment of polymeric NMs is still dependent on a case-by-case evaluation, and it should comprise the evaluation of parameters, such as the route of administration and dose, among others, to define the required tests for the hazard assessment .The obstacles identified in this review prevent the identification of toxicity trends and the generation of a useful database where we can rely for the Safe-by-Design. Only by performing SJ gathered the information, analyzed, and wrote the first draft of the manuscript. SJ, MS, CS, GB, PW, and OB defined the subjects for discussion. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


During pregnancy, the hormonal systems of the mother and fetus are intricately interconnected to ensure normal fetal development. Accordingly, maternal alcohol consumption during pregnancy can interfere with fetal development, not only directly, through adverse effects exerted by alcohol that crosses the placenta and enters the fetal bloodstream, but also indirectly, by disturbing the functions and interactions of maternal and fetal hormones. In both the mother and the fetus, alcohol exposure can impair the functioning of the hypothalamic-pituitary-adrenal axis, which regulates the body’s response to stress; the hypothalamic-pituitary-gonadal axis, which controls reproductive functions; and the hypothalamic-pituitary-thyroid axis, which regulates the metabolism of almost all tissues. In addition, alcohol can interfere with the activities of growth hormone and insulin-like growth factors, which promote body growth and activity. Some of the effects of maternal alcohol consumption on fetal hormone systems may contribute to the adverse effects observed in children with fetal alcohol syndrome and related disorders. The endocrine  system plays a critical role in maintaining the body’s internal equilibrium . Through the release of hormones, the endocrine system regulates functions as diverse as reproduction, stress response, metabolism, growth, and behavior. Two structures—(1) a brain region called the hypothalamus and (2) the pituitary gland, which is attached to the base of the brain through the pituitary stalk—work together to control the activity of most endocrine glands in the body, including the adrenal glands, gonads , and thyroid gland. In turn, each of those glands produces one or more hormones  that control various physiological activities in the body. Alcohol can alter the activities of all components of the endocrine system by acting either directly on the endocrine glands and/ or on the hypothalamus or pituitary.In a pregnant woman, alcohol-induced alterations in endocrine activity may affect not only her health but also her ability to maintain a successful pregnancy. Moreover, alcohol consumption during pregnancy can directly affect fetal development because alcohol readily crosses the placenta. Finally, alcohol consumed during pregnancy may alter fetal development indirectly by disrupting the normal hormonal interactions between the mother and the fetus. This article reviews the multiple hormonal effects of alcohol use during pregnancy on the mother and fetus. The article incorporates findings from both human and animal studies, because although the amount of data from human subjects continues to grow, research using animal models of prenatal alcohol exposure has greatly expanded scientific understanding of alcohol’s effects on endocrine function.During pregnancy, the placenta plays a pivotal role in maintaining pregnancy and affecting fetal development. Until the fetal endocrine system is functional, the placenta acts as a miniature endocrine system, producing hormones such as human chorionic gonadotropin, chorionic thyrotropin, and chorionic corticotropin. Through its hormone production, the placenta regulates fetal growth, maturation, and nutrient utilization. In addition to its endocrine activity, the placenta acts as a partial barrier, or filter, between the maternal and fetal blood, allowing the transfer of some maternal hormones and other substances  from the mother to the fetus while preventing the transfer of others. As a result of these placental functions, the fetus is exposed to three sets of hormones: (1) those secreted by the placenta, (2) those produced by the mother, and (3) the fetus’s own hormonal secretions.Alterations in placental functioning, including the production and activity of placental hormones, may affect fetal growth and development and increase the risk of spontaneous abortion. To date, little information is available on the direct effects of alcohol on the placental hormones in humans. Animal models have extended researchers’ limited understanding of the placenta’s role in the effects of prenatal alcohol exposure.Historically, alcohol research in humans has been conducted primarily in men. Only during the past two decades have researchers begun to analyze alcohol’s effects on women  functions. The maternal endocrine system undergoes numerous changes that are geared toward maintaining the pregnancy and providing support for the fetus. Any disruption of the maternal hormone balance can lead to poor pregnancy outcome, including fetal birth defects. As the following sections describe, alcohol can interfere with maternal endocrine functions through numerous mechanisms also see .The concentration of a given hormone in the blood depends on many factors, including the rate at which the hormone is produced and secreted, its distribution in the body, and the rate at which it is removed from the blood. Alcohol can interfere with a hormone’s function by altering each of those factors. In addition, total hormone concentration does not necessarily correspond to the concentration of active hormone in the blood, because proteins called binding proteins, or globulins, may attach to and temporarily inactivate a hormone. Alcohol also may alter the levels of these proteins, thereby modifying endocrine activity and, during pregnancy, inducing complex consequences for both mother and fetus.The hypothalamic-pituitary-adrenal (HPA) axis is a hormone system that plays an essential role in the body’s response to stressful events see , p. 172.In the short term, glucocorticoids mobilize the body’s energy resources to respond to stress. This mobilization occurs at the expense of energy-dependent functions, such as digestion, growth, and reproduction. When the stressful situation is prolonged  or occurs frequently, the resulting metabolic effects and redistribution of resources may have pathological consequences, including the development of ulcers, growth retardation and underdevelopment  in children, and suppression of the immune system. Alcohol consumption also activates the HPA axis and stimulates glucocorticoid release, similar to the effects of stress. .The basal or resting activity of the HPA axis  increases during pregnancy. This increase results in higher CRH and ACTH levels and, consequently, elevated glucocorticoid levels in the blood of pregnant females compared with nonpregnant females. Researchers have investigated alcohol’s impact on HPA activity in both pregnant and nonpregnant females. Studies of pregnant rodents found that alcohol consumption further stimulated an already activated HPA axis. Thus, compared with pregnant females on non-alcohol-containing diets, pregnant females receiving an alcohol-containing diet exhibited increases in adrenal gland weight, resting glucocorticoid levels, and the HPA response to stress  axis control reproductive functions and behavior. The HPG axis is activated by the secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus. GnRH, in turn, stimulates the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary. Both LH and FSH regulate the development, growth, maturation, and reproductive functions of the gonads and stimulate the production of sex hormones, including estrogens and androgens . Both estrogens and androgens activate numerous processes in the maturing organism, such as the onset of puberty, the development of secondary sex characteristics, and the behaviors associated with reproduction. In the developing fetus, androgens also have an organizing function, affecting not only the testes and ovaries but also the size and function of different brain regions.Researchers have clearly documented the adverse effects of chronic alcohol consumption on women’s reproductive functioning. For example, alcoholic women have a higher frequency of menstrual abnormalities, such as irregular menstruation and the cessation of ovulation, than do nonalcoholic women . Those mStudies of alcohol’s effects on HPG activity in pregnant women found that alcohol use altered the levels of sex hormone-binding globulin (SHBG), a protein that binds to and temporarily inactivates androgens, thereby regulating the balance between biologically active and inactive androgens . During 4) and triiodothyronine (T3). Of those two hormones, T3 is substantially more active, and much of the body’s T4 is converted into T3 in the liver and other tissues. Reductions in thyroid hormone levels, a condition called hypothyroidism, can result in serious consequences, the extent of which depends on the time during the person’s life when thyroid function becomes impaired.The hypothalamic-pituitary-thyroid (HPT) axis regulates the rate of metabolism in the body and is essential for the normal growth and development of almost every organ system. The HPT axis is activated by the secretion of thyrotropin-releasing hormone (TRH) from the hypothalamus, which stimulates the release of thyroid-stimulating hormone (TSH) from the pituitary. TSH, in turn, regulates the growth and activity of the thyroid gland and the secretion of two structurally related thyroid hormones—thyroxine , liver, muscle, bone, heart, and lungs. In the absence of growth hormone, both animals and humans show stunted growth. Conversely, the hormone’s presence results in increased growth as well as enhanced organ size and function. Growth hormone is produced in the pituitary gland. Secretion of the hormone is stimulated primarily by growth hormone-releasing hormone (GHRH), which is released from the hypothalamus. An inhibiting hormone that is also secreted by the hypothalamus—somatostatin—cooperates with GHRH to regulate growth hormone secretion. In addition, estrogens and androgens promote and glucocorticoids inhibit growth hormone release, demonstrating the interactive nature of the endocrine system.Although growth hormone exerts some of its effects by acting directly on its target tissues, small proteins called insulin-like growth factors (IGFs), or somatomedins, mediate other effects. In adults, two forms of IGFs exist—IGF-1 and IGF-2—which are produced primarily in the liver. These growth factors, whose production is induced by growth hormone, act on various tissues to mediate the effects of growth hormone. In addition, IGFs are thought to play an important role in the regulation of fetal growth and development.Studies in human alcoholics have not yet identified characteristic alcohol-induced alterations in growth hormone secretion and activity, at least in part because confounding factors  also influence growth hormone secretion. Animal research has shown that pregnant alcohol-consuming rats exhibit reduced growth hormone levels , which is associated with characteristic patterns of abnormal facial structures, growth retardation, and developmental abnormalities of the central nervous system (CNS). FAS was first associated with maternal alcohol consumption more than 25 years ago . More reThe type and extent of the alcohol-induced fetal damage is partly related to the level and pattern of fetal alcohol exposure. For example, lower levels of prenatal alcohol exposure are required to induce neurodevelopmental effects than to induce physical or growth effects . ResearcThe type of fetal damage that results may also be related to the timing of alcohol exposure . The critical period is the time during which an organ system is undergoing crucial steps in development and/or maturation and consequently is most vulnerable to the disruptive effects of any agent that causes abnormal fetal development or birth defects. For example, because the development of the facial and skull bones occurs during the first 3 months of gestation in humans, alcohol exposure during the first trimester can affect those structures and result in the characteristic facial abnormalities observed in children with FAS. Conversely, alcohol exposure during the second or third trimester is more often associated with growth retardation and neurological defects, because fetal growth and brain development happen more rapidly during those gestational stages. If maternal alcohol consumption occurs during all three trimesters, the fetus is exposed to alcohol during the critical periods for the development of facial characteristics, growth patterns, and CNS function and thus may develop full FAS.Prenatal alcohol exposure may adversely affect the fetal endocrine system and, consequently, the functioning of numerous organ systems. The endocrine activities of both mother and fetus change throughout gestation. For example, whereas the transfer of maternal hormones across the placenta and/or placental hormone production is essential during early pregnancy, the activity of the fetal endocrine system becomes more pronounced and important later in gestation. Consequently, alcohol’s impact on fetal endocrine activity may occur through different avenues at different time periods. Because of their numerous effects on physiological processes, alcohol-induced alterations in hormone levels likely mediate some of the effects of prenatal alcohol exposure.The HPA axis is essential for life because it affects the metabolism and activity of numerous systems  and ensures homeostasis in response to stress. In the fetus, the first cells to become functional in the pituitary are those that release ACTH. By 9 weeks of gestation, the human fetal pituitary gland contains measurable ACTH levels. Because maternal ACTH cannot cross the placenta into the fetal circulation, the fetal HPA system is controlled by a placental hormone called human chorionic corticotropin until the fetal hypothalamus and pituitary have fully matured. Maternal glucocorticoids , however, can cross the placenta and enter the fetal circulation.As noted previously, alcohol consumption activates the HPA axis and stimulates glucocorticoid release. Therefore, alcohol consumed during pregnancy will activate the maternal HPA axis and result in increased glucocorticoid levels. Those glucocorticoids can cross the placenta, resulting in elevated glucocorticoid levels in the fetal blood, thereby signaling the fetal HPA axis to decrease its activity. At the same time, however, alcohol in the maternal blood also crosses the placenta and directly activates the fetal HPA axis. Such conflicting messages may alter the development of the fetal HPA axis by disrupting communication among the CNS, hypothalamus, pituitary, and adrenal glands.To investigate alcohol’s effects on the fetal HPA axis, researchers have compared HPA activity in newborn rodents that were prenatally exposed to alcohol with newborn control animals that were not exposed to alcohol. In those studies, alcohol-exposed neonates showed evidence of prenatal HPA activation, such as elevated corticosterone levels in the blood and brain for several days after birth see . ThereafAfter the first few weeks of life, the pattern of reduced responsiveness to stress is reversed. In fact, adolescent and adult animals prenatally exposed to alcohol typically display increased HPA activation in response to various types of stress as indicated by the following observations see .In response to stress such as immobilization, ether, or cold, alcohol-exposed animals show elevated corticosterone, ACTH, and/or β-endorphin levels compared with control animals.Animals prenatally exposed to alcohol show increased ACTH and corticosterone levels following one-time  exposure to drugs such as alcohol and morphine compared with control animals.Stress-induced elevations in ACTH and corticosterone levels appear to be prolonged in alcohol-exposed animals following immobilization stress, suggesting that those animals may be less able to inhibit HPA activity or recover from stress than control animals.Excessive HPA activity may have profound consequences throughout the animal’s life, including possible impairments in growth or immunity resulting from a redistribution of energy resources. Interestingly, the effects of prenatal alcohol exposure may differ between male and female offspring and may also depend on the nature and intensity of the stressor applied, the hormone analyzed , and the time course of hormone measurement. For example, when animals were exposed to repeated immobilization stress, males prenatally exposed to alcohol showed increases in β-endorphin levels, whereas females showed increases in ACTH levels. In contrast, control males and females showed habituation  in their HPA responses to this repeated, predictable stressor . This exSimilar to the findings in animals, a recent study in human infants whose mothers drank heavily at conception found greater increases in cortisol levels in response to stress  compared with control infants . FurtherDuring gestation, the HPG axis influences not only the development of the reproductive system but also the organization of the CNS.Research conducted in animal models has shown that prenatal alcohol exposure adversely affects male offspring by altering the structure and function of the fetal testes as well as other components of the urogenital tract see . For exaIn the fetus, alcohol decreases the number of testosterone-producing cells in the testes and reduces testosterone production by inhibiting certain enzymes.At birth, animals prenatally exposed to alcohol exhibit decreased brain levels of the active form of testosterone  and decreased testosterone levels in the blood compared with control animals.Adult male animals prenatally exposed to alcohol exhibit lower testis and prostate weights as well as decreased levels of testosterone and LH (which is released from the pituitary to stimulate testosterone formation in the testes).Alcohol-induced reductions in testosterone levels may alter testosterone’s effects on brain organization during gestation and may explain the differences in brain structure and sex-typical behavior seen between animals prenatally exposed to alcohol and control animals. For example, the SDN–POA in the hypothalamus is smaller in males prenatally exposed to alcohol than in control males . These aChanges in HPG activity and in behaviors controlled by the HPG axis also have been noted in female rodents prenatally exposed to alcohol, including the following see :Increased levels of the pituitary hormone prolactin and decreased levels of LH in the blood from approximately 22 to 35 days of age (the elevated prolactin levels may persist into adulthood)Delays in puberty and in displaying sexually receptive behavior, possibly resulting from the hormonal changesImpaired maternal behavior Deficits in sexual behavior and earlier cessation of estrus cycling , indicating that prenatal alcohol exposure may accelerate the aging process of the female reproductive system .Limited data are available on the association between prenatal alcohol exposure and HPG activity in humans. The available information indicates that prenatal alcohol exposure may slightly delay puberty in males, although the time of onset of puberty still is generally within normal limits in those adolescents . More exNormal functioning of the HPT axis is critical for growth and development as well as for regulation of the body’s overall metabolic rate. Reductions in thyroid hormones during the first 2 years of life may be particularly devastating, because those hormones play a crucial role in CNS development. Accordingly, untreated hypothyroidism in infancy results in growth retardation  and mental retardation. During the first weeks of gestation, the transfer of maternal thyroid hormones to the fetus may play an important role in fetal development. By 11 to 12 weeks of gestation, however, the thyroid of the human fetus can produce and secrete its own hormones. Maternal alcohol consumption may reduce the availability of thyroid hormones to the fetus either indirectly, by inhibiting the transport of maternal hormone across the placenta, or directly, by interfering with the function of the fetal thyroid once it is active.Studies in animal models have demonstrated that following prenatal alcohol exposure, the growth of the thyroid gland is retarded (for a review, see An analysis of numerous studies on children prenatally exposed to alcohol, however, showed no long-term changes in thyroid function or thyroid hormone levels . The disGrowth hormone and IGFs play a central role in promoting body growth and development. Accordingly, alcohol-induced impairment of this hormone system during fetal development could have severe consequences. Animal studies have shown that newborn animals prenatally exposed to alcohol had reduced growth hormone levels compared with control animals . More reGrowth hormone normally is released from the pituitary periodically throughout the day. Growth hormone release increases in response to various factors, such as changes in blood sugar or insulin levels, fasting, or exercise. In children with FAS, growth hormone responses to stimulation by such factors were normal, as were growth hormone levels during sleep (for a review, see Alcohol consumption during pregnancy disrupts the normal functioning of both the maternal and the fetal endocrine systems and may disturb the normal maternal-fetal endocrine balance. Those alterations may adversely affect the development and organization of multiple systems in the fetus and likely mediate some commonly observed effects of pre-natal alcohol exposure. The exact mechanisms underlying alcohol-induced fetal damage have not been fully delineated. The tremendous impact of prenatal alcohol exposure on fetal development is not surprising, however, considering the interrelationship of the maternal and fetal endocrine systems, the complex role of the placenta, and the numerous direct and indirect effects of maternal alcohol consumption on both the mother and the fetus. Thus, although numerous factors likely play a role in alcohol-induced fetal damage, the disruption of hormonal influences on the developing fetus may explain at least some of the effects of prenatal alcohol exposure. An increased understanding of endocrine function during pregnancy; of the development of the fetal endocrine system; and of the processes influenced by maternal, placental, and fetal hormones may offer new insights into the adverse effects of prenatal alcohol exposure and possible methods of attenuating those effects.


Disease ecology emphasizes how ecological interactions between microparasites (pathogenic microbes), macroparasites , and animal hosts help understanding transmission of diseases and parasites within the epidemiological landscape. Diseases and parasites are ecological and evolutionary forces at all biological levels of organizations from organisms, populations, communities to ecosystems and there is an increasing interest to investigate them in an evolutionary ecological perspective.Integrating evolution, co-evolution into the ecology of transmission in a spatial context poses many challenges. To tackle these challenges disease ecologists use a wide varieties of methods and tools such as molecular approaches developed from pathogens screening, high-throughput technologies, population genetics, phylogenetics and phylogeography, quantitative epidemiology, population dynamics, theoretical epidemiology, spatial analyses, and landscape ecology.This special issue calls into presenting advances and identifying gaps in the disease ecology and evolutionary ecology of diseases, using wild mammals and their pathogenic bacteria, viruses, parasites, and vectors as so many models. This special issue is a collection of studies in disease ecology that contribute to Conservation Medicine  and One Szentiványi et al. Viruses, bacteria, blood protists, and fungi have been detected in bat flies that show physiological consequences on bats and their ectoparasites. The authors recommended additional studies to understand the interlinkages between bat hosts, ectoparasites, and their associated microparasites. McKee et al. examined Bartonella from European bats and their ectoparasites using network analysis, Bayesian phylogenetics, and tests on co-phylogenetic association. The authors were able to disentangle the processes, ecological, or evolutionary, that contribute to shape the interactive communities. Bat phylogeny and bat roost sharing help to explain the evolutionary patterns of vector-borne diseases.In two studies presented in this special issue, bats and their parasites, vectors, and microbes were investigated. Information on bats, bat flies (obligate hematophagous ectoparasites of bats) and their microparasites was synthesized by Kosoy and Goodrich reviewed published studies on the phylogenetic sister clades Bartonella and Brucella that infect wild carnivores to analyse and compare the ecology of these two clades of bacteria in closely related host species. Bartonella species were much reported in every sampled wild felid species, whereas among Brucella studies only few of them have reported Brucella in felids by detection of antibodies. The authors stressed that wild carnivores often carry the same microparasites as the domesticated cats and dogs, merely exposure is related to differences in biology, distribution, and historical interactions. In a comprehensive review, André synthesized the actual knowledge on the diversity of the tick-borne bacteria of species from Ehrlichia, Anaplasma and “Candidatus Neoehrlichia sp.” in wild carnivores worldwide and discussed consequences for human and animal health as well as wildlife conservation. The author emphasized the importance of Whole Genome Sequencing and Next Generation Sequencing (NGS) technologies to better understand the importance of wild carnivores in the transmission of several agents such as Anaplasmataceae.Carnivores and bacterial diseases were the topic of two studies. Maina et al. who summarized and discussed the actual knowledge of the epidemiology and distribution of Rickettsia asembonensis, a well-characterized rickettsia of the Rickettsia felis-like organisms, worldwide, as well as its arthropod hosts. The authors emphasized the need to conduct further analyses, functional and structural, to find out differences and/or similarities between R. asembonensis and other rickettsial species, and to better characterize the current/potential arthropod vectors with other flea-borne rickettsial species (R. felis and R. typhi), but also non-rickettsial pathogens such as Yersinia pestis, the agent of the plague.Flea-borne rickettsial disease was further explored by Sariyeva et al. investigated the role of gray marmots in the maintenance of highly virulent strains of Y. pestis in endemic foci of the Tien Shan Mountains, Kyrgyzstan. Plague circulates incessantly in populations of gray marmots, their fleas and other rodent species, stressing the importance of significant changes in rodent communities during the previous two decades. Biggins and Eads reviewed hypotheses regarding the epidemiology of Y. pestis using recent data from North America supporting maintenance of Y. pestis by persistent transmission. They proposed a maintenance mechanism, the Synergistic Positive Feedback cycles, that facilitates periodic epizootic eruptions “in place” resulting in sudden outbreaks that spread rapidly in time and space involving flea vectors, hosts, and the plague bacterium. The authors stressed that the absence of plague epizootics may reduce public health risk, but may still have ecologic impact on wild mammalian populations.Tangtrongsup et al. who investigated the prevalence of intestinal parasites of Giardia duodenalis and Cryptosporidium spp. in captive agile gibbons (Hylobates agilis), lar gibbons (H. lar), and pileated gibbons (H. pileatus) at the Krabokkoo Wildlife Breeding Center, Thailand. The authors stressed the improvement of hygiene management to prevent potential transmission between gibbon and human.Studies on the parasites and diseases find application in conservation medicine as exemplified by Takhampunya et al. conducted an intensive study in populations of humans, animals, and vectors in Northern Thailand where scrub typhus is highly endemic. Leptospira spp., Bartonella spp., Rickettsia spp., and Orientia tsutsugamushi were detected using NGS in the studied populations. The authors confirmed the transmission of several bacterial diseases in the area, some of which are known to cause severe illness in human populations. Ruiz-Arrondo et al. outlined the benefits and the limitation of the entomological surveillance programme of mosquitoes implemented by the Government of La Rioja (Northern Spain). In order to implement a One Health approach, the surveillance programme should screen wild birds for flaviviruses and sentinel horses. Better coordinating efforts from biologists, epidemiologists, and veterinarians would be an added value to enable ecological data to be operationalised to inform human, animal, and ecosystem health.Finally, two studies contributed to the One Health approach. Using a metagenomic approach, SM and MK have served as editors of the Research Topic and have co-written the editorial.MK was employed by company KB One Health LLC. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PME gene transcription; moreover, the enzymatic activity of PMEs is modulated and controlled by PME inhibitors (PMEIs), which are also induced in response to pathogenic attack.Until recently, plant-emitted methanol was considered a biochemical by-product, but studies in the last decade have revealed its role as a signal molecule in plant-plant and plant-animal communication. Moreover, methanol participates in metabolic biochemical processes during growth and development. The purpose of this review is to determine the impact of methanol on the growth and immunity of plants. Plants generate methanol in the reaction of the demethylation of macromolecules including DNA and proteins, but the main source of plant-derived methanol is cell wall pectins, which are demethylesterified by pectin methylesterases (PMEs). Methanol emissions increase in response to mechanical wounding or other stresses due to damage of the cell wall, which is the main source of methanol production. Gaseous methanol from the wounded plant induces defense reactions in intact leaves of the same and neighboring plants, activating so-called methanol-inducible genes (MIGs) that regulate plant resistance to biotic and abiotic factors. Since PMEs are the key enzymes in methanol production, their expression increases in response to wounding, but after elimination of the stress factor effects, the plant cell should return to the original state. The amount of functional PMEs in the cell is strictly regulated at both the gene and protein levels. There is negative feedback between one of the MIGs, aldose epimerase-like protein, and  Plants are the source of over a million metabolites , includiIn general, the source of metabolic methanol formation in plants is the demethylation of macromolecules Figure . If the Pectin methylesterases of higher plants encoded by a multigenic family (67 putative isoforms in Arabidopsis)  and are Arabidopsis thaliana RESISTANCE METHYLATED GENE 1 and WRKY22 in response to biotic stress  transcripts  in suspension culture and to be metabolized to [3-13C]serine, [13CH3]methionine, and [13CH3]phosphatidylcholine in addition to the induction of the methyl-β-d-glucopyranoside de novo synthesis  and Bemisia tabaci (whitefly)  . Moreovehitefly) . It has hitefly) . The sighitefly) . Among thitefly) . Moreovehitefly) . While tA. thaliana infection by the necrotrophic pathogen Botrytis cinerea, PME activity and pectin demethylesterification are dynamically modulated by PMEIs (In general, prolonged and continuous exposure to stress factors, such as prolonged mechanical wind impact, accompanied by the synthesis of PMEs, causes irreversible changes in woody plants  like conby PMEIs .AELP involved in intercellular transport and possibly controlling the transport and metabolism of sugars (PME expression. The proposed model for the feedback regulation of gene expression involving PMEI and AELP suggests that when the cell wall is mechanically damaged, the PME genes are activated (Figure PMEI genes and activation of the expression of the AELP gene through its methanol-inducible promoter. As shown in Figure PME gene (AELP gene. In accordance with this scheme, AELP proteins are likely to behave as transcription factors by inhibiting the expression of the PMEs in the nucleus and not in the apoplast.Recently, another PME activity controlling mechanism was identified: the methanol-inducible gene f sugars  negatived Figure ; synthesPME gene  and reduThus, the proposed model assumes the regulation of PMEs functioning at both the gene and protein levels.PME, which has been observed in transgenic tobacco plants (AELP (PMEI (Recent research has strengthened our view that endogenous metabolic methanol in plants not only is a by-product of biochemical processes of plant life but also performs an important signaling function. Influencing genes involved in the plasmodesmata gating and the intercellular transport of macromolecules, methanol participates in the controlled growth and development of plants . Since to plants , leads to plants . At the ts (AELP  and PMEILP (PMEI , lowerinYD, ES, and TK conceived the concept, prepared, revised, and finalized the manuscript. All the authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The current role of computational approaches is providing innovative, incremental, and complementary data-driven solutions for the prediction, simplification, and characterization of cancer and intrinsic mechanisms, and to promote new data-intensive, accurate diagnostics and therapeutics.The ability to understand the complexity of cancer-related data has been prompted by the applications of (1) computer and data sciences, including data mining, predictive analytics, machine learning, and artificial intelligence, and (2) advances in imaging technology and probe development. Computational modelling and simulation are systematic and cost-effective tools able to identify important temporal/spatial patterns (and relationships), characterize distinct molecular features of cancer states, and address other relevant aspects, including tumor detection and heterogeneity, progression and metastasis, and drug resistance. These approaches have provided invaluable insights for improving the experimental design of therapeutic delivery systems and for increasing the translational value of the results obtained from early and preclinical studies. The big question is: Could cancer theranostics be determined and controlled  The highly intricate nature of cancer makes the approaches to managing and rationalizing large dimensional cancer data notably different from those used in other types of diseases ,2,3. CanAlthough cancer is related to genetic mutations in cells, the interactions between cells and the surrounding medium affect cancer growth and tissue invasion. In order to develop accurate models to describe this highly complex disease, different biological and physiological scales have to be considered and incorporated into mathematical and computational models supporting the rational therapy design. Several approaches have thus provided tailor-made drug treatments towards specific cancer cells, reducing side effects. In this context, different theranostic agents have been developed to selectively deliver the active drug to the tumor site and to simultaneously monitor the therapeutic efficacy by, e.g., constructing tumor imaging frameworks. However, literature regarding cancer theranostics is lacking in comprehensive and systematic approaches to: (1) fully inspect the relevant interaction patterns and synergistic effects, (2) evaluate tumor heterogeneity and data-intensive theranostics technologies, (3) confirm the effectiveness of therapeutics, and (4) compare and validate specific mechanistic models. Fundamental aspects on the cellular and molecular basis of cancer have also been explored through the establishment of relevant biological networks ,15,16,17In silico approaches, including simulation and modelling . The rational and multiscale integration of different types of omics data is essential to elucidate the most relevant factors that lead to the disease and to prioritize treatment targets . Coordinin silico approaches for dealing with information on cancer development, progression, and treatment has been described in an uncorrelated manner in the current state of the art, making the clear distinction and the establishment of relations between them difficult tasks.A wide range of In spite of that, a distinction can be made considering the target properties or phenomena to be evaluated at four main scales: the biomolecular, cellular, intercellular/tissue, and macroscopic scales. When the focus is to study the structure and dynamics of proteins, lipids, and other relevant molecules, a biomolecular scale can be considered. At the cellular scale, relevant cell functions, mechanisms, and responses are assessed, while works focusing on cells transformations, such as those occurring in the malignant processes, or in the cell–cell and cell–environment interactions fall into the intercellular/tissue scale. Finally, the tumor morphology, vascularization, shape, and progression are assessed at the macroscopic scale.in silico approaches applied to different systems at different scales are presented.In what follows, some selected examples of One of the emerging cancer theranostic tools is the use of microRNAs (miRNAs). These small endogenous noncoding RNA molecules regulate gene expression by targeting messenger RNAs (mRNAs), being powerful regulators of the different cellular processes involved in the initiation and development of different types of cancer, including breast ,90, prosThe fact that miRNAs can be found in biofluids makes them attractive non-invasive tools for cancer detection and monitoring. Moreover, the differential expression of miRNAs has been correlated with cancer development and progression. As miRNAs regulate specific genes, including tumor suppressors and oncogenes, they can be used as therapeutic agents inducing the increase or decrease of the expression level of a given miRNA. When the goal is the inhibition of an oncogenic miRNA, mimics should be used. These are synthetic oligonucleotides identical to endogenous miRNAs able to restore the downregulated activity of miRNA tumor suppressors. Another possibility is the use of miRNA sponges, which are oligonucleotide constructs able to remove the excess of oncogenic miRNAs .Bertoli et al. have explored the role of miRNAs in breast ,96 and pIn two studies ,96, the A collection of several computational approaches for identifying cancer-associated miRNAs have been recently reviewed by Cantini and co-workers . The autThe alterations in the information flow over cellular networks, and the structure/function of a protein and the respective interactions with small molecules, are essential for defining and predicting drug response phenotypes, as well as describing the effect of the designed pharmaceutical agents on multiple targets and pathways ,99.Some authors  have proOne of the most compelling and effective tools for the stoichiometric or dynamic simulation of biological networks , and forThe complexity of signaling networks and the respective impact on cellular functions have also been reported. The activity of the epidermal growth factor (EGF) and nerve growth factor (NGF) in rat pheochromocytoma has been simulated by Brown et al. . The EGFFaura and co-workers  developein silico methods based on pathway cross-talk inhibition offer invaluable strategies to identifying customized and effective treatments, especially for heterogeneous cancer diseases, such as breast cancer. The use of other oncogenic features, such as gene mutations and the rational identification of biomarkers for pathway addiction, as well as the cross-talk between parallel signaling pathways for identifying patients prone to respond to specific inhibitors, are much needed [Cross-talk among signaling pathways plays an essential role in cancer drug resistance, especially in receptor-targeted therapies, and may occur at multiple levels . Howeverh needed .Identifying effective drug combinations for treating complex diseases remains a challenge given the large number of possibilities. These have been predicted using different approaches including mathematical modeling, stochastic search, global gene expression, and targeted phosphoproteomics profiling . The mecin silico using pathway analysis, which have allowed prioritizing potential drug pairs [The combination of drugs to improving treatment of advanced solid tumors is of paramount importance to bypass drug resistance, promoted by compensatory signaling mechanisms which counterbalance the therapeutic effects and lead to treatment failure ,110. Theug pairs ,109,110.ug pairs  as potenTumor progression and metastatic dissemination in different cancers have also been the focus of systematic characterization and prediction. Relevant patterns related to dynamic alterations of glioma histological features, radiotherapy efficacy, and survival time have been predicted in cases involving glioma cell proliferation. The velocity of glioblastoma growth and the respective size, as well as the gradient of cells migrating from the border of the tumor, have been determined  for moniThe correlation between glioma cells and vasculature has also been established based on a model for describing proliferation-invasion-hypoxia-necrosis-angiogenesis. This mathematical model allowed for discriminating histological grades and stereotypical alterations between the tumor grades, and predicting invasion kinetics and malignant scenarios such as hypoxia and necrosis .Zhou et al.  designedThe fundamental concepts and the critical role of multiscale cancer modeling for providing a quantitative understanding of cancer initiation, progression and treatment, have been introduced and discussed by Deisboeck et al. . This apMore recently, Na and Choi  have desThe four groups identified based on the cellular heterogeneity in the tumor microenvironment c–e possein silico models is provided in ref. [A detailed description of the extensive efforts in the field of cancer immunotherapy personalization aiming at integrating clinical data into  in ref. . In whatin silico, indicating a potential minimization of side-effects. This approach was inspired in a previous study conducted by Pappalardo et al. [Different studies based on agent-based models  and delao et al. , focusedPappalardo et al.  have appThe difficulty in anticipating clinical outcomes according to each patient needs has also fostered the development of innovative modeling approaches capable of analyzing the entire or a specific part of the immunotherapy system, and predicting individual therapy results. These approaches have been formulated based on the integration of patient information with dynamical models representing the impact of different treatment scenarios. Agur and co-workers  have recIn another study, the cancer-immune dynamics resulting from interleukin-2 (IL-2) or adoptive cell transfer (ACT) immunotherapy was described ,120, sugFor example, the method developed by Cappuccio and co-workers ,127 for A similar approach  was implNew insights on prostate cancer immunotherapy have been provided by Agur and co-workers  using a An algorithm including information on the preparation, personalization and prediction of a refined treatment, and also on the monitoring stages has been used to validate iteratively personalized models using data from patients, collected as training sets. This algorithm possesses a validation criterion to determine when the personalized model can predict individual treatment results under different treatment procedures ,132.Some authors have selected key parameters ,134 for Other approaches based on the malignant glioma model have been used to explain the treatment failure in slowly growing cancers, characterized by higher initial tumor load or reduced cytotoxic-T-lymphocyte efficacy. With no exception, the desired response can be obtained by increasing the amount of transferred cytotoxic-T-lymphocytes and using the patient’s parameters ,134.Very recently, Baker and co-workers  have useIt is well known that theranostic approaches refer to systems and strategies in which disease diagnosis and therapy are combined through the administration within a single formulation of a drug and an imaging agent, or a material with an intrinsic ability to be used for imaging purposes, such as gold- and iron oxide-based nanoparticles. The combination of diagnostic and therapeutic agents allows to assess the efficacy of drug targeting or off-target accumulation, to monitor therapeutic efficacy enabling a real-time adjustment of the treatment.In silico approaches can extensively contribute to the development and optimization of these systems, providing a set of numerical modeling methods and computational technologies that support the design of drug and therapeutic systems, allow the prediction of biological interactions and responses, simulate reactions, and estimate optimal parameters [rameters ,137.Many of the developments in this area have been devoted to the design and optimization of nanotechnology-based systems. The contribution of modeling and simulation techniques is quite vast, relying on the (1) rational design of nanoparticles with optimized characteristics , (2) modelling of drug loading and release profiles, (3) optimization of the internalization processes and interaction with the biological membranes and systems, and (4) improvement of nanoparticles bioactivity, targeting and toxicity, among others ,137.2 nanoparticle surface, besides providing a way to discern smaller nanoparticles from the larger aggregates, also enhanced visible light triggered DNA damage upon cancer targets through localized production of reactive oxygen species. Resorting to quantum mechanics calculations at the Density Functional Theory (DFT) level using the B3LYP functional and the polarizable continuum model (IEFPCM) to obtain the free energies of solvation, the authors explored the coordination modes of alizarin blue black B to the TiO2 surface, and found that the more favorable interaction occurs through the alizarin blue black B sulfonate group, while the metal center maintained the distorted geometry .in silico, in vitro, and in vivo studies, the authors were able to find out an efficient internalization into breast cancer cells and the excellent drug loading and release behavior of the chemotherapeutic drug doxorubicin (DOX). Using MD simulation the authors explored the interaction between a DPPC bilayer and a magnetite nanoparticle functionalized with a branched polysaccharide consisting of l-arabinose, d-galactose, l-rhamnose, and d-glucuronic acid, with a grafting density of three chains/nm2 and chain lengths of 6.5 nm  or external triggers  for transport of therapeutic agents and to the selected target has been essential for the timely detection, diagnosis, and treatment of cancer.Biomarkers have been used as input information in artificial neural networks for establishing cancer types and the respective malignancy Other authors  have perThe used of multi-imaging modalities have revealed important biochemical signatures, the size of the tumors and tracking the respective progression . The useComputational approaches have also contributed to the accurate planning of experiments for delivering increasing doses of radiation therapy to specific target volumes in cancer patients ,164,165.Recent advances in magnetic resonance on-line imaging and in the use of implanted markers have allowed enhancing the precise on-time tumor localization, reducing the radiation doses in the surrounding organs.Molecular imaging has provided relevant information on the tumor phenotype determining treatment decisions and the refinement of the applied therapy by providing predictive data on the treatment outcomes. The delineation of tumor sub-volumes for dose optimization and administration and the identification of spatial patterns of radioresistance are also key applications of molecular imaging ,167,168.Different machine learning and artificial intelligence algorithms have provided tools for the automated assessment of tumor response, which have reduced the number of tasks and the variation between readings and increased productivity and efficiency . AdvancePhenotypic alterations exhibited by human cancers can be observed noninvasively by medical imaging and processed by radiomics, the high-throughput mining of quantitative image features for medical knowledge extraction. Radiomics have allowed improving cost-effective decision support in cancer treatment by (1) converting imaging data into a high-dimensional mineable feature space using several algorithms, and (2) incorporating, diagnostic, prognostic, and predictive signatures. A comprehensive quantification of tumor phenotypes by applying radiomics analysis of a large number of quantitative image features has been proposed by Aerts and co-workers . The selThe association of radiomics with clinical factors, prognosis, and gene-expression levels, using large amounts of features and with external/independent validation cohorts of patients, has been evaluated. The authors have shown the translational capability of radiomics in these two cancer types and the successful quantification of a general prognostic cancer phenotype with potential to be employed on other cancer types. The correlation between a prognostic radiomic signature, capturing intratumor heterogeneity and gene expression patterns, was established based on a radiogenomics analysis .Gillies et al.  have proDifferent grades of prostate cancer have also been discriminated by radiomics in magnetic resonance imaging , contribAlso evaluated was the genetic expression classification of the most lethal high-grade serous ovarian cancer. A three-dimensional representation was used for discriminating primary and metastatic lesions. It was found that the longer the distance between a metastatic lesion and the primary tumor, the larger the heterogeneity of the texture between these lesions . It was Xi and co-workers  proposedRanjan et al.  developein silico models mimicking the mechanistic details of cancer processes is still a fertile ground of research, from the theoretical, empirical and clinical perspectives. The integration of in silico/in vitro/in vivo data in preclinical development and the coupling between nanotechnology, molecular imaging, and computer science have offered an invaluable opportunity of attaining effective solutions for the prevention, diagnosis and therapy of cancer. Although there is a general consensus that computational approaches are important tools for the sustained development of accurate and personalized cancer therapies, some concerns still remain pertaining to whether or not those approaches provide reliable support to clinical translation. Comprehensive understanding of cancer-related complications and specificities , and of tumor genotype, phenotype, metabolomics, and microenvironment, as well as systematic rationales of the involved mechanisms, are still much needed. This means that molecular diagnostics, chemo- and bioinformatics, imaging and big data analytics, and clinical data must be routinely implemented in order to obtain more accurate diagnostic and therapeutic pathways to effectively boost longevity while maintaining the quality of life.The development and improvement of