This paper presents an open-source environment for development, tuning, and performance evaluation of magnetic, angular rate, and gravity-based (MARG-based) filters, such as pose estimators and classification algorithms. The environment is available in both ROS/Gazebo and MATLAB/Simulink, and it contains a six-degrees of freedom (6 DOF) test bench, which simultaneously moves and rotates an MARG unit in the three-dimensional (3D) space. As the quality of MARG-based estimation becomes crucial in dynamic situations, the proposed test platform intends to simulate different accelerating and vibrating circumstances, along with realistic magnetic perturbation events. Moreover, the simultaneous acquisition of both the real pose states (ground truth) and raw sensor data is supported during these simulated system behaviors. As a result, the test environment executes the desired mixture of static and dynamic system conditions, and the provided database fosters the effective analysis of sensor fusion algorithms. The paper systematically describes the structure of the proposed test platform, from mechanical properties, over mathematical modeling and joint controller synthesis, to implementation results. Additionally, a case study is presented of the tuning of popular attitude estimation algorithms to highlight the advantages of the developed open-source environment. Providing accurate pose estimates  is a crucial task in the control of agile mobile systems, such as robots. Since the controller and estimator algorithms are linked in closed-loop, therefore, the estimator algorithm should meet important design requirements. Such requirements ensure that the controller algorithm successfully stabilizes the system in the close vicinity of the desired state based on the estimation results ,2,3. If The relative localization problem is solved with microelectromechanical systems-based (MEMS-based) sensors, such as accelerometers, magnetometers, and gyroscopes, in today’s embedded systems. These sensors form the inertial measurement unit (IMU), where a microcontroller processes the sensor data, executes filtering algorithms and functions as an attitude and heading reference system (AHRS) . At the Deterministic approaches use solely gravity and magnetic field observations to solve the Wahba’s problem and provide attitude estimation ,15. AmonThe performance of each technique discussed in the previous subsection heavily depends on the core parameters that characterize the filter structure. These parameters should be selected carefully in order to both meet the design requirements and provide accurate and robust attitude estimates in the intended application. If the intended application is expected to contain different system conditions , then the problem of selecting the proper parameters becomes even more crucial, since the system dynamics influences significantly the MARG-based state estimation process. Usually, it is difficult to derive the appropriate filter parameters ; moreover, the determination process requires particular equipment and measurement methods. As a result, engineering intuition-based filter tuning is usually performed, which yields only a compromise solution between filter dynamics and accuracy. However, it is also common to use optimization-based techniques to maximize the filter performance ,25,27,29Both approaches discussed above require an environment, which enables the evaluation of filter performances. Namely, the true system states need to be obtained simultaneously with the estimation results in order to determine the estimation quality. The literature offers various solutions for such environments, from camera-based techniques ,30, overAs a result of the investigation, this paper proposes a novel test environment for effective development of MARG-based algorithms. This test environment includes a six-degrees of freedom (6 DOF) mechanism to execute various external accelerations and vibrations, thereby simulating different system conditions. An MARG unit is attached to the end of the defined kinematic chain of joints; therefore, the environment simultaneously supplies the raw MARG sensor data, along with the true pose (position and orientation) states, during the motion of the system. Moreover, the test environment includes an artificial magnetic perturbation algorithm to generate realistic magnetic disturbance effects during the execution of different test scenarios. This complete environment allows to simulate various real word scenarios and thereby enables both the development and testing of any filter structure. Additionally, the tuning of filter parameters can easily be performed, since the true states of the system are provided in the environment, and even numerical optimization can be employed to obtain maximized filter performance. The author made both the ROS/Gazebo and MATLAB/Simulink implementations publicly available in the The remainder of the paper is organized as follows. To be able to effectively develop, test, and evaluate different relative localization-type algorithms, a test environment is required to be designed. This test environment should both allow to simulate various realistic system behaviors  and contain realistic sensor models which include manufacturing errors and noise sources. Additionally, the test environment should provide the real system states (ground truth position and orientation), along with the raw, noisy, uncalibrated sensor data, during the execution of different system conditions. These features both enable the evaluation of filter performances and foster the development of novel techniques.A test environment that satisfies the aforementioned requirements is depicted in The 6 DOF test bench consists of three prismatic joints and three revolute joints. The prismatic joints maintain the desired spatial coordinates of the MARG unit. Moreover, by sliding back and forth, up, and down the sensor frame in the 3D space different accelerating system conditions are simulated via these joints. The revolute joints control the orientation (instantaneous Euler angles) of the sensor frame. These joints can execute both fast and slow rotation motion to simulate vibration and oscillation effects. A plate is attached to the end of this kinematic chain which contains the MARG unit  or angular velocity (revolute joints) is zero, i.e.,ith joint as:If the above condition is satisfied, then the sliding mode is activated and viscous friction acts on the joints, i.e.,ints see . If the The evaluation of Equation (1) results in the motion equations of the system in the following form:Proportional-integral-derivative (PID) controllers maintain the position regulation of the 6 DOF test bench. The prismatic joints are actuated with force inputs, while torque signals are supplied to the revolute joints. As a result, six force/torque action-type controllers are applied to perform both translational and rotation motion with the test bench in the 3D space. The control requirements are summarized in q) in closed-loop. First, the damping ratio Hith joint is given as:ith element of the mass matrix in Equation (3), and ith joint. By differentiating Equation (15), the equivalent third-order system is obtained, and the PID controller parameters can be derived:As a result, the closed-loop dynamics of the ith PID controller is straightforward. Based on the comparison of the reference system The derivation of Since the extra time constant in the third-order reference system . In the experimental results, the reference values are supplied with approximately 1-s period for the prismatic joints, while the revolute joints are required to track The right side of the first row highlights the performance of actuated revolute joints. Since these joints intend to simulate different oscillation magnitudes; therefore, the joints are characterized by faster dynamics. Based on the figure, it can be concluded that both high and low frequency oscillations are successfully realized by these joints. The reference joint coordinates Ωk=IM. Moreover, each sensor model provides the measurements as the sum of the real physical quantity, bias term (Each sensor model in Equation (18) is characterized by scale factor and misalignment errors. These errors represent the imperfection of manufacturing by the corresponding matrices ocedures ,40,41.The aforementioned MARG model enables the generation of realistic raw MEMS sensor data during the motion of the 6 DOF test bench. Therefore, the effects of both external accelerations and sensor frame oscillations are appropriately represented by Magnetic fluctuations cannot be generated by Equation (18) in the simulation environment. However, random magnetic perturbations commonly occur in real world scenarios, where the magnetometer output gets disturbed by ferromagnetic objects . TherefoAn additional algorithm is utilized to generate these artificial magnetic perturbations. The algorithm calculates the output  in three steps as follows.m of length m) is produced with Equation (19), where ith waveform, Generate an artificial signal by combining five fundamental waveforms. In this algorithm, a square signal, a sawtooth wave, two sinusoidal signals and a triangle wave are combined to generate the artificial signal m) and calculate the analytic signal m is calculated, a zero vector h of length m and h is obtained [Use the artificial signal ) has been implemented with a Simulink S-function block. Each joint is controller in closed-loop; therefore, six position input effort output-type controllers have been implemented (Equation (14)) to maintain the desired joint coordinates. The PID parameters have been obtained using Equation (17). The MARG unit is connected to the end of the kinematic chain and is composed of accelerometer and gyroscope Simulink blocks, along with a custom-made magnetometer model (see Equation (18)). In the current implementation, it is assumed that the sensor models are calibrated (bias terms are zero vectors); moreover, zero scaling and no misalignment are considered in Equation (18). However, these values can easily be modified in the corresponding Simulink sensor blocks, if more advanced problems need to be examined. controller manager ROS package. Effort action-based controllers are defined to drive the joints in closed-loop. The ros controllers meta-package provides the environment to implement these low level controllers. As it is given in Equation (14), these controllers are driven with desired position inputs and supply torque outputs to the joints. The state vector joint state controller, which is a sensor controller and supplies the true states of the system continuously . The MARG unit is connected to the body plate of the test platform and is implemented with Gazebo sensor plugins [In ROS/Gazebo environment, the dynamics of the 6 DOF test bench is implemented in a Universal Robotic Description Format (URDF) file . This XM plugins . The posThis section intends to show that the proposed environment fosters the effective analysis of MARG-based algorithms. Namely, the test platform enables (i) to observe the impact of different dynamic conditions on the estimation performance, (ii) the tuning of filter parameters, (iii) to detect robustness issues, and, finally, (iv) the complete development and evaluation MARG-based algorithms.The case study demonstrates the performance evaluation and tuning of attitude filters in the following subsections. As it was discussed in the introduction, the task of attitude estimators is to provide both accurate and robust estimation results even if external disturbances  occur during the motion of the dynamical system. Therefore, the performance of these filters can be tested in such environment where the aforementioned disturbances commonly appear with different magnitudes and frequencies. The proposed test environment is completely suitable for the simulation of these system conditions.Three popular attitude filters are discussed in the analysis, namely the ECF , GDA 2020, and sThe algorithm in Reference  derives n matrix . The derThe algorithm in Reference  also useThe EKF employs the gyroscope-based quaternion propagation to obtain the a priori state estimate H denote the Jacobians of state dynamics and measurement update equations, respectively, while P is the error covariance and G is the Kalman gain. Moreover, Q and R denote the state and measurement noise covariance matrices, which mainly determine the performance of the EKF.In Equation (26), In the conducted experiments, the 6 DOF test platform executed various system behaviors in order to evaluate the filter convergences in different scenarios. External accelerations and sensor frame oscillations have been generated by supplying random desired spatial coordinates to the PID controllers of the three prismatic joints. Simultaneously, diverse sinusoidal signals as reference joint coordinates were supplied to the PID controllers of the three revolute joints to maintain a variety of sensor frame oscillations, where both the amplitude and frequency coefficients were randomly generated. As a result, the closed-loop system of the 6 DOF mechanism executed an extensive range of dynamic motions in the 3D space. Moreover, the proposed magnetic perturbation algorithm generated random pose-based disturbance events, as well. During these system motions, the test environment recorded the true system states (ground truth position and orientation of the sensor frame), along with the raw, noisy MARG sensor data. Therefore, the collected database enabled to both evaluate and tune the attitude estimator algorithms.The MARG sensor data (the output of the simulation environment) drove the attitude filter algorithms (Equations 24)–(26). The outputs of attitude filters were observed; moreover, the tuning of each filter was performed based on the heuristic techniques developed in References ,2,54. Fi4–(26). TIt can be observed that the test environment effectively fostered the tuning procedure of each filter algorithm. The initial filter outputs (pink curves) contained notable estimation errors; therefore, the default filter parameters did not contribute to an acceptable filter performance during the executed dynamic motions. However, the tuning of filter parameters could easily be performed in the environment, which resulted in improved filter performances. The effects of external accelerations, vibrations, and magnetic perturbations can also be observed in the measurement results. These external disturbances significantly decrease the performance of each filter during the first 12 s in the experiment. These effects can be handled more effectively in adaptive filter structures. The analysis of adaptive filters exceeds the scope of this article, but, in fact, the test environment aims to effectively support the development and evaluation of such novel techniques.k). Moreover, a cumulated performance index can be defined with Equation (27) to quantify the overall performance (N is the length of the measurement). The evaluation of these performance indexes is summarized for each filter in The test environment also enables the evaluation of filter convergence in terms of mean squared error (MSE) and standard deviation (STD) of attitude estimation errors  in the development of novel algorithms.This paper presented an universally applicable test environment for development, testing, and performance evaluation of MARG-based filtering techniques, such as pose estimators and classification algorithms. The test environment contains a closed-loop controlled 6 DOF dynamical system to alter the pose of an MARG unit in the 3D space and thereby simulate various system behaviors. Effort action-based joint controllers are employed to execute realistic external accelerations and vibrations by the 6 DOF platform; moreover, an artificial magnetic perturbation algorithm is applied to generate realistic magnetic disturbances. The simulation environment executes the desired dynamical motions and simultaneously supplies the true system states , along with the raw sensor measurements. As a result, the proposed environment provides effective help in the development of filtering techniques, since a big variety of system conditions can be both simulated and evaluated. The environment has been implemented in both ROS/Gazebo and MATLAB/Simulink and is publicly available online. Future work will include the analysis of advanced conditions, where the impact of static friction, sensors frame misalignment, and scaling errors will also be evaluated during the state estimation procedures.


With the development of molecular high-throughput assays (i.e. next generation sequencing), the knowledge on the contribution of genetic and epigenetic alterations to the etiology of inherited endocrine disorders has massively expanded. However, the rapid implementation of these new molecular tools in the diagnostic settings makes the interpretation of diagnostic data increasingly complex.This joint paper of the ENDO-ERN members aims to overview chances, challenges, limitations and relevance of comprehensive genetic diagnostic testing in rare endocrine conditions in order to achieve an early molecular diagnosis. This early diagnosis of a genetically based endocrine disorder contributes to a precise management and helps the patients and their families in their self-determined planning of life. Furthermore, the identification of a causative (epi)genetic alteration allows an accurate prognosis of recurrence risks for family planning as the basis of genetic counselling. Asymptomatic carriers of pathogenic variants can be identified, and prenatal testing might be offered, where appropriate.omic approaches should be embedded in interdisciplinary discussions using all available clinical and molecular data.The decision on genetic testing in the diagnostic workup of endocrine disorders should be based on their appropriateness to reliably detect the disease-causing and –modifying mutation, their informational value, and cost-effectiveness. The future assessment of data from different  Genetic disorders compose a substantial fraction of human diseases, and it is estimated that nearly 5% of live births have a genetically driven illness recognizable until the 25th year of life . HoweverMEN2 variants). This risk can also be determined in relatives of mutation carriers, and the knowledge on an inherited genetic variant is the basis to advice the patients´ family in respect of family planning and prenatal testing. Finally, the precise determination of the molecular alteration causing the endocrine disorder allows to understand its pathophysiology and thereby to develop and apply an adapted therapy.In clinical endocrine practice, genetic testing is primarily requested to confirm a suspected clinical and endocrine diagnosis, in particular in case the clinical features are ambiguous. Additionally, it also contributes to the identification of presymptomatic individuals. Thereby, their risk to develop an inherited endocrine disorder can be predicted, and prophylactic measures might be taken ) have become increasingly available for clinical use at reasonable costs, and significant progress has been achieved regarding the detection rate in human genetic diagnostic testing , 3. As ahttps://endo-ern.eu) summarizes the current role of genetic testing in the diagnostic workup of (inherited) endocrine disorders and emphasizes the chances and advantages of modern genetic tests as well as the accompanying challenges and limitations. The paper will mainly refer to molecular germline variants and congenital disorders and will not cover genetic testing of somatic variants in tumors, which requires different considerations.This position paper of the European reference networks on rare endocrine conditions  to get an up-dated overview on available diagnostic tests and therapies.Due to the permanent improvement in diagnostic testing and increasing number of genetic factors associated with endocrine disorders, this review can only provide an overview on testing strategies and available tests. The authors therefore kindly ask the readers to visit curated and public databases like orphanet  and recording of the medical history by using a standardized and curated terminology (e.g. Human Phenotype Ontology (HPO)), which helps to specify the order for genetic testing Fig. . In manyAMH gene should be sequenced, whereas analysis of the AMH-R gene is indicated in case AMH is normal/high in PMDS.An example for the need of precise clinical data as the basis for targeted genetic testing is short stature  where thThe central role of the precise molecular diagnosis as a decision aid for personalized clinical managements has meanwhile been shown for a broad range of endocrine disorders Table , rangingATP channel mutations, where biallellic mutations in either ABCC8 or KCNJ11 predict a diffuse type, while a single paternally inherited recessive mutation is highly suggestive of a focal type and may entail specific imaging and curative treatment . SRS is characterized by severe intrauterine and postnatal growth retardation, body asymmetry, feeding difficulties, relative macrocephaly at birth and characteristic facial features , but which differ from these rules. Mitochondrial inheritance is one example, as the respective disorders follow and exclusively maternal transmission pattern. Other examples of non-Mendelian inheritance are imprinting disorders like the Prader-Willi and Silver-Russell syndromes, in which the sex of the parent transmitting the molecular basic mechanism contributes to the phenotypic expression , assyndrome .Though the majority of pathogenic variants consist of pathogenic variants affecting only single nucleotides , there are further types of molecular alterations which can be associated with endocrine disorders Table . SNVs asIn the majority of known inherited endocrine disorders, the variant is either inherited and follows a classical Mendelian trait  or arises de-novo . However, in the latter case these variants as well as epimutations might arise after fertilization, and can therefore occur as somatic mosaicism, meaning that not all cells of an organism carry the variant. In case of mosaicism, the ratio of cells with different (epi)genotypes can differ considerably, as demonstrated for Silver-Russell syndrome and McCune Albright syndrome e.g. , 23)(Tab(Tab23]). In someUntil recently, the detection of genomic variants of different sizes and nature often required the application of a step-wise process due to the limitations of the tests, accordingly this procedure was expensive and time-consuming. The parallel analysis of several genes, or even the comprehensive analysis of the whole genome by NGS, is a quantum jump in routine molecular diagnostics. In heterogeneous disorders with hundreds of genes known to cause similar and overlapping phenotypes Table , these fThe decision on a genetic testing algorithm to confirm the clinical diagnosis of an endocrine disorder should consider both the range and types of pathogenic variants, as well as the appropriateness of the tests Tables , 2:MEN1 gene) or von Hippel-Lindau syndrome (VHL gene), single gene testing is recommended. In some genetically heterogeneous disorders like Beckwith-Wiedemann syndrome and transient neonatal diabetes mellitus, a step-by-step molecular analysis might be meaningful, starting with the most frequent alterations. On the other hand, this stepwise strategy might not be useful in case of a suspected disorder with a genetically heterogenous background, variable expression and/or incomplete penetrance like permanent neonatal diabetes mellitus or hypothyroidism . HoweveIn summary, specific tests targeting variants and genes will also be applied in the future in disorders with a small spectrum of mutations testing of which provides a high detection yield, whereas NGS based assays are used in genetically heterogeneous entities.The chosen assay also influences the source and amount of the patients´ sample. For conventional cytogenetics, viable cells are required, whereas most molecular tests, including NGS, are based on genomic DNA which can be principally isolated from all freshly drawn or archived tissues. However, the molecular strategies also differ in the need of DNA.With the increase of biological data ascertained by high-throughput omics technologies, the demand of databases on biological information has increased and the number of repositories is permanently growing (Nucleic Acids Research references 180 databases ). These The freely or commercially available in-silico tools to predict the pathogenicity of a genomic variant mainly rely on its biochemical, structural, and functional properties, and its evolutionary conservation across species. Recent studies that compared the performance of the major prediction tools applied in genetic diagnostic testing have revealed a diverse picture of their reliability (for review: ). TherefSegregation analysis seeking for the association of a genetic variant with the phenotype in a family is an appropriate tool to corroborate its pathogenicity.The use of NGS gene panels allows to select and target specific groups of genes, and for some disorders it is therefore the first choice of testing. In contrast, NGS-based approaches covering hundreds of unselected genes or even the whole genome might lead to the inclusion of factors in the diagnostic setting that are functional candidates. The huge number of variants obtained by exome or genome-wide approaches reflect the variability and complexity of the human genome. In fact, the majority of variants represent non-pathogenic polymorphisms which also occur in control cohorts Fig. . Thus, tThe combination of these information should support the laboratory to delineate the pathogenicity of a genomic variant. With the guidelines for interpretation of genomic variants suggested by the American College of Medical Genetics , a widelAnother challenge in the diagnostic use of WES/WGS is the handling of incidental findings, i.e. genetic alterations associated with conditions or diseases unrelated to the patient’s present condition for which current tests are being performed but with important clinical ramifications. To circumvent these putative outcomes, which are difficult to manage in routine diagnostic workup and counseling, to reduce the costs for consumables and to avoid excessive amounts of data, targeted NGS panels have been established as an appropriate tool for NGS-based genetic testing Table . HoweverLaboratories offering genetic tests should implement a quality management system . It shouWith the publication of guidelines for NGS testing, the European Society of Human Genetics has undertaken an essential step towards an international standard of NGS-based diagnostics .omic sources, future diagnostic approaches will additionally become more comprehensive [The implementation of NGS assays in DNA testing has significantly increased its diagnostic yield , 32, butehensive .The growing knowledge on the contribution of genetic factors to endocrine disorders and the rapid implementation of new molecular tools in the diagnostic settings makes the interpretation of diagnostic data increasingly complex. Therefore, the data assessment should be embedded in interdisciplinary discussions using all available clinical and molecular information. Therefore, the metabolic and hormonal assessment remain fundamental. However, WES, WGS and further NGS formats are indispensable tools to identify new pathophysiological mechanisms of human disorders and to improve diagnostic algorithms. In the future, nearly all genetic alterations will be addressable by comprehensive NGS approaches.Finally, the knowledge on the genetic cause of a disease does not only allow a precise clinical management, but it also helps to avoid invasive and expensive diagnostic tests which burden the patient, and lead to a faster diagnosis allowing an earlier and therefore more effective medical intervention (for review: ). The ea


The innate immune system constitutes the first line of defense against invading pathogens, regulating the normal microbiota and contributes to homeostasis. Today we have obtained detailed knowledge on receptors, signaling pathways, and effector molecules of innate immunity. Our research constellation has focused on ways to induce the expression of antimicrobial peptides (AMPs), the production of oxygen species (ROS and NO), and to activate autophagy, during the last two decades. These innate effectors, with different mechanisms of action, constitute a powerful defense armament in phagocytes and in epithelial cells. Innate immunity does not only protect the host from invading pathogens, but also regulates the composition of the microbiota, which is an area of intense research. Notably, some virulent bacteria have the capacity to downregulate innate defenses and can thereby cause invasive disease. Understanding the detailed mechanisms behind pathogen-mediated suppression of innate effectors are currently in progress. This information can be of importance for the development of novel treatments based on counteraction of the downregulation; we have designated this type of treatment as host directed therapy (HDT). The concept to boost innate immunity may be particularly relevant as many pathogens are developing resistance against classical antibiotics. Many pathogens that are resistant to antibiotics are sensitive to the endogenous effectors included in early host defenses, which contain multiple effectors working in cooperation to control infections. Here, we review recent data related to downregulation of AMPs by pathogenic bacteria, induction of innate effector mechanisms, including cytokine-mediated effects, repurposed drugs and the role of antibiotics as direct modulators of host responses. These findings can form a platform for the development of novel treatment strategies against infection and/or inflammation. After Alexander Fleming first discovered penicillin, several generations of different types of classical antibiotic drugs were developed. Most of these antibiotics target a specific molecule or an essential mechanism needed for survival of the bacterium. However, the targets of antibiotics can be altered by mutations, rendering them ineffective in controlling the infection. Bacterial enzymes can also degrade active antibiotics, and membrane proteins can pump the antibiotic drug out of the bacterial cell and thus prevent it from reaching the intended target. Genes encoding the degrading enzymes and the membrane pumps can be located on plasmids that are horizontally transferred between bacterial strains , 2. ExceVarious alternative strategies have been suggested, including blockage of host microbe interactions, inhibition of virulence factors, and the development of bacterial phage therapy . All theWe have developed a strategy based on direct stimulation of host cells by activation of endogenous defense mechanisms, such as antimicrobial peptides (AMPs), reactive oxygen species (ROS and NO) and autophagy, in both epithelial cells and macrophages. These are early immune mechanisms aiming to limit the spread of pathogens by killing them –8; Figur. PathogeAlternative strategies based on direct application of AMPs have been in development. First, topical treatment using modified peptides on skin infections, relying on direct antimicrobial activity have been presented . HoweverMycobacterium tuberculosis (Mtb) with a beneficial outcome , which are effective in animal models , 13. In  outcome . An addi outcome . Our aimin vitro . These cOur focus in this review will be induction of antimicrobial effector mechanisms in mucosal epithelial cells and phagocytes of the macrophage lineage.Innate immunity constitutes the first line of defense and includes specific cells that produce various effector molecules to activate mechanisms resulting in the elimination of pathogens. Different cells of hematopoietic origin constitute the effector cells of innate immunity, such as NK cells and dendritic cells, as well as the professional phagocytic cells monocytes/macrophages and granulocytes. Furthermore, the epithelial cells, while of non- hematopoietic origin, are of fundamental importance, making up a vital surface layer and working as a continuous defense barrier. In our models, the target cells of innate induction have been epithelial cells and macrophages , 14. EpiAMPs and reactive radicals, such as nitric oxide (NO) and oxygen species (ROS) with the capacity to eliminate invading pathogens are some of the effector molecules produced by epithelial cells and phagocytes. The combination of these effector systems characterized best in phagocytes, and a similar system operates on epithelial surfaces. Together these effectors work in cooperation in either an additive or a synergistic manner as an efficient armament against microbes. Notably, these effector molecules are evolutionary ancient and have co-evolved with the natural microbiota and have done so without selecting for invasive resistant bacterial strains. This is in contrast to the situation with classical antibiotics that have been only been used for several decades and their usage has selected multiple resistant strains.Innate effectors, such as AMPs, are not only included in the armament for killing pathogenic intruders but work also as modulators of cell activity. In this respect, AMPs resemble cytokines and chemokines by activating and recruiting different immune cells. Interestingly, this dual activity has been described for several AMPs, indicating a common character. Some AMPs constitute links to adaptive immunity for specific responses and memory functions. As an example, the human cathelicidin LL-37 is chemotactic to human T cells , and humIt is of importance to consider the turnover and differentiation of epithelial cells and phagocytes, since the cells have to reach maturity for full activity. Epithelial cells are derived from basal cells in the lungs  and fromNeutrophils are potent phagocytes loaded with a dazzling array of antimicrobial effector molecules. These cells, of hematopoietic origin, differentiate from the bone marrow and enter the circulation as mature phagocytes. The lifetime of the neutrophils is short in the circulation. If they get alarmed through chemotaxis by bacterial compounds, such as N-formyl-MetLeuPhe (fMLF), they will transmigrate from the blood to the site of infection loaded with effectors and exert their functions by phagocytosis of microbes or by release of antimicrobial molecules. It should be mentioned that mouse neutrophilic granulocytes do not produce alpha-defensins, thus questioning results from mouse models as responses in human with reference to studies of these AMPs .As indicated above, mature epithelial cells and phagocytes are constantly alert and ready to kill microbes by constitutive expression of genes encoding antimicrobial effectors. However, both epithelial cells and phagocytes must be activated to enhance the expression of innate immune effectors. Several pathogens suppress these immune effectors as part of their virulence in order to invade the body.The gut microbiota plays a major role in the maintenance of the overall health of the host. AMPs and additional innate effector molecules are generally produced by epithelial cells as well as by circulating inflammatory cells. The epithelial-derived AMP-based defense system has co-evolved with diverse microorganisms and has been shown to regulate the composition of the commensal microbiota , 29. ExpDifferent bacterial species thrive in the luminal milieu of the intestinal tract and the microbiota of the gut has evolved by metabolic adaptations. The microbial ecosystems produce a spectrum of metabolites within the human host with diverse functions. Many of these metabolites with specific functions have been characterized and have the ability to limit the growth of surrounding bacteria. The natural microbiota lacks virulence factors needed to break through the epithelium . In addiPathogens exhibit different metabolic adaptation compared to the natural microbiota, and several pathogens have evolved strategies, making them resistant to AMPs [reviewed in Duperthuy ]. These Shigella infection (Shigella spp. down-regulated CAP-18 (rabbit cathelicidin) in the large intestine, at the site of infection (Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) suppress LL-37 and HBD-1 expression in intestinal epithelial cells through the actions of cholera toxin (CT) and labile toxin (LT), respectively  also can downregulate CAP-18 in the small intestinal epithelia  expression in human biopsies of patients with early stage of nfection . Plasmidnfection . As a prnfection . Interesnfection . Later, ectively . A similal tract . In expepithelia , 40.These studies led to the conclusion that counteracting or blocking pathogen-mediated down-regulation of endogenous AMPs could be used for treatment of infections. Various small molecular compounds are known to induce endogenous AMPs, where immunomodulatory properties could be harnessed to treat infections. Host directed therapy (HDT) against pathogens by boosting the expression of endogenous AMPs is thus considered as a novel alternative for treatment of infectious diseases .in vitro and in vivo. By this strategy multiple peptides with different killing mechanisms together with NO and ROS would be engaged, making selection of resistant bacterial strains unlikely. These small inducing molecules can enter the blood stream and have systemic effects, as well as also boosting innate defenses at mucosal surfaces and in phagocytic cells  . The actammation .Butyrate also affects various immune cells in the gut with beneficial effects of the host, balancing inflammatory reactions to homeostasis of host microbe interactions in the gut . The speThe concentration of butyrate in the gut is 2.3–26.1 mmol/kg, while it is 1–64 μM  in the bButyrate is a foul-smelling compound and is not possible to use it as an oral drug. Therefore, we searched for alternative compounds and identified phenyl-butyrate (PBA), a drug already approved for treatment of urea cycle disorders by virtue of the ammonium scavenging capacity . ImportaClostridium difficile in a mouse model . Subsequently, we showed Entinostat to be effective in animal models for Shigella and Vibrio cholera infections , a broader effect of FXR was seen to regulate innate immunity in small intestinal epithelial cells  receptor via upregulation of Ca++ channels, resulting in a normalized lysosomal acidification .Ciprofloxacin, one of the best-studied antibiotics of the quinolone class, was developed as a potent inhibitor of topoisomerase 2 in bacteria. It is active against both gram-positive and gram-negative bacteria. It is widely used by virtue of its good and broad intracellular penetration effects and low adverse event profile. However, resistance against ciprofloxacin has emerged, and consequently the recommendations regarding empirical use of this drug have been changed. In addition, potentially serious adverse side effects are also increasingly acknowledged. These side effects include cardiac arrhythmias, tendon ruptures, and the selection for Ciprofloxacin has a direct bactericidal effect against bacteria via the SOS-response, which releases large amounts of ROS, causing DNA-damage, and bacterial death . ConsequMycoplasma spp., atypical mycobacteria and Legionella spp. It also has been extensively used as a prophylactic drug in patients with asthma and bronchiectasis. The prophylactic use of AZM was further substantiated recently in a randomized and placebo-controlled trial, where the number of asthma exacerbations was reduced, and the quality of life was increased (Azithromycin (AZM) is a macrolide antibiotic drug targeting the 50S subunit of the bacterial ribosome. It is widely used against respiratory tract infections, since it is also active against atypical bacteria, including ncreased . A Cochrncreased . The drancreased .Pseudomonas infections in the lung via direct inhibition of the inflammasome , which has an impact on the immune responses during TB-infection at several levels. One key mechanism appears to involve direct interactions between RIF and nuclear receptors. For example, it has been shown that RIF activates pregnane X receptor (PXR) that can promote TB growth in macrophages via increased efflux of TB-drugs out of the cell . This lel growth . A recenl growth . These fl growth , which cInitial innate defenses at epithelial surfaces are complex and include AMPs as important effectors. Many of the surface effectors including ROS and NO are also present in phagocytes, the second line of early defenses. The expression of AMPs is included in the differentiation program of innate immune cells but is also influenced by signals from the environment. Over the last decade, it has emerged that metabolites from bacteria and nutritional components are influential environmental mediators for the expression of the innate immune effectors. These innate components constitute an important barrier against invading pathogenic microbes, preventing them from entering the internal host environment. The strength of the barrier is decisive for keeping microbes at bay and the environmental signals are important for a tight barrier. Several pathogens suppress the expression of barrier components and thereby manage to open an entrance into the body, leading to infections. Details behind the interactions between bacteria and host cells must be resolved for each pathogen and much work remains to be done. However, powerful analytical models based on gene deletions in bacteria and host cells from different organisms are developing fast and open up for novel treatment approaches. A detailed knowledge on pathogen-mediated suppression of innate immunity and the discovery of counteracting compounds is the focus of our research. For example, inducers of innate effector mechanisms may be used alone or in combination with antibiotics. Such approaches would likely reduce the spread of resistance strains. Future research efforts on the interaction between multidrug-resistant bacteria and the host is warranted. The use of host directed therapy holds the promise to promote health and reduce the spread of antibiotic-resistant strains.PB, RR, BA, and GG planned the outline, wrote, and edited the manuscript. RSR edited and made the figures. All authors contributed to the article 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.


Given the latest updates in scientific publications about the impact of COVID-19 on oral health, such as the role of salivary glands as potential reservoirs for SARS-CoV-2 , we aimeBased on the recent data, topical and systemic corticosteroid therapy is not recommended for COVID-19 infection. Hence, positive patients with immune-mediated oral conditions  may present exacerbations of these manifestations during the period of viral infection. Correspondingly, multidrug therapies in patients positive for SARS-CoV2 and hospitalization conditions could also result in oral implications such as opportunistic infections, xerostomia, traumatic ulcerations due to orotracheal intubation, and periodontal disease .Due to the pandemic scenario of COVID-19 in Brazil and following the recommendations of the Federal Council of Dentistry  and the Aiming to bridge the gap between healthcare services and the population, telemedicine has been recognized and regulated in Brazil  as a tooThe systems available for executing telemedicine and/or teledentistry are still restricted, but represent an easy, viable, and accessible tool, useful for both health professionals and patients 9. This However, one limitation of this technology includes poor resolution of the images provided by the patient. The images must have an adequate resolution, without alterations with digital filters and must encompass the entire area of the mouth to be analyzed. These specifications will allow the correct evaluation by the oral medicine specialist. It is worth stressing that this diagnostic tool is palliative. Therefore, it is not possible to be used in all cases.Thus, telemedicine is not a substitute for face-to-face consultation, and is mainly aimed at supporting the Brazilian Unified Public Health System (SUS) during the COVID-19 pandemic. Whenever physical consultation is required after teleorientation, the health measures of the regulatory agencies must be followed.


The topics of this Special Issue included: (1) sources of natural products used in the treatment of gout; (2) isolation and characterization of natural products useful in gout control; (3) natural products which increase uric acid excretion and/or reduce uric acid absorption; (4) natural products with antioxidant and anti-inflammatory effects in gout; (5) natural products which reduce uric acid biosynthesis or enhance its degradation; (6) comparison of natural and synthetic drugs in gout treatment; (7) safety of gout control with natural products; (8) chemical modifications of natural products useful in gout control to improve their efficacy and/or safety. For this Special Issue, eleven articles were published which are briefly described as follows.Gout is a common inflammatory arthritis characterized by elevated plasmatic levels of uric acid. Several risk factors were identified, including diets rich in seafood and meat, alcohol consumption, and obesity. In this disease, when uric acid levels in the body increase due to increased formation and/or decreased excretion, the solubility limits of sodium urate are exceeded and precipitation occurs, particularly within joints, synovial fluid, and periarticular tissues. The deposits of urate crystals can initiate attacks of acute painful gouty arthritis which can evolve to chronic gout when permanent erosive joint deformity emerges. The estimated prevalence of this condition is increasing in many developed countries, particularly in men and postmenopausal women. Therefore, preventive and therapeutic strategies have been considered to avoid and treat this disease. In this context, the control of gout has been mainly performed by reducing the inflammation , as well as by reducing the uric acid formation  and by favouring its excretion . However, these approaches only have relative success and can originate several side effects, particularly in chronic use. For these reasons, over the years, researchers have been searching for alternative therapeutic strategies, particularly involving the use of natural products. Therefore, this Special Issue aimed to collect original research as well as review articles and meta-analysis addressing the use of natural products as well as extracts or isolated compounds in gout control. Accordingly, researchers were invited to contribute with manuscripts with in vitro and/or in vivo studies, including xanthine oxidase inhibition and/or in vivo antihyperuricemic effect. In some cases, a partial phytochemical characterization was performed and potential mechanisms of action were explored. Of these, several works involve natural products from traditional Chinese medicine.The majority of the published works of this Special Issue focuses on plant extracts from different regions of the world, and their potential interest in gout control was explored by Euphorbia hirta L. (Ara Tanah) aiming to improve the antigout activity of extracts from the whole plant excluding roots. For this, response surface methodology and liquid-chromatography mass spectrometry analysis were performed. The authors established the best extraction conditions for total flavonoid content, total phenolic content, and in vitro xanthine oxidase inhibitory activity as well as the optimized levels of these compounds and its bioactivity. In addition, the main phytochemical compounds in the optimized E. hirta extract were determined, also aiming to be useful for further development of antigout products.Abu Bakar et al. reported the optimization of conditions to extract phytochemical compounds from Aristolochia baetica and Aristolochia paucinervis) were explored by Bourhia et al. These plants have been largely used in folk medicine to treat several diseases, including rheumatologic conditions, namely, those caused by hyperuricemia. These authors found that the two studied plants contain different classes of secondary metabolites, including polyphenols and flavonoids. In addition, radical scavenging effects were observed as well as cytotoxicity in cancer cell lines. In an in vivo study, no signs of toxicities nor mortalities were observed on oral-treated mice with 2000 mg/kg of the two investigated exacts.The phytochemistry, antioxidant activity, antiproliferative effect, and acute toxicity of a methanolic extract of the roots of two Moroccan Aristolochia species (Paullinia pinnata (Sapindaceae), a plant used to treat various diseases including arthritis, improve monosodium urate-induced gouty arthritis in rats. In addition, they evidenced the analgesic, anti-inflammatory, and antioxidant effects of these extracts by in vivo studies. Specifically, these authors demonstrated that both extracts significantly reduced monosodium urate-induced inflammation and hyperalgesia in both ankle and paw. In addition, a significant decrease of synovial myeloperoxidase was observed as well as nitric oxide and malondialdehyde reductions in serum, spinal, and left and right hemispheres and an increase of superoxide dismutase activity.Tseuguem et al. demonstrated that aqueous and methanol leaf extracts of Dendrobium candidum leaves in rats with hyperuricemia induced by a high-purine diet were evidenced by Lou et al. In fact, after administration of this extract during 9 weeks to model rats, liver and kidney function biochemical parameters, especially serum uric acid levels, were significantly improved. In addition, a reduction of xanthine oxidase and adenosine deaminase activities in liver was observed. Moreover, according to histological analysis, this extract significantly prevented kidney and liver from damage and intestinal injury. Furthermore, a reduction in inflammation in these hyperuricemic rats by inhibiting the expression of both NF-κB and TLR4 proteins was also reported.The beneficial effects of a macroporous resin extract of Tu-Teng-Cao is a drug used in traditional Chinese medicine which has been widely applied in the clinical treatment of arthritis. In this context, Yao et al. evidenced that a Tu-Teng-Cao extract alleviates monosodium urate-induced acute gouty arthritis in rats by inhibiting uric acid and inflammation. Indeed, the treatment significantly attenuated the degree of ankle swelling, inflammation, and dysfunction index as well as the levels of proinflammatory cytokines in the joint fluid of the rat model of acute gouty arthritis. In addition, significant antihyperuricemia activity was also evidenced, and histological evaluation showed that the Tu-Teng-Cao extract relieved several signals of pathological damage. Moreover, this extract alleviated swelling, inflammation, and bleeding of the renal corpuscle and convoluted tubules of rats.in vivo and in vitro experiments. The investigators of this study observed that Huzhentongfeng could significantly suppress the paw swelling and neutrophil infiltration induced by monosodium urate intra-articular injection in rats compared with the control group. In addition, inhibition effects on the secretion of several inflammatory cytokines  were evidenced. Moreover, this product could prevent the oligomerization of ASC adapter proteins and had antioxidant effects in cell-free and cell-based tests.Huzhentongfeng is an extract from four Chinese medical herbs used in the treatment of gout. The suppressive effect of this extract on experimental gouty arthritis was explored by Wu et al. by means of α in the ankle joint. Therefore, it was concluded that Qu-Zhuo-Tong-Bi may be a promising herbal formula for the prevention and treatment of gouty arthritis in humans.Qu-Zhuo-Tong-Bi is an empirical traditional Chinese medicine prescription for the clinically treatment of acute gouty arthritis without serious adverse effects. In this context, Lv et al. studied the anti-inflammatory and analgesic effects of this product on acute gouty arthritis and the recurrent attack in model rats, as well as potential underlying mechanisms of action. These researchers observed that Qu-Zhuo-Tong-Bi extracts can suppress ankle swelling and synovial inflammation in the monosodium urate-induced gouty arthritis rat model, alleviate the acute attack, and prevent the recurrent attack of gouty arthritis. In addition, this treatment significantly decreased both mRNA and protein levels of NLRP3, as well as the production of IL-1 and TNF-The use of isolated compounds from natural and semisynthetic origin is also presented in two articles of the Special Issue.Wortmannin is a steroidal metabolite associated with several bioactivities, including anti-inflammatory effect. However, the mechanisms of action of this compound are not completely explored. Therefore, Mehran et al. presented multispectroscopic and molecular docking studies of the binding interaction between wortmannin and calf thymus DNA. Considering the observed results, these authors suggested that this molecule may exert its biological effects, at least in part, via interaction with DNA. In addition, it was demonstrated that wortmannin interacts with this biomolecule in the non-intercalating mode, which is considered as a new mechanism of action.in vitro xanthine oxidase inhibition and in vivo hypouricemic effect on animal models. The analysis of data collected from the past six years demonstrated that a large number of compounds are being explored in in vitro studies and also in in vivo biological evaluations, and very promising molecules are being developed. In addition, it was verified that molecules from natural sources or their mimetics and semisynthetic derivatives constitute the majority of compounds being explored at the moment by means of in vitro and in vivo studies. Furthermore, as frequently the observed in vitro XO inhibition results do not have a clear correspondence with the in vivo hypouricemic effects, it is necessary to perform other complementary studies to better establish the pharmacological profile of these molecules under development.A review article, from Serrano et al., presented an integrated overview of recent studies with focus on isolated molecules with Finally, the analysis of existing clinical data on the use of natural products in gout control was also explored in two review articles.The effectiveness of cherries in reducing uric acid and gout was studied by Chen et al. in a systematic review of the clinical evidence in this point. After application of the search strategy, six clinical studies reporting decreases in the incidence and severity of gout following the ingestion of cherries or products containing cherries were included in this review. Despite the described positive effects of cherry intake in gout, it was not possible to conduct a meta-analysis due to the lack of relevant studies and to the high degree of variation in the methodologies and metrics used in the studies included in this review. Therefore, additional more rigorous and larger clinical trials are needed, including long-term follow-up studies.A systematic review and meta-analysis of randomized controlled trials on natural product dietary supplements on patients with gout was presented by Yang et al. After application of the search strategy and establishment of the clinical outcomes, nine randomized controlled trials were included in the review, and meta-analysis concerning the efficacy and safety of these supplements were performed. The authors observed that natural product dietary supplements appeared to be superior to control groups in affected joint pain, swelling, and activity limitation, while not in decreasing seric uric acid and C-reactive protein levels or the incidence of adverse effects. However, due to poor trial quality and the absence of standardized evaluation criteria, the current existing evidence is insufficient to allow a definitive statement about the efficacy and safety of these supplements. Hence, further larger and more rigorously designed randomized controlled trials are clearly needed in the future.Thus, with contributions from research groups from diverse countries, this Special Issue presented recent experimental findings and reviews on natural and semisynthetic products with relevant potential for the prevention and treatment of gout.


Similarly, neuronal activity in the PVT contributes to depression-like behavior, through yet undefined subregions. The posterior PVT in particular demonstrates a role in anxiety-like behavior, generally promoting but also inhibiting this behavior. This subregion is also especially responsive to stressors, and it functions to suppress the stress response following chronic stress exposure. In addition to participating in unconditioned or primary emotional responses, the PVT also makes major contributions to conditioned emotional behavior. Neuronal activity in response to a reward-predictive cue can be detected throughout the PVT, and endogenous activity in the posterior PVT strongly predicts approach or seeking behavior. Similarly, neuronal activity during conditioned fear retrieval is detected in the posterior PVT and its activation facilitates the expression of conditioned fear. Much of this involvement of the PVT in arousal and valence has been shown to occur through the same general afferents and efferents, including connections with the hypothalamus, prelimbic and infralimbic cortices, nucleus accumbens, and amygdala, although a detailed functional map of the PVT circuits that control emotional responses remains to be delineated. Thus, while caveats exist and more work is required, the PVT, through its extensive connections with other prominent nuclei in the limbic system, appears to be an integral part of the emotional processing network.The paraventricular nucleus of the thalamus (PVT) has for decades been acknowledged to be an important node in the limbic system, but studies of emotional processing generally fail to incorporate it into their investigational framework. Here, we propose that the PVT should be considered as an integral part of the emotional processing network. Through its distinct subregions, cell populations, and connections with other limbic nuclei, the PVT participates in both major features of emotion: arousal and valence. The PVT, particularly the anterior PVT, can through its neuronal activity promote arousal, both as part of the sleep-wake cycle and in response to novel stimuli. It is also involved in reward, being both responsive to rewarding stimuli and itself affecting behavior reflecting reward, likely  While the paraventricular nucleus of the thalamus (PVT) has for decades been acknowledged to be an important node in the limbic system . While the anterior and posterior halves of the PVT both appear to be involved in these behaviors, their relative contributions to each behavior demonstrate significant variation, suggesting that there may be a gradient of cells across the antero-posterior PVT axis that participate in them.The PVT shows a clear role in arousal, with slightly more evidence being generated from research investigating the anterior PVT than the posterior PVT. In work on circadian arousal, early research in rats demonstrated that levels of c-Fos  are increased in the PVT in the dark (active) phase relative to the light phase  or stimulation of the middle PVT . Conversely, in response to purportedly rewarding stimuli, including access to a female conspecific  or thermoneutral zone, dopamine D2 receptor-expressing PVT neurons, which in the mouse are enriched in the posterior PVT, show a decrease in activity, as measured by calcium transients  axis (adrenocorticotropic hormone (ACTH) and corticosterone) to restraint stress, following repeated exposures to this stressor . While the anterior and posterior halves of the PVT both appear to be involved in these behaviors, their relative contributions to each behavior again demonstrate significant variation.fos is increased in the anterior but not posterior PVT to a food-predictive cue in rats that have attributed incentive salience to the cue (Flagel et al., Research on conditioned reward and motivation suggests that while the entire PVT is affected by and can affect the behavioral response to reward-predictive cues, activation of the anterior PVT generally inhibits motivated or seeking behavior while activation of the posterior PVT may instead promote it (but see Otis et al., via projections to the nucleus accumbens shell, as the inhibition of lever-pressing from PVT photoactivation is recapitulated by photoactivation of the anterior PVT-to-nucleus accumbens shell pathway and reversed by its photoinhibition (Do-Monte et al., In contrast to research on endogenous neuronal activity, research using experimenter-induced changes in neuronal activity suggests that approach behavior is affected by the anterior rather than the posterior PVT. Cue-induced sucrose magazine entries are not affected by chemogenetic inhibition of both anterior and posterior rat PVT (Choi et al., via a prelimbic-to-PVT-to central amygdala pathway. In turn, activation of the PVT and this pathway appears to facilitate the expression of conditioned fear following this same delayed timeline. Freezing to a conditioned tone during a fear retrieval test conducted 24 h but not earlier after conditioning is diminished by pharmacological inhibition, using microinjection of muscimol into the rat dorsomedial nucleus which spreads into the PVT (Padilla-Coreano et al., via the central amygdala, serves to promote the expression of conditioned fear.The PVT also shows a clear role in conditioned fear, particularly fear retrieval, with significant research on this topic focused on the posterior PVT. Neurons in the PVT show increased activity to a conditioned fear tone, but only starting more than 6 h following tone-shock pairings, and lasting at least 7 days. For example, in the rat PVT, more neurons show electrophysiological responses to a conditioned fear tone tested 24 h but not 2 h after conditioning, and levels of c-Fos are elevated in a fear retrieval test conducted 7 days but not 6 h following conditioning (Do-Monte et al., While much progress has been made in delineating the functional contribution of the PVT to emotional processing, some of the data as a whole remain equivocal, and, as such, many outstanding questions remain. The contribution of the specific PVT subregions does vary, based on the particular behavioral test employed and even within the same assay, suggesting that the exact role of the PVT may depend less on subregion and more on cell type. Very recent research has demonstrated the existence of antero-posterior gradients of two well-defined neuronal subtypes (Gao et al., via specific populations of cells distributed throughout its subregions. Similarly, PVT neuronal activity appears to affect depression-like behavior, through yet undefined subregions and cell populations. The posterior PVT in particular also demonstrates a role in anxiety-like behavior, generally promoting but also inhibiting this behavior. While this subregion is also especially responsive to stressors, it appears to function to suppress the stress response following chronic stress exposure. Following conditioning, the posterior PVT again plays a major role in emotional behavior. Neuronal activity in response to a reward-predictive cue can be detected throughout the PVT, but endogenous activity in the posterior PVT is a robust predictor of approach or seeking behavior. Similarly, neuronal activity during conditioned fear retrieval is detected in the posterior PVT and its activation appears to facilitate the expression of conditioned fear. Much of this involvement of the PVT in arousal and valence has been shown to occur through the same general afferents and efferents; however, a detailed functional map of the PVT circuits that control emotional responses, particularly those involving the posterior PVT, remains elusive. Afferents from the hypothalamus affect the involvement of the PVT in arousal, anxiety-like behavior, and the response to stress, and afferents from the prelimbic cortex affect its involvement in the expression of conditioned fear. In turn, through efferents to the nucleus accumbens, the PVT affects arousal, reward-related behavior, anxiety-like behavior, and motivation, and through efferents to the amygdala, it affects these same behaviors as well as the expression of conditioned fear. Finally, the PVT also affects arousal through efferents to the infralimbic cortex. It may be that some of these projections originate from the same PVT cells, but that remains to be determined. Thus, while caveats exist and more work is required to define its exact role, the PVT, through its extensive connections with other prominent nuclei in the limbic system, appears to be an integral part of the emotional processing network.On the whole, however, evidence supports a strong role for the PVT in multiple aspects of emotional processing, demonstrating that it is both responsive to and itself affects both arousal and valence . The PVTJB, NM, and W-JG wrote and edited the manuscript. All authors contributed to the article 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.


Organelles are involved in many cell life activities, and their metabolic or functional disorders are closely related to apoptosis, neurodegenerative diseases, cardiovascular diseases, and the development and metastasis of cancers. The explorations of subcellular structures, microenvironments, and their abnormal conditions are conducive to a deeper understanding of many pathological mechanisms, which are expected to achieve the early diagnosis and the effective therapy of diseases. Organelles are also the targeted locations of drugs, and they play significant roles in many targeting therapeutic strategies. Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical tool that can provide the molecular fingerprint information of subcellular compartments and the real-time cellular dynamics in a non-invasive and non-destructive way. This review aims to summarize the recent advances of SERS studies on subcellular compartments, including five parts. The introductions of SERS and subcellular compartments are given. SERS is promising in subcellular compartment studies due to its molecular specificity and high sensitivity, and both of which highly match the high demands of cellular/subcellular investigations. Intracellular SERS is mainly cataloged as the labeling and label-free methods. For subcellular targeted detections and therapies, how to internalize plasmonic nanoparticles or nanostructure in the target locations is a key point. The subcellular compartment SERS detections, SERS measurements of isolated organelles, investigations of therapeutic mechanisms from subcellular compartments and microenvironments, and integration of SERS diagnosis and treatment are sequentially presented. A perspective view of the subcellular SERS studies is discussed from six aspects. This review provides a comprehensive overview of SERS applications in subcellular compartment researches, which will be a useful reference for designing the SERS-involved therapeutic systems. Subcellular compartments, including cell membrane, nucleus, mitochondrion, lysosome, and endoplasmic reticulum (ER), etc., are the essential functional subunits within eukaryote cells Given the essential roles of these cellular compartments in cellular activities, it can be speculated that any dysfunction or disruption of the structures and functions of subcellular compartments can result in abnormal intracellular homeostasis and various cellular disorders. For example, disturbances to the nucleus may lead to the abnormal regulation of cell activity and even cause cell death. Various diseases, such as cancers, heart disorders, and brain disorders, are also related to nuclear dysfunction Besides, targeting therapeutic agents at specific subcellular organelles is the key to maximize the therapeutic index, completely eradicate tumors, and prevent invasion, metastasis, and recurrence. In the context of cancer therapy, the biggest bottleneck is how to deliver the drugs to the lesion locations, since the simple transportations of therapeutic agents into the tumors or cancer cells are not efficient and enough to yield the expected therapeutic outcome in situ characterization techniques have been involved due to the merits of monitoring the dynamic changes of a living cell. Most of them are microscopic techniques since the cells and bacteria are in a micrometer size range. Established microscopic technologies, e.g., electron microscope At present, the organelle-related analyses mainly focus on two aspects: (1) the detections of structures and compositions of organelles, and (2) the explorations of microenvironments of organelles  and the dynamic changes of their structures and microenvironments during therapeutic processes. Traditional subcellular compartment analytical methods are protein immunoblotting Raman spectroscopy is one kind of vibrational spectroscopies, which can provide native and rich chemical information of cellular compositions, including nucleic acids, proteins, and lipids. Compared to broad fluorescence bands, Raman bands are narrow and easy to identify. Besides, water has fewer impacts on the Raman spectra. Therefore, compared to infrared spectroscopy, Raman spectroscopy is more suitable for the detections of biological systems.7 incident photons, limiting its application in biofields. One of the remarkable achievements in the developing history of Raman scattering is the discovery of SERS, first reported by Fleishmann et al. in 1974 However, Raman signals are too weak to be detected because only one Raman photon can be excited out of 10In this review, SERS as a useful analytical tool in subcellular components based detection and therapy were retrospected and summarized Scheme . Two comSERS has gained ever-increasing attention, recently focusing on cellular studies. This high interest arises mainly from the uniqueness of SERS (molecular specificity and great sensitivity two-in-one) and highly matching between the SERS specialties and the demands of cellular/subcellular investigations. SERS exhibits applicability in subcellular detections from the following aspects.(1) SERS has an acceptable spatial resolution level for main cell compartments. The cells and prominent organelles, such as cell nuclei (1-20 μm), mitochondria (0.5-1 μm), and lysosome (less than 1 μm), are all at the micrometer level via the commercial confocal Raman microspectrometers. SERS micro-imaging not only can provide two-dimensional information, but also can reveal surface, interface, or even layer-by-layer information of cells and organelles, enabling three-dimensional chemical structure reconstruction (2) The localized field enhancement effect allows for selective measurements of aimed locations. SERS is a highly surface-dependent effect  in-situ way.(3) SERS can explore living cells in an SERS features the common points of optical approaches that are non or less invasive and show minimum biological sample damage Subcellular SERS studies follow the conventional SERS detection strategies, which can be sorted as direct/indirect measurements. In some descriptions, they are mentioned as label-free/labeling methods, as shown in Scheme The label-free method discloses the molecular vibration information of analytes by placing them near the SERS substrates, or the analytes directly contacting the SERS substrates Besides the label-free SERS, another SERS analytical strategy is the SERS labeling method. The SERS reporters and the recognition ligands are modified on the surface of plasmonic NPs, and these integrated complexes are called SERS tags or SERS labels The quantitative sensing mechanisms of SERS nanosensors are mainly cataloged in two types: (1) intensity (ratio)-dependent SERS nanosensors. By constructing or destroying the hotspot structures of the targets, or the signal changes relative to the internal standards, the “SERS on” or “SERS off” is entirely dependent on the concentration of analytes SERS labeling methods exhibit broad usage in multi-component detection and imaging For subcellular targeting researches, delivering the SERS-active nanoplatforms into specific subcellular compartments is one of the most critical steps. For nanomaterials internalization, phagocytosis and pinocytosis are two major cellular internalization mechanisms for exogenous NPs. Most particles will arrive in the lysosome or cytosol eventually. To help the SERS-active nanoplatforms arriving in our interested organelles, so far, two main strategies have been employed and developed to deliver nanocarriers to specific subcellular organelles, noted as passive targeting and active targeting mechanisms.Passive targeting refers to the selective accumulation of NPs or nanosystems in specific organelles due to the physical, chemical, and pharmacological factors of the organelles  and the inherent properties of NPs, including size, shape, and surface charge. For example, small molecules such as water and some ions can freely enter and exit the nucleus since the nuclear pores on the surface of the nuclear membrane are about 9 nm in size Developing universal targeting strategies that are independent of the inherent natures of NPs and organelles is in great demand. Active targeting by modifying targeting ligands such as aptamers and targeting peptide sequences on the surface of the nanomaterials has been extensively exploited, in which the specific targeting peptide sequences are the most commonly used targeting ligands. The surface decoration of the cell-penetrating peptide  is helpful for the internalization of nanomaterials. RGD is known to target αvβ6, an RGD receptor, as well as other αv integrins on the cell surface and assists the receptor-mediated endocytosis in cancer cells Shi's group reported a variety of nuclear targeting nano-delivery systems by conjugating the nucleus targeting peptide on the surface of the nanomaterials for cancer targeting therapy In order to obtain the mitochondrion-targeting, the mitochondrion-targeting peptide (MLALLGWWWFFSRKKC) At present, the modification of nanocarriers with intelligent response-type lipophilic amines is the most effective strategy for realizing the long-stay of nanocarriers in the lysosome and prohibiting the nanocarriers from escaping from lysosomes. Lysosomes give an acidic microenvironment, which makes amino groups protonated. Thus, NPs with the protonated amino groups easily penetrate the lysosomal membrane, resulting in abundant accumulation of NPs in lysosomes. For other organelles except lysosomes, the microenvironment is in a neutral condition, which can't trigger the protonation reactions, and thus the lysosome-selective targeting is realized. 4-(2-hydroxyethyl) morpholine and N, N-dimethylethylenediamine are two commonly used lysosomal targeting groups, and both of them have been used for the surface decoration of nanomaterials in vivo cell SERS measurements Optical fiber tip Since these internalized MNPs are exogenous matters for cells, they will bring certain interference to the cells. Commonly, for the intracellular SERS measurements, the AuNPs are more acceptable than AgNPs for the consideration of better biocompatibility of Au, especially for the nanotherapeutic experiments in living bodies. So far, Au nanorods (AuNRs) The concentration of the SERS-active nanoprobes should be assessed before intracellular SERS measurements, by testing the cellular viability after the cells co-culture with nanoprobes for different time . The cell viability can be simply determined by using the MTT -2,5-diphenyltetrazolium bromide) or WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5--2H, tetrazolium monosodium salt) etc. assays, in which the cells were properly metabolized by a dye. They undergo visible color changes that are monitored by spectrophotometer, while the cells incapable of metabolizing the dye remain colorless. Also, flow cytometry is another powerful tool for cell viability tests. Taking spherical AuNPs as an example, over 85% of cellular viability based on the MTT assay is considered low/no toxicity and widely acceptable for intracellular experiments. Above 90% of cell viability is preferred. Otherwise, they will cause severe cell apoptosis. However, in order to achieve high SERS sensitivity, more SERS nanoprobes will be helpful. Thus, a trade-off concentration of nanoprobe is expected.SERS tags are usually functionalized by both Raman reporters with distinguished signatures and the functional coating layers that endow the SERS tags with biocompatibility/stability and/or targeting bioconjugation. In many reported SERS nanoprobes, the surface coating of polyethylene glycol (PEG) is preferred, which offers protection from the chemical and physical environments, minimizing non-specific binding and prolonging blood circulation lifetimes in living body experiments The addition of functional coating molecules onto the NP surface tends to affect colloidal stability and brings irreversible NP aggregation, especially in the centrifugation step. This issue also limits the large-scale SERS tag synthesis. How to avoid the colloid aggregation during the SERS nanoprobe preparation is very skillful and bothered each beginner. Thus, the ligand amount must be strictly controlled, and the surface decoration of colloids needs well designed.The surfaces of MNPs are active for the unspecific bindings of uninterested molecules. To avoid these SERS background signals, commonly used biocompatible protective layers - silica or bovine serum albumin (BSA) coatings Similar to the photobleaching of the fluorescent dyes used for imaging and bio-labeling, the SERS nanoprobes also face the fading problem. The irregular variation or fluctuation of SERS signals may cause the SERS blinking effect, which is the typical behavior of a single molecule caused by the diffusion effects of analytes (Brownian diffusion) A silica shell can also be a protective layer to maintain colloidal stability and prevent interaction with the external environments. The primary strategy for silica shell decoration is achieved by the mercaptotrimethoxysilane or aminopropyltrimethoxysilane (APTMS) and then reacted with sodium silicate or tetraethylorthosilicate. A remarkable example is the shell-isolated NPs . In the absence of Au cages, the cells could alive under the laser power of 6.0 W/cm2 for 5 min, but they started to die at a laser power of 1.5 W/cm2 if the Au cages exist. In order to avoid the photodamage on the biosamples, a defocusing way can be applied to minimize the effects of laser damage if the vertical spatial resolution can be ignored The local photothermal energy not only burns the Raman reporters but also directly damages the cells and tissues during the SERS measurements. Though the photodamage effect of MNPs can be utilized for killing cancer cells according to their large absorption cross-sectional area of near-infrared light, in most SERS measurements of living cells or bodies, photodamage is undesirable. Laser power should be optimized for safety. Lim et al. In this section, more detailed examples applying SERS label-free and labeling technologies for subcellular compartment detections will be provided, including subcellular macromolecular profiling, subcellular imaging, subcellular environments, cell membrane detections, and isolated organelles.-1  to the target locations. The surface of the cell membrane is rich in receptors and targets. In 2010, Yan et al. obtained the SERS spectra of the cellular surface of the tumor and nontumor cell lines under the assistance of the drop-coating AgNPs on a silicon wafer  NLS peptide (CGGPKKKRKVGG) on the AuNRs through a thioalkyl-triazole linker To obtain the SERS information of the entire mitochondria, Karatas et al. isolated mitochondria from A549 lung cancer cells and mixed them with AuNPs to obtain the SERS spectra of mitochondria Nanomaterials enter cells through the clathrin-mediated endocytosis pathway, and they will accumulate in the lysosome or cytoplasm at the first stage To simultaneously observe and compare the molecular information of different organelles, Xu and Liang et al. designed and prepared the nuclear, mitochondrial and lysosomal targeting AuNR nanoprobes for recording the SERS spectra of three organelles  that can specifically identify the target proteins were then used for amplifying SERS signal by forming nanoaggregates. This method was used for in situ imaging of sialic acid (SA) on the target protein epithelial cell adhesion molecule (EpCAM), as well as their dynamic changes during drug treatment Via the plasmonic coupling of these two kinds of nanoprobes in a single core-multi satellite nanostructure, they realized the sensitive SERS imaging of SA on the surface of living cells. Moreover, they also designed a micro-competition system for the simultaneous quantification of multiple glycans on cell surfaces with SERS The expression of the cell surface species, such as glycoproteins and glycolipids, can be the indexes of various diseases. in situ study sialoglycan levels in different cell types and the dynamic expression processes of them under the physiological conditions via the MPBA@AgNPs SERS nanosensor Based on the recognition of the benzoic group with SA, Xu et al. developed a 4-mercaptophenylboronic acid (MPBA)-functionalized AgNP-based SERS nanosensor to in situ detections of the subcellular microenvironments will facilitate the understanding of the mechanisms of subcellular related diseases during the occurrence and development processes. In this section, we will introduce some recent progress in SERS tag-based subcellular microenvironment sensing, especially intracellular pH detections.Subcellular microenvironments such as pH, the content of ROS, biomolecules, and redox potential are important parameters for ensuring normal cell growth, proliferation, and others In 2020, Choi et al. reported an efficient SERS-based immunoassay technique for protein quantification and imaging. Their method can reliably estimate the protein level at a sub-picomolar detection limit A series of SERS nanosensors with high sensitivity and organelle-targeting ability have been developed for monitoring the subcellular pHs via the aptamer hybridization in vitro labeling detections. Gogotsi et al. in-situ determination of the molecular composition of the organelles. Compared with the intracellular SERS applied by nanoprobes, the nanotip-based method can arrive at interesting locations. However, since the micromanipulation under the microscope has limited imaging resolution and low contrast for other organelles except for the cell nucleus, these optrodes are only applicable for detecting cell nucleus, cytoplasm, and extracellular regions.In addition to those nanoprobes that should be internalized in cells for subsequent subcellular environment analyses, many needle-like optrodes and nanopipettes were utilized for tracing the single-cell low-copy proteins and components. Wang et al. decorated the outer surface of glass nanotubes with Au and Raman-active molecules to detect cancer biomarker, free iron ions and hemoglobin in a single cell in situ SERS, there is still a problem unsolved on the difficulty to assign these bands because the intracellular biological environment is incredibly complicated, and there are some interference signals from other biomolecules. Therefore, some ex-situ strategy has emerged for structural analysis and quantitative analysis of the isolated organelles.Although the rich and dynamic biomacromolecular information can be obtained by in situ with the assistant of mitochondrion-targeting nanoprobes, compared with the isolation-based ex situ method Culha et al. investigated the feasibility of SERS to understand the mode of interaction of AuNPs with the extracted mitochondria. They found that the AuNPs did not cause significant damage to mitochondria  in situ SERS strategy to selectively measure intracellular biomolecules was compared with the ex-situ SERS strategy Nucleus is a place of genetic material storage and possesses a high-density place in one cell. Cytobiological methods for cell nucleus related studies start with the extraction processes of intranuclear components with many cell lysis buffers, following by the structural characterizations and quantitative analysis of the extracted components. TheAt present, with a deeper understanding of the structure and function of organelles, the designs of the organelle targeting platforms based on chemotherapy, photothermal therapy (PTT), photodynamic therapy (PDT), and other multi-mode therapeutic platforms have attracted much attention. The most significant advantage of SERS technology in this field is that it can realize the real-time monitor of the biomolecular changes and the quantitative assay of small molecules in living cells.It is unique to look at the process and mechanism of disease treatments from the organelles' Ramanomics. In 2013, the El-Sayed group developed a nuclear targeting plasmon-enhanced single-cell imaging technology to study the molecular changes of the nucleus, before and after the cells were treated with anticancer drugs in situ SERS Moreover, the therapeutic plasmonic SERS nanoprobes with organelle-targeting features were designed to study the different responses of cancer cells and normal cells upon to PTT. The nanoprobe composed of porous Ag/Au nanoshells and carbon dots, was prepared for the PTT of cells  at organelle level (nucleus and mitochondrion) to assess the heat tolerance mechanism by In addition to PTT, changes in the SERS spectrum of the nucleus during PDT were also investigated. The B16 cell line (a murine melanoma cell line) was chosen. The results evidenced the protein degradation and the DNA fragmentation in the therapeutic process, which is meaningful not only for exploring the PDT molecular mechanisms but also for the assessment of the dosage of photosensitive reagents Besides phototherapies, electrical stimulation is another clinically permitted therapeutic mode. Its curative effect was also revealed by subcellular SERS, reported by Jin et al. Chemotherapy is an important way for tumor treatments. Xu et al. used AuNRs-based nuclear targeting probes to study the effects of DNA targeting molecules (Hoechst33342) and anticancer drugs  on the nucleus In addition to directly detecting the changes of biomolecules in organelles, detecting the intracellular microenvironment during treatments can help us better understand the therapeutic mechanism and the important role of organelles in the therapeutic processes. In 2016, Yang's group used the AuNRs with the decoration of pH-responsive molecules and a layer of the peptide as a protective layer to carry out the SERS detection for lysosomal pH during PTT -1), which eliminates interference possibility from cellular molecules. By using these Au@Ag NPs, the ROS dynamic changes at mitochondria during the PTT process could be revealed by the Raman mapping. Results show that after irradiation by an 808 nm laser, the SERS intensities of the peak at 2226 cm-1 gradually decreased due to the ROS production during the PTT process. This study also demonstrated that during the PTT process, the ROS-induced cell death is a major reason in comparison to the heat-induced cell death.Besides the pH environment, ROS changes are also crucial for the understanding of the mechanism of PTT and PDT. Recently, an interference-free SERS-active nanoprobe for intracellular ROS detection was developed by Xu et al As a powerful spectroscopic tool, SERS has been employed to detect the organelles and components at the subcellular level, and reveal the action mechanisms of some therapeutic means through the molecular fingerprint information. By integrating the diagnosis and therapy, SERS nanotags could track the release of the drugs in cells and realize tumor treatment at the same time.via the environmental pH changes during cell endocytosis In 2016, Cui et al. fabricated a multifunctional nanocarrier for tracking the intracellular Dox release process based on the acidic environment in lysosomes, achieving the killing effect on cancer cells. Furthermore, the detailed release process and the carrier distribution were recorded by SERS  in vivo treatment of tumors in-situ SERS reporting glucose level in cells based on a ''turn-off'' mechanism (intensity decrease) of the Raman reporter (MBN) induced by the H2O2 etching effect on the AgNPs. Thus, the enzymatic activity of GOx and the degradability of the nanoreactor in an acidic environment can be monitored; that is, this nanosensor can immediately feedback the curative effect by SERS In addition to the implementation of tracking drug delivery by SERS, some cancer cell treatment strategies with SERS tags were also developed to support therapy or feedback therapeutic efficacy. For example, Wang et al. prepared a novel traceable and targeted drug delivery nanosystem based on Au nanoflowers (AuNFs). This nanosystem has a high drug encapsulation capability and an intra-lysosome pH-controlled release feature. The AuNF-based nanocarriers were applied for efficient intracellular SERS imaging-guided chemo-phototherapy, showing great potential in facilitating the accurateThis review summarized recent progress in subcellular compartment studies using SERS as a powerful analytical tool. The applications of the SERS-based detection schemes in subcellular structures studies were reviewed Table , includiLooking back on the above publications, people have made progress in many aspects; however, many efforts should be exerted no matter on the bio-SERS experimental aspects or on the extensive SERS applications in the subcellular studies, because this subject is full of challenges and many technical problems still exist. Many new technologies are involved in Raman analytical methods, and their integration will bring a brighter future for intracellular SERS studies. In the following, we listed six unsolved problems in the intracellular SERS experiments. According to these problems, a perspective for future studies is given. And some of them are already on the way to reality.SERS tags and nanoprobes are all plasmonic MNP-based. So, to prepare SERS nanoprobes, we firstly synthesize metallic Au or Ag NPs. The shape and size are different in trials, which is one of the trouble issues leading to the poor repeatability and low accuracy of the SERS results. A robust method to synthesize monodisperse MNPs with high reproducibility and stability is in great demand. Except for many parameters, such as reagents, temperature, pH, concentration, charging sequence and mixing time, etc., the synthesis method is also essential. The seed-growth method in vivo due to SERS tags showing excellent signal intensity. Some organelle targeted-based drug release methods are also designed based on this point The SERS-based platform has been successfully used in the diagnosis and treatment of cancer in situ hybridization methods, have been successfully applied for illuminating genome-scale and transcriptional activity in a single cell by fluorescent imaging Because the targeting ability of the nanoprobes is one of the key issues for ensuring the reproducibility and accuracy of intracellular SERS, more specific targeting ligands and techniques are required to reduce the off-target effect of the nanoprobes. This progress often depends on the discoveries in bionics and the imitation of physiological processes such as viruses and bacteria. Recently, many precise diagnosis and medical methods, for example, gene-editing technique, click chemistry approach and fluorescence There are still too few molecules available for intracellular detection. Compared with fluorescent sensing molecules, the choice of responsive Raman reporting molecules is very limited, especially in the detection of small molecules in cells. Also, it is difficult for small molecules to trigger an effective signal amplification mechanism. So, it is still challenging to sense small intracellular molecules, as well as cellular metabolites. More efforts are required to involve in in the synthesis of sensing probe molecules and Raman reporters.Since the limited surface area of a SERS-active NP, except giving the signal reporter with a larger Raman scattering cross-section and preferably having a band in bio-window range, the Raman reporters are expected to endow multi roles, for example, the bridging linker for identifying elements, the hydrophilic/hydrophobic regulator, the surface blocker and protector, and the modular for the plasmon feature of NPs, etc. The binding ways to metal are not limited to thoil, amino, and carboxyl groups anymore. In a recent study, Li and Tang et al. 2/sr, Raman-based subcellular imaging requires a relatively long exposure or integration time, making it difficult to image a subcellular compartment whose cellular changes can occur on the scale of milliseconds. The current SERS imaging speed seriously limits the detection in a high temporal resolution. Some promising improvements were achieved by using highly bright SERS nanotags. In 2019, Ye et al. 9, the detection sensitivity down to a single-NP level, high-resolution cell imaging within 6 s and high-contrast. These nanoprobes may open new opportunities for rapid ultrasensitive biosensing and bioimaging. In addition to the highly bright SERS nanotags, Brolo's group Due to the Raman scattering cross-section of the molecules is about 10-30 cm11/cm2, 20 to 35 nm gap), Dana et al. Besides the imaging speed, spatial resolution is always pursued by researchers. Supra-resolution fluorescence imaging technology has become influential in visualizing the organelles and their interactions. Inspiringly, supra-resolution SERS imaging is already on the way. Using a high-density, uniform hot spot substrate (hot spot density 10Establishing a consolidated SERS database is imperative toward the practical application of SERS. Most subcellular SERS studies are currently based on the workgroup-specific database obtained under different technical and biological parameters or the experience summed up by the predecessors for cellular analysis. This hinders validation in large-scale studies and implementation in clinics. It can't work for the information of unknown samples, or selectively identifying molecular changes resulting from cellular processes in large and multidimensional data sets. Following SERS methodology standardization for subcellular studies, the most critical step is to establish a consolidated subcellular biomolecules SERS database. This database will provide the comprehensive spectral profiles of organelles, including the data of subcellular structures in different cells and the same subcellular compartment at different physiological states, for instance, in response to various stresses. It will be a reliable tool for the identification, discrimination of subcellular compartments, in-depth deciphering of mechanism, and even prediction.via label-free SERS strategy, reasonable analysis and processing of the obtained data is still a challenge, no matter in the cellular level or in the sub-cellular level. Therefore, combined with advanced algorithms for demultiplexing or decoding the spectra of components will be promising in the near future, e.g., PCA-LDA Although the sensitivity of Raman spectroscopy is sufficiently high to identify significant cellular macromolecular information and their variations existing between different cellular samples and different cellular processes