In this review, we highlight the recent findings about this non-canonical sensory mechanism, as well as its involvement in metabolic functions and bacterial motility. Additionally, we explore some of the specific proteins and protein-protein interactions that mediate protein activity sensing and their downstream effects. The complex sensory activities covered in this review are important for bacterial navigation and gene regulation in their dynamic environment, be it host-associated, in microbial communities or free-living.Bacteria have evolved complex sensing and signaling systems to react to their changing environments, most of which are present in all domains of life. Canonical bacterial sensing and signaling modules, such as membrane-bound ligand-binding receptors and kinases, are very well described. However, there are distinct sensing mechanisms in bacteria that are less studied. For instance, the sensing of internal or external cues can also be mediated by changes in protein conformation, which can either be implicated in enzymatic reactions, transport channel formation or other important cellular functions. These activities can then feed into pathways of characterized kinases, which translocate the information to the DNA or other response units. This type of bacterial sensory activity has previously been termed  Bacteria face constant challenges in their changing environments. Nonetheless, bacteria can adapt and ensure their survival by employing several strategies. They can modify their cellular activities and, for instance, suppress the synthesis of certain proteins, or in turn they can produce enzymes that are required to metabolize available nutrients. Additionally, some bacteria can transition into more resistant phases  in the absence of appropriate nutrition . AnotherVast repertoires of sensory systems that mediate these diverse and temporally distinct responses have evolved in bacteria. Well-characterized kinase-dependent systems important for sensing and signaling are two-component systems (TCSs), consisting of a sensor kinase or multi-kinase networks  and a coCanonical TCSs comprise one or several regulatory kinases and a cognate response regulator, which is phosphorylated upon sensor activation . CanonicWe would also like to mention quorum sensing as one important subsystem of bacterial sensory activity. Quorum sensing is mediated by autoinducer (AI) molecules in concert with specific receptors, which allow bacteria to communicate in a population [summarized in two recent reviews ]. The fiprotein activity sensing . These co-regulatory processes frequently involve the maintenance of metabolic homeostasis. Mostly, they occur at the bacterial membrane, involving transport proteins of metabolically important substrates and their regulation, thus playing a role in the co-regulation of metabolic functions by membrane-bound TCSs. Some protein activities, possibly linked to freely diffusing metabolic substrates, also contribute to cytoplasmic sensing as highlighted below.The concept of co-opting accessory proteins such as transport proteins for sensory purposes in the downstream activation of TCSs has been described in several bacterial species, primarily flux sensing, involves protein conformational changes upstream of a TCS cascade  flagellar genes is regulated by the cytoplasmic TCS FlgS-FlgR and its impact on σ54-dependent transcription  is a central metabolic function for carbohydrate uptake in many bacteria that also has diverse roles in regulation, including motility and taxis .protein activity sensing by the activity of a membrane transport system, which is metabolically active and at the same time relays information to transcriptional regulators. In many Proteobacteria, also a truncated, secondary PTS exists (PTSNtr), which only acts in regulation. In E. coli, PTSNtr interacts with the potassium transporter TrkA and the sensor kinase KdpD, influencing the activity and expression of these and other target proteins  interacts with the bifunctional ppGpp synthase/hydrolase SpoT1, a key enzyme and regulator of the stringent response , because it can initiate a dual response not only on the level of motility in the short-term but also by influencing metabolic gene regulation in the medium temporal range.Conversely, TlpD had an effect on transcriptional regulation, impacting the expression of numerous genes involved in redox homeostasis and metabolism . TherefoTaken together, emerging evidence shows the presence of a functional coupling, either by direct interaction or regulatory effects of metabolic enzymes, between electron transport chain components and other transport systems to regulators of metabolic functions, motility and flagella, including the bacterial taxis systems. We argue that this link is crucial in order to connect the energy-consuming functions of the flagellar apparatus to the sum of metabolic activities providing the necessary energy. This link might have been selected during evolution to provide useful feedback responses from metabolism to motility and taxis and vice-versa, in order to adjust motility functions, such as flagellar rotation, number of flagella, or the functionality and amount of chemotaxis receptors to the levels in energy and metabolic activity. These connections underline how important it is for bacteria to efficiently transmit and integrate information on the intracellular energy and nutritional levels into their sensory circuitry. Although the function and evolutionary aspects of the activity sensing processes related to motility are not entirely clear, they definitely highlight the importance to better understand the mechanisms behind the sensing of the intracellular energy state, which may depend on protein activities and protein-protein interactions.A discussion about protein activity sensing in metabolism and motility also requires the mention of the term moonlighting proteins. The term was coined about two decades ago , to descE. coli, H. pylori and other organisms including Homo sapiens . AcnB has been described as a moonlighting protein with dual functions in  sapiens . Several sapiens . Under o sapiens . This abunctions . By RNA himurium . Concordum  encoding for chemotactic-related genes (V. cholerae, genes from cluster II encode proteins responsible for chemotactic behavior in most environmental conditions. Meanwhile, cytoplasmic receptors associated with proteins encoded by genes from cluster I are specifically induced under low oxygen conditions (In order for bacteria to thrive in different habitats, efficiency in energy sensing and the integration of multiple signals are essential. For pathogenic and non-pathogenic bacteria, the regulatory properties and survival strategies have been evolved to cope with complex and variable environments. While in the early days of pathogen research, researchers focused on the effect of virulence factors in host damage, recently the combination of bacterial traits is viewed more in the context of optimizing the metabolic situation inside the host niche to the benefit of specific bacteria . This reed genes . In V. cnditions , leadingnditions . Moreovenditions . Other snditions , and insnditions .Numerous examples now illustrate the role of non-regulator proteins with important primary functions in membrane transport or metabolism as cofactors in central regulatory processes, for example those governing bacterial metabolism, taxis and motility. However, some of the important mechanisms of their sensory and regulatory functionality, in the context of their structure and possible modifications, remain unknown. More examples of complex sensory perception and signal integration by activity sensing and protein-protein interactions are to be discovered in all bacterial taxons. Further research is required to decipher the mechanisms behind temporal and spatial protein activities and localization dynamics, and how those feed into their activities as sensors, cofactors or transmitters of signals. The value of such functions can also be appreciated by mathematically modeling their influence on the activity kinetics of sensory systems and their output. A greater understanding of these processes will be beneficial to the study of bacterial sensing, inter-species communication and their proliferative capacities in the context of hosts and other relevant environments.All authors wrote the manuscript jointly. AA and CJ revised the manuscript critically for timely content and structure. CJ developed the initial concept of the article and acquired funding. All authors edited, revised, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bladder cancer is a fatal cancer that happens in the genitourinary tract with quite high morbidity and mortality annually. The high level of recurrence rate ranging from 50 to 80% makes bladder cancer one of the most challenging and costly diseases to manage. Faced with various problems in existing methods, a recently emerging concept for the measurement of imaging biomarkers and extraction of quantitative features called “radiomics” shows great potential in the application of detection, grading, and follow-up management of bladder cancer. Furthermore, machine-learning (ML) algorithms on the basis of “big data” are fueling the powers of radiomics for bladder cancer monitoring in the era of precision medicine. Currently, the usefulness of the novel combination of radiomics and ML has been demonstrated by a large number of successful cases. It possesses outstanding strengths including non-invasiveness, low cost, and high efficiency, which may serve as a revolution to tumor assessment and emancipate workforce. However, for the extensive clinical application in the future, more efforts should be made to break down the limitations caused by technology deficiencies, inherent problems during the process of radiomic analysis, as well as the quality of present studies. Bladder cancer ranks ninth of the most common malignancies and the 13th most common predisposing cause of cancer-related mortality all over the world, with over 357,000 new cases and over 130,000 deaths annually .Clinical decision and follow-up management of bladder cancer predominantly depend on the presence or absence of muscle invasion and accurate grade of malignancy and also take specific pathological types into consideration , 4.However, all aspects in the management of bladder cancer including tumor staging, diagnosis, treatment, and prognostic evaluation have still been limited by various factors. The gold standard nowadays for bladder cancer detection is telescopic checking of the bladder (cystoscopy) . ConsideApart from the difficulties in accurate tumor detection and clinical grading, the prediction of treatment efficacy is also a major obstacle in the management of bladder cancer. It has been widely accepted that patients with nonmuscle-invasive bladder cancer  are mostly at early stage and are advised to be treated with TURBT followed by treatment applying Bacillus Calmette-Guérin (BCG) , whereasConfronting the above problems and limitations, a novel concept of radiomics has emerged for solving the issues of the generalization of precision medicine and how it can be applied in the field of bladder cancer monitoring. It is a high-throughout quantitative feature extraction method to mine the information contained in the multimodality medical images including computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasonography (US) , then coIn this paper, we review the present studies in association with our topic and discuss the promising usages and hidden challenges of this novel method adapting acute imaging analysis, combining radiomics with ML in the precise management of bladder cancer. This review paper considers and discusses the issues as follows:The concept of radiomics and its significanceWorkflow of radiomicsClinical applications of the combination of radiomics with ML in bladder cancer managementChallenges and future directions.The meaning of “precision medicine” indicates that the reasonable strategies of treatment are singled out according to the characteristics of different subtypes. It has substantially changed the treatment strategies in the recent 10 years. To make a precise treatment for an individual, accurate detection, characterization, and monitoring after treatment are very important. Unfortunately, the current tumor assessment is far from our expectation because of variable technology deficiency. One important problem is that radiologists usually use subjective, qualitative features to make tumor assessment, which make the results less reproducible and more unstable. Besides, with the rapid development of gene therapy and immunotherapy, gene expression signature and immune phenotype are also essential parts for a comprehensive tumor assessment. Current evaluation for gene expression and immune phenotype is most based on the biopsy, which is invasive and expensive, let alone the result is confused because of intratumoral heterogeneity. Thus, the demand for a non-invasive, cheap, and stable method to assess and monitor tumor has never been greater.The computational medical imaging, also known as radiomics, was first invented by Lambin in 2012 , 17. It Radiomics is a multidisciplinary-based technology. There are four main steps to complete a radiomics program :(1) Image acquisition and preprocessing(2) Volumes of interest (VOIs) segmentation(3) Feature extraction and quantization(4) Model building.Image acquisition is the first and an important step in radiomics.There are two common formats of the image data recorded, including Picture Archiving and Communication System (PACS) and the Digital Imaging and Communication in Medicine (DICOM), and they are used in most of the medical institutions, which provide a great convenience for the radiology study.The accuracy and reproducibility of the final radiomics model lie on the quality of image acquisition. However, there is no guideline nor consensus on image acquisition. As a result, the acquisition strategies in different research teams can be distinctive, which can cause heterogeneity among separate studies. Meanwhile, it is difficult for researchers to take labeling, annotation, segmentation, and quality assurance seriously. Because these processes require well training, wasting both time and money.Various imaging modalities such as modern CT, MRI, PET, and US scanners allow for acquisition and image reconstruction in wide variations . The radThe region for image data capture is defined as “The Volume of Interest” (VOI). VOIs segmentation is the core step of radiomics study because it determines which volume is analyzed within a medical image.The ideal VOI includes the complete information for the target lesions, nothing more nor less. Unfortunately, usually, it is hard for the radiologists to make it because many tumors have indistinct borders . BesidesVOIs can be created manually, automatically, or semiautomatically. In the past 5 years, most current radiomics study created VOIs manually. However, it is time-consuming and laborious when utilizing big data in radiomics study. Thus, many pieces of research try to create VOI automatically. There are lots of algorithms on VOI creation. A common segmentation algorithm is the “seed method.” The radiologist will place some seeds in VOI, and the computer will create VOI automatically . UnfortuFeature extraction is the next step after a VOI is reasonably segmented, which is the essence in the workflow of radiomics. It mainly performs the extraction of high-throughput data of quantitative imaging features to identify VOIs and the selection of useful information to aid in the discrimination of normal and abnormal images.The quantitative data usually can be classified into four types:Shape characteristics: Description of the shape and geometric features of target lesions. For example, the volume of VOI, maximum diameter along with different orthogonal directions, maximum surface area, tumor compactness and sphericity.(1) First-order statistical characteristics: Description of the distribution of individual but do not describe its spatial arrangement, including the mean, median, maximum and minimum of voxel intensity, skewness (asymmetry), kurtosis (flatness), uniformity, and randomness (entropy).(2) Second-order statistical characteristics: Also known as texture features. It is calculated from a statistical correlation between adjacent voxels, usually by Gray-Level Co-Occurrence Matrix (GLCM) ((3) x (GLCM) . TextureHigh-order statistical characteristics: Quantitative data from the filter or mathematical transformation on images, including fragment analysis, Minkowski function, wavelet transform, and Laplace transform of gaussian filtered image. The aim of filer and mathematical transformation is to identify repetitive or non-repetitive patterns, suppress noise, or highlight details.(4) To conclude, the building of radiomics model involves three main aspects, including the selection of radiomic features, choice of ML methods, and final validation of the developed model. Image analysis software can provide many features, so it is vital to select essential features for further study. At the same time, model selection is as important as feature selection. Selecting a suitable model can help obtain a reliable and stable result, which is very important for clinical decision making. Complete radiomics analysis should include validation, and both internal validation and external validation are indispensable.a priori selection of features on those definitions and selects targeted parameters  , which iModel validation is a dispensable step in model building, which serves as a useful tool to assess the performance and applicability of the developed radiomics model. To make sure that the model is effective for all of the targeted patients, not only the patients selected in the model building process, the internal and/or external validation should be tested. Typically, model performance is measured according to its discrimination, which can be expressed in the form of the receiver operating characteristic (ROC) curve or be calculated as the area under the ROC curve (AUC) in a quantitative way. ROC curve can easily display the ability of disease recognition at any threshold. When comparing two or more models, the ROC curves can draw each model in the same coordinate to identify advantages and disadvantages visually.Accurate grading is of vital importance in the follow-up management of bladder cancer and serves as the starting point of radiomics applied in bladder cancer as shown in Recently, there have also been notable advances in MRI technology. Wang et al.  built anUndoubtedly, clear discrimination between NMIBC and MIBC is crucial for pretreatment decision, posttreatment prognosis, and lasting period consequent management of bladder cancer patients. Considering the large percentage of diagnostic errors caused by conventional cystoscopic examination , researcWith regard to the reflection on the heterogeneity of cancer caused by analyzing radiomic features extracted from the whole tumor tissue, Zheng et al.  suggesteRealizing the high risk of metastasis and mortality of MIBC that is in need of immediate treatment to improve the living quality of patients while faced with the various kinds of ML models emerging, researchers have begun to seek for the urgently needed strategy. A confirmative research was conducted for selection in which seven models including radial basis function (RBFN), back-propagation neural network (BPN), extreme learning machine (ELM), regularized ELM (RELM), SVM, naive Bayes (NB) classifier, and k-nearest neighbor (KNN) were ever explored and compared to predict the 5-year mortality of 117 MIBC patients who had undergone RC . The eveRegarding the importance of preoperative prediction of the risk of bladder cancer recurrence, Xu et al.  developeAll of these studies showed the great potential of radiomics aided by ML in the improvement of tumor classification and prognosis prediction of bladder cancer, overcoming the defects traditionally and obviously improved the living quality of bladder cancer patients. Great progression as radiomics made in the prognosis of bladder cancer, the correlations between the imaging features and genomic signatures have rarely been explored up to now in the field of bladder cancer management. Confronting the blank of related researches, Lin et al.  put forwOnce a definite diagnosis is made and the tumor subtype is identified, early evaluation of therapeutic efficacy and response can aid in clinicians'decision on whether to discontinue chemotherapy at an optimal phase. In the research conducted by Cha et al. , the feaRegarding the aggressiveness of MIBC, studies were conducted to assess whether a CT-based decision-support system could improve identification of patients who have complete or partial response to neoadjuvant chemotherapy . This stUp to now, there has been a large number of studies to illustrate the rapid development of radiomics and ML algorithms, as well as the effectiveness of their combination in the full-cycle management of bladder cancer, but we also need to realize the limitations when applied in actual use comprehensively.First, the defects in the aspect of inherent technologies should be recognized. Usefulness as quantitative features based on medical images show in the management of bladder cancer patients, their tendency of causing significant errors and overestimation has also been reported . It is pSecond, limitations mentioned in current articles that have already been published should be highlighted and overcome. Quite a large percent of studies are conducted in a single institution, and the sample size is too small to be convincing. Apart from that, some studies lack external validation for model development. For the extensive clinical application, the studies should be designed in a much more comprehensive and delicate way to raise the reliability of research results. To achieve future optimization of this novel method, more investigations should be carried out to test the potential of optimizing the predictive model by the combination of imaging biomarkers with other non-imaging biomarkers.Third, it has been a long-lasting debate whether such AI-supported systems are much smarter than clinical practitioners , 51. HowMoreover, the application of radiomics in bladder cancer remains at the macroscopic level. Although we have witnessed the breakthrough of various researches from basic tumor detection to accurate grading, even recent studies have moved toward the prediction of treatment outcomes. However, gene therapy and immunotherapy, which involve gene expression signature and immune phenotype, have been introduced as revolutionary tools for comprehensive tumor assessment and divert our attention to the microscopic level. A novel concept named “radiogenomics” perfectly presents the integration of genomics with radiomics and serves as an alternative to the invasive biopsy . TremendLG and YC collected and reviewed the literature, and wrote the manuscript. JL helped with the writing design and revised the manuscript. PZhao, PZhan, and RA provided helpful comments on the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Elucidation of the pathophysiological mechanism of central post-stroke pain (CPSP) is essential to the development of effective therapeutic modalities for CPSP. However, the pathophysiological mechanism of CPSP has not yet been clearly elucidated. The recent development of diffusion tensor tractography (DTT), derived from diffusion tensor imaging (DTI), has allowed visualization and estimation of the spinothalamic tract (STT), which has been considered the most plausible neural tract responsible for the pathogenesis of CPSP. In this mini-review, six DTT studies in which CPSP due to STT injury in stroke patients was demonstrated are reviewed. The information provided in the reviewed studies suggests that DTT is useful in the elucidation of the pathophysiological mechanism associated with CPSP. We believe that the reviewed studies will facilitate neurorehabilitation of stroke patients with CPSP. However, DTT studies of CPSP are still in the beginning stage because the total number (six studies) of the reviewed studies is very low and half were case reports. Therefore, further studies involving large numbers of subjects are warranted. Stroke is a leading cause of mortality and morbidity in adults. Central post-stroke pain (CPSP) is a neuropathic pain caused by cerebrovascular insult to the somatosensory pathway in the brain . CPSP, wFor management of CPSP, medications including antidepressants, antiepileptic drugs, and opioids have commonly been used for a long time , 9. ReceSeveral theories including central sensitization, neural excitability changes due to disinhibition, alteration of the somatosensory function, changes in thalamic function, and inflammation of an involved neural tract have been suggested as pathophysiological mechanisms for CPSP , 23–26. Precise evaluation of the STT had been impossible because it cannot be clearly identified from adjacent neural structures by conventional brain magnetic resonance imaging. In contrast, the recent development of diffusion tensor tractography (DTT), derived from diffusion tensor imaging (DTI), allows us to visualize and estimate the STT three dimensionally at the subcortical level . TherefoIn this mini-review, DTT studies that demonstrated CPSP because of STT injury in stroke patients are reviewed. Relevant studies in the period from 1990 to 2018 were identified using the following electronic databases: PubMed, Google Scholar, and MEDLINE. Additionally, the following keywords/abbreviations were used to search the databases: DTI, DTT, CPSP, STT, thalamocortical tract, spino-thalamo-cortical pathway, stroke, intracerebral hemorrhage (ICH), and cerebral infarct. This review was limited to studies of humans with stroke. We selected relevant studies according to the flow diagram shown in DTT for reconstruction of the neural tracts usually employs a combined region of interest (ROI) method that reconstructs only neural fibers passing more than two ROI areas. The ROI areas and reconstruction conditions for the neural tracts are well-defined for each neural tract –36. ManyThe main advantage of DTT over DTI is that the entire neural tract can be evaluated in terms of DTT parameters. Among the various DTT parameters, fractional anisotropy (FA), mean diffusivity (MD), and tract volume (TV) are most commonly used to evaluate the state of the STT in patients with brain injury , 37, 38.However, several limitations of DTT should be considered –41. FirsThe first study after introduction of DTI was reported by Seghier et al. . A 65-yeSubsequently, Goto et al.  investigwww.fmrib.ox.ac.uk/fsl]). The laterality index / was estimated to determine the asymmetry of DTT parameters between hemispheres. Only the laterality index for the TV of the STT in the CPSP group was lower than that of the non-CPSP group without differences in other DTT parameters . The decrease in TV without changes in the FA or MD values in the CPSP group indicates partial injury of the STT. Therefore, the authors concluded that partial injury of the STT appeared to be a requirement for the development of CPSP in patients with ICH  using DTT for the STT and MLP . Thirty with ICH . The relIn 2012, Hong et al. investigated the prevalence of CPSP according to the integrity of the STT in patients with ICH  . They reDuring the same year, Jang et al.  investigRecently, Jang et al.  reportedIn this mini-review article, six DTT studies of CPSP related to STT injury in stroke patients were reviewed. The information provided in these reviewed studies suggests that the STT injury is an important pathophysiological mechanism of CPSP and that DTT is useful for elucidation of the pathophysiological mechanism associated with CPSP in stroke patients. However, only six studies on this topic have been reported to date, half of which were case reports. In addition, the two studies at the beginning of the series did not appear to reconstruct the STT precisely; moreover, reliability in terms of the repeatability and reproducibility of the reconstruction method for the reconstructed neural tracts was not demonstrated , 23. TheSJ: study concept and design, manuscript development, writing, and funding. JS: study support. SL: critical revision of manuscript for intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The development of the fetal heart is exquisitely controlled by a multitude of factors, ranging from humoral to mechanical forces. The gatekeeper regulating many of these factors is the placenta, an external fetal organ. As such, resistance within the placental vascular bed has a direct influence on the fetal circulation and therefore, the developing heart. In addition, the placenta serves as the interface between the mother and fetus, controlling substrate exchange and release of hormones into both circulations. The intricate relationship between the placenta and fetal heart is appreciated in instances of clinical placental pathology. Abnormal umbilical cord insertion is associated with congenital heart defects. Likewise, twin-to-twin transfusion syndrome, where monochorionic twins have unequal sharing of their placenta due to inter-twin vascular anastomoses, can result in cardiac remodeling and dysfunction in both fetuses. Moreover, epidemiological studies have suggested a link between placental phenotypic traits and increased risk of cardiovascular disease in adult life. To date, the mechanistic basis of the relationships between the placenta, fetal heart development and later risk of cardiac dysfunction have not been fully elucidated. However, studies using environmental exposures and gene manipulations in experimental animals are providing insights into the pathways involved. Likewise, surgical instrumentation of the maternal and fetal circulations in large animal species has enabled the manipulation of specific humoral and mechanical factors to investigate their roles in fetal cardiac development. This review will focus on such studies and what is known to date about the link between the placenta and heart development. Since David Barker first documented the relationship between infant birth weight and adult onset disease , single umbilical artery ligation (SUAL), maternal hyperthermia in sheep, unilateral uterine artery ligation in guinea pigs and bilateral uterine artery and vein ligation in rats; 2. Those that reduce oxygen availability for transfer to the fetus ; and 3. Those that alter nutrient availability for the fetus . However, maternal hypoxia may result in reduced maternal food intake or alterations in the placenta's ability to deliver nutrients to the fetus. Likewise, altering the maternal diet may affect placenta development and thus decrease oxygen transfer capacity. For simplicity sake, the animal models have been divided into those that report fetal hypoxaemia  and also take a year to reach adulthood. Further, there are a limited number of facilities in the world that allow for postnatal longitudinal studies. The sheep placenta is cotyledonary in nature, composed of many individual placentomes, which form at sites in the uterus called caruncles. There is no trophoblast invasion of maternal vessels, and an epithelial layer separates maternal blood from the trophoblast  and lifespans, which allows for a high throughput of pregnancy, postnatal and intergenerational studies. The rodent and guinea pig placentae are discoid in nature, trophoblast invade and remodel the uterine vasculature to promote blood flow and the trophoblast is directly bathed in maternal blood, which is similar to the human   mother , which subsequently increases placental vascular resistance and reduces gas and nutrient exchange between the fetal and maternal circulations  and low-protein diets (6–9%) in mice, rats and guinea pigs, typically reduce placenta weight, as well as regional weights and/or volumes of the transport labyrinthine zone (Lz) and endocrine junctional zone (Jz) Table . These cIn addition to undernutrition, excess calories during pregnancy can also affect the placenta and offspring heart. Maternal high-fat or high-fat/high-sugar diets have been associated with both unchanged . A selection of these genes are listed in Table Hey1/2, Mekk3, Gab1, Hai1, Flrt2, Phd2, Cited2, Ovol1, Vcam1, Mmp14/16). Malformations of the heart and placenta are the most commonly cited reasons for mid-gestational lethality. Heart defects also arise at around day 10 of pregnancy, when organogenesis becomes highly dependent on placental function. Previous work has largely focussed on assessing the impact of a genetic manipulation on either the formation of the placenta or the fetal heart, rather than considering an interaction between the two. In spite of several of the genes listed in Table Studies performed in genetically-modified mice have started to provide novel insights into the regulation of, and relationship between, fetal heart development and placental formation Table . Indeed,Hoxa13, results in defective vascularization and formation of the placental labyrinthine (exchange) zone , nuclear hormone receptors (Pparg), mitogen-activated protein kinase signaling pathway  and protein modification machinery (Senp2) in the placenta also appear to be required for fetal heart development. Loss of any of these genes leads to reduced vascularization and development of the placental labyrinthine zone  and downstream signaling pathways, including extracellular signal-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K). The carunclectomy model in sheep (Jones et al., Cortisol is an important regulatory signal during fetal development, which amongst other important roles, acts to mature the cardiovascular system in preparation for birth (for review, Fowden and Forhead, In vitro, T3 inhibits the proliferation of cardiomyocytes isolated from hearts either before or during the prepartum surge in T3 concentration (Chattergoon et al., Thyroid hormones, especially T3, promote the maturation of a range of organs (For review, Forhead and Fowden, Epidemiological and clinical studies suggest a link between placental morphology and increased risk of cardiovascular disease in adult life. The mechanistic basis of this relationship has not been fully elucidated. However, experimental animal models and studies in genetically-modified mice, have provided novel insights into the relationship between placental formation and fetal heart development and the role humoral and mechanical forces play in the development of both of these organs Figure . FurtherEC, KB, and AS-P contributed equally to reviewing the literature and writing and editing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The intrinsically complex nature of fish and seafood, as well as the complicated organisation of the international fish supply and market, make struggle against counterfeiting and falsification of fish and seafood products very difficult. The development of fast and reliable omics strategies based on spectroscopy in conjunction with multivariate data analysis has been attracting great interest from food scientists, so that the studies linked to fish and seafood authenticity have increased considerably in recent years. The present work has been designed to review the most promising studies dealing with the use of qualitative spectroscopy and chemometrics for the resolution of the key authenticity issues of fish and seafood products, with a focus on species substitution, geographical origin falsification, production method or farming system misrepresentation, and fresh for frozen/thawed product substitution. Within this framework, the potential of fluorescence, vibrational, nuclear magnetic resonance, and hyperspectral imaging spectroscopies, combined with both unsupervised and supervised chemometric techniques, has been highlighted, each time pointing out the trends in using one or another analytical approach and the performances achieved. The demand for fish and seafood products has increased notably during the last years, mostly as a consequence of the new special attention paid by consumers towards healthier food. The technological development that has invested the whole fisheries sector has additionally contributed to overcome the well-known obstacles to export fish and seafood worldwide, deriving from the high vulnerability of the products, to the point that today more than 35% of all caught and cultured fish is traded across national boundaries . The groIn this scenario, what is remarkable is that consumers demand not only for more fish, but for even safer and higher-quality fish, whilst the deliberate or accidental lack of transparency about the identity of products and fraudulent or negligent activities continue to grow. Based on what has been recently reported by the Food and Agriculture Organization, fish and related products have become among the most vulnerable to fraud category of food. Nevertheless, the effective monitoring of illicit practices in the fisheries sector is hampered by the increasing spread of highly processed fish products, in which the presence of different types of fraud can be hidden with ease .Tetraodontidae, Molidae, Diodontidae, and Canthigasteridae families) or the replacement of certain kinds of raw fish fillets with gastro-intestinal toxic fish  are just some of many examples. Likewise, occurrence of some harmful marine biotoxins may be linked to the geographical distribution of the producing organisms [The voluntary substitution of commercially valuable fish species with lower quality ones, represents the most recurrent form of fish fraud, although substitution can also take place accidentally when species look so similar that they are mistaken for each other. The geographical provenance and the production process are other current authenticity topics concerning fish and seafood products, whose falsification which is hard to bring to light, has a negative economic impact. Despite being economically motivated, mislabelling concerning these issues may occasionally represent a risk to public health. The illegal commercialisation of poisonous fish species , mid-infrared (MIR), Raman), fluorescence or absorption ultraviolet-visible (UV–Vis), and nuclear magnetic resonance (NMR) spectroscopies, together with hyperspectral imaging (HSI) spectroscopy, represent the most used techniques, even if they are still being developed.Based on this background, the present review article has been designed to highlight the uses and developments of fast and reliable omics strategies based on UV–Vis, NIR, MIR, Raman, NMR, and HSI spectroscopies, with the attempt to address the key authenticity challenges within the fish and seafood sector. To this end, a brief discussion concerning basilar concepts underlying these techniques has been provided, and has been accompanied by a short overview about the implementation of several chemometric tools, in order to highlight the potential benefits in extracting relevant information from spectral data.The main body of this review focuses specifically on the application, over the years, of spectroscopy and chemometrics to distinguish products in accordance with the species, production method (wild or farmed), farming system , geographical provenance (different FAO areas and countries of origin), and the processing technique (fresh or fresh/thawed) that at present, correspond to the key authenticity concerns for which there must be ongoing and effective monitoring.Spectroscopy is the study of electromagnetic radiation interacting with matter, which can be absorbed, transmitted, or scattered on the basis of both the specific frequency of the radiation and the physical/chemical nature of the matter. When absorbed, radiation leads to a change in the energy states of atoms, nuclei, molecules, or crystals that make up matter, inducing an electronic, vibrational, or rotational transition, depending on the energy of the incident radiation . When thFor the sake of conciseness, the main features related to spectroscopic techniques used mostly in the food authentication field are summarised in UV–Vis spectroscopy involves the electronic excitation of molecules containing specific chromophore groups, which results from the absorption of photons at two wavelength regions of the electromagnetic spectrum. In the absorption mode, the amount of light retained by the sample is measured, while in the fluorescence mode the amount of light emitted after absorption is taken into consideration . TypicalInfrared spectroscopy involves three different sub-regions of the electromagnetic spectrum, namely NIR, MIR, and FIR, whose absorption by samples results in vibrations of atoms in molecular bonds . These vThe NIR spectrum of food samples results from absorption by molecular bonds containing prevalently light atoms and it is characterised by the presence of broad and overlapping overtone and combination bands ,23. By cRaman spectroscopy is a molecular vibration technique based on the inelastic Raman scattering, a physical effect that comes with molecular vibrations and triggers a change in the polarizability of the molecule . In partFor a long time after its discovery, Raman spectroscopy has been poorly exploited in food applications, by reason of several analytical disadvantages and interference see . These dHSI is a technique cobbling together spectroscopy and computer vision to give useful information concerning the physicochemical characteristics of samples in relation to their specific spatial distribution. Briefly, HSI systems provide several hyperspectral images of the tested sample, corresponding to three-dimensional data containers, of which each sub-image is a map showing spatial distribution of the sample constituents in relation to each single wavelength ,30.Over the recent years, the steady usage growth of HIS technology in the field of food research has been mainly driven by the availability of different instrumental configurations that exploit fluorescence, absorbance, or light scattering phenomena. On the other side, application of spectral imaging technologies is not at all widespread in the food industry, due to a variety of factors ranging from high costs and low availability of instrumentations, to the computation speed and necessity of expertise by users .NMR spectroscopy is a very versatile technique for food analysis and its untargeted applications have become very popular. The first reason for NMR popularity is that the composition of the matter under study can be perfectly mapped out by the overall NMR spectral profiles, thus giving a comprehensive view for the identification of all major and minor food components . At the On the basis of the physical state of the matter and on the intended aim of NMR application, different methodologies involving the use of NMR have been optimized. Among these, high-resolution NMR, low-field NMR, solid-state NMR, liquid-state NMR, and NMR imaging are the most used ones, any of which requires specific instrumentation and different approaches to sample preparation, data acquisition, and processing .Raw spectra resulting from spectroscopic analyses are usually characterised by broad and unresolved bands containing too much information, some of which are certainly useful and need to be retained, but some of which hamper the correct data interpretation and need to be removed. Recent advances in chemometrics have marked an important milestone in spectra analysis, since they have simplified the identification of hidden interrelations between variables providing the key for discrimination and classification of samples ,35. In oBased on the explorative or predictive nature of the methodology, qualitative chemometric techniques are usually classified into unsupervised and supervised techniques. While unsupervised techniques are independent of prior knowledge of class membership of samples to perform classification, supervised techniques call for such knowledge. Brief descriptions of the principles behind the chemometric techniques which are being used to a greater extent are provided below.Pre-treatment of spectral data is recognized as being fully integrated into the chemometric set-up itself. Prior to the development of chemometric models, raw spectroscopic data are suggested to be pre-processed by applying some corrections, aimed to enhance spectral properties and minimize the fraction of systematic variation which does not contain relevant information to the discrimination of samples. One such systematic variation is the sum of different physical effects which arise during instrumental acquisition of spectra , which are responsible for the appearance, especially in solids samples, of multiplicative, additive, and non-linearity effects  .Thus, pre-processing algorithms are usually classified into signal correction methods , differentiation methods , and filtering-based methods  . While sA more detailed description of spectral pre-processing techniques can be widely found in the literature ,39,40. EUnsupervised methods look at the study of variability among samples for the purpose of identifying their natural characteristics and possible similarities among them, without the need to provide any information about the class to which samples belong.Between the various available techniques, principal component analysis (PCA) is the most used one. PCA is a quite basic projection method able to reduce the original correlated variables into a smaller number of new uncorrelated latent variables , containing as much systematic variation as possible of the original data . Score pHierarchical cluster analysis (HCA) is another frequently employed unsupervised method, based on the splitting of samples into different clusters. This splitting is based on the degree of analogy among samples and it is generally performed by evaluating the Mahalanobis or Euclidean distance between the same samples. The hierarchical approach followed is thus aimed at constructing a ladder, in which the most closely related samples are first classified into small groups, and then progressively assembled into bigger groups including less similar samples . ResultsSupervised techniques require the previous knowledge of the class membership of the samples tested, which can be used to develop predictive models able to discriminate and classify future unidentified samples. There are several different chemometric techniques belonging to the category of the supervised methods, most of which require a training set (to find classification rules for the sample), and a test set (to assess the predictability of the model developed) .Linear discriminant analysis (LDA) and quadratic discriminant analysis (QDA) are variance-based methods which use Euclidean distance to find those combinations of the original variables determining maximum separation among the different groups of samples . Both teK-nearest neighbors (k-NN) clustering is one of the simplest method to discriminate samples on the basis of the distance among them. After choosing the adequate number of k-neighbor samples, the algorithm identifies the k-nearest samples of known class membership to select the classification of unknown samples. This method, unlike LDA and QDA, does not require any prior assumption and its success is independent of the homogeneity of sample numbers in each tested class .Among supervised machine learning approaches, support vector machines (SVM) are particularly advantageous when samples classification is complicated by non-linearity and high dimensional space. The core of the method is the use of specific functions for pattern analysis , through which the margin of separation between classes is maximised and complex classification problems that are not linear in the initial dimension  are resolved .Similarly, artificial neural networks (ANN) is a machine learning method characterised by the ability to adapt to the data, providing classification also in the presence of non-linearity input–output relationships. Structured and organized in a less complex way than SVM, ANN usually generate a more rapid response at a lower computational cost; these efforts, however, are counterbalanced by a reduction in accuracy ,44. NeveSoft independent modelling of class analogy (SIMCA) is an alternative pattern recognition method which first performs individual PCA on the samples for each class they must be assigned to, in order to compress original variables into a smaller number of new principal components. Principal components and critical distances computed are then used to delineate a confidence limit for each class. Unknown samples are then assigned to the class to which they get close by projection into the resulting multidimensional space . SIMCA iRegression-based supervised discriminant analyses exploit specific classification algorithms to model the interrelations existing between measured variables  and qualitative parameters , such that maximum separation between the different groups of samples is achieved. Partial least square-discriminant analysis (PLS-DA) and orthogonal partial least square-discriminant analysis (OPLS-DA) belong to this category of techniques. PLS-DA involves a standard PLS regression to find interrelations between the X-matrix (containing measured variables) and Y-matrix  by building new variables (latent variables). These interrelations allow not only to classify new samples into one of the Y-groups based on measured spectrum, but also to identify variables that mostly contribute to the classification. Although PLS-DA has the advantage of modelling noisy and highly collinear data efficiently, the technique is often unsuccessful when the non-related  variability in the X-matrix is substantial, since it hinders the correct interpretation of the results . This drSpectroscopic and chemometric analyses have been used over the years for many applications in fishery research, those in the authentication field being among the most promising ones. Some of the works concerning the flexibility of spectroscopy in fish and seafood analysis have already been reviewed by different authors ,47,48,49Therefore, in the following section, more attention has been paid to the resolution, on a case-by-case basis, of the weightiest authentication issues in the fish and seafood sector, namely species substitution, geographical origin falsification, production method or farming system misrepresentation, and fresh for frozen/thawed product substitution, each time pointing out the trends in using one or another method as well as the discrimination performances achieved, which are considered to be the most intuitive parameters used for chemometric models diagnostics. An overview of the most frequently investigated authentication issues in the fishery sector and the trend of using each spectroscopic technique over the years by the scientific community are plotted in Substitution or counterfeit of high-value fish species with low-value ones has many quality and safety implications. Therefore, the confirmation of scientific and commercial names declared on the label through the use of rapid and low-cost methods is increasingly popular in food research.Callinectes sapidus) with blue swimmer crabmeat (Portunus pelagicus) in 10% increments, taking into consideration their different commercial values [An early study explored Vis-NIR spectroscopy as a tool to detect the counterfeit of Atlantic blue crabmeat  from those of haddock (Melanogrammus aeglefinus) was recently made [Based on the fact that the past several years have seen a sharp rise in the interest towards the portability of instruments, which may provide greater flexibility especially in on-line, in-line, and at-line routine quality control, a study performed by O’Brien et al. (2013), explored the ability of a hand-held NIR spectrometer to give positive results of discrimination between high-value and low-value whole fish and fish fillet species . In parttly made . Raw filtly made . SIMCA ctly made . As for Mylopharyngodon piceus), grass carp (Ctenopharyngodon idellus), silver carp , bighead carp (Aristichthys nobilis), common carp (Cyprinus carpio), crucian (Carassius auratus), and bream (Parabramis pekinensis), were also investigated by NIR spectroscopy [Different species of freshwater fish of the Cyprinidae family, namely black carp (troscopy . Fish satroscopy .Argyrosomus argentatus), hairtail (Trichiurus haumela), and red coat (Nemipterus virgatus) [Zhang et al. (2017) attempted to classify marine fish surimi by 1100–2500 nm NIR spectroscopy, according to the species by which products were composed, namely white croaker (irgatus) . Accordiirgatus) . HoweverSpecies authenticity was also studied by comparing FT-NIR and FT-MIR spectra of red mullet and plaice fillets  to those of Atlantic mullet and flounder fillets  . LDA andThe usefulness of NIR spectroscopy was explored to identify different fish species used to make fishmeal under industrial conditions. The 1100–2500 nm raw or second derivative NIR spectra of samples containing salmon, blue whiting, and other  fish species were elaborated by PCA, LDA, and DPLS (PLS-DA). Models developed correctly classify, on average, more than 80% of the fish meal samples into the three groups assigned according to the fish species .Salmo salar) mini-burgers with different percentage  of Rainbow trout (Onconrhynchus mykiss). The resulting 11 formulations of salmon burgers were grouped into 11 distinct clusters, even when the samples were stored for different periods of time before acquisition [In contrast to the multiple applications of NIR spectroscopy, only one study explored the discrimination abilities of MIR spectroscopy . This stuisition .−1  recorded by a Raman spectrometer equipped with a 532 nm laser exciting source [Trachurus trachurus), European anchovy (Engraulis encrasicolus), Bluefish , Atlantic salmon , and flying gurnard (Trigla lucerna) samples. In this case, however, the study was less rapid and more elaborate since the spectral acquisition was performed on the previously extracted lipid fraction of fish [Only two applications of Raman spectroscopy concerning fish species authentication are available. The aim of the first study was to discriminate 12 different fish fillets of different species by using pre-treated Raman spectra in the range 300–3400 cmg source . HCA anag source . Similar of fish .Gadus morhua), haddock (Melanogrammus aeglifinus), saithe (Pollachius virens), and pollack (P. pollachius), were subjected to 13C-NMR spectroscopic analysis of phospholipid profiles, in order to authenticate samples according to the species [13C-NMR spectral peaks provided 78% and 100% of the correctly classified samples, respectively [Muscle lipids of four different species of fish belonging to the Gadoid family, namely cod  proposed a comparison between the classification performances of wild and farmed European sea bass obtained by three different NIR spectroscopic/chemometric approaches, and the classification performances obtained using only chemical and morphometric features . The useMore recently, the systems behind the production of European sea bass, was also investigated by applying unsupervised PCA and supervised OPLS-DA to 1100–2500 nm NIR spectra . PCA buiAuthentication by NIR and SIMCA analysis of European sea bass raised in extensive ponds, semi-intensive ponds, intensive tanks, and intensive sea-cages, was also performed both on fresh fillets and freeze-dried fillets . AuthorsFollowing these principles, NIR spectroscopy was also used to directly authenticate freeze-dried rainbow trout fillets by rearing farm and, at the same time, to check whether NIR discriminating capability changed between raw and cooked freeze-dried fillets . Rainbow1H) NMR spectroscopy can be used to analyse lipid mixtures such as fish oil, requiring simple preparation of samples and short time of spectra acquisition and providing a great deal of useful information [1H-NMR to identify the production process or the farming system were made. One of the earliest studies used SVM to elaborate 1H-NMR spectra, and it was highly effective in predicting the wild or the farmed origin of salmon from different European countries [1H-NMR fingerprinting of lipids from gilthead sea bream with more complex chemometric data analyses [1H-NMR spectral profiles returned, in fact, a clear separation between wild and farmed samples, which was found to be linked to methyl and methylene protons, together with methylene and methyne protons in unsaturated fatty acids [Several applications of NMR spectroscopy aimed at authenticating the production process or the farming system were found in literature. In particular, proton  of these products [n−3 polyunsaturated fatty acids) was also found to play a special role in the discrimination between wild and farmed specimens of gilthead sea breams [1H-NMR spectroscopy. Just like SIMCA classification did in the previous study, OPLS-DA also led to a perfect separation of samples, but with the great advantage of being able to highlight the most effective variables in discrimination in the simplest of ways.If the previous studies were performed on fresh raw fish, other studies were intended to evaluate any differences in classification outcomes deriving from various degrees of fish processing. Lipids extracted from different types of processed Atlantic salmon products  were subjected to products . SIMCA aproducts . A highea breams . The inf1H-NMR molecular profiles of gilthead sea bream fish specimens produced according to different farming systems, have also been investigated, to seek out differences among three different kinds of aquaculture practices , but also any variations in the molecular patterns after a 16-day storage time under ice [The nder ice . PCA-sco13C) NMR instead of 1H-NMR. Authors combined 13C-NMR spectra of muscle lipids of Atlantic salmon with PNN and SVM chemometric elaborations, to discriminate between farmed and wild samples and obtained excellent discrimination performances  [13C-NMR signals being generally much weaker than those provided by 1H-NMR , useful and complementary information can be obtained by this technique.Another different NMR approach retrieved from the published literature concerned the use of carbon-13 (ctively) . DespiteProving the geographical origin authenticity of fish and seafood often involves the use of multi-disciplinary and cross-disciplinary approaches which take account of the environmental and genetic backgrounds affecting fish final characteristics . SeveralUnlike the other authentication issues discussed above, NIR spectroscopy has been less explored for fish geographical origin identification. The reason, probably, is the great difficulty experienced in modelling total variability of NIR spectra and uniquely steering it to provenance, since provenance is the sum of a huge amount of different intrinsic or extrinsic factors .Apostichopus japonicus) come from, were also identified by using NIR spectroscopy [A traceability model able to predict the geographical origin of Chinese tilapia fillets coming from four different Chinese provinces, was developed by NIR spectroscopy . SIMCA atroscopy . In thistroscopy .The last available application of NIR spectroscopy concerned the authentication of European sea bass according to Western, Central, or Eastern Mediterranean Sea provenances, by using OPLS-DA as a classification technique . Results1H-NMR and SVM of spectra extracted from samples coming from Canada, Alaska, Faroes, Ireland, Iceland, Norway, Scotland, and Tasmania. SVM returned a low degree of misclassification (4.6%) and, thus, an excellent correct classification rate for all the salmon samples [13C-NMR coupled with PNN or SVM. This time, although the PNN- and SVM-based approaches used returned different correct classification rates , a comparable classification accuracy between the two methodologies approaches was observed [1H-NMR lipid fingerprint, elaborated by LDA or PNN, allowed also to differentiate 76.2–100% of wild and farmed gilthead sea bream samples coming from Italy, Greece, Croatia, Turkey, and the Mediterranean Sea (for wild specimens), with better classification rates when PNN was applied [1H-NMR lipid fingerprint [Masoum et al. (2007) proposed a method for the origin authentication of Atlantic salmon based on  samples . Likewis samples . Here toobserved . The 1H- applied . Farmed gerprint . In thisgerprint .1H-NMR dealt with the geographical authentication of bottarga, a fish-derived product consisting of salted and dried mullet  roe [One last interesting application of lus) roe . Low-molFish is commonly processed by freezing in order to be preserved from deterioration. Frozen fish, however, is usually characterised by much lower quality and commercial value compared to fresh fish. Therefore, fraudulent practices consisting in the substitution of fresh with frozen/thawed products are not uncommon events . ConsideFront-face fluorescence spectroscopy is one of the earliest spectroscopic techniques historically applied to differentiate fresh from frozen/thawed fish. It has been demonstrated that typical changes in fluorescence spectra of aromatic amino acids, nucleic acids, and nicotinamide adenine dinucleotide (NADH) occur during storage, as a consequence of several reactions involving free amino acids and carbonyl compounds of reducing sugars, formaldehyde (produced from trimethylamine oxide), and malondialdehyde (produced from oxidation of fish lipids during storage). Therefore, changes in fluorescence of fish samples may be considered as fingerprints for fresh and aged fish fillet identification . The fluMore applications of IR spectroscopy are reported in the published literature. Uddin and Okazaki (2004) used NIR reflectance spectroscopy on dry extract of horse mackerel specimens to evaluate freshness . Both PCSolea vulgaris) samples [Another study was directed to compare classification ability of Vis-NIR (380–1080 nm) and NIR (1100–2500) spectroscopy in authenticating fresh and frozen/thawed swordfish and, through the application of PLS-DA, it was found that in this case, Vis-NIR spectra gave better results in the external validation (≥96.7% of correctly classified samples) . Althoug samples . Again,  samples , while b samples .Ottavian et al. (2013) proposed an interesting three-step approach based only on NIR spectra and latent variable modelling techniques to develop a species-independent classifier able to simultaneously discriminate between fresh and frozen/thawed fish and, remarkably, overall classification accuracy of the method ranged between 80% and 91%, based on the strategy adopted and the instrument used . By contFinally, one single application of Raman spectroscopy to the authentication of fresh fish is now available . Lipid fDiscrimination between fresh and frozen/thawed cod fillet was studied by Vis-NIR/HSI, using both a handheld interactance probe and an imaging spectrometer  . SpectraNMR spectroscopy is considered to be a useful and suitable tool for the discrimination of fresh from frozen/thawed fish, since NMR signals are sensitive enough to changes in water mobility and its interaction with other molecules . NMR speThe food scientists’ interest towards the development of reliable methods for the resolution of several food authenticity issues is well documented by the increasing number of scientific works which, albeit through different methodologies, have attempted to address the same problems. It is clear from the analysis of the latest literature that spectroscopy combined with chemometrics is just one of the many untargeted strategies adopted: chromatographic, MS-based, as well as bio-molecular and sensory techniques have been already widely exploited and have demonstrated their exceptional multipurpose qualities for fish authenticity testing ,107,108.These techniques are known to share certain common disadvantages, such as the long time needed for analysis, high costs of the equipment, the need of sample preparation prior to analysis, destructiveness, and the demand for qualified personnel. On the other hand, as they become more consolidated within the research community, these techniques excel by their higher accuracy, specificity, and sensitivity compared to spectroscopic ones, to the point that many of them are used in food official controls. Despite this, the attractiveness of spectroscopy and chemometrics is evidenced by not only by the large literature provided in the present review, but also by several other applications covering a wide range of food and foodstuffs: fruits and vegetables, honey, wine, edible oils and fats, cereal and cereal-based products, milk, and dairy products ,113,114 Having said that, some critical reflections should be made about the problems related to the use of spectroscopy and chemometrics, which still have not been overcome. In accordance to what has been already reported and to our opinion, the research papers analysed were found to be highly variable to each other in terms of analytical set-up . This variability, as easily understood from A critical and objective evaluation of these works is also severely hampered by a lack, in certain cases, of comprehensive data with regards to the validation of the results. Alongside the internal cross-validation, the external validation of the qualitative chemometric model is, in our opinion, a crucial point in assessing the overall goodness of the classifiers and avoiding misleading interpretations. The last aspect which should be emphasised is that a detailed description of the characteristics of the sample dataset was not often reported and the lack of standardisation of external factors , may have interfered with spectral analysis, possibly affecting the robustness of the model. In this scenario, a recommendation for future works is to consider the intrinsically natural variability of the fish products , and to organise the sampling in such a way that as much of the expected variability of samples is collected during the calibration stage. That way, the robustness of the models can make their way to the spread of applications also in the industrial sector.As a final remark, no technique should be universally regarded as the optimal solution. However, the possibility of using UV, IR, Raman, and NMR spectroscopies with no distinction for food authentication purposes is still an obstacle to overcome, and therefore, in accordance to our experience, untargeted NIR spectroscopy represents the most versatile option thanks to its high sensitivity to organic molecules of food, cost-effectiveness, and ease of use. Additionally, the use of NIR spectroscopy with supervised chemometric method, able to separate relevant from non-relevant spectral variation like OPLS-DA, should be encouraged since the interpretability of results is enhanced.Recent increases in the complexity and competitiveness of the fishery and seafood sectors, have resulted in the presence, on the international market, of a huge variety of fresh and processed products, but at the same time, have meant that the risk of fraud deriving from substitution among look-alike products is now exponentially higher than it was even a few years ago. Thus, ensuring the truthfulness of fish and seafood claims concerning their quality and origin, has become an exceptionally important topic, firstly with a view to enable consumers to make informed decisions.The overview presented in this review clearly highlights the effective support provided by analytical approaches based on spectroscopy and multivariate data analysis for the evaluation and monitoring of fish and seafood products authenticity. Fluorescence, vibrational, NMR, and HSI spectroscopic applications have been discussed, with an accent on the trends toward their use for several authentication purposes. In this connection, IR spectroscopy has been the most exploited technique, especially in studies concerning species and fresh for frozen/thawed products substitutions. NMR, instead, has shown many applications in the field on the production method, farming system, and geographical origin identification. By contrast, Raman and HIS have provided very encouraging results in some fish authentication fields, but their overall potential has so far been largely ignored.Rapidity, non-destructive nature, ease of use, and high-throughput measurements make the spectroscopic non-targeted approach an ideal tool for quality control operations, especially in the context of daily routine and screening analysis in the food industry, and as a possible substitute of traditional analytical techniques. Thanks to the technological development of the spectroscopic instrumentation, the availability of miniaturized and portable devices on the market is rapidly growing, and this will contribute to an additional growth of applications in the food sector. On the other hand, these analytical strategies in the official control of foodstuffs are still far from being effectively applied, largely due to the need of a strict validation to assure further reliability and robustness of results before implementation as standalone tools. For these reasons, standardisation of the working conditions, optimisation of the chemometric software, and creation of large databases for data-sharing and for encouraging greater cooperation between food scientists, represent important current research fields and future challenges to be faced.


Background: Catatonia is a syndrome comprising psychomotor, behavioral, and autonomous symptoms which may occur in the context of severe schizophrenic, affective, and other mental disorders or medical conditions. Treatment options include high dose benzodiazepines (lorazepam) and electroconvulsive therapy (ECT) with some evidence for the effectiveness of glutamate antagonists. However, due to a lack of randomized controlled studies in this severely ill population, evidence base is weak.Methods: On occasion of the case of a patient with treatment resistant catatonia in schizoaffective disorder, we developed the hypothesis of vagus nerve stimulation (VNS) being a potential therapy for treatment resistant catatonia.Results: Based on a selective literature search, we found a remarkable overlap of the pathophysiology of catatonia on the one hand and the putative mechanisms of action of VNS on the other hand in several domains: functional brain imaging, involved neurotransmitter systems, clinical, and theoretical. We thus decided to use VNS as a single subject clinical trial. During the 1-year-follow-up, we observed a fluctuating, but ultimately marked improvement of both catatonic symptoms and general psychopathology.Conclusions: We assume there is a sufficient hypothetical corroboration for the potential effectiveness of VNS as a long-term treatment in predominantly catatonic syndromes. This hypothesis could be tested in proof-of-concept clinical trials. Catatonia is a clinical syndrome with predominant psychomotor symptoms , which may be accompanied by affective, behavioral, and autonomous disturbances , 2. AccoVagus nerve stimulation (VNS) is an add-on treatment for treatment resistant depression with further potential applications in other psychiatric conditions . A recenA 46-year-old female patient diagnosed with schizoaffective disorder (this case was already described as part of a case series in Methfessel et al.; writtenThe pivotal stimulus for the idea to use VNS in treatment-resistant catatonic patients came from clinical experiences in patients with major depression. If these patients respond to ECT but experience early and frequent relapses, this might constitute an indication for additional VNS therapy. The study by Aaronsen et al.  found nuProceeding from these clinical considerations, we searched the scientific literature to identify possible overlaps between the pathophysiology of catatonia and putative mechanisms of action of VNS. Findings from different domains, especially brain imaging and involved neurotransmitter systems, confirm such an overlap and corroborate the potential utility of VNS in treatment resistant catatonia. For reasons of clarity, the corresponding findings will be depicted in Starting from a difficult-to-treat clinical case with treatment resistant catatonia and schizoaffective disorder, a hypothesis was developed postulating the potential effectiveness of VNS in catatonic syndromes. A selective literature search detected overlaps between the pathophysiology of catatonia on the one hand and the putative mechanisms of action of VNS on the other hand. Several lines of evidence corroborate the theoretical utility of VNS in catatonia. Findings from different domains, i.e., brain imaging, neurotransmitter systems, pharmacology, clinical, and theoretical considerations, ultimately led to the first-ever treatment trial of VNS in catatonia. Ultimately, a marked improvement of the patient's catatonic symptoms and general psychopathology could be observed and was followed by a fluctuating course after discharge from hospital with further need for intense and adapted maintenance treatment including ongoing VNS, frequent maintenance ECT and pharmacotherapy alongside general psychiatric and psychological care. However, both the clinical course in our patient, and the theoretical underpinnings of the hypothesis presented above are deemed sufficiently robust to justify further research on the role of VNS in catatonia. This hypothesis could be tested in proof-of-concept clinical trials. In doing so, both the obvious clinical heterogeneity and the presumably heterogeneous pathogenesis of catatonic syndromes will have to be considered as VNS might well be a treatment option for specific subgroups of patients with catatonia. These populations would have to be refined using clinical and/or biological markers as reliable predictors of VNS response. Due to the problems in recruiting these mostly severely affected patients, this will only be possible in larger multicenter studies.DZ developed the hypothesis and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RCW declared a past co-authorship with the author DZ to the handling editor.


In fact, these stem cell-derived neural aggregates resemble the three-dimensional (3D) cytoarchitectural arrangement of the brain overcoming not only the unrealistic somatic flatness but also the planar neuritic outgrowth of the two-dimensional (2D) in vitro cultures. Despite the growing use of cerebral organoids in scientific research, a more critical evaluation of their reliability and reproducibility in terms of cellular diversity, mature traits, and neuronal dynamics is still required. Specifically, a quantitative framework for generating and investigating these in vitro models of the human brain is lacking. To this end, the aim of this review is to inspire new computational and technology driven ideas for methodological improvements and novel applications of brain organoids. After an overview of the organoid generation protocols described in the literature, we review the computational models employed to assess their formation, organization and resource uptake. The experimental approaches currently provided to structurally and functionally characterize brain organoid networks for studying single neuron morphology and their connections at cellular and sub-cellular resolution are also discussed. Well-established techniques based on current/voltage clamp, optogenetics, calcium imaging, and Micro-Electrode Arrays (MEAs) are proposed for monitoring intra- and extra-cellular responses underlying neuronal dynamics and functional connections. Finally, we consider critical aspects of the established procedures and the physiological limitations of these models, suggesting how a complement of engineering tools could improve the current approaches and their applications.Cerebral (or brain) organoids derived from human cells have enormous potential as physiologically relevant downscaled  Known as brain or cerebral organoids, these constructs mimic the 3D structure of the brain  thanks tin vitro cultures, have been applied to study brain organoids, and most investigations are geared toward assessing their reliability as novel and more accurate human brain models. In particular, advanced imaging techniques . Then, we review the computational models employed to assess organoid formation, organization, and resource uptake . In the sections on Structural Characterization and Integrated Electrophysiological Approaches, the experimental approaches currently provided to characterize the structure and function of cellular networks within brain organoids are discussed, focusing on methods for studying the 3D architectural of single neurons and their morphological and electrophysiological connections at cellular and subcellular resolution. Finally, in the Conclusion, we suggest how the potential of these imaging, computational and electrophysiological tools can be combined with bioprinting, fluidics and biomaterial engineering within an integrated experimental and theoretical framework so as to establish a quantitative, reproducible and accurate in vitro model of the human brain for a diverse range of applications.Neuroscientists are aware of the urgent need of improving brain organoids in terms of reproducibility and oxygen/nutrient supply as well as of fully characterizing their structural and functional features  for the assessment of model goodness through a quantitative comparison with their Brain organoids are different from classical 3D cultures of neurons, which are known as neurospheres, neural spheriods or neuro-aggregates. The latter are generated from differentiated neural cells or their progenitors. The cells are usually cultured in non-adherent plates and they cluster together, growing in suspension rather than on the base of the plate. On the other hand, brain organoids originate from  stem cells which are cultured in conditions that promote differentiation and self-organization such that the cells spontaneously develop into various brain regions resembling the developing human brain. Hence, we can define an organoid as a mini-organ (or organ sub-region) which resembles the essential, albeit immature, structural features of its upscaled counterpart . Brain oAn established protocol for generating human brain organoids has been described by The protocol developed by Lancaster and co-workers paved the way for several other studies. Most of them observed that brain organoids become necrotic in their core at later stages of culture and healthy neurons are found only along the perimeter . RecentlAlthough, a wide variety of chemical patterning factors have been used to drive the neuronal differentiation of specific brain sub-regions  such as in vitro models characterized by co-culturing “building blocks”  describing distinct areas of the human brain , most of which are not well understood or easily observable . Since il trials .in vitro systems). One year later, using a carefully-judged combination of image processing tools and computational models of oxygen transport and consumption, Unfortunately, little effort has been made in implementing models of organoid growth and differentiation, and even less for brain organoids. In particular, it has been recently demonstrated by in silico and the in vitro outcomes, the same group demonstrated that the computational model can simulate the developing morphology of the organoid, suggesting that cell proliferation and a single inhibitory protein is enough to achieve organoid morphogenesis  to resemble the main architectural features of the brain allows the characterization of the mechanisms involved in brain formation such as proliferation, lineage specification and organ homeostasis. To this end, in vitro models. Such models are also instrumental for understanding the complex mechanisms underlying organogenesis in a dish and wider collaborations between in silico and in vitro modelers should be encouraged. Finally, customized versions of the computational and theoretical frameworks established in The combination of experimental procedures and computational models as proposed in the studies by Berger, Buske and Dahl-Jens and their respective co-workers offer several advantages for optimizing the design of more physiologically relevant Cerebral organoids resemble the main structural features of the human brain recapitulating its 3D cytoarchitectural arrangement. Characterizing the three-dimensional structural organization of the cells as well as neuronal shape, size, complexity and distribution and their physical/morphological cell-cell connections within organoids is important for establishing similarities with the human brain. Quantitative and precise morphometric measures  are eveNeuroanatomical features  are generally extracted using optical methods such as confocal, multi-photon and light sheet microscopy, suitable for imaging within samples at cellular (sub-micrometric) resolution . ConversOrganoid diameters range from 1 to 3 mm, as represented in Although, they were originally developed for whole animal perfusion, clearing protocols can be adapted for use on vessel-free, unperfusable samples, such as brain organoids, obtaining unprecedented representations of their 3D cellular structure. For instance, Renner et al. identified internal connections between the cortical areas which appear isolated in 2D non-clarified sections using the SWITCH clearing method and subsequent in-depth confocal imaging . Howeverin vivo animal models) are often qualitative and performed by visual inspection  imaging has been applied to the human brain organoids (2+ ions with genetically encoded calcium indicators or small molecules based on the aminopolycarboxylic acid BAPTA (Current/voltage-clamp and optogenetic methods can be used to investigate the neuronal dynamics involved at the single cell level. In order to functionally characterize neural aggregates, Calcium (Carganoids . Studiesrganoids  showed fid BAPTA . CalciumAlthough, calcium imaging allows functional characterization of single neurons and small aggregates, this approach does not provide a more complex analysis of the dynamics involved at the whole network scale. Technologies such as MEAs may be used to acquire the electrophysiology from multiple recording sites, as well as to record cell responses evoked by chemical  or electPrevious studies focusing on complex networks at the whole-brain scale of human neuroimaging  and at ain vitro model of the human brain. However, since this technology is relatively young (ad hoc work-flow overcoming the aforementioned limitations by combining the functional methodologies and imaging techniques illustrated in The development of stem cell-based organoids is one of the most fascinating and promising techniques for providing a physiologically relevant downscaled ly young , the genly young . As a reAll authors contributed to the preparation and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Introduction: PV implantation is indicated for severe PV regurgitation after surgery for congenital heart defects, but debates accompany the following issues: timing of PV implantation; choice of the approach, percutaneous interventional vs. surgical PV implantation, and choice of the most suitable valve.Timing of pulmonary valve implantation: The presence of symptoms is class I evidence indication for PV implantation. In asymptomatic patients indication is agreed for any of the following criteria: PV regurgitation > 20%, indexed end-diastolic right ventricular volume > 120–150 ml/m2 BSA, and indexed end-systolic right ventricular volume > 80–90 ml/m2 BSA.Choice of the approach: percutaneous interventional vs. surgical: The choice of the approach depends upon the morphology and the size of the right ventricular outflow tract, the morphology and the size of the pulmonary arteries, the presence of residual intra-cardiac defects and the presence of extremely dilated right ventricle.Choice of the most suitable valve for surgical implantation: Biological valves are first choice in most of the reported studies. A relatively large size of the biological prosthesis presents the advantage of avoiding a right ventricular outflow tract obstruction, and also of allowing for future percutaneous valve-in-valve implantation. Alternatively, biological valved conduits can be implanted between the right ventricle and pulmonary artery, particularly when a reconstruction of the main pulmonary artery and/or its branches is required.Hybrid options: combination of interventional and surgical: Many progresses extended the implantation of a PV with combined hybrid interventional and surgical approaches. Major efforts have been made to overcome the current limits of percutaneous PV implantation, namely the excessive size of a dilated right ventricular outflow tract and the absence of a cylindrical geometry of the right ventricular outflow tract as a suitable landing for a percutaneous PV implantation.Conclusion: Despite tremendous progress obtained with modern technologies, and the endless fantasy of researchers trying to explore new forms of treatment, it is too early to say that either the interventional or the surgical approach to implant a PV can fit all patients with good long-term results. The pulmonary valve (PV) regurgitation following the surgical repair of congenital heart defects is generally tolerated quite well in the immediate post-operative period and for the first few years . HoweverPV implantation reduces the right ventricular end diastolic volume, preventing pathological right ventricular remodeling, and restores the right ventricular function, preventing progressive dilatation toward right ventricular failure , 11–16.timing of PV implantation, particularly in asymptomatic patients , 13– 24choice of the approach, percutaneous interventional , 25–38 vchoice of the most suitable valve , 45–53.With regard to the need for a PV implantation in the presence of severe PV regurgitation, extensive debates accompany the following issues:In the presence of PV regurgitation, the presence of clinical symptoms clearly corresponds to class I evidence of indication for PV implantation in both the European  and NortPV regurgitation > 20%2 BSAindexed end-diastolic right ventricular volume >120–150 ml/m2 BSAindexed end-systolic right ventricular volume > 80–90 ml/mIn asymptomatic patients the criteria for indications for PV implantation, and particularly the choice of the best timing, are less clearly defined . Generalmorphology and size of the right ventricular outflow tract, in particular in the presence of a previously implanted trans-annular patchmorphology and size of the pulmonary arteries in the case a required enlargement or reconstructionpresence of residual intra-cardiac defectspresence of extremely dilated and dysfunctional right ventricle , 55–57The choice between the approach with percutaneous interventional , 25–38 vIf reconstruction of the pulmonary artery branches, or closure of residual intra-cardiac defects, or remodeling of a hugely dilated right ventricle, are required in association with the PV implantation, and these cannot be accomplished by a procedure of interventional cardiology, then the indication is given for surgical implantation of a PV or a right-ventricle to pulmonary artery valved conduit , 39–44.Regarding the choice of the most suitable valve to implant in PV position, the first choice is not for a mechanical prosthesis, despite the proven longevity, because of the risks of thromboembolic phenomena and the requirement for life-long anticoagulation , 48, witThe Trifecta®  valve, tri-leaflet stented pericardial valve designed for implantation in aortic position, with a supra-annular sewing cuff, pericardial-covered posts, pericardial leaflets taking origin from the exterior of the valve construct, increasing the effective orifice area for any external diameter, provided good results in PV position , 52.These characteristics should provide a reduced rate of structural valve deterioration over time, particularly because previous studies showed that PV implantation performed in young patients, with relatively smaller size biological valves, correlated with a higher risk of valve failure and need for early valve re-replacement , 46.Regarding the size of the valve to implant in PV position, our choice is to implant a relatively large size of the biological prosthesis, adequate for the effective orifice area of the native PV indexed for the patient BSA . This chAlternatively, biological valved conduits, such as homografts, bovine jugular veins, or Hancock® conduits , can be used as PV, with the implantation accomplished between the right ventricle and pulmonary artery. As this surgical approach is more extensive than the simple PV implantation, the choice of right ventricle to pulmonary artery biological conduit for PV regurgitation is particularly utilized when the reconstruction of the main pulmonary artery and/or its branches is required , 53, 58.Since the first clinical report of interventional implantation of a pulmonary valve in a right ventricle to pulmonary artery conduit with valve dysfunction  and our The major efforts have been made to extend the PV implantation in the presence of dilated right ventricular outflow tract, previously the major limit to the use of interventional technique –67. ThesAll the above reported techniques, very recently reported in the literature, clearly express the attempt of overcoming the current limits of percutaneous PV implantation, namely the excessive size of a dilated right ventricular outflow tract and the absence of a cylindrical geometry of the right ventricular outflow tract as a suitable landing for a percutaneous PV implantation.Despite the tremendous progress obtained with the availability of modern technologies, and the endless fantasy of researchers trying to explore new forms of treatment, at the moment it is too early to say that either the interventional or the surgical approach to implant a PV can fit all patients with good long-term results.AC provided the conception and design of the article, drafted the work and revised for the content, and agreed to be accountable for all aspects of the work.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.