Mass Spectrometry Imaging (MSI) has emerged as a powerful imaging technique for the analysis of biological samples, providing valuable insights into the spatial distribution and structural characterization of lipids. The advancements in high-resolution MSI have made it an indispensable tool for single-cell or subcellular lipidomics. By preserving both intracellular and intercellular information, MSI enables a comprehensive analysis of lipidomics in individual cells and organelles. This enables researchers to delve deeper into the diversity of lipids within cells and to understand the role of lipids in shaping cell behavior. In this review, we aim to provide a comprehensive overview of recent advancements and future prospects of MSI for cellular/subcellular lipidomics. By keeping abreast of the cutting-edge studies in this field, we will continue to push the boundaries of the understanding of lipid metabolism and the impact of lipids on cellular behavior. The significance of cellular biology has skyrocketed in the realm of biological sciences, particularly when it comes to analyzing cellular processes at a single-cell level, such as determining cell fate, aging, differentiation, and proliferation . Cells aMass spectrometry imaging (MSI) has been developed as a powerful imaging tool for analyzing biological samples at the molecular level . It alloSingle-cell lipidomics is a rapidly growing field that aims to study the lipid composition of individual cells. The significance of single-cell lipidomics lies in its ability to shed light on the biochemical processes occurring within cells and the vital role played by lipids in these processes. In addition to its fundamental importance, single-cell lipidomics also has numerous applications in the study of diseases and pathological conditions. For instance, lipid peroxidation, a chemical reaction that occurs when lipids in the cell membrane are oxidized by free radicals, leading to the formation of highly reactive and potentially harmful compounds known as lipid peroxidation products, has been implicated in several diseases and conditions, including neurodegenerative diseases, cardiovascular disease, cancer, and aging ,14. FurtLipids also play a key role in the functioning of organelles, which are subcellular structures that perform specific functions within the cell. The main organelles found in these cells include the mitochondria, nucleus, Golgi apparatus, endosomes (E), endoplasmic reticulum, lysosomes (LE), and peroxisomes. The study of organelle lipidomics, which involves the analysis and characterization of lipids in specific organelles, offers numerous benefits compared to examining the entire cell. The relationship between the distribution of lipids in the organelles of animal cells are shown in For single-cell/organelles MSI, the cells and organelles used in an experiment are mainly from cell cultures and tissues . DespiteThe tissue microenvironment contains richer biological information, which helps to study diseases more intuitively. Hence, it is meaningful to image a whole tissue section at a single-cell scale, which maintains the relationships of intercellular interactions in the tissue microenvironment. For whole tissue sections, the steps of sample preparation are the same as traditional MSI. Samples were obtained by cutting ~10 μm sections from frozen tissue or paraffin-embedded (FFPE) tissue. A high resolution MSI technique is the cornerstone of cell imaging for whole tissue sections, as the cells are crowded on the sections and too large a pixel may contain multiple cells.The average cell size ranges from 20 to 30 μm for organisms such as live cells and skin cells, with exceptions such as microglia, whose typical diameter measures 7 to 12 μm. The size of cellular organelles is even more diminutive, with structures like the endoplasmic reticulum and Golgi apparatus measuring less than 1 μm in diameter. Consequently, high spatial resolution mass spectrometry imaging (MSI) instruments are required to investigate the lipidome at a cellular or subcellular level. Microscope mode MSI utilizes a broad laser beam to irradiate the sample in a manner similar to that of a conventional microscope light source. The simultaneous acquisition and preservation of the distribution pattern of specific ions during ionization is a distinguishing aspect of this method, enabling resolution without the limitations imposed by ionization techniques ,25. AddiThe resolution of laser-based MSI can vary based on the type of laser focusing used. There are two main categories: far-field technology and near-field technology. Near-field methods involve pixel ablation at distances from the sample surface that are much smaller than one wavelength , while far-field methods ablate pixels at distances larger than this value . Far-field technology has its resolution limited by optical diffraction, whereas near-field technology does not have this limitation. Within near-field technology, there are further subcategories including apertureless tip enhancement techniques and aperture tip desorption techniques . The groMatrix-assisted laser desorption/ionization (MALDI) is a most widely used ionization technique of far-field-based MSI . The funP. caudatum . I. I53]. I nuclear  marker,  nuclear . The tas nuclear . This meThe utilization of image-based analysis strategies is commonly known for its high-throughput nature. However, in the context of Mass Spectrometry Imaging (MSI), the imaging process for the analysis of the lipidome of individual cells on a pixel-by-pixel basis can be rather lengthy. This is particularly true for microprobe-mode MSI, where higher resolution results in a prolonged imaging process and the requirement for ultra-high resolution when imaging at the cellular/subcellular level. Moreover, the implementation of tandem mass spectrometry further limits imaging throughput. Efforts have been directed towards accelerating high-resolution MSI, such as the implementation of compressed sensing, and dynamic sparse sampling ,56, withSensitivity refers to the ability to detect low levels of analytes within the sample and is often quantified by the limit of detection (LOD). High sensitivity is of utmost importance in high spatial resolution mass spectrometric imaging (MSI) due to its ability to detect molecular species with low abundance within cells and subcellular structures. This attribute is particularly relevant in biological systems where the molecular composition of cells and subcellular structures can exhibit significant variability, and where low-abundance molecular species can have a critical impact on cellular processes. The specific roles played by different isomers of phosphatidylcholine (PC) in cell signaling and membrane organization have been well documented in the literature. These lipid isomers can be distinguished through tandem mass spectrometry. However, the high spatial resolution of the instrument poses a challenge in terms of the abundance of ions that can be analyzed in a single pixel during MS analysis. The higher the resolution, the fewer ions that are available for analysis, making it difficult to perform multiple MS/MS analyses. The success of wide-coverage MS/MS analysis at the single-cell scale heavily relies on the sensitivity of the instrument. This is because the sensitivity of the instrument determines the minimum amount of sample that can be analyzed in a single pixel, as well as the accuracy and reliability of the results. A more sensitive instrument can detect low-abundance species, improving the coverage of lipid structure identification and enabling a more comprehensive analysis of cell signaling and membrane organization. In the context of single-cell or organelle analysis, sensitivity and the low abundance of molecules can significantly limit the variety of lipids that can be structurally identified. However, merely increasing sensitivity is not enough, as traditional data-dependent acquisition (DDA) tandem mass spectrometry analyzes only a few ions within a narrow isolation window at a time to identify their structures, while other analytes are wasted and can only be analyzed in subsequent injections. The development of data-independent acquisition (DIA) tandem mass spectrometry methods has increased the utilization rate of analytes with lower abundance to some extent, as multiple ions can be fragmented concurrently. However, the obstacle to the application of DIA lies in how to analyze the obtained mass spectra, and various deconvolution methods have been proposed to make it possible to use DIA mode MSI for the analysis of lipid spatial distribution ,66. In aThe integration of MSI with other imaging modalities offers a more comprehensive insight into cellular information, especially when MSI alone is not yet capable of providing sufficient detail for single-cell imaging. As previously mentioned, MSI faces difficulties in characterizing the size, shape, and boundaries of cells in tissue sections, which can be easily achieved through other imaging techniques such as fluorescence microscopy. However, the implementation of multimodal imaging presents several challenges, including the requirement for the preservation of the molecular composition of cells and the avoidance of significant ion suppression. The integration of multiple sets of data generated from different imaging modalities requires accurate alignment at the micron level, given the micron-scale resolution of cellular structures.2O cluster GCIB-SIMS and C60 SIMS. The multimodal MSI offers the potential to provide a more comprehensive understanding of cellular biology. However, it also presents a number of challenges that must be addressed in order to obtain accurate and reliable results. The development of robust and reliable multimodal imaging methods will significantly enhance our ability to study cellular biology at a high level of detail.The integration of multiple ionization methods of MSI has the potential to yield a more comprehensive analysis of substances. While each single ionization technique has its limitations, the combination of multiple MSI modes provides a broader scope of substance analysis. For instance, MALDI excels in detecting large molecules such as proteins, however it lacks the necessary spatial resolution. Conversely, SIMS boasts a high spatial resolution, yet the molecular ions produced are low in abundance and biased towards smaller molecules like metabolites. By integrating MALDI-MSI with SIMS-MSI, researchers have been able to enhance the information content and image quality of mass spectrometry imaging ,70. FurtIn recent years, advancements in mass spectrometry imaging (MSI) of high spatial resolution and sensitivity have paved the way for analysis in cellular/subcellular lipidomics. With improved techniques and methods for data analysis, MSI is now a powerful tool for complete cellular lipidomics analysis. By providing in-depth information on the spatial distribution and structure of lipids in individual cells, MSI has the potential to greatly deepen our understanding of metabolic diversity within tissues and the crucial role of lipids in regulating cellular behavior. The detailed characterization of biomolecules from single cells or organelles in tissues also requires tandem MS imaging, which may become the next climax of MSI. Certainly, instrumental and methodological innovation are necessary to support such research efforts. However, limitations such as the speed and sensitivity of MSI imaging, as well as the challenge of assigning measurement data to individual cells, may present barriers to the widespread application of MSI in cellular lipidomics. Nevertheless, these limitations also present opportunities for future development and improvement in the field, particularly in the development of computational methods . The int


Recent evidence has supported the hypothesis that amyotrophic lateral sclerosis (ALS) is a multi-step disease, as the onset of symptoms occurs after sequential exposure to a defined number of risk factors. Despite the lack of precise identification of these disease determinants, it is known that genetic mutations may contribute to one or more of the steps leading to ALS onset, the remaining being linked to environmental factors and lifestyle. It also appears evident that compensatory plastic changes taking place at all levels of the nervous system during ALS etiopathogenesis may likely counteract the functional effects of neurodegeneration and affect the timing of disease onset and progression. Functional and structural events of synaptic plasticity probably represent the main mechanisms underlying this adaptive capability, causing a significant, although partial and transient, resiliency of the nervous system affected by a neurodegenerative disease. On the other hand, the failure of synaptic functions and plasticity may be part of the pathological process. The aim of this review was to summarize what it is known today about the controversial involvement of synapses in ALS etiopathogenesis, and an analysis of the literature, although not exhaustive, confirmed that synaptic dysfunction is an early pathogenetic process in ALS. Moreover, it appears that adequate modulation of structural and functional synaptic plasticity may likely support function sparing and delay disease progression. Severe loss of spinal and bulbar motoneurons (MNs) is the main feature of different MN diseases, including amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy. ALS is a rare but severe and fatal disease with an incidence of approximately 2 people per 100,000 ,4,5.Despite considerable efforts in ALS research, the causes of the disease are still unknown and no effective cure is available, but many recent data are challenging the vision of a simple MN disease, suggesting that ALS could be caused by a network failure ,7.Although MN loss is the critical hallmark of ALS, given the specific vulnerability of these cells, other cell populations are involved, including corticospinal neurons as well as glial and muscle cells ,8,9,10, By modeling ALS incidence data, recent studies by Al-Chalabi and colleagues have proposed that ALS is a six-step process whereby the onset of symptoms occurs only after being exposed to a sequential series of risk factors ,15 and gIt is known, for instance, that an important role during pathogenesis could be exerted by plasticity occurring within neuronal circuitries. Similar to other well-known homeostatic systems, including those regulating blood pressure, glucose metabolism, or temperature, there are a variety of mechanisms regulating the activity of neural networks within an adequate range. One of these mechanisms is known as homeostatic synaptic plasticity, which serves as a modulator of synaptic strength in order to prevent excessive potentiation or depression of synapses within a given neuronal network, thus maintaining the efficiency and fidelity of signaling and avoiding cell suffering ,24,25,26These mechanisms may be responsible for adaptive responses that compensate for the progressive loss of function caused by neurodegeneration, thus delaying symptom onset ,35,36,37This comprehensive review is an overview of the experimental findings exploring the controversial role of synaptic plasticity and its failure in ALS, employing either human patients and autoptic samples or animal models of ALS. Despite the limitations of animal models, which only partially recapitulate the human disease, these studies have the advantage of better dissecting pathological events at the cellular and molecular levels and, importantly, facilitating observation of the pathological processes occurring during the presymptomatic stage. The events occurring at early stages during pathogenesis are extremely important because they demonstrate the resiliency of neural circuits, and they are probably the most important processes to be studied in order to dissect the nature of compensatory plastic changes during disease.Structural and/or functional modifications of specific synapses or large neuronal networks have been documented as disease-linked mechanisms either in patients or in different experimental models of ALS. These pathological changes of synaptic connectivity were found at all levels of the voluntary motor system, from the sensorimotor cerebral cortex down to the neuromuscular junction (NMJ) and skeletal muscle . DespiteSevere functional impairment and cell death take place in the cerebral cortexes of patients affected by ALS-FTD, but also in ALS patients with or without cognitive or behavioral symptoms ,4,5,93. Alterations of connectivity and synaptic function in the brain have also been reported in several animal models recapitulating different pathological aspects of ALS, and these studies have also proposed different cellular and molecular mechanisms. An increase of intrinsic cortical excitability has been reported in mice overexpressing an SOD1 mutation, but the pattern and timing of the cell-type specific alterations appeared to be intricate . In partMany other studies have proposed various molecular mechanisms of hyperexcitability and cortical dysfunction using different ALS mouse models. For instance, dysregulation of FUS protein and its cytoplasmic accumulation are signs of ALS and ALS/FTD, and mutations of the gene can cause the disease ,95. NormTDP-43 proteinopathy is present in the majority of ALS cases . Like FUTaken together, this collection of studies demonstrates the complex involvement of structural and functional plastic changes within cortical networks, which can either represent a piece of the pathogenic process, a compensatory mechanism demonstrating the functional resiliency of neural circuits, or both. Considering the central role of corticospinal neurons in the context of ALS pathogenesis, further research efforts are required to clarify the link between synaptic functions and the selective vulnerability of these neurons.The hippocampus and its connections with other brain areas are fundamental for the normal functioning of learning and memory processes, as well as for mood and other cognitive functions. Together with the above-described cortical modifications, functional deficits, cell death and plastic changes are also present in the hippocampal circuitry in different neurodegenerative disorders characterized by cognitive dysfunctions, including Alzheimer’s disease ,100,101,Neurogenesis is present in adult mammals but restricted to a few CNS areas, including the hippocampus. Adult hippocampal neurogenesis is an important form of plasticity occurring in the dentate gyrus, which is linked to learning and memory  and a vaMany other studies have used different animal models to dissect the role of hippocampal synaptic function and plasticity in ALS, applying a deeper mechanistic approach and obtaining more cellular and molecular details. For instance, alterations of synaptic functions in the hippocampus and neocortex have been reported in mice carrying an FUS mutation as a result of dysregulation of RNA trafficking and alterations of protein homeostasis, mitochondrial function, and axonal protein synthesis, thus causing dysfunctions in brain connectivity and cognition ,50,51,52ALS and FTD are also linked to TDP-43 proteinopathy, which is present in both familial and sporadic cases, where cytoplasmic aggregates of abnormally phosphorylated and ubiquitinated TDP-43 are present in neurons and glial cells of various CNS areas, thus causing neurodegeneration due to both toxicity and loss of function of mutant TDP-43 . In factSimilar considerations may arise from other studies showing an increase of hippocampal LTP in presymptomatic SOD1 transgenic mice ,67,68, wAs described above, ALS is a complex neurodegenerative disease characterized by both motor and non-motor dysfunctions, and functional impairments and neurodegeneration can be present at all levels of the central and peripheral nervous systems. However, the onset of disease is usually defined by focal neuromuscular deficits, with muscle weakness and atrophy accompanied by the death of defined subpopulations of spinal or brainstem MNs. Thus, the onset may be spinal, with muscle weakness affecting arms or legs, or bulbar, with early symptoms of dysarthria or dysphagia ,2. ThereStudies involving animal models have further investigated these initial findings obtained from patients. For instance, disease-linked changes in MN soma size were also observed in SOD1 transgenic mice. In particular, an enlargement of MN soma size has been observed as early as the presymptomatic stage of disease, followed by a dramatic size reduction below control levels at the end stage ,33, whicClear evidence of compensatory synaptic plasticity has been reported in the spinal cord of SOD1 transgenic mice, where cholinergic C-type synaptic terminals to the soma and proximal dendrites of MNs were preserved and increased in size, whereas the number of S- and F-type boutons progressively decreased together with MN degeneration, starting at the presymptomatic stage ,74. C-boAgain, structural and functional synaptic modifications can represent either a piece of the pathogenic mechanism or a trait of compensatory capability (or both). In any case, this capability could be considered as a putative therapeutic target under certain conditions.The ability of surviving MNs to compensate for the loss of an MN subpopulation and maintain motor function has also been demonstrated in a mouse neurotoxic model of MN loss. In this model, the partial removal of lumbar MNs caused an acute functional impairment that was followed by recovery, which conversely may have been linked to events of synaptic plasticity  occurring within the spinal circuitries and regulated by several factors, including TDP-43, sonic hedgehog, and other factors known to also regulate neural development and stem cell functioning ,79,80,81ALS patients frequently develop dysphagia and dysarthria as a consequence of bulbar MN death ,2, whereNMJ degeneration is an early event in ALS pathogenesis, starting before the onset of symptoms, and such alterations of the NMJ have been reported in several studies involving different animal models ,86,113. The mechanisms controlling NMJ synaptic modifications include the participation of Schwann cells ,117. In The temporal sequence of functional and morphological alterations of the neuromuscular systems would suggest a dying-back pattern of neurodegeneration, as NMJ alterations usually appear before MN death . HoweverRemarkably, a recent study using mouse and Drosophila models of MN loss provided direct evidence of NMJ homeostatic plasticity, which was mediated by the evolutionary conserved presynaptic ENaC channel. In fact, the deletion of the gene encoding ENaC in these models prevented NMJ plasticity and severely increased MN loss and disease progression . Other sStructural and functional synaptic alterations and plasticity may occur at all levels of the central and peripheral nervous systems and at all stages of ALS disease. Despite evidence showing that certain cell populations are more vulnerable to cell stress and degeneration, it remains unclear which of these cell types will degenerate first. The historical controversy between the dying-back and dying-forward hypotheses of ALS pathogenesis is still open ,131,132.Large cortical areas are subjected to anatomical and functional alterations in ALS, as both reductions  and incrThe NMJ is another crucial crossroad for pathological and adaptive processes in ALS. NMJ alterations and synaptic detachment are early events that often precede MN degeneration, and this is the main evidence supporting the dying-back hypothesis ,91,113, Controversial results also have been reported about hippocampal plasticity. In particular, reduced neurogenesis ,60, impaThe body of evidence presented here, although obviously incomplete, demonstrates that the roles of synaptic function and plasticity are extremely important in the context of ALS pathogenesis and that this research field is promising and currently growing. Importantly, activity-dependent synaptic plasticity has been implicated in the compensatory processes occurring after a traumatic brain or spinal cord insult, and therapeutic strategies aimed at potentiating this spontaneous capability of the nervous system have proven to be beneficial ,137,138.From the analysis of the literature presented here, it appears that synaptic function is a central aspect of ALS pathogenesis. Further efforts are necessary to understand how to correctly modulate these mechanisms in order to reduce the pathological aspects of the altered synaptic plasticity, thus contributing to reducing the rate of neurodegeneration and/or supporting the compensatory processes that are capable of sustaining function sparing, thereby delaying symptoms, reducing the severity of functional deficits, and prolonging survival.Until the identification of etiological factors and discovery of reliable early biomarkers or predictors, it is evident that therapies can only start after the onset of symptoms. Therefore, even the most effective therapeutic approach would only slow down the progression of disease and make the compensatory changes more efficient and long-lasting in order to overcome the functional deficits despite the permanent neuronal and synaptic loss. In this context, a better understanding of the role of early synaptic alterations, including cortical hyperexcitability and connectome alterations that can be revealed by non-invasive methods such as fMRI, would represent a future direction towards the development of methods for early presymptomatic diagnosis. Coupling early diagnosis with an effective therapy capable of stimulating synaptic plasticity would represent an ideal future scenario.


Despite recent improvements in cancer diagnostics, 2%-5% of all malignancies are still cancers of unknown primary (CUP), for which the tissue-of-origin (TOO) cannot be determined at the time of presentation. Since the primary site of cancer leads to the choice of optimal treatment, CUP patients pose a significant clinical challenge with limited treatment options. Data produced by large-scale cancer genomics initiatives, which aim to determine the genomic, epigenomic, and transcriptomic characteristics of a large number of individual patients of multiple cancer types, have led to the introduction of various methods that use machine learning to predict the TOO of cancer patients. In this review, we assess the reproducibility, interpretability, and robustness of results obtained by 20 recent studies that utilize different machine learning methods for TOO prediction based on RNA sequencing data, including their reported performance on independent data sets and identification of important features. Our review investigates the strengths and weaknesses of different methods, checks the correspondence of their results, and identifies potential issues with datasets used for model training and testing, assessing their potential usefulness in a clinical setting and suggesting future improvements. Cancer is the leading cause of death worldwide, and the overall burden of cancer incidence and mortality is expected to increase due to the growing population, aging inhabitants, and changes in the prevalence of risk factors . Key facThe development of array- and next-generation sequencing (NGS)-based whole-genome profiling techniques has enabled the rapid molecular characterization of cells or tissues, inspiring the establishment of several large-scale cancer genomics initiatives, such as ICGC and TCGA . These iVarious methods that utilize machine learning to predict the TOO based on NGS data have been developed recently, although the clinical use of such methods is still limited . AdvantaIn this minireview, we aim to assess the reproducibility, interpretability, and robustness of different NGS-based TOO prediction methods, including reported performance on independent data sets and identification of important features. Since the accuracy of the models depends on the input data type , we haveWe have conducted a literature search to identify studies that used RNA-Seq data to train machine learning models for cancer classification and TOO prediction. Studies that relied exclusively on miRNA sequencing were excluded from further analysis. Based on the aforementioned criteria, we selected 20 recently published studies for analysis.The majority of studies were based on deep learning, using neural networks of different architectures . SeveralThe models were trained on datasets comprising 9 to 40 cancer types (with a median number of 32 cancer types), and the number of sample points used for training ranged from 1,960 to 20,918 . All of the selected studies trained their models on either TCGA or ICGC data, with some studies including cancer sequencing data produced in-house  or data We have compared the classification accuracy of various models, which we defined as the number of correct predictions divided by the number of total predictions. In the cases where the classification accuracy was not reported, we have calculated it from the results described in the original publication. Cases where no prediction could be made were counted as incorrect predictions. Accuracy was the most commonly used measure of predictive performance in the studies surveyed in this minireview. While this measure can be influenced by class-imbalanced data, the influence of training dataset composition on measures of predictive performance is outside of the scope of this minireview.ρ = 0.0591, p-value = 0.7989). The prediction accuracy varies by tumor type, with some tumor types being more frequently mispredicted. Patterns of more frequent misclassifications among groups of cancers arising from the same organ , and/or among cancers represented by a small number of samples in the training set , as noted in multiple studies (In general, the analyzed models achieve high cross-validation prediction accuracy in the range of 73%–99% (with a median cross-validation accuracy of 95.5%;  studies . All of Out of the studies included in this minireview, only seven tested the performance of the developed models on an independent test set. Overall, the predictive accuracy of models on independent test data was lower than the cross-validation accuracy calculated on the data used for training the model, with only two studies attaining a prediction accuracy on an independent test set that was comparable to or higher than the one obtained with cross-validation . Jones eInterestingly, when testing the accuracy of the same model on test sets composed of both primary and metastatic samples, metastatic samples showed a lower prediction accuracy. For example, Furthermore, Several methods employed feature selection approaches to identify the most informative sets of genes for TOO prediction using different strategies to choose relevant genes. Two studies utilized various subsets of top-selected genes based on the Gini index from Random Forest models . SimilarTo estimate the correspondence of informative gene sets identified by various studies, we extracted gene lists provided by the studies that employed feature selection on the primary dataset. Studies selected between 36 and 4,861 unique genes, with the highest number of overlapping genes found between gene lists identified by We mapped the extracted gene lists to hallmarks of cancer  and GeneKLK3, a serine protease used as a serum marker in prostate cancer screening and disease monitoring, and PRAC, a highly expressed gene in prostate cancer (NAPSA, an aspartic proteinase expressed in type II pneumocytes. Its expression can be used to distinguish pulmonary lesions originating from primary lung adenocarcinoma or other primaries (IGFBP1, a hepatocyte-derived secreted protein required for normal liver regeneration by inhibiting proapoptotic signals (We further examined genes proposed by different studies as potential cancer signatures. Well-known prostate cancer signatures, including e cancer , have berimaries . Additio signals , as an iDPYS, were also identified by three more recent studies (BEST1 and new prostate and gastric biomarker SI, were either not found by other studies or were only identified by  studies  as imporDEFB1 gene was detected by four different studies ( studies , howeverIn this minireview, we analyzed 20 recent studies that employed machine learning to predict the TOO of cancers based on NGS data. Our goal was to assess their performance, reproducibility, interpretability, and robustness. We found that all of the analyzed methods exhibited very high prediction accuracy, ranging from 73% to 99%. This performance represents an improvement over currently used microarray-based methods, which have a prediction accuracy of 54%–100% . These fHowever, while the overall prediction accuracy of the models is high, it varies by tumor type, with tumors originating from the same organ or tumors that are underrepresented in the training set being more frequently mispredicted. This suggests that the accuracy depends more on the composition of the training set than on the method used for training the model. Researchers should, therefore, aim to assemble balanced datasets for model training and include as many samples of rare and underrepresented cancers as possible.Furthermore, most of the analyzed studies did not employ an independent test set, and the ones that did mostly showed a reduction in accuracy, especially for test sets obtained from metastatic patients or FFPE samples. Since CUP patients are, by definition, metastatic patients and FFPE tissues are still the most commonly available sample type for RNA-Seq, due to the cost-effectiveness of storage , both meIdentification of features important for prediction, implemented by several of the analyzed studies, could lead to novel biomarker discovery and discovery of genes whose expression is dysregulated in cancer, expand our current knowledge of mechanisms of cancer development and progression, identify potential actionable targets, and inspire novel treatment strategies. Indeed, most of the studies that employed feature selection identified at least 25% of actionable targets among their set of selected genes and showed that some of those genes are already known cancer signature genes. However, the overlap of gene lists provided by different studies is quite low, indicating that these results should be interpreted with caution. It is important to note that the majority of the analyzed studies employed filter methods, which select relevant features based on their intrinsic characteristics. Some used wrapper methods, where features are added or removed iteratively and scored based on their impact on the machine learning model’s performance, or embedded approaches, which combine properties of both filter and wrapper methods.Filter methods, while computationally less demanding, typically do not consider the subsequent classification model, often resulting in inferior performance compared to wrappers. In contrast, wrappers can be susceptible to overfitting and sensitive to parameter adjustments . RecentlFor example, in a study using ten microarray datasets for cancer classification, Gene Selection Programming , a metho


Glioblastoma (GBM) is a highly aggressive brain tumor, and its treatment is further complicated by the high selectivity of the blood–brain barrier (BBB). The scientific community is urgently seeking innovative and effective therapeutic solutions. Liposomes are a promising new tool that has shown potential in addressing the limitations of chemotherapy, such as poor bioavailability and toxicity to healthy cells. However, passive targeting strategies based solely on the physicochemical properties of liposomes have proven ineffective due to a lack of tissue specificity. Accordingly, the upregulation of transferrin receptors (TfRs) in brain tissue has led to the development of TfR-targeted anticancer therapeutics. Currently, one of the most widely adopted methods for improving drug delivery in the treatment of GBM and other neurological disorders is the utilization of active targeting strategies that specifically target this receptor. In this review, we discuss the role of Tf-conjugated liposomes in GBM therapy and present some recent studies investigating the drug delivery efficiency of Tf-liposomes; in addition, we address some challenges currently facing this approach to treatment and present some potential improvement possibilities. In normal, healthy cells, the process of cell growth and division is intricately controlled by hundreds of genes that promote proliferation, suppress it, and/or signal when cells should undergo apoptosis. Cancer cells are genetically mutated, rendering them unresponsive to many signals that control cellular growth and death refer to  1,2]. ,2. 1,2].Chemotherapy is a frequently employed treatment modality for cancer. It employs cytotoxic drugs which interfere with DNA synthesis and cell division to kill fast-growing cells . HoweverLiposomes are nanosized to microsized synthetic spherical vesicles made up of a phospholipid bilayer surrounding an aqueous spherical core, similar in structure to that found in cells ,13. EachMoreover, liposomes can be used to deliver drugs to specific cells or tissues as they can be targeted to bind to selected cells by modifying their surface with ligands that bind to specific receptors on the target cells, a technique known as active targeting. A wide range of molecules can be used to functionalize the surfaces of liposomes, including carbohydrates, proteins, peptides, aptamers, monoclonal antibodies (mAbs), and more ,23,24,25While extensive research has explored various aspects of GBM-targeted therapy, the majority of the existing literature focuses on targeting the TfR with a wide array of ligands. Building upon these reviews ,31,32, t+3) ions by binding to TfRs on the cell surface; the Fe+3/Tf/TfR complex then enters the cells through clathrin-mediated endocytosis [Transferrin (Tf) is an 80 kDa glycoprotein found in the blood that plays a crucial role in the transportation of iron throughout the body. It is primarily synthesized by the liver and secreted into the bloodstream. Tf binds to and carries iron ions from areas of absorption, such as the intestines, to areas of utilization, such as the bone marrow, where iron is necessary for the production of new red blood cells ,34. The ocytosis ,36,37. Two types of Tf receptors exist, labeled 1 and 2 . TfR1, also known as CD71, is the major TfR isoform and is widely expressed in many cell types. It plays a crucial role in the uptake of Tf-bound iron into cells. TfR1 is responsible for the endocytosis of Tf and its subsequent internalization into the cell, along with bound iron. It is highly expressed in cells with a high demand for iron, such as developing red blood cells, actively dividing cells, and liver and bone marrow cells. TfR2, the second TfR isoform, shares some structural similarities with TfR1 ,38,39. HWhen the Tf ligand binds to either of the TfRs, the complex is internalized through clathrin-mediated endocytosis (CME), which is one of the major pathways for receptor-mediated endocytosis and involves the formation of specialized vesicles called clathrin-coated vesicles (CCVs). These vesicles are formed at specific plasma membrane regions known as clathrin-coated pits (CCPs) ,42. ClatInitiation: CME is triggered by the binding of ligands, such as proteins, growth factors, aptamers, hormones, etc., to specific cell surface receptors. This interaction induces receptor clustering and the recruitment of signal-transducing adaptor proteins to the cytoplasmic domain of the receptors.Assembly of the clathrin coat: Adaptor proteins, such as AP2 (adaptor protein 2), bind to the cytoplasmic tails of the clustered receptors and recruit clathrin molecules to the CCPs. Clathrin is a triskelion-shaped protein that assembles into a lattice structure around the CCPs, providing structural support and driving vesicle formation.Formation of the clathrin-coated vesicle: the forming vesicle is constricted and eventually pinches off, forming a clathrin-coated vesicle containing the cargo molecules from the extracellular space.Vesicle fusion and cargo sorting: The clathrin-coated vesicles fuse with early endosomes, and the cargo molecules are delivered to their respective destinations. As cargo molecules enter early endosomes, they are either directed to recycling pathways back to the plasma membrane or sent to late endosomes and lysosomes for degradation.The attachment of Tf to a drug-loaded liposome surface and the binding of the Tf to a TfR induces the uptake of the liposomes into a cell. There are several methods to conjugate ligands onto liposomes, depending on the nature of the ligand and the desired conjugation strategy. The conjugation between the ligand and the phospholipid membrane occurs either directly or through a spacer molecule. Below are some commonly used methods of ligand attachment ,43,44,45Conjugation of the targeting moiety prior to the formation of liposomes: this approach involves the synthesis and purification of the lipid-moiety pair prior to the preparation of the liposome. Post-insertion method: In this method, liposomes are prepared separately and then the targeting ligands are conjugated onto the surface of the liposomes after their formation. Ligands can be attached to the liposomal surface through various chemical reactions, such as amine coupling, thiol-disulfide exchange, or carbodiimide chemistry refer to . Care muNon-covalent incorporation: ligands can sometimes be placed on the surface of liposomes via non-covalent interactions, such as electrostatic interactions and hydrophobic interactions.Click chemistry: This refers to a class of high-yield bioorthogonal reactions  that can be used to conjugate ligands onto liposome surfaces. Examples of click chemistry reactions include azide-alkyne cycloaddition (CuAAC) and strain-promoted azide–alkyne cycloaddition (SPAAC). Streptavidin–biotin interaction: biotinylated lipids can be incorporated into liposomes, and ligands that are conjugated with streptavidin can be attached to the liposomal surface via the strong and specific interaction between streptavidin and biotin.The choice of conjugation method depends on various factors such as the nature of the ligand, the stability requirements, the desired density of ligands on the liposomal surface, and the intended application of the ligand-modified liposomes ,46. It iThe hallmarks of cancer include uncontrolled cell division, evasion of growth suppressors, metastasis, and the ability to stimulate the growth of new blood vessels (angiogenesis) ,54,55,56It is important to note that the relationship between iron and cancer is complex and multifaceted, and research is ongoing to fully understand its implications. The role of iron in cancer development and progression can vary depending on the specific context, and it is a subject of further investigation in the field of cancer research. Due to the overexpression of TfRs on different cancer cells and their specific pathway characteristics, TfRs have emerged as a promising therapeutic approach for treating cancer. Surface conjugation with Tf molecules has been extensively studied and accounts for approximately 43% of the strategies utilized .INK4a gene, and PTEN gene deletion [Glioblastoma multiforme (GBM) is the most aggressive type of glioma, which is a group of tumors that originate from glial cells or their precursors in the central nervous system (CNS). Gliomas account for 80% of malignant brain tumors ,68. The deletion ,73,74,75deletion ,77. Howedeletion ,78. The deletion ,79. TherOne increasingly prominent strategy involves the use of targeted nanoparticles, which utilize ligands specific to receptors for receptor-mediated uptake ,81. AmonLam et al.  conducteIn another study, Jhaveri et al.  encapsulLi et al.  preparedIn another study, Lakkadwala et al.  conducteDespite the promising results reported on Tf-conjugated liposomes in GBM therapy, some challenges need to be addressed. For instance, many NPs-based experimental approaches do not reach clinical evaluation due to the complications introduced by the BBB . There aWith regard to toxicity, all the components making up a drug delivery system should be non-toxic and approved by the proper authorities . This isAs nanotechnology advances, a wide range of new materials is being developed for potential use in drug nanocarriers. However, obtaining clinical approval for these new materials can be a time-consuming process. As a result, researchers tend to concentrate on using materials that are already known and approved, which hinders the translation of novel materials into clinical practice ,94. The With respect to in vivo targeting ability, when NPs are administered systemically to transport substances across the BBB, there is a possibility that they may become coated with serum albumin, immunoglobulins, and other blood components. This coating can cause the NPs to be engulfed by macrophages and results in a substantial accumulation of NPs in the liver and spleen. Consequently, only a small portion of the drugs actually reach the brain tumors, leading to a significant reduction in the effectiveness of the therapy ,98. CoatVarious strategies have been proposed to mitigate the nonspecific adsorption of the protein corona. These include surface modification with stealth polymers such as PEG and the use of biomimetic membrane camouflage. Another intriguing approach involves pre-coating the NPs with specific proteins or peptides to regulate the formation of the protein corona. For instance, iron-mimicking peptides such as T10 and CTR can be used to facilitate the recruitment of the Tf corona onto NPs in situ. Upon systemic administration, these peptides bind to the naturally occurring Tf present in the bloodstream, forming a corona around the NPs’ surface. This in turn enables the selective transportation of NPs across the BBB and their internalization into the targeted tumor cells. Since these peptides attach to the nonbinding regions of the Tf molecule, this binding event does not disrupt the biological function of endogenous Tf ,90,110.Intranasal (IN) delivery has been extensively researched as a method of delivering drugs for various medical conditions. Recently, there has been a particular focus on using IN delivery to bypass the BBB and directly target the brain, and treat central nervous system (CNS) disorders. The nasal route offers several advantages for drug delivery because it is easily accessible, highly vascularized, and allows for lower medication doses because it can circumvent hepatic metabolism; the aforementioned advantages make IN particularly relevant in GBM therapy. The irritation to the nasal cavity and mucociliary clearance associated with IN delivery can be overcome by using nanocarriers, which are biocompatible formulations and are easily absorbed by the nasal mucosa ,112. SemAnother method proposed to facilitate the transport of chemotherapeutic agents across the BBB involves the disruption of the BBB by using focused ultrasound (FUS) ,90. ReseMoreover, Tf as a targeting moiety against the TfR suffers from certain limitations such as loss of specificity due to abundance of endogenous Tf. The presence of plasma Tf molecules can compete with Tf-conjugated NPs for binding to the TfR, leading to a decrease in targeting specificity and therapeutic efficacy. Furthermore, the TfR is also expressed on the surfaces of normal cells; therefore, Tf-conjugated liposomes may enhance the off-target uptake of chemotherapeutics and cytotoxicity to these normal cells . The offIn recent years, single chain fragment variables (scFvs) have emerged as an alternative to antibodies to target the TfR. These scFvs retain the binding specificity of antibodies while demonstrating improved pharmacokinetic properties. Their smaller size allows for better tissue penetration and faster clearance from blood and tissues. Although antibodies and their fragments offer several advantages, their use in clinics would result in a substantial rise in treatment expenses. As a result, there have been extensive efforts to explore more economical alternatives ,120,121.Recently, NPs have emerged as a promising therapeutic alternative capable of overcoming the shortcomings of chemotherapy, mainly poor biodistribution and significant toxicity to healthy tissues. However, passive targeting strategies based solely on NPs’ physicochemical properties have proven ineffective due to the lack of tissue specificity. To address this issue, active targeting strategies based on the overexpression of the TfR in brain tissue were developed. Currently, targeting the TfR remains one of the most popular approaches for enhancing the delivery of anti-cancer therapeutics to GBM. Despite scientific advancements, the progress in clinical trials has been limited primarily due to the high costs and complex production methods associated with NP-based therapeutics. Further research is necessary to optimize TfR targeting and to explore the full potential of TfR-targeting liposomes for drug delivery in GBM. Such advancements would significantly impact cancer research and ultimately contribute to improving the quality of life and survival chances of GBM patients.


Understanding the subtle signs of carpal instability and other unique injury patterns in the wrist is a critical skill for radiologists. Proper patient management and outcomes are directly dependent on the accurate interpretation of wrist imaging studies. This review will provide a detailed overview of typical imaging features of carpal trauma and instability, management, and complications, using multimodality imaging and original medical illustrations. A detailed overview of the osseous, ligamentous, arterial anatomy of the wrist, arcs of Gilula, and zones of vulnerability will be provided. Carpal fractures, dislocations, special radiographic views, and imaging pearls will be discussed. Instability patterns and the myriad of associate abbreviations  will be clarified. Expected outcomes, potential complications, and management will be reviewed. The carpus is a complicated structure composed of multiple anatomic structures which coordinate together for proper biomechanical function. With so many integral structures allowing for a comprehensive range of motion, direct trauma and injury to any of these structures may lead to wrist instability or dysfunction, which can eventually progress to disability .Investigation of the carpus following trauma and instability symptoms presents a challenge for radiologists, given the complexity of the involved structures and the importance that the report has for patient outcomes. Proper patient management and outcomes are intrinsically tied to the accurate interpretation of wrist imaging studies. Recognition of carpal instability and its associated fractures on initial imaging studies in the acute setting is paramount to the diagnosis and management of these injuries, although diagnostic wrist arthroscopy also plays an important role. Failure to identify these injuries can contribute to long-term complications, including but not limited to degenerative arthritis, nonunion, and even avascular necrosis . These cIn this article, we discuss the clinically relevant anatomy of the carpus, illustrate common carpal fracture and dislocation patterns on imaging, detail imaging features of carpal instability, and review expected clinical outcomes, potential complications, and common management strategies.Proximal (first) arc—smooth line along the proximal surfaces of the scaphoid, lunate, and triquetrum.Middle (second) arc—smooth line along the distal surface of the scaphoid, lunate, and triquetrum.Distal (third) arc—smooth line along the proximal surface of the capitate and hamate, closely paralleling the second arc.The wrist includes the distal ends of the radius and ulna, eight carpal bones, and proximal bases of the metacarpal bones . The carIn a biomechanically normal wrist, there should be no irregularities in the contour of these arcs when traced. Any step-offs/disruptions in these lines suggest a ligamentous injury or fracture at the site of the broken arc . ExceptiThe intrinsic (interosseous) and extrinsic (capsular) ligaments of the wrist are essential for stability and mobility. Intrinsic ligaments are defined as those attachments that connect one carpal to another. These include the scapholunate ligament, lunotriquetral ligament, midcarpal ligaments, and distal carpal row interosseous ligaments, of which the scapholunate and lunotriquetral are the most important. In function, the scapholunate ligament resists volar flexion of the scaphoid and dorsal extension of the lunate. The lunotriquetral ligament balances proximal carpal row motion in the opposite direction. Extrinsic ligaments are defined as those that connect the carpal bones to adjacent structures, such as the metacarpals and distal radius and ulna. The extrinsic ligaments provide gross stability to the wrist, whereas the intrinsic ligaments contribute more towards fine wrist motion .The carpal bone blood supply to the scaphoid, lunate and capitate is unique in that the arteries enter the distal half of each bone. This is important as this puts the proximal portion of the bone at risk for ischemic injury and avascular necrosis when the bone is fractured. The arterial anatomy consists of the radial and ulnar artery branches in addition to the anterior interosseous branches supplying the arches. The radial artery traverses the lateral aspect of the wrist and provides blood supply largely to the dorsal structures of the hand. The ulnar artery traverses along the medial aspect of the wrist in the Guyon Canal, composed of the deep palmar branch that continues in the Guyon Canal while the superficial palmar branch that exits the canal and crosses the surface of the hypothenar muscles and deep to the palmar aponeurosis, providing blood supply to the digits. Although variable in its presentation, the superficial branch is particularly vulnerable to injury and can be compressed against the hook of the hamate .A basic standard radiographic series of the wrist includes PA, lateral, and oblique views. These views are sufficient to identify most pathology, however the use of stress radiographs can provide additional valuable information. Commonly obtained stress views of the wrist include clenched fist and ulnar deviation views, both of which allow for a more sensitive dynamic assessment of scapholunate instability. The ulnar deviation view also tensions the scaphoid into a more upright position for better visualization of its long axis, similar to an angulated scaphoid view.Lateral wrist radiographs provide a wealth of information in the assessment of carpal alignment but are susceptible to suboptimal projection at the time of imaging which frequently limits assessment. A well-positioned lateral wrist radiograph should demonstrate the volar surface of the pisiform midway between the ventral surface of the capitate and distal scaphoid. When this has been accomplished, intercarpal alignment is most accurately demonstrated. In this scenario, with the wrist in the neutral position (neither flexed nor extended) a line that bisects the center of the articular surface of the radius drawn along the long axis of the radius should align with the axis of the lunate and capitate. Deviation of the lunate or capitate away from this line is an indicator of carpal instability . When thPlain radiographs are the most common initial imaging modality for identifying carpal fractures and instability, and cross-sectional imaging is usually not required. However, in the setting of uncertainty or negative radiographs, obtaining a computed tomography (CT) or magnetic resonance (MR) imaging can add further details to the trauma evaluation of the wrist. In one study, plain radiographs had a sensitivity and specificity of 69.7% and 83.3% for the detection of carpal fractures in the emergency setting, in contrast to CT scans having a sensitivity and specificity of nearly 100% when used in conjunction with a physical exam and clinical assessment . CT shouIndeterminate initial radiographic findingsDetermination of extent of fracture/dislocation for preoperative surgical planningHigh index of suspicion for carpal fracture with negative initial radiographic findingsTraumatic carpal fractures and dislocations are common in young, active patients. These types of injuries cause pain, disability, and decreased productivity. Conventional radiography is still the main imaging modality for evaluating and identifying suspected carpal traumatic injury . HoweverMultidetector CT can easily show the extent of carpal fractures and dislocations, often showing fractures that are not visible on radiographs . In addiThe most common carpal bone to fracture is the scaphoid. Classically, patients present pain in the anatomical snuff box after a hyperextension trauma. This is a common occult fracture on plain radiographs, with up to 65% going undetected and thus misdiagnosed clinically as a sprain . InadequThe majority of scaphoid fractures (70%) occur through the waist of the scaphoid, with 10% of scaphoid fractures involving the distal third, and 20% involving the proximal third  2,14]. . 14]. WiCommon scaphoid fracture complications include avascular necrosis and nonunion . AvasculConservative management of scaphoid fractures consists of cast immobilization across the wrist, including the first metacarpophalangeal joint. This is the most widely used management strategy for scaphoid fractures, particularly when the fracture is stable ,5,19. LoThe next most common carpal fracture is the triquetral fracture. Most of these occur on the dorsal aspect of the triquetrum and are best identified on the lateral radiograph at the dorsal aspect of the triquetrum and are best identified as the so-called “pooping duck sign”. These result from compression of the ulnar styloid process and the dorsal aspect of the triquetrum during forceful ulnar deviation and wrist hyperextension ,5. TriquTrapezium fractures are divided into body and ridge fractures. Body fractures are often associated with fractures of the base of the first metacarpal . These aThe pisiform has the potential to fracture in a multitude of patterns depending on the mechanism of injury. A direct blow is the most common, while a transverse fracture can be seen with resisted hyperextension, as the pisiform exists as a sesamoid within the flexor carpi ulnaris tendon ,11. All Like the trapezium, hamate fractures are subdivided into body and hook fractures. Body fractures occur with a variety of fracture patterns without any specific difference in management related to the fracture types . For nonFractures of the capitate usually occur in conjunction with other fractures. Although isolated fractures do occur, they are quite rare. The proximal pole of the capitate, like the scaphoid, is vascularized via a retrograde blood supply from the distal pole and is consequently vulnerable to avascular necrosis of the proximal component . CapitatLunate fractures are categorized as volar pole, dorsal pole, transverse, osteochondral, and transarticular . Volar pThe trapezoid is the least commonly fractured carpal bone, as it exists in a relatively protected location . FracturCarpal and carpometacarpal dislocations and fracture-dislocations are infrequent but severe injuries, which can be overlooked both clinically and radiographically . This haNormal alignment of the carpus should always be assessed on neutral lateral view , as prevThe scapholunate angle is formed between a line drawn along the axis or volar surface of the scaphoid and a line through the axis of the lunate, with the normal range being 30–60°  [3]. TheThe intrinsic ligaments of the wrist are deep, interosseous ligaments spanning between adjacent carpals. Many have components of both true ligaments (with collagenous fibers and fascicles) and functionally less important areas of fibrovascular or fibrocartilaginous tissue. These are intimately related to the extrinsic ligaments in most cases. The key intrinsic ligaments are the scapholunate ligament (SLL), the lunotriquetral ligament (LTL), and the scaphotrapeziotrapezoid ligament (STTL) . The scapholunate ligament can be thought of as having three separate components: proximal, dorsal, and volar  2,14]. ,14. 2,14The lunotriquetral ligament can also be thought of as having three separate components as well: volar, dorsal, and proximal  8,14]. ,14. 8,14The extrinsic ligaments are those that arise from the radius and ulna and insert onto the carpal bones ,26. MoreThe dorsal capsular ligaments  include The volar radiocarpal ligaments include the radial collateral ligament (RCL), radioscaphocapitate ligament (RSCL), short radiolunate ligament (SRL), and long radiolunate ligament (LRL) . The RCLThe volar ulnocarpal ligaments  include Lastly, the volar midcarpal ligaments include the arcuate and deltoid ligaments that are further subdivided into radial and ulnar limbs. The scaphocapitate ligament (SCL) arises from the ulnar aspect of the distal pole of the scaphoid, deep to the RSCL, and extends to the volar capitate body. It represents the radial limb of the arcuate ligament. The triquetrohamocapitate ligament (THL) extends from the triquetrum to the capitate, across the hamate. It represents the ulnar limb of the arcuate ligament. Some describe this as the distal band of the palmar scaphotriquetral ligament, which is another mid-carpal capsular ligament superficial to the SCL and THL ,27. The In general terms, carpal instability (CI) may be categorized as dissociative (CID), non-dissociative (CIND), combined/complex (CIC), or adaptive (CIA) . These gThe various patterns of carpal instability exist as a spectrum which ranges from frankly obvious instability (acute fracture-dislocation) all the way to nearly normal with only a clinical complaint and normal imaging and physical exam. The Watson classification categorizes carpal instability in relation to imaging in five stages. Stage 0 is termed ‘normal’, described as an asymptomatic patient with normal function of the carpus. Stage 1 is termed ‘pre-dynamic’, described as wrist pain with symptoms of instability; however, the imaging is normal. Stage 2 is termed ‘dynamic’, described as pain with dynamic or stress imaging demonstrating an imaging abnormality. Stage 3 is termed ‘static’, described as pain with abnormal imaging without stress. Stage 4 is termed ‘posttraumatic arthritis’, which includes scaphoid nonunion advanced collapse (SNAC) and scapholunate advanced collapse (SLAC) .Perilunate injury involves both intrinsic and extrinsic ligaments and results in a fairly predictable order of progression when the wrist is loaded in extension, ulnar deviation, and intercarpal supination. The typical history is a fall onto a dorsiflexed wrist , and the injury progresses through four distinct stages depending on the magnitude of the traumatic force that it sustained. The first stage consists of scapholunate dissociation a. As theThe previously mentioned ligamentous perilunate injuries are considered “lesser arc” injuries, but each stage may occur as a greater arc injury, which has a trans-osseous  path of biomechanical failure  instead Management of stage two and three perilunate dislocation consists of immediate closed reduction in which axial traction is used and posteroanterior pressure applied while the hand moves from extension to flexion to bring the capitate back into the lunate fossa. An audible clunk usually signifies reduction. Management of stage four injuries consists of closed reduction with axial traction and anteroposterior pressure applied while the hand moves from flexion to extension to bring the lunate back into the lunate fossa between the capitate and radius . Of noteDorsal intercalated segment instability (DISI) occurs when there is disruption of the SLL . The “inSimilar to the role that disruption of the SLL plays in DISI, volar intercalated segment instability (VISI) deformity occurs when there is disruption of the lunotriquetral ligament (LTL) . BecauseMidcarpal instability (MCI) is a confusing, uncommon and somewhat poorly understood topic with a wide spectrum of information regarding its etiology, mechanism, terminology, and treatment . It is aIntrinsic MCI is further broken down into palmar, dorsal, and combined . In palmThe role of imaging in patients with carpal fracture and instability has never been greater, continuing to be integral in the diagnosis and direction of care. Long-term outcomes are heavily tied to the radiologist’s ability to adequately identify and characterize the extent of the carpal instability and injury, ensuring the correct treatment plan is initiated, particularly in those that require corrective surgical management. With the goal of decreasing missed diagnoses and poor clinical outcomes, the importance of the radiologist’s comprehension of the different carpal instability patterns remains crucial.


This review examines the utilization of telemedicine in sarcoma care during the COVID-19 pandemic. The authors conducted a systematic literature review of peer-reviewed studies following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The findings demonstrate the potential of telemedicine to improve clinical and psychological outcomes for sarcoma patients. This development builds on the progress of tele-oncology and telerehabilitation have shown significant progress over the past decade, with the COVID-19 outbreak exerting an accelerating effect on their adoption. Telehealth applications in sarcoma care include remote consultations, psychological support and virtual symptom assessment. Both healthcare providers and patients have reported satisfaction with telehealth services, at levels comparable to in-person visits. The study concludes that telehealth offers opportunities for tailored, individualized care and is likely to play a crucial role in sarcoma patient management in the post-COVID-19 era.Background: The COVID-19 pandemic has created a challenging environment for sarcoma patients. Most oncology societies published guidelines or recommendations prioritizing sarcoma patients and established telehealth as an efficient method of approaching them. The aim of this review is the assessment of current evidence regarding the utilization of telemedicine in diagnosis, treatment modalities, telerehabilitation and satisfaction among sarcoma patients and healthcare providers (HP). Methods: This systematic review was carried out using the databases PubMed and Ovid MEDLINE according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Results: The application of telemedicine to the management of sarcoma has yielded improved clinical and psychological outcomes. Specifically, significant progress has been demonstrated in the areas of tele-oncology and telerehabilitation during the last decade, and the COVID-19 outbreak has accelerated this transition toward them. Telehealth has been proven efficient in a wide spectrum of applications from consultations on physical therapy and psychological support to virtual care symptom management. Both HP and patients reported satisfaction with telehealth services at levels comparable to in-person visits. Conclusions: Telehealth has already unveiled many opportunities in tailoring individualized care, and its role in the management of sarcoma patients has been established in the post-COVID-19 era, as well. Telemedicine is a compound word composed of tele-  and medicine. It is not certain when telemedicine was initiated as a concept and practice, but it has been reported that in 1906, Eindhoven, the inventor of the electrocardiogram, published a paper highlighting its potential telemedical applications .The potential of telemedicine in cancer care has been recognized over the last few decades. On the one hand, telemedicine was seen as a way to bridge the gap between the increasing number of cancer patients and the limited number of cancer specialists. On the other hand, particular components of specialized cancer investigation and care are greatly suitable for a remote approach. An additional aspect of telemedicine with a major potential in oncology, a field that increasingly relies on the constant renewal of treatment guidelines, is the remote education of healthcare practitioners .In early 2020, the scientific community and the general public were astonished to learn about the successful telemedical management of venous thrombosis during space flight . Only a During the pandemic, tele-oncological services were deployed in the entire spectrum of cancer care from primary prevention and screening to the follow-up of patients undergoing surgery, radiation therapy (RT), chemotherapy or immunotherapy. In this sense, telemedicine contributes to both safe and effective cancer care in the sense that follow-up was not interrupted, while vulnerable cancer patients were not exposed to the risk of contracting infections and concomitant complications. Examples of tele-oncology during the pandemic include the use of virtual tumor boards, remote monitoring of chemotherapy, and telepsychiatry for managing anxiety and depression. Relevant scholarship has also touched upon the facilitators and barriers to telemedicine adoption, which include access to technology and digital literacy, data protection and reimbursement policies .Evidence from interviews involving healthcare workers and patients suggested that tele-oncology was superior to standard care in terms of convenience and reduced travel time and costs. Providers also reported improved patient engagement and satisfaction with telehealth, while survivors reported feeling safer and more comfortable at home. However, they also faced technical difficulties and limitations in physical assessments. Some providers also reported feeling less connected to their patients and were concerned about missing important nonverbal cues. On these grounds, it is expected that telemedicine will continue to play an important role in cancer care, particularly for survivors and for patients residing in remote communities .Tele-oncology for sarcomas often requires a multidisciplinary approach for proper management. Sarcomas are a diverse group of tumors that can arise from bone or soft tissue, and they represent less than 1% of all adult cancers and 15% of all pediatric cancers. The prognosis for sarcoma patients is highly dependent on the stage and histologic subtype of the tumor, with 5-year survival rates ranging from less than 10% for advanced metastatic disease to over 80% for early-stage disease ,10. One In a world gradually adopting tele-oncology, the need for ongoing evaluation and improvement of telehealth technologies and workflows is pivotal. The aim of this review is the assessment of current evidence regarding the utilization of telemedicine in diagnosis, treatment modalities, telerehabilitation and satisfaction among sarcoma patients and healthcare providers (HP).The systematic review followed the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The protocol has not been registered. Data collection has been performed using the PubMed and Ovid MEDLINE databases with a defined search strategy from 2010 to date, according to the PRISMA recommendations . Moreoven = 327) and screening titles and abstracts (n = 901), we deemed 176 studies eligible for full-text assessment. After removing those of them whose full text was not available in English or at all (n = 11), we examined the full text of 165 studies and eventually included 141. A PRISMA search flow diagram describing the search and selection process in detail can be found in Our search yielded 1228 potentially eligible studies. After removing duplicates  were observed in the mean weekly imaging case volumes after the pandemic onset, with a total volume decline of 24,383  in 2020 versus 33,913  in 2019. However, the impact on imaging volume varied by modality type. The greatest declines occurred in nuclear medicine (85%) and magnetic resonance imaging (MRI 74%), followed by ultrasound (US 64%), computed tomography (CT 46%) and X-ray (22%) ..133].On 5 May 2023, the Director General of the WHO declared the end of the COVID-19 pandemic as a global health emergency . By thatThe opportunities of telemedicine include (a) improved access to specialists for people residing in remote areas; (b) remote and potentially more regular consultations and monitoring, given the decrease in transportation time and costs; and (c) pragmatic and/or home-based clinical trials, where patients can be monitored in their daily living settings in accordance to the principles outlined through the concept of living labs ,135. TheThe aforementioned advantages apply in the context of pandemic preparedness and broader health crises. In the event of emerging epidemics, telecare, a concept that is actively researched by health bodies such as the WHO or regulatory authorities such as the European Commission, could provide for the continuity of care for patients with sarcoma. As a matter of fact, both telemedicine and non-communicable diseases (NCDs) such as cancer consist of the cornerstones of the EU4Health Programme, the European strategy for containing the cross-border transmission of pathogens .Broader crises with dire implications in healthcare, such as war, have shown that telemedicine holds great potential in cancer care for internally displaced populations and refugees. The effectiveness of telemedicine intervention in this case is certainly subject to limiting factors such as the availability of telecommunications and injuries sustained within the conflict either on the patients’ or the healthcare providers’ side . These lOverall, telemedicine has the potential to improve the diagnosis and care of sarcoma patients by improving access to care, reducing treatment delays, and improving patient outcomes. However, more research and guidance in the form of practice guidelines is needed to fully understand the potential benefits and limitations of telemedicine and champion good remote practices in sarcoma care.The COVID-19 pandemic has been associated with a detrimental impact on health services. On the other hand, every cloud has a silver lining, and thus, the COVID-19 outbreak has accelerated the adoption of telehealth and has taken the digital transformation of medicine to a whole new level. The development of telehealth has the potential to rejuvenate daily practice given that its implications will affect a wide spectrum of cancer patients either under active treatment or surveillance. This new reality can apply even to malignancies of surgical interest, such as sarcomas. The maintenance and evolution of telemedicine can be proven beneficial to this special patient population in terms of QoL beyond the pandemic, as well. However, further investigation is required to assess sarcoma patients’ experience of the emerging electronic platforms, as well as to establish their non-inferiority compared with conventional clinical practice.


Non-surgical approaches have been proposed in the management of mandibular fractures, especially in children, but there is a lack of clear guidelines on the clinical indications of conservative approaches. The aim of this scoping review is to provide the available evidence of the role of the orthodontist in the management of mandibular fractures. The PRISMA-ScR guidelines were followed to select eligible articles from the PubMed, Scopus, and Web of Science databases according to precise inclusion criteria. The research questions were formulated as follows: “what is the scientific evidence concerning the rule of orthodontists in the management of mandibular fractures” and “the preferential use of the direct bonding technique with orthodontic brackets rather than rigid arch bars”? Seventeen articles were included. Five articles presented the use of removable acrylic splints or functional appliances, six articles concerned the employment of cemented acrylic or rigid splints, and six articles described the management of mandibular fractures in adults and children using orthodontic brackets or mini-screws. Most of these techniques have been employed in children and growing subjects, while fewer data were available regarding conservative treatments in adults. Preliminary evidence suggests that condylar and some minor parasymphyseal fractures in children may be managed with conservative approaches. In adults, minor condylar and stable body mandibular fractures with minimal displacement have been reduced similarly. However, there are no sufficient elements that could suggest the preferential use of orthodontic brackets over rigid arch bars in adults. Further randomized and non-randomized clinical trials with long follow-ups will be needed to better define the clinical indications of the orthodontic approaches in the management of mandibular fractures based on severity, location, and age. The mandible is a U-shaped bone that connects to the cranial base at the temporomandibular joints and functions as an interface with the maxilla through the oral occlusion ,3,4,5,6.The mechanism of the injury is crucial to evaluate since it gives the doctor a hint as to whether there are any concurrent injuries that can impede healing or call for a different type of therapy ,17,18,19Open reduction internal fixation (ORIF) has emerged as the gold standard of treatment for achieving anatomic reduction for a range of mandibular fractures, including condylar head fractures, despite the therapeutic value of a closed reduction of mandible fractures with mandibulomaxillary fixation (MMF) ,24. ORIFA separate discussion should occur regarding children and growing subjects. The occurrences of facial fractures reach a peak throughout puberty and adolescence due to increased sport activity ,47,48,49In this regard, the aim of this scoping review is to provide the available evidence of the role of the orthodontist in the management of mandibular fractures.This scoping review followed the PRISMA-ScR guidelines . Using aIn the present scoping review, the selection of articles was based on precise inclusion and exclusion criteria. In particular, articles in English, without timeline restrictions, were only included if they presented at least one conservative method of orthodontic mandibular fracture management. With regard to the study design, the following publications were taken into consideration: randomized and non-randomized controlled clinical trials, cohort studies, case-control studies, cross-sectional studies, retrospective studies, case reports, and case series. On the other hand, all articles not relevant to the orthodontic management of mandibular fractures, not available in English and with the full text, and some study designs  were excluded.Two independent reviewers were involved in the article selection processes. After removing duplicates, papers were initially screened for titles and abstracts. The full-text versions of all articles satisfying the inclusion criteria were further analyzed. After comparing the final results, if any disagreement occurred a third reviewer was involved. The following information was taken from the chosen articles: the study and the year of publication, the aim, the sample (including age), the design of the study, the site of the fracture/s, the approach, and the main findings.The search strategy led to the identification of a total of 1256 records in the PubMed (n = 552), Scopus (n = 497), and Web of Science (n = 207) databases . Next, 5Regarding the study designs, most of the articles were case reports ,66,68,71In contrast to the intermaxillary fixation (IMF) approaches, which are usually managed by a maxillofacial surgeon, such as conventional or mini-screw-retained arch bars and occlusal splints fixed with circummandibular wiring, from the results of the present review, among the conservative IMF methods that are manageable by orthodontists and pediatric dentists, the following approaches emerged: (1) mini-screws and elastics ,66,70; . From thMany authors presented the use of acrylic splints that were removable ,68,72 orQadri GW et al.  employedOther authors described fracture reduction with the use of fixed orthodontic appliances or brackets ,66,69,71The aim of this scoping review is to give scientific evidence concerning the role of orthodontists in the management of mandibular fractures. The results of the present review show that conservative closed approaches that are manageable by orthodontists may be a good alternative in some types of mandibular fractures, especially in children and adolescents, while few studies have discussed the use of these methods in adults.Most mandibular fractures need to be reduced and fixed in order to promote primary or secondary bone healing  with an intermediate callous healing phase. Intermaxillary fixation (IMF) with arch bars, occlusal splints, orthodontic brackets, or mini-screws may be used to execute this type of fixation. Instead, open reduction and internal fixation (ORIF) with plates and screws applied directly to the fracture site avoids the intermediate callous phase, prevents micromotions, and allows primary bone healing ,73,74. OThe IMF approaches include the conventional ,62,67,69A prospective comparative study  that enrThe single patient with a condylar fracture in the case report of Chen et al.  was manaThe management of mandibular fractures in children is different compared to adults for several reasons. Firstly, children, compared to adults, tend to present greenstick fractures with little to no displaced bone ,73,74. CCazzolla et al.  presenteAnother retrospective study included in the present review  reportedThe case series of Agarwal et al.  presenteThe results of the present review reveal that orthodontists could be a point of reference in the management of some types of mandibular fractures. However, currently the studies have mainly been conducted on children and growing subjects, and the follow-up was often not sufficient for monitoring until the completion of growth. Few studies on the conservative management of mandibular fractures included adult subjects. Furthermore, most of the studies included in this review were case reports or case series, and there were some retrospective studies. Therefore, further randomized and non-randomized clinical trials with long-term follow-ups are recommended in both growing subjects and adults in order to define the treatment guidelines and the clinical indications of the conservative orthodontic approaches for the management of mandibular fractures.From the clinical perspective, the orthodontic management of mandibular fractures should be differentiated in terms of the patients’ ages and the sites of the fractures.In children affected by condylar fractures, orthodontists or pediatric dentists should be asked to use removable acrylic splints and functional exercises or intermaxillary fixation using brackets and elastics. Furthermore, in the case of minor parasymphyseal fractures, clinicians could use acrylic splints or cemented rigid splints.In adult subjects with minor condylar fractures, the use of orthodontic brackets or mini-screws and elastics by orthodontists has been described, whereas some minor mandibular fractures with minimal displacement have been alternatively managed with cemented acrylic splints and elastics. However, most of the articles were case reports and case series, and due to the lack of a sufficient number of clinical studies, it is not yet possible to suggest the preferential use of conservative orthodontic IMF over rigid arch bars and ORIF in adults. Therefore, further clinical studies with long follow-ups in both children and adults are needed to more precisely define the clinical indications of conservative orthodontic approaches in the management of mandibular fractures based on severity, location, and age.