The updated 4th edition WHO classification of lymphoid malignancies released in 2016 contains pivotal new terminology and information that is important for radiologists to understand. In spite of these updates, some lymphoproliferative disorders included in this update have been rarely discussed in the radiology literature. Many of these disorders have distinct clinical and imaging features, overlapping with more common disorders, thus causing a delay in diagnosis and management. Early diagnosis of many of these disorders is key as many of these are potentially treatable because early intervention may be lifesaving. The purpose of this manuscript is to provide guidance for radiologists regarding certain rare variants of B-cell lymphoproliferative disorders, in terms of clinical, histologic, and imaging findings, as well as to incorporate the new and updated terminology of some disorders included within the 4th edition of the WHO classification of lymphoid neoplasm.Lymphoproliferative disorders (LPD) are conditions characterized by the uncontrolled proliferation of B or T-cell lines. They encompass a wide spectrum of abnormalities, which may be broadly classified as reactive processes or malignant diseases, such as lymphoma, based on their cellular clonality and clinical behavior. While some of these disorders are rare, they may be encountered sporadically in clinical practice, causing diagnostic dilemmas owing to overlap in their clinical and imaging features with more common disorders. The updated 4th edition WHO classification of lymphoid neoplasms was released in 2016 to incorporate the rapid clinical, pathological, molecular biology and cytogenetic advances of some of these disorders. Despite these updates, very little information is presented in the literature from the radiology perspective. The aim of this article is to familiarize radiologists and other physicians with certain rare variants of B-cell lymphoproliferative disorders with a focus on imaging features of these disorders, as well as to provide an overview of some important updates contained within the new WHO classification of lymphoid neoplasms. Lymphoproliferative disorders (LPD) are heterogenous conditions characterized by the uncontrolled proliferation of B or T-cell lines. They encompass a wide spectrum of abnormalities with varied clinical, pathologic, and imaging features. They are broadly classified as reactive or malignant diseases based on their cellular clonality and clinical behavior and include lymphomas and other hematologic malignancies . LymphomImaging plays a pivotal role in the management of lymphomas, most notably when using 18F-FDG PET/CT. The use of 18F-FDG PET/CT changes the staging of disease in nearly one-third of cases, frequently resulting in tumor upstaging by detecting occult sites not seen on conventional imaging with modalities such as CT or MRI. 18F-FDG PET/CT can clinically help to further subclassify lymphomas into aggressive or indolent subtypes based on standardized uptake values (SUVs) and the heterogeneity of disease involvement. Aggressive lymphomas often display higher SUVs >10–13) at baseline, with disease manifesting with focal increased metabolic activity in nodal or extranodal sites  at basel. During In 2016, the World Health Organization (WHO) updated the 2008 classification of tumors of hematopoietic and lymphoid tissue. This new update accounts for significant advances in knowledge of lymphoma behavior since 2008 and clarifies the pathogenesis and molecular biology of different types of lymphoproliferative disorders including lymphomas ,6. TablePrimary central nervous system lymphoma (PCNSL), including the extreme rare variant lymphomatosis cerebri (LC), is a rare subtype of NHL with exclusive involvement of the central nervous system (CNS), representing 1% of all intracranial malignancies . LC implThe majority of PCNSLs including LC are diffuse large B-cell lymphomas (DLBCLs), with the remaining cases representing low-grade BCL 8%), Burkitt (5%), or TCL (2–3%) %, Burkit. MicroscClinically, in both PCNSL and LC, the median age of presentation is 65 years, and in PCNSL most patients present clinically with focal neurological deficits. However, the most common presenting symptoms of LC are the impairment of cognitive functions with rapidly progressing dementia, behavioral abnormalities, and, less commonly, focal neurological deficits. The diagnosis of LC is usually delayed as clinical manifestations of LC are vague and overlap with imaging features of other disorders. The prognosis of LC is usually poor given the rapidly progressive disease process, and most LC patients do not survive beyond six months of initial presentation .The classic imaging appearance of PCNSL is a focal solitary mass or multiple masses within the brain parenchyma. On CT, PCNSL often shows homogenously hyperdense lesions due to high tumor cellularity with homogenous enhancement and a predilection for the deep gray matter and deep nuclei, periventricular trigon, and corpus callosum that often abuts the sub ependymal/ventricular or meningeal surfaces . InvolveSpecial sequences such as apparent diffusion coefficient (ADC) maps help differentiate PCNSL from high-grade tumors such as glioblastoma multiforme (GBM). PCNSL demonstrates marked diffusion restriction on diffusion-weighted images (DWI) with lower ADC values compared to GBM due to tumor restriction in lymphoma cells. The main role of 18F-FDG PET/CT in CNS lymphoma is to differentiate PCNSL vs. disseminated systemic lymphoma involving the CNS . AdditioAutoimmune lymphoproliferative syndrome (ALPS) is a lymphoproliferative disorder that may be interpreted as malignant lymphoma. ALPS results from inherited mutations in the apoptosis signaling pathways ,23. ALPSClinically, ALPS presents in childhood and results in massive hepatosplenomegaly, chronic lymphadenopathy, thymic enlargement, and, in most patients, autoimmune phenomena such as hemolytic anemia .Imaging is not necessary to establish a diagnosis of ALPS but might aid in its diagnostic evaluation. Stability in the size of lymphadenopathy and hepatosplenomegaly over many years may suggest ALPS from other malignant lymphoproliferative diseases  Figure .Once the diagnosis of ALPS is established, baseline and periodic follow-up CT scans should therefore be obtained to document the stability of lymphadenopathy and hepatosplenomegaly. 18F-FDG PET/CT may also be helpful because mild to moderate FDG uptake is typical of ALPS compared to the high FDG uptake characteristic of malignant lymphomas. By evaluating for areas of high FDG uptake, 18F-FDG PET/CT may help in the early detection of lymphoma transformation and demonstrate the best site for biopsy, when indicated . AwarenePrimary mediastinal large B-cell lymphoma (PMLBCL) is an uncommon B-cell lymphoma, previously classified as a subtype of DLBCL but now considered an aggressive neoplasm that originates from thymic B cells, constituting less than 3% of all NHL cases . ClassicThe median age of presentation with PMLBCL is slightly older than CHL, commonly affecting middle-aged women in their third or fourth decade. PMLBCL has a more favorable survival than other subtypes of aggressive DLBCLs, and poorer prognosis compared to CHL. The 5-year survival rate seen in cases of PMLBCL is 85% . PatientThe genetic profile of PMLBCL is more similar to CHL than other BCLs . It is cOn imaging, patients typically present with a large ill-defined anterior mediastinal mass that often measures more than 10 cm in diameter with local infiltration of the underlying mediastinal structures ,30,31. OThe differential diagnosis of PMLBCL includes other anterior mediastinal masses such as CHL, germ cell tumors, and large thymic tumors, which can display necrosis and calcifications on CT . DiffereThe use of 18F-FDG PET/CT is essential in the evaluation of patients with PMLBCL to reveal sites of disease not visible on CT and to provide more accurate staging and radiation field planning . 18F-FDGLymphomatoid granulomatosis (LG), also known as angiocentric lymphoma, is a rare lymphoproliferative disorder. It results from the uncontrolled proliferation of EBV-infected B cells with prominent T-cell reactivation. Liebow et al.  first deLG is more common among immunocompromised patients, affecting males more than females, and is most common in the fifth or sixth decade of life . AlthougImaging findings of LG include bilateral diffuse reticulonodular opacities and mass-like lesions involving mainly the middle and lower lung zones with predominantly central involvement and relative sparing of the periphery. On CT, the reticulonodular opacities represent multiple small nodules that have irregular margins. Nodules typically show an interseptal and peribronchovascular distribution. Nodules tend to coalesce over time, forming larger masses that may manifest as coarse irregular opacities surrounded by ground-glass opacities.The differential diagnosis for these imaging findings is broad and includes pulmonary sarcoidosis, lymphocytic interstitial pneumonitis (LIP), atypical infections, and hypersensitivity pneumonitis. LG is usually not associated with mediastinal nodal enlargement or air trapping and the clinical course tends to be rapidly progressive and non-responsive to antimicrobial treatment. Such findings may be beneficial in excluding sarcoidosis and LIP as well as atypical infections ,42. SyncWith 18F-FDG PET/CT, LG may show central FDG uptake within pulmonary nodules and masses. Peripheral uptake in the walls of cavitary pulmonary lesions  has also been reported. The appearance of coarse linear opacities may represent a healing phase of nodules and masses . LG shouLymphoplasmacytic lymphoma (LPL) is a rare indolent BCL characterized by overlapping features between B-cell lymphoproliferative disorders and multiple myeloma (MM). LPL is characterized by IgM paraproteinemia and rarely IgG or IgA production. WM is usually reserved for patients presenting with LPL in addition to bone marrow infiltration. It remains controversial whether LPL and WM are different manifestations of a single disease or two unique entities . WM is cL265P mutation as a driver event in the pathogenesis of WM that occurs in 95% of patients. This specific biomarker is currently used to distinguish this malignancy from other overlapping BCLs, such as marginal zone lymphoma, as these disorders all share elevated IgM levels [Histologically, WM shows mixed small B lymphocytes and plasma cells known as lymphoplasmacytic cells . SimilarM levels . LPL hasM levels .WM is characterized by a wide spectrum of clinical presentations varying from bone marrow and organ infiltration by malignant lymphoplasmacytic cells to elevated IgM paraprotein, causing anemia, hyperviscosity, extramedullary disease, and peripheral neuropathy. A substantial number of patients, however, are asymptomatic at the time of diagnosis. In the absence of features of other lymphoproliferative disorders, LPL is a diagnosis of exclusion that may be reached after the elimination of other small B-cell IgM-secreting lymphomas and MGUS or myeloma .Multimodality imaging, including CT, MRI, and/or 18F-FDG PET/CT, is essential for initial diagnosis, especially in extramedullary disease presentation. Imaging can be helpful in determining optimal biopsy sites, as well as the assessment of response to therapy and monitoring disease progression or transformation. Unlike MM, WM is not associated with definite osseous lytic lesions, a feature that can distinguish WM from MM. WM usually manifests as osteopenia, marrow space widening, and endosteal erosions ,53 Figu.WM patients can also present with splenomegaly with or without focal lesions and lymphadenopathy typically within the axillary, retroperitoneal, and/or inguinal nodal stations . CranialIn 2016, the WHO released an update to its lymphoid neoplasm classification including various lymphoproliferative disorders, and this update is intended to account for the rapid changes in our understanding of lymphoma behavior, and to incorporate the increased high-end technology advances within this field, to study molecular drivers and cytogenetics of these disorders. Despite these updates, many of these disorders are only addressed in a few case reports within the radiology literature. Radiologists must stay abreast of these updates as they play a major role in the management of these disorders as they are often the first to suggest the diagnosis, help with disease staging, assess response to therapy, and identify disease relapse.


Genome-scale metabolic models (GEMs) are powerful tools for understanding metabolism from a systems-level perspective. However, GEMs in their most basic form fail to account for cellular regulation. A diverse set of mechanisms regulate cellular metabolism, enabling organisms to respond to a wide range of conditions. This limitation of GEMs has prompted the development of new methods to integrate regulatory mechanisms, thereby enhancing the predictive capabilities and broadening the scope of GEMs. Here, we cover integrative models encompassing six types of regulatory mechanisms: transcriptional regulatory networks (TRNs), post-translational modifications (PTMs), epigenetics, protein–protein interactions and protein stability (PPIs/PS), allostery, and signaling networks. We discuss 22 integrative GEM modeling methods and how these have been used to simulate metabolic regulation during normal and pathological conditions. While these advances have been remarkable, there remains a need for comprehensive and widespread integration of regulatory constraints into GEMs. We conclude by discussing challenges in constructing GEMs with regulation and highlight areas that need to be addressed for the successful modeling of metabolic regulation. Next-generation integrative GEMs that incorporate multiple regulatory mechanisms and their crosstalk will be invaluable for discovering cell-type and disease-specific metabolic control mechanisms. Cellular metabolism is a fundamental biological process used by all living organisms to generate and expend energy for growth . AlthougMost of our understanding of metabolism comes from cumulative data from biochemical experiments carried out over the past century. Although experimental evidence has been insightful, this type of investigation is often restricted to studying an isolated metabolic pathway in a limited number of conditions. To overcome this limitation, modern approaches seek to study metabolism from a systems biology perspective ,7,8. In While GEMs in their most basic form can model cellular metabolism reasonably well ,14,15, tWhile this enumeration of regulatory mechanisms is not exhaustive, these six mechanisms represent major ways through which cellular regulation affects metabolism. Of note, various methods have focused on integrating omics data directly into GEMs to improve model accuracy. Several excellent reviews have already covered this topic in greater detail ,21,22,23Before discussing how regulatory mechanisms have been integrated into GEMs, we begin with a brief overview on mathematical methods relevant to the algorithms discussed throughout this review .S, where rows signify metabolites and columns represent reactions. For each reaction (column), the stoichiometric coefficients of all metabolites involved in the reaction are set to a non-zero value. Specifically, positive coefficients indicate the production of a metabolite, while negative coefficients indicate the consumption of a metabolite. A key assumption of FBA is steady-state metabolism in which each metabolite’s production and consumption is balanced equally [S represents the stoichiometric matrix, v is the vector of reaction fluxes, and b, representative of changes in metabolite concentrations, is set to a zero vector to mathematically reflect steady-state metabolism. Since the number of reactions exceeds the number of metabolites, the system is underdetermined, and a large solution space exists; however, this can be narrowed by imposing additional constraints and narrowing the flux bounds. In FBA, linear optimization techniques are applied to solve for a flux distribution that optimizes an objective function (objZ). To reflect evolutionary pressure, growth is typically maximized by defining a biomass objective function that consists of biomass precursors [objZ is the objective function, biomassv is the flux through a user-defined biomass reaction, v is the vector of all reaction fluxes, lb is a vector of the lower bound flux limit, and ub is a vector of the upper bound flux limit. Constraint-based modeling (CBM) is the standard framework for reconstructing and analyzing GEMs, primarily through the addition of model constraints . The mos equally . Mathemaecursors . Other oecursors . These civ represents the flux through reaction i, n is equivalent to the total number of reactions, ilb is the lower flux bound for reaction i, and iub is the upper flux bound for reaction i. In FVA, the objective function (objZ) serves as a reference to find the possible lower and upper flux limits for Equation (3a) and Equation (3b), respectively. A different method, called parsimonious FBA (pFBA), also improves GEM simulation accuracy by determining the feasible flux ranges based on the assumption that cells maximize efficient enzyme usage to promote their growth [iv is the flux through reaction i, n is the total number of reactions, biomassv is the flux through the biomass reaction, biomass,lbv is the lower limit of the flux through the biomass reaction, ilb is the lower flux bound for reaction i, and iub is the upper flux bound for reaction i. Of note, the flux solutions achieved using pFBA tend to emphasize a small number of high-flux reactions due to the underlying assumptions of pFBA.Although FBA can generate flux solutions that match experimental data ,49, thisr growth . This asTranscriptional regulation describes the diverse ways in which gene transcription into messenger RNA (mRNA) is regulated by the cell. mRNA transcripts carry gene-encoded instructions for protein production to the ribosome, where translation occurs. Since gene transcription is required to express the proteins that participate in metabolic reactions, the association between genes, proteins, and reactions is strongly dependent on transcriptional regulation. Transcriptional regulation is often represented through transcriptional regulatory networks (TRNs), which describe how the transcription of target genes is influenced by regulatory genes and environmental perturbations ,52. In tregulatory flux balance analysis (rFBA). This algorithm is an expansion of FBA in which metabolic models with Boolean TRNs are used to predict steady-state flux distributions over a series of time intervals [E. coli growth predictions from GEMs across numerous metabolic environments.The simplest TRN is a Boolean network in which genes assume one of two states: active or inactive. This status is based on multiple factors including the activity of regulating genes and environmental conditions . Booleanntervals . To modesteady-state rFBA (SR-FBA), which simulates steady-state metabolism more comprehensively by incorporating both metabolic and regulatory constraints into a single optimization problem [integrated FBA (iFBA), which incorporates kinetic models and ordinary differential equations (ODEs) [While rFBA marked an important step forward in modeling the transcriptional regulation of metabolism, it is limited by its selection of only one valid metabolic state for each interval when multiple could occur. This limitation was addressed by  problem . Insteads (ODEs) ; howevertoolbox for integrating genome-scale metabolism, expression, and regulation (TIGER) was developed [FlexFlux is a similar platform but improves upon TIGER in terms of usability as TRNs and GEMs can be inputted in a standardized file format [FlexFlux also enables the integration of multi-state regulatory networks and allows the conversion of discrete regulatory states into continuous intervals. In order to facilitate the combination of Boolean TRNs and GEMs, a platform called eveloped . TIGER ae format . FlexFluProbabilistic regulation of metabolism (PROM) is a method that more comprehensively addressed these limitations by shifting the discrete paradigm to a continuous model of transcriptional regulation [Mycobacterium tuberculosis (M. tb), leading to enhanced simulation accuracy using the M. tb GEM [While Boolean TRNs have been shown to improve flux simulations, there are several limitations endemic to this framework. These include an overly simplistic model of metabolism, a theory-driven approach, and the need for extensive literature searching to define regulatory rules . This wagulation . Using T. tb GEM .Transcriptional regulated FBA (TRFBA) is a method that represents TRNs continuously and improves upon PROM by reducing the amount of transcriptomics data required to build continuous TRNs [E. coli, only TRFBA is more accurate than pFBA when applied to S. cerevisiae [CoRegFlux was developed to address this limitation [learning cooperative regulation networks (hLICORN), CoRegFlux determines TF–target gene relationships from transcriptomics data in the absence of a pre-existing TRN before predicting fluxes.Advancing the continuous TRN paradigm, several other algorithms have been developed. ous TRNs . Of noterevisiae . Howevermitation . Using lIntegrated deduced and metabolism (IDREAM) is a method that functions similarly to continuous TRN methods such as PROM but alternatively integrates an environment and gene regulatory influence network (EGRIN) [optimization of regulatory and metabolic networks (OptRAM) is an in silico strain design algorithm that optimizes the production of desired metabolites and has successfully been applied to S. cerevisiae [ (EGRIN) ,55. EGRI (EGRIN) . Buildinrevisiae .While TRN modeling and integration into GEMs has advanced considerably, these integration methods fail to account for additional regulatory mechanisms such as signaling or allostery. This limits the extent to which they accurately model cellular metabolism, as significant numbers of metabolic fluxes are unable to be explained by transcriptional regulation alone , necessiPost-translational modifications (PTMs) induce chemical changes onto proteins that alter their function, interaction, and localization ,58. GiveRegulated metabolic branch analysis (RuMBA) introduced one way to study PTMs in bacterial cells [Flux space shift (FSS) analysis was developed to address this specific issue by focusing on differential enzyme regulation for optimal growth in certain nutrient conditions [al cells . This menditions .comparative analysis of regulators of metabolism (CAROM) is a data-driven method that integrates omics data to decipher how both microbes and mammalian cells allocate different types of regulation, including PTMs [In contrast to RuMBA and FSS, ing PTMs . CAROM aprotein-specific information matrix (PSIM) is a genome-scale model that focuses on the protein secretory system [The methods discussed above were specifically designed to study how PTMs influence metabolic activity by introducing new CBM constraints. In contrast, some other studies have investigated the role of PTM regulation in metabolism indirectly. For instance, y system . Given ty system , the mody system . This woEpigenetics refer to biochemical changes that are transiently passed down to successive generations without modifying the DNA sequence. This enables eukaryotic cells to have identical genomes yet different phenotypes. While DNA cytosine methylation, non-coding RNAs, and histone modifications all play crucial roles in the mechanisms of epigenetics ,69,70, hepigenome-scale metabolic network model (EGEM) was developed [GEMs potentially offer a framework for measuring how metabolism interacts with epigenetics ,78. Howeeveloped . This moeveloped .fast reconstruction of compact context-specific metabolic networks for the integration of transcriptomics data (FASTCORMICS) method incorporates microarray data to existing GEMs [While EGEM mechanistically accounts for epigenetic regulation, recent studies have tried to model the resulting transcriptional impact of epigenetic regulation on metabolism. The ing GEMs . Althouging GEMs .dynamic flux activity (DFA) approach constrains GEMs with time-course metabolomics [As mentioned above, histone PTMs and metabolic states are naturally intertwined because of the small molecules required for covalent modifications. However, the process of epigenetic modification occurs in a highly dynamic fashion. Therefore, the changes in metabolite concentrations over time are important data that can provide better understanding of this interplay. The bolomics ,82. Chanbolomics . AlthougEnzyme activity is also influenced by the structural stability of the protein and its interactions with other proteins. Protein–protein interactions (PPIs), which entail physicochemical contacts between proteins, govern a wide range of cellular processes including but not limited to signal transduction , moleculIn the context of metabolism, PPIs play a major role in regulating reduction–oxidation  reactions  and induE. coli growth and metabolism in extreme temperatures by integrating protein structure information [E. coli GEM with protein structural information, Chang et al. not only simulated growth rate patterns resembling what was observed experimentally, but they also revealed metabolic mechanisms that may confer thermotolerance for heat-adapted E. coli strains. These findings were supported by follow-up experiments that showed increased growth for E. coli cultured in conditions supplemented with metabolites normally impacted by heat stress. Other than direct PPI integration into GEMs, several groups have integrated protein stability data to more accurately model metabolism using GEMs. Protein structural integrity is critical for proper functionality. One of the earliest studies by Chang et al. investigated ormation . This inE. coli and Thermotoga maritima to investigate how growth is limited by protein instability due to heat stress. The authors further showcased how GEM-PRO models can be used for applications at the intersection between systems and structural biology. However, GEM-PRO has not yet been widely applied since its introduction as it requires an abundance of protein structure information, which is not readily available nor attainable for most organisms. Although structural information may be derived from homology modeling, this method can often compromise the overall quality of the model.A successive study by the same group formally introduced the computational process of integrating protein structure information into GEMs . The appAllosteric regulation describes the modulation of enzyme activity via effectors, which are ligands or metabolites that bind to an enzyme at a location other than the active site. Upon binding to an enzyme, effectors can either enhance  or attenuate  activity through the associated reaction. This form of regulation is often depicted as “feedback” or “feedforward loops’’ depending on whether the effector metabolite is produced downstream or upstream of the reaction catalyzed by the regulated enzyme, respectively. Allosteric regulation enables biochemical adaptation to rapid changes in the cellular environment, particularly metabolite concentrations. Similar to PPIs/PS, allosteric regulation plays a key role in cell signaling, biomolecular transport, and metabolism ,95.allosteric regulation FBA (arFBA), which was designed to incorporate allosteric regulation when solving for flux predictions. This method introduces a regulation (R) matrix  that characterizes effector–reaction relationships. A value of 1 indicates reaction activation via effector binding, while a value of −1 indicates reaction inhibition (systematic identification of meaningful metabolic enzyme regulation (SIMMER), contextualizes experimental data via Michaelis–Menten rate law equations. The authors also used an extended S. cerevisiae GEM to infer fluxomics-constrained genome-scale metabolic fluxes via FVA. Both studies revealed distinct ways that metabolism is regulated depending on the environmental conditions. However, they both investigated allosteric regulation of metabolism at a small scale (≈100–300 reactions).Although allosteric regulation plays a major role in regulating metabolism, this regulatory aspect has not been widely considered in genome-scale metabolic modeling studies. In fact, Machado et al. was the first group to integrate genome-scale allosteric regulation with metabolic models . Specifihibition . In a tab vector in CBM). More importantly, signaling systems such as phosphorylation of the G-protein coupled receptor can respond to environmental perturbations quickly, but the reset of the system via receptor internalization can be slow [The signaling pathways enable cells to quickly respond to fast-changing environmental and extracellular signals for a wide range of behaviors, from chemotaxis to developmental processes. Generally, a cascade of proteins participates in the process. For example, the Wnt signaling pathway plays an important role in embryonic development by controlling body axis formation . This pa be slow . In othe be slow ,104.To address this issue of timescales, metabolic modeling with ODEs enables the computation of metabolite concentrations as a function of time. However, this approach is limited by high computational costs, difficulty in finding unknown kinetic parameters, and the inability to connect to phenotypic properties. As with TRN mechanisms, dynamic kinetic modeling with ODEs can capture signaling mechanisms in ways that traditional modeling methods fail to achieve. To leverage the advantages of both GEMs and ODEs, two integrated models, iFBA   is the first approach for addressing this challenge of curating GEMs integrated with regulation [S. cerevisiae and greatly improve the model’s accuracy. Nevertheless, unlike the curation of traditional metabolic network GEMs, for which a comprehensive suite of tools exists, the curation of integrated GEMs has not been automated yet, and GEMINI has been applied only to TRNs.Integrated GEM model development involves an iterative process of incorporating regulatory information and conducting regular quality checks until a high-quality model is achieved. Once a draft model is reconstructed, the curation of these models poses additional challenges . Errors gulation . GEMINI GEM with enzymatic constraints using kinetic and omics, is one method that could be leveraged to integrate enzyme kinetic information into GEMs [S. cerevisiae GEM, yielding an updated model with kinetic constraints that generated more accurate simulations compared to previous models. Although GECKO demonstrated the value of incorporating kinetic constraints into GEMs, these parameters are not widely available for most enzymes. However, this limitation could be remedied by methods such as DeepEC (Deep learning of Enzyme Commission numbers) [Modeling regulatory mechanisms characterized by dynamic enzymatic activity, such as allostery and PTMs, will further require the inclusion of kinetic parameters that relate enzyme concentrations to metabolic flux changes. GECKO, or nto GEMs . This menumbers) , which acomputation of microbial ecosystems in time and space (COMETS) [3-dimensional dynamic flux balance analysis (3DdFBA) [dynamic FBA (dFBA) [Another major area for improvement involves the ability to computationally model metabolism at the spatiotemporal level. This is because regulatory events often occur at different timescales (milliseconds vs. hours), and the degree of regulation can be distributed unevenly depending on the cellular objective . For exa(COMETS)  and 3-di(3DdFBA) , which sA (dFBA) . It is iOver the past three decades, GEMs have become integral for modeling metabolism at a systems-level perspective. GEMs have enabled the study of metabolic rewiring in response to environmental factors and paved the way for metabolic engineering in agricultural, industrial, and medical applications ,116,117.Within this review, we covered six regulatory mechanisms relevant to metabolism: TRNs, PTMs, epigenetics, PPIs/PS, allostery, and signaling. We discussed how various methods have integrated these regulatory mechanisms into GEMs, thereby improving the accuracy in model simulations. We also highlighted areas for improving the integration of regulatory information into GEMs. These include the continued incorporation of highly curated experimental data on regulatory mechanisms, iterative curation of integrated GEMs, addition of dynamic constraints to capture spatiotemporal metabolic changes, and supplementation with AI methods that can provide missing information on protein structure , biomoleLooking to the future of metabolic modeling, the successful construction of next-generation GEMs and ultimately whole cell models for complex organisms  relies o


Muscular dystrophies are a heterogeneous group of inherited diseases characterized by the progressive degeneration and weakness of skeletal muscles, leading to disability and, often, premature death. To date, no effective therapies are available to halt or reverse the pathogenic process, and meaningful treatments are urgently needed. From this perspective, it is particularly important to establish reliable in vitro models of human muscle that allow the recapitulation of disease features as well as the screening of genetic and pharmacological therapies. We herein review and discuss advances in the development of in vitro muscle models obtained from human induced pluripotent stem cells, which appear to be capable of reproducing the lack of myofiber proteins as well as other specific pathological hallmarks, such as inflammation, fibrosis, and reduced muscle regenerative potential. In addition, these platforms have been used to assess genetic correction strategies such as gene silencing, gene transfer and genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as well as to evaluate novel small molecules aimed at ameliorating muscle degeneration. Furthermore, we discuss the challenges related to in vitro drug testing and provide a critical view of potential therapeutic developments to foster the future clinical translation of preclinical muscular dystrophy studies. Muscular dystrophies (MDs) are inherited disorders characterized by progressive skeletal muscle degeneration, clinically resulting in weakness in specific muscle groups, loss of ambulation, breathing and swallowing difficulties and, in most cases, reduced life span . On a hiMDs are characterized by the genetic and clinical heterogeneity of causative genes, pattern of inheritance, age of onset, rate of progression and type of muscle affected . CardiacDMD gene (Xp21) that lead to the loss (DMD) of dystrophin or reduction (BMD) in its production. While thousands of pathogenic mutations have been described, most DMD-causing mutations are located in a “hotspot” region between exons 45 and 55 of the DMD gene, encoding the central rod domain of the protein .,34.33,34CMs may be used to model cardiovascular features of MDs, but they are among the most difficult cell type to differentiate from iPSCs. Signals induced by nodal, BMP and fibroblast growth factors (FGFs) are first required to generate cardiac mesoderm-like cells . The resThe discovery of iPSCs paves the way for their use in translational research as a patient-specific tool for high-throughput drug screening and toxicity analyses as well as for the in vitro assessment of disease pathologies before the onset of typical secondary effects of inflammation. Thus, these cells replace primary cell cultures and immortalized myogenic cell lines.Before the advent of iPSCs, drug screening models were based on patient samples or immortalized myogenic human and rodent cell lines. However, neither of these models are ideal. While cells derived from humans can be used to directly model the effects of drugs on humans, their expansion capabilities are more limited than those of in vitro cell lines; immortalized cell lines may, in turn, exhibit genetic and metabolic anomalies. These drawbacks limit the ability of these models to faithfully simulate human diseases. iPSCs can overcome these constraints, providing a promising and valuable tool for both therapeutic purposes and basic scientific research .iPSCs were employed for drug screening purposes by Abujarour and colleagues  37]. Th. Th37]. Later, Choi and colleagues established a human DMD model using iPSCs  38]. In. In38]. 2+ levels and reductions in Caspase-3 activation and apoptosis were observed. These data support the idea that membrane fragility causes intracellular calcium overload even in the early stages of differentiation, suggesting the potential of membrane sealants in the treatment of DMD.Concomitantly, other researchers established an iPSC-derived DMD CM model of dilated cardiomyopathy that was used to test the ability of an amphiphilic surface copolymer, Poloxamer 188 (P188), to repair disrupted cellular plasma membranes  39]. Th. Th39]. mdx mice, showed functional recovery following perfusion with nicorandil. These results support the hypothesis that nicorandil protects against stress-induced cardiac injury and has potential as a new therapeutic agent for DMD cardiomyopathy.DMD CMs also exhibited decreased levels of endothelial and neuronal nitric oxide synthases (NOSs), with consequentially increased susceptibility to stress-induced injury . Indeed,A key aspect of drug screening is the scalability of the cell cultures. From this perspective, Uchimura and colleagues were able to myogenically differentiate human iPSCs by overexpressing tetracycline-inducible MyoD in a large-scale culture (384-well microplates) with a consistently high myotube differentiation efficiency, facilitating high-throughput drug screening .mdx mice revealed that treatment with these two molecules significantly improved their pathological muscle features, supporting their therapeutic potential. Furthermore, fenofibrate mitigated the mitochondrion-induced programmed death of DMD CMs. These data indicate the feasibility of drug screening with hiPSC-derived DMD models.Recently, a DMD hiPSC-derived myoblast drug screen identified compounds that improved the DMD phenotype in rodent models  41]. Th. Th41]. In 2019, Kokubu and colleagues established a drug screening platform based on myocytes from MM-derived iPSCs  42]. Th. Th42]. Exon skipping represents a very promising therapeutic strategy for modifying molecular diseases. This approach, which to date has been applied mainly to DMD, aims to eliminate the “molecular defect” by directly modifying the dystrophin-encoding mRNA, correcting the genetic mutation that alters the ORF. This method allows the production of a shorter, less active but still functional protein, which appears to be capable of reducing the clinical manifestations of the disease and slowing ambulatory and pulmonary function decreases after long-term use .Exon skipping is carried out using short fragments of RNA called ASOs, which target pre-mRNA sequences . DystropDMD gene after treatment with an ASO targeting exon 51  harboring several mutations (exon 47–50 or 48–50 deletions) across the  exon 51  44]..DMD geneThe efficacy of exon skipping was also tested in a DMD model based on iPSC-derived myotubes .2+ overflow and a reduction in creatin kinase (CK) secretion in DMD iPSC-derived myotubes in which dystrophin expression was restored by ASO transfection . CRI. CRI3]. cas3, which encodes a large protein with a helicase possessing a single-stranded DNA (ssDNA)-stimulated ATPase activity. The signature gene for type II CRISPR/Cas systems is cas9, which encodes a multidomain protein that combines all the functions of effector complexes and the target DNA cleavage and is essential for the maturation of the crRNA. Type III systems possess the signature gene cas10. CRISPR/Cas systems and subtypes were thoroughly discussed in . Th. Th62]. The CRISPR/Cas9 approach was also used to correct LGMD R1 iPSCs, demonstrating the rescue of the calpain 3 gene in myotubes in vitro  63]. Th. Th63]. These results serve as proof-of-concept for the combination of genome editing and iPSC technologies to foster autologous cell therapies for LGMDs.DMPK with a novel artificial 3′ UTR in the cells. In this way, DMPK haploinsufficiency may be prevented. Possible limitations include the fact that the novel 3′ UTR may lead to abnormal regulatory features of DMPK transcripts and that pathogenic repeats are still present in the genome, which may negatively affect the replication and heterochromatinization of the locus. Furthermore, the persistence of (CUG)n repeat transcription may result in RAN translation. The authors hypothesized that corrected DM1 hiPSC-derived skeletal muscle progenitor cells can be used as a cell source for transplantation.Notably, genome editing can potentially be used to permanently correct the defective gene in DM1 . Xia andDMPK gene with dual sgRNA/CRISPR/Cas9 prevented nuclear focus formation and splicing alterations, reverting DM1 iPSCs to a normal phenotype [Other studies demonstrated that removal of the CTG expansion repeats from the 3′- or 5′-UTR of the henotype ,66,67. SDMPK gene normalization. Xia and colleagues frequently observed the inversion of flanked CTG repeats, although these results are unpublished [However, doubts exist regarding the DNA-directed strategy despite the promising results obtained by these groups in terms of ublished . Such fiDespite potential concerns and the need for further research, these studies potentially validate the use of CRISPR/Cas9 for gene editing in subjects with dominant MDs.In the last decade, iPSC-based disease models have offered unprecedented possibilities for establishing high-throughput drug discovery and safety test platforms. Recently, even more opportunities have been provided by 3D organoids and personalized organs-on-chips in association with genome editing, high-throughput imaging and omics methods and the incorporation of artificial intelligence data analysis methods.The major critical aspect of disease modeling is the ability of the platform to faithfully recapitulate the relevant pathophysiology and neuropathological features. Demonstrating the sufficient quality and differentiative capacity of iPSCs is a prerequisite for high-throughput phenotypic analyses.Generating iPSCs from somatic cells remains one of the challenges associated with iPSC-based models. The selection of the cellular reprogramming method is of crucial importance, as it can influence the final MD phenotype observed in vitro. The first key step is the selection of the donor cell type, usually dermal fibroblasts from punch skin biopsies or mononuclear cells from peripheral blood . ResearcConcerning iPSC reprogramming, the initial methods were based on the use of retroviral and lentiviral vectors to deliver reprogramming factors. This entailed the risk of inactivating tumor suppressor genes and/or activating oncogenes due to insertions at specific sequences as well as the risk of iPSC alterations due to the constitutive expression of reprogramming factors. To circumvent this contingency, transient, integration-free methods have been developed to deliver reprogramming factors. Among them, viral delivery and transient transfection methods using Sendai virus, adenovirus, episomal plasmids and synthetically modified mRNAs have been the most widely used . This stThroughout the in vitro culture period, iPSCs can acquire chromosomal abnormalities, genetic instability, copy number variants and loss of heterozygosity, and these phenomena need to be evaluated and minimized.Often, iPSC-based disease modeling studies include the comparative analysis of one or a few patient-derived iPSC lines with their respective sex-matched controls  to evaluate the pathological phenotype. These results can be biased by the genomic background of the subject or by the features of the single clone; therefore, the use of relatively large cohorts of patient-derived iPSCs is necessary for the acquisition of robust in vitro data.The acquisition of a precise and mature phenotype represents another challenge regarding iPSCs. iPSC-derived cells often exhibit immature features similar to their respective embryonic or fetal phenotypic cells or harbor a heterogeneous mixture of phenotypic subtypes at the end of the differentiation protocol. This can cause analytical variation and make analyzing the late-onset phenotype difficult.Moreover, muscles comprise a complex system that includes cranial, trunk, and limb muscles, each of which have different developmental origins and programs. Muscles themselves are composed of slow or fast myofibers, characterized by different types of myosin heavy chain genes. This complexity makes it considerably challenging to faithfully mirror muscle pathophysiology in vitro. Indeed, muscle innervation is needed to carefully reproduce the phenotype. Late-onset disease phenotypes represent another obstacle, as they are difficult to reproduce in culture. In the future, the development of 3D cultures such as organoids derived from iPSCs might provide an effective platform to study diseases at the tissue or organ level, including spinal cord/muscle innervation, even for a long period . The so-Altogether, these findings suggest that iPSC technology will become increasingly relevant for muscle disease modeling and therapeutic development in the future.The development and optimization of iPSC-based muscle platforms as representative models for human studies have allowed scientists to unravel disease-specific mechanisms. Further advances may finally facilitate the complete elucidation of key pathogenetic events and thereby lead to the development of effective treatments for muscular dystrophies. iPSC models can provide a useful platform for evaluating the efficacy/side effects of therapeutic technologies, such as small molecules, ASOs, gene transfer, and CRISPR/Cas9 genome editing. In addition, genetically corrected lines could represent a valuable cell source for autologous ex vivo gene therapies for the regenerative treatment of MDs. The increasing capacity to manipulate genomes has the potential to accelerate the translation of new therapeutic strategies from in vitro models to clinical settings.Overall, iPSC technology, combined with gene silencing, transfer and editing techniques, is potentially revolutionary and might bridge the gap between preclinical and clinical trials, enabling more reliable testing models for precision medicine and drug discovery.


Healthcare settings, including nuclear medicine (NM) departments, promptly adjusted their standard operating procedures to cope with the unprecedented crisis caused by coronavirus disease 19 (COVID-19) pandemic. Nuclear thyroidology has adopted changes and predicated on a careful risk–benefit analysis, in order to prevent a potential spread of the virus while being at the same time effective, safe and preserving their quality of essential services. Since most thyroid nodules (TNs) are benign, and malignant neoplasms are characterized by an indolent natural history, it is generally safe to delay diagnostic and therapeutic procedures. In this respect, the main adjustments that nuclear thyroidology has adopted are summarized into the following: general workplace adjustments including remote work for NM staff; postponing appointments for consultation, diagnostic and therapeutic purposes and rescheduling based on individualized risk stratification; telemedicine; preparation for possible issues on radiopharmaceuticals synthesis and delivery; preventing measures and protocols to minimize or avoid potential COVID-19 infection of patients and medical staff. This document should be considered as updated guidance on how clinical management of TNs and thyroid cancer has been altered, remodeled and adapted to the new circumstances in the COVID-19 era, based on the rapidly growing volume of scientific information regarding the new coronavirus. Several reports have been issued to serve as guidance on all aspects of NM practice, including nuclear thyroidology , 2, withThe risk for serious disease and death in COVID-19 cases increases with advancing age and presence of comorbid health conditions. Cancer patients are regarded as a highly vulnerable population to infection and development of more severe symptoms, which is possibly due to weakened immune systems, caused directly by tumor growth and indirectly by effects of anticancer treatment. However, there is scarce evidence about the impact of COVID-19 infection in patients with suspicion or a history of thyroid cancer. Indeed, early data from China suggest that COVID-19 does not worsen the outcome of cancer, but as the pandemic continues, with investigations and treatment delay, morbidity and mortality from thyroid cancer may increase . In this131I administration), as well as the vast majority of those with suspicious thyroid nodule(s) (TNs) or neck lymphadenopathy, are not considered at higher risk of viral infection. Of course, there are some exceptions including transient reduction in myeloid and lymphoid cell lineages after ablative therapy which can last up to 1 year, as well as bone marrow suppression following prolonged high dose of radioiodine which has been described, and may increase the risk for COVID-19 severity and complications .Reports from China and the United States describing clinical, demographic characteristics and outcomes of patients with cancer and COVID-19 examined risk factors for mortality, documenting a higher rate of death due to COVID-19 and severe outcomes in patients with cancer. Despite thyroid cancer’s indolent nature, more recent studies have shown thyroid cancer as a frequent diagnosis among patients admitted to hospitals with COVID-19. However, these results are preliminary, and no causal association has yet been documented among thyroid cancer and COVID-19 infection. They are mainly attributed to the high prevalence of thyroid cancer and nodules , 8. To tications . Patient et al. identified genes modulated by COVID-19 infection that are implicated in oncogenesis, including E2F transcription factors and RB1, laying the foundations for further research. Although recent data indicate that COVID-19 infection stimulates inflammatory–immune responses in thyroid gland, and extensive injury to the follicular epithelium and parafollicular cells has been described, there is no evidence yet associating SARS-CoV-2 with tumorigenesis in thyroid gland . FNA of most asymptomatic TNs with no history of neck irradiation, with no familial history of thyroid cancer or multiple endocrine neoplasia syndromes, with favorable clinical and sonographic characteristics (using an acceptable scoring such as thyroid imaging reporting and data system (TIRADS)), without suspicion of medullary thyroid cancer (MTC) (e.g. with family history of MTC) or anaplastic thyroid cancer (ATC) and without compressive symptoms due to thyroidal masses is recommended to be postponed and rescheduled during restrictive quarantine phase  and FNA cytology for diagnostic or follow-up reasons for patients with suspicion or history of TNs or thyroid cancer, during the acute phase of the pandemic. Exceptions are:Postponement and rescheduling or teleconsultation of scheduled outpatient clinical examination/imaging/functional test  suggesting large goiter should undergo imaging for further assessment ;patients with history, clinical characteristics and laboratory examinations indicating aggressive thyroid disease, for example, anaplastic, medullary, metastatic or other diseases, for example lymphoma;pediatric patients with non-incidental cervical findings;high-risk patients (according to the American Thyroid Association (ATA) 2015 risk stratification system for recurrence in thyroid cancer patients , heart failure, chronic kidney disease, prior coronary artery disease, renin–angiotensin–system blocker usage and low lymphocyte were positively associated with mortality in patients with TC and COVID-19 infection. Moreover, they showed that radioactive iodine (RAI) treatment and cumulative iodine activity did not negatively affect the severity and mortality of COVID-19 in this patient group.Cancer patients are regarded as a highly vulnerable group in the current COVID-19 pandemic, showing deteriorating conditions, higher risk for ICU admission and poor outcomes from the infection. However, we need to acknowledge that malignant diseases are heterogenous and divided into different groups, especially according to cancer type, stage and clinical profile of each patient. Concerning thyroid cancer (TC) and COVID-19 outcomes and mortality, there are scarce published data. Among them, Sahinhttps://www.eurothyroid.com/news/covid-19-thyroid-diseases.html, Accessed: June 7, 2021). All the aforementioned cases that are at a higher risk of severe/critical infection with COVID-19, including patients with comorbidities, should be shielding and self-isolating. They should systematically follow general principles to help prevent the spread of airway and chest infections, including handwashing and respiratory hygiene and register for support when needed . Moreover, acknowledging specific clinical characteristics of patients with malignant neoplasms as well as the risk for severe COVID-19 infection, the National Comprehensive Cancer Guidelines prioritize COVID-19 vaccinations for patients with cancer. For patients with solid tumor malignancies, the panel recommends vaccination, even if the patients are receiving cytotoxic chemotherapy, targeted therapy, checkpoint inhibitors and other immunotherapy or radiation .The minority of TC patients with advanced or metastatic disease who are receiving multikinase inhibitors (such as lenvatinib or sorafenib), chemotherapy or external beam radiotherapy are at an increased risk of developing adverse events , resulting in a high probability of severe illness from SARS-CoV-2  or with rapidly growing TNs/ cancer;patients with history, clinical characteristics and laboratory examinations indicating aggressive thyroid disease, for example, anaplastic, medullary, metastatic or other diseases, for example lymphoma;pediatric patients with worrisome rate of progression (e.g. children of younger age or intermediate or high ATA pediatric risk for recurrence level) , 24;patients who have been fully vaccinated.All the aforementioned cases should be prioritized for  thyroidectomy and lymph node neck dissection during the pandemic, always with precautions and risk/benefit assessment. A COVID-19 prevention protocol should be followed during surgery , 26. PatIn less acute pandemic period, all surgeries are recommended to be performed, using a strict preventing–screening protocol as previously mentioned since recent data have shown that head and neck surgery during the pandemic is safe even when prolonged and complex.131I) therapy, either as remnant ablation or as adjuvant treatment (as defined in the Martinique principles (Postponement of RAI (inciples ), especiinciples . The nee131I therapy for which a patient has already begun pre-treatment such as administration of redifferentiating agents or thyroxine withdrawal.Patients who have been fully vaccinated.All the aforementioned cases should be prioritized for radioiodine therapeutical administration during the pandemic, always with precautions and risk/benefit assessment. A preventing protocol should be proceeded before any RAI treatment procedure.Multidisciplinary approach is considered an indispensable condition for achieving safe and effective management of acutely unwell patients. Emergencies related to thyroid gland diseases are infrequently observed in clinical practice . TN/cancer-related emergencies are caused by overt dysfunction of the gland, which are so severe that require admission to ICU frequently. They include compressive symptoms caused by huge goiter and aggressive thyroid tumors, hypothyroid coma and thyrotoxic storm. MTC and ATC, as well as extensive metastatic disease of DTC, should also be considered as cases that require early diagnosis and prompt treatment, since they are characterized with unfavorable prognosis.99mTc SPECT/CT scintigraphy or PET/CT, which only serve as adjunctive imaging methods in the diagnostic algorithm of goiter-related compressive phenomena, no other NM modalities could be of additional value in the diagnostic algorithm of such cases. Acknowledging the exceptional conditions under which a patient with severe compressive symptoms should be managed in the COVID-19 era, we recommend that SPECT and PET/CT should be substituted by other imaging modalities that do not include the administration of radiopharmaceuticals and are less time-consuming. Urgent surgery represents the gold standard treatment in patients with rapidly increasing neck masses associated with stridor with resultant morbidity. Patients requiring notwithstanding surgical intervention include those with evidence of aerodigestive tract compromise/invasion; recurrent laryngeal nerve palsy due to malignancy; locoregional metastasis; large, compressive tumors; clinical concern for example, rapid growth, poorly differentiated tumors; MTC or ATC. A preventing protocol should be proceeded with any surgery. However, in case of severe life-threatening symptoms, patients can be excluded from the preventing protocol.The diagnosis of goiter-related compressive phenomena is based on symptoms and signs complained by patients. Imaging examinations either confirm the clinical suspicion or show the surgeon goiter’s extension toward the neighboring structures, and particularly cleavage plans. The clinical picture is characterized by dyspnea with hypoxemia and respiratory acidosis, stridor, dysphonia, dysphagia and difficult or impossible extension/flexion of the neck. Rarely, superior vena cava syndrome may occur. Except for radioiodine or Most TNs are benign, while malignant neoplasms are characterized by an indolent natural history. In this respect, most nuclear thyroidology (diagnostic and therapeutic) procedures are not urgent. There is little evidence that early detection and treatment of DTC significantly alter disease outcomes, which are not the case for ATC and MTC that need to be managed without delay. Therefore, for most cases, it is generally safe to delay diagnostic and therapeutic procedures including thyroidectomy and RAI treatment. However, it is vital for higher-risk cases to be early recognized and treated properly. Risk stratification as proposed by the latest ATA guidelines, TIRADS as well as dynamic clinical evaluation is a sine qua non, to ensure that patients with characteristics that imply an unfavorable prognosis, will be prioritized for diagnostic and therapeutic procedures during the pandemic. Very low, low and intermediate risk for recurrence patients can safely postpone diagnostic and therapeutic protocols, as described above. Personalized risk assessment should be performed to justify a delay in surgery even for high-risk patients with thyroid cancer. Except for local COVID-19 prevalence, tumor aggressiveness expressed as rapidly progressive disease, big invasive regional metastatic lesions, distant metastases or suspicion of tumor dedifferentiation should be taken into consideration. Priority for urgent intervention is placed on the need for control of high-volume neck disease and airway protection . A stricA priori patient notification is useful offering time for better preparation of possible questions to be referred. A convenient tool for institutions is to offer online-specific frequently asked questions on issues of possible patient information concerning, for example the risk of RAIT or surgery postponement. Numerous online information from different societies/groups specifically relating to COVID-19 and TNs and cancer are currently available, where patients could be referred to .After an individual risk–benefit analysis, diagnostic and therapeutic interventions may proceed for selective cases, despite the threat of COVID-19 infection (as stated in the previous section). All remaining patients can be safely reviewed remotely based on risk, in predefined time intervals, with an ‘open line’ for the patient to report abnormalities. Exceptions are fully vaccinated patients who can safely undergo diagnostic and therapeutic workup without delay. Proper remote consultation should be carefully planned since it is time-consuming. The challenge not only for NM but also for all medical specialties during the pandemic is to ensure essential services are provided while keeping the patients and staff as safe as possible by reducing the potential exposure to COVID-19. Several reports, reviews, protocols and guidelines have been issued since the onset of the pandemic in the past year. Clinical practice has been altered, remodeled and adapted to the new circumstances in the COVID-19 era. The main adjustments that nuclear thyroidology has adopted are summarized into the following: general workplace adjustments including remote work for NM staff (at least for a group of them); postponing appointments for consultation, diagnostic and therapeutic purposes and rescheduling based on individualized risk stratification; telemedicine; preparation for possible issues on radiopharmaceuticals synthesis and delivery, preventing measures and protocols to minimize or avoid potential COVID-19 infection of patients and medical staff , 29, 30.In addition to these suggestions, treating physicians should bear in mind the possibility that a treated patient could potentially be infected before, during or after hospitalization. Thus, the patient may require further specialized/acute care within or outside radioprotected areas (i.e. ICU) after discharge. Therefore, and to the extent possible, discharge rules should be applied more strictly and according to national rules , so that other healthcare practitioners and the general public remain protected in such a scenario, as already stated in our original paper.Due to the emerging nature of the COVID-19 crisis, this document is not based on extensive systematic review or meta-analysis but on rapid expert consensus. The document should be considered as guidance only; it is not intended to determine an absolute standard of medical care. Healthcare staff needs to consider individual circumstances when devising the management plan for a specific patient.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this guidance.This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.


Ischemic stroke and traumatic brain injury (TBI) are among the leading causes of death and disability worldwide with impairments ranging from mild to severe. Many therapies are aimed at improving functional and cognitive recovery by targeting neural repair but have encountered issues involving efficacy and drug delivery. As a result, therapeutic options for patients are sparse. Neurotrophic factors are one of the key mediators of neural plasticity and functional recovery. Neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) serve as potential therapeutic options to increase neural repair and recovery as they promote neuroprotection and regeneration. BDNF and NGF have demonstrated the ability to improve functional recovery in preclinical and to a lesser extent clinical studies. Direct and indirect methods to increase levels of neurotrophic factors in animal models have been successful in improving postinjury outcome measures. However, the translation of these studies into clinical trials has been limited. Preclinical experiments have largely failed to result in significant impacts in clinical research. This review will focus on the administration of these neurotrophic factors in preclinical and clinical stroke and TBI and the challenges in translating these therapies from the bench to the clinic. Ischemic stroke, a leading cause of disability worldwide, results from limited blood flow to the brain due to the blockage or narrowing of arteries. Unfortunately, there are a lack of therapeutic options that can effectively minimize damage or aid in recovery following brain injury from ischemic stroke. The pharmacologic standard of care involves clot breakdown with the thrombolytic agent tissue plasminogen activator (tPA) and/or prevention of further ischemia using anticoagulants such as aspirin. After a stroke, tPA is often the best available option for patients but must be administered within the first 3 hours, or potentially up to 4.5 hours . The shoNTR, which induces apoptosis, and in some cases may promote neuronal survival and neurite growth during neurodevelopment [One avenue of stroke research has involved exploring the use of neurotrophin treatments as a mechanism for neural repair and enhanced recovery. Neurotrophins are a family of growth factors that play important roles in the survival and function of neurons. There are four known members of the neurotrophin family of growth factors in mammals: brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin 4 (NT-4) . Neurotrelopment . Therefoelopment . Of thesThis review will outline the current state of preclinical and clinical research surrounding the potential for BDNF and NGF to become treatment options for patients following stroke. These neurotrophins promote neuroprotection and regeneration and have been examined to determine their role in neural repair as well as their ability to improve functional recovery in preclinical and, to a lesser extent, clinical studies. Direct and indirect methods of increasing levels of these neurotrophins in animal models have demonstrated promise in improving outcome measures following brain injury. Unfortunately, the translation of these preclinical studies into clinical trials has been limited. This review will focus on both direct administration of exogenous neurotrophic factors and indirect methods of modifying endogenous neurotrophic factor levels in the central nervous system after stroke, and it will also examine the challenges involved in moving BDNF and NGF-related treatments from the bench to the clinic.Currently, a need exists for the development of additional therapies that are effective for improving recovery from ischemic injury. While there have been promising advances in preclinical studies, many of these therapies have failed to translate clinically. There are several challenges that may contribute to this lack of translation. First, many treatments have poor pharmacokinetic profiles . Second,In vitro experiments demonstrate that the molecule LM22A-4, a selective small-molecule partial agonist of TrkB, can trigger the downstream activators of the TrkB receptor [Neurotrophins can form protein-antibody complexes which may affect their tissue distribution, metabolism, and elimination. Additionally, peptidases and proteases in the blood can degrade neurotrophins, leading to reduced bioavailability, as evidenced by poor tissue distribution and short half-lives. An increased dose would be required to compensate for its poor bioavailability. This, in turn, can trigger adverse effects. Administration of neurotrophins can cause immunogenicity which can manifest in adverse effects including hypersensitivity and anaphylactic shock . In ordereceptor . AlthougAttempts to use BDNF and NGF as therapeutics for central nervous system (CNS) disorders typically utilize central administration routes that bypass the BBB, including intracerebroventricular (ICV) injection, intraparenchymal injection, and intranasal administration . This isIndirect modification is achieved by the administration of therapeutics that elicit an increase in neurotrophins in the CNS, including drugs classified as NMDA receptor antagonists, cholinesterase inhibitors, statins, and sigma-1 receptor agonists. NMDA receptor antagonists, including memantine, ketamine, and dextromethorphan, have been used in humans as experimental therapeutics for stroke. NMDA receptor antagonism is a mechanism of action in several Alzheimer's disease therapeutics, including memantine. In addition to NMDA receptor antagonism, memantine was found to increase BDNF levels in macaques, as measured by upregulated mRNA and protein expression of BDNF . BecauseIn vitro studies have shown that neural progenitor cells are capable of releasing neurotrophins including BDNF, NGF, and NT-3 [Stem cell therapy is another method currently being studied for its potential to increase BDNF levels. and NT-3 . Furtherand NT-3  while moand NT-3 –25.Several case studies examining the administration of neurotrophic factors, specifically NGF, were published during the 1980s and 1990s but resulted in a lack of consensus on measurable outcomes of recovery. Trials involving central administration of NGF to aid recovery after cerebral ischemia were preceded by its use in Parkinson's disease and Alzheimer's disease . ClinicaMany of the ongoing clinical studies are aimed at using rehabilitation methods such as exercise, transcranial magnetic stimulation (TMS), or hyperbaric oxygen to increase neurotrophin levels, specifically BDNF, and NGF . An earlval66met single nucleotide polymorphism (SNP), which is common in humans, particularly in Asian (40-50%) and Caucasian (25-32%) populations [val66met SNP is linked to a decrease in activity-dependent BDNF release (but not constitutive BDNF release) and reduced cortical plasticity [val66met polymorphism may play a role in neurological and neuropsychiatric disorders. The inconsistency in results is complicated by variations in the genetic model used, age, sex, ethnicity, and other factors [The beneficial impact of treatment effectiveness is further limited by the presence of BDNF polymorphisms. One of the more studied genetic variants of BDNF is the ulations . The valasticity . It is pasian 25-% populat factors –36.NCT03686163, has recruited 106 participants to evaluate the effects of intranasal NGF for acute ischemic stroke (20 μg/day) for two weeks beginning at least 72 hours poststroke. Results are expected in late 2020. Additionally, there are several ongoing or recruiting trials seeking to evaluate the potential to enhance stroke recovery of therapeutics that can influence BDNF levels in the CNS, including memantine for enhanced stroke recovery (NCT02144584) and evaluation of memantine vs. placebo on ischemic stroke outcome (NCT02535611) as well as use of donepezil in combination with transcranial direct current stimulation and intensive speech therapy (NCT04134416). Given the abundance of preclinical research using BDNF and BDNF-enhancing therapeutics, it is notable that BDNF itself is not used as a potential stroke therapeutic in current clinical trials. This may be partially due to its severe limitation in crossing the blood-brain barrier and the resulting challenges related to drug delivery and direct central administration in humans [There is currently only one clinical trial evaluating central administration of exogenous neurotrophic factors . This trNCT025356 as well Although administration of neurotrophic factors to treat stroke in a clinical setting has not been a primary focus of recent literature, there is substantial interest in understanding the role that endogenous neurotrophic factors play in recovery following other forms of injury such as TBI to optimize recovery. TBI occurs following a bump, blow, or jolt to the brain that causes brain edema and results in neuronal cell death. Treatment options for TBI are similarly lacking, with typical immediate interventions including hyperosmolar therapy to relieve intracranial pressure , 38 and κB, and moreover, this response was inhibited when TrkB receptor inhibitor was coadministered [κB, and an increased expression of TrkB post-TBI. Further, these effects were reversed via the administration of a TrkB receptor antagonist. To address BDNF's difficulty in crossing the BBB, Kim et al. injected BDNF-filled nanoparticles via IV three hours post-TBI injury in mice [The direct administration of BDNF as a therapeutic option post-TBI has centered around the role of BDNF in promoting an anti-inflammatory cytokine milieu. In fact, BDNF has been implicated in downregulating the inflammatory response in other pathological states besides TBI/stroke. For example, in female rats inoculated with Streptococcus pneumoniae meningitis, intracisternal BDNF infusions were associated with a significant decrease in inflammatory cytokines, including IL-1B, TNF-a, and NF-nistered . To specnistered . It was  in mice . This rein vitro study, NGF attenuated the robust proinflammatory response to LPS-induced monocytes significantly decreasing NF-κB, IL-1b, and IL-6 mRNA levels [Similar to BDNF, NGF has also been studied in the context of taming the post-TBI inflammatory response as well as its neuroprotective effects. Many TBI research studies focus on the role NGF plays in synaptic transmission, by promoting a more anti-inflammatory cytokine milieu. In an A levels . In a siA levels . It was A levels . These pA levels .in vitro studies demonstrate the potential of these two neurotrophins to improve recovery, the lack of clinical research and the lack of consensus among clinical research outcomes emphasize the gap in the translation of BDNF and NGF research from bench to clinic. From these studies, it is clear that neurotrophins, specifically BDNF and NGF, administered directly and indirectly have a growing role in increasing neurogenesis and functional recovery after stroke and TBI and that BDNF and NGF have a synergistic role in motor learning and cognitive recovery. Bearing these partial successes in mind, the lack of clinical research due to the ineffectiveness of treatment options serves as an impetus for the necessity of further investigation not only to identify but also to resolve the roadblocks of the translation of this research.Determining the role of BDNF and NGF in stroke and TBI is complex and often contradictory. While animal and


Photodynamic therapy (PDT) is a promising non-invasive phototherapeutic approach for cancer therapy that can eliminate local tumor cells and produce systemic antitumor immune responses. In recent years, significant efforts have been made in developing strategies to further investigate the immune mechanisms triggered by PDT. The majority of in vitro experimental models still rely on the two-dimensional (2D) cell cultures that do not mimic a three-dimensional (3D) cellular environment in the human body, such as cellular heterogeneity, nutrient gradient, growth mechanisms, and the interaction between cells as well as the extracellular matrix (ECM) and therapeutic resistance to anticancer treatments. In addition, in vivo animal studies are highly expensive and time consuming, which may also show physiological discrepancies between animals and humans. In this sense, there is growing interest in the utilization of 3D tumor models, since they precisely mimic different features of solid tumors. This review summarizes the characteristics and techniques for 3D tumor model generation. Furthermore, we provide an overview of innate and adaptive immune responses induced by PDT in several in vitro and in vivo tumor models. Future perspectives are highlighted for further enhancing PDT immune responses as well as ideal experimental models for antitumor immune response studies. Photodynamic therapy (PDT) is a cancer modality that combines three essential components of a photosensitizer (PS), harmless light, and molecular oxygen . It is bWhen the immune monitoring function becomes dysfunctional, tumor cells can continue to grow and form malignant tumors ,7. AdditAlthough the antitumor immune effects of PDT have been thoroughly investigated, there is a dire need for more relevant models capable of recapitulating the heterogeneity and the microenvironment of the in vivo human tumor, thus allowing for more predictive in vitro evaluation of PDT-induced antitumor immunity . As tradCell culture is an indispensable tool in the preclinical drug development process . Two-dimIn recent years, 3D tumor cultures have emerged as excellent cancer models, which can bridge between preclinical and clinical studies for better evaluation of novel anticancer drugs . SpheroiSeveral studies have compared 2D and 3D cell cultures in terms of morphology, cell survival, proliferation, differentiation, cellular responses to stimuli, cell–cell signaling mechanisms, cell rigidity, metastatic and invasive characteristics of tumor cells into adjacent tissues, together with angiogenesis stimulation and immune checkpoints evasion, response to therapeutics, gene expression, and general functional properties .Over the recent years, several types of 3D tumor models have been established to generate spheroids in oncology. The main 3D culture models of cancer include multicellular tumor spheroids (MCTS), tumor tissue explant, and tumor on a chip . This seThe hanging drop technique is a simple scaffold-free approach in which small droplets of the cell suspension are placed on a reversed upside-down culture lid . This me1O2, causing a phototoxic effect via apoptosis and paraptosis pathways in MCTS [This technique is a simple method that prevents cells from attachment and matrix deposition on modified non-adherent culture surfaces such as well plates, flasks, or dishes ,33. The  in MCTS . This ty in MCTS . However in MCTS .The four most common types of bioreactors used for 3D spheroid production include spinner flask bioreactors, rotational culture systems, perfusion systems, and mechanical force systems . The bioA hydrogel is an aqueous medium-inflated network of physically or chemically cross-linked polymer molecule . The hydIn the magnetic levitation-based method, the cells are combined with magnetic particles and subjected to magnetic force to surmount the gravitational force, allowing them to levitate and form cellular aggregates . This meIdeal anticancer treatment should induce local tumor regression and systemic antitumor immune responses capable of obliterating distant metastases with minimal toxicity to healthy tissues . From thThe innate arm of the immune response system eliminates pathogenic agents by phagocytes , the complement cascade, and natural killer (NK) cells . FollowiThe initiation of acute inflammation by PDT attracts neutrophils to the irradiated tumor area and secretes chemokines and granule proteins to promote DCs maturation and activation . ActivatSeveral studies demonstrated that PDT stimulates both innate and adaptive immune responses that play a major role in tumor eradication . Table 2Abdel-Hady et al.  provided2+, which can potentially lead to apoptosis [PDT-induced cell death depends on the intracellular localization and binding sites of the PSs . Photoexpoptosis ,76. PDT poptosis ,78. TherThe ER is a vital organelle that performs several essential cellular processes and can influence cancer pathogenesis . ER streHowever, one major problem that could potentially hamper the application of these PSs is their inherent low absorbance wavelength that cannot reach deep-seated cancer tissues . As a reMitochondria are vital subcellular organelles that undertake critical roles in metabolism, including cellular proliferation. Their actions are complexly interlinked with signaling pathways and the apoptotic process . Thus, gIn addition to improved ROS generation, PDT-triggered mitochondrial apoptosis stimulates signal transduction pathways, which also promotes immune responses . StudiesNanomedicine is a rapidly evolving field that is transforming cancer diagnosis and treatment . NanoparThe application of nanotechnology has also provided many novel strategies for delivering multiple immunostimulatory agents in a well-coordinated and targeted manner ,97,98. INPs are ideal PS delivery platforms and immunostimulatory agents due to their tunable size and surface chemistry, which allows for multiple modifications with various ligands, making them suitable for intracellular targeting . In thisTo bridge the gap between preclinical studies and clinical trials, it remains imperative to evaluate the potency of PDT-induced immunity using tumor models that can readily recapitulate human response . Since 2In this sense, the development of 3D tumor models that closely resemble human tumors and the microenvironment can add a significant value in predicting the clinical efficacy of PDT and PDT-induced immune responses in vivo . FollowiAlthough several pieces of evidence have established the capacity for PDT to obliterate the primary tumors and provide durable antitumor immune responses, some limitations were reported ,110 due Several adjuvants have been developed to enhance anticancer agents: Toll-like receptor (TLR) agonists, such as Bacillus Calmette–Geurin , imiquimod (TLR-7), and CPG oligodeoxynucleotide , stimulate innate and adaptive immune responses . They inTumor ICD is initiated by the release of DAMPs, such as CRT, HSP70, HMGB1, or ATP, which can promote immune maturation and activation at the targeted tumor region through their interactions with PRRs . It was The tumor microenvironment (TME) has a strong immunosuppressive effect, which is the main reason behind the low efficiency of cancer therapies that act by stimulating immunity against cancer . TME is Tumor cells can be differentiated from normal cells by their overexpression of tumor-specific antigens . The iniIn recent years, PDT has gained a great deal of attention due to its ability to eradicate primary tumors. It generates systemic and long-lasting antitumor responses that combat metastases and tumor recurrence. Although clinical evidence is very scarce, numerous preclinical studies showed that PDT modality could potentially become a potent therapeutic option for cancer treatment. Therefore, researchers have explored several approaches aimed at overcoming the immunotolerance in treated tumors, attenuating the immunosuppressive TME and establishing a robust and systemic adaptive immune response that can obliterate distant tumor lesions. Combinational therapies that synergize with the immunostimulant role of PDT may pave a way for the successful utilization of PDT as a mainstay treatment in the clinical arena. Additionally, the improved intracellular delivery of PS and immunostimulatory agent integrated with nanotechnology is a great stride forward in enhancing the efficacy of PDT and may endorse the rational design of a PDT regimen. A physiologically relevant tumor model that closely resembles the 3D architecture and functional properties of the solid human tumor is essential for the evaluation of tumor mass and immune system interaction as well as PDT-triggered antitumor immunity. Currently, the majority of PDT and immunity experiments have been performed on traditional 2D monolayer cell cultures. Several lines of evidence suggest that promising drug candidates fail clinical trials, thereby hampering the discovery of potent therapeutics. This is because the cellular environment of 2D cell cultures does not resemble that of real tumors. Thus, to validate in vitro data, in vivo studies have been intensely investigated, using animal models that may closely reflect what happens in humans. As a matter of fact, tumors are artificially generated within a short period in these xenografts’ models. Furthermore, these animals are immunosuppressed and contain immune cells that differ from their human counterparts. To minimize the number of animals used, and perhaps replace these costly and cumbersome animal studies, 3D tumor models have been developed. Three-dimensional (3D) cell cultures may bridge between preclinical and clinical studies, since they are scientifically accurate and simulate different aspects of human tumors. Thus, spheroids have a significant value in predicting the clinical efficacy of anticancer drugs. The overall findings of this review concluded that very few studies in relation to PDT-induced antitumor immunity were conducted within 3D tumor models . Thus, t


Rheumatic disease occurring as a paraneoplastic manifestation such as dermatomyositis is well recognised, whilst the symptoms of cancer may frequently mimic the presentations of common rheumatic disorders. It is essential to recognise these differential diagnoses to avoid delay in the detection of malignancy.We present an unusual case of rheumatoid arthritis where, conversely, the diagnosis of inflammatory arthritis was delayed due to the attribution of musculoskeletal symptoms to existing malignancy undergoing treatment.This case highlights the importance of careful history and examination.Cancer and inflammatory disorders may co-exist and delayed diagnosis of either condition can result in increased morbidity.A 65-year-old male presented to Ear, Nose and Throat (ENT) clinic with a facial lump in association with swelling in his throat, tongue and neck associated with a decrease in appetite and weight loss. He reported an active lifestyle, including mountain climbing until a month prior. He was a non-smoker with alcohol intake of less than 10units/week. Other medical history included osteoarthritis of his left knee which did not limit function.Imaging of the head and neck confirmed a mass in the oropharynx, tonsils, and tongue muscles. Subsequent biopsy revealed moderate to poorly differentiated squamous cell carcinoma (SCC) of the left tonsil.11 months prior to this presentation, he had developed pain and swelling involving knees, shoulders, and small joints of hands. His mobility became progressively impaired eventually requiring a wheelchair for any outdoor excursion.The systemic and musculoskeletal symptoms were attributed to a paraneoplastic manifestation of his malignant process.A rheumatology opinion was eventually requested in view of the persistence of joint symptoms despite effective cancer treatment with radical radiotherapy. He described early morning stiffness lasting more than 3 hours. Synovitis was evident at his hands, wrists and knees, whilst shoulder movement was restricted, resulting in a high disease activity score (DAS) of 7.44. Blood workup showed normal rheumatoid factor and cyclic citrullinated peptide (CCP). Acute phase markers were elevated with ESR 99 mm/hr, CRP 79 mg/l and ferritin 1194 ng/ml. Ultrasound (US) hands confirmed widespread active synovitis involving bilateral wrist and metacarpophalangeal joints. X-rays showed only degenerative changes. A diagnosis of seronegative inflammatory arthritis was established, and he was initiated on hydroxychloroquine with reducing regime of prednisolone. Methotrexate (MTX) was briefly deferred pending oncology advice. Following initiation of MTX he improved dramatically, returning to normal mobility, and steroids were successfully tapered off.Our patient had symptoms of inflammatory arthritis prior to identification of a SCC with local spread; however, his systemic and musculoskeletal features were initially regarded as paraneoplastic.Paraneoplastic arthritides encompass the musculoskeletal manifestations of malignancy. This includes polyarthritis, inflammatory myopathies, hypertrophic osteoarthropathy and palmar fasciitis. Symptoms may precede or occur concurrently with cancer presentations and often respond to treatment of the underlying cancer. Persistence of musculoskeletal symptoms may be a clue to co-existence of a rheumatic diagnosis. Reassessment of history and examination can guide physicians to a concurrent second diagnosis when clinical progress is not as expected.Our case also highlights the question of safe use of immunosuppressive drugs in rheumatic patients with cancer and whether they may promote or induce malignant disease. Literature is challenging to assess as many systemic inflammatory disorders, including RA, have an established increased risk of certain cancers, including lymphoproliferative disease. He was managed by prednisolone and hydroxychloroquine, followed rapidly by MTX, leading to dramatic symptomatic improvement.Low-dose MTX (25—30mg weekly) is considered first-line treatment in RA. Of the commonly used anti-rheumatic therapies it has the least evidence to suggest a potential increased malignancy risk. Other anti -rheumatic drugs such as Tumour Necrosis Factor (TNF) inhibitors also have a favourable risk profile regarding cancer development. Most observational studies evaluating the use of biologic therapy in RA patients with previous solid tumours do not show an increased risk of recurrence.Coordination of care with the patient and their oncologist is essential in the management of cases of individuals with rheumatic disease and concomitant cancer.Cancer is often considered with certain rheumatic presentations but sometimes it is equally important to look for concomitant rheumatic disease when patients with cancer have musculoskeletal symptoms.As cancer can mimic the early systemic features of arthritis and patients will often delay seeking medical advice, clinicians should be alert to the possibility of co-existent disease and ensure careful assessments at clinical encounters.The treatment of rheumatic disease in a patient with cancer may be of equal importance to the individual resulting in reduced quality of life, sometimes with greater impact than that of the cancer itself.Many anti-rheumatic therapies have a favourable profile regarding the risk of de novo malignancy and recurrence.For patients with rheumatic disease and active or previous cancer, clinical decision-making should carefully weigh up the risks and benefits of treatments for both conditions. Therapeutic options should be considered between rheumatologist, oncologist, and patient, taking into account prognosis, quality of life and patient choice.