Dysregulation of this pathway and changes in the ratios of these metabolites have been associated with numerous neurotoxic and inflammatory states, many of which present simultaneously with chronic pain symptoms. While further studies utilizing biomarkers to elucidate the kynurenine pathway’s role in chronic pain are needed, the metabolites and receptors involved in its processes nevertheless present researchers with promising sources of novel and personalized disease-modifying treatments.Chronic pain is a pressing medical and socioeconomic issue worldwide. It is debilitating for individual patients and places a major burden on society in the forms of direct medical costs and lost work productivity. Various biochemical pathways have been explored to explain the pathophysiology of chronic pain in order to identify biomarkers that can potentially serve as both evaluators of and guides for therapeutic effectiveness. The kynurenine pathway has recently been a source of interest due to its suspected role in the development and sustainment of chronic pain conditions. The kynurenine pathway is the primary pathway responsible for the metabolization of tryptophan and generates nicotinamide adenine dinucleotide (NAD Chronic pain is characterized as pain that persists for over twelve weeks despite medication or treatment . ChronicIn the United States, chronic pain is amongst the most commonly reported chronic conditions . In the According to recent studies, chronic pain continues to inflict enormous strain on public health, with total societal costs reaching as high as USD 635 billion a year in the United States alone . The ecoDespite the massive amounts spent on treatment, chronic pain is rarely resolved with total alleviation, as current medical approaches are limited in their efficacy . The cruThe diagnostic tools currently used to evaluate pain are highly influenced by subjective assessments, inevitably restricting the effectiveness of corresponding treatment modalities. In comparison to other pain therapeutics, opioid analgesics continue to display a relatively high level of effectiveness for treating chronic pain . HoweverA major contributing factor behind the relative lack of success in finding alternative medications to replace opioids is our somewhat nominal understanding of the biochemical mechanisms that underlie chronic pain. A deeper comprehension of the underlying biochemical mechanisms that govern the subjective experience of chronic pain would open the door to the development of novel and personalized non-opioid treatments that precisely regulate pain pathways . In ordeA biomarker is defined as a substance in an organism that can be accurately and reproducibly measured to indicate regular biological processes, pathological biological processes, or pharmacological responses to a treatment . BiomarkA further complicating circumstance in the search for chronic pain biomarkers is the logicality of the search itself . It has As of late, a particular mechanism of interest to chronic pain researchers has been the kynurenine pathway (KP) of tryptophan metabolism . ResearcTryptophan (Trp) is an essential amino acid that is metabolized by way of two distinct branches known as the serotonin and kynurenine pathways (KP), illustrated in N-formylkynurenine, which is primarily driven by the enzymes indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) [N-formylkynurenine is subsequently broken down by kynurenine formamidase to produce kynurenine (KYN). KYN can then be further metabolized through three sub-branches. The first branch mainly occurs in microglia and involves the degradation of KYN into 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid (3-HAA) by kynurenine 3-monooxygenase (KMO) and kynureninase, respectively [+) [+ synthesis.In mammals, the KP is started via the conversion of Trp into se (TDO) . N-formyectively . 3-HAA cectively . During ectively . Quinolively [+) . This afThe second branch of the KP also occurs in microglia, but is initiated by kynureninase activity on KYN . SimilarThe third branch of the KP involves the synthesis of kynurenic acid (KA) from KYN in peripheral skeletal muscle and astrocytes . KA is c+, a crucial chemical in cellular energy generation [Out of the various branches that KYN metabolism is composed of, the two routes that respectively result in the metabolites QA and KA appear to be especially implicated in chronic pain states, with the former metabolite acting as a precursor for the coenzyme NADneration . In addineration . The con+, with QA as an intermediate byproduct [N-methyl-D-aspartate (NMDA) receptor agonist in the brain [During physiological states, the favored route of the KP results in the eventual conversion of KYN into NADyproduct . QA is ayproduct . More sphe brain . QA alsohe brain .The vast majority of QA present in the body is produced by microglia and macrophages, hence making QA a particularly sensitive marker of chronic systemic inflammation . Product2+) into the neuron [2+ activate enzymatic pathways that degenerate critical proteins in the cell and increase intracellular nitric oxide levels, ultimately resulting in an apoptotic response by the neuron. Abnormally high levels of QA can cause hindered neuronal cell function or apoptotic death . When exe neuron . ExcessiAnother mechanism of neurotoxicity exhibited by QA is its detrimental effects on the glutamate–glutamine cycle within astrocytes . The gluChronic pain is one of the most common comorbidities of persistent inflammatory states. The substantial involvement of the KP in chronic inflammation mechanisms has consequently made its metabolites compelling targets for biomarker investigation in previous studies. Of the KP metabolites, elevated QA has been found to be the most common abnormal biomarker amongst chronic pain subjects. In a retrospective analysis of 17,834 unique chronic pain samples, elevated QA was observed in 29% of patients, the highest out of eleven compounds in a pain-specific biomarker panel that evaluated essential micronutrients for nerve health , chronic inflammation (QA and KA), oxidative stress/damage , and neurotransmitter turnover (5-hydroxyindoleacetate and vanilmandelate) . In a su+ is converted into kynurenic acid (KA) via kynurenine aminotransferase (KAT) enzymes [Conversely, kynurenine that is not used to produce NAD enzymes . KA is a enzymes . KA’s me50 = 7.9 to 15 µM) [50 = 200 to 500 µM) as well. Since the NMDA receptor is the ionotropic glutamate receptor subtype that is most permeable to Ca2+, its agonism is often implicated as the main driver of excitotoxic states [At elevated micromolar concentrations, KA is a broad-spectrum ionotropic glutamate receptor antagonist at α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), NMDA, and Kainate glutamate receptors . Howevero 15 µM) . KA was c states . Therefo2+ channels in astrocytes and sympathetic neurons, resulting in decreased intracellular levels of cyclic adenosine monophosphate (cAMP) and Ca2+, respectively [KA has also demonstrated agonist activity at certain G-protein-coupled receptors (GPCRs) as well . Of partectively . Furtherectively . KA-GPR3ectively .2+ concentration, elevated levels correlate with the activation of crucial inflammatory transcription factors such as NF-κB [2+ channels, which in turn reduces inflammatory signal secretion. Similarly, KA may also interfere with inflammation generation via KA-GPR35-mediated inhibition of the PI3K/Akt and MAPK pathways, both of which are essential for producing an inflammatory response [With the aforementioned effects of KA and GPR35 interaction in mind, it is possible that KA-GPR35 signaling may indirectly lead to the attenuation of the inflammatory response . In a pras NF-κB . Thereforesponse ,64. In cresponse . KA-GPR3response . See TabAs previously discussed, persistent inflammatory states are highly correlated with chronic pain. In patients who experience comorbid pain with various psychiatric conditions, diminished levels of neuroprotective metabolites have been frequently identified . KA’s fuSince KA and QA generation respectively occur within diverting branches of the KP, circulating levels of KA and QA can often share an inverse relationship to each other . In addip < 0.001), indicating that the greater the pain intensity is, the lower the Trp plasma levels are. Conversely, the results also showed a statistically discernible positive relationship between pain intensity and the kynurenine/Trp ratio (p < 0.001), indicating that elevated pain correlates with increased kynurenine levels. In another study by Staats Pires et al. 2020, individuals with chronic back pain had considerably higher IDO levels and kynurenine/Trp ratios than their healthy counterparts [As per the earlier overview of the KP, it is well recognized that IDO is one of the primary enzymes that drives the conversion of Trp to KYN. In accordance with this characteristic, prior clinical studies have discovered a correlation between elevated IDO levels and the presence of chronic, inflammation-based pain. In a study carried out by Barjandi et al. 2019, a sample size of 113 female patients was examined, 40 of which had fibromyalgia, 17 with temporomandibular disorders myalgia, and 56 were healthy controls . For allterparts . TogetheAs previously discussed, it is important to note that Trp acts as the precursor for the neurotransmitter serotonin . Since IIn addition to enhancing our comprehension and our ability to more accurately diagnose chronic pain conditions, the successful identification of mechanistic chronic pain biomarkers would also facilitate the development of novel, personalized, non-opioid therapies that can act as disease-modifying agents. Biomarkers would also grant researchers and healthcare professionals the ability to monitor the effectiveness of these novel modulating treatments over extended periods of time. Objective biomarkers are foundational to the practice of personalized medicine, and identifying and verifying biomarkers to aid in the diagnosis and treatment of chronic pain conditions would substantially lower healthcare expenses worldwide. While the discovery of any pain-specific biomarker is a meaningful contribution to our continuous pursuit towards further understanding the pathophysiology of chronic pain, efforts must be geared towards the most critical and influential biomarkers: those that can be modified to alter the course of a disease.


Patients receiving intravenous (IV) antibiotics in the outpatient setting require intensive laboratory monitoring and frequent clinical intervention. Infectious Diseases (ID) clinics often provide this care due to the need for dedicated staff for lab surveillance and follow-up scheduling. Before April 2023, our OPAT program required a minimum of 3 (non-physician) clinic staff who were tracking patients using a patchwork of paper lists, Electronic Health Record (EHR) “sticky-notes,” and telephone encounter notes.From September 2022 to April 2023, infectious disease stakeholders at MaineHealth were engaged in a specialty-specific EHR optimization program and were asked to generate a wish list of improvements. The development of a reliable, user-friendly OPAT tracking tool was identified as a key priority. Dedicated IT analysts searched publicly available databases of tools for our EHR  and reviewed existing, local reporting tools to identify solutions. Through an iterative process with ID stakeholders, IT analysts, and informatics specialists, a reporting tool was developed and implemented for all ID specialty offices.Stakeholders noted three key priorities for OPAT tracking - 1) identifying recently discharged patients on OPAT 2) flagging patients due for interventions , and 3) easy viewing of individual patient data to identify unique circumstances and home agency information. A locally-built innovative Reporting Workbench report for tracking pediatric kidney transplant patients served as a model for our build, as no identified tools in the EHR database matched our stakeholder needs.OPAT DashboardA view of the OPAT dashboard highlighting the ability to track patient follow-up, outstanding labs, antibiotic courses, and end-of-therapy plans. The dashboard contains many areas for customization and because it is built as a report within the EMR, it can be customized to particular work-flows .OPAT Enrollment WidgetBased on feedback from stakeholders, the enrollment form for this OPAT tracking dashboard allows significant flexibility for lab collection reminders, comments that display in the dashboard, and other functionality. Several fields also do not display on the dashboard for simplicity, but are viewable to users when individual patients are selected in the dashboard.In the early stages of using this tool, clinic staff have reported increased satisfaction with the new OPAT tracker. The development of this tool highlights the benefit of frequent, collaborative interaction between clinical IT teams, physician informaticists, and subject-matter experts who use the system. The success of this OPAT tracking tool has prompted us to investigate rolling out a similar tool for additional programs including HIV/PrEP care and Hepatitis C treatment.All Authors: No reported disclosures


Penicillin allergies are commonly reported in children but rarely true and perpetuate the use of alternative antibiotics. Leading allergy societies recommend a proactive approach to penicillin allergy delabeling. We use patient journey mapping to identify opportunities to improve the delabeling of penicillin allergies in children before, during, and after a child’s initial reaction to penicillin.Mapping was conducted via a secondary analysis of a qualitative study consisting of 18 interviews with parents recruited via social media reporting a penicillin allergy in their child. Two researchers coded interviews using a directed content analysis to capture parent’s experiences before, during, and after their child’s penicillin reaction. We describe the common experiences of parents ( >9 participants).Pre-reaction, parents reported a lack of awareness regarding potential reactions children may experience when taking penicillin. Intra-reaction, parents reported their child’s provider suspected their child’s reaction was due to a penicillin allergy, wrote the child’s reaction in the medical record, and suggested or advised that penicillin be avoided in the future without mention of the availability of penicillin allergy testing. Post-reaction, parents remained unaware of the availability of penicillin allergy testing and uninformed of the implications of penicillin allergies on their child’s care. To better engage parents in penicillin allergy testing, participants suggested that providers inform parents of: 1) potential reactions their child may experience at the time of penicillin prescription (pre-reaction); 2) the availability of penicillin allergy testing to confirm the presence of true allergy (intra-reaction); and 3) penicillin allergy testing procedures and the benefits of penicillin allergy testing for their child’s health (post-reaction), see Table 1.Using patient journey mapping, we describe the common experiences of parents concerning their child’s penicillin allergy, gaps in care parents experienced, and suggestions offered by parents to address their unmet care needs. These findings may inform future efforts to improve the delabeling of penicillin allergies in children.Sherry Pagoto, PhD, Astellas Pharmaceuticals: Scientific Advisor|Weight Watchers, International,: Scientific Advisor


Chronic hepatitis B virus (HBV) infection leads to the development of cirrhosis and hepatocellular carcinoma. Lifelong treatment with nucleotides/nucleoside antiviral agents is effective at suppressing HBV replication, however, adherence to daily therapy can be challenging. This review discusses recent advances in the development of long-acting formulations for HBV treatment and prevention, which could potentially improve adherence. Promising new compounds that target distinct steps of the virus life cycle are summarized. In addition to treatments that suppress viral replication, curative strategies are focused on the elimination of covalently closed circular DNA and the inactivation of the integrated viral DNA from infected hepatocytes. We highlight promising long-acting antivirals and genome editing strategies for the elimination or deactivation of persistent viral DNA products in development. Hepatitis B virus (HBV) in the human population has existed for 2000–3000 years , and theThe WHO has set ambitious targets to eliminate HBV as a public health threat by 2030. HBV “service targets” include further improvement in childhood vaccine coverage , major increases in birth-dose vaccine coverage or other methods to prevent mother-to-child HBV transmission (38–90%), improved diagnosing (5–90% of people with chronic HBV were diagnosed) and successful treatment (1–80% of eligible people were treated). These service improvements are aligned with “impact targets” of a 90% reduction in new HBV infections and a 65% reduction in HBV-related deaths (when compared to estimates from 2015). Nevertheless, preliminary data suggests that global HBV-related mortality is projected to increase by 39%, from 850,300 deaths in 2015 to 1,109,500 deaths in 2030, if the current diagnosis and treatment timing are not improved  .Exciting progress has been made in developing LA therapies for treating HBV infection, and these drugs hold great promise for the future of HBV treatment, including long-acting TFV for the prevention of mother-to-child transmission ,134,135.The ideal goal of antiviral therapy is to achieve a cure, which largely depends on suppressing the pool of cccDNA and HBsAg. HBV cure strategy can be broadly categorized into two types: functional cure and sterile cure. A functional cure for HBV involves the loss of HBV surface antigens (HBsAg), with or without the development of antibodies against HBsAg (anti-HBs), as well as undetectable serum DNA and the persistence of cccDNA with low or no transcriptional activity, allowing for treatment to be stopped. In a functional cure, the activated immune system should be able to control the remaining infected cells. However, a sterile cure of HBV would require the complete elimination of the cccDNA ,137.The recent advancement in technology has led to the development of new gene editing tools capable of mutagenizing or completely degrading the long-lived forms of HBV DNA ,139,140.Gene editing systems utilize engineered DNA-binding nucleases to generate site-specific insertion-deletion (indels) mutations. Their mode of action has advanced from induction of double-stranded break followed by spontaneous DNA repair to a less invasive and more precise single nucleotide modification ,144. MegToday, these setbacks have been largely resolved using cheap, efficient, and highly adaptable engineered RNA-guided CRISPR systems. Originally identified as a bacteria’s immune defense system, CRISPR technology is now widely recognized as a promising tool to permanently eliminate HBV expression with high specificity. Generally, the wild-type (WT) CRISPR system is a ribonucleoprotein (RNP) complex that comprises a nuclease enzyme (Cas) joined to a user-defined guide RNA (gRNA) by a tracRNA (trans-activating CRISPR RNA). Although more permissive variants have been developed, the WT CRISPR gene editing activity is restricted to genetic sequences that contain Protospacer Adjacent Motif (PAM) and this motif sequence varies between all Cas nuclease subtypes . The preIn gene-based therapy, the objective is to harness the specificity of an RNA-guided CRISPR system to permanently inactivate persistent HBV DNA forms without editing the host genome or creating new active viral species. The WT Cas may induce double-stranded breaks (DSB) in the host genome resulting in a lethal frameshift or chromosomal recombination following the error-prone NHEJ repair ,151. AlsLess invasive Cas-derived base editors and prime editors capable of creating single nucleotide alterations without inducing DSBs were created to address these concerns . Base edYan and his team first discovered rewriting the genetic code of episomal cccDNA into a dysfunctional phenotype using CBEs. They successfully introduced premature stop codons in the overlapping regions of polymerase and surface genes, which significantly decreased the expression of HBsAg and HBV polymerase in naturally infected HEPG2-NTCP cells . In theoAn even more relevant and promising finding was presented at the recently concluded HBV 2022 International Meeting. In this elegant study, LNP-delivered CBE mRNA led to a sustained loss of key viral proteins (HBsAg and HBeAg) and HBV DNA resulting in an impeded viral rebound in lamivudine-pretreated primary human hepatocyte (PHH) and mice harboring cccDNA surrogate compared to only lamivudine treated groups . The robIt has been previously reported that cell-derived nanoparticles called exosomes can successfully package and deliver cas9 RNPs to HBV-expressing cells where they demonstrate significant DSB-induced viral gene knockdown . Wang etThe prevention of end-stage liver diseases associated with chronic HBV is based on antiviral therapy with the goal of achieving a cure, which largely depends on suppressing the pool of cccDNA and HBsAg. HBV cure strategy can be broadly categorized into two types: functional cure and sterile cure. A functional cure for HBV involves the loss of HBsAg, with or without the development of antibodies against HBsAg, as well as undetectable serum DNA and the persistence of cccDNA with low or no transcriptional activity, making the treatment able to be stopped. In a functional cure, the activated immune system should be able to control the remaining infected cells. However, a sterile cure of HBV would require the complete elimination of the cccDNA ,137.Achievement of HBV functional cure focuses on eliminating HBsAg and suppression/elimination of cccDNA. Approved IFN/PEG-IFN therapy mainly has immunostimulatory effects and can induce HBsAg negativity, but the success rate is low and adverse events are common. Alternatively, HBsAg secretory inhibitor can also be used to prevent the secretion of HBsAg. Considering HBsAg can also use canonical host-cell secretory pathways, this strategy may also be unsuccessful. Nevertheless, Rep2139, an inhibitor of HBsAg secretion, when combined with PEG-IFN-α induced a significant decline in viremia and seroconversion in favorable HBsAg responders ,166.NAs aim to inhibit HBV DNA replication and reduce HBV DNA and ALT levels but rarely reduce cccDNA pool in the liver. Although currently approved NAs cannot alone achieve a functional cure, the chances of achieving a long-lasting functional cure for hepatitis B, with no rebound after stopping therapy, would be higher using a combination of immunomodulators, such as IFN-α and two or more antivirals that can target different steps in the virus replication cycle. A combination of virus entry blockers and maturation inhibitors, such as oral CAMs can be very effective in this regard . A combiWhen patients need treatments, the liver cccDNA pool is already established. Due to its long half-life and in the absence of massive liver regeneration, it has been mathematically modeled that long-term treatment with NAs would be necessary to significantly impact this pool ,147. TheThe road to achieving a higher functional cure rate for HBV and prevention of end-stage liver diseases is challenging. It is apparent that neither antiviral medications nor immunomodulators can achieve a functional cure on their own, indicating that further improvements are necessary to increase the likelihood of a functional cure. These improvements may include developing long-acting medication to provide sustained viral suppression and improve the chances of effectively reducing HBsAg level and silencing the cccDNA.


The global impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to considerable interest in detecting novel beneficial mutations and other genomic changes that may signal the development of variants of concern (VOCs). The ability to accurately detect these changes within individual patient samples is important in enabling early detection of VOCs. Such genomic scans for rarely acting positive selection are best performed via comparison of empirical data with simulated data wherein commonly acting evolutionary factors, including mutation and recombination, reproductive and infection dynamics, and purifying and background selection, can be carefully accounted for and parameterized. Although there has been work to quantify these factors in SARS-CoV-2, they have yet to be integrated into a baseline model describing intrahost evolutionary dynamics. To construct such a baseline model, we develop a simulation framework that enables one to establish expectations for underlying levels and patterns of patient-level variation. By varying eight key parameters, we evaluated 12,096 different model–parameter combinations and compared them with existing empirical data. Of these, 592 models (∼5%) were plausible based on the resulting mean expected number of segregating variants. These plausible models shared several commonalities shedding light on intrahost SARS-CoV-2 evolutionary dynamics: severe infection bottlenecks, low levels of reproductive skew, and a distribution of fitness effects skewed toward strongly deleterious mutations. We also describe important areas of model uncertainty and highlight additional sequence data that may help to further refine a baseline model. This study lays the groundwork for the improved analysis of existing and future SARS-CoV-2 within-patient data. Despite its tremendous impact on human health, a comprehensive evolutionary baseline model has yet to be developed for studying the within-host population genomics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Importantly, such modeling would enable improved analysis and provide insights into the key evolutionary dynamics governing SARS-CoV-2 evolution. Given this need, we have here quantified a set of plausible baseline models via large-scale simulation. The commonly shared features of these relevant models—including severe infection bottlenecks, low levels of progeny skew, and a high rate of strongly deleterious mutations—lay the foundation for sophisticated analyses of SARS-CoV-2 evolution within patients using these baseline models.The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019 is the most impactful human pathogen to arise thus far in the 21st century. Since its emergence, SARS-CoV-2 has been directly responsible for nearly 8 million deaths as of December 2022 . HoweverUnderstanding the evolutionary dynamics of SARS-CoV-2 and predicting new variants of concern (VOCs) that may result in waves of increased infection and mortality remains vital. Although the tools for studying these questions as framed via interhost spread of SARS-CoV-2 have been well-developed , the abiFor the viral population within a single patient, these episodic, beneficial mutations may be expected to modify patterns of genomic variation in a manner that would deviate from background patterns produced under constantly operating evolutionary processes  in plausible models, consistent with other recent results for SARS-CoV-2  and the A total of 12,096 model–parameter combinations were simulated  in five Within these required parameter sets, there are a total of 112 extended models ; the number of models that cleared the filtering requirement varied considerably between the required parameter sets. Briefly, 9 of the 18 required parameter sets had bottleneck models consistent with the empirical data , 11 of the 18 had consistent recombination-only models, 10 out of 18 had consistent recombination + progeny skew models, and all 18 had consistent full models. Generally, if a simpler model could be accepted, then more complicated models extending that model also tended to produce plausible results. The distribution of these nonrejected models is more fully detailed in The evaluation of intrahost population genomic data from SARS-CoV-2 patient samples has the potential to detect signatures of positive selection and allow for the early identification of VOCs. However, it is important to recognize that levels and patterns of genetic variation in a population are the result of a variety of factors including mutation, recombination, reproductive dynamics, purifying and background selection, and the history of bottlenecks and growth characterizing patient infection. One of the key methods of differentiating between these alternative explanations is through comparisons between empirical data and plausible simulated data . HoweverOur study supports three main conclusions that help to define a feasible parameter space governing intrahost SARS-CoV-2 evolutionary dynamics. First, our results support recent conclusions that SARS-CoV-2 transmission is generally associated with severe infection bottlenecks of one or a few virions . SpecifiSecondly, our results highlight the importance of considering the pervasive effects of purifying and background selection, via a consideration of the DFE, in constraining levels of variation segregating above the 2% frequency threshold. The widespread production of strongly deleterious mutations appears necessary to explain the apparent contradiction between the high mutation rates of RNA viruses  and the Ξ; Finally, lower progeny skew values produce more plausible models. Specifically, models with progeny skew and containing either lower probabilities of skewed offspring events and/or lower burst sizes were overrepresented amongst acceptances . Reproduction of SARS-CoV-2 within a host cell is generally nonlytic and instead involves release of new virions through continuous budding . The proFurther model differentiation is limited by current data available for intrahost variation of SARS-CoV-2. The 2% allele frequency cutoff used in this study mirrors the cutoff used in several previous studies . AlthougThese general bioinformatic recommendations assume that sequencing data will effectively sample intrahost variation without bias. However, most SARS-CoV-2 sequencing protocols rely on targeted PCR or probe-based enrichment of the SARS-CoV-2 genome to reduce background contamination of host nucleotides. Targeted enrichment approaches have been widely used in biology and are usually sensitive to any standing genetic variation that impacts the binding efficacy of enrichment probes or primers . Indeed,In addition to limitations relating to the detection of rare alleles, our study was also unable to consider the impacts of host compartmentalization  on intrahost genetic diversity. Compartmentalization may be an important factor for SARS-CoV-2 evolution in regard to the production of viral reservoirs in prolonged infections or infection of immune-compromised patients . HoweverWith that said, based on the currently available data, our study has successfully quantified areas of the SARS-CoV-2 evolutionary parameter space that are most plausible. In addition, the simulation framework presented here may be utilized to compare against future sequencing studies, which will likely enable a further narrowing of likely models. However, even the relatively broad parameter space here identified may be utilized to more effectively screen for newly emerging positively selected mutations  and in so doing reduce the traditionally high false-positive rates traditionally associated with such selection scans . This woWe used the SLiM software package v4.0.1,  to condu9–1011 . Owing to this scaling of population size, we scaled up the range of mutation and recombination rates accordingly to maintain a constant population-scaled product with the effective population size. The duration of infection was chosen to represent a range of potential times from initial infection to sampling, that is, including both time from initial infection to symptom onset and time from symptom onset to sampling. Given that most empirical studies report sampling in terms of days from symptom onset and variation therein  as the max rate (Ξ = 0.0001) as the minimum rate . Finally, we parameterized the DFE as the ratio of strongly deleterious relative to neutral mutations .To model recombination, we used a single parameter for the genome-wide recombination rate. For each virion, the simulation chose another random virion to serve as the recombination partner, and the simulation used this recombination partner for all progeny produced by the focal virion in that tick cycle, including those experiencing progeny skew. We used a multiple-merger coalescent model of progeny skew  which remax rate  or the pmum rate . The geoSimulations used a common, burn-in source population which was created using a full-model simulation run for 25,000 ticks at the highest value of carrying capacity and the mid-level value for all other parameters. We replicated each parameter combination five times. Therefore, we ran a total of 12,096 model–parameter combinations .Using the scikit-allel package  and custevad204_Supplementary_DataClick here for additional data file.


Fluorescence microscopy techniques have been widely adoptedinbiology for their ability to visualize the structure and dynamicsof a wide range of cellular and subcellular processes. The specificityand sensitivity that these techniques afford have made them primarytools in the characterization of protein localizations within cells.Many of the fluorescence microscopy techniques require cells to befixed via chemical or alternative methods before being imaged. However,some fixation methods have been found to induce the redistributionof particular proteins in the cell, resulting in artifacts in thecharacterization of protein localizations and functions under physiologicalconditions. Here, we review the ability of commonly used cell fixationmethods to faithfully preserve the localizations of proteins thatbind to chromatin, undergo liquid–liquid phase separation (LLPS),and are involved in the formation of various membrane-bound organelles.We also review the mechanisms underlying various fixation artifactsand discuss potential alternative fixation methods to minimize theartifacts while investigating different proteins and cellular structures.Overall, fixed-cell fluorescence microscopy is a very powerful toolin biomedical research; however, each experiment demands the carefulselection of an appropriate fixation method to avoid potential artifactsand may benefit from live-cell imaging validation. When feasible, fixed-cell imagingexperiments have been suggested to be supplemented by live-cell imagingto ensure that fixation does not introduce artifacts.1Visualizing the subcellular locationsof proteins is essentialfor understanding their functions and regulation. Fluorescence microscopyis among the most popular methods for characterizing protein localizationsin the cell, which affords high specificity and sensitivity withoutrequiring complicated sample preparation. Since fluorescence microscopyfeatures an inherent trade-off between spatial and temporal resolution,precisely determining the locations of highly dynamic proteins isoften difficult in living cells. Fixation provides an attractive wayto preserve snapshots of cells, allowing for high-spatial-resolutionmeasurements of protein localizations that are too dynamic to be characterizedin living cells. Although fixation has been thought to faithfullypreserve live-cell conditions, an increasing number of works havedemonstrated that fixation can sometimes artificially redistributeproteins in the cell, where the artifacts depend on the fixation protocolbeing used and the protein or cellular structure being studied.In this Review, we discuss works that characterizethe effect offixation on subcellular protein localizations and develop methodsto reduce fixation artifacts. We first briefly introduce three commonways to label a protein in the context of fixed-cell fluorescencemicroscopy—immunostaining, genome editing to label an endogenousprotein, and exogenous expression of a protein fused to a fluorescenttag—along with two widely used classes of fixatives, e.g.,cross-linking  and coagulating (organic solvents) fixatives.We then review studies that report fixation-introduced artifacts whileimaging a variety of proteins and structures in the cell, includingchromatin-binding proteins, liquid–liquid phase separation(LLPS) droplets, membrane receptor clusters, the Golgi apparatus,the endoplasmic reticulum (ER), mitochondria, and ciliary proteins.The qualities of fixation for different proteins are summarized in 9 to knock-in the cDNA of a fluorescent tag intothe genomic sequence that encodes the target protein. Genome editingtakes a significantly longer time  than immunostaining(hours) and requires verification that tagging the endogenous proteindoes not affect its functions, but in return, enables live-cell imagingof the protein at native expression levels. Cell fixation and permeabilizationare not required for visualizing an endogenous protein that is labeledvia genome editing, but these treatments can be necessary for morecomplex experiments, such as simultaneously imaging the protein andother biomolecules, e.g., nucleic acids, through fixed-cell basedfluorescence in situ hybridization (FISH) methods.10 Expressing an exogenous protein fused to a fluorescenttag is another strategy for labeling and imaging the protein in thecell, which demands controls similar to the genome editing-based labelingstrategy to guarantee that the protein’s function is unaffectedby tagging. The exogenous expression strategy generally takes moretime than immunostaining, but less time than genome editing. Thisstrategy sometimes requires selecting cells with exogenous expressionlevels comparable to the protein’s native expression levelsto study protein functions under near physiological conditions. Likethe genome editing-based strategy, exogenous expression enables live-cellimaging of proteins of interest, but cell fixation and permeabilizationis unavoidable in specific applications, including simultaneouslyimaging the protein and nucleic acids. In short, while each of thethree methods has its own benefits, pitfalls, and unique sets of considerations,they are all susceptible to fixation artifacts.Labeling a proteinof interest is a critical step of studying theprotein using fluorescence microscopy. Among numerous labeling methods,three strategies are broadly applicable to any protein of interestin the cell, including immunostaining, genome editing to label anendogenous protein, and expressing an exogenous protein fused to afluorescent tag. Immunostaining uses a fluorescently labeled antibodytargeting the protein of interest and is, by nature, a fixed-celltechnique, as cells must be fixed and permeabilized to allow the antibodyto enter the cell and bind to its target protein 1. While in situ. Upon being dissolved in water, PFA is depolymerized to monomericformaldehyde (FA) that is practically capable of cross-linking biomolecules.Compared with GA, FA has a smaller molecular weight and thus diffusesthroughout the cell more quickly. In contrast, being a dialdehydeconsisting of a pentane with formyl groups at C-1 and C-5, the bifunctionalityof GA allows it to form inter- and intramolecular covalent cross-linksmore quickly and over longer distances than FA. PFA and GA are oftenused in combination to better preserve subcellular localizations ofproteins like membrane receptors,12 tubulins,13 and some cytoplasmic proteins.14 It is theorized that PFA and GA used together can achievea more stable fixation output due to their complementary fixationrates and distances.16 In contrast, coagulating fixativesincluding methanol, ethanol, and acetone denature and precipitateproteins through rapid dehydration, a process generally faster thancross-linking using aldehydes. However, they are known to extractvarious biomolecules including lipids and cytosolic and nuclear proteins,and the rounds of dehydration and rehydration can noticeably alterthe appearance of many subcellular structures.18Two classes of chemical fixativesare broadly used in fixed-cellfluorescence microscopy: cross-linking fixatives, i.e., aldehydesincluding paraformaldehyde (PFA) and glutaraldehyde (GA), and coagulatingfixatives, i.e., organic solvents including methanol, ethanol, andacetone. Aldehydes react with proximal amino acid side chains to formmethylene bridges, cross-linking proteins 20 In addition, the structural stability affordedby cross-linking fixatives, or their combination, comes at the costof restricted access to epitopes and autofluorescence of the aldehydesthemselves.23 In contrast, organic solvents precipitate out lipidsthroughout the entire cell, fixing the cell and permeabilizing itsmembranes in one step. They also preserve epitopes well and do notexhibit significant autofluorescence compared with aldehydes.20Immunofluorescence introduces additional considerations, wherecell fixation must be followed by permeabilizing cell membranes toallow for antibody access and preserve the epitopes to allow for antibodybinding. Cross-linking fixatives themselves do not permeabilize cellmembranes; thus, permeabilization is typically performed after cross-linkingusing detergents, e.g., saponin, Triton X-100, and Tween-20, whichremove lipids and cholesterol from the membrane.Nevertheless, there iscurrently no universal, artifact-free fixationmethod that perfectly preserves the appearance of cells. Differentproteins and subcellular structures have been found better preservedby different cell fixation protocols. Below, we review the effectsof different fixation methods on various systems of interest in thecell and discuss the live-cell experiments used to identify the fixationartifacts.24 The localizations of chromatinarchitectural high mobility group (HMG) proteins are also thoughtto play a role in the regulation of mitotic chromatin.26 A large body of work used immunofluorescence to show that TFs andHMG proteins appear to be evicted from mitotic chromatin. However,more recent reports suggest that these observations are inconsistentwith live-cell fluorescence images and can largely be attributed tofixation artifacts.Numerous proteinsare associated with chromatin in mammalian cells.Characterizing where and when they bind on chromatin is critical tounderstanding their functions. For example, during mitosis when transcriptionis stopped and chromatin condenses, it is thought that mitotic bookmarking,i.e., the association of a small number of critical transcriptionfactors (TFs) to mitotic chromatin, might enable daughter cells toreestablish their intended transcriptional program after mitosis.5 contrary to previous findings based on immunofluorescencethat endogenous HMGB and HMGN proteins dissociate from mitotic chromatin.28 Consistently, fluorescently labeled HMGB1, HMGB2, HMGN1, and HMGN2strongly colocalize with Hoechst-labeled condensed chromatin in livemouse 3T3 cells, which become largely excluded from the compactedchromatin upon cell fixation with PFA. The authors verified that thediscrepancy was not a result of fluorescent tagging of proteins ortheir ectopic expression levels. Interestingly, however, fixing cellswith a methanol/acetone mixture temporarily preserved the HMGB–chromatinassociation, though it ultimately caused nearly all fluorescence signalto be lost by the end of the fixation protocol, proving to be an inappropriatefixative. Because the fixation artifact was only observed upon PFAand FA treatments, the authors argued that (para)formaldehyde-basedfixation may prevent HMG–chromatin binding by distorting thechromatin structure or the structure of the HMG proteins themselves.Specifically, after fusing high mobilitygroup box (HMGB) and highmobility group N (HMGN) proteins to a fluorescent tag and expressingthem in HeLa cells, Pallier et al. found that the proteins bind tochromatin throughout early to late phases of mitosis in live cells,32 Teves et al. imaged a variety of endogenously expressed Halo-taggedTFs, including SRY-box transcription factor 2 (Sox2), octamer-bindingtranscription factor 4 (Oct4), estrogen related receptor beta (Esrrb),Krüppel-like factor 4 (Klf4), specific protein 1 (Sp1), forkheadbox protein O1 (Foxo1), and forkhead box O3 (Foxo3a), in live mouseembryonic stem cells and showed that they are all enriched at themitotic chromatin but released into the nucleoplasm upon cell fixationwith PFA at different concentrations and methanol. It is noteworthythat among all the TFs investigated in this work, Sox2 is the onlyone that has an HMG domain, suggesting that the reported fixationartifact is not limited to chromatin-associated proteins with HMGdomains. Other works have found similar artifacts where TFs that associatewith mitotic chromatin in live cells are evicted upon fixation, includingGATA binding protein 1 (GATA1) upon fixation by 0.4% PFA,6 forkhead box protein A1 (FoxA1) upon fixationby 3.7% FA,7 and hepatocyte nuclear factor1β (HNF1β) upon fixation by 4% FA.8 Notably, in all the studies discussed here, the cross-linking fixatives(various concentrations of PFA and FA) but not the coagulating fixativesall induced similar fixation artifacts where TFs were evicted frommitotic chromatin. While fixation using a methanol/acetone solutiontemporarily preserve the binding of HMGB1 to mitotic chromatin,5 fixation using 100% methanol still resulted insignificantly reduced Sox2 enrichment on mitotic chromatin.2 Interestingly, cell fixation by both −20°C and room temperature methanol was shown to completely preservebinding of HNF1β to mitotic chromatin.8 The difference in fixation outcomes could be due to the differentproteins of interest, the different fixation protocols used, or thecombination of the two, yet these findings do not clearly suggestany one coagulative fixation protocol over another.The fixation-induced release of mitotic chromatin-associated proteinsis not unique to HMG proteins upon PFA fixation. Instead, this artifacthas been found replicated across many TFs upon cell fixation by bothcross-linking and coagulating fixatives. Whereas previous works usedimmunofluorescence to show that most TFs are excluded from chromatinduring mitosis,2 twostudies by Festuccia et al. showed that the mitotic chromatin bindingactivities of Esrrb can be preserved by fixation with 3.7% glyoxalalone and by fixation with 2 mM disuccinimidyl glutarate (DSG) followedby 4% FA.34 Glyoxal is a bifunctional cross-linker witha faster fixation rate than PFA and it has been increasingly usedin recent super-resolution studies.35 DSGis also a bifunctional cross-linker and, like GA, is able to cross-linkproteins over longer distances than FA alone.37 While the interesting findings by Festuccia et al. demonstrate thepromise of fixation protocols using glyoxal and DSG combined withFA, the fixation quality is possibly dependent on the protein of interest.It is recommended that researchers carefully choose a fixation protocolfor a specific chromatin-associated protein and compare against live-cellimages if fixed-cell techniques are required to investigate the proteinlocalization.It is noteworthythat some cross-linking fixatives are reportedto preserve the binding of specific proteins to mitotic chromatin.Whereas Teves et al. demonstrated that Esrrb is evicted from mitoticchromatin upon fixation with 4% PFA,39 While rigorous characterization of LLPS in vivo remains a question under active investigation,41 detection of discrete puncta that have a spherical shape, undergofusion and fission, and dynamically exchange biomolecules with thesurrounding according to fluorescence recovery after photobleaching(FRAP) is often considered evidence of putative LLPS in living cells.While such diverse measurements have been widely used for studyingproteins under overexpression conditions, far fewer approaches areavailable to probe LLPS under physiological conditions. Detectionof local high-concentration regions or puncta of an endogenously expressedprotein using immunofluorescence of fixed cells has been used in anincreasing number of studies as evidence of LLPS.46 This assumes that fixation faithfully preserves multivalentinteractions and LLPS formed in living cells. Surprisingly, however,our recent study suggested that this is not always true. We expressedin human U2OS cells various fluorescently labeled proteins that areknown to undergo LLPS, including the IDRs of fused in sarcoma (FUS),Ewing sarcoma breakpoint region 1/EWS RNA binding protein 1 (EWSR1),and TATA-box binding protein associated factor 15 (TAF15), and wecompared the appearance of their LLPS puncta in living cells and afterfixation using cross-linking fixatives, e.g., PFA at different concentrationsand a combination of PFA and GA. We found that fixation can both enhanceand diminish the appearance of LLPS in living cells by significantlychanging specific proteins’ puncta sizes and numbers. Althoughsome of our tested proteins, i.e., EWS::FLI1, full-length FUS, andTAF15 fused to ferritin heavy chain (FTH1), do not have their punctatedistributions significantly changed upon cell fixation, this workpresents a caveat in studying LLPS using fixation-based methods includingimmunofluorescence and again highlights the need to use live-celltechniques to verify that fixation does not artifactually redistributeproteins with a LLPS potential in the cell.Over the past decade,LLPS has garnered much interest in the biologyresearch community as a potential mechanism by which biomoleculesmay rapidly self-organize in the complex cellular environment. Unlikemany membrane-bound structures where biomolecules are contained bya membrane with protein channels that allow specific biomoleculesto pass in and out, LLPS is driven by high levels of transient, selective,and multivalent interactions between the intrinsically disorderedregions (IDRs) of proteins comprising the droplet.a priori how fixationwith a given protocol can affect the subcellular localization of aparticular protein, an increasing number of reports have shown thatproteins undergoing faster binding/unbinding events are more poorlypreserved by fixation. Chromatin-associated proteins have providedprominent examples. Schmiedeberg et al. compared wild-type methyl-CpGbinding protein 2 (MeCP2), a DNA-binding protein, and mutants withweakened and more transient chromatin interactions. They found thatonly the wild-type and mutants with chromatin-binding residence half-timeslonger than 5 s as measured by FRAP were well-preserved by 4% PFAfixation.3 Consistently, it has been shownthat many stable chromatin-binding proteins are well preserved byfixation, including the histone protein H2B,4 TATA-bindingprotein (TBP),2 and CCCTC-binding factor(CTCF).48 In contrast, Sox2, which more transientlybinds to mitotic chromatin than H2B according to FRAP and single-particletracking (SPT) measurements, is artificially released to the nucleoplasmupon fixation with PFA.2 These findingscollectively suggest that proteins that bind to chromatin with longerresidence times are better preserved by fixation. Similarly, we recentlyreported that proteins have their intracellular LLPS puncta betterpreserved by fixation if their multivalent protein–proteininteractions underlying LLPS are less dynamic. Specifically, we comparedHalo-tagged TAF15 and TAF15-FTH1 fusion proteins with both SPT andfixed-cell imaging assays. We found that TAF15 binds to its LLPS punctawith much shorter residence times than TAF15-FTH1. While the punctaof TAF15 have their sizes and number significantly reduced upon PFAfixation, the appearance of LLPS of TAF15-FTH1 was well preservedby fixation with 4% PFA. These results suggest that both chromatin-associatedproteins and proteins with a LLPS potential tend to have their intracellulardistributions better preserved by fixation if they have slower binding/unbindingrates to interaction partners.While it is difficult to know Understanding the mechanism underlyingthis trend can help informpotential ways to mitigate fixation artifacts. Teves et al. proposeda model that explains the mislocalization of specific TFs from mitoticchromatin by first noting that these TFs rapidly exchange betweenchromatin-bound and freely diffusing states and that cross-linkingfixation methods rely on the diffusion of aldehydes that permeatethe cell from the membrane inward, forming a cross-linking gradient2A. Since1 Predictions based on our model are consistentwith experimental observations8 that more dynamic protein-binding events are more poorly preservedby fixation and further suggest that the severity of fixation artifactsis dependent on the overall rates of fixation relative to proteinbinding, rather than protein binding and unbinding rates themselves.This is consistent with the observation that while PFA artificiallyevicts Esrrb from mitotic chromatin, glyoxal, a cross-linker witha faster fixation rate than PFA, faithfully preserves this mitoticbinding activity.34 Furthermore, our model explains PFA/FA fixation-induced depletionof TFs from mitotic chromatin as that the fixation rate of TF moleculesbound to chromatin is slower than that of unbound molecules, whichis consistent with the explanation using the cross-linking gradientmodel.In principle, this model of fixation is applicablenot only toTFs dynamically interacting with mitotic chromatin but also generallyto proteins that dynamically interact with a centrally located structurein the cell, e.g., LLPS puncta in the nucleus, where we would expectthat the cross-linking gradient would fix the freely diffusing proteinmolecules near the cell periphery first and deplete the puncta asa result. However, this is inconsistent with our observation thatthe LLPS puncta of specific proteins can increase in number and becomelarger upon fixation. The discrepancy motivated us to propose a newmodel of fixation where we take into consideration the dependenceof fixation rates on different proteins and their microenvironments,which results in potentially different fixation rates for proteinsbound to LLPS puncta and for proteins freely diffusing outside ofpuncta 2B.We quMany cellular proteins besides those bound to chromatinor undergoingLLPS are known to have their distribution affected by fixation. Whilethe balance of fixation and protein binding dynamics are predictedto play a role in these fixation artifacts according to above-discussedmodels, some of the artifacts are also thought to be caused by mechanismssuch as incomplete fixation and disruption to organelle morphology.In addition, some of the reported fixation artifacts remain unexplained,and the underlying mechanisms are under active investigation.11 Accordingto SPT and FRAP measurements, many membrane molecules including transmembraneproteins and lipids remain mobile upon fixation with only PFA, butnot with a combination of PFA and 0.2% GA.12 In immunofluorescence experiments involving cell fixation by PFAalone, membrane receptors remain largely mobile after incomplete fixationand adding a specific receptor’s secondary antibody causesaccretion of the unfixed receptor, resulting in artifactual clusteringof the receptor. These findings highlight the importance of live-cellcontrols for fixed-cell studies, measuring postfixation diffusionin assessing the quality of fixation, and carefully selecting an appropriatefixation protocol, which could vary with the biomolecule of interest.One group of proteins reported to have fixation artifacts are membranereceptors, which are typically dispersed across the cell membraneand cluster in response to stimuli to trigger critical signaling pathways.Because the clustering events are quite transient, characterizingthem in live cells can be difficult, making fixed-cell imaging attractive.However, membrane proteins and receptors have been found to artifactuallycluster in immunofluorescence images. Specifically, Stanly et al.showed that the transmembrane receptors lymphatic vessel endothelialhyaluronan receptor 1 (LYVE-1), cluster of differentiation 31 (CD31),and cluster of differentiation 44 (CD44) diffusely localize in livecell membranes but artifactually cluster upon fixation with both 1%and 4% PFA and that this postfixation redistribution can be preventedusing 0.2% GA in addition to PFA.18 This screening approach provides useful information on which organellesand subcellular structures are and are not well preserved by differentfixation protocols. For example, while the localizations of proteinmarkers of endoplasmic reticulum (ER) and Golgi apparatus were robustlypreserved by all fixation protocols tested, those of mitochondriawere poorly preserved by coagulative fixatives. Specifically, mitochondriain cells appeared to collapse into aggregates near the nucleus afterbeing dehydrated with ethanol and methanol. The authors concludedthat fixation via PFA followed by permeabilization via Triton X-100is generally the best fixation protocol, though it is still not idealfor certain proteins of interest; for example, a combination of FAand Triton X-100 is not suitable for immunostaining of lipid droplet-associatedproteins.49 Also, the study by Stadleret al. would have benefited from a comparison of the fixed-cell dataagainst the live-cell localizations of the proteins investigated.Artifacts in fixed-cell imaging are also reported to occur dueto the physical deformation of organelles or subcellular structuresof interest in response to fixation. In search of a single fixationprotocol that is suitable for proteome-wide localization studies usingimmunofluorescence, Stadler et al. compared the effects of 6 fixationprotocols, involving both coagulative and cross-linking fixatives,on the localizations of 18 proteins that are associated with differentorganelles and subcellular structures.50 Although the authors did not compare fixed-cell immunofluorescenceto live-cell images, their work revealed major discrepancies betweendifferent fixation protocols. For example, fixation of the cilia markerprotein adenylyl cyclase 3 (ADCY3) with a combination of 4% PFA and4% sucrose and with 4% PFA diluted in cytoskeletal buffer yieldeddifferent results. While the PFA–sucrose method showed themarker localizing to cilia, the PFA–cytoskeletal buffer methodinstead revealed the presence of the marker in mitotic spindles. Whilethey recommend fixation using 4% PFA diluted in cytoskeletal bufferor at least the use of cytoskeletal buffer as a general starting point,they conclude that there is no optimal fixation protocol among thosethey tested and that different fixation protocols may need to be usedeven for the same protein, depending on its subcellular localizationof interest. The mechanism underlying the differential effects ofthe investigated fixation protocols is still unknown.Recent work examining the effects of fixation on the localizationsof ciliary proteins highlights that different fixation protocols canbe required even for proteins associated with the same organelle.Primary cilia are sensory, microtubule-based organelles that protrudefrom surface of cells and play a role in cell signaling. Precise measurementof the localizations of ciliary proteins has been the first and criticalstep of understanding their extraciliary functions in signaling. Huaand Ferland investigated this by measuring the localizations of 8ciliary proteins using immunofluorescence based on both coagulativeand cross-linking fixation protocols.While applying fluorescencemicroscopy to fixed cells is a powerfulway to study the subcellular localizations of proteins, care needsto be taken to ensure that the chosen fixation protocol faithfullypreserves those protein localizations. Live-cell imaging of the proteinunder investigation after labeling it fluorescently, whether by genomeediting to label an endogenous protein or by expressing an exogenousprotein fused to a fluorescent tag, is often a helpful control toensure that fixation is not introducing artifacts. Since there currentlyis no universal, artifact-free fixation protocol, it is often necessaryto choose an appropriate fixation protocol on a case-by-case basisdepending on the protein being studied. We discuss different mechanismsunderlying fixation artifacts, including incomplete fixation causingthe artifactual clustering of membrane receptors, coagulating fixativesdeforming mitochondria, and slow fixation relative to the bindingand unbinding dynamics of proteins associated with subcellular structuresincluding chromatin and LLPS droplets. However, many other known fixationartifacts occur for yet unknown reasons, e.g., aldehyde- and organicsolvent-based fixation protocols failing to preserve the localizationsof ciliary proteins.1 This idea is consistentwith several earlier works. Notably, Teves et al. showed that highpressure freezing followed by freeze substitution, a standard fixationtechnique used for electron microscopy that slows down biomolecularmotions, manages to partially preserve binding of Sox2 to mitoticchromatin, which is artificially released to the nucleoplasm uponPFA fixation.2 It will be of great futureinterest to develop novel fixation methods with significantly fasterfixation rates than biomolecular interactions to eliminate many fixationartifacts in the cell.Our recent work suggests that a fast overallfixation rate relativeto protein binding/unbinding rates can minimize the dynamics-dependentfixation artifacts.


Macrophages are critical regulators of tissue homeostasis but are also abundant in the tumor microenvironment (TME). In both primary tumors and metastases, such tumor-associated macrophages (TAMs) seem to support tumor development. While we know that TAMs are the dominant immune cells in the TME, their vast heterogeneity and associated functions are only just being unraveled. In this review, we outline the various known TAM populations found thus far and delineate their specialized roles associated with the main stages of cancer progression. We discuss how macrophages may prime the premetastatic niche to enable the growth of a metastasis and then how subsequent metastasis-associated macrophages can support secondary tumor growth. Finally, we speculate on the challenges that remain to be overcome in TAM research. Macrophages are tissue-resident immune cells that emerge from multiple waves of hematopoiesis during embryonic development to seed their organs of residency . In miceDuring organogenesis, macrophages undergo tissue imprinting whereby embryonic progenitors first acquire a core macrophage differentiation program including pattern recognition and cytokine receptors . Then, tIn the most basic sense, oncogenesis occurs as a result of an accumulation of mutations in oncogenes that permit normal cells to overcome restrictions on cellular replication such that they can grow without restraint to form a tumor. However, it is not only the tumor that has pathological consequences; other components that form the tumor microenvironment (TME) influence oncogenesis and cancer progression . The TMEMacrophage diversity is broadly conceptualized through the prism of two antagonistic polarization states: pro-inflammatory M1 and anti-inflammatory M2 macrophages . Upon th+ TAMs have also been shown to preferentially localize at the invasive margins of primary tumors and metastases in the MN-MCA1 murine model of cancer [Although TAMs undoubtedly exhibit immunosuppressive properties, the M1/M2 paradigm does not fully reflect the extent of macrophage and TAM heterogeneity  and the f cancer , therefoCsf1op), the major macrophage growth factor, and genetically modified to develop mammary cancer had a delay in the development of metastatic carcinomas, therefore showing the involvement of macrophages in malignant progression of breast cancer [TAMs were originally considered remnants of an abortive immune response against the tumor . Howevert cancer . These sTAMs promote vascularization to supply oxygen and nutrients to cancer cells in the well-described process of angiogenesis . Numeroureg) recruitment. This phenomenon has been highlighted in ovarian cancer [regs can then deactivate cytotoxic T cells directed against tumor cells [Another prominent function of TAMs in primary tumors is their role in establishing and maintaining an inflammatory environment. Examples of such TAM-derived inflammatory factors favoring tumor development are multiple and include CXCL8 in endometrial cancer , IL-6 inn cancer , nasophan cancer , and livn cancer , where tor cells . TAMs caor cells , 49, 50,or cells . It is wor cells  or PD-L1or cells  and coulor cells . In a sior cells .TAMs also promote epithelial to mesenchymal transition (EMT), a process during which epithelial-like, early proliferating cancer cells lose the capacity for cell–cell adhesion and adopt a fibroblast-like phenotype with invasive and migratory properties , 56. EMTTAMs are also involved in active ECM remodeling, collaborating notably with cancer-associated fibroblasts (CAFs) to promote tumor cell intravasation . Indeed,+ TAMs upon TGF-β stimulation in the TME. Attracted by fibroblast-derived CXCL12, these TAMs migrate toward the vascular niche, where they adopt a perivascular TAM phenotype and disrupt the junctions between endothelial cells, which allow tumor cells to intravasate into the circulation [EMT and ECM remodeling precede the intravasation of tumor cells into the circulation and their subsequent dissemination to distal organs. This key event in metastasis formation occurs at sites known as “tumor microenvironment metastasis (TMEM) doorways”, characterized by the dynamic association between one endothelial cell, one TAM and one cancer cell –82. TAMsculation –85. Of nculation , 86.H2-CD4+ T cells stimulate EGF production by TAMs, and depletion of CD4 + T cells or IL-4-neutralizing antibody treatment reduces the metastatic burden [In addition, activation of a paracrine loop also allows cancer cells that produce CSF-1 and TAMs that produce EGF to migrate together toward TMEM doorways. Thus, blocking CSF-1 or EGF receptors reduces cancer cell migration and invasiveness in breast cancer rodent models . Furtherc burden . CollectIn the late 19th century, Paget proposed the “seed and soil” theory of metastasis  in whichCx3cr1CreERT2-based fate mapping mouse model challenged with ovarian cancer cells, which has the capacity to form metastases in the omenta [+ CD163+ omentum macrophages were shown to favor metastatic dissemination from ovaries to omenta, and their depletion resulted in reduced ascitic volume and metastatic invasion of this organ [Investigations are ongoing to understand how macrophages shape the premetastatic niche and whether their origins have a differential impact. This latter question has been approached using the e omenta . In thisis organ . To date+ exosomes bind specifically to liver-resident macrophages (known as Kupffer cells), whereas ITGα6β4+ and ITGα6β1+ exosomes recognize lung fibroblasts and epithelial cells.Mechanistically, pre-metastatic niche development and macrophage imprinting within that niche have been shown to be dependent on extracellular vesicles (EVs) that originate from the primary tumor and circulate within the blood. Of note, EVs have been classified based on their specific size and biogenesis and encompass microvesicles (150–1000 nm) arising from membrane budding, which are involved in local communication , and sma+ cytotoxic T cells from the pre-metastatic niche [The mechanisms of action of tumor-derived EVs are only beginning to be elucidated. For example, macrophage migratory inhibiting factor (MIF) produced by primary pancreatic tumors and delivered by EVs remotely induces liver Kupffer cell production of TGF-β . This pric niche , 98. Theic niche . Further+ Kupffer cells [+ T cells and improves the growth potential of disseminated tumor cells. Others have shown that EVs from lung adenocarcinoma notably induce upregulation of CD206, PD-L1 and GLUT1 by lymph node CD68+ macrophages. GLUT1 expression by macrophages increases their glucose uptake, and this glycolytic shift favors the establishment of the pre-metastatic niche [In the liver, EV lipid cargo is handled by a specific subset of CD206er cells , 101, leer cells . This phic niche . In lineic niche , redefinic niche , these fThese emerging findings, which place macrophages at the forefront of pre-metastatic niche establishment, can also be envisaged in the context of the macrophage network between distal tissues, which has been demonstrated to play a notable role in the context of myocardial infarction . Indeed,Finally, it should be noted that while EVs are scrutinized for their role in the priming of premetastatic niches, tumor cells can also prime distal macrophages via their release of free enzymes such as lysyl oxidase (LOX) .Macrophages continue to support metastasis development after tumor cell migration has occurred. This is evident based on the finding that inhibiting TAM recruitment to a metastatic site results in a lower metastatic burden, as shown for lung –110 and Once the secondary tumor is established, macrophages deemed metastasis-associated macrophages (MAMs) in the literature  maintain+ and CD4+ lymphocytes and a reduced metastatic burden [Many studies have described the recruitment of CCR2-expressing monocytes to the metastatic niche upon CCL2 production by stromal cells, which gives rise to MAMs , 116. Thc burden .The liver is the most common site for metastasis, likely due to its dense blood vessel architecture, with the portal vein supplying a large amount of blood and hepatic sinusoids offering a secondary network with lower pressure and thus more time for migrating tumor cells to attach to the organ . As show+ macrophages display metabolic, immunosuppressive and matrix remodeling features that accumulate in metastases [After the liver, the lungs constitute the second most frequent site of metastases. Exosomal priming of lung macrophages promotes the development of the pre-metastatic niche by inducing T-cell suppression  and neuttastases .In this review, we have highlighted various facets of tumor-associated macrophage biology that influence different steps of cancer development. The versatility of TAM functions is evident; thus, it is difficult to identify one unified target that might be of clinical benefit –130. TheTo argue for this improved consideration of TAM heterogeneity, we have discussed the extent of TAM heterogeneity, with TAMs actually encompassing spatiotemporally unrelated macrophage populations within primary tumors, distal healthy tissues and metastasis sites. It remains to be fully deciphered how fundamental determinants of macrophage biology, such as their origin, their local environment and the time spent in the tissues, differentially influence tumor progression in these three different contexts .To tackle these fundamental questions, our methodology needs to evolve. Many studies have relied on mouse models of cancer thus far, but we should acknowledge the inherent limitations of these systems. Orthotopic models, such as the widely used canonical B16 melanoma model, are convenient and easily combinable with knock-in or knock-out animals; unfortunately, this type of model is quite different from the natural disease course of cancer. Indeed, while the primary TME can be more-or-less recapitulated depending on the models, these systems completely bypass the key step of pre-metastatic niche priming owing to their fast-developing nature. This feature disconnects these models from patient contexts in which, as previously stated, metastasis remains the main cause of death. Genetic models closer to what is observed in patients do exist but are usually less convenient due to their lower penetrance and often asynchronous tumor emergence, limiting reproducibility and the establishment of robust conclusions. In contrast, patient biopsies represent invaluable samples and are extensively used; however, disease genesis is difficult to determine from one end-point sample from one location, either the primary tumor or metastasis, and only limited information can be extracted from the analysis.Considering these issues, meaningful alternatives are needed to better understand the roles of macrophages in every step of the disease process. There are many avenues to be explored, and the recent increase in single-cell omics technologies offering snapshots of tissue activity at an unprecedented resolution will no doubt enable the precise identification of targets during disease development. These approaches now need to be coupled with models that consider disease dynamics, from the initial acquisition of oncogenic mutations to metastasis and multiorgan failure. The most recently developed spatial transcriptomic technologies allow for the identification of pathways that are activated in TAMs but also in all the other cells from the TME while conserving its architecture. These technologies can even be applied to fixed samples, allowing the retrospective analysis of hundreds of thousands of samples from cancer patients stored in hospitals worldwide. The increase in immunometabolism research should also reveal novel insights into macrophage activity within the TME, which could lead to the development of a new generation of metabolite-targeted therapies to reprogram TAMs into anti-tumor cells. It is up to us to make fruitful use of this wealth of information to generate knowledge that will inform the precise design of innovative TAM-related immunotherapies.


Managing multiple pregnancies is challenging and requires careful evaluation. Point of care ultrasound (POCUS) has emerged as a potentially crucial tool in assessing suspected first-trimester pregnancies. However, its role in evaluating multiple pregnancies remains uncertain. We present the case of a 36-year-old Ghanaian female who presented with acute vaginal bleeding after undergoing in vitro fertilization. A bedside transabdominal POCUS identified four intrauterine gestations with fetal poles and cardiac activity, suggesting a quadruplet viable pregnancy. A subsequent transvaginal ultrasound confirmed the findings. The patient was discharged with a follow-up appointment with an Obstetrician-Gynecologist. This case highlights the significance of POCUS in early pregnancy diagnosis, facilitating accurate identification and appropriate referral for further management. It also demonstrates the utility of POCUS in determining gestational age and viability. To our knowledge, no published case reports specifically address the diagnosis of a quadruplet pregnancy, emphasizing the role of POCUS in optimizing care for high-risk multiple pregnancies. Point of care ultrasound (POCUS) has evolved into an invaluable tool for numerous healthcare professionals, spanning from the emergency department (ED) to the internal medicine ward A 36-year-old Ghanaian female presented to our tertiary academic hospital's emergency department with a chief complaint of vaginal bleeding. She disclosed having undergone in vitro fertilization (IVF) one-month prior in Ghana due to a 6-year history of infertility. At triage, her vital signs were temperature of 36.9 °C, heart rate of 83 bpm, blood pressure of 122/84 mmHg, respiratory rate of 18 bpm and SpO2 of 99%. Physical examination revealed a non-tender abdomen and a closed cervical os with no active bleeding. In order to further evaluate the patient's condition, an obstetric POCUS examination was performed using a 3-5MHz curvilinear transducer. The POCUS examination identified the presence of four distinct live intrauterine gestations, each displaying a fetal pole and cardiac activity .A serum beta human chorionic gonadotropin level subsequently drawn revealed a value of 377, 520 IU/L (<5 UI). A comprehensive obstetric ultrasound was further requested and confirmed the four viable intrauterine gestations, with estimated gestational ages ranging from 8 weeks 0 days to 8 weeks 2 days. There was no evidence of heterotopic ectopic pregnancy. Following the diagnosis, the patient was discharged from the emergency department with an urgent appointment booked with the on-call Obstetrician-Gynecologist for further management and follow-up. Due to the POCUS and comprehensive ultrasound exams identifying multiple gestations, we were able to provide the patient urgent follow up for her high-risk pregnancy. Typically, first trimester patients with vaginal bleeding at our center are referred to our Early Pregnancy Assessment Unit (EPAU), which does not always necessitate a call to the on-call Obstetrician-Gynecologist. Additionally, we hereby declare that the referred patient provided us with her verbal consent and had the opportunity to review this manuscript.Multiple pregnancies including quadruplet pregnancies are rare and associated with increased risks of adverse maternal and neonatal outcomes Our case file highlights the utility of POCUS in providing crucial information regarding gestational age and viability in a patient presenting with first-trimester vaginal bleeding. Prior to our examination, the patient was unaware that she was pregnant. Furthermore, POCUS facilitated early diagnosis and referral for high-risk prenatal care. Early initiation of prenatal care has been associated with positive outcomes, including reduced neonatal and infant mortality rates and decreased incidence of low birth weight th week of pregnancy, particularly when the fetal heart rate becomes detectable around the 8th week It is important to acknowledge the limitations of transabdominal obstetric POCUS. Firstly, it should be noted that transabdominal POCUS cannot definitively rule out ectopic pregnancy. A recent systematic review encompassing both transabdominal and/or transvaginal POCUS examinations performed by emergency physicians reported a sensitivity of only 90% To our knowledge, this is the first case report to identify four live intrauterine gestations using obstetric POCUS. Utilizing POCUS in patients with high-risk multiple pregnancies who have undergone assisted reproduction can aid in the assessment and management of potential complications. In this case, POCUS correctly identified multiple viable live intrauterine gestations and helped facilitate appropriate prenatal care and follow-up. The authors have no disclosures related to this work. Video S1