Methane is the final product of the anaerobic decomposition of organic matter. The conversion of organic matter to methane (methanogenesis) as a mechanism for energy conservation is exclusively attributed to the archaeal domain. Methane is oxidized by methanotrophic microorganisms using oxygen or alternative terminal electron acceptors. Aerobic methanotrophic bacteria belong to the phyla Proteobacteria and Verrucomicrobia, while anaerobic methane oxidation is also mediated by more recently discovered anaerobic methanotrophs with representatives in both the bacteria and the archaea domains. The anaerobic oxidation of methane is coupled to the reduction of nitrate, nitrite, iron, manganese, sulfate, and organic electron acceptors  as terminal electron acceptors. This review highlights the relevance of methanotrophy in natural and anthropogenically influenced ecosystems, emphasizing the environmental conditions, distribution, function, co-existence, interactions, and the availability of electron acceptors that likely play a key role in regulating their function. A systematic overview of key aspects of ecology, physiology, metabolism, and genomics is crucial to understand the contribution of methanotrophs in the mitigation of methane efflux to the atmosphere. We give significance to the processes under microaerophilic and anaerobic conditions for both aerobic and anaerobic methane oxidizers. In the context of anthropogenically influenced ecosystems, we emphasize the current and potential future applications of methanotrophs from two different angles, namely methane mitigation in wastewater treatment through the application of anaerobic methanotrophs, and the biotechnological applications of aerobic methanotrophs in resource recovery from methane waste streams. Finally, we identify knowledge gaps that may lead to opportunities to harness further the biotechnological benefits of methanotrophs in methane mitigation and for the production of valuable bioproducts enabling a bio-based and circular economy. It has a higher heat retentive capacity compared to CO20 years . FurtherMethane originates from abiogenic, thermogenic and biogenic microbial sources. In this work, we focus on biogenic methanogenesis by methanogenic archaea; which liberate methane as the end-product from biological decomposition of organic matter as a mean of energy conservation . Methano4 year–1 from all sources (ranging from 594 to 881 Tg CH4 year–1) (4 year–1 (ranging from 500 to 798 Tg CH4 year–1) leaving a positive net average balance of 112 Tg CH4 year–1 escaping into the atmosphere (Most recent estimations from data over the past decade (2008–2017) indicate a total average net methane production of 737 Tg CH year–1) , whereasmosphere . Howevermosphere . Similarmosphere  , a limited number of archaeal groups are known to produce methane in the final stage of AD, namely methanogenesis, using COfixation . Despitefixation .Until very recently, only seven orders from the Euryarchaeota phylum were recognized as methanogenic archaea: Methanobacteriales, Methanocellales, Methanococcales, Methanomicrobiales, Methanomassiliicoccales, Methano- sarcinales, and Methanopyrales . Today, Methanotrophic microorganisms oxidize methane to harness energy under oxic and anoxic conditions using a range of diverse electron acceptors. Methanotrophy was initially reported in 1906 as an oxygen-dependent process and for almost a century, aerobic methanotrophy was considered as the only biological pathway to oxidize methane and that all methanotrophs belonged to the Proteobacteria phylum . HoweverMethylococcaceae and Methylothermaceae), Alphaproteobacteria , and the Verrucomicrobia phyla (family Methylacidiphilaceae)  harbor only the sMMO (Methylocella) rely on lanthanide metals for the second step of methanol oxidation as the catalytic driving force in MDHs from the XoxF family . These enzymes oxidize methane to methanol, and subsequently, methanol dehydrogenases (MDHs) further oxidize methanol to formaldehyde . After filaceae) . Verrucoilaceae) , whereasilaceae) . While mthe sMMO . Proteobthe sMMO . All pMMthe sMMO  which rethe sMMO . StrikinF family . For a dMethanosarcina barkeri are known to oxidize methane through extracellular electron transfer (EET), referred as electrogenic anaerobic methane oxidation (The Anaerobic Oxidation of Methane (AOM) was first observed in marine sediments where methane consumption was linked to the presence of sulfate . Howeverxidation .Methanoperedenaceae), was identified from anoxic sediments in agricultural soil  . After iral soil . The bacoxyfera”  was idenoxyfera” . “Ca. M. enzymes . The pre enzymes   from the Euryarchaeota phylum and named “Candidatus Methanoperedens nitroreducens,” capable of coupling the reduction of nitrate to ammonia with nitrite as intermediate, to the oxidation of methane (nitrate-dAOM) via the reverse last step from methanogenesis  in the absence of nitrate aerobic methanotrophs is attributable to their metabolic versatility, thriving in broad temperature (4–95°C) and pH (1–8) ranges . ParticuMethylobacter) may possess high-affinity cytochromes , enabling methane oxidation at low oxygen levels in the nM range  encode relatively higher hemerythrins and hemerythrin-containing domains compared to than alphaproteobacterial methanotrophs (Methylobacter) present at the oxic-anoxic interface, and alphaproteobacterial methanotrophs present and stratified at the oxic water layer  have been implicated for both gammaproteobacterial  and alphaproteobacterial (Methylosinus) methanotrophs under hypoxia  excrete methane-derived organic compounds  during oxygen-limited growth (Methylobacter to hypoxia (Methylosinus species) have been co-cultivated alongside strictly anaerobic methanogens under oxygen limitation  differ vastly in regard to taxonomy (75–92% 16S rRNA similarity) , cell shobiaceae .Methylomirabilis bacteria and “Ca. M. nitroreducens” in a widely diverse range of environments such as, sandy riverbeds  as the most abundant element on earth by mass, is widely spread in marine  and fres Fe-dAOM . In thisreported . In addi Cadagno . A folloulbaceae . The debductases .Methanoperedens archaea and/or Methylomirabilis bacteria, are available , part of the archaellum. Two of the four flaB genes were highly expressed during Mn-dAOM experiments in “Ca. M. manganicus,” suggesting the involvement of these conductive appendices in electron transfer. In addition to archaellum-mediated AOM, Humic-dAOM has been reported in an enrichment culture of both N-dAOM microorganisms where AQDS and anthraquinone-2-sulfonic acid (AQS) acted as electron acceptors for methane oxidation  from parent pre-enriched inoculum of “Ca. M. ferrireducens” (25% enrichment) and a minimal proportion of “Ca. M. manganicus” , pollutants are transported from households and/or industrial discharge into wastewater treatment plants (WWTPs). Since modern WWT developed in the late 1800s and formally implemented in the early 1900s, organic carbon removal is performed through microbial aerobic oxidation commonly known as activated sludge (AS) . The AS The complexity of the microbial communities in AD, the large volumes of wastewater, and the variations in organic matter load; pose particular disadvantages prompting to bioaugmentation protocols for biogas. Bioaugmentation is needed to remediate the variation in methane content, and the presence of toxic bioproducts such as hydrogen sulfide . Carbon 2) covering a large portion of the entire nitrogen cycle in one treatment process (2 (2O), a potent GHG, is produced and released to the atmosphere and current estimates report that 5% of the total N2O annual emissions come from WWT [Nitrogen as a form of pollution, is mainly represented by ammonia and has traditionally been removed through the canonical nitrification-denitrification process (N/dN). In N/dN, ammonia is oxidized to nitrate and subsequently reduced dinitrogen gas   performed by anammox bacteria from the phylum Planctomycetes, discovered and isolated from sewage sludge . Soon afx (PN/A) . The PN/x (PN/A) . For a fThe need for innovative methane removal technologies originates because despite the significant advancements in AD and the possibility of recovering biogas for energy production from waste, some challenges remain. Waste and wastewater treatment technologies remain with open loops that call for novel microbiology-based technologies for GHG mitigation . BiologiWith the evidence of methane emissions from WWT, current trends include attempts to estimate the contribution through the implementation of methodologies aiming to accurately determine the footprint of WWT . CurrentMethanoperedens archaea has been proposed as a decoupling mechanism from Methylomirabilis bacteria Aerobic ammonia-oxidizing bacteria (AOB) oxidize half of the ammonia available to nitrite , requiring half the aeration energy compared to N/dN .(ii)Anammox bacteria, oxidize the remaining ammonia using the nitrite produced by AOB. Anammox however, produces nitrate as a side reaction during carbon fixation , 6B.(iii)Methane is oxidized by nitrite-dAOM bacteria using nitrite most likely under competition with anammox bacteria. Both producing nitrogen gas as final nitrogen-product .(iv)Methylomirabilis bacteria to oxidize methane using nitrate  and exhibit an estimated yield at steady state of 0.077 ± 0.027 Cmole CH4 mole–1, as determined by indirectly comparing the expected biomass decay versus the total biomass content while monitoring side populations through fluorescent microscopy  of N-dAOM microorganisms are lower compared to canonical aerobic heterotrophic microorganisms. N-dAOM microorganisms have doubling times estimated from weeks to months based on the quantification of 16S ubations . Recentlcroscopy . For “Ca 20 days . For com 20 days . Anammox 20 days . Table 1®, and PN/anammox  expressed the genetic capacity to counteract the adverse effects of oxygen-stress upon 5% v/v oxygen exposure  have been proposed as an ideal setup for the enrichment of N-dAOM microorganisms, usually accompanied by anammox bacteria . MBRs en systems , but theIn this section, relevant aspects affecting the performance and growth of N-dAOM microorganisms were described. Currently the advancement of N-dAOM-based technology is faced with bottlenecks in the following aspects:(i)Most work relies on methane-rich artificial gas mixtures (>90 percent). There are no studies with real wastewater, except for one study where in a 50 L pilot plant, dissolved methane was removed  coupled to denitrification . However the role of N-dAOM microorganisms in a dual post-treatment containing oxic and anoxic compartments was not fully characterized next to the use of synthetic media . The bio(ii)Studies to optimize fundamental aspects of growth and enrichment conditions are lacking. Currently, evidence on the role of iron, copper, and lanthanide is available ; however(iii)Methylomirabilis bacteria compete with aerobic methane oxidizers in regard to their methane affinities Bioreactor configuration is a limiting factor, where membrane-based bioreactors rely on biofilm formation rich in N-dAOM microorganisms which lacks spatial niche differentiation . This prAfter highlighting the diverse factors affecting the performance of N-dAOM microorganisms and knowledge gaps in the research, it is evident that reactor configuration and more importantly the synergistic combination with other processes are crucial research questions. Exploring these aspects can reveal the true value whether N-dAOM microorganisms could be applicable and biotechnologically beneficial. Anaerobic microbes and processes in anoxic compartments are undoubtedly applicable under granular configurations where both aerobic and anaerobic processes occur simultaneously in WWT . Thus reCurrently natural gas is the primary source of methane for industrial and chemical production of methanol and other industrial compounds. However natural gas reservoirs are finite and is thus a non-renewable fuel source. Furthermore, the industrial production of methanol and olefines is not environmentally friendly, requiring large amounts of energy, metal catalysts, and high temperature and pressure conditions . MethanoThe fate and use of methane can follow several paths, and depends on infrastructure, policy, and the adoption of a waste-to-value culture . The pro2 exhaust waste streams through unsustainable thermochemical processes, at a rate of 110 million metric tons per year. Methanol is one of the most important building blocks for nearly 50000 different chemicals. Approximately one third of it is used for formaldehyde production, followed by synthesis of olefins for the plastic industry  have become an important biopolymer in the past years as a replacement to chemically produced plastics . PHA is Haloferax mediterranei and Alcaligenes latus produce high PHA levels, reaching more than 50% of the cells’ dry weight. Genetically modified Escherichia coli expressing PHA biosynthesis genes , accumulate PHA from lactose -3-hydroxybutyl-CoA, and finally the polymerization into the final PHA molecule, respectively  and carbon (methane or methanol) source  absence of ammonia or nitrate as nitrogen source, (ii) copper deprivation and (iii) a regime of fed-batch to induce storage of carbon in the cell. ) source . In genes as PHB . Anothers as PHB . Inheren®). However, more research on lower quality biogas use is needed to exploit emerging opportunities with biogas as a substrate while mitigating GHG emissions from waste management.The use of augmented biogas from wastewater is already applied at full-scale . SCP can be purified from all methanotrophs as all carbon fixation pathways yield structural proteins that form new cells during microbial growth, and its use dates back to the early 1960s as the first bio-based product from methane .All proteins are formed by polymers of amino acids and depending on their quaternary structure and function, harbor cofactors such as vitamins and metals, making them a suitable feed source for diverse animals regardless of their origin . ProteinIdeal SCP methanotrophic strains should have a fast growth rate with high protein production capacity and easy to cultivate. Furthermore, the methanotrophs should be able to tolerate a wide range of pH and temperatures. The use of methane from natural gas for the production of SCP, is already approved for animal feed in agriculture . Current® and FeedKind® respectively. The methanotrophs used are commonly composed of a mix culture with unknown ratios. Methylococcus capsulatus or Methylomonas sp. as the most common strains described in the literature. For more examples of SCP from bacteria at an industrial scale, see Methylococcus capsulatus with algae C. sorokiniana, has been proposed and research under conditions with supplementation of artificial waste biogas (60% methane) for SCP production showed promising results aerobic methanotrophs have been identified in widespread environments as key drivers of Earth’s carbon fluxes, playing a crucial role as the sole biological methane sink. Methanotrophs determine the fate of methane in both natural and human-impacted ecosystems, where the mitigation of greenhouse gas emissions is recognized as one of the most important environmental development goals. Importantly, methanotrophs have the potential to transform methane into valuable products.Application of methanotrophs for both climate change mitigation and resource recovery in a circular economy face parallel challenges, namely limitations inherent to using methane as a substrate, and the need to revolutionize current chemical-based industrial practices to bio-based solutions. Past research on methanotroph ecology, physiology, and genomics yielded exciting discoveries. Future research direction on novel engineering solutions incorporating methane waste streams from human-influenced ecosystems (WWT), could lead to a new era of methanotrophic biorefineries, move waste management and production systems close to zero net emissions, and reconcile human intervention in the carbon cycle.SGC, AV, and AH wrote the manuscript. MP, MAH, and HN contributed to the critical review and streamlining of the manuscript, SGC and AH coordinated the manuscript conceptualization. All authors contributed to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bisphenol A is an extremely high-volume chemical widely used in polycarbonate plastics, the linings of food and beverage tins, and shopping receipts. Canadians are ubiquitously exposed to bisphenol A and research shows that exposure at environmentally relevant doses causes endocrine disruption. Recent risk assessments and exposure estimates by the European Food Safety Authority have guided increased restrictions around the use of bisphenol A and established a lower tolerable daily intake, while the CLARITY-BPA program in the United States identified several adverse effects below this exposure level. Within the context of bisphenol toxicity and international regulation, this paper describes the need for revised bisphenol A risk assessments in Canada. Completed in 2008, the most recent bisphenol A risk assessment conducted by Health Canada does not include risks from alternative bisphenols or non-dietary exposure. It also does not account for the additive effects caused by simultaneous exposure to multiple endocrine-disrupting chemicals. Bisphenol A (BPA) is an extremely high-volume chemical that has been used for over sixty years to produce many items familiar to Canadians A 1]. Ex. Ex1]. EAlthough its estrogenic properties had been previously documented, BPA caused little concern during the first thirty years of widespread use. Then, in 1993, the first data were published demonstrating that BPA can leach from polycarbonate plastic and drive estrogenic responses within cells in culture . FollowiFor several years considerable discrepancy existed around the estrogenicity of BPA. While studies detected adverse outcomes in test animals exposed to doses as low as 5 ng/kg bw/day, weak estrogenic effects were initially observed in cell culture and in other in vitro experiments . The Can50) was 10−5 M [−6 M [Beyond its estrogenic effects, BPA has also been shown to bind additional nuclear receptors and alter epigenetic modifications. BPA has displayed anti-androgen activity through in vitro assays in yeast, where the half maximal effective concentration  . CLARITYIn 2019, results from the guideline studies were made publicly available . At the While the confounding variables outlined above likely diminished differences between control rats and rats dosed with BPA, both the guideline and academic studies identified numerous significant adverse endpoints following BPA exposure A,B. For In 2008, Canada took the lead in regulatory action around BPA. Since that time the Canadian government has done considerably less than foreign regulatory agencies to update and refine the risks of bisphenol exposure. Canadians need a re-assessment of the literature supporting the endocrine-disrupting properties of BPA. When the most recent risk assessment was completed in Canada, mechanisms rationalizing the low dose effects of BPA in test animals were unclear. We now know that BPA can display strong estrogenicity in various tissues through non-canonical estrogen signaling pathways that are particularly detrimental to developing organisms. The current TDI of BPA in Canada is six-fold higher than the TDI established by the European Food Safety Authority (EFSA), while the results from CLARITY-BPA report adverse effects following doses much lower than the latter. Canadians also need risk assessments for alternative bisphenols, most notably bisphenol S and bisphenol F. Alternative bisphenols are now widely used to replace BPA in polycarbonate, resins and thermal paper. Unfortunately, their physiochemical properties and toxicity appear to be similar to BPA suggesting that products made with these newer chemicals are no safer than the products comprising polycarbonate or BPA-containing resins which were banned more than a decade ago . FinallyBisphenol A is a high-volume chemical that has been used extensively for several decades resulting in nearly ubiquitous exposure to Canadians. In the early 1990s, the estrogenic properties of BPA were realized leading to the restricted use of this chemical in products intended for infants. Since the most recent risk assessment of BPA in Canada, a growing body of peer-reviewed research has characterized molecular mechanisms of BPA toxicity and consistently reported adverse events following low dose exposure in test animals. The EU has reduced the TDI of BPA to 4 μg/kg bw/day and further restricted its use in many products. While any resulting regulatory action is still uncertain, results of the CLARITY-BPA study in the United States indicate that BPA causes adverse effects at doses as low as 2.5 μg/kg bw/day. While factors such as contamination and animal stress likely diminished the differences observed between control and BPA-treated animals in this study, 2.5 μg/kg bw/day is 10-fold lower than the current TDI in Canada, supporting the need to urgently re-assess safe exposure levels for Canadians. In response to restrictions and concerns around BPA, alternatives such as BPS are now widely used but are likely not safer alternatives. Through the Chemicals Management Plan, Health Canada has recently proposed subgrouping 343 BPA structural and functional analogs for future problem formulation . As stat


Little is known about parent and family well-being after acute neonatal seizures. In thus study, we aimed to characterize parent mental health and family coping over the first two years after their child’s neonatal seizures. Parents of 303 children with acute neonatal seizures from nine pediatric hospitals completed surveys at discharge and 12-, 18- and 24-months corrected age. Outcomes included parental anxiety, depression, quality of life, impact on the family, post-traumatic stress and post-traumatic growth. We used linear mixed effect regression models and multivariate analysis to examine relationships among predictors and outcomes. At the two-year timepoint, parents reported clinically significant anxiety (31.5%), depression (11.7%) and post-traumatic stress (23.7%). Parents reported moderately high quality of life and positive personal change over time despite ongoing challenges to family coping. Families of children with longer neonatal hospitalization, functional impairment, post-neonatal epilepsy, receiving developmental support services and families of color reported poorer parental mental health and family coping. Parents of color were more likely to report symptoms of post-traumatic stress and positive personal change. Clinicians caring for children with neonatal seizures should be aware of lasting risks to parent mental health and family coping. Universal screening would enable timely referral for support services to mitigate further risk to family well-being and child development. Neonatal seizures are a medical emergency. The prognosis following acute neonatal seizures can be difficult to predict and may include epilepsy and cerebral palsy ,2. The bWe previously characterized the association between acute symptomatic neonatal seizures and parent and family well-being at discharge from neonatal hospitalization . More thGiven the strong influence of parent and family well-being on child mental health and development ,8, and tLongitudinal examination of the well-being of families was a pre-specified secondary aim of a comparative effectiveness study examining the safety of early discontinuation of antiseizure medication after acute symptomatic neonatal seizures . FamilieApproximately half of the participants were enrolled during NICU admission, with the remainder recruited prior to age 24 months from the first Neonatal Seizure Registry cohort  or from One parent or other legal guardian per family completed a suite of validated survey instruments near the time of discharge from the NICU and when their child reached 12-, 18- and 24-months corrected age. Parents completed the surveys online or by telephone interview with a trained research assistant.Measures of parent and family well-being included the following well-validated scales. Three scales measured parental mental health symptoms or family coping difficulties: the Hospital Anxiety and Depression Scale (HADS) ; Impact Demographic variables included child insurance type (public or private), child race and ethnicity and parent-reported maternal education level. These were obtained by medical record review or parent report. Clinical course variables obtained by medical record review included gestational age at birth (with preterm birth defined as <37 weeks completed gestation), complex medical condition , seizure etiology , discharge on antiseizure medication and age at discharge from the neonatal seizure admission.Variables related to the child’s neurological outcome included diagnosis of epilepsy or cerebral palsy, and functional neurodevelopment at corrected age 24 months as measured with the Warner Initial Developmental Evaluation of Adaptive and Functional Skills (WIDEA-FS) . A childTM Software version 9.4 . Univariate statistics described the sample. To examine changes in well-being over time, we used a series of linear mixed effect regression models, one for each well-being outcome, utilizing restricted maximum likelihood fitting and the Kenward–Roger degree of freedom adjustments. We included person-specific intercepts and slopes over time, and controlled for institution, time point of measurement and which caregiver  responded. Factors of interest were maternal education and child race and ethnicity , insurance type (public vs. private), neonatal seizure etiology, length of neonatal seizure hospital stay, antiseizure medications maintained (vs. discontinued) upon hospital discharge, functional impairment, receiving developmental support services, diagnosed with epilepsy by the measurement timepoint and diagnosed with cerebral palsy by 24 months. Backward stepwise selection was used to determine which factors of interest were retained in the final models.Study personnel who coded clinical variables were blinded to the results of parent well-being assessments. All analyses were conducted with SASWe also conducted a single, multivariate analysis utilizing all six outcomes  measured at discharge and 12, 18 and 24 months. To make the outcomes more comparable in this multivariate analysis, we first converted them to Z-scores and aligned the directionality so that lower scores indicated better well-being for all outcomes. The multivariate analysis allowed us to assess whether an exposure had similar or differential effects on the well-being outcomes. When the effect was similar, it allowed us to pool across outcomes to increase precision.Linear mixed models were also used for these analyses. However, in this case, we used empirical (robust) standard errors to accommodate arbitrary forms of correlations across the six well-being outcomes and across the three time points. We included predictors of outcome type , exposure  and data collection time point, as well as the interaction of exposure and outcome type (to assess similarity of the association across the six outcomes). We first ran the full model, then removed non-significant terms, one by one, in an order that took statistical significance and practical/clinical importance into account.p-values and presented the results graphically by showing the effect estimates plotted versus outcome type (“interaction plots”). If there was not an interaction, then we calculated overall estimates, essentially averaging over outcome types.Once non-significant terms were removed, we considered each of the exposures in turn. If an interaction with parent or family well-being outcome was present (indicating that differences between categories of the exposure were not the same across outcome types), we then reported interaction p < 0.01 as statistically significant and results with 0.01 < p < 0.05 as nominally significant.Given the large number of comparisons, we report results with Of the 305 infants enrolled , two were excluded because they did not meet entry criteria. The final sample included 303 primary caregivers. In 227 (75%) cases, the respondent at all time points was the mother and, in 27 (9%) cases, it was the father. In the remaining 49 cases, the respondent varied across the timepoints between mother and father or, in a few cases, another legal guardian . Frequencies of IES scores above the cut-off suggestive of or probable for post-traumatic stress disorder (PTSD) decreased slightly from 27.4% at 12 months (first measurement) to 23.7% at 24 months . The IOFLinear mixed effect regression models for each of the six well-being outcomes, adjusted for institution, time point of measurement and caregiver respondent revealed different associations based on the outcome of interest .p < 0.0001). HADS anxiety and depression scores were nominally worse for parents whose children had been discharged on antiseizure medication at 18 months compared with the 24-month time period . Parents of BIPOC children had nominally better anxiety scores  compared with parents of white, non-Hispanic children.HADS anxiety and depression scores were significantly worse for parents of children with functional impairment compared with parents of children without functional impairment  compared with parents of children without an epilepsy diagnosis, significantly worse for parents of children receiving developmental support services  compared with parents of children who did not and nominally significantly worse for parents of children with functional impairment compared with parents of children without functional impairment . Parents of BIPOC children reported significantly worse post-traumatic stress symptoms compared with parents of white, non-Hispanic children .Post-traumatic stress scores were significantly worse for parents of children with a diagnosis of epilepsy , significantly worse for families with children who had post-neonatal epilepsy compared to families of children without epilepsy  and nominally worse for parents of children who received developmental support services and  compared with those whose children did not receive developmental support services. Parents of children who had longer neonatal hospitalizations reported significantly worse impacts on their family  compared with parents of children with shorter neonatal hospitalizations.The IOF scores were significantly worse for parents of children with functional impairment compared with parents of children without functional impairment  compared with parents of children without functional impairment. WHOQOL-BREF scores were also nominally worse for parents of children with longer neonatal hospitalizations  than for parents of children with shorter neonatal hospitalizations.WHOQOL-BREF scores were nominally worse for parents of children with functional impairment  than parents of white, non-Hispanic children. Parents whose children had public insurance also had nominally better PTGI scores  than parents of children with private insurance.Parents of BIPOC children had significantly better PTGI scores (9.6 points better, p < 0.001 between the following predictors and well-being outcomes, indicating that these variables had differential effects across well-being outcomes: functional impairment, epilepsy diagnosis, child receiving developmental support services, child race/ethnicity and length of stay for child’s neonatal hospitalization. p < 0.03; see In the multivariate modeling, we assessed whether the variables of interest had similar or differential effects across all well-being outcomes. After controlling for study site, measurement time point and caregiver respondent, linear mixed modeling revealed significant interactions, all This multicenter study provides a robust characterization of parent mental health and family coping over the first two years after the birth of a child with acute symptomatic seizures. We found that approximately one-third of parents experienced clinically important symptoms of depression at their child’s neonatal hospital discharge and about 10% of parents continued to have depressive symptoms 12 to 24 months later. While more than half of the parents experienced clinically important symptoms of anxiety at their child’s neonatal discharge, almost one-third of parents continued to experience clinically important anxiety 12 to 24 months later. Moreover, clinically important post-traumatic stress symptoms were found in over one-quarter of parents as their child’ reached one-year corrected age, and a similar percentage reported symptoms as their child reached two years corrected age. Impact on the family was moderate and stable over the two-year measurement period, which indicated ongoing stressors and difficulty coping with family life related to the child’s healthcare needs or uncertain prognosis. Notwithstanding, parents reported moderately high quality of life and positive personal change after traumatic stress that was stable over time; however, the wide standard deviations indicate variability in these outcomes. Given the high proportion of parents who experience mental health symptoms at discharge and the risk of persistent parental mental health symptoms, universal mental health education and screening is warranted prior to hospital discharge. Moreover, parent and family coping and resilience should be revisited at children’s and parents’ follow-up primary and specialty care visits to ensure that families are connected to necessary services .We also found significant associations between several clinical factors that differentially influenced parent well-being outcomes over time. Children’s level of functional impairment, diagnosis of epilepsy and need for developmental support services were consistently associated with poorer parental mental health and family coping. Previous research has documented relationships between parent mental health symptoms such as anxiety, depression and post-traumatic stress and poor family functioning when children have neurological morbidities or developmental problems resulting from neonatal conditions ,19,20, iThe differential impact of the child’s condition on family well-being over time for children who developed epilepsy is consistent with qualitative research studies that included parents of young children with epilepsy that described the chronic and unpredictable acute nature of the condition and chronic trauma for families ,24. AddrRecent systematic reviews of depression  and postOur findings should be considered within the context of several study limitations and strengths. First, although we included a broad range of medical and socio-demographic predictors, other unmeasured characteristics such as socioeconomic status or caregiver coping style may also influence family well-being. Although the sample was diverse with respect to child race and ethnicity, we did not have a sufficient sample size to conduct comparisons by specific racial or ethnic groups. Because of the strong influence of structural racism on child and family well-being in the US , furtherIn summary, our findings add to the evidence of the vulnerability of families of children with long-term health conditions and need for greater access to mental health screening and services. Further research regarding interventions to prevent or mitigate threats to family well-being are needed to enable children with neonatal seizures to reach their full developmental potential and quality of life.


Mycobacterium leprae), apart from their own end-organ toxicities. Most ENL therapeutic guidelines are primarily designed for acute episodes and there is scarcity of literature on management of patients with chronic and recurrent ENL. It is difficult to predict which patients will develop chronic or recurrent ENL and plan the treatment accordingly. We need simple point-of-care or ELISA-based tests from blood or skin biopsy samples, which can help us in identifying patients who are likely to require prolonged treatment and also inform us about the prognosis of reactions so that appropriate therapy may be started and continued for better ENL control in such patients. There is a significant unmet need for research for better understanding the immunopathogenesis of, and biomarkers for, ENL to improve clinical stratification and therapeutics. In this review we will discuss the potential of neutrophils (polymorphonuclear granulocytes) as putative diagnostic and prognostic biomarkers by virtue of their universal abundance in human blood, functional versatility, phenotypic heterogeneity, metabolic plasticity, differential hierarchical cytoplasmic granule mobilization, and their ability to form NETs (neutrophil extracellular traps). We will touch upon the various aspects of neutrophil biology relevant to ENL pathophysiology in a step-wise manner. We also hypothesize about an element of metabolic reprogramming of neutrophils by M. leprae that could be investigated and exploited for biomarker discovery. In the end, a potential role for neutrophil derived exosomes as a novel biomarker for ENL will also be explored.Erythema nodosum leprosum (ENL), also known as type 2 reaction (T2R) is an immune complex mediated (type III hypersensitivity) reactional state encountered in patients with borderline lepromatous and lepromatous leprosy (BL and LL) either before, during, or after the institution of anti-leprosy treatment (ALT). The consequences of ENL may be serious, leading to permanent nerve damage and deformities, constituting a major cause of leprosy-related morbidity. The incidence of ENL is increasing with the increasing number of multibacillary cases. Although the diagnosis of ENL is not difficult to make for physicians involved in the care of leprosy patients, its management continues to be a most challenging aspect of the leprosy eradication program: the chronic and recurrent painful skin lesions, neuritis, and organ involvement necessitates prolonged treatment with prednisolone, thalidomide, and anti-inflammatory and immunosuppressive drugs, which further adds to the existing morbidity. In addition, the use of immunosuppressants like methotrexate, azathioprine, cyclosporine, or biologics carries a risk of reactivation of persisters ( Mycobacterium leprae where the clinical spectrum of the illness is dictated by the host immune response to the pathogen  and integrin α4β1 (VLA4) gradually decreases while expression of toll-like receptor 4 (TLR4) and CXCR2 and other chemokine receptors progressively increases  tied to interleukin (IL)-23 and IL-17 production by lymphocytes. The short-lived CD66bncreases –39. Throemergency granulopoiesis” occurs as the earliest protective innate response, with increased production of IL-23 and IL-17 in the inflamed milieu providing increased G-CSF and IL-6 leading to appearance of immature neutrophils with lower buoyancy in circulation, better defined as Low Density Neutrophils (LDNs)  .Neutrophilic leukocytosis as an indicator of systemic inflammation is one of the earliest features noted in ENL patients. Among various parameters used for monitoring neutrophil counts, the neutrophil-to-lymphocyte ratio (NLR) has been recognized as a unique, stable marker reflecting underlying acute inflammatory response. NLR is considered an easy, inexpensive, and reproducible parameter associated with clinical outcome and disease severity –45. In rp <0.001) for ENL diagnosis that easily differentiated ENL patients from those with Type 1 reaction with high validity cut-off of 2.95, high diagnostic accuracy  and an area under curve value of 0.796 , a marker of neutrophilic activation, is upregulated by a direct effect of interferon-gamma (IFN-γ) and G-CSF on precursor cells in bone marrow. Increased CD64 expression on circulating neutrophils as well as increased mRNA expression  for ENL .Bioinformatic pathway analyses of the gene expression profiles from ENL skin lesions have observed a role of an integrated axis comprising of TLR2/FcR activation/neutrophil migration/inflammation as a mechanism of neutrophil recruitment in ENL providing a deeper insight into neutrophil biology . Transcrbright/CD62Lbright), banded neutrophils (CD16dim/CD62Lbright), and a third subset comprising of CD16bright/CD62Ldim neutrophils exhibiting immunosuppressive properties  and CD11b (tertiary granules) could be explored for their role in ENL progression.After encountering micro-organisms, neutrophils destroy them intracellularly as well as extracellularly: in their fight with microbes, dead neutrophils provide chromatin and proteins to form NETs and, live neutrophils create a cytoneme network .NETs play a role in autoimmunity and thrombosis by accelerating the inflammatory response either as danger-associated molecular patterns or complement activators –76. Theyde novo expression of disease-specific protein fingerprints which could accelerate the inflammatory response .Investigations are required to elucidate the frequency and clinical significance of LDNs as well as expression of various activation surface markers to test their potential as a biomarker for ENL.The potential prognostic serum levels of NETs/NETs associated proteins should be evaluated in prospective follow up studies of ENL patients and its correlation with disease activity tested in larger cohorts. This could prove helpful in determination of the duration of therapy.M. leprae interferes with metabolic pathways in neutrophils to precipitate ENL in LL patients merits further exploration.How M leprae-specific transcriptional-reprogramming” in neutrophils/NETs. Subsequent translation can yield altered molecular configurations that can be packaged into exosome vesicles and released out of NETs; such NET-associated exosomes may serve as potential “messengers/transmitters” that may cross talk with other active immune cells/tissue resident cells/Schwann cells resulting in the progressive fulminant multi-organ pathology seen in ENL.We support investigations into the concept of “M. leprae infection?What are the signals that cause neutrophil activation and subsequent development into LDNs following How do the newly recognized neutrophil subsets/subtypes/subpopulations (identified by phenotypic markers and segregated on basis of density centrifugation) contribute to ENL pathophysiology?Do these neutrophil subpopulations exhibit any differences in granule protein content and secretion in the context of ENL susceptibility?M. leprae alter metabolic configuration of neutrophils? How does it alter its metabolic configuration to precipitate immunological exacerbation in LL patients?Does M. leprae use its weaponry (lipid virulence factors) to exploit various host biological pathways to rewire the host innate immune/cytokine responses in ENL?How does Many aspects related to ENL pathophysiology remain enigmatic. Answers to these questions have the potential to facilitate discovery of clinical useful biomarkers in the near future.M. leprae infection, more research is required to shed light on the signaling pathways governing these perturbations and to elucidate the intricate interactions among various metabolic programming pathways in neutrophils, with the hope that this basic research can translate into the development of biomarker discovery and novel therapeutics and diagnostic strategies. This might help to identify patients who are prone to develop recurrent or chronic/recalcitrant T2R and initiate them on to targeted therapy.Profound metabolic changes are posited to occur in neutrophils during normal physiological processes and under various pathophysiological conditions including Neutrophils are the key players in ENL and have a potential role as biomarkers of disease outcome or as therapeutic targets. However, there is still much work to be done before they might be used as validated prognostic markers. Moreover, newer therapeutic agents targeting particularly neutrophil migration and mobilization without causing global immunosuppression could be designed.Exploiting various aspects of neutrophil biology  can leadWe envision that liquid-biopsy based exosomal biomarkers for ENL can be developed and validated in the near future with the potential for enhanced early detection, improved diagnosis, disease outcome predictability, and therapeutic window determination.-omics” studies could provide scientists and clinicians with a powerful tool to understand the various aspects of neutrophil biology involved in development and progression of ENL. It will provide a great opportunity and hope to identify biomarkers specific for different cellular processes/states along with those indicating efficacious therapeutic intervention.Moreover, an integration of information from different “SS and KS: literature search, manuscript preparation, and experimental studies. MS: literature search and manuscript review. TN and SD: definition of intellectual content, manuscript editing, manuscript review, and clinical studies. RM: definition of intellectual content and manuscript review. SC: concepts, design, literature search, manuscript preparation, manuscript editing, and guarantor. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study provides evidence that acute lethal TBIs can be detected to a high degree of statistical accuracy using forensic biochemistry. The high inter-individual correlations of biomarkers may help to estimate the CSF concentration of an unknown biomarker, using extrapolation techniques.Diagnosing traumatic brain injury (TBI) from body fluids in cases where there are no obvious external signs of impact would be useful for emergency physicians and forensic pathologists alike. None of the previous attempts has so far succeeded in establishing a single biomarker to reliably detect TBI with regards to the sensitivity: specificity ratio in a post mortem setting. This study investigated a combination of body fluid biomarkers (obtained post mortem), which may be a step towards increasing the accuracy of biochemical TBI detection. In this study, serum and cerebrospinal fluid (CSF) samples from 30 acute lethal TBI cases and 70 controls without a TBI-related cause of death were evaluated for the following eight TBI-related biomarkers: brain-derived neurotrophic factor (BDNF), ferritin, glial fibrillary acidic protein (GFAP), interleukin 6 (IL-6), lactate dehydrogenase, neutrophil gelatinase-associated lipocalin (NGAL), neuron-specific enolase and S100 calcium-binding protein B. Correlations among the individual TBI biomarkers were assessed, and a specificity-accentuated threshold value analysis was conducted for all biomarkers. Based on these values, a decision tree modelling approach was performed to assess the most accurate biomarker combination to detect acute lethal TBIs. The results showed that 92.45% of acute lethal TBIs were able to be diagnosed using a combination of IL-6 and GFAP in CSF. The probability of detecting an acute lethal TBI was moderately increased by GFAP alone and considerably increased by the remaining biomarkers. BDNF and NGAL were almost perfectly correlated ( Traumatic brain injury (TBI) substantially contributes to the global injury burden with a continuously increasing prevalence over the last quarter of a century . ApproxiHowever, despite numerous investigations of fluid TBI-related biomarkers, neither a single characteristic biomarker specific to TBI, nor a preferential body fluid has been established to date. Individual markers suffer either a poor sensitivity or a poor specificity, even though the respective counterpart often seems to be excellent . This faThe overall aim of this study is to determine the combination of the aforementioned forensically investigated biomarkers (obtained post mortem) to detect lethal acute TBIs with the highest possible accuracy. This study aims to provide novel insights into the investigation of lethal TBIs for the emerging field of post mortem biochemistry ,17,18,19Patient-specific serum and cerebrospinal fluid (CSF) sample data was extracted from the authors’ previous publications ,7,8,9. TSamples taken from CSF were included in the statistical analyses of the given study, as serum samples were only recommended for TBI detection in a minority of previous investigations ,7,8,9. SFirst, receiver operating characteristic (ROC) curves were created independently for CNS biomarkers and acute-phase proteins, including all 100 aforementioned cases, to identify sensitivity and specificity when investigating fatalities by single-marker measurements. Moreover, the ROCs were used to establish potential threshold values based on conservative estimations to discriminate between TBI cases and controls using bootstrap replicate confidence intervals. Positive likelihood ratios were calculated for each individual biomarker to assess their utility to screen for acute lethal TBIs. The accuracy was calculated as the sum of the true positive and true negative results, divided by the number of included cases. ROC curves were calculated to demonstrate individual diagnostic ability. However, using this maneuver to rank/rate the diagnostic ability between markers does not maximize the potential of each marker to work as a group.k = 5) cross-validation for the constructed tree models. This maneuver resulted in a total of 53 TBI and control cases (excluding the remaining samples of the two cohorts where few marker levels were randomly missing) and was done to assess the accuracy and misclassification rate of combined biomarker applications in TBI screening. It was determined whether there was any significant diagnostic improvement when combining different markers compared to the diagnostic accuracy of the analyses of individual markers.Rather than comparing individual markers to find a single best marker, a classification tree model considers all the markers together and find the most effective way of differentiating/classifying TBI and controls. This would help to provide higher diagnostic accuracy than individual markers. Therefore, a classification tree model was performed based on all individual cases, where all eight biomarkers were analyzed using statistical learning analytical techniques performed with R. Tree models involve segmenting the predictor space into simple regions by building splitting rules which can be summarized in a decision tree. Single biomarkers/predictors would be a one-node classification tree model with the cut-point/threshold being the one obtained in the ROC. When considering multiple biomarkers/predictors, statistical learning methods were used to fit the optimal decision tree model. Initially, the recursive binary splitting method expanded the decision tree to be as large as possible. This would cause overfitting and an increase in variance. The expanded decision tree was subsequently pruned down using the cost complexity pruning method, which minimizes the turning parameter. The misclassification rate, an estimation of test error in statistic learning methods, was determined using K-fold  of the ROC curves of all individual biomarkers were overlapping . The accCombining IL-6 and GFAP helps correctly classifying lethal acute TBIs in more than 90% of cases. A combination of IL-6 and GFAP correctly classified 49 out of the given 53 cases, resulting in an accuracy of 92.45%, and a complimentary rate of misclassification of 7.55% by deploying the decision tree modelling approach . To achip = 0.049), LDH (p = 0.024) and NGAL (p = 0.002)  . The latgression A accordip = 0.021) and NSE (p = 0.033) (p = 0.049) (p < 0.001)  . Moreove= 0.049) . A moder< 0.001)  with an gression B accordiS100B was the only biomarker investigated to present totally independent changes from other co-analyzed biomarkers in acute lethal TBIs.The great value of continuing research related to TBI beyond the sole determination of death is exemplified by the chronic neurodegenerative disorder named chronic traumatic encephalopathy. The underlying disease linked to repetitive TBI remains obscure for clinicians, and can therefore only be diagnosed post mortem . Vice veFor the cause of death determinations, forensic biomarkers to detect lethal acute TBIs could be of great advantage in cases for which external impact signs are missing. The assessment of eight TBI-related biomarkers performed here enables forensic pathologists to diagnose an acute lethal TBI with an accuracy of 92.45%, using CSF levels of IL-6 and GFAP with the cut off values stated in this study. Interestingly, both markers recently also showed promising results in the microscopic investigation of their neuronal and astroglial expression in fatally injured human brain tissue compared to controls . When thThe physiological level of IL-6 in antemortem CSF was stated to be 1 to 23 pg/mL, which was established based on 121 patients with no present or previous inflammatory disease . HoweverThis study revealed that combining the acute phase protein IL-6 with the CNS marker GFAP increases the accuracy of detecting a lethal acute TBI and, therefore, our above-stated hypothesis can be accepted. Apart from its occasional presence in neurons ,30 and tAccording to the positive likelihood ratio presented in this study, GFAP moderately increases the probability of the presence of an acute lethal TBI by 30% , if the The given study highlights the potential to increase the diagnostic accuracy of a medical condition based on biomarker combinations in body fluids that are routinely sampled and stored during forensic autopsies . AlthougOn a greater scale, being able to assess a multitude of diseases from body fluids in combination with advanced computer evaluations of virtual autopsies  could deIt should be noted that the routine applicability of post-mortem biochemical observations will likely be limited to the early phase after death due to tissue degradation or the stability of the biomarker in the respective body fluid . The incIn collaboration with biochemistry units, post mortem biochemistry could be established as an additional diagnostic tool for external autopsies, which are performed away from the forensic departments by independent investigators, and in the long-term increase diagnostic accuracy. The majority of biomarkers can be measured with minimal effort in a time-efficient and cost-effective way using automated analyzers. Biomarkers appear to be economically superior compared to other methods, such as forensic imaging. Additionally, future studies should explore the strategy to combine forensic biochemistry with minimal invasive autopsies  to furthFirst, the study is limited by its sample size. Second, unswayable factors such as the ambient temperature at the time of death and the freezing of the used body fluids for storage purposes might have impacted degradation processes and, thus, the biochemical observations in the given study. However, these limitations would also apply in further practical uses, i.e., the markers presented here seem robust enough to allow for clinical application. Third, undetected neurodegenerative conditions might have impacted the concentrations of the here investigated TBI-related biomarkers , althougThis study provides evidence that acute lethal TBIs are detectable with high statistical accuracy (up to 92.45%) using forensic biochemistry. The high inter-individual correlations of biomarkers may help to estimate the CSF concentration of an unknown biomarker based on other markers. CNS biomarkers can be combined and interpreted together, and this may increase the diagnostic accuracy of acute lethal TBI. The methods applied here can also be used to stratify and prioritize some methods/samples over others due to increased reliability, which is applicable beyond the forensic investigation of traumatic fatalities in the future.


Pax3 lineage, and FAPs from the mesoderm, while craniofacial muscles have MuSCs of the Mesp1 lineage and FAPs from the ectoderm-derived neural crest. Both in vivo and in vitro data revealed distinct patterns of proliferation and differentiation in these craniofacial muscle stem/progenitor cells. Additionally, the proportion of cells of different embryonic origins changes throughout postnatal development in the craniofacial muscles, creating a more dynamic niche environment than in other muscles. In-depth comparative studies of the stem cell biology of craniofacial and limb muscles might inspire the development of novel therapeutics to improve the management of myopathic diseases. Based on the most up-to-date literature, we delineated the pivotal cell populations regulating craniofacial muscle repair and identified clues that might elucidate the distinct embryonic origin and injury response in craniofacial muscle cells.Craniofacial muscles emerge as a developmental novelty during the evolution from invertebrates to vertebrates, facilitating diversified modes of predation, feeding and communication. In contrast to the well-studied limb muscles, knowledge about craniofacial muscle stem cell biology has only recently starts to be gathered. Craniofacial muscles are distinct from their counterparts in other regions in terms of both their embryonic origin and their injury response. Compared with somite-derived limb muscles, pharyngeal arch-derived craniofacial muscles demonstrate delayed myofiber reconstitution and prolonged fibrosis during repair. The regeneration of muscle is orchestrated by a blended source of stem/progenitor cells, including myogenic muscle satellite cells (MuSCs), mesenchymal fibro-adipogenic progenitors (FAPs) and other interstitial progenitors. Limb muscles host MuSCs of the  The emergence of craniofacial muscles, together with the skull and sensory organs derived from the placodes and neural crest, contributed to the development of the vertebrate head, an evolutionary novelty . The musUnlike the extensively studied limb and trunk muscles, the characteristics of craniofacial muscles have only been investigated in recent years. Researchers have gradually realized that craniofacial muscles have disparate evolutionary origins and undergo distinct developmental trajectories from limb and trunk muscles . MoreoveSkeletal muscle regeneration is orchestrated by the interactions among multiple resident muscle cells . Of thesIn this review, we aim to provide an up-to-date discussion of the unique properties of the major cell groups in craniofacial muscles, ranging from their embryonic origin to injury response.Pax7  derived from the prechordal mesoderm  Mesmesoderm ; (2) Mesl arches ; (3) Mes somites . Thus, tal field . The criogenesis .Pax3/Pax7 lineage  MuSCs associated with the pharyngeal arch muscles, which include the jaw muscles, facial expression muscles and pharynx- and larynx-associated muscles, which are of the Mesp1/Isl1 lineage; and (3) MuSCs associated with the tongue muscles, which are of the Mesp1/Pax3 lineage  ext lineage . In addiker MyoD . In contal MuSCs . The de istories .Because skeletal muscle regeneration largely resembles embryonic muscle development, a significant disparity exists between adult myogenesis in craniofacial and limb muscle regeneration. Craniofacial MuSCs exhibit distinct behaviors both in a homeostatic state and in response to acute and chronic stimuli ,b.in vivo and in vitro analyses provided various evidence to elucidate the mechanisms underlying this observation. The proportion of MuSCs in adult uninjured EOM is five times higher than that in typical limb muscles . PICs are bipotent progenitor cells that give rise to both smooth muscle and skeletal muscle in vitro. Although PICs are not of the Pax7 lineage, they require Pax7 for myogenic specification. In addition, a subgroup of PICs share overlapping surface markers with FAPs (Sca-1 and Pdgfrα) and possess fibrogenic and adipogenic capacity, while the Pdgfrα- PICs are myogenic (In the attempt to identify specific stem cells responsible for muscle regeneration, myogenic .Twist2+ cell. Unlike MuSCs, which contribute to the formation of all types of myofibers, Twist2+ cells are type IIb/x fiber specific myogenic progenitors (Twist2+ cells do not express the MuSCs marker Pax7 but can initiate myogenesis both in vitro and in vivo (Twist2 expression and began to express Pax7 (Twsit2+ cells is their opposite roles in muscle regeneration and myofiber size maintenance. MuSCs exert minimal effects on muscle fiber size, as evidenced by the unaffected sarcopenia in Pax7-ablated mice (Twist2+ cells are required for type IIb/x fiber size maintenance, but their ablation does not affect muscle regeneration (Twist2+ cells undergo a pre-myogenic state but are insufficient to guide full regeneration (Twist2+ cells are specifically excluded from the tongue musculature (Another type of interstitial stem cell with myogenic potential is the genitors . Remarka in vivo . Once coess Pax7 . A notabted mice . In contneration . The hypneration . It is iculature . Since tculature , furtherIn contrast to trunk/limb muscles, which are ancestral muscles essential to the support and locomotion of the entire body, craniofacial muscles have long been regarded as a variation on the general body muscle scheme. This variation is manifested in many aspects, including developmental origin, myogenic regulatory trajectory, susceptibility to muscular diseases and regenerative capacity. This discrepancy between craniofacial and trunk/limb muscles may to related to their different embryonic origins and to the Hox gene, which conveys positional memory . StudiesXC and JL conceived the idea of writing this manuscript. XC drafted the manuscript. JL drew the figure and critically revised the manuscript. BS critically revised the manuscript. XC and BS provided the funding support. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Early cancer detection should lead to an overall stage shift, less-intensive treatments and better patient outcomes. Current recommended screening programmes are limited to a handful of individual cancers. A multi-cancer early detection test that simultaneously detects and localises multiple cancers could reduce the morbidity and mortality associated with cancer. However, for effective large-scale population screening, an MCED test should balance sensitivity and specificity, and be able to predict TOO,1 while minimising testing-associated potential risks, including overdiagnosis. Clinically relevant positive predictive value is essential,1 as the goal is to facilitate early intervention. A single test to screen for multiple cancers allows aggregation of prevalence rates, which allows such an MCED test—with a very low false positive rate (FPR) and moderate sensitivity across a broad range of cancer types—to detect many cancers that otherwise would go undetected until later stages when signs or symptoms are more likely to appear.3A paradigm shift is underway from early detection of individual cancers to simultaneous early detection of multiple cancers with a single blood draw. This is possible through analysing circulating tumour cell-free DNA (cfDNA) and leveraging genomic technologies and machine learning, ranging from logistic regression to neural networks, to simultaneously detect multiple cancers and accurately predict the tissue of origin (TOO).1 To assess performance of the machine-learning classifier for detection of >50 cancers and TOO prediction in the second CCGA substudy, samples from 6689 participants  were divided into training and validation sets.1 In the validation set, specificity was 99.3% .1 Stage I–IV sensitivity was 76.4% in a prespecified subset of 12 high-signal cancers  and 54.9% in all cancers. Sensitivity increased with increasing disease stage: in prespecified cancers, from 39% in stage I to 92% in stage IV; in all cancers, from 18% in stage I to 93% in stage IV. TOO was predicted in 96% of samples with a cancer-like signal, of which 93% were accurate.1 This study demonstrated that informative methylation patterns can be leveraged for early multi-cancer detection, including detection of lethal cancers without recommended screening programmes.Simultaneous detection of multiple cancers using cfDNA should include provisions for tumour localisation to guide diagnostic evaluation. The Circulating Cell-free Genome Atlas (CCGA) Study (NCT02889978) is a prospective, case-controlled study with longitudinal follow-up designed for discovery and validation of such a blood test. Biospecimens were collected from patients with newly diagnosed cancer before therapeutic intervention, and from healthy controls. Assessment of three prototype assays  in the first substudy led to a focus on methylation for further assay development. More than 100,000 key methylation regions were selected as containing the most informative cancer-/tissue-specific methylation patterns versus noncancer controls. A robust targeted methylation assay for plasma cfDNA fragments was designed to detect and localise multiple cancers across stages with high specificity, as appropriate for a population-based screening programme.Study limitations were noted. Stage and cancer distribution probably affected aggregate sensitivities. The FPR may have been overestimated because complete 1-year follow-up to confirm noncancer status was not available for all noncancer participants. Additionally, analyses are ongoing to improve TOO prediction accuracy. These issues will be addressed through two ongoing studies in asymptomatic intended-use screening populations: STRIVE (NCT03085888), a case-cohort study of ~100,000 women undergoing mammography screening; and SUMMIT (NCT03934866), a study of ~25,000 smokers and former smokers at high risk of lung cancer. PATHFINDER (NCT04241796), a prospective interventional study of ~6200 participants, will evaluate the clinical implementation of MCED testing by returning test results to clinicians and participants, ascertaining participant-reported outcomes and perceptions of the test and tracking diagnostic pathways toward resolution of a signal-detected test result.4 Study comparisons are challenging because of differences in participant eligibility, cancers targeted for detection, methodology and performance metrics variability. For example, alternative approaches have been described that simultaneously detected 16 cancers based on analysis of tumour-specific mutations in cfDNA combined with protein biomarkers , as well as 5 cancers  or 12 cancers  based on DNA methylation signatures8 , combined with the ability to detect nearly any cancer type, may be the most effective approach.MCED tests should be intended to complement recommended screening approaches by identifying cancers they do not cover. To screen for cancers with low occurrence in the general population, many people must be screened to detect a single cancer. Screening for all cancer types simultaneously would allow the prevalence of rare and common cancers to be combined, thus decreasing the number needed to screen. Additionally, combining high specificity and aggregate tumour prevalence is expected to result in positive predictive values exceeding those seen with single-cancer screening approaches that rely on high sensitivity but lower specificity.Collectively, an opportunity exists for MCED testing to reduce cancer mortality and treatment costs, improve the effectiveness of cancer screening and complement single-cancer screening approaches. We are optimistic that findings from additional trials in intended-use screening populations will identify proper workflows for test implementation and facilitate adoption of MCED tests in clinical practice.


The SERT binding index (BI) was obtained at baseline, 24 h post stimulation protocol, one month, and three months post stimulation by a [11C]DASB PET scan. It was found that one day of active aHF-rTMS (five sessions) did not result in significant SERT BI changes at any time point. For the 20 sessions of active aHF-rTMS, one month after stimulation the SERT BI attenuated in the sgACC. No significant SERT BI changes were found after 20 sessions of sham aHF-rTMS. A total of four days of active aHF-rTMS modified sgACC SERT BI one month post-stimulation, explaining to some extent the delayed clinical effects of accelerated rTMS paradigms found in human psychopathologies.Repetitive transcranial magnetic stimulation (rTMS) is thought to partly exert its antidepressant action through the serotonergic system. Accelerated rTMS may have the potential to result in similar but faster onset of clinical improvement compared to the classical daily rTMS protocols, but given that delayed clinical responses have been reported, the neurobiological effects of accelerated paradigms remain to be elucidated including on this neurotransmitter system. This sham-controlled study aimed to evaluate the effects of accelerated high frequency rTMS (aHF-rTMS) over the left frontal cortex on the serotonin transporter (SERT) in healthy beagle dogs. A total of twenty-two dogs were randomly divided into three unequal groups: five active stimulation sessions (five sessions in one day,  The serotonin transporter (SERT) plays a crucial role in human major depression, one of the most common mental illnesses worldwide. Selective serotonin re-uptake inhibitors (SSRIs), the first line pharmacotherapeutic modality for depression, exert their function by modulating the serotonin 5-HT) level through blocking of the serotonin transporter (SERT) . SERT is-HT levelAlthough SSRIs are considered as the first line antidepressant treatment and are also used for a variety of anxiety disorders, not all patients respond well, and treatment resistance is not uncommon ,12,13,14Recently, accelerated (a)rTMS protocols have been introduced, showing similar but faster clinical effects in depressed patients. Here, instead of applying rTMS only once per day, and this during four to six weeks, arTMS protocols deliver the same number of stimuli and sessions spread over only a couple of days . Immedia11C]-DASB binding alterations occurred in the regions with high SERT density. In line with antidepressant intake, a higher dose resulted in higher occupancy of the SERT binding sites methyl triflate, was performed in order to synthesize the SERT ligand [11C]DASB. Radiochemical purity of more than 99% was achieved DASB PET scans: baseline, 24 h post stimulation, 1 month, and 3 months after the last aHF-rTMS treatment session was applied .All dogs underwent four DASB after one month, though significance vanished after stringent post-hoc testing. Nevertheless, our results suggest delayed decreased SERT binding in the sgACC, a key region involved in the therapeutic response of antidepressant therapy in humans and effects. This delayed decrease in SERT BI may point to the presumably therapeutic effect as this is similar to SSRI treatment, where meaningful clinical effects are to be expected after a couple of weeks and not immediately. In addition, these preliminary findings suggest a similar working mechanism of aHF-rTMS, as compared to pharmacotherapy; further research is needed to explore the exact pathways of the (accelerated) rTMS effects on the serotonergic system. Given the similarities between the human and the canine species on the specific anatomical localizations, our observations also indicate that aHF-rTMS treatment may be a valid option to treat dogs diagnosed with mood and anxiety disorders.Only the active 20 session aHF-rTMS protocol resulted in delayed changes in SERT BI, measured with [


Bladder endometriosis is an infrequent cause of a focal bladder mass and may masquerade as a neoplasm on ultrasound imaging. This diagnostic dilemma can be resolved with a multiparametric MRI, which shows characteristic hemorrhagic signals of this entity. We present an unusual case of bladder endometriosis where the patient complained of lower abdominal pain without urinary symptoms and was found to have a bladder mass on ultrasonography. This mass was further investigated by an MRI of the pelvis, which revealed characteristic features of endometriosis; the diagnosis was confirmed on subsequent laparoscopy. Endometriosis is a commonly encountered chronic benign gynecological condition characterized by the presence of functional endometrial glands and stroma-like lesions outside the uterus. In rare cases, the involvement of the urinary system due to endometriosis has been observed. However, the most common site of the urinary system involvement due to this entity is the posterior wall of the urinary bladder, which is in close proximity with the uterus, and hence vulnerable to direct spread from the uterus . In thisA 31-year-old unmarried female presented with complaints of lower abdominal pain and severe dysmenorrhea. Her pain had gradually increased in intensity over time. Clinically, the patient was vitally stable, and her abdomen was soft and non-tender. A review of her records showed that she had undergone two previous ultrasounds; one had been performed nine years back in 2012, which had shown large multiloculated cystic lesions in both adnexa and cul-de-sac with low-level internal echoes suggestive of bilateral ovarian endometriomas. At least two intramural fibroids had also been identified in the uterine body. Subsequently, she had undergone a laparoscopic cystectomy, confirming endometrioma. She had later undergone laparotomy for myomectomy, adhesiolysis, and bilateral ovarian cystectomy. Her follow-up/second ultrasound had been done in 2016, which had shown new development of multiple uterine fibroids without any ovarian pathology.Based on her current symptoms, she again underwent an ultrasound pelvis after an interval period of five years, which demonstrated a bulky anteverted and anteflexed enlarged uterus with multiple intramural and subserosal fibroids of variable sizes. The urinary bladder base revealed a slightly lobulated well-defined hypoechoic immobile mass without any significant vascularity, measuring 22 x 20 x 36 mm Figure . Both ovThe MRI pelvis showed an enlarged uterus with multiple uterine fibroids and an irregular mixed signal intensity mass along the posterior wall of the urinary bladder. This lesion was showing intravesical extension and was posteriorly in contact with the uterus with indistinct fat planes Figure . On careEndometriosis is a benign condition characterized by the presence of heterotopic endometrial tissue that structurally and functionally behaves as endometrial glands or stroma . It has The patients commonly present with infertility or pain-related symptoms, including cyclic pelvic pain, dysmenorrhea, dyspareunia, dysuria, and dyschezia . BladderVarious theories have been proposed in the literature regarding the cause of endometriosis, and the most preferred one is retrograde menstruation. It has been found that during menstruation, there is spillage of endometrial cells in the anterior cul-de-sac, which is the most dependent portion, just like the posterior cul-de-sac, hence representing a favorable site for deep endometriosis. It is proposed to perform imaging before or during menstruation as the lesions become more perceptible .Laparoscopic evaluation and histological confirmation are the gold standard methods for the diagnosis of endometriosis . ValuablOn ultrasound, endometriotic nodules appear as well-defined, solid, hypoechoic, irregular masses. They may contain low-level echoes or small cystic spaces and often show no blood flow on color Doppler. Anteverted-retroflexed uterus ("question mark sign") is often seen with severe posterior compartment deep infiltrating endometriosis. The "kissing ovaries" sign signifies ovaries that are adherent to one another posterior to the uterus and are frequently seen with bilateral endometriomas . Deep inMRI is a robust imaging technique for the assessment of bladder endometriosis. Whenever there is a high suspicion of cancer, MRI is a reliable modality with its superior tissue characterization, better multiplanar capability, higher contrast resolution, and better delineation of bladder wall layers, in comparison with ultrasonography. The typical appearance of bladder endometriosis on MRI is usually of low signal intensity on T2 weighting with intermediate signal intensity on T1 weighting, and foci of high signal intensity on T1 and T2 weighting . AccordiFew "pearls of wisdom"/recommendations regarding the use of MRI for the diagnosis and characterization of pelvic endometriosis are presented here. (a) T1-weighted fat-suppressed sequences should be included in all-female pelvis MR examinations because this sequence is very helpful in detecting small endometriomas and differentiating them from mature cystic teratomas. Peritoneal endometriotic deposits usually appear bright on T1-weighted images, representing blood products, and hence the best sequence to pick this abnormality is the T1-weighted fat-saturated sequence. (b) It must be taken into account that endometriomas and endometriotic foci can demonstrate restricted diffusion and mimic pelvic malignancies. (c) The ancillary finding of hematosalpinx and endometriomas with "T2 shading" sign  are suggestive of pelvic endometriosis. These ancillary findings were not present in our case to support the diagnosis. (d) Solid deep infiltrating endometriosis, as seen in our case, can involve the uterus, cul-de-sac, pelvic ligaments, anterior rectosigmoid colon, bladder, as well as surgical scars. The best sequence to detect deep infiltrating endometriosis and associated fibrosis is the T2-weighted MR sequence without fat suppression. These lesions demonstrate ill-defined margins and T2 hypointense signals as a result of fibrosis along with foci of T2 hyperintensity representing ectopic endometrial glands. These typical MR signal characteristics can help in establishing the diagnosis ,14.This case report highlights the importance of a collaborative effort involving clinicians and radiologists in patient management. In a nutshell, our report illustrated typical MRI features of bladder endometriosis in a patient with atypical clinical presentation of the disease. Familiarity with imaging techniques and background knowledge of this rare entity are of paramount importance to reach an early diagnosis and avoid unnecessary interventions, thereby reducing morbidity and mortality among patients.


An 83-year-old Caucasian male presented with a history of asymptomatic yellow-orange macules and plaques concentrated on his trunk and proximal extremities that have been slowly progressing for the past three years. A punch biopsy revealed the presence of eosinophilic amorphous and fissured material within the superficial and interstitial dermis consistent with nodular amyloidosis. With the lack of concurrent systemic symptoms and negative systemic laboratory workup, the patient was diagnosed with disseminated primary localized cutaneous nodular amyloidosis (PLCNA). Due to the possibility of developing systemic progression, serial monitoring was recommended. This case highlights an under-reported and unusual presentation of a widely distributed form of PLCNA compared to the more common localized nodular and plaque variants. There are different subtypes of primary cutaneous amyloidosis (PCA), which include: macular, lichen, and nodular . PrimaryAn 83-year-old Caucasian male with a past medical history of hypertension, hyperlipidemia, gastroesophageal reflux disease, and two facial basal cell carcinomas presented with asymptomatic, scattered yellow-orange-hued non-scaly macules and thin plaques located on the chest, upper arms, and back coalescing into a reticular pattern Figures -1B.He reported the lesions began on his chest and gradually spread to cover a significant portion of his trunk and proximal extremities over the past three years. The patient denies any history of illicit substance use and has a family history of breast cancer in his sister. The patient recalls no preceding trauma and his age-appropriate cancer screenings are up to date. Complete blood counts, as well as a comprehensive metabolic panel, erythrocyte sedimentation rate, and urinalysis, were all within acceptable ranges. Serum protein electrophoresis, serum immunofixation, and urine protein electrophoresis were also within normal limits. Histopathology from two separate skin biopsies showed a homogenous collection of eosinophilic material within the superficial dermis along with a similar appearing substance encompassing dermal blood vessels and surrounding interstitium Figures -2B. The diagnosis of disseminated primary localized cutaneous nodular amyloidosis (DPLCNA) was confirmed with crystal violet staining which further accentuated the purple amorphous material Figures -3B. SincThere are different subtypes of primary cutaneous amyloidosis (PCA), which include: macular, lichen, and nodular amyloidosis . PCA is The precise pathogenesis of PCA is not well understood. Cutaneous macular and lichen amyloidosis originate from degenerated keratinocyte intermediate filaments while nodular amyloidosis consists of immunoglobulin ƴ light chains and β2-microglobulin, which are thought to be produced by plasma cells in the region of the cutaneous deposits .Clinically, PLCNA presents as a single or multiple yellow-brown waxy nodules or plaques with well-defined borders. These plaques may occur focally and occur most commonly on the trunk and extremities with subtle areas of purpura . HighligRoutine histopathology of PLCNA with hematoxylin and eosin staining typically presents with a pale pink, sometimes fissured, amorphous material in the superficial and deep dermis which can sometimes be found amongst dermal blood vessels and surrounding interstitium -5. WithiTreatment for PLCNA is only required pending the development of systemic manifestations. If the lesions are cosmetically disfiguring or symptomatic, they can be treated via surgical excision, resurfacing lasers, or other destructive methods . All patWe present an under-reported subtype of primary cutaneous nodular amyloidosis with a very distinct disseminated appearance. Currently, there is no standard of care for patients diagnosed with DPLCNA. Due to the innumerable lesions, a destructive method was not reasonable or desired by our patient. With a lack of systemic manifestations, treatment with active surveillance on a semi-annual basis was advised. To monitor for systemic progression, we propose a semiannual physical evaluation for abrupt disease morphologic progression with assessment for macroglossia, carpal tunnel, or signs of restrictive cardiomyopathy. In addition, we suggest a yearly laboratory evaluation to monitor for alterations in his complete blood counts, comprehensive metabolic panel, urinalysis, serum and urine protein electrophoresis, and serum immunofixation. Further research should be conducted due to the rarity of DPLCNA, the lack of knowledge known about this variant, and potential differences in progression to systemic disease.


A 32-year-old woman with chemorefractory mullerian adenosarcoma showed clinical benefit in response to administration of lenvatinib plus pembrolizumab. In this case report, we describe the course of her illness and her response to this treatment. Mullerian adenosarcoma is a rare gynecological cancer characterized by a biphasal aspect, consisting of a benign epithelial proliferation in addition to a malignant stroma . While lA 32-year-old primiparous woman sought medical attention for the appearance of a rapidly enlarging and painful pelvic mass 6 months after vaginal delivery, accompanied by irregular uterine bleeding. She had a history of infertility attributed to endometriosis and treated with hormonotherapy (dienogest). She was a nonsmoker and had no history of drug or alcohol abuse. The physical examination showed a woman measuring 160 cm with a weight of 85 kg, approximately. Imaging studies revealed a large abdominopelvic mass of malignant appearance with no lymphadenopathy and no distant metastases. During preparation for elective surgery, she presented for severe abdominal pain to the Emergency Department where the diagnosis of an intraabdominal perforation was entertained. An urgent laparotomy was immediately performed, resulting in the palliative resection and debulking of a perforated mass of pelvic origin with extensive involvement of the abdominal cavity.KRAS mutation (p.Gly12Asp). PD-L1 expression on tumor cells was 0%.The pathology report described a 24 cm tumor with honeycomb appearance and cystic degeneration, made up of benign mullerian-type glands admixed with a malignant stromal/mesenchymal hypermitotic and cytologically atypical component, diagnostic of mullerian adenosarcoma with extensive high-grade sarcomatous stromal overgrowth and without heterologous components. The malignant cells stained with ER, PR, cyclin D1 and WT1, suggestive of a gynecologic origin. It was not possible to determine whether the primary site was uterine or extra-uterine. Immunohistochemistry showed no mismatch repair (MMR) protein loss. An inhouse next-generation sequencing platform of the tumor designed to detect DNA and RNA alterations in 52 genes  only revThe patient was started on doxorubicin for residual disease and received six cycles, which resulted in disease stabilization, as best response. Radiotherapy also had to be given for a bleeding intra-rectal lesion. At progression, chemotherapy was changed to gemcitabine/docetaxel, with disease stabilization; this treatment had to be stopped for toxicity (fatigue and thrombocytopenia) after 4 months. Stability was maintained for another 5 months with anastrozole. For major disease progression with the appearance of new lesions and partial bowel obstruction, ifosfamide was given but, despite short-term stabilization, did not prevent further progression. On the basis of the success of lenvatinib/pembrolizumab in endometrial cancer, this regimen was prescribed to the patient after informed consent through a compassionate program. A clinically meaningful tumor reduction was observed, although not qualifying for partial response per RECIST criteria, after 9 weeks of treatment. Regrowth of a tumor implant was seen after 18 weeks, which did not respond to tumor embolization. Frank disease progression was evident with new tumor deposits after 8 months of lenvatinib/pembrolizumab. Two cycles of trabectedin were unsuccessful and the patient died after opting for palliative care. Survival was 29 months between surgery and death. The various tumor sizes through treatment stages are listed in Adenosarcoma and endometrial adenocarcinoma are considered separate diseases, although endometrioid carcinoma mixed with clonally related adenosarcoma is reported . Lenvati