Cancer Discovery paper, Stephanie Dobson et al identified rare relapse-fated subclones in B-ALL and showed that they possess an intrinsic drug tolerance as well as metabolic and stemness signatures. The identification of these subclones and associated pathways will be instrumental to target relapse-fated cells, thereby preventing their full evolution into relapse clones.In a recent 1Clonal evolution is a major driver of disease progression and relapse in hematological malignancies. In this process, genetic and epigenetic mechanisms intervenient to shape leukemia composition, favoring the emergency of multiple co-existing leukemic clones, including those ultimately driving relapse. In acute lymphoblastic leukemia (ALL), where a branching evolution takes place, the clone driving relapse is not derived from a major diagnostic clone but rather from a minor ancestral one.3 Nevertheless, the molecular determinants that confer a diagnostic subclone a relapse fate before its evolution into a drug-resistant relapsing clone following chemotherapy exposure remain unclear.In the attempt to understand how cytotoxic chemotherapy shapes clonal evolution and drug resistance, numerous studies have compared diagnosis and relapsed samples.Cancer Discovery paper, Dobson et al undertook a strategy which enabled the identification and further characterization of rare relapse-fated subclones.4 The authors performed whole exome sequencing of 14 paired diagnosis and relapse B-ALL patients samples encompassing distinct cytogenetic subtypes. This analysis allowed them to identify somatic single nucleotide variants, insertion-deletion mutations and DNA copy number alterations in bulk patient samples at diagnosis as well as at relapse. In parallel, diagnosis and relapsed samples were injected at limiting dilution doses in immunodeficient mice. In this way, the leukemic variants identified in the primary samples could be next target sequenced in the engrafted mice. By comparing the variant allele frequencies of the leukemic variants in diagnosis/relapse patient samples and xenografts, the authors were able to observe distinct clonal compositions in distinct xenografts which were issued from the same diagnostic sample. This indicates that the distinct leukemia-initiated cells from which these grafts originated were derived from genetically diverse subclones present at diagnosis. Hence, by performing large-scale xenograft assays at limiting dilution and combining them to genomic analysis, the researchers managed to capture a wide range of diagnostic subclones, which were endowed of distinct competitive repopulation capacity, immunophenotype, migration and proliferation capacities. Importantly, as evidenced by population phylogenetic analysis and mutation evolutionary trees, the diagnostic subclones captured by limiting dilution patient-derived xenografts (PDXs) included diagnosis relapse-initiating clones (dRI), namely very minor latent subclones present in the diagnostic samples and fated to relapse.In the recent Why these clones were fated to relapse and other ones not? The experimental evidences provided by Dobson et al indicate that dRI possess at least 3 major features. The first one is an intrinsic drug tolerance. When compared to PDXs issued from major diagnostic clones, at least some of the dRI-derived PDXs analyzed showed reduced sensitivity to standard chemotherapy drugs, such as dexamethasone, vincristine and L-asparaginase. Hence, albeit this analysis included a limited number of samples, it suggests that drug tolerance arises prior chemotherapy exposure.Second, dRI subclones possess a mitochondrial metabolic signature. Transcriptional analysis of diagnostic-derived PDXs and dRI-derived PDXs revealed that the latter is enriched with pathways involved in aminoacid metabolism, Krebs cycle, oxidative phosphorylation and lipid metabolism. Interestingly, these pathways were commonly enriched in dRI-derived PDX and relapse-derived PDX, suggesting that a metabolic rewiring underlies the road to relapse.Last but not the least, hematopoietic stem cell genes, chromatin remodeling and cell stress responses signatures, such as unfolded protein responses, were also commonly enriched in dRI-derived PDX and relapse-derived PDX, suggesting their involvement in the evolution to relapse.Taken together, these data suggest a model whereby relapse fate arises stochastically prior chemotherapy exposure from genetic and/or epigenetic mechanisms that contribute to rewire the cellular metabolism, confer stemness properties and modulate stress response pathways Fig. . In this5 and/or facilitate their interaction with protective bone marrow niches, and/or to provide them with the self-renewal capacity to initiate relapse following dormancy. In addition, metabolic changes might be instrumental to provide drug tolerance and/or to enable them to be counter-selected during chemotherapy exposure.6 Similarly, stress responses might be necessary to guarantee the survival of dRI subclones following drug treatment. Further experiments will be necessary to fully elucidate these questions and to understand which genetic and/or epigenetic mechanism favors the emergency of dRI subclones. Nevertheless, the pathways identified by Dobson et al will be instrumental to target relapse-fated subclones, thereby preventing their full evolution to relapse.How do the pathways identify shape the relapse fate? Stemness features might contribute to keep dRI dormantIn conclusion, thanks to a titanic experimental strategy, Dobson et al managed to a find the needle into the haystack; and of foremost, important therapeutic implications.


Mal de débarquement syndrome (MdDS) is a motion-induced disorder of oscillating vertigo that persists after the motion has ceased. The neuroimaging characteristics of the MdDS brain state have been investigated with studies on brain metabolism, structure, functional connectivity, and measurements of synchronicity. Baseline metabolism and resting-state functional connectivity studies indicate that a limbic focus in the left entorhinal cortex and amygdala may be important in the pathology of MdDS, as these structures are hypermetabolic in MdDS and exhibit increased functional connectivity to posterior sensory processing areas and reduced connectivity to the frontal and temporal cortices. Both structures are tunable with periodic stimulation, with neurons in the entorhinal cortex required for spatial navigation, acting as a critical efferent pathway to the hippocampus, and sending and receiving projections from much of the neocortex. Voxel-based morphometry measurements have revealed volume differences between MdDS and healthy controls in hubs of multiple resting-state networks including the default mode, salience, and executive control networks. In particular, volume in the bilateral anterior cingulate cortices decreases and volume in the bilateral inferior frontal gyri/anterior insulas increases with longer duration of illness. Paired with noninvasive neuromodulation interventions, functional neuroimaging with functional magnetic resonance imaging (fMRI), electroencephalography (EEG), and simultaneous fMRI-EEG have shown changes in resting-state functional connectivity that correlate with symptom modulation, particularly in the posterior default mode network. Reduced parieto-occipital connectivity with the entorhinal cortex and reduced long-range fronto-parieto-occipital connectivity correlate with symptom improvement. Though there is a general theme of desynchronization correlating with reduced MdDS symptoms, the prediction of optimal stimulation parameters for noninvasive brain stimulation in individuals with MdDS remains a challenge due to the large parameter space. However, the pairing of functional neuroimaging and noninvasive brain stimulation can serve as a probe into the biological underpinnings of MdDS and iteratively lead to optimal parameter space identification. Mal de débarquement syndrome (MdDS) is a disorder of persistent oscillating vertigo that follows entrainment to passive motion such as during water, air, or land travel . Symptom18F18-FDG PET and were compared with 20 age-, sex-, and handedness-matched healthy controls. These participants underwent structural and resting-state fMRI on the same day immediately before the PET scan. After age and gray matter volume were corrected, as well as concurrent measures of anxiety and depression, a single cluster of hypermetabolism (z > 3.3) at Montreal Neurological Institute (MNI) coordinates  mapping to the junction between the left entorhinal cortex and amygdala was revealed , whereas the right entorhinal cortex–hippocampus bias toward spatial processing may be used to navigate based on an awareness of an environmental map . As a hypothesis, a shift to a semantic memory-based egocentric strategy for navigation may be more useful when external sensory stimuli cannot be processed reliably.While it may be premature to assign the functional relevance of the left-sided bias of the differential metabolic activity in this study to the semiology of MdDS, both the entorhinal cortex and the hippocampus to which it projects do exhibit some lateralization of function. Both the left and right entorhinal cortices activate during object recognition and spatial processing tasks, with the left side biased toward object recognition and the right side biased toward spatial processing . This isOne concern related to the discovery of a limbic focus of altered activity in MdDS is the potential confound of concurrent depression and anxiety. In order to determine whether these metabolism findings could be related to concurrent anxiety or depression, the anxiety and depression subscores of the Hospital Anxiety and Depression Scale (HADS) were used as nuisance regressors and were additionally used in multiple regression analyses to determine which brain regions correlated most strongly with these scores . DepressA second PET study of relevance to MdDS compared baseline cerebral metabolic differences in fishermen who were prone to land sickness vs. those who were not . In thisz > 3.3 were the right superior frontal gyrus and the left SPL. Structural imaging was reported as normal, but areas of difference in metabolism were not corrected for differences in gray matter volume.In the individuals who were prone to developing land sickness, there was hypometabolism in the right cerebellar inferior semilunar lobule (HVIIA), uvula, nodulus, and tonsil with relative hypermetabolism in the bilateral prefrontal and occipital cortices, specifically in the left superior occipital, superior, and inferior parietal lobules (SPL and IPL), and bilateral superior frontal gyri including the dorsolateral prefrontal cortex (DLPFC). The foci that survived a statistical threshold of Reduced vestibulocerebellar metabolism in land sickness-prone individuals was postulated to relate to suppression of visual–vestibular inputs during continuous vestibular activation during wave-motion exposure, while higher metabolism in the occipital cortices in these individuals was postulated to relate to greater visual dependence with perhaps a heightened ability to suppress low-frequency vestibular signals that can trigger motion sickness. Similarly, higher prefrontal activity was postulated to be involved in the regulation of mood and anxiety. Along with the increases in metabolism in the SPL, this higher prefrontal activity may contribute to enhanced visual orientation and visuospatial attention required to suppress motion sickness but could potentially lead to more land sickness.Given the inclusion of different sexes and symptom states, it is not possible to directly compare the results of these two PET studies. Land sickness is well-recognized as a common phenomenon affecting over 70% of otherwise healthy individuals and is not considered to be pathologic –32. ConsLow-frequency spontaneous oscillations in cortical activity can be measured through blood oxygen level-dependent (BOLD) signals to reveal functionally connected regions , 36. FunThe DMN is activated during self-referential information processing and is composed of key nodes in the medial prefrontal cortex (mPFC), posterior cingulate cortex (PCC), precuneus, intraparietal sulcus (IPS), and hippocampal formation (HF+), which includes the entorhinal cortex , 42. TheThe SN has major hubs in the anterior insula and the anterior cingulate cortex (ACC) and serves to shift attention to behaviorally relevant stimuli whether internal or external . The ECNResting-state fMRI data acquired in 20 individuals with MdDS and 20 healthy controls were used to calculate the degree of functional connectivity between the entorhinal cortex/amygdala area of hypermetabolism and specific areas of the neocortex . PearsonComparisons between individuals with MdDS and healthy controls using this seed-based analysis revealed two distinct patterns of connectivity differences. First, individuals with MdDS showed decreased connectivity between the entorhinal cortex/amygdala seed and the left superior medial gyrus, left middle frontal gyrus, left superior and middle temporal gyri, FEF, and right insula, while showing increased connectivity with areas V1, V5, SPL, middle temporal gyrus, and right amygdala . The gen®) guidance. The goal was to determine resting-state functional connectivity change as a function of symptom change as well as baseline measures of connectivity that correlated with treatment response.Change in functional connectivity measured by fMRI between the entorhinal cortex/amygdala and the whole brain as a function of response to transcranial magnetic stimulation (TMS) was assessed in 20 right-handed women who underwent five sessions of prefrontal repetitive TMS (rTMS)  . SymptomOf the 20 individuals, six reported improvement, eight reported no significant change, and six reported worsening using a cutoff of 10 points on the visual analog scale . NotablyThis study was one of the first to show that symptom improvement in MdDS corresponds to a general decrease in functional connectivity between posterior sensory processing regions and the entorhinal cortex. The higher baseline connectivity between posterior parietal and occipital cortex with the entorhinal cortex in individuals with MdDS compared with healthy controls may have been reversed in treatment responders . These d3. VBM analysis was performed on 28 individuals with MdDS compared with 18 healthy age- and sex-matched controls . VBM is an imaging tool used to make voxel-by-voxel comparisons between segmented brain tissues . These mcontrols . Individt > 3.0, p < 0.005uncorr). Decreased volumes relative to controls were noted in the following areas: the bilateral middle orbital gyri, left pgACC, left middle temporal gyrus, left calcarine gyrus, and right inferior frontal gyrus (IFG) in the cerebrum and the left cerebellar lobule VIIa Crus II in the cerebellum. These analyses were done with nuisance covariates of age, and the anxiety and depression subscores of the HADS. Specific interrogation of motion-sensitive area V5/MT and the parieto-insular vestibular cortex (PIVC) were assessed because both regions receive vestibular projections and show volume changes after peripheral vestibular injury , and right temporal lobe in the cerebrum and in bilateral hemispheric lobules VIIIb and IX, left Crus I, VIIa/VIIb, and VIIIa in the cerebellum . Both IFG/anterior insular (AI) regions increased in volume with duration, with left and right correlation coefficients of +0.440 and +0.427, respectively (p < 0.05corr)  included the bilateral postcentral gyri, left superior occipital gyrus (V3a), right Heschl's gyrus, right cuneus, left middle occipital gyrus (V5/MT), left amygdala, left SPL, left lingual gyrus (V3v/V4v), bilateral calcarine gyri, left middle and inferior temporal gyri, and right temporal pole. In the cerebellum, the bilateral anterior cerebellum (lobules I–IV), left hemispheric lobule IX, and vermian lobule IX (uvula) increased in volume with duration. Cerebral volume decreases with duration included the bilateral middle ACC and the left middle frontal gyrus (area of the DLPFC). The right cerebellar lobule VIIIa/b was the only cerebellar region to decrease in volume with duration of illness  . Other c illness . As a caThe most significant volume-related markers of MdDS were correlations of decreased pgACC volume and increased IFG/AI volumes with duration of illness. The pgACC is functionally connected to the limbic system, specifically reducing amygdala activity and activating in tasks related to emotional conflict regulation, fear extinction, and planning of responses to threats –59. The A role for the cerebellum in MdDS may be related as much to its connectivity with resting-state networks as it does to specific pathways within the vestibular system. Bilateral cerebellar lobules VIII/IX showed increased volume compared with controls. Lobule VIII is functionally connected to the premotor cortex and is thus relevant for the planning of motor movements , 62. TheA case was reported of a marker of transient MdDS using an EEG-based source localization method called standardized low-resolution brain electromagnetic tomography (sLORETA) . sLORETAIn the same studies that evaluated rTMS over DLPFC, functional connectivity with high density EEG was recorded before and after the intervention. Measurement of this connectivity was reflected through independent component phase coherence (ICPC), a calculation of synchronicity at individual frequency bands  on an independent component level rather than an individual channel level . ICPC caSimilar to fMRI analyses, baseline high ICPC values correlated with greater treatment response; high ICPC values across all frequency bands portended better treatment response . This maIn addition to the above ICPC studies that have revealed biomarker information at the EEG sensor level, investigations of EEG source level computations have been able to reproduce resting-state networks that spatially correspond to the standard template of brain networks –73. Our Simultaneously acquired fMRI-EEG data on individuals with MdDS have provided cross-modal validation of biomarkers found separately, showing that brain network-level changes associated with clinical effects are consistent across modalities of different temporal resolutions , 72–74. Our multimodal imaging data of EEG and fMRI have shown that improvement in symptom severity was correlated with a reduction in connectivity involving multiples nodes of the DMN, including the entorhinal cortex, precuneus, IPLs, and medial frontal gyrus. Specifically, in the positive responders to rTMS, a significant reduction of symptoms was associated with a reduction in connectivity in the medial frontal gyrus—a key node of the DMN. In the DLPFC stimulation protocol, we have shown that the stimulation did not lead to increased connectivity between the stimulation site and entorhinal cortex itself, however, as we had hypothesized based on lower prefrontal-to-limbic connectivity in MdDS , 49. RatCombined EEG-fMRI studies have suggested a strategy of using multimodal data to guide rTMS in MdDS. These investigations have indicated that connectivity within the DMN, measured by fMRI as well as EEG, can be an imaging-based symptom biomarker. Treatments guided by the modulation of this biomarker may be informative in trials of brain stimulation, potentially not limited to rTMS. More importantly, although fMRI reveals symptom-related connectivity, an EEG-based targeting strategy would be compatible with simultaneous rTMS and may provide instantaneous feedback in trial sessions of stimulation protocols. Biomarkers from the two modalities can be integrated to guide rTMS targets. For example, the high spatial resolution of fMRI can be used to capture a disease-modifying network involving deep cortical or subcortical structures, whereas connectivity involving superficial nodes of these networks can be identified with EEG, which has high temporal resolution, potentially through a matching procedure with the fMRI-measured network as described in our approaches. New stimulation protocols could therefore be designed to promote network modulation in desired directions.MdDS represents a model of the human brain's entrainment to motion that results in a persistent sense of oscillating vertigo. The anatomical substrates for this perception have been interrogated with multiple neuroimaging modalities in order to evaluate metabolism, functional connectivity, brain volume, and synchronicity. A developing model for MdDS hypothesizes that at least one hub of the neural network that contributes to persistence of symptoms includes the left entorhinal cortex and amygdala. Through their reciprocal inhibitory action on the prefrontal cortex, these structures may wield outsized effects on both task-specific and amodal resting-state networks. Symptoms that are common in MdDS such as cognitive difficulty and feeling of sensory overload might be attributed to inefficiencies in toggling between resting states, e.g., DMN and ECN, and in dysfunction in neural substrates that filter irrelevant sensory stimuli, e.g., SN. A general neuroimaging feature of improved symptoms appears to be desynchronization of medium- and long-range connections, particularly between the parieto-occipital cortex and limbic areas as well as between frontal and parieto-occipital cortices. As further probing of these connections occurs through modulating symptoms with noninvasive brain stimulation, a more detailed understanding of the core neural components that drive persistence MdDS should emerge.YC, LD, and HY were all involved in the manuscript preparation, figure generation, and review process. 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.


Chlamydia trachomatis serovar B, and (ii) in vivo, by using a guinea pig model of Chlamydiacaviae-induced inclusion conjunctivitis. The mAb26 has been superior in comparison with mAb51 in the prevention of chlamydial infection in HCjE cells. The mAb26 has conferred ≈40% inhibition of the infection, compared to less than 5% inhibition in the presence of the mAb51. In vivo, mAb26 significantly diminished ocular pathology intensity in guinea pigs infected with C. caviae compared to either the mAb51-treated or sham-treated guinea pigs. Our data provide insights that tetanus immunization generates antibodies which induce heterologous chlamydial immunity and promote protection beyond the intended target pathogen.Vaccines can have heterologous effects on the immune system, i.e., effects other than triggering an immune response against the disease targeted by the vaccine. We investigated whether monoclonal antibodies (mAbs) specific for tetanus could cross-react with Chlamydia and confer heterologous protection against chlamydial infection. The capability of two tetanus-specific mAbs, namely mAb26 and mAb51, to prevent chlamydial infection has been assessed: (i) in vitro, by performing a neutralization assay using human conjunctival epithelial (HCjE) cells infected with  Chlamydia trachomatis, and Borrelia burgdorferi are still unavailable for a variety of reasons, which include but are not limited to (i) pathogen complexity, (ii) pathogen novelty, (iii) absence of efficient delivery system and adjuvants, (iv) lack of knowledge on the mechanisms of protective immune response induction, (v) lack of relevant/suitable animal models, and (vi) ethical issues for conducting clinical trials .,36.35,36In the context of the prevention of chlamydial infection, mAb26 was shown to be superior in both in vitro and in vivo settings compared to mAb51. In a setting that mimics the natural chlamydial infection, e.g., by using a relevant ocular epithelial cell line HCjE to investigate the ability of mAb26 and mAb51 to inhibit the infection, we showed that the mAb26 efficiently neutralized chlamydial EBs in solution and inhibited the infection in vitro. We have also shown that cross-reactive antibodies generated after tetanus immunization can interfere with chlamydial infection in vivo by alleviating the clinical picture of ocular chlamydial infection in guinea pigs. The finding that mAb26, topically applied during the acute phase of infection to the animals, was able to reduce the chlamydial infection in vivo is important and might have clinical relevance. The genital and ocular guinea pig models are established and relevant animal models to investigate chlamydial infection ,70,71,72Many processes in nature are extremely complex and very rationally designed during evolution. We suggest that certain low-affinity antibodies generated after TTd immunization, which cannot provide protection against TeNT intoxication per se, such as mAb26, could act as positive binding modulators by contributing to resistance to heterologous infectious agents. More research is needed to understand how such cross-reactive antibodies shape the chlamydial infection.We are planning to investigate further whether the active immunization with tetanus toxoid could alleviate the chlamydial infection. Characterizing the interactions of tetanus-specific and Chlamydia cross-reactive mAbs, aiming to primarily determine their fine epitope specificity, could contribute to research aimed at formulating a vaccine that will effectively protect against chlamydial infections. Additionally, defining the precise mechanisms underlying this heterologous immunity could also pave the way to a better definition of the mechanisms that chlamydial infection exerts on specific mucosal surfaces.Vaccination against tetanus disease, due to heterologous effects, might contribute to the immunity to chlamydial infections. Elucidation of mechanisms underlying heterologous impact could provide important indications for designing the efficient vaccine formulation against chlamydial infections.


In rural Ethiopia, 72% of women are exposed to lifetime intimate partner violence (IPV); IPV is most prevalent during pregnancy. As well as adversely affecting women’s physical and mental health, IPV also increases the risk of child morbidity and mortality associated with maternal depression, thus making antenatal care an important opportunity for intervention. Adapting generic, task-shared, brief psychological interventions for perinatal depression and anxiety to address the needs and experiences of women affected by IPV may improve acceptability to women and feasibility for health workers. This randomised controlled feasibility trial will compare brief problem solving therapy (PST) specifically adapted for pregnant women experiencing IPV (PST-IPV) with standard PST and enhanced usual care to determine the feasibility of a future fully powered randomised controlled trial.Seventy-five pregnant women scoring five or more on the Patient Health Questionnaire, endorsing a tenth question about functional impact and reporting past-year IPV, will be recruited from antenatal care clinics in predominantly rural districts in Ethiopia. Consenting participants will be randomised to either four sessions of PST-IPV, four sessions of standard PST or information about sources of support  in a three-arm design. The interventions will be delivered by trained, supervised antenatal care staff using a task-sharing model. Assessments will be made at baseline and after 9 weeks by masked outcome assessors and will include measures of depression symptoms (primary outcome), post-traumatic stress, anxiety symptoms, functional impact, past-month IPV and hypothesised mediators (secondary outcomes). A mixed-method process evaluation will determine the feasibility of a future randomised controlled trial, assess the feasibility, acceptability, fidelity and quality of implementation of PST-IPV, generate testable hypotheses about causal mechanisms, and identify potential contextual factors influencing outcomes.Despite mental health being a critical concern for women experiencing IPV, there is limited evidence for brief, task-shared psychological interventions adapted for their needs in low- and middle-income countries. Contextually tailored interventions for pregnant women experiencing IPV in low- and middle-income countries require development and process evaluation. This randomised controlled feasibility trial will yield results on the feasibility of conducting a fully powered trial, relevant to researchers, primary and antenatal care clinicians in resource-limited settings.PACTR202002513482084. Prospectively registered on 13 December 2019.Pan-African clinical trials registry:  Intimate partner violence (IPV) refers to behaviour by a partner or ex-partner that causes  physical, sexual or psychological harm. IPV includes physical aggression, sexual coercion, psychological abuse and controlling activity . It is hThe relationship between IPV and mental health is bidirectional, such that IPV increases a woman’s risk of mental disorders, which in turn increases a woman’s vulnerability to (further) IPV. For example, IPV is associated with subsequent depressive symptoms, suicide attempts  and alcoThere is limited evidence on mental health interventions tailored to the needs of pregnant women experiencing IPV , especiaA recent systematic review confirmed the paucity of intervention research addressing the mental health of both survivors and perpetrators of IPV in LMICs ; no studI2 49.3%) and consistent but non-significant differences in PTSD, depression and psychological distress symptom improvements [We recently investigated whether IPV exposure moderates the efficacy of generic psychological interventions in LMICs. Our meta-analysis of 15 studies that provided data showed that women reporting IPV demonstrated greater improvements in anxiety symptoms than women not reporting IPV following generic psychological interventions  in this context.Determine whether the PST-IPV intervention and processes of recruitment, randomisation, follow-up and evaluation are feasible and acceptable to pregnant women and health workers in rural Ethiopia. This includes estimating parameters to inform the design of a future RCT.Refine the PST-IPV intervention and study design in response to a mixed-method process evaluation, which assesses the feasibility, acceptability, fidelity and quality of the intervention, explores causal mechanisms, and identifies contextual factors that may influence outcomes .In this randomised feasibility trial and process evaluation of perinatal PST-IPV in rural Ethiopia, we aim to determine the feasibility and acceptability of the intervention and study design to inform a future fully powered RCT. Specific objectives are to:In this three-arm feasibility trial, we will randomise eligible women  to PST-IPV, standard PST or enhanced usual care (information about relevant sources of support). All arms represent additions to standard clinical care, which does not currently provide any routine interventions for perinatal depression or IPV.This study is linked to a two-arm feasibility trial that will randomise eligible women  to standard PST or information only, the protocol of which is reported separately  see Fig. .Fig. 1FThe study will be conducted in selected health facilities in the predominantly rural areas of Gurage zone  and Silt’e zone  in the Southern Nations, Nationalities, and Peoples’ Region of Ethiopia . PrimaryThis study builds on the programme for improving mental health care (PRIME) study , which iA systematic review and meta-analysis , furtherAdaptation for IPV focused on international guidelines , which rAs a feasibility trial, this study is not powered to detect intervention efficacy, but rather to estimate feasibility parameters to inform a future RCT, and test intervention and research protocols. With a total sample size of 75 , a drop-out rate of 30% can be estimated to within a 95% confidence interval of ±3%. To estimate the standard deviation of the primary feasibility outcome measure to inform a future RCT sample size calculation, recommended feasibility trial sample sizes range between a total of 24 and 50 across two arms , 57.Research staff will consult health care workers based in primary hospitals and/or health centres in Gurage and Silt’e zones to identify potentially eligible women meeting those inclusion criteria about which information is held for screening. Potentially eligible women will be approached through ANC clinics and HEW home visits by research staff and provided with written and verbal information in Amharic, before being invited to give written, informed consent to initial screening. Non-literate women will signify their consent by finger print. A high school-educated assistant will act as a witness to confirm that the information sheet has been read aloud correctly to non-literate women. Women screened as eligible to participate in the randomised feasibility trial will then be invited to give written, informed consent to participate. As with screening, non-literate women will signify their consent to participate in the trial by witnessed finger print.Due to potential risks of abusive partners learning of women’s involvement, information sheets, consent forms and other study paperwork will be kept in the locked research office after being read and signed but will be accessible to women at their request. Due to logistical challenges affecting women’s daily lives in this setting, it is not possible to give prospective participants 24 h to decide whether to take part. However, a minimum of 30 min will be allowed for the woman to make up her mind about participating in the study.Consecutive, potentially eligible women who provide informed consent will be screened for depressive symptoms and a history of past-year IPV after their ANC appointment. Depressive symptoms will be screened for using the locally validated  nine-iteAll screened women scoring five or more on the PHQ-9 , 64 and Consenting women who disclose any past-year IPV during screening will be randomised to one of PST-IPV, standard PST or enhanced usual care, whereas consenting women reporting no IPV on baseline measurements will be randomised to one of standard PST or enhanced usual care for the separate, nested PST feasibility trial see Fig. .All women screened as eligible to participate will be informed about the study and given the opportunity to ask questions before being invited to consent to participate in the randomised feasibility trial. Women consenting to participate will first receive their routine clinical care and then participate in a baseline interview using fully structured questionnaires. If screening or this assessment identify any sources of concern , the researcher will discuss these with the woman and explain the need to share them with her health care professional, before involving them. To be eligible to participate in the PST-IPV components of the overall study, women must meet the eligibility criteria (see below).Speak Amharic Are aged 16 years or overAre between 12 and 34 weeks gestation of pregnancyAre intending to reside in the study area for the duration of the studyScore 5 or more on PHQ-9 with functional impairment (tenth question)Report IPV in the past year (in a current or previous relationship) during screeningConsent to participate, including to accept enhanced usual care or to attend four sessions of PST-IPV or PST (if randomised to a treatment arm)Women can be included if they:Women are required to speak Amharic, the official language of the region, so that they can access all translated study materials, verbally or in writing. Relationships are defined as any romantic or sexual interaction within or outside marriage.PHQ-9 has been validated in the study area in both primary care  and ANC Are acutely unwellRequire emergency treatmentAre identified by the ANC provider during pre-screening as having possible psychotic symptomsAre unable to understand the interview Expect to move away from the study area before the study is completedWomen will be excluded if they:All women endorsing question nine of PHQ-9 about suicidal thoughts or self-harm behaviours will receive the Amharic  Mini-IntOtherwise eligible women reporting no past-year IPV exposure will be invited to participate in the separate, nested feasibility trial of standard PST for depression but will not be eligible for this study of PST-IPV.Interested women who cannot read will be read aloud the information sheet. If unable to write, participants will record a thumb print, signed by a literate witness (a high school-educated assistant) after confirming that the information sheet has been read aloud correctly; otherwise, participants will provide written informed consent. Study documents will be kept in the locked research office but will be accessible to women at their request. The information sheet includes details of how participants can access compensation, if required.All participants will be reimbursed for their time attending research interviews in addition to transport costs and any additional expenses incurred. Participants will be reimbursed for transport costs to attend PST or PST-IPV sessions but will not be compensated for their time during these sessions. This is to determine the acceptability of psychological intervention sessions to women and enable calculation of drop-out rates. A fund will be available to assist participants disclosing IPV who wish to access support services .Research staff will telephone the Centre for Innovative Drug Development and Therapeutic Trials for Africa data manager, based at the College of Health Sciences, Addis Ababa University, Ethiopia, who is not otherwise involved in this study, to allocate each participant to a study arm  using a random number list generated by the trial statistician (GM), who is not otherwise involved in data collection processes. The data manager will telephone a separate member of research staff based in Silt’e, who will inform a clinician trained to deliver PST-IPV or standard PST when a new participant has been allocated to that trial arm. Research staff will provide participants with an unmarked card indicating their enrolment in the trial and agree a date and time to attend their first session of PST-IPV or standard PST. When they attend, they will be expected by their allocated clinician. For participants allocated to enhanced usual care, their antenatal care provider will provide them with information about sources of support and the research assistant will arrange a follow-up appointment 9 weeks later.Women who do not attend their appointed date and time will be contacted using the telephone number (where applicable) and/or household details they have provided at trial enrolment or through their allocated HEW, up to a maximum of three attempts.Given qualitative differences between PST and PST-IPV, clinicians delivering the intervention cannot be masked to participant allocation. To avoid contamination, PST-IPV, standard PST and enhanced usual care will be delivered by different practitioners who have either attended a PST-IPV or a standard PST training course, or received basic training to provide enhanced usual care. Post-intervention outcome assessments will be conducted by trained, independent assessors (working in separate offices from practitioners delivering interventions and not travelling together) masked to intervention allocation, with a minimum post-high school diploma level of education. Independent assessors will document any incidents of unmasking. Data analysts will also be masked to intervention allocation.Participants randomised to PST-IPV or standard PST will attend four intervention contacts within a maximum of 8 weeks. Where birth occurs before the end of treatment, the feasibility of continuing PST-IPV or standard PST post-natally will also be assessed.Both manualised interventions will be delivered by trained health workers, such as nurses and midwives, supervised by Psychology Masters-level qualified research staff, with access to a project mental health professional for clinical concerns. Responses to concerns and risk incidents will follow standard operating procedures (SOPs). Government-employed health workers trained to deliver the interventions will be paid for their time.Both PST-IPV and standard PST intervention arms follow the same structure of four sessions, based on the model successfully employed in South Africa and adapted for this rural Ethiopian setting , 66. SesPST-IPV content and materials are adapted to address the needs and experiences of women affected by IPV, whilst standard PST content and materials are generic. Adaptations for women experiencing IPV include training staff using content and materials from the new WHO curriculum on caring for women subjected to violence , attentiParticipants randomised to PST-IPV or standard PST will attend a total of six contacts: one baseline pre-intervention research assessment, four intervention contacts and a follow-up research assessment contact 9 weeks after recruitment. The feasibility of follow-up 9 weeks after recruitment will be assessed and may change, depending on gestation of pregnancy at the time of enrolment.One-third of participants will be randomised to information only about sources of support   which haAnxiety symptoms using the Generalised Anxiety Disorder 7 (GAD-7) scale Disability using the 12-item Ethiopian adaptation  of the WIPV: participants will be asked about past-month IPV at baseline and 9-week follow-up to determine whether the frequency of different types of IPV  change during the study periodSelf-efficacy using an adapted self-efficacy scale validated in Ethiopia for sexual health research Mastery (the extent to which the person considers themselves in control of forces affecting their lives in important ways ) using aPerceived social support using the Oslo Social Support Scale 3 (OSSS-3)  previousHealth care-seeking behaviour using the client service receipt inventory  adapted Adverse events or unanticipated harms taking place during the study periodObstetric and neonatal outcomes: HEWs, who maintain maternal care records, will report the location of birth and any stillbirths and early neonatal deaths among study participants, attendance at subsequent ANC appointments and medication prescriptions for physical and mental health, as they are not reliably self-reported ; where wAttitudes towards gender roles using the WHO Attitudes Towards Gender Roles questionnaire At baseline and 9-week follow-up, the following will be measured using Amharic-translated scales. If scores are normally distributed, mean differences will be compared between arms using Acceptability of PST-IPV and of the study design to women and health workers in terms of uptake, completion and drop-out ratesAdoption by health workersAppropriateness from women and health workers’ perspectivesFeasibility in terms of practical organisationFidelity of deliveryCostSafetyThe mixed-method process evaluation will assess variables associated with intervention implementation processes. Recommended intervention process outcomes (for implementation) include acceptability, adoption, appropriateness, feasibility, fidelity, implementation cost, penetration and sustainability . In thisScreening rates and reasons for refusal (acceptability)Recruitment rates Numbers of eligible women at each clinical site (appropriateness)Proportions of eligible women agreeing to participate (acceptability)Willingness to be randomised and comprehension by participants of randomisation Session duration Take-home activity completion Drop-out rates Follow-up rates (feasibility of retention)The following administrative data will be collected, which will inform the feasibility outcome measures shown in brackets:Staff willingness to participate at each site and retention in the study Optimal recruitment procedures (feasibility)Acceptance of randomisation at each site Research staff will document key site features to enable comparison of process data, and keep field journals noting:The acceptability and burden on women and staff of the intervention itself (including which components are experienced as helpful or unhelpful), randomisation, outcome measures and follow-up will be assessed through qualitative interviews with a purposively sampled sub-group of approximately six women (three per intervention arm) and six staff (three per intervention arm). Interviews will also explore feasibility and acceptability of the study’s SOP, which dictates the management of safety, confidentiality and ethical concerns, risk disclosures during sessions and research contacts. Interviews will be conducted in Amharic by research staff who did not have extended contact with participants during the trial in a private location convenient to them. Participants will be compensated for their time, and interviews will be audio-recorded, transcribed and translated into English.Health workers trained to deliver PST-IPV and standard PST will receive monthly supervision with a local psychiatric nurse. Health workers will audio-record PST-IPV and standard PST sessions with the participant’s consent for review by their supervisor and to inform discussions of challenging cases during supervision sessions.A random sample of these audio recordings will be evaluated using the ENACT scale  to assesRecordings will also be evaluated for intervention fidelity and session completion using a checklist of session components. Supervision records and therapist logs will also be reviewed for intervention fidelity and contamination between arms . The freOutcome assessors will receive supervision from a Psychology Masters-qualified supervisor. Supervision logs will include discussing any items which are poorly understood or completed, and reviewing the time taken for outcome assessment to assess its acceptability and feasibility.We will follow the Consolidated Standards of Reporting Trials (CONSORT) extension to randomised pilot and feasibility trials .We will review process evaluation data for indications of sub-group differences in uptake, recruitment and retention of participants by site, health worker, recruitment method/site, participant  and health worker characteristics . Quantitative scale results will be analysed using STATA .Feasibility studies are not powered to calculate effect sizes but standard deviations and drop-out rates will be calculated on the intention-to-treat sample  to inform sample size calculation for a future RCT, for outcome measures, recruitment rates and drop-out rates. Quantitative process data will determine future RCT design improvements, such as session number and duration, recruitment, outcome measures, and strategies preventing contamination. The extent of missing data will be evaluated as part of the process evaluation. For clinical outcome measures, missing data will not be imputed. Rather, standard deviations will be calculated using data from participants for whom outcome measures are available.We will apply the framework approach to thematic analysis of qualitative interviews  and triaStrategies to ensure data protection, quality assurance and dissemination of results are outlined in Supplementary file This study involves recruitment of potentially vulnerable women, disclosure of IPV, and detecting depression, anxiety and PTSD symptoms. The SOP therefore outlines actions to mitigate potential risks. If participants disclose current exposure to IPV, research staff will be trained to listen non-judgementally, offer privacy and confidentiality, and information about local agencies who can provide assistance  on 13 December 2019. Recruitment of participants has not yet commenced. Recruitment of participants is anticipated to complete by 31 December 2020. Any proposed changes to the protocol will be submitted to King’s College London and Addis Ababa University ethics review boards and updated on the pan-African clinical trials registry. Research staff will inform trial participants, where required, and changes will be discussed in the ultimate results publication. The trial sponsor is King’s College London, De Crespigny Park, Denmark Hill, London SE5 8AF, UK.Additional file 1: Supplementary file 1. Provisional theory of change map for PST-IPV. Supplementary file 2. Example information sheet about local sources of support provided to all participants. Supplementary file 3. Data protection, quality assurance and dissemination plans. Supplementary file 4. Data collection forms. Supplementary file 5. Ethical approval. Supplementary file 6. (a) Participant information sheet. (b) Participant consent form. (c) Health worker information sheet and health worker consent form.


The traditional view of protein aggregation as being strictly disease-related has been challenged by many examples of cellular aggregates that regulate beneficial biological functions. When coupled with the emerging view that many regulatory proteins undergo phase separation to form dynamic cellular compartments, it has become clear that supramolecular assembly plays wide-ranging and critical roles in cellular regulation. This presents opportunities to develop new tools to probe and illuminate this biology, and to harness the unique properties of these self-assembling systems for synthetic biology for the purposeful manipulation of biological function. Proteins are the workhorse molecules of the cell, driving virtually every function and developmental program in biology. Amazingly, many of these critical molecules readily aggregate and assemble inside of living cells through interactions amongst unfolded and folded domains. This can occur aberrantly and lead to disease; but there is also accumulating evidence that aggregation phenomena can be regulated by the cell and used to carry out important and beneficial biological functions ranging from molecular scaffolding to memory –4. MoreoProtein components can self-assemble into higher-order complexes or assemblies within the cell. A common feature of many of these proteins is the presence of intrinsically disordered regions (IDRs). IDRs are protein sequences that do not adopt a single three-dimensional structure, but instead endow proteins with flexibility to adopt a range of states, from unstructured to partially structured . Due to At the dynamic end of the spectrum are supramolecular assemblies based on phase separation. Biological phase separation has generated tremendous excitement as recent discoveries point to its critical role in many cellular processes, such as the formation of organelles and regulatory complexes, as well as in human diseases . Our goaPhase separation is a physical phenomenon that occurs when a solution of molecular components spontaneously separates or demixes into co-existing (lower free energy) phases, such as when oil is mixed with water . In cellAs discussed more below, these dynamic assemblies can help to compartmentalize biological reactions and in some cases can even be composed of distinct, non-miscible sub-compartments with different protein compositions . In geneAt the other end of the spectrum are solid structures, such as amyloid fibers. Amyloids are highly stable assemblies composed of protein molecules organized in a cross-β-sheet lattice of indefinite length . AmyloidProteins with high propensity to form amyloids contain disordered regions enriched in hydrophobic and polar amino acid residues. However, amino acid composition alone is insufficient to predict amyloid-forming capacity, as the position within the protein sequence also matters. Collectively, these insights have been used to predict amyloid-forming sequences in the proteomes of organisms , 35.Amyloid fibrils are extraordinarily robust biomaterials. They are protease-, heat-, and detergent-resistant and can have stiffness comparable to that of spider silk and collagen fibers . Both prAmyloids have been linked to many human diseases, ranging from diabetes to systemic amyloidoses , 40. PerPrions are a unique class of aggregating proteins. Prion proteins, first discovered as the causative agents of transmissible neurodegenerative disorders in mammals , haMost known prions form amyloid aggregates, templating the amyloid conformation onto newly synthesized proteins. Conversion between the prion and soluble conformation is a reversible process . HoweverSaccharomyces cerevisiae may harbor many more uncharacterized prionoid elements that appear to be independent of Hsp104, and instead dependent on other chaperones , which is formed by self-templated aggregation of the translation termination factor Sup35 [PSI+] strains are referred to as weak and strong [PSI+] have lower efficiency of translational read-through relative to strong [PSI+], and thus a weaker phenotype. However, weak [PSI+] fibers are thermodynamically more stable, with larger and more rigid amyloid cores compared to strong [PSI+]. This structural difference results in less efficient fragmentation by the chaperone machinery, and consequently the generation of fewer seeds, slower templating of new protein to the prion form, and less stable inheritance , formed by the translation termination factor Sup35  [MOT3+] [GAR+] [SWI+] prion formed by the yeast Swi1 protein represents an intriguing potential example of bet-hedging. As the main subunit of the SWI/SNF chromatin remodeling complex, Swi1 serves as a global transcriptional regulator. When Swi1 adopts its prion form, a variety of phenotypes can be induced, such as growth phenotypes on alternative carbon sources, sensitivity to antifungal agents, and, importantly, abolished adhesion to other cells or substrates [SWI+] cells, an isogenic population can effectively safeguard against unpredictable environments via these diverse and potentially beneficial phenotypes, for example, by providing an opportunity for non-adherent [SWI+] cells to disperse to new locations to ensure re-population and survival [Other notable examples of yeast prions that enable epigenetic switching to endow cells with beneficial phenotypes in specific metabolic and environmental conditions include [URE3] , [MOT3+] [MOT3+] , and [GA] [GAR+] . In partbstrates . By mainsurvival .Clostridium botulinum global transcriptional terminator Rho  [RNQ+]   and [RNQ] [RNQ+] .One approach often used by synthetic biologists to engineer new synthetic protein systems is to leverage the functional modularity of certain protein domains. Fortunately, many PrDs have been found to be modular, which allows transfer of prion-forming capability onto new proteins by simply fusing them to PrDs . These fEscherichia coli CsgA protein, responsible for curli amyloid fiber formation, to the mussel foot protein (Mfps) in order to create a synthetic protein capable of generating a fibrillary structure of adhesive proteins that outperforms mussel foot in adhesion to underwater surfaces  or [PRION+] phenotypic state. Second, cells can reversibly transition between the two stable states in all-or-none fashion [PSI+]-inducing factor under the control of a temperature-sensitive promoter, cells could be programmed to remember a short exposure to elevated temperature over ten generations later. These types of prion-based memory elements could be deployed in populations to record and report on environmental variables experienced in natural or industrial contexts, such as in industrial bioreactors.Yeast prions have several properties that, in principle, make them excellent candidates for building stable synthetic memory . First,  fashion . Third,  fashion . These c fashion . SpecifiPrion conformations and their associated phenotypes are inherited in a dominant non-Mendelian fashion . As suchProtein self-assembly remains a little explored and exploited mechanism for synthetic biology and cellular engineering; but it could offer many advantages, such as the ability to program emergent nonlinear behaviors and catalyze drastic cellular changes with a relatively small set of constituent parts. However, the unique properties of these systems can also make them difficult to design and manipulate. For example, challenges associated with designing and manipulating disordered and self-assembling proteins include: (1) The high false positive rate of structure prediction algorithms. This necessitates experimental validation of assembly formation for each predicted protein or domain. (2) Predicting and designing the stoichiometry of disordered protein assemblies. This will require an increased understanding of the basic biophysics of assembly formation, or either focusing on applications that are insensitive to stoichiometry or using structured protein domains to introduce well-defined molecular interactions. (3) Lack of methods for precisely controlling formation and dissociation of assemblies. Here, the use of engineered post-translation modifications known to modulate assembly formation could be highly advantageous, but will require the development of synthetic tools for exacting control over these signaling events. Overall, through the combination of synthetic biology manipulation, quantitative studies, and an increased understanding of their underlying biophysics, we can make possible an era of creating designer protein assemblies for application and study.


Coatings deposited utilizing different thermal spray variants have been widely used for diverse industrial applications. Today, various coating techniques belonging to the thermal spray family and spanning a vast cost–quality range have been embraced by the industry to either extend the longevity of components or enhance their performance, especially when these parts routinely operate in harsh conditions. The current state-of-the-art route for depositing the ceramic coatings is usually atmospheric plasma spraying (APS), while metallic and intermetallic coatings are sprayed by high-velocity oxy-fuel (HVOF) methods. Between them, the above spray methods and the vast portfolio of commercially available spray grade powders are capable of providing coatings that can combat premature surface degradation when industrial components are exposed to wear, corrosion, oxidation, high thermal load, etc. The immense versatility of the technique has already led to its numerous industrial uses, ranging from the advanced gas turbine requirements to the relatively more mundane needs of sectors such as textile, mining, pulp and paper and petrochemical sectors.However, efforts continue to explore new potential applications to further expand this envelope. Two of the papers in this Special Issue focusing on impact wear  and tribTraditionally, thermal sprayed coatings have been realized employing powder feedstock, with the particle size typically being in the 10–100 µm range, with the lower end of this range being preferred for high melting point materials such as ceramics. Use of such feedstock, now commercially available for an exhaustive spectrum of material chemistries, results in splat sizes that are several tens of microns and consequently in coarse-structured coatings. However, there is growing interest in realizing fine-structured coatings using submicron and nanosized powders that can potentially yield enhanced functional performance. Such a feedstock injection methodology constitutes the basis for Suspension Plasma Spraying (SPS), which has been found capable of producing coatings with tailored microstructures, including the extremely porous to the very dense, vertically cracked, columnar, etc., and thus are not easily realizable when using a typical spray-grade powder feedstock. With the above approach providing a convenient pathway to deposit fine-structured coatings, thermal spraying with suspensions is perhaps the next frontier. A vast majority of interest in SPS has hitherto been driven by the excitement of obtaining columnar thermal barrier coatings (TBCs). One of the papers in this Special Issue investigates SPS-derived double-layered Gadolinium Zirconate/Yttria-Stabilized Zirconia (YSZ) coatings . The rap2O3—YSZ coating in bovine serum solution [It is also relevant to point out that the advent of axial injection capable plasma spray systems is a potential game-changer for use of liquid feedstock in the form of suspensions or solution precursors. This is by virtue of the fact that axial feeding enables far more intimate contact between the liquid feedstock and the plasma plume to facilitate thermal energy transfer and enable effective utilization of plasma energy. This advantage, which manifests in the form of higher throughputs, longer stand-off distances etc., has been harnessed in a couple of the above-mentioned studies ,9. The fsolution .Admittedly, it was not possible to include in this Special Issue other key areas that, too, continue to play a crucial role in the continued development of thermal spraying. These include, for example, evolution of new torch designs, advanced characterization of coatings, novel approaches for in-flight diagnostics and modelling of coating formation. The use of artificial intelligence/machine learning and data-driven modelling approaches, as illustrated in one of the papers , is also


The article is aimed at analyzing the available research and comparing the properties of bio-inductive materials in direct and indirect pulp capping procedures. The properties and clinical performances of four calcium-silicate cements , a light-cured calcium silicate-based material  and an enhanced resin-modified glass-ionomer (ACTIVA BioACTIVE) are widely discussed. A correlation of in vitro and in vivo data revealed that, currently, the most validated material for pulp capping procedures is still MTA. Despite Biodentine’s superiority in relatively easier manipulation, competitive pricing and predictable clinical outcome, more long-term clinical studies on Biodentine as a pulp capping agent are needed. According to available research, there is also insufficient evidence to support the use of TheraCal LC or ACTIVA BioACTIVE BASE/LINER in vital pulp therapy. The major challenge for the modern approach in restorative dentistry is to induce the remineralization of hypomineralized carious dentine, and therefore, protecting and preserving the vital pulp. Traditionally, deep caries management often resulted in pulp exposure and subsequent root canal treatment. The promotion of biologically-based treatment strategies has been advocated for partial caries removal aimed at avoiding carious pulp exposure. Indeed, recent consensus reports have stated that the complete or nonselective carious removal is now considered overtreatment . ManagemSix different bioactive materials have been described and compared: four calcium-silicate cements—ProRoot MTA , MTA Angelus , RetroMTA  and Biodentine ; a light-cured calcium silicate-based material TheraCal LC ; and a resin-modified glass-ionomer (RMGIC) with improved resilience and physical properties—ACTIVA BioACTIVE  .The article is aimed to review the properties of bio-inductive materials applied for direct and indirect pulp capping and to compare their clinical performances.The indirect pulp capping (IPC) procedure is generally used in deep cavity preparations, with or without carious dentine remaining which is in close proximity to the pulp but showing no visible pulp exposure ,12. AccoOne-stage selective carious-tissue removal includes the application of a biomaterial onto a dentine barrier after either firm or soft dentine is left only on the pulpal aspect of the cavity, whilst peripheral carious dentine is removed down to hard dentine on the same visit permanent restoration is being placed. On the other hand, stepwise excavation involves re-entry after 6–12 months after application of a biomaterial in an indirect, two-stage, selective carious-tissue removal technique. The first stage involves selective carious removal to soft dentine, to an extent that facilitates proper placement of a temporary restoration, and the second stage includes removal to firm dentine. Eventually, final placement of a permanent restoration is performed. Tooth disinfection with NaOCl or chlorhexidine digluconate 2% solution, as a less aggressive alternative, sterile technique, and rubber dam use, are recommended at all times throughout the management of deep caries whether the pulp is exposed or not ,16,17,18According to recent position statement of the ESE, deep caries management should focus on avoidance of pulp exposure by the means of choosing selective one-stage carious-tissue removal or stepwise excavation treatment, rather than redefined indirect pulp capping procedure .Direct pulp capping (DPC) treatment is used when the vital asymptomatic pulp is visibly exposed due to caries or trauma, or due to a misadventure during tooth preparation or caries removal . It inclIn contrast to pulp capping procedures, which does not involve any pulp tissue removal, partial pulpotomy removes 2–3 mm of the pulp tissue at site of exposure. In practice this technique is used for removing the superficial layer of infected tissue in case of asymptomatic carious pulp exposure or when pulp has been exposed to the oral environment. The pulp capping procedures protects the tissue, but may not reverse a superficial inflammatory processes; therefore, it is recommended that 2–3 mm of tissue is removed in a partial pulpotomy procedure. The question about choosing the proper treatment arises when we take into consideration that visual analysis to distinguish between inflamed and noninflamed pulp tissue may not be sufficiently accurate . If hemo2) has been considered the gold standard. Long term clinical observations of calcium hydroxide are incomparable to any other bioactive material, as the first reports of successful pulpal healing using Ca(OH)2 were published between 1934 and 1941 . Fo. Fo59]. The first formulation of MTA was grey, but due to the reported discoloration of teeth , an alte2 in a randomized clinical trial, confirmatory evidence emerged for the superior performance of MTA as a DPC agent when evaluated in a practice-based research network . Ad. Ad26]. Lack of cytotoxicity and biocompatibility are the significant factors referring to pulp capping agents which directly affect the clinical outcome. Based on evaluation of pulpal responses in dog partial pulpotomy cases, complete dentin bridge was observed only in 33% of specimens . It was TheraCal LC exhibits higher bond strength values than Biodentine when layered with either composite or glassionomer cement ,129. To Although sufficient bioactivity, superior handling properties and superior quality of bonding with the final overlaying restoration could justify the use of TheraCal LC as the IPC agent, further in vitro and in vivo studies are required. Furthermore, TheraCal cannot be recommended for DPC .ACTIVA BioACTIVE-BASE/LINER  was launched in 2014 claiming the strength, aesthetics and physical properties of composites and increased release and recharge of calcium, phosphate and fluoride in comparison with glassionomer (GI). On the other hand, it has been proven that the fluoride ion release of ACTIVA is lower than that of the conventional GI  and also than that of RMGI cement  ,131. TheThe company markets this product as a “light-cured resin-modified calcium silicate” (RMCS) combining uncompromised attributes of both composite and GI . DespiteComparing to both MTA and Biodentine, ACTIVA BioACTIVE represents a favorable setting time with no delay placing final restoration . The matThe bioactive properties of ACTIVA BioACTIVE products are based on a mechanism whereby the material responds to pH cycles and plays an active role in releasing and recharging of significant amounts of calcium, phosphate and fluoride . Some auDespite the fact that ACTIVA BioACTIVE products are RMGICs, the laboratory and clinical findings indicate that the self-adhesive ability of the material is not elucidated . The afoConsidering the material’s characteristics, the application of ACTIVA BioACTIVE in direct and indirect pulp capping might be justified. Nevertheless, to this day there is insufficient evidence and there have been too few clinical studies to support its reliability in vital pulp therapies. Moreover, the resin in pulp capping materials such as ACTIVA BioACTIVE BASE/LINER would presumably shift the regeneration-inflammation balance towards the latter. As in the other light-cured tricalcium silicate, incomplete resin photopolymerization may lead to free monomers’ release, and consequently to pulpal toxicity . HoweverDevelopment of minimally invasive biologically based therapies aimed at preservation of the pulp vitality remains the key theme within contemporary clinical endodontics. The present findings confirm that both MTA and Biodentine are reliable materials in the matter of inducing dentin bridge formation while keeping a vital pulp in both direct and indirect pulp capping procedures ,112,140.Future in vitro and in vivo studies are necessary to validate the clinical importance of the new generation of light-cured resin-modified calcium silicates; namely, TheraCal LC and ACTIVA BioACTIVE BASE/LINER. In addition, more studies are needed to support use of these materials in VPT other than indirect pulp capping. RMGIC’s superior handling properties, quality of bonding with final overlaying restoration and the possibility of immediate restoration placement, might result in more predictable treatments from both a histological and a clinical perspective. Therefore, those materials should constitute the objects of future studies, especially in terms of cytotoxicity, quality of induced dentin bridge and protocols for higher bond strength to tooth structure and final restoration.


Due to higher vulnerability and immunogenicity of extended criteria donor (ECD) organs used for organ transplantation (Tx), the discovery of new treatment strategies, involving tissue allorecognition pathways, is important. The implementation of machine perfusion (MP) led to improved estimation of the organ quality and introduced the possibility to achieve graft reconditioning prior to Tx. A significant number of experimental and clinical trials demonstrated increasing support for MP as a promising method of ECD organ preservation compared to classical static cold storage. MP reduced ischemia–reperfusion injury resulting in the protection from inadequate activation of innate immunity. However, there are no general agreements on MP protocols, and clinical application is limited. The objective of this comprehensive review is to summarize literature on immunological effects of MP of ECD organs based on experimental studies and clinical trials. The remarkable evolution of solid organ transplantation (Tx) has led to improved overall outcomes for patients with terminal organ dysfunction. However, ischemia–reperfusion injury (IRI) in combination with early immune activation remains a significant challenge limiting the potential of this therapy , 2. IRI For more than 50 years, static cold storage (SCS) was the gold standard method for organ preservation until the interest in the concept of organ machine perfusion (MP) was renewed . To dateMP is a promising tool to reduce the gap between organ demand and supply that is resulting in a dramatic prolongation in waiting times and associated with increased morbidity and mortality for patients on the waiting list for Tx . In an eex vivo -γ, and C-X-C motif chemokine ligand (CXCL)-8], leading to a donor-derived leukocyte mobilization and removal prior to kidney Tx . More reAlthough the primary results are encouraging, more research focusing on the reduction of immunogenicity of ECD organs is needed.Currently, there is no general consensus on the standardized pretreatment of ECD livers in order to improve Tx outcomes . ExperimIn several studies in DCD rat models, a reduction in IRI in liver tissue was evident after HMP when compared to SCS , 61, 62.SNMP and NMP significantly ameliorated hepatic damage in DCD livers compared to SCS in animal models , 68, 72.The currently ongoing VITTAL trial aims to improve the suitability of non-transplantable livers in the UK by monitoring their function during NMP followed by Tx of the sufficiently improved graft , 80. We Experimental and clinical studies of ECD lungs and MP are compiled in Short-term HMP could resuscitate ischemically damaged DCD lungs and ameliorate IRI. In a canine model of MP, HMP improved the ATP production by the mitochondrial electron transport chain, leading to a significant decrease in oxidative damage and production of pro-inflammatory cytokines (IL-6 and TNF-α) after reperfusion compared to SCS . Moreoveex vivo NMP and attenuated the subsequent IRI after Tx (NMP was able to modulate pro-inflammatory gene expression and reduce pulmonary dysfunction, edema, pro-inflammatory cytokines, and the number of neutrophils in animal DCD lungs , 83. Morafter Tx .The use of MP in reconditioning of ECD donor lungs for Tx is currently under investigation in clinical trials , 86, witCurrently, clinical evidence of MP in ECD heart grafts is limited . HMP impex situ MP for ECD pancreas graft for whole-organ Tx (There is a limited number of studies evaluating the safety and feasibility of organ Tx . HMP of organ Tx , 90. Mororgan Tx . Even 24organ Tx . Since tMP allows successful utilization of more vulnerable and immunogenic otherwise discarded ECD organs. It has been shown that MP not only reduces the levels of pro-inflammatory cytokines and positively influences gene expression related to hypoxia during reperfusion but also induces donor-derived leukocytes, including dendritic cell-generating non-classical monocytes, mobilization, and removal prior to Tx. Moreover, MP was able to protect against epithelial and Kupffer cell activation and to reduce recipient T cell infiltration of the donor graft. More recently, novel methods such as viral vector delivery during MP to allografts are under investigation . This biAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


We describe addiction consult services (ACS) adaptations implemented during the Novel Coronavirus Disease 2019 (COVID-19) pandemic across four different North American sites: St. Paul’s Hospital in Vancouver, British Columbia; Oregon Health & Sciences University in Portland, Oregon; Boston Medical Center in Boston, Massachusetts; and Yale New Haven Hospital in New Haven, Connecticut.ACS made system, treatment, harm reduction, and discharge planning adaptations. System changes included patient visits shifting to primarily telephone-based consultations and ACS leading regional COVID-19 emergency response efforts such as substance use treatment care coordination for people experiencing homelessness in COVID-19 isolation units and regional substance use treatment initiatives. Treatment adaptations included providing longer buprenorphine bridge prescriptions at discharge with telemedicine follow-up appointments and completing benzodiazepine tapers or benzodiazepine alternatives for people with alcohol use disorder who could safely detoxify in outpatient settings. We believe that regulatory changes to buprenorphine, and in Vancouver other medications for opioid use disorder, helped increase engagement for hospitalized patients, as many of the barriers preventing them from accessing care on an ongoing basis were reduced. COVID-19 specific harm reductions recommendations were adopted and disseminated to inpatients. Discharge planning changes included peer mentors and social workers increasing hospital in-reach and discharge outreach for high-risk patients, in some cases providing prepaid cell phones for patients without phones.We believe that ACS were essential to hospitals’ readiness to support patients that have been systematically marginilized during the pandemic. We suggest that hospitals invest in telehealth infrastructure within the hospital, and consider cellphone donations for people without cellphones, to help maintain access to care for vulnerable patients. In addition, we recommend hospital systems evaluate the impact of such interventions. As the economic strain on the healthcare system from COVID-19 threatens the very existence of ACS, overdose deaths continue rising across North America, highlighting the essential nature of these services. We believe it is imperative that health care systems continue investing in hospital-based ACS during public health crises.  The Novel Coronavirus Disease 2019 (COVID-19) pandemic forced hospital inpatient addiction consult services (ACS) to rapidly evolve as hospitals modified operations to care for people with COVID-19 and protect staff and other patients from infection. When hospital systems are overwhelmed there is a risk that people with substance use disorders (SUD) will be further deprioritized as well-known structural barriers to treatment, namely stigma, lack of clinician training in addiction, and clinician discomfort, may be exacerbated [It is critical to continue to prioritize and address substance use in the hospital setting as people with SUD may be particularly vulnerable to the psychosocial and medical effects of the COVID-19 pandemic. People with SUD may be at increased risk of relapse and acquiring infections such as HIV and HCV due to social distancing mandates causing isolation and stress, as well as disruptions to harm reduction and treatment services. Additionally, factors including homelessness and increased rates of incarceration place people with SUD at higher risk of contracting COVID-19, and higher rates of medical comorbidities also increases their risk of severe COVID-19 infection . AlarminCOVID-19 introduces challenges and some potential opportunities in caring for hospitalized people with SUD. In this perspective piece, we describe real world ACS adaptation experiences from: St. Paul’s Hospital in Vancouver, British Columbia; Oregon Health & Sciences University (OHSU) in Portland, Oregon; Boston Medical Center (BMC) in Boston, Massachusetts; and Yale New Haven Hospital (YNHH) in New Haven, Connecticut instituted during the COVID-19 pandemic. We reflect on our experiences to inform ACS responses during this current and for future public health emergencies and highlight areas in need of research.In British Columbia, the first case of COVID-19 was reported on January 28th, 2020. Increasingly strict physical distancing regulations were implemented in the following months, but due to generally low overall provincial case counts hospital personnel continued to deliver in person care at St. Paul’s throughout the pandemic. Oregon confirmed its first case of COVID-19 on February 28th, and by March 13th OHSU had implemented COVID-19 modified operations including restricting all patient visitors and encouraging staff who could to utilize telemedicine to preserve personal protective equipment (PPE) and promote physical distancing. In Massachusetts the first case of COVID-19 was reported on February 1st, and in March BMC rapidly reorganized by shifting outpatient care to predominately telemedicine and recommending that inpatient hospital staff when possible work from home with patient visits being conducted by phone to similarly conserve PPE and protect staff from infection. Connecticut confirmed its first case of COVID-19 on March 8th, with comparable rapid reorganization occurring at YNHH in March to support PPE conservation and infection control.Policy changes to medications for opioid use disorder (MOUD) access and administration were rapidly enacted in all settings to help fight the spread of COVID-19. In British Columbia increased flexibility for patients accessing MOUD were first recommended on March 17th, 2020, with interim clinical guidance on the prescribing of pharmaceutical alternatives to illicit opioids (“safe supply”) issued shortly afterwards . Such poAcross most of our respective ACS, early in the pandemic, patient visits shifted to primarily telephone-based consultation paired with ACS staff working from home to conserve PPE and promote physical distancing . At all sites temporary isolation and recovery unites were eventually established for people unable to safely isolate (e.g. person’s experiencing homelessness) to recover from COVID-19 and reduce transmission in the shelter systems, on the street, and within crowded housing. These units offered some patients with SUD a place to go following their acute care hospitalization. The establishment of the units required intensive ACS support to aid with the rapid development of SUD protocols to ensure withdrawal and SUD treatment in these units, care coordination for hospitalized patients, expansion of telemedicine consultations, coordination with opioid treatment programs to deliver methadone, and SUD care continuation post discharge.At BMC peer mentors and social workers also increased discharge outreach for high-risk patients, in some cases supported by the hospital providing prepaid cell phones for patients without phones. Cell phones provided to patients were flip phones pre-loaded with 1500 min; capabilities included calling, texting and internet access. At OHSU, lack of patient phones was a common barrier to telemedicine follow up and outreach early in the pandemic; however, through ACS advocacy with payers, pre-paid phones were made available later in the pandemic. At YNHH and St. Paul’s resources for phones were not provided to patients which limited access to both inpatient and outpatient telemedicine.Despite the increased impact of COVID-19 on people with SUD, the pandemic’s financial impact to the health service sector has threatened financial stability of ACS teams. Inpatient ACS remain limited across the continent and historically have operated on extremely restricted or unstable budgets. YNHH and BMC ACS teams were asked to redeploy physician staff for COVID-19 initiatives, necessitating back up coverage or a reduction in ACS services. At YNHH, hiring freezes halted the filling of open positions. At OHSU and BMC, ACS expansion efforts have been significantly delayed or cancelled. This is despite a perceived surge of hospital SUD related admissions in all settings following the first wave of the pandemic. In the US, billing for services was also limited without in-person consultation in the early pandemic, so many ACS saw a temporary reduction in revenue.Our experiences, across four hospitals in North America, highlight critical challenges to providing care to hospitalized people with SUD during the COVID-19 pandemic and responses to these challenges (Table We believe that ACS were essential to hospitals’ readiness to support systematically marginalized patients during the pandemic. ACS supported SUD management and care coordination in COVID-19 isolation and recovery units and led regional SUD care coordination efforts. ACS further leveraged the interdisciplinary team approach during the pandemic where social work and peer services enhanced inpatient and post discharge outreach. Qualitative studies evaluating the impact of this active outreach strategies on the hospitalization experience would be helpful to gain a nuanced understanding of the pros and cons of more active outreach. Additionally, research evaluating the impact of contact with ACS during the pandemic on substance use and other health-related outcomes could quantify its role in supporting hospitalized people with SUD during public health crises. For example, we encourage other ACS leaders and addiction clinicians to track metrics that may be impacted by ACS, such as rates of discharges against medical advice among COVID-19 positive patients with SUD who connected with ACS versus those that did not.Regulations loosened during the COVID-19 pandemic, for example tele-buprenorphine access in the US and safer supply programs in Canada, resulted in perceived increased engagement in care during hospitalization as some barriers preventing patients from accessing ongoing care were reduced. Due to The Controlled Substances Act, safe supply programs currently undergoing evaluation in Canada remain illegal in the US. While studies evaluating the impact of the regulatory changes to safe supply in Canada and MOUD in the US are in progress, we believe that, in the absence of evidence, diminishing barriers in the community to MOUD and harm reduction are critical and had positive impacts on the inpatient management experience.Despite regulatory changes and rapid adaptations, our hospital-based ACS teams were not able to compensate for the larger systemic gaps in the substance use treatment system that persisted and were undoubtedly exacerbated by COVID-19. Ongoing studies at our institutions and others will guide future evidenced based ACS adaptations, but while we wait for empirical data, we hope the experiences shared here, from four ACS impacted early in the COVID-19 pandemic, may serve as a roadmap for other areas who are more recently facing a surge of COVID-19 admissions. As the economic impact threatens the very existence of ACS, overdose deaths continue rising across North America, highlighting the essential nature of these services . In ligh


Halophilic bacteria are remarkable organisms that have evolved strategies to survive in high saline concentrations. These bacteria offer many advances for microbial-based biotechnologies and are commonly used for industrial processes such as compatible solute synthesis, biofuel production, and other microbial processes that occur in high saline environments. Using halophilic bacteria in electrochemical systems offers enhanced stability and applications in extreme environments where common electroactive microorganisms would not survive. Incorporating halophilic bacteria into microbial fuel cells has become of particular interest for renewable energy generation and self-powered biosensing since many wastewaters can contain fluctuating and high saline concentrations. In this perspective, we highlight the evolutionary mechanisms of halophilic microorganisms, review their application in microbial electrochemical sensing, and offer future perspectives and directions in using halophilic electroactive microorganisms for high saline biosensing. SpeGeobacter sulfurreducens and Shewanella oneidensis •Cl2 (PVP-Ru(bpy)2Cl2) in an MFC-based setup for self-powered, environmentally friendly monitoring of COD, resulting in higher sensitivity (by one order of magnitude) compared to the MFC setup with an air-breathing cathode. A linear relationship between the coulombs of charge and COD was established and determined to be ~10,000 mg COD L−1. Additionally, as part of their self-powered biosensor design, the researchers entrapped the bacterial cells  in alginate-capsules. As such, this biosensor design offers a means for applications as a biosensor platform in high-salt solutions.In their design, the researchers utilized a disposable cathode based on [Ru(bpy)In addition to the study presented above, several microbial fuel cells in high saline environments have been developed for the treatment of wastewater and power production from urine ,61,62,63In the last two decades, microbial whole-cell biosensors have shown great potential for use in areas of environmental monitoring and biomedical diagnostics ,50,65. WHalomonas elongate strain OUT30018 for the detection of metals in high-salt environments and sites [Acinetobacter baylyi Tox2 with the host A. baylyi strain ADP1 for the detection of cytotoxicity levels of environmental, seawater samples contaminated with heavy metals  [A. baylyi Tox2 as a rapid and sensitive biosensor element to monitor seawater cytotoxicity. Therefore, microbial whole-cell biosensors offer a means for heavy metal detection and monitoring. The biosensing constructs in bacteria are capable of producing qualitative and quantitative outputs as a response to heavy metals [Many hypersaline sites are naturally enriched with heavy metals . While tnd sites . This bi Cu, Cd) . This sty metals . Future Given these examples, a major future trend in microbial biosensors is to design and develop sensing platforms for applications in extreme, high saline conditions, allowing for practical detection devices. Since most normal bacteria cannot survive such extreme environments and have relatively narrow ranges of environmental tolerance , the selIsolation of both halophilic and electroactive bacteria allows for the characterization of these unique traits and physiologies, as well as a fundamental understanding of these metabolic processes that can help shape future studies for saline microbial electrochemical systems and sensing . Many elWith novel electroactive and environmental tolerant bacterial strains being studied and advances in next-generation sequencing technologies, genetic mechanisms for these traits are being found and applied in model microbial systems. Genetic engineering for electroactive halophilic bacteria has been accomplished by introducing genes for outer membrane cytochrome proteins for EET at the cellular surface and biosynthetic pathways for the synthesis of electron mediator molecules such as phenazines ,75,76,77Halanaerobium praevalens [Additionally, with the advancement of knowledge of microbial electrochemical systems, biofilm formation has been found to be paramount to the success of these systems . Quorum aevalens . Quorum Another interesting aspect for future studies is to examine and characterize halophilic enzymes isolated from salt-loving bacteria for the design of enzymatic-based biosensors. Enzymes isolated from extremophilic bacteria, called extremozymes, have been of interest for many industrial applications in extreme conditions . GenomicMicrobial electrochemical biosensors offer a great opportunity for environmental monitoring and degradation applications. The major limitations of these technologies can be the durability and stability of the electroactive bacteria since they are the biocatalysts in these sensing systems . By utilizing halophilic bacteria and their mechanisms, microbial electrochemical biosensors can be greatly enhanced to improve the overall sensor performance. The few pioneering studies in this field reviewed herein, along with this perspective, aim to point out the many exciting future directions that can be gained from these studies. These intriguing areas focused on heavy-metal sensing, extremely tolerant bacterial cells, and extremozymes will allow for the design and development of novel biosensors for extreme environmental monitoring.