We found that the overall expression levels of these 83 genes explain approximately 60% of Ca2+ signal entropy. The average contribution of each single gene was 17%, revealing a large degree of redundancy between genes. Using different heuristics, we estimated the dependency between the size of a gene set and its information content, revealing that on average, a set of 53 genes contains 54% of the information about Ca2+ signaling. Our results provide the first direct quantification of information content about complex cellular phenotype that exists in mRNA abundance measurements.Quantifying the dependency between mRNA abundance and downstream cellular phenotypes is a fundamental open problem in biology. Advances in multimodal single‐cell measurement technologies provide an opportunity to apply new computational frameworks to dissect the contribution of individual genes and gene combinations to a given phenotype. Using an information theory approach, we analyzed multimodal data of the expression of 83 genes in the Ca 2+ dynamics response to ATP.The dependency of cellular signaling dynamics variability on transcriptional state was estimated by applying information theory estimation to paired multimodal measurements of mRNA abundances and cellular cytosolic Ca Cellular phenotypes that emerge over long timescale, for example, cellular differentiations, have a longer timescale than the lifetime of mRNA molecules that potentially determine the emerging phenotypes. In these cases, mRNA abundances themselves change dynamically adding additional complexities. (ii) Quantification of importance of mRNA abundance requires paired measurements of mRNA and the emerging cellular phenotype in question, measurements that are technically challenging due to the destructive nature of mRNA quantification methods. (iii) The statistical measures needed to answer these questions, entropy and mutual information, are notoriously hard to infer. Below, we discuss how these challenges could be addressed to provide direct quantification of the information content in mRNA abundance.2+ signaling is a useful model system to quantify the dependency between mRNA abundance and emerging cellular phenotypes. Ca2+ signaling is a system in which the emerging phenotype is faster than changes in mRNA abundance. This timescale separation allows us to assume mRNA abundances are at a quasi‐steady state and do not change significantly during the experiment. The dynamics of the Ca2+ signaling response to ATP is a well‐studied model system for environmental sensing, featuring one of the most ubiquitous and multifunctional pathways across cell types. An important role of Ca2+ signaling is the coordination of responses to changes in extracellular environment. In the physiological context of tissues, cell lysis causes an unusual local increase in extracellular ATP, among other molecules. This type of damage sensing relies on the purinergic cell surface receptors, P1 and P2, which detect adenosine and ATP, respectively . PLCβ cleaves PIP2 into IP3 and DAG, which facilitate signaling by binding to their respective receptors, IP3R and DAGR. The IP3R is embedded in the membrane of the endoplasmic reticulum and functions as a gated Ca2+ channel that releases Ca2+ into the cytoplasm upon IP3 binding. Cytoplasmic Ca2+ concentrations are kept relatively low at 50–100 nM and spike up to 1uM during signaling with significant and rapid fluctuations producing unique dynamics in every cell  have many emergent features such as oscillations caused by coupling between positive and negative feedback loops  has been developed as a high‐throughput, single‐cell method for accurately counting large numbers of transcripts , and a stronger relationship between G and Ca2+ is equivalent to a greater distance between the joint and marginals: IGCa2+=DKLℙ∥ℚ. Using the Donsker‐Varadhan representation of the DKL, the model parameters θ are optimal when gradient ascent has maximized EℙTθ−logEℚeTθ≤DKLℙ∥ℚ, where E denotes the expected value.This estimate represents a lower bound on the mutual information. MINE is highly flexible because it makes almost no assumptions about the structure of the data. MINE searches through a large function space for the optimal transformation function to encode the data types assuming there are enough samples to constrain the model. The result is a lower‐bound estimate of the mutual information between paired modalities of almost any dimensionality and complexity.New analytical frameworks have emerged for understanding complex dependencies in multimodal measurements with intractable data distributions. Information theory provides a powerful analytical framework for understanding the relationship between system structure and output , indicating a high degree of redundancy  measures the information overlap between genes by subtracting I and I from I. A gene pair with negative SRI means that the sum of the mutual information between each gene and Ca2+ was greater than the gene pair, so the genes must contain some of the same information (redundant). A positive SRI indicates that there is more information about Ca2+ in the gene pair than in the sum of the individual genes (synergistic). An SRI of 0 describes a pair of genes that are either generally uninformative or are independent, containing unique and nonoverlapping information about Ca2+. Calculating SRI between all gene pairs reveals that most pairs are significantly redundant . Each set size was sampled four times. For random sets, 53 genes contained 54% of the phenotypic information. To understand the upper and lower bounds on information in each set, we performed two directed heuristic searches. The directed searches first picked the most (least) informative gene, and then tested every possible addition to the set to add the member that contributed the most (least) information until the sets were of maximum size. The upper bound in green shows that the information quickly plateaus as the best 12 genes contain 54% of the phenotypic information, and all further additions contribute minimal additional information. Compared to random gene sets, using only the most informative combination of genes dramatically reduces the number of genes required to recapitulate most of the phenotypic information from 53 to 12. The lower bound in purple shows the unique information per gene given the set, sorted from least to greatest. Because the lower bound always adds the least informative and most redundant genes first, the last genes contain the most unique information. PPP3CA is the first gene added to the upper bound and the last gene to be added to the lower bound, which means it must have both the most absolute information and the most unique information. Interestingly, the growth of information in the least informative set was approximately linear, meaning that there is always some unique information in every gene. The slope of the lower bound is 0.03 bits, which represents the average unique information per gene.To explore the mutual information between Caions Fig . Calcula2+, we can also estimate the “redundancy explained” of all sets of genes of a given size according to equation 2+, we ca2+ signals to compare with differential entropy  resembles the curve in Fig Finally, we calculated the mutual information between transcript principal components and Cafrom Fig  and undefrom Fig . In agreet al, 2+ signaling. We found that approximately 60% of Ca2+ signal information exists in transcript counts, which is 2.5 (±0.4) bits. Furthermore, the framework we developed provides key information about information synergy and redundancy can be used to quantify the joint information in sets of genes, and reveals how overall dependency changes with the size of the set. On average, genes were found to contain 61% redundant information with each other, although nearly all genes contained some unique information. Genes that appeared to contain little phenotypic information individually were in fact the most synergistic and became informative in pairs. The unique information present in gene sets is best visualized in Fig The complexity of biological regulation is staggering. While many details about biological networks are known, the gaps in our knowledge make some simple questions very challenging to answer. For example, to what degree does abundance of one set of molecules matter? Specifically, does the abundance of mRNAs matter for the regulation of complex cellular phenotypes such as signaling response to a ligand? Here, we provide a framework for answering such questions through the combination of paired single‐cell data and application of recent advances at the interface between machine learning and information theory  bits of mutual information is that transcripts can differentiate approximately six distinct states of Ca2+ signaling dynamics. An important feature of our analysis is that all inference was done relaying on natural heterogeneity without any experimental perturbation to gene expression circumventing compensation and nonlinear dependencies that are common pitfalls of perturbation analysis  demonstrate that with minor adaptations for bias removal, MINE can robustly estimate mutual information between two high‐dimensional vectors containing 100+ features. The generalizability of this framework provides a new tool to put weights and interpretable numbers on different “arrows” within complex biological regulatory networks. Importantly, such “arrows” do not necessarily represent direct mechanistic steps. There are numerous reactions that occur posttranscriptionally to determine Ca2+ signaling in response to activation of GPCR in a clonal population of MCF 10A cells. In previous work, we estimated that a cellular population is composed of multiple subtypes  and not to individual columns, preserving relative magnitude across genes and timepoints.Transcript counts from the 83 genes and 314 timepoints of CaH) of the normalized power spectral density (P), calculated here using the Fourier transform. Although the calculation requires a sampling frequency (fs), the result does not change above a sufficiently large value.Spectral entropy of a signal is defined as the Shannon entropy . Then, I was calculated, and equation  as if all individual sets contain unique information:This metric represents the amount of extra information assuming no redundancy between elements. Equation  first cak = set size, n = total number of genes.k out of the maximum, which is the NRI at k = 1 minus the full mutual information. The Redundancy Explained (RE) is calculated as follows:From equation , we can Evan Maltz: Conceptualization; software; formal analysis; validation; investigation; visualization; methodology; writing – original draft; writing – review and editing. Roy Wollman: Conceptualization; formal analysis; supervision; funding acquisition; validation; methodology; writing – original draft; project administration; writing – review and editing.The authors declare that they have no conflict of interest.Appendix S1Click here for additional data file.


The purpose of this study was to analyze the relationship between tibiofemoral rotation and patellar maltracking in patients with recurrent patellar dislocation.A total of 143 consecutive knees (118 patients) with clinically diagnosed recurrent patellar dislocation from January 2018 to December 2019 were retrospectively analyzed. Patellar tilt angle and bisect offset index were recorded on axial CT to assesses the severity of patellar maltracking. Tibiofemoral rotation angle is measured by comparing the angle between the posterior femoral and tibial condylar lines on three‐dimensional CT. The Pearson correlation was calculated to investigate the association between tibiofemoral rotation angle and patellar maltracking. Patients were divided into the rotation group (≥15°) and control group (<15°) based on the value of tibiofemoral rotation and a further comparison was performed. To further clarify the complicated relationship among tibial tubercle‐trochlear groove (TT‐TG), tibial tubercle‐posterior cruciate ligament distance (TT‐PCL), tibiofemoral rotation, and patellar maltracking, patients were divided into four subgroups according to the value of TT‐TG and TT‐PCL.vs 121.2, p < 0.001) and patellar tilt angle  were significantly higher in the rotation group than that in control group. For patients with a TT‐TG distance of >20 mm, the increased TT‐TG distance was mainly caused by tibiofemoral rotation angle in group C  and predominantly induced by tibial tubercle lateralization in group D . Bisect offset index and patellar tilt angle were significantly higher in the group C than group D.The mean preoperative tibiofemoral rotation angle was 12° ± 6° . Pearson correlation between patellar maltracking parameters  and various bony deformities found that the tibiofemoral rotation angle was moderately correlated with bisect offset index  and patellar tilt angle . Further results demonstrated that bisect offset index (152.1 The increased tibiofemoral rotation angle is associated with patellar maltracking in patients with recurrent patellar dislocation. Patients with increased tibiofemoral rotation angle usually have more severe patellar maltracking. The most important finding of this study is that the increased tibiofemoral rotation angle is associated with patellar maltracking in patients with RPD. Tibiofemoral rotation‐induced TT‐TG abnormality deserves more attention in clinical practice, because these patients usually have more serious patellar maltracking compared with tibial tubercle lateralization‐caused TT‐TG abnormality. Therefore, the purpose of this study was: (i) to report the value of tibiofemoral rotation angle in patients with recurrent patellar dislocation; and (ii) to analyze the relationship between tibiofemoral rotation angle and patellar maltracking. We hypothesized that the increased tibiofemoral rotation angle is a potential contributing factor for patellar maltracking in patients with recurrent patellar dislocation.A total of 169 patients were diagnosed with RPD and surgically treated in our institution from January 2018 to December 2019. The inclusion criteria were: (i) recurrent patellar dislocation; (ii) record of preoperative J‐sign; (iii) presence of preoperative imaging. The exclusion criteria were: (i) revision surgery; (ii) acute first‐time dislocation; (iii) lack of preoperative CT imaging. Finally, a total of 143 knees in 118 patients who met the criteria were enrolled in the study. This study was approved by our Institutional Ethics Board .Patellar maltracking was characterized by the bisect offset index (BOI) and patellar tilt angle on CTCT scans were performed on an Aquilion One scanner  for all patients with RPD during a maximum quadriceps contraction preoperatively. The Digital Imaging and Communications in Medicine data from the hip‐knee‐ankle CT scan were reconstructed into three‐dimensional models with Mimics Research 20.0  to measure the rotational parameters of the lower extremity according to the method described previouslyThe knee radiographs included the anteroposterior view and the lateral view with 30° of knee flexion. The Caton‐Deschamps Index (CDI) was used to measure patellar height, and patella alta was defined as CDI ≥1.2et al.CT scans were performed for all patients with RPD during a maximum quadriceps contraction preoperatively. All patients were examined with computerized axial tomography for the measurement of the TT‐TG distance according to the method described by Camp To further clarify the complicated relationship among TT‐TG, TT‐PCL, tibiofemoral rotation, and patellar tracking, patients were divided into four subgroups according to TT‐TG and TT‐PCL .t‐test or Mann–Whitney U‐test was used. The Pearson correlation  or Spearman's rank correlation coefficient  was calculated. A comparison of patellar maltracking was performed between the rotation group and control group. p values <0.05 were considered significant and all p values were two‐tailed.Statistical analyses were performed with the SPSS 20.0 software package . Pearson's chi‐square test or Fisher's exact test was used to compare categorical variables. For comparisons of continuous variables, Student's A total of 143 knees in 118 patients with RPD were included in the present study. One hundred and four patients experienced patellar dislocations during activities of daily living and 14 patients with competitive sports. No patients showed limited knee ROM preoperatively.r = 0.462, p < 0.001) and BOI    and patellar tilt angle  were significantly higher in the rotation group  than that in control group   and patellar tilt angle  between these two subgroups. For patients with a TT‐TG distance of >20 mm, Group C had normal TT‐PCL and increased tibiofemoral rotation, in contrast, Group D had increased TT‐PCL and relatively normal tibiofemoral rotation. In other words, the increased TT‐TG was mainly caused by tibiofemoral rotation angle in Group C and was predominantly induced by tibial tubercle lateralization in Group D. Further comparison showed that the BOI  and patellar tilt angle  were significantly higher in the Group C than Group D.The results of comparison among the four subgroups were showed in Table The most important finding of this study is that the increased tibiofemoral rotation angle is associated with patellar maltracking in patients with RPD. Tibiofemoral rotation‐induced TT‐TG abnormality deserves more attentions in clinical practice, because these patients usually have more serious patellar maltracking compared with tibial tubercle lateralization‐caused TT‐TG abnormality.et al. compared the degree of tibiofemoral rotation in different populations and found that tibiofemoral rotation was correlated with the severity of patellar dislocation, with the greatest value in fixed or obligatory dislocation patientset al. analyzed the influence of rotational limb alignment parameters on patellar kinematics, which demonstrated that tibiofemoral rotation produced a significant influence on patellar medial‐lateral shift in cadaveric kneesTibiofemoral rotation angle, as an important component of rotational parameters of the lower extremity, has been found to be higher in patients with RPD than that in healthy populationet al. found that TT‐TG distance had no linear correlation with tibial tubercle lateralizationet al. also declared that an increased TT‐TG may result from the true lateralization of the tibial tubercle, an increased tibiofemoral rotation, or bothet al. stated that tibial tubercle osteotomy could increase external torsion of the tibia and exacerbate symptoms in the presence of underlying rotational malalignmentet al. reported the long‐term results of tibial tubercle osteotomy in 39 patients and found that the osteoarthritic changes of the patellofemoral joint had advanced in up to 42% of patientsTT‐TG distance has long been thought to reflect the lateralization of the tibial tubercle and is commonly used as an indicator for tibial tubercle medicalization. However, recent studies found that the TT‐TG was in fact an amalgamated measure of true lateralization of the tibial tubercle and the tibiofemoral rotationet al. evaluated the role of TT‐TG distance in patellofemoral kinematics using dynamic CT and found that an association existed between TT‐TG distance and the patellar tracking parametersThe correlation between TT‐TG and patellar maltracking has been reported. Williams There were several limitations of this study. First, patellar maltracking was assessed using static CT measurements, which may underestimate or overestimate the severity of the true patellar maltracking. Second, in the present study, patients are divided into rotation group  and control group , which is without theoretical justification because little research on this has yet been done and further studies are needed to confirm what is the threshold of pathological tibiofemoral rotation angle. Third, although the present study found a potential correlation between tibiofemoral rotation and patellar maltracking, we did not further investigate the potential causes of increased tibiofemoral rotation and how to correct this rotational deformity.The increased tibiofemoral rotation angle is associated with patellar maltracking in patients with recurrent patellar dislocation. Patients with increased tibiofemoral rotation angle usually have more severe patellar maltracking. Therefore, tibiofemoral rotation angle should be measured routinely preoperatively to assess the patellar tracking more accurately.This study was funded by Capital's Funds for Health Improvement and Research (2020–1‐2075) and Beijing JST Research Funding (QN202202).The authors declare that they have no conflict of interest.Guan Wu participated in study design, data collection, and drafted the manuscript. Hui Zhang and GuanYang Song carried out the radiological measurements. Tong Zheng participated in the data collection and statistical analysis. YanWei Cao carried out the radiological measurements. Yue Li participated in the study design and data collection. Zhi Jun Zhang conceived of the study, and participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.All procedures performed in this retrospective study were in accordance with the ethical standards of the Beijing Jishuitan hospital, and this study was performed after obtaining approval from our institutional review board .All patients provided informed consent before participating in this study.All patients provided informed consent for publication.The datasets used or analyzed during the current study are available from the corresponding author on reasonable requestYes


Those bereaved by suicide are a high‐risk group of adverse health outcomes and suicidal behavior, yet little is known about the experiences and support needs of these individuals in the UK.We conducted a national cross‐sectional study using an online survey and analyzed the experiences of 7158 participants who had been bereaved or affected by suicide.Suicide had a major impact on 77% of participants, including those who had lost a friend and those exposed to suicide at a professional level. Mental and physical health problems linked to the suicide were reported in half. Adverse social outcomes and engaging in high‐risk behaviors following the suicide were common. Over a third reported suicidal ideation and 8% had attempted suicide as a direct result of the suicide loss. Most had not accessed support services, with the majority viewing provision of local suicide bereavement support as inadequate.Our results highlight the need for a multi‐disciplinary approach in postvention and the provision of proactive outreach to support those bereaved by suicide. Postvention efforts need to acknowledge the death of a friend by suicide as a significant loss. Suicide is a major public health problem with over 700,000 people estimated to die by suicide globally each year  that can occur in those bereaved by suicide which can lead to acute psychological distress  group. The survey was administered through an online survey platform “SelectSurvey” and was open between July 2017 and August 2018. The survey included 71 questions, predominantly checkbox questions but some free‐text questions to elicit more details on the experiences of being bereaved or affected by suicide. Findings related to the qualitative sections of the survey will be reported elsewhere. The participant information sheet provided broad definitions of being bereaved by suicide  or affected by suicide . Sections of the survey covered socio‐demographics; suicide in the workplace; general details of the suicide ; the impact of suicide ; and support services . All participants were advised to refrain from answering any questions found to be too sensitive, if they became distressed. Where participants had experienced multiple suicides, we asked them to provide responses based on one death by suicide. This was to enable the research team to understand the totality of the lived experience of one single suicide and avoid examination of the effects of multiple exposures to suicide. The full survey is available from the corresponding author.Inclusion criteria were those aged 18 and above; resident in the UK; and who perceived themselves as bereaved or affected by suicide. Participants were recruited via social media platforms ; authors' affiliation websites ; advertising flyers via newspapers, radio, and TV; academic conferences; and by word of mouth.n = 630) or not meeting the eligibility criteria, i.e., being aged under 18 (n = 64) or living outside of the UK (n = 193).A total of 9744 people opened the online questionnaire, 1699 (17%) of whom did not answer any of the survey questions. Of the remaining 8045, 887 (11%) were excluded due to missing data (The survey was completed online or a paper version was available for those without internet access. Participants provided electronic consent prior to beginning the survey (or written consent for those completing a paper version). All responses were anonymous. At the end of the survey, participants were directed to a website listing details of key support resources and contact details of suicide bereavement organizations to minimize any potential distress. No compensation was offered for participation in the study and respondents could discontinue the survey at any point. The study was approved by the University of Manchester Research Governance and Ethics committee .p < 0.05 interpreted as statistically significant. Stata 15.0 was used for all statistical analysis  were used for demographic and categorical data. Pearson's chi‐square tests for differences in proportions were used for pairwise comparisons. We removed cases where data were not known for that item. Statistical tests were two sided, with In total, 7158 people responded to the sections on being bereaved or affected by suicide and these represented our final sample. Responses were received across the UK: 84% from England, 7% Scotland, 5% Wales, 3% Northern Ireland, and <1% from the Isle of Man and the Channel Islands.p < 0.001). For a fifth  of respondents the suicide had occurred less than a year ago, 2051 (36%) between 1 and 5 years ago, 891 (15%) over 5 but <10 years ago, 883 (15%) between 10 and 20 years ago, and 702 (12%) over 20 years ago. Three‐quarters (75%) were in full‐time employment or self‐employed. Of the employed responders, 2318 (43%) were categorized as professionals; 788 (15%) were managers, directors and senior officials; 698 (13%) were administrative and secretarial; and 691 (13%) were in caring and leisure occupations.Table Two thirds  of respondents reported being bereaved or affected by one single suicide, while a third  had experienced more than one suicide, ranging from 2 to 70 deaths. Four hundred and seventy seven (7%) respondents experienced between four and 70 deaths by suicide. Exposure to high numbers of deaths by suicide was common among occupations such as health professionals , caring personal services , and protective services . The occupation with the highest exposure to suicide was crime scene examiner. Participants from Northern Ireland were more likely than those from other UK countries to have experienced more than one suicide (44% vs. 32%).In total, 5499 participants provided information on their relationship to a person who had died by suicide. The most common relationship was the death of a friend 19%), followed by a parent (16%), sibling (16%), or a son/daughter (14%)  of respondents reported the suicide had a major impact on their lives, particularly those who had lost a family member (95%). Only 20 (<1%) reported no impact from the death by suicide. Adverse social life events following the death by suicide were reported by over a third (39%) of respondents. The most common were family problems, relationship breakdown, and financial difficulties  as did those aged under 25 . Differences between risk behaviors and the personal relation to the deceased were observed, with those bereaved by a parent  or a spouse/partner  more likely to have partaken in high‐risk activities. Those who had been bereaved or affected by suicide for longer than a year were significantly more likely than other participants to have engaged in high‐risk behaviors .Of 5470 respondents, 1641 (30%) reported they had engaged in high‐risk behaviors following the suicide, 1055 of whom provided specific details. The most common related to alcohol and drug misuse , recklessness with finances , sexual promiscuity , lack of road safety , and aggressive behavior . Men more often reported high‐risk behaviors  reported suicidal ideation and 382 (8%) of 4818 respondents made a suicide attempt following the person's death. These proportions were similar for men and women (Table n = 2864) of participants had accessed support from one or more services following the death by suicide. This was most commonly from their GP , private counseling/bereavement support , or online support , but also included suicide bereavement services , self‐help groups , and information leaflets . Most  reported their preferred time for being approached and offered support was within a week of the death; a quarter  preferred to be approached between 1 week and 1 month after the suicide. Of 808 respondents who provided information on support provided by their employer, around half  had been offered support.Forty percent (n = 192) reported there were no local support services available to them. Overall, the provision of suicide bereavement support in the local area was viewed as inadequate by 2876 (62%) of 4621 respondents.Reasons for not accessing support were provided by 1575 participants, and included having supportive families and friends , feeling able to cope alone , and being unaware of available services . Twelve percent  should be sufficiently trained and equipped with the skills to recognize and address the complex grief, trauma, and adverse behavioral responses associated with suicide bereavement. Other agencies likely to be in contact with people recently bereaved by suicide including GPs, frontline staff, funeral directors, and social support services, also need the knowledge to signpost support effectively. This is in line with the quality standard on suicide prevention by the UK National Institute for Health and Care Excellence NICE,  which acIn addition to responding to immediate support needs, ongoing multi‐agency care and proactive outreach should be provided to ensure risk can be safely managed over time. Interventions such as the Australian “StandBy” response service, which provides immediate support and clear pathways to care for people impacted by suicide, has been shown to reduce levels of suicidality and social isolation associated with suicide bereavement . Future service development needs to consider the recommendations of those with lived experience and recognize the prolonged levels of distress that can occur from this unique grief. A multi‐agency approach combining efforts from voluntary sector organizations, mental and physical healthcare providers, social care, and community services could help develop a sustainable model of resourcing at local, regional, and national levels. Ultimately, developing tailored evidence‐based programs for all people affected or bereaved by suicide may help to reduce the short‐ and long‐term negative outcomes following a death by suicide. However, a comprehensive research agenda is required to monitor the standards and ensure services deliver equitable postvention support.The differential experiences of those who have been affected or bereaved by suicide suggests interventions need to be multi‐dimensional, but more research is needed to ascertain the most effective methods of delivering support to different groups. A number of core standards have recently been recommended by the Support After Suicide Partnership to inform services and ensure effective evidence‐based support can be delivered to those bereaved .


STMN2, UNC13A, and others. UNC13A is a major ALS/FTD risk gene, and the genetic variations that increase the risk for disease seem to do so by making the gene more susceptible to cryptic exon inclusion when TDP‐43 function is impaired. Here, we discuss the prospects and challenges of harnessing these cryptic splicing events as novel therapeutic targets and biomarkers. Deciphering this new cryptic code may be a touchstone for ALS and FTD diagnosis and treatment.Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two devastating human neurodegenerative diseases. A hallmark pathological feature of both diseases is the depletion of the RNA‐binding protein TDP‐43 from the nucleus in the brain and spinal cord of patients. A major function of TDP‐43 is to repress the inclusion of cryptic exons during RNA splicing. When it becomes depleted from the nucleus in disease, this function is lost, and recently, several key cryptic splicing targets of TDP‐43 have emerged, including  Nuclear depletion and cytoplasmic aggregation of TDP‐43 is a pathological hallmark in more than 97% of ALS cases and nearly 50% of cases with FTD (known as FTLD‐TDP).Amyotrophic lateral sclerosis (ALS) is a fatal adult‐onset neurodegenerative disease characterised by selective motor neuron degeneration, which causes progressive muscle weakness, leading to paralysis and eventually death from respiratory failure.2Among all these diverse roles, a major regulatory function of TDP‐43 has recently emerged—as a negative regulator of cryptic exons during splicing.3STMN2, which encodes stathmin 2, a protein that regulates microtubule stability in neurons. It was one of the most downregulated genes in their data sets. They quickly realised that the STMN2 gene harbours a cryptic exon (exon 2a) that is normally not included in mature STMN2 mRNA. The first intron of STMN2 contains a TDP‐43 binding site, suggesting that TDP‐43 normally functions to repress inclusion of this cryptic exon. When TDP‐43 is lost or its function is impaired, exon 2a is incorporated into mature mRNA. Unfortunately, this exon harbours a stop codon and a premature polyadenylation signal , which results in truncated STMN2 mRNA and protein. Aberrant splicing and reduced STMN2 protein levels seem to be a major feature of familial and sporadic ALS cases (except those with SOD1 or FUS mutations).STMN2 splicing. STMN2 cryptic exon inclusion also seems to play a role in FTD. Truncated STMN2 RNA is elevated in postmortem brain tissue from FTLD‐TDP‐43 cases but not in controls or cases with progressive supranuclear palsy (PSP), a different type of neurodegenerative disease with tau pathology instead of TDP‐43 pathology.STMN2 levels correlate with the levels of phosphorylated TDP‐43 protein and with an earlier age of FTD symptom onset.STMN2 cryptic exon inclusion may even herald TDP‐43 inclusion formation, being present in a subset of the most vulnerable neurons even in the absence of TDP‐43 nuclear depletion or cytoplasmic aggregation. This point is important because these TDP‐43‐dependent cryptic exon splicing events in ALS and FTD might now allow the field to be able to detect the earliest events of TDP‐43 pathology and will suggest not only disease mechanisms but also novel therapeutic targets. The discovery of STMN2 cryptic exon splicing in ALS and FTD shines a bright light on one key mRNA target. However, could there be more out there beyond STMN2? If so, it will lead to many new pathways for defining disease mechanisms and innovating sensitive biomarkers and therapeutic strategies.A breakthrough came in 2019 when two teams discovered an important human cryptic splicing target of TDP‐43.4STMN2 cryptic exon and dozens of novel cryptic exon events. Perhaps most surprisingly, we found UNC13A to be one of the most significantly alternatively spliced genes. UNC13A immediately caught our attention because it is one of the top genome‐wide association study (GWAS) hits for ALS and FTD‐ALS.Our group and another team recently identified dozens of novel cryptic exon splicing events regulated by TDP‐43 in human neurons, including at least one event that is directly connected to ALS and FTD by human genetics.UNC13A mRNA, which introduces a premature stop codon, leading to a truncated protein. There are TDP‐43 binding sites in the intron harbouring this cryptic exon, suggesting that TDP‐43 directly regulates this splicing event. Indeed, depletion of TDP‐43 from human neuronal cells or motor neurons differentiated from induced pluripotent stem cells (iPSCs) caused inclusion of this cryptic exon in UNC13A. We also analysed a series of postmortem samples from the Mayo Clinic Brain Bank and from the New York Genome Center and found inclusion of this novel UNC13A cryptic exon in FTLD‐TDP patient brains and ALS cases.UNC13A cryptic exon expression levels are increased in the frontal cortices of patients with FTLD‐TDP, which is also positively correlated with phosphorylated TDP‐43 levels.UNC13A as a consequence of TDP‐43 depletion.UNC13A gene as a consequence of TDP‐43 dysfunction.UNC13A mRNA and protein, likely because the mRNA is degraded by a process of nonsense‐mediated decay  ,UNC13A variants.UNC13A that increase cryptic exon inclusion are associated with decreased survival in patients, we strongly suspect that therapeutic strategies aimed at blocking this single splicing event could have a substantial therapeutic benefit (discussed below). ALS and FTD patients without the risk SNPs also have some cryptic splicing events , so it is likely that therapeutic reduction of UNC13A cryptic splicing will be beneficial in these patients as well.UNC13A as a biomarker and therapeutic target in ALS and FTD (discussed below). However, we think this might be just the tip of the iceberg—we have dozens of novel TDP‐43 cryptic splicing events in hand, and we strongly suspect that there will be even more to discover. Are some of these also connected by human genetics to ALS and FTD? Can we decipher an FTD/ALS “code” comprised of specific patterns of cryptic exon splicing? In a broader sense, we now have a highly sensitive way of detecting the cellular consequences of TDP‐43 loss of function, even before frank TDP‐43 nuclear depletion and cytoplasmic aggregation. This will empower much more sensitive ways of visualising and studying ALS and FTD mechanisms and pathology in human patient samples.The discovery by both teams of a novel TDP‐43‐dependent cryptic splicing event in a bona fide FTD‐ALS risk gene now opens up many exciting new directions for validating 6UNC13A cryptic exon inclusion event, which leads to decreased expression of UNC13A mRNA and protein,STMN2 gene is able to rescue the motor phenotype in mice deficient for Stmn2 (Stmn2‐/‐).How can we translate the discovery of TDP‐43‐dependent cryptic splicing targets into therapeutic strategies for ALS and FTD? Because TDP‐43 loss of function and gain of‐ function might both play a role in disease and given TDP‐43's essential role in cellular function,UNC13A and STMN2, ASOs can be designed to specifically target and block the cryptic splicing sites on the pre‐mRNA. Indeed, the remarkable therapeutic effects of the splice‐skipping ASO nusinersen in SMASTMN2 and UNC13A), then it seems feasible to design multiple ASOs targeting these regions. However, if many of the TDP‐43 splicing targets are important for disease, then other more global approaches will be needed to correct these events.Another approach could be to reduce or prevent cryptic splicing events from happening, even in the face of TDP‐43 dysfunction. Antisense oligonucleotides (ASOs) are chemically modified oligonucleotides that alter RNA targets, including triggering their degradation or modulating pre‐mRNA splicing.As genome editing approaches, including RNA editing, have become optimised for clinical application,7ECD.C9ORF72 geneIn addition to serving as therapeutic targets, these cryptic exon inclusion events can simultaneously be harnessed as biomarkers for diagnosis and prognosis and for evaluating the efficacy of experimental therapeutics.These new reagents will play an outsize role in detecting disease early, monitoring its progression and assessing whether candidate therapies are having an effect. Efforts should be launched to establish novel tools to detect cryptic proteins and peptides and to verify them in human samples. Then, with these in hand, we will need to evaluate the diagnostic efficacy of each marker for ALS and FTD and build prognostic biomarker panels. We envision a panel of high‐quality reagents to detect cryptic exon events in ALS and FTD biospecimens and then perform detailed clinical assessments. This will give the field an unprecedented lens through which to view disease—even in parts of the brain where TDP‐43 is still apparently in the nucleus but dysfunctional. It may give us a glimpse at the early events of pathogenesis and help stratify ALS and FTD patients for the design of more effective clinical trials.STMN2 cryptic exon is highly abundant,STMN2 has remained elusive. Other cryptic peptides may not be translated, owing to nonesense‐mediated mRNA decay (NMD), or may be cleared immediately and thus impossible to detect. Moreover, even if they are translated, they may be very low in quantity or may not be released outside the cell. The success of these biomarker efforts also hinges on the ability to produce high‐quality cryptic peptide‐specific antibodies. Methods are now available to sensitively detect changes in RNAs expressed in the brain by noninvasively analysing the blood,STMN2 and UNC13A) will provide more sensitivity and specificity than relying on single cryptic splicing events. If a cryptic peptide can be identified in serum or CSF, it might reflect the earliest signs of TDP‐43 dysfunction (even before cytoplasmic aggregation).There will be challenges along the road to cryptic exon biomarker development. It might be difficult to detect putative cryptic exon‐derived peptides (cryptic peptides). For example, even though the 8UNC13A, cryptic exon inclusion and human geneticsSTMN2 cryptic splicing on axon regeneration,STMN2 and UNC13A is the right place to start. Meanwhile, efforts should be underway to identify additional candidate genes that may serve as therapeutic targets and biomarkers.The emergence of aberrant splicing events as a disease mechanism in ALS and FTDAaron D. Gitler is a scientific founder of Maze Therapeutics.


Protection from radiation damage is an important adaptation for phototrophic microbes. Living in surface, shallow water, and peritidal environments, cyanobacteria are especially exposed to long‐wavelength ultraviolet (UVA) radiation. Several groups of cyanobacteria within these environments are protected from UVA damage by the production of the pigment scytonemin. Paleontological evidence of cyanobacteria in UVA‐exposed environments from the Proterozoic, and possibly as early as the Archaean, suggests a long evolutionary history of radiation protection within this group. We show that phylogenetic analyses of enzymes in the scytonemin biosynthesis pathway support this hypothesis and reveal a deep history of vertical inheritance of this pathway within extant cyanobacterial diversity. Referencing this phylogeny to cyanobacterial molecular clocks suggests that scytonemin production likely appeared during the early Proterozoic, soon after the Great Oxygenation Event. This timing is consistent with an adaptive scenario for the evolution of scytonemin production, wherein the threat of UVA‐generated reactive oxygen species becomes significantly greater once molecular oxygen is more pervasive across photosynthetic environments. The efforts to escape harm can take three basic forms: responding to UV radiation by moving away from it, blocking it before it can do damage, or repairing the damage after the fact. Most organisms have some capacity to heal radiation damage that has already occurred, as exemplified by widespread DNA repair mechanisms  around 2.3–2.4 Ga, during the “Great Oxygenation Event” (GOE). At that time, mass‐independent sulfur fractionation, possible in an atmosphere with oxygen levels below 10−5 PAL, ceased irreversibly  and most UVB (280–315 nm) radiation; a further increase to 1 PAL changes relatively little  ebo genes that transport an intermediate synthetic product into the periplasm for final assembly, and (3) additional copies of genes also present elsewhere in the genome that partake in making precursors—such as tryptophan   Of the six Scy proteins . A BLAST search for ScyC using default parameters returned 71 closely related cyanobacterial sequences and one slightly more distant sequence from Methylocaldum marinum, a methane‐oxidizing gammaproteobacterium . All other hits had an E‐value above 1 and were not included as potential homologs.To gather Scy protein amino acid sequences for the analysis, we searched the NCBI non‐redundant protein database . Especially for ScyB, the chosen cutoff also corresponds to a substantial decrease in sequence similarity. Altogether, 81 sequences were selected for ScyB and 157 for ScyA, with the higher number reflecting the presence of two copies in most taxa. Methylocaldum marinum appears in each case as the closest non‐cyanobacterial sequence to the query.Other Scy proteins did not present as such isolated clusters in the sequence space. Thus, similarity cutoffs were more subjectively established where hits became generally non‐cyanobacterial  contained detected homologs to all three Scy proteins, with incomplete sets of Scy proteins appearing in some Nostocales as well as unclassified Cyanobacteria.2.2Pleurocapsa sp. CCALA 161, Scytonema hofmanni UTEX B 1581, and Chlorogloeopsis sp. Cgs‐089 were absent from the database and omitted, while Aphanothece hegewaldii CCALA 016 was absent and replaced with the closely related strain A. hegewaldii SAG 253.80. To better resolve basal relationships between Cyanobacteria in the tree, Gloeobacter violaceus CCALA 980 (lacking Scy proteins) was added as a closer outgroup than M. marinum. Where taxa had multiple slightly divergent copies of 16S rRNA, the sequence with the highest number of copies was chosen as the representative sequence.To construct a reference species tree including the taxa on the protein trees, pre‐aligned 16S rRNA sequences from these taxa were collected from the SILVA database . We did not assume any invariant sites in the alignment. We proceeded similarly to the protein trees, building a Bayesian phylogenetic tree using PhyloBayes 4.1 . This departure from the species tree is also the feature of note on the automated protein tree‐species tree reconciliation  and further recent duplications. Unfortunately, the inference of this ancient duplication could not be tested by the automated protein tree‐species tree reconciliation, which failed to find any temporally feasible solution, likely due to the increased complexity of the ScyA tree together with the inherent phylogenetic uncertainty in the reconstruction.Calothrix and Cylindrospermum and the other members of Myxococcales and Acidobacteria. Of the sequences in the tree, these bear the least similarity to the query and were included by the conservative cutoff in order not to exclude other sequences relevant to the study (see “Sequence collection” above). Their placement on long branches indeed reflects a distant relationship with the main cluster of included sequences, but the placement of each group within cluster Y is robust to the exclusion of its sister , perhaps an unreliable placement among other long branches in this part of the tree. In fact, with a slightly different taxon sampling it moves basal to Cluster X  includes all descendants of the first scytonemin‐capable common ancestor of Clade 10, Clade 11, and Clade 9. How old is this clade and does the beginning of scytonemin production correlate with known atmospheric or planetary changes in Earth history? The congruence of Scy protein trees to published species trees of Cyanobacteria permit divergence time estimates from the latter to be mapped to the former.However, existing age estimates for events in cyanobacterial evolution vary substantially based on dataset, clock model, and fossil calibration choices (Sánchez‐Baracaldo, More recently, cyanobacterial divergence times have been estimated using a set of horizontal gene transfer events that impose novel constraints on both posterior age estimates and evolutionary model choice (Fournier et al., 4Phylogenies of scytonemin biosynthesis enzymes and molecular clock studies of Cyanobacteria indicate that scytonemin production evolved in the Paleoproterozoic, after the GOE and the establishment of the ozone layer. This suggests that cyanobacteria only adapted to become more resistant to UVA after a UV‐protective atmosphere was present. Thus, the threat of UVA is curiously inverted compared to UVB and UVC: once the surface flux of short‐wavelength UV wanes and perhaps allows for easier growth in exposed environments, UVA becomes a threat and its abatement comes under a stronger selective pressure (see Table It is entirely possible that mechanisms were in place to protect cyanobacteria from short‐wavelength UV radiation even before the GOE: for example, mycosporine‐like amino acids may have been in use for protection against UVB already, as their broad taxonomic distribution (Sinha et al., Even if Archean atmospheric oxygen levels had not yet accumulated to a sufficient extent to induce widespread damage to cells via UVA radiation, cyanobacterial mats may have experienced localized oxygen concentrations high enough to be harmful in this regard. Such a scenario could even have selected for microbial mat ecologies to include mutualistic bacteria performing aerobic respiration, which would prevent the accumulation of oxygen to harmful levels. This has previously been proposed with regard to mitigating oxidative stress induced by cellular processes (Taverne et al., In today's ocean water, about 10% of incident UVA radiation penetrates to more than 10 m of depth (Tedetti & Sempéré, There are other reasons to anticipate the appearance of scytonemin not just after the GOE, but soon after. Some geochemical evidence suggests that while oxygen levels were generally higher in the Proterozoic than before the GOE, they first hit a peak in the immediate aftermath of the GOE and then decreased again, forming an “overshoot” overlapping with the Lomagundi carbon isotope excursion and subsiding with it before 2.0 Ga (Partin et al., In any case, this perspective also suggests that scytonemin appearance could be used as a time calibration on cyanobacterial evolution, much like has been proposed previously for heterocyst development (Schirrmeister et al., It is notable that we found no evidence of any group acquiring the capacity for scytonemin production by horizontal gene transfer, otherwise a common process facilitating adaptive radiations in the microbial world. Widespread acquisition of scytonemin production might have been hindered by the relatively complex genetic machinery involved, though a transfer of the complete scytonemin operon seems feasible. Alternatively, the consignment of scytonemin to a particular clade of Cyanobacteria might reflect unique UVA pressures they face, growing in highly exposed environments with the presence of oxygen.Chroococcidiopsis that have retained scytonemin in that environment. Some relatively shallow losses after billions of years of unbroken vertical inheritance might be explained by the relatively stable oxygen concentrations over the Phanerozoic (Lyons et al., Multiple independent losses of the genetic ability to produce scytonemin are inferred within the scytonemin clade; however, any ecological pattern related to differential UVA exposure that explains the observed retentions and losses of scytonemin synthesis is not obvious. Both loss and retention seem to have occurred widely whether the cyanobacteria in question have unicellular or multicellular lifestyles and whether they live in fresh water or the sea—or under a layer of rock, as is the case for Regardless of sporadic losses, this phylogenetic evidence reflects a general vertical inheritance of scytonemin production within Cyanobacteria, with an origin during a time of major redox transition in Earth history following the GOE. This pattern reflects a persistent need for protection from UVA in an oxygenated world.The authors declare that there is no conflict of interest.Appendix S1 Supporting InformationClick here for additional data file.


A cost of reproduction may not be observable in the presence of environmental or individual heterogeneity because they affect the resources available to individuals. Individual space use is critical in determining both the resources available to individuals and the exposure to factors that mediate the value of these resources (e.g. competition and parasitism). Despite this, there has, to our knowledge, been little research to understand how between‐individual differences in resource acquisition, caused by variation in space use, interact with environmental variation occurring at the population scale to influence estimates of the cost of reproduction in natural populations. We used long‐term data from the St. Kilda Soay sheep population to understand how differences in age, relative home range quality, and average adult body mass, interacted with annual variation in population density and winter North Atlantic Oscillation index to influence over‐winter survival and reproduction in the subsequent year, for females that had invested into reproduction to varying degrees. Our results suggest that Soay sheep females experience costs both in terms of future survival and future reproduction. However, we found little evidence that estimated costs of reproduction vary depending on relative home range quality. There are several possible causes for the lack of a relationship between relative home range quality and our estimate of the costs experienced by females. These include the potential for a correlation between relative home range quality and reproductive allocation to mask a relationship between home range quality and reproductive costs, as well as the potential for the benefit of higher quality home ranges being offset by higher densities. Nevertheless, our results raise questions regarding the presence or context‐dependence of relationships between resource access and the estimated cost of reproduction. Female St. Kilda Soay sheep anad lamb. We focus on females only given that we cannot accurately quantify resource allocation by males into the rut, that greater allocation of resources during this time does not necessarily translate into greater paternity, and that male space use varies markedly before and during the rut. We used data from the long‐term individual‐based study of St. Kilda Soay sheep. Soay sheep are believed to descend from some of the first domesticated sheep in Europe , and that were known or believed to be dead by the spring of 2020 (see below). For each year of an individual's life, we classified their reproductive status as follows. First, we assigned individuals as having bred or not bred  rather than the ad hoc bandwidth recommended by Kie  for each individual, we year‐centred these measures so that each individual's annual home range quality is expressed relative to the average home range quality in that year.To prevent individuals from having multiple observations with the same coordinates which can make home range estimation problematic , prime‐aged adults (2–7 years old), or old adults (>7 years old). We estimated an individual's lifetime mean residual body mass by taking August mass measurements for each individual in each year they were weighed , extracting the residuals from a linear model with body mass as the response variable and foreleg length and age (linear and quadratic terms) as explanatory variables, and averaging these for each individual.https://crudata.uea.ac.uk/cru/data/nao and averaged values from December to March of each year to obtain a measure of weather severity in the winter following each reproductive bout.We included variables describing population density and winter weather in our models to understand how the inclusion of between‐year environmental variation affected our estimates of the costs of reproduction and whether there was any evidence that costs were more pronounced when conditions were poor. To capture variation in population density, we used the number of adults (> 1 year old) in the Village Bay area recorded across the 10 August censuses in the year of the reproductive bout. Owing to sheep being marked with unique ear tags, double counting of individuals is very unlikely. In addition, because we use 10 censuses to derive summer density estimates, it is also unlikely that individuals would have been missed entirely. To characterize variation in the severity of winter weather conditions, we used the North Atlantic Oscillation index (NAO), which captures the effect of winter weather on ungulate survival and reproduction . However, it is important to note that in analyses considering individuals that weaned a singleton versus those that weaned twin(s), we excluded yearling females as they never bear twins.To understand how estimates of the cost of reproduction are affected by accounting for variation in individual resource access and environmental conditions, we first used three models to understand how accounting for individual variation in long‐term body mass or relative home range quality, and between‐year variability in density and winter weather affected our estimates of reproductive costs. The first model contained only the measure of reproductive status and female age (three‐level factor)  and annual environmental conditions (population density and NAO) to understand how reproductive costs were mediated by variation in resource access and environmental conditions (models 4 – 8 in Tables c) to compare the relative support for models in each model set. All data manipulation and analysis were carried out in R version 4.0.3 using sf (version 1.0–2), adehabitatHR (version 0.4.18), dplyr (version 1.0.4), and lme4 (version 1.1–25) packages.In each case, we used generalized linear mixed models with a binomial distribution and logit link function and included the year of breeding and individual identity as random effects. All covariates were scaled to a mean of zero and standard deviation of 1 prior to analysis to enable direct comparisons, and we used Akaike's Information Criterion corrected for small sample size , suggesting a small cost of reproduction in terms of reduced overwinter survival. This difference increased slightly to 4% when we considered females that had invested into lactation versus those that had not . Weaning twins was not more costly than weaning a singleton lamb . Including variables describing individual heterogeneity  and variation in annual environmental conditions improved model fit , and that the benefits of being relatively heavy were particularly pronounced for yearlings (regardless of reproductive status) and old breeding females  with those that had not bred, we found that non‐reproductive females were actually less likely to reproduce in the subsequent year, regardless of whether we included information on individual resource acquisition or annual environmental conditions (Models 1–3 in Tables c units of this model). Reproductive probability was reduced at high density for both females that had twinned and those that reared singletons, but this reduction was stronger for those providing care to two lambs : AgreeTables S1–S7Click here for additional data file.


In recent years, structural and functional alterations in the cerebellum have been reported in autism spectrum disorder (ASD). Intriguingly, recent studies demonstrated that the social behavioral profile of individuals with cerebellar pathologies is characterized by a theory of mind (ToM) impairment, one of the main behavioral hallmarks of ASD. The aim of the present study was to compare ToM abilities and underlying cerebello‐cortical structural patterns between ASD individuals and individuals with cerebellar atrophy to further specify the cerebellar role in mentalizing alterations in ASD. Twenty‐one adults with ASD without language and intellectual impairments (based on DSM‐5), 36 individuals affected by degenerative cerebellar damage (CB), and 67 healthy participants were enrolled in the study. ToM abilities were assessed using the reading the mind in the eyes test and the faux pas test. One‐way ANCOVA was conducted to compare the performances between the two cohorts. Three‐dimensional T1‐weighted magnetic resonance scans were collected, and a voxel‐based morphometry analysis was performed to characterize the brain structural alterations in the two cohorts. ASD and CB participants had comparable ToM performance with similar difficulties in both the tests. CB and ASD participants showed an overlapping pattern of gray matter (GM) reduction in a specific cerebellar portion (Crus‐II). Our study provides the first direct comparison of ToM abilities between ASD and CB individuals, boosting the idea that specific cerebellar structural alterations impact the mentalizing process. The present findings open a new perspective for considering the cerebellum as a potential target for treatment implementation. The present work will critically advance current knowledge about the cerebellar role in ToM alterations of ASD, in particular, elucidating the presence of common cerebellar structural abnormalities in ASD and cerebellar individuals that may underlie specific mentalizing alterations. These findings may pave the way for alternative therapeutic indications, such as cerebellar neuromodulation, with a strong clinical impact.The present work will critically advance current knowledge about the cerebellar role in theory of mind alterations of autism spectrum disorder (ASD), in particular, elucidating the presence of common cerebellar structural abnormalities in ASD and cerebellar individuals that may underlie specific mentalizing alterations. These findings may pave the way for alternative therapeutic indications, such as cerebellar neuromodulation, with a strong clinical impact. Autism spectrum disorder (ASD) is a clinically complex and heterogeneous condition whose pathogenesis remains unclear    with the picture of the eye‐region expression. The test consists of 36 photos of actors' eyes, and for each, the participants have to choose from four words the one that best describes what the person in the photograph is thinking or feeling  and affective (question 6) components of ToM. The detection of faux pas requires both an understanding of false or mistaken beliefs and an appreciation of the emotional impact of a statement on the listener. The participants read 10 stories that contained a social faux pas and 10 control stories that contained a minor conflict but in which no faux pas was committed. A faux pas occurs when a speaker says something without considering that the listener might not want to hear it or might be hurt by what has been said. After each story, the participant was asked whether anyone said anything that they should not have said. When a faux pas was identified, further clarifying questions were presented to evaluate the understanding of the mental states and emotions of the agents involved in the stories.Each faux pas story question correctly answered was scored as one, resulting in a maximum score of six for each correctly answered faux pas story. The no faux‐pas stories were given a score of two if they were correctly identified as not containing a faux pas. Two additional control questions were asked for all 20 stories to confirm that the participant had a factual understanding of the stories  for mood  or age , while there was a significant difference in age between the CB and ASD groups . For this reason, one‐way ANCOVA with age as a covariate was applied to compare each variable in a between‐group design. When significant differences were observed (p < 0.05), post hoc comparisons were performed using Fisher's least significant difference. Partial eta‐squared values were calculated as a measure of effect size, and the results were interpreted using Cohen's  was compared with the HP group for the variables education and age. No significant differences were found in education .2, slice thickness = 1 mm) scans were acquired to perform voxel based morphometry analysis. To characterize the brain anatomy and determine the presence of macroscopic structural abnormalities, the TSE scans of patients were visually inspected by an expert neuroradiologist. For the HP group, conventional MRI images were inspected to exclude participants with any pathological conditions based on the inclusion criteria.For each group, the MRI protocol was acquired at 3 Tesla  and included dual‐echo turbo spin echo (TSE)  and fast‐Fluid Attenuated Inversion Recovery (FLAIR)  as conventional MRI scans. 3D‐modified driven equilibrium Fourier transform (MDEFT) . One CB (CB32) and two ASD individuals were excluded for the presence of artifacts due to movement. Thus, a total of 32 CB and 19 ASD individuals underwent the MRI protocol. Additionally, for the MRI analysis, two separate groups of 39 and 31 matched HPs (MRI‐HP 1 and 2) were recruited as controls for the CB and ASD group, respectively. The demographic characteristics of the MRI groups are reported in Table http://www.fil.ion.ucl.ac.uk/spm/]). The Spatially Unbiased Infratentorial Template (SUIT) toolbox   cluster‐level correction (clusters formed at p < 0.001 at uncorrected level).Voxel‐based morphometry (VBM) was performed on cerebellar modulated GM maps entered into a voxel‐wise two‐sample t‐test model to separately compare the cerebellar GM volumes between the CB group and MRI‐HP group 1  and the ASD group and MRI‐HP group 2 . The analysis was restricted only to the voxels of the cerebellum by using an explicit exclusion mask. The results were considered significant at http://www.fil.ion.ucl.ac.uk/spm/), Wellcome Trust Centre for Neuroimaging, Institute of Neurology, University College London, UK) .To investigate the presence of accompanying cortical atrophy, whole‐brain VBM was also performed in both groups. The 3D‐T1 volumes were segmented into GM maps and registered to MNI space utilizing the “New Segment” and “DARTEL” routines in SPM8 (www.fmrib.ox.ac.uk/fsl/), applied to the modulated GM maps. Spearman's correlations were computed for the relationship between these volumes, expressed in mm3, and impaired ToM scores in both groups.Based on MRI analysis results, the mean cerebellar and cortical GM values only from clusters that were significantly altered in CB and ASD compared to HP were extracted and correlated with impaired ToM scores. The lobular volumes were extracted using the “fslstats” command line from the FMRIB software library ]. Post hoc comparisons showed that both the CB and ASD groups had significantly lower scores than the control group , while no significant difference was found between the CB and ASD groups (p = 0.68)  Figure .p = 0.000, ηp2 = 0.14)] and cognitive component scores ]. No group effect was found in the no‐faux pas total  or affective component scores . Post hoc comparisons showed that both the CB and ASD groups had lower scores than the control group in the faux pas total  and cognitive component scores , while no significant differences were found between the CB and ASD groups in either total (p = 0.88) or cognitive component scores (p = 0.99)  Figure . Mean anFor both experimental groups, no significant direct correlation was obtained between test scores and VAS mood scores, and no significant inverse correlation was obtained between test scores and total scores on the motor scale Table . These rp < 0.05 FWE voxel‐level correction . The results showed a pattern of structural alterations in the CB individuals compared to the control group at the level of the cerebellar hemispheres. In particular, the CB individuals showed a pattern of reduced GM volumes both at the level of the anterior and posterior cerebellar portions with significant peaks at the level of the right and left lobules I–IV and right lobule VI, with an extension to the right Crus I–II. The second cluster of reductions in GM was found at the level of the left Crus I and Crus II . For detailed statistics, see Table Based on the VBM results, Spearman's correlations were performed between impaired task performances (RME and FP) and reduced cerebellar GM volumes in each group. In the CB group, correlations between impaired ToM scores and reduced cerebral GM volumes were also tested. Finally, the relationship between ToM performance and GM volume in the overlapping right Crus II region was examined including the participants of both groups in the correlational analysis. All the analyses performed did not show any significant relationship , planning, abstract reasoning, and processing of emotional stimuli (BA11) (Coricelli, In order to understand why the cerebellum is a critical node for mentalizing processes, the sequence detection theory (Leggio & Molinari, Precisely, the idea is that the cerebellum produces internal models of mental processes that occur during social interactions and for which the prediction of sequential events is crucial (Leggio & Molinari, The RME and faux pas tasks used in this study measure different aspects of mentalizing but they both require advanced ToM capacities. Indeed, the RME is considered an advanced ToM task (Baron‐Cohen et al., In line with all these observations, we speculate that the structural alterations in cerebellar Crus II, known to be strictly related to more advanced ToM features (Van Overwalle & Mariën, To conclude, some potential limitations need to be discussed. The first limitation of this research concerns the heterogeneity in the CB sample. Moreover, it must be considered that the choice of grouping cerebellar individuals with different aetiologies is related to the rarity of this neurodegenerative condition, which clearly affects the inclusion rate and makes it difficult to find large numbers of cerebellar individuals with the same diagnosis. Another issue that merits discussion is that we did not find a direct link between the observed patterns of cerebral and cerebellar atrophy and altered performances in the ToM tasks. While the lack of correlations is a challenge to our hypothesis, the heterogeneity of the CB group and, together, the relatively small sample size of ASD, are likely to prevent from the detection of correlational results. Indeed, along with the CB group heterogeneity, it has to be considered that a high intersubject variability also characterizes the ASD population (Simmons et al., In terms of future developments and perspectives, these findings contribute to further elucidating the role of cerebello‐cerebral circuits in social deficits, opening a new perspective to consider the cerebellum as a potential target for treatment implementation.Recent studies have shown the rehabilitation potential of tDCS in stroke patients (Ayache et al. 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.The experimental procedures were approved by the ethics committee of the IRCCS Santa Lucia Foundation. In accordance with the Declaration of Helsinki, written informed consent was obtained from each participant.Silvia Clausi, Giusy Olivito, and Maria Leggio contributed to conception and design of the study. Silvia Clausi, Giusy Olivito, Libera Siciliano, Michela Lupo, and Roberto Baiocco contributed to conducting the research and investigation process, performing the experiments or data/evidence collection. Giusy Olivito acquired the MRI protocol, processed, and analyzed the MRI data. Fiorenzo Laghi and Maria Leggio contributed to the management, coordination responsibility, and supervision of the research activity planning and execution. Silvia Clausi and Giusy Olivito wrote the first draft of the manuscript. Maria Leggio supervised development of the work. All co‐authors contributed to final editing and critical revision of the original manuscript.Table S1: Correlations between impaired ToM task performances and regions of reduced GM volumes.Click here for additional data file.


Genetic evaluations in sheep have proven to be an effective way of increasing farm profitability. Much research has previously been conducted on producing within‐country genetic evaluations; however, to date, no across‐country sheep genetic evaluations have been produced between Ireland and the UK. The objective of the present study was to examine the feasibility of an across‐country genetic evaluation of live body weight and carcass composition traits for Texel sheep raised in Ireland and the UK. The benefit of genetic selection based on across‐country genetic evaluations, in comparison with within‐country genetic evaluations, was also quantified. Animal traits included early‐life and postweaning live body weights, and muscle and fat depth ultrasound measurements. Irish and UK data were combined, common animals with progeny with records in both countries were identified and a series of bivariate analyses were performed separately for each trait to produce across‐country genetic evaluations. Fixed effects included contemporary group, age at first lambing of the dam, parity of the dam (Ireland), dam age at lamb's birth (UK), a gender by age of the lamb interaction, a birth type by rearing type of the lamb interaction and country of birth of the lamb. Random effects included the animal additive genetic, dam maternal, litter common environment and residual effect. The model for postweaning weight, muscle depth and fat depth included only the animal additive genetic and litter common environmental random effects. Genetic correlations between the two countries ranged from 0.82 to 0.88 for the various traits. Across‐country breeding values were estimated for all animals and response to selection was predicted using the top 10 and top 20 sires in both within‐ and across‐country analyses for the two countries. Overall, results showed that rates of genetic gain could potentially increase from between 2.59% and 19.63% from selection based on across‐country genetic evaluations compared to within‐country evaluations alone. Across‐country evaluations are feasible and would be of significant benefit to both the Irish and UK sheep industries. In order to realize these potential gains though, there would need to be a switch in emphasis by sheep breeders towards using objective traits as their primary selection criteria. Growth and carcass composition traits are of high economic importance worldwide  and the UK (Agriculture and Horticulture Development Board—AHDB) resulting in a high volume of data being available particularly for live body weight and carcass composition traits measured on certain common breeds. Therefore, it could be of significant advantage to pool these data together into an across‐country evaluation system to allow breeders to more accurately compare and select animals across country.The objective of the present study was to assess the feasibility of combining lamb live body weight and carcass phenotypic records and pedigree data from Ireland and the UK in order to develop an across‐country genetic evaluation system for pedigree Texel sheep. An additional objective was to quantify the potential benefit of selection based on across‐country genetic evaluations in comparison with within‐country genetic evaluations.22.1http://www.sheep.ie) and AHDB, the UK national Sheepbreeder database (https://ahdb.org.uk/beef‐lamb). The study focussed on purebred Texel lambs. Three live body weight traits, namely preweaning weight , weaning weight  and postweaning weight  and two carcass composition traits, namely muscle depth  and fat depth  from Ireland were examined. Similar traits were examined in the UK data including early‐life body weight , postweaning weight , muscle depth  and fat depth . In both cases, muscle and fat depth were assessed with ultrasound measurements.All data used in the present study were obtained from Sheep Ireland, the Irish national database  and dam parity (Ireland) were subsequently categorized as 1, 2, 3, 4 or ≥5. Age at first lambing was defined as the age of the ewe at her first lambing. In Ireland this was whether the dam first lambed down as a ewe lamb (8–18 months of age) or a hogget (18–28 months of age) whereas in the UK data, this ranged from 1 to 3 years. Birth type was defined as the number of lambs born per lambing event; only lambs born with a birth type between 1 and 4 were retained for analysis. Rearing type was defined as the number of lambs reared per litter; only lambs with a rearing type of between 1 and 3 were retained for further analysis. Records from lambs that were artificially reared, reared by a non‐genetic dam or born as a result of embryo transfer were discarded. For all traits, lambs were allocated to a contemporary group of flock‐by‐week of weighing. Contemporary groups were only retained for further analysis if they contained five or more records. Following all data edits described 33,371 early‐life/preweaning weight records, 33,868 early‐life/weaning weight records, 25,293 postweaning weight records, 21,429 muscle depth records and 21,309 fat depth records remained across both countries ; Dam age, age of the lamb's dam at lambing (UK data only); Sex*Age, the interaction between the sex of the lamb and age of the lamb at recording; Birth type*Rearing type, the interaction between the birth type and rearing type of the lamb; Country, country of birth of the lamb; Animal, random additive genetic effect of the animal (lamb) including all pedigree available; Dam, random maternal effect of the lamb's dam; Litter, random common environmental effect amongst lambs in the same litter; and e, random residual effect.Where The model was first applied to Irish and UK data, separately, after removing the country effect, to derive within‐country estimates of variance components and animal breeding values of individual animals in each country. Subsequently, bivariate analyses were conducted on joint across‐country data to estimate the genetic correlations between countries and breeding values of all animals in the combined dataset. In the latter analyses, all variance component estimates across country were fixed to the previously calculated within‐country estimates to allow for a direct comparison of estimated breeding values from within‐country and across‐country evaluations. Residual covariance estimates due to dam and litter effects as well as between countries were fixed to zero as no animal had phenotypic records in both countries. Estimated breeding values (EBVs) and accuracies of EBVs were derived for all animals and were expressed on the scale of each country.All these analyses were conducted with the ASReml software  and bivariate (across‐country) analyses are also summarized in Table Genetic correlation estimates between the two countries were stronger than 0.80 in all cases except when weaning weight from Ireland was combined with early‐life weight from the UK. In this case, the weak genetic correlation (0.38) suggests that the across‐country evaluation for these traits would not be beneficial. Therefore, no further analyses were conducted for this trait combination. However, the other trait measured in Ireland at an early growth phase, preweaning weight, was highly correlated with UK early‐life weight. In the latter case, as well as for all carcass traits the strong genetic correlation estimated between the two countries warrants possible benefits from a joint genetic evaluation. Strong genetic correlations between traits also indicate that limited re‐ranking of sires would be expected between the two countries.3.1In order to further examine and quantify the benefit of across‐country genetic evaluation, predicted response to sire selection within and across country was estimated for each trait separately Tables , 3, 4, 5Difference between response to selection based on across‐ versus within‐country evaluation would be expected to be mainly due to difference in EBV accuracy and selection intensity. The average sire EBV accuracy ranged between 0.53 and 0.70 in both within‐ and across‐country genetic analyses. Although there was no overall increase in accuracy for across‐country genetic analyses there was an increase seen for sires with a substantial number of progeny in both countries. Expectedly, selection intensity was always higher when sires were selected based on the wider pool of the across‐country evaluations in comparison with selecting from the within‐country evaluations.After the minimum accuracy threshold of 0.65 was imposed there were between 119 to 369 sires remaining in the within‐country analysis and 182–473 sires remaining for the across‐country analysis, depending on the trait. Across‐country evaluations were of benefit to both Ireland and the UK for all traits studied with a potential increase in predicted genetic gain between 2.59%  genetic evaluations have already proven their worth in both the beef and dairy cattle industries with the development of Interbeef and Interbull, respectively. However, to date no international genetic evaluations have been conducted for sheep in the northern hemisphere. Interbull provides genetic evaluations for a multitude of traits for dairy cattle including production, fertility, health and conformation traits Mark, , 2005. I4.1Connectedness amongst sheep populations in different countries is a key component in the feasibility of conducting international genetic evaluations. This is because bias in EBV estimation is reduced as connections between flocks and separate management units are increased (Hanocq et al., 4.2In general heritability estimates derived in the present study were higher in Ireland than in the UK. There were substantial differences between heritability estimates for postweaning weight and muscle depth between countries with Irish heritability estimates 10–12% higher than UK for these traits, respectively. These parameters are generally consistent, though, with the scientific literature (Safari & Fogarty, The benefit of conducting international evaluations is dependent on the magnitude of the genetic correlation between countries for a trait (Mulder et al., 4.3When genetic correlations between countries are sufficiently strong, combined selection of animals across country should always be on a par if not superior to within‐country selection (Smith & Banos, Pooling data from different countries and combining in an international dataset gives rise to a greater number of selection candidates, thus increasing selection intensity. As accuracy levels remained relatively stable in within‐ and across‐country genetic evaluations, selection intensity was deemed to be one of the most influential factors in increasing the rate of genetic gain achieved per year.Predicted response to selection derived in the present study demonstrates the benefits for individual traits separately. At a practical level, sires are selected based on overall selection indices rather than individual trait EBV estimates. As the gain differs according to the different traits, when all growth and carcass trait EBVs are combined it is unlikely that the expected increase in genetic gain predicted on a single trait basis would be realized, whether this is operated within‐ or across‐country selection. This is an area that should be explored in the future work.5Strong links and genetic correlations between Ireland and the UK were found that would facilitate a joint genetic evaluation for Texel sheep across the two countries. Through the combination of data and pedigree records across country the present study has demonstrated that a considerable improvement can be achieved in the rate of genetic gain through the informed selection of breeding stock regardless of the country of origin. However, the actual rate of genetic gain achieved will be highly dependent on breeders future selection decisions ‐ whether this is based on the traditional method of selection using phenotype alone or if the incorporation of genetic evaluations can become the norm.None declared.


The immune system can influence cancer development by both impeding and/or facilitating tumour growth and spread. A better understanding of this complex relationship is fundamental to optimise current and future cancer therapeutic strategies. Although typically regarded as a localised and immunosuppressive anti‐cancer treatment modality, radiation therapy has been associated with generating profound systemic effects beyond the intended target volume. These systemic effects are immune‐driven suggesting radiation therapy can enhance anti‐tumour immunosurveillance in some instances. In this review, we summarise how radiation therapy can positively and negatively affect local and systemic anti‐tumour immune responses, how co‐administration of immunotherapy with radiation therapy may help promote anti‐tumour immunity, and how the use of immune biomarkers may help steer radiation therapy‐immunotherapy personalisation to optimise clinical outcomes. Radiation therapy (RT) is central to the management of an estimated 50% of cancer patients, either on its own or in combination with surgery and/or systemic treatment, in both curative and non‐curative settings.+ helper T cells, CD8+ cytotoxic T cells, natural killer (NK) cells and dendritic cells are associated with more favourable treatment outcomes and improved prognosis due to their ability to attack and destroy cancer cells.Tumour‐driven perturbations of the immune system can affect both its innate and adaptive components, with the TME being the site and orchestrator of subsequent immunological responses.Given the increasing appreciation of the importance of the immune landscape in cancer prognosis, there is a growing need for therapeutic modalities that can rejuvenate the immune response to attack and destroy the tumour.+ helper T cells (TH) and CD8+ cytotoxic T cells (CTL), which mediate and influence tumour‐specific destruction.1apresentation of the tumour antigens by cell surface major histocompatibility complex (MHC) molecules,bupregulation of cell surface T cell costimulatory molecules, andcmigration of mature APCs to the draining lymph node where they can interact with circulating T cells; anduptake of tumour antigens by antigen‐presenting cells (APCs) at the tumour site and maturation of APCs in response to the release of damage‐associated molecular patterns (DAMPs)2APC selection of T cells with relevant tumour‐specific T cell antigen receptors (TCR) and subsequent activation of these T cells through costimulatory molecules resulting in proliferation and differentiation into effector T cells.Central to the complex immune response against cancer is the generation of tumour‐specific effector T cells, such as CD4Effector T cells then migrate to the tumour site to mediate anti‐tumour immune responses Fig.  where a —‘don't eat me signal’—and its loss on aged or damaged cells leads to homeostatic phagocytosis.Radiation therapy induces cancer cell death through a number of mechanisms,+ T cell activation.Tumour ICD, including by RT, also results in the release of high‐mobility group box protein 1 (HMGB1) and heat shock proteins (HSP) as well as accumulation of fragmented DNA in the cytoplasm of tumour cells.DNA damage responses to RT also results in interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) production by NK cellsCollectively, these mechanisms result in enhanced tumour cell recognition by the immune system and generation of effector T cells such as CTL and TH1 cells that can help mediate tumour destruction  towards a TH type 2 phenotype (TH2)—typically associated with a pro‐angiogenic, pro‐inflammatory and immunosuppressive microenvironment favourable for tumour growth.Collectively, RT can induce conditions favouring the recruitment of TH2 cells, MDSCs, M2 macrophages and Tregs as well as upregulation of immune checkpoint inhibitors (ICIs) in the TME that can reduce the cytotoxic immune responses against the tumourIn addition to RT‐mediated immune modulatory effects having either a positive or negative influence on anti‐tumour immunity, some of the induced responses reflecting the inflammatory state of the TMEAn equilibrium (or lack thereof) of these overlapping immune‐mediated pathways govern the anti‐ and pro‐tumorigenesis responses. Strategies aiming to tip this precarious balance present therapeutic challenges and opportunities for enhancing anti‐tumour immune responses.The earliest indication of the existence of important synergy between RT and the immune system is founded on Mole's 1953 report highlighting that local RT could induce systemic effects resulting in tumour reduction at distant sites,Over the last decade and a half, ICIs have disrupted the systemic treatment paradigm for several malignancies. While they can provide durable cancer control, overall, they appear to benefit only a small proportion of patients. Of the 43.63% of US cancer patients identified to be eligible for ICIs in 2018, only 12.46% were estimated to respond to the treatment.Understanding the key mechanisms that govern resistances to ICIs as well as systemic effects of RT will involve a closer study of the immune contexture in an attempt to improve clinical outcomes. At the preclinical stage, this requires RT studies to be conducted in immune‐competent subjects to ensure the intricate and dynamic nature of the interactions between cancer and immune cells is preserved. Secondly, clinically relevant surrogate markers of immune modulation need to be systematically assessed. Together, this will ensure a systematic and data‐driven approach to unravelling the immune‐mediated outcomes of RT. Here, we highlight some immunotherapy approaches that may work well with RT towards generating clinically relevant anti‐tumour immunity Fig. .Immune checkpoint inhibitors act by preventing the activation of the pathways, which blocks T cells from destroying cancer cells.The toxicity profile of the combination of RT and ICI is also an active area of research. Although a systematic review demonstrated comparable grade 3–4 toxicities compared with ICI alone in central nervous system melanoma metastases, NSCLC and prostate cancer,Cell‐lysing chimeric antigen receptors (CAR)‐T cells are cancer patient‐derived T lymphocytes that are engineered to target the corresponding ligand‐expressing cancer cells.Combining RT with CAR T cells presents exciting possibilities to expand the current role of RT from a bridging or salvage therapy following relapse to more sophisticated applications such as eliminating immune suppressive cells like MDSCs, Tregs and cancer‐associated fibroblasts (CAFs) that are often enriched in the TME. These approaches warrant a better understanding of the RT parameters needed to achieve these precise biological effects.The roots of this therapy can be traced back to William Coley in 1891, when he demonstrated tumour regression in patients with sarcoma inoculated with live and inactivated bacteria.There is clear interdependence between inflammation and angiogenesis: tumour‐derived immune cells release pro‐angiogenic factors that promote the growth of new blood vessels, and the new blood cells facilitate the migration of immune cells from circulation into the tumour.Anti‐angiogenic strategies have been evaluated based on the premise that tumours secure vascular supply through the expression of immune system derived pro‐angiogenic growth factors, such as those of the VEGF family. VEGF ligands are expressed in most solid cancers,Combining RT with angio‐regulators is an attractive strategy to modulate the TME by affecting angiogenesis, but biomarker‐driven systematic studies of the effect of dose and scheduling are needed before this can be translated in the clinic.Toll‐like receptors (TLR) are pattern recognition receptors usually expressed on macrophages and dendritic cells. They play a vital role in innate and adaptive immune responses. Immunostimulatory properties of TLR agonists can enhance anti‐tumour ICD. HMGB1 modulation, for example, has been proposed as a potential strategy to improve RT outcomes, and various HMGB1‐targeted therapeutics are currently in development.Macrophages are myeloid cells, and their recruitment at sites of injury is associated with important tissue repair functions.+ T cells, increases the expression of MHC‐I on tumour cellsRetinoic acid is a steroid hormone important in regulating mucosal immunity in the gut and promoting myeloid differentiation.Nanoparticles are synthetic material with overall dimensions in the nanoscale (<100 nm). In modern medicine, they are utilised in various clinical applications ranging from imaging to drug or gene delivery into the tumours.Given the immune system is intertwined with cancer developmentThere are an increasing number of reports on using blood immune biomarkers to predict RT efficacy in a variety of cancers, including in the treatment of glioblastoma,Perhaps the most challenging aspect of using immune features as dynamic biomarkers is their known variations in concentration in human blood and the presence of various factors that may influence their signature. Ethnicity,As the field of immune‐based biomarkers matures, there is potential for identifying a few key parameters that could be useful for RT personalisation.For well over a century now, RT has established itself as a highly effective cancer treatment modality in both curative and non‐curative clinical settings. Although it was recognised quite early on that some interplay exists between the immune system and anti‐tumour RT responses,Radiation therapy can have both favourable and unfavourable effects on anti‐tumour immunity, and the interactions between RT and the immune system are complex, often overlapping, and on many occasions paradoxical.Exploitation of the potential synergism between RT and other immune‐modulating modalities in reversing the immunosuppressive effects of certain cancers on the TME and beyond warrants understanding of how best to combine these treatments, how to sequence them and how to select the optimal RT dose‐fractionation schedules and target volumes. Biologically relevant biomarkers diagnostic of tumour‐induced immune polarisation and predictive of treatment response are needed to guide the systematic evaluation of the plethora of possible combinatorial strategies leading to improved personalisation of treatments.The survival benefits attributed to local treatments in the recent clinical studies in the setting of metastatic disease are very promising.This work is partly supported by the Radiation Oncology Private Practice Fund, Canberra Health Services; and Research and Innovation Fund, Centre for Health and Medical Research, ACT Health, ACT Government.All data analysed for this study are present in the submitted files and/or stored in an institutional public repository server.