Here, we highlight current approaches and challenges as well as novel insights in the process of GPCR sensor development, and discuss possible future directions of the field.Neurochemicals have a large impact on brain states and animal behavior but are notoriously hard to detect accurately in the living brain. Recently developed genetically encoded sensors obtained from engineering a circularly permuted green fluorescent protein into G-protein coupled receptors (GPCR) provided a vital boost to neuroscience, by innovating the way we monitor neural communication. These new probes are becoming widely successful due to their flexible combination with state of the art optogenetic tools and  A variety of neurochemicals, including transmitters, modulators, peptides and hormones, are constantly released within the brain during neuronal and glial communication, and play a fundamental role in coordinating physiological brain functions. Tools for monitoring the dynamic changes of individual neurochemicals are therefore highly desirable. For decades fast-scan cyclic voltammetry and microdialysis have been the gold-standard techniques used for measuring the extracellular concentrations of neurochemicals during animal behavior . HoweverOptical microscopy techniques in combination with genetically encoded sensors are emerging as a powerful solution that can elegantly answer this need. In particular the recently introduced genetically encoded sensors based on a single circularly permuted green fluorescent protein (cpGFP) engineered into G-protein coupled receptors (GPCR), which for simplicity here we refer to as “GPCR sensors,” can enable the detection of neuromodulatory molecules at high resolution in awake behaving animals . One of in vivo sensing of dopamine  as well as small portions of TM5 and TM6 of the D1 dopamine receptor (DRD1) , while iIn all cases, the final goal is to maximize the coupling between receptor conformational change and the fluorescence response of cpGFP. This process, builds on previous knowledge established for genetically encoded sensors of similar design  and mostin vivo application, they may represent an ideal starting point for further optimization through targeted mutagenesis at a reduced number of sites, and thus could provide a useful shortcut in a process that is otherwise very costly and time-consuming. Considering that the ICL3 is an extremely variable region among different class-A GPCRs  into other GPCR subtypes made it possible to rapidly generate preliminary sensors for norepinephrine, serotonin, melatonin, and opioid neuropeptides . Althoug-A GPCRs  both in -A GPCRs , and tha-A GPCRs , the preThe concept of versatile engineering is somewhat reflected in the field of GPCR crystallography, where long and disordered ICL3 sequences are counterproductive and are thus commonly replaced with small fusion proteins  with robotic cell-picking, which was successfully used to evolve voltage sensors derived from integral membrane proteins . HoweverAlthough all attention of current GPCR sensor screening approaches focuses on obtaining sensors with the largest maximal fluorescence changes, this property is not by itself sufficient to guarantee success when testing the probe in the living brain. The apparent affinity of the probe is another important factor to be considered when imaging neuromodulators. Owing to its design each probe can only reliably work within a narrow concentration range. Because the levels of release for endogenous neuromodulators are variable among different brain regions, mostly depending on the abundance of neuromodulatory projections in the area, multiple probes with complementary affinity ranges need to be developed in order to most sensitively detect the same neuromodulator in different regions. Up to now, sensors with different affinity ranges have been obtained mostly by using different receptor subtypes as a starting point  , and in A deeper understanding of the basic photophysical properties of GPCR sensors may lead to novel applications based on different fluorescence properties  and open new research directions. In fact, while intensity-based fluorescence readouts can provide valuable information about the dynamics of biological systems using simple instrumentation, they suffer from limitations  that make them unsuitable for quantitative measurements of analyte concentrations . These dAll these advantages have sparked a growing interest in techniques like fluorescence lifetime imaging (FLIM)  or fluorOn the other hand, the field of FLIM sensors based on circularly permuted fluorescent proteins (cpFPs) is still largely underdeveloped. While some groups have reported the use of cpFP sensors with FLIM readout , the senIgnoring for simplicity excited-state dynamics and multiphoton effects, the photophysical basis of sensor function can be described by two limit cases . In the Careful photophysical studies  have shoIt is reasonable to imagine that in the near future the development of GPCR sensors will also expand toward both red and near-infrared (NIR) wavelengths, by taking advantage of a large variety of fluorescent proteins available at these wavelengths . This hain vivo imaging  for the technology described in this paper. The remaining 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 recent introduction of direct acting antivirals for the treatment of hepatitis C virus (HCV) has dramatically improved treatment options for HCV infected patients. However, in the United States (US) treatment uptake has been low and time to initiation of therapy has been long. We sought to examine provider perspectives of facilitators and barriers to HCV treatment delivery.From June to August 2019, we conducted in-depth, semi-structured interviews with medical staff providing HCV care as part of a university medical center in Los Angeles, CA. In order to understand the HCV treatment process, we interviewed key staff members providing care to the majority of HCV patients seeking care at the university medical center, including hepatologists and infectious disease specialists as well as key nursing and pharmacy staff. The interviews focused on workload and activities required for HCV treatment initiation for non-cirrhotic, treatment naïve patients.Providers noted that successful HCV treatment delivery was reliant on a care model involving close collaboration between a team of providers, in particular requiring a highly coordinated effort between dedicated nursing and pharmacy staff. The HCV care team overwhelmingly reported that the process of insurance authorization was the greatest obstacle delaying treatment initiation and noted that very few patient level factors served as a barrier to treatment uptake.In the US, prior authorization for HCV treatment is a requirement for most public and private insurance plans. In an era with access to therapies that allow for a cure—and until revocation of prior authorization for HCV treatment is a reality—implementing strategies that can expedite authorization to accelerate treatment access are critical. Not only will this benefit patients, but it has the potential to help expand treatment to settings that are otherwise too resource strained to successfully deliver HCV care. It is estimated that 2.4 million people in the United States (US) are infected with hepatitis C virus (HCV), making it one of the most common blood borne infections in the US , 2. TranAcute HCV infection is typically asymptomatic or mild , 8. HoweTreatment initiation following HCV diagnosis has been low with a number of studies indicating that less than a third of patients initiated HCV treatment with DAA-containing regimens –20. FollCurrent treatment guidelines in the US recommend eight week treatment with Glecaprevir/pibrentasvir or twelve week treatment with Sofosbuvir/velpatasvir for HCV genotype-1, treatment naïve, non-cirrhotic patients, with both regimen yielding comparable treatment outcomes , 26, 27.The study was approved by the institutional review board at the University of California, Los Angeles. The need for consent was waived by the ethics committee. From June to August 2019, we conducted in-depth, semi-structured interviews with medical staff providing HCV care as part of a university medical center and the affiliated primary care and specialty clinics in Los Angeles, CA. The health care system which serves as the setting for this study consists of approximately sixty outpatient care clinics and two hospitals and serves an estimated 500,000 patients per year . The majority of patients receive HCV care through specialty care clinics (hepatologist) with a lesser number receiving care through infectious disease specialists. Within the system, delivery of HCV care is dependent both on providers (physician and physician extenders) and other medical staff, with heavy reliance on medical assistants, nursing staff, as well as pharmacy staff. Given this team approach, the medical staff play a critical role in the HCV continuum of care including patient intake, assessment of treatment readiness, uptake and adherence issues, as well as follow-up to ensure that treatment is completed.In order to understand the full spectrum of the HCV treatment process, we interviewed all key staff members (n = 6) providing care to the majority of HCV patients within this health system, including two hepatologists, one infectious disease specialist, one nurse who serves as the primary HCV care coordinator in the hepatology clinic, and one clinical pharmacist who oversees HCV treatment coordination within the pharmacy. The nurse and pharmacist were the only two staff members within the system to serve as HCV care coordinators, while the hepatology clinic is staffed by six physicians. The two physicians interviewed as part of this study were chosen given their specialization in HCV care and their higher HCV patient load. In addition, while non-specialist physicians  also provide HCV care within this system, the number of HCV patients any given provider serves is significantly limited by comparison and we were interested in understanding barriers and facilitators to care in a setting specifically focused on HCV, potentially removing barriers that result from lack of specialization in delivering HCV care.The 30-minute interviews were conducted in person, one-on-one, and used a semi-structured, open-ended script. The interview guide was informed by Andersen’s behavioral model of health services utilization [Interviews followed established qualitative interview methodology combining open-ended questions as well as structured probes to explore factors in each of the three domains within the Andersen behavioral model . Each inWe found that the process of HCV treatment initiation involves a highly coordinated effort between dedicated nursing and pharmacy staff. Once a physician has determined treatment is indicated, the nursing coordinator starts the process by ensuring that all laboratory testing and assessments have been completed. A common missing laboratory component, which is a requirement for insurance approval is HCV genotype testing. Consequently, it is not unusual for the nurse to initiate follow-up testing and patient coordination to ensure that all necessary lab results for insurance authorization are in place.Once all the requisite material are ready, the nurse coordinator prepares a pre-authorization packet which consists of all relevant paperwork including findings from the clinical evaluation, laboratory results, and administrative requirements necessary for insurance authorization. The pre-authorization packet is sent to a dedicated in-house pharmacist who serves as the pharmacy ‘case manager’ in order to usher the approval process through the system. The steps can include verifying coverage, outreach to patient assistance programs to mitigate cost of treatment including those whose health insurance may not cover the entirety of the cost, as well as follow-up calls to ensure that medications have been ordered and shipped. The pharmacist estimates that each case requires several phone calls and can take several hours to manage these efforts. Both the nurse and the pharmacist noted that there were no differences in the process or time required to submit insurance authorization paper-work for either the eight week or twelve week regimen.Once insurance coverage has been authorized, the nurse coordinates with the patient to ensure medication pick-up/delivery with additional calls to verify treatment start date. This in turn will allow the nurse coordinator to schedule a follow-up visit after four weeks of treatment for laboratory testing.Beyond coordinating the start of treatment, the nurse remains available throughout the course of treatment to address any patient concerns and continues to manage the process by tracking shipment dates for refills, ensuring patients are available to receive refills, and coordinating and scheduling follow-up visits. Not surprisingly, this requires significant effort with multiple phone calls, multiple stakeholders, and much time in order to ensure successful treatment initiation and compliance. Given the workload, the nurse coordinator estimates a maximum possible patient load of fifteen patients at any given point in time.A summary of the findings related to the facilitators and barriers of HCV treatment delivery are presented in Beyond insurance, the medical team noted that pill burden can be a deciding factor among the minority of patients who have a choice between eight or twelve week treatment. For instance, providers felt that patients taking other medications seem to have a preference for the longer treatment regimen (Sofosbuvir/velpatasvir for twelve weeks) given the one pill per day dosing as compared to the larger daily pill burden for the shorter, eight week treatment (Glecaprevir/pibrentasvir for eight week with three pills per day). Providers also felt that age affected treatment preference, with younger patients preferring the shorter treatment regimen as compared to older patients, including ‘baby boomers,’ a generational cohort highly impacted by HCV which constitutes a large proportion of patients receiving HCV care . The treOverall we found that the process of insurance authorization for HCV treatment—even in the context of a well-resourced health system—to be laborious and serve as one of the greatest obstacles to initiation of shorter HCV treatment regimen. We also found that a model of care involving close collaboration between a full team of providers is key to the success of treatment delivery, which has allowed for linkage to care and functional cure in 94% of HCV patients within this health system . In an eIn 2020 the US Preventative Task Force updated its HCV screening guidelines from a ‘risk-based’ screening approach to recommend one-time screening for HCV infection for adults 18 to 79 years, regardless of risk , 35. ThiPrior authorization for medications were initially put into place to address higher cost prescriptions that may have lacked medical justification and directed to newer high cost drugs with limited benefit . When DAUntil the revocation of pre-authorization for HCV treatment is a reality, strategies that improve efficiency in the process are critical. Introducing non-clinical staff to serve as insurance authorization coordinators can help decrease nursing staff workload. Task-shifting administrative roles has the potential to free resources, allowing for increased sustainability of a program that is heavily reliant on a treatment coordinator. That task-shifting may also allow scaling of HCV treatment from specialty clinics into other settings such as primary care, where providers may not have nursing time available to devote to the process. Second, access to treatment may be increased by expansion of insurance coverage to allow for a choice of treatment , especially in cases where a shorter treatment regimen has the chance of improving compliance such as those who are unstably housed or have competing substance use issues. Additionally, ensuring prompt and continuous access to medications by eliminating the need for refills by dispensing the full course of treatment when possible can help streamline the process from both the provider and patient perspective.Several limitations of this study should be noted. First, the sample size is relatively small, therefore additional barriers and facilitators of HCV care not mentioned by the providers we interviewed would not be detected. However, the primary theme around the resource intensive nature of the prior authorization process was repeated by all interviewees which allowed for “saturation” around this theme. Second, the findings were in the context of a well-resourced large healthcare system and may not be relevant to other health settings. Nonetheless, our results demonstrate that despite improvements in treatment for HCV, much work remains for improving the continuum of HCV care. Changes in policies around prior authorization for curative HCV treatment have the potential to increase treatment capacity allowing us to get one step closer to the treatment goal of 80% of eligible patients, ultimately helping to accelerate the drive toward HCV elimination targets .


Traumatic brain injury (TBI) is the leading cause of long-term disability in working age adults. Recent studies show that most acute TBI patients demonstrate vestibular features of dizziness and imbalance, often from combined peripheral and central vestibular dysfunction. Effective treatment for vestibular impairments post-TBI is important given its significant adverse impact upon quality of life and employment prospects. The most frequent peripheral vestibular disorder in acute TBI is benign paroxysmal positional vertigo (BPPV), affecting approximately half of acute cases. Although there is effective treatment for idiopathic BPPV, there are no high-quality clinical data for post-TBI BPPV regarding its prevalence, natural history, which treatment is most effective and when is the best time to treat. In particular, observational studies suggest post-TBI BPPV may be recurrent, indicating that hyperacute treatment of BPPV may be futile. Given the potential hurdles and the lack of accurate post-TBI BPPV data, the current study was designed to provide information regarding the feasibility and optimal design of future large-scale prospective treatment studies that would compare different interventions and their timing for post-TBI BPPV.A multi-centre randomised mixed methods feasibility study design was employed. We aim to recruit approximately 75 acute TBI patients across a range of clinical severities, from three major trauma centres in London. Patients will be randomised to one of three treatment arms: (1) therapist-led manoeuvres, (2) patient-led exercises and (3) advice. Participants will be re-assessed by blinded outcome assessors at 4 and 12 weeks. Acceptability of the intervention will be obtained by patient interviews at the end of their treatment and therapist interviews at the end of the study. Primary outcomes relate to feasibility parameters including recruitment and retention rates, adverse events and intervention fidelity. We will also aim to provide a more accurate estimate of the prevalence of BPPV in TBI cases on the trauma ward.The multi-centre nature of our feasibility study will inform the design of a future prospective treatment trial of BPPV in acute TBI. Important parameters we will obtain from this study, key for designing a future prospective treatment study, include estimating the prevalence of BPPV in TBI patients admitted to UK major trauma wards, and elucidating both patient and care-provider barriers in delivering BPPV treatment.ISRCTN91943864. Registered on 10 February 2020.ISRCTN,  Traumatic brain injury (TBI) is the most common cause of long-term disability in working age adults . Indeed,The impact of dizziness on TBI survivors can be varied and burdensome, affecting physical, psychological and socioeconomic domains . Acute aThe most frequently diagnosed cause of dizziness following TBI is benign paroxysmal positional vertigo (BPPV), theorised to be due to mechanical displacement of calcium carbonate ‘crystals’ or otoconia Fig.  during tRobust clinical guidelines  direct tIndeed, despite the relatively high prevalence of post traumatic BPPV, emerging evidence suggests patients are not routinely assessed during the acute stages of their injury . Our pilIn summary, BPPV seems to affect the majority of acute TBI patients with possible long-lasting physical, psychosocial and economic consequences. Despite this, for a variety of reasons acute assessment and treatment is sub-optimal, whilst insufficient data exists to support decisions regarding effective treatment.The primary aim of this study is to evaluate the feasibility of conducting a future randomised controlled trial (RCT) investigating the effectiveness of treating BPPV in acute TBI.To establish trial recruitment rates, including the willingness of patients to be randomised to a treatment armTo determine trial retention rates, the fidelity of the intervention, its acceptability to participants and clinicians and any missing dataTo explore the incidence of adverse eventsPrimary objectives focus on feasibility outcomes and will aid the design of a future definitive trial:An estimate of the prevalence of BPPV in acute TBICalculating effect sizes for primary and secondary outcome measures to thereby inform sample size estimates for future studiesExamining the relationship between vestibular perception, balance impairment and clinical outcomeSecondary objectives will also support the design of a future RCT. They include:Currently, it is not routine practice in all major trauma centres to assess for or treat BPPV in acute TBI. In order to implement any long-term changes to screening and/or treatment behaviours, it is necessary to identify any possible barriers to such changes in clinical practice. Therefore, a feasibility study utilising both quantitative and qualitative methods was deemed appropriate.Phase 1 will consist of a feasibility trial involving approximately 75 patients randomised to one of the three arms.Phase 2 will qualitatively explore the experiences of patients and clinicians participating in the trial and their views regarding progression to a randomised controlled trial. Patients who decline to take part will be invited for their feedback to enhance recruitment for a future trial.This multi-centre, mixed methods randomised feasibility trial will comprise a randomised trial with two intervention arms and one comparator arm, and a subsequent qualitative study Fig. . ReportiTrauma care in England is delivered through specialist centres . Indeed,Inclusion criteria consists of patients over the age of 18 with a non-penetrating head injury, able to provide informed consent (or with a consultee willing to provide advice), inpatient on a trauma or outlying ward and a sufficient grasp of English to complete outcome measures. Patients will be excluded if they have a current history of substance abuse, are medically unstable, are pregnant, have vascular or orthopaedic injuries precluding neck extension, have active psychiatric disease or a history of psychotic disease, have a Glasgow Coma Score less than 14, or have a current prescription of phenytoin.Eligible patients who consent to take part in the study will be assessed by a ward therapist for BPPV using a Dix Hallpike manoeuvre Fig. , which iThose diagnosed with BPPV will be randomised to one of three intervention arms Fig. . PatientTherapist-led repositioning manoeuvresParticipants will be randomly allocated to one of three arms: (1) therapist-led repositioning manoeuvres, (2) patient-led exercises, or (3) advice.2.Patient-led exercisesRepositioning manoeuvres are physical movements of the head and body typically used to treat idiopathic or traumatic BPPV . Ward thPatient-led or Brandt-Daroff exercises are a form of physical self-treatment involving repeated movements of the head and trunk prescribed by clinicians as an alternative to, or in tandem with, repositioning manoeuvres. The National Institute of Clinical Evidence suggest Brandt-Daroff exercises as a treatment option for idiopathic BPPV , althoug3.AdviceWard therapists will teach participants and carers (where required) the Brandt-Daroff exercises. A repeat diagnostic test will be completed. Following treatment, participants will be instructed to complete the exercises twice per day for 2 weeks, using a standardised, written exercise sheet. Participants will be asked to complete a home diary to record their adherence with the exercises.Ward therapists will provide participants with verbal and written advice. This will comprise ensuring adequate fluid intake, taking precautions when mobilising independently on the ward or at home and trying to ensure normal head and body movement patterns. Advice will be provided in the course of the participant’s hospital stay, during a scheduled therapy session. Such advice is based on evidence suggesting dizzy patients who avoid head movements may be prone to motion sensitivity, which can delay recovery and contribute to long-lasting subjective dizziness and imbalance , 32. A bFidelity monitoring is an important part of any rehabilitation research intervention, enabling investigators to differentiate between comparator and experimental interventions and ensuring any procedures are carried out as defined in the protocol or guidelines . In thisBaseline demographic data will be recorded including age, gender, factors relating to the head injury, functional status, falls and past medical history. Primary outcome measures relate to feasibility objectives: number of eligible patients, time taken to fulfil recruitment target, trial dropout rates, variability in intervention fidelity, proportion of outcome measures completed and any adverse events noted throughout the trial.Timepoint 0—during hospital stay, following consent, randomisation and prior to treatmentTimepoint 1—at 4 weeks following treatmentTimepoint 2—at 12 weeks following treatmentSecondary outcomes will be collected at three timepoints:Secondary outcomes comprise the diagnostic test for BPPV  to explore the frequency and recurrence rate of BPPV, in addition to a range of self-reported measures explored below.Self-reported disability pertaining to dizziness will be evaluated using the Dizziness Handicap Inventory (DHI), a 25-item questionnaire with high test re-test reliability and low error of measurement score . DizzineCognitive function will be screened using the Montreal Cognitive Assessment (MoCA), a standardised test of visuo-spatial and executive function, naming, memory, attention, language, abstraction and orientation, routinely used in clinical settings . Health Three further questionnaires will be used at timepoints 1 and 2 to explore functional recovery, return to work and quality of life. The Glasgow Coma Outcome Score is a tool extensively used in phase III trials as a primary measure of recovery following TBI . The extObjective balance outcomes  and modified Dynamic Gait Index (mDGI)) will be measured at all three time points. The mCTSIB evaluates how participants are able to use sensory inputs during static balance in four conditions; feet together and eyes open or closed on a hard or soft surface for 30 s. This test has demonstrated excellent reliability and validity in those with vestibular disorders . The mDGAs this is a feasibility study, no formal sample size calculation has been undertaken. However, Sim and Lewis  recommenA sample of up to ten patients who declined to take part in the study will be interviewed to examine their reasons for declining and therefore optimise recruitment strategies for a future study. Approximately ten therapists and 18 patients participating in the trial will also be interviewed at the end of the trial to explore intervention acceptability. Sample sizes for the qualitative component were drawn up following consultation of relevant guidance  which adThe aim of this phase will be to explore the feasibility of the trial from a participant and therapist perspective using face to face, individual interviews. A topic guide will be used to structure the interviews and will examine participants’ and therapists’ views on the study design and their experience of recruitment processes, assessments, outcome measures, the intervention itself and follow-up assessments. Data from this phase will be utilised to refine the study procedures to maximise recruitment for a subsequent RCT.Potential participant interviewees will be purposively sampled at the 12-week follow-up timepoint. A sample of approximately 18 participants  will explore the thoughts and views of those taking part in the trial. Additionally, up to 10 participants who declined to participate in the study will be invited to take part in individual interviews to explore reasons for non-participation. They will be interviewed face to face or by telephone, according to preference. Up to ten therapists participating in the trial across all centres will be asked to consent to individual interviews.α = .025) based on the Bonferroni correction. We do not expect to demonstrate significant results from this data; rather, it will be used to calculate effect sizes for future prospective treatment studies.Results will be reported descriptively to chart demographic and observational data. Participant recruitment/retention data will be reported using pilot and feasibility CONSORT guidelines . An estiQualitative data will be transcribed verbatim and analysed using a Framework approach , 54. ThiA steering group comprising of an external chair, statistician, lay members and ward therapists from all sites discussed and provided input into the protocol at the planning stage. The steering group will meet throughout the duration of the trial to monitor recruitment and any adverse events. Additionally, a data monitoring committee will have access to unblinded data and will therefore be able to oversee the safety of the trial participants. Trial cessation criteria include any patient safety concerns, the futility of treatment in a specific intervention arm and recruitment of 75 patients.Written data will be stored securely at individual NHS centres. Study data will be collected and managed using REDcap electronic data capture tools hosted at Imperial College London , 56. RED60% of those screened eligible for inclusion.Initially 30% of eligible patients are expected to consent to participate in the study, rising to 50% of those eligible consented as the research team gain confidence with consenting procedures.≤ 40% drop out rate.Trial progression criteria will be used to determine whether it is appropriate to move forwards to a RCT. Criteria include:Thresholds for participants consenting have been informed from previous feasibility studies in acute TBI , 58 and This feasibility trial was designed following discussions with patients from a specific brain injury charity. Patient members form an important component of the steering group. Feedback from patient members was invited on the proposed trial design, protocol and participant facing documents, such as information sheets and topic guides for interviews. Patients and the public will play a central role in planning dissemination of results and future steps.This paper describes the rationale and the methods of a mixed methods feasibility trial for managing BPPV in acute TBI. The results of this feasibility trial will not only inform the design and development of a future RCT, but will also highlight and describe the prevalence of BPPV in this population. Long-term implications of diagnosing and treating BPPV early on in acute TBI may have a positive impact on patient centric outcomes including reduced symptom load, attenuated falls risk and a shortened delay in return to work. Additional service benefits include a reduced hospital length of stay, improved rehabilitation outcome and a lessened burden on outpatient resources following discharge.


G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins which conduct a wide range of biological roles and represent significant drug targets. Most biophysical and structural studies of GPCRs have been conducted on detergent-solubilised receptors, and it is clear that detergents can have detrimental effects on GPCR function. Simultaneously, there is increasing appreciation of roles for specific lipids in modulation of GPCR function. Lipid nanoparticles such as nanodiscs and styrene maleic acid lipid particles (SMALPs) offer opportunities to study integral membrane proteins in lipid environments, in a form that is soluble and amenable to structural and biophysical experiments. Here, we review the application of lipid nanoparticle technologies to the study of GPCRs, assessing the relative merits and limitations of each system. We highlight how these technologies can provide superior platforms to detergents for structural and biophysical studies of GPCRs and inform on roles for protein-lipid interactions in GPCR function. G protein-coupled receptors (GPCRs) constitute a family of over 800 integral membrane proteins, unified by a shared seven transmembrane helix architecture  are increasingly used as platforms for biophysical and structural studies of GPCRs. These technologies feature nanoscale (~ 10 nm in diameter) lipid bilayers stabilised by amphipathic protein sequences or synthetic amphipathic polymers     (POPG). In both cases, the monomeric, reconstituted receptor was functional with respect to ligand binding and nucleotide exchange at the Gs heterotrimer. Since these initial studies, a wide selection of GPCRs have been reconstituted into nanodiscs and studied by a range of biochemical and biophysical methods , an NMR study of the deuterated receptor demonstrated greater constitutive activity and slower conformational exchange between receptor active and inactive states in a nanodisc compared to detergents  and phosphatidylinositol (PI) lipids, and zwitterionic phosphatidylethanolamine (PE) and PC lipids, all with dioleoyl (DO) acyl chains , increasing nanodisc PG content increases Gq coupling , NMR experiments on the nanodisc-reconstituted receptor were used to demonstrate that polyunsaturated docosahexaenoic acid (DHA) acyl chains shift the receptor conformational ensemble towards states more productive in G protein binding, which correlates with greater observed stimulation of Gs by the receptor in nanodiscs containing DHA chains , reconstitution into POPC/POPG nanodiscs facilitated coupling to β-arrestin1, which was weak in detergent  demonstrate the advantages of nanodiscs over detergent solutions for structural studies of GPCRs. For both the M2 muscarinic receptor (M2R) and βNanodiscs can therefore provide a superior environment for biophysical and structural studies of GPCRs compared to detergents and have provided considerable insight into lipid modulation of GPCRs. However, there are several limitations to the technology and practical aspects to consider for successful nanodisc reconstitution of GPCRs.The primary issue with nanodiscs is that the reconstitution process requires a detergent-solubilised GPCR. As such, any GPCR that is to be reconstituted into a nanodisc must first pass the gauntlet of detergent solubilisation, which may involve the loss of too much receptor material/activity for the reconstitution to be viable. One solution to this issue is to reconstitute the receptor immediately after membrane solubilisation and before purification and then to purify the receptor from other membrane proteins in nanodiscs; this has been demonstrated for two class B GPCRs , which may be difficult to separate from those produced by the reconstituted GPCR.2AR and is selectively enriched amongst lipids retained by this receptor upon solubilisation  lipids have been shown to play roles in receptor-arrestin interactions . Even then, lipids that are sufficient to reconstitute/modulate receptor activity in vitro may not necessarily be lipids that act functionally in vivo. It is also apparent that one lipid may have different effects on different aspects of receptor activity; for example, PE favours an inactive receptor conformation but promotes G1AR construct was reconstituted with dimyristoyl PC lipids , which features hydrophobic styrene and hydrophilic maleic acid moieties that are typically present with gross ratios of 2:1 or 3:1 styrene/maleic acid Fig. . The resSeveral alternative amphipathic polymers have been applied to and developed for membrane protein studies     from HEK293 membranes (Hall et al. 2AR from HEK293 membranes. Whilst the material was not purified, detailed analysis of the stability and activity of the receptor in the resulting nanoparticles showed that receptor ligand binding affinity was similar in DIBMA nanoparticles, membranes and detergent, whilst receptor thermostability was improved by 10° in DIBMA nanoparticles versus detergent (Harwood et al. q and Gs heterotrimers to a degree that is comparable to higher concentrations of detergent-purified NTSR1.In addition to SMA, the SMI, DIBMA and PMA polymers have recently been applied to solubilisation of GPCRs. The acid-compatible SMI polymer was used to solubilise ATo date, studies using amphipathic polymers have established that they are a viable means for detergent-free production of functional GPCRs in lipid nanoparticles from yeast, insect and human cell expression systems, which can be analysed by biochemical and biophysical techniques. However, there are a number of practical considerations when using amphipathic polymers for formation of GPCR lipid nanoparticles.2+ and Ca2+ which precipitate SMA may be required in buffers for purification or activity assays of GPCRs and may also directly modulate GPCR activity (Ye et al. Firstly, as previously discussed, there are well-established limitations of the SMA polymers that may inhibit their application to the solubilisation and purification of GPCRs. Divalent cations such as MgEscherichia coli, the pattern of pH dependence of KcsA solubilisation was quite different to that of model membrane solubilisation at lower polymer concentrations; this is proposed to result from polymer interactions with charged regions of membrane proteins that are absent from model membrane studies (Kopf et al. solubilisation conditions for a receptor may not translate to optimal purification conditions for that receptor, especially if excess free polymer is present (Kopf et al. Irrespective of the kind of polymer used, polymer solubilisation of membranes can be very sensitive to factors such as experimental temperature and salt concentration, so some effort is required to determine optimal conditions for solubilisation and subsequent purification. Studies on simplified model membrane systems show that many variables affect SMA solubilisation kinetics, including properties of the membrane bilayer such as fluidity, lateral pressure and charge density, as well as experimental salt concentration, polymer concentration (Scheidelaar et al. Finally, there are still aspects of the physical characteristics of polymer lipid nanoparticles that are unclear. Some studies have shown that rapid exchange of lipids between individual SMA and DIBMA lipid nanoparticles is possible (Hazell et al. 2 added during complexing was observed bound to the receptor. Nonetheless, there is increasing appreciation of the importance of the lipid environment in structural and biophysical studies of GPCRs, and as the amount of literature published in this area increases, it is anticipated that detergent studies will be increasingly complemented by studies in lipidic environments such as nanodiscs and polymer lipid nanoparticles.It should first be noted that despite the advantages of both lipid nanoparticle technologies in providing lipid context to GPCR studies, detergents clearly play an important role in studies of GPCRs. Detergent solubilisation is a necessary step in nanodisc reconstitution, and if a receptor is stable and demonstrates the desired functionality in detergent, then, depending on the question to be addressed, reconstitution into nanodiscs or attempts at detergent-free purification with polymers may be harder to justify. In some cases, if there is prior information regarding functionally important lipids, it may be possible to re-introduce specific lipids in detergent-solubilised form to improve the function of the receptor. Such an approach was successful for a recent cryo-EM structure of the NTSR1-β-arrestin1 complex (Huang et al. Nanodiscs and rHDLs represent a well-validated means to study GPCRs in a defined and experimentally tractable lipid environment. This nanodisc environment can support higher receptor activity than detergents and allow for receptor functions that benefit from the absence of detergent, the presence of a bilayer structure, the presence of specific modulatory lipids or a combination of these factors. Nanodiscs have proved an excellent means for assessing the roles of specific lipids on GPCR function and have added considerable detail to the literature on GPCR-lipid interactions. Conversely, the need to reconstitute functionally supportive lipid environments in a bottom-up manner means that we must be cautious to assume that the lipids chosen necessarily reflect important/annular lipids in vivo, and optimisation of nanodisc reconstitution may be laborious. Saposin A nanoparticles may offer a viable alternative to MSP nanodiscs in such cases. Typically, nanodisc reconstitution will be most suitable for receptors that are stable in detergent, and thermostabilisation of the receptor sequence may be required to achieve this. Moving forward, techniques such as native mass spectrometry may be able to provide complementary ‘top-down’ information on GPCR-lipid interactions; a recent study clearly identified lipids that associate with several different GPCRs after solubilisation (Yen et al. Polymer lipid nanoparticles can potentially overcome the disadvantages of nanodiscs, namely, they can be formed without detergents and rely on the receptor itself to organise its lipid environment rather than the experimentalist. Reports to date show at least four kinds of polymer can be used to solubilise functional GPCRs, and the potential advantages of polymer-lipid nanoparticles for studies of GPCRs clearly justify further use of these systems. Firstly, they can potentially allow poorly thermostable receptors to be studied in lipid environments without need for thermostabilising mutations of the receptor sequence, by negating the need for the detergents. Secondly, polymer-lipid nanoparticles can potentially preserve native-like protein-protein and protein-lipid interactions which would otherwise be perturbed by detergents. The demonstration that SMA can solubilise a GPCR heterodimer from liposomes suggests that it may be possible to isolate complexes of GPCRs with other integral or peripheral membrane proteins formed in biological membranes without detergents. Likewise, it is possible to analyse the lipid content of protein-containing polymer lipid nanoparticles (Dörr et al.


Prostate cancer continues to exert a significant public health burden across the globe with hundreds of thousands of new diagnoses per year. There have been many advances in prostate cancer treatment that have dramatically improved the outlook for a lot of patients, especially by targeting a key factor in prostate cancer development called the androgen receptor. However, with increasing of targeted therapies we see a shift in the spectrum of treatment resistance disease. Molecular signatures are essentially maps of the potential for tumor evolution. By analyzing patient and pre-clinical model derived data, we may put together lists of genetic determinants of cancer progression and predict if patients will be prone to develop aggressive disease. In this manuscript we are reviewing some of the ways that these signatures are generated and discuss the advantages and disadvantages of their utility in personalized medicine.Prostate cancer is a high-incidence cancer that requires improved patient stratification to ensure accurate predictions of risk and treatment response. Due to the significant contributions of transcription factors and epigenetic regulators to prostate cancer progression, there has been considerable progress made in developing gene signatures that may achieve this. Some of these are aligned to activities of key drivers such as the androgen receptor, whilst others are more agnostic. In this review, we present an overview of these signatures, the strategies for their derivation, and future perspectives on their continued development and evolution. This is supported by the accumulation and analysis of a huge amount of data derived from genomic studies that include the human genome project and the cancer genome atlas. In this review, we provide a comprehensive overview of the biology involved in transcription factor-mediated prostate pathophysiology and the tools that have led to several transcriptional signatures in clinical or research use whilst exploring the utility of such signatures.Prostate cancer (PCa) is a high-incidence malignancy that results in 360,000 deaths annually and is the second most common cause of death amongst men despite significant advances in its treatment . The bioTranscription factors (TFs) bind onto specific DNA sequences to regulate gene expression. TFs are regularly altered in expression and activity in various cancer types as a result of mutations, amplifications or deletions, chromosomal abnormalities, or chromatin landscape remodeling . Cancer Because TFs regulate target gene expression, target gene expression can be consequently used as a proxy for TF activity  and the As introduced in the previous section, TFs and epigenetic regulators may affect an array of biological processes/hallmarks of cancer or, instead, have greater bearing on one major biochemical pathway contributing to tumorigenesis. TFs such as the AR and FOXA1 and epigenetic modulators such as EZH2 have certainly been shown to be pleiotropic, directly affecting multiple biological functions and, as such, producing multiple phenotypic changes. On the other hand, TFs such as E2F, SOX9, TWIST1, and ASCL1 affect single  processes such as proliferation, differentiation, and lineage determination. A short summary of TFs important in PCa pathophysiology can be seen in The importance of TFs (and the AR in particular) in PCa can be illustrated by (1) its effectiveness as a therapeutic target, (2) its activity as a prognostic biomarker, and (3) the effect of its targeting on the evolution of PCa.Upon androgen  stimulation, the AR regulates gene transcription through crosstalk with other transcription factors, nuclear translocation, and binding to androgen response elements to regulate prostate development . TranscrThe AR is most highly expressed in luminal epithelial cells, which produce prostate-specific antigen (PSA), a major diagnostic biomarker in PCa. Its transcriptional activity supports a specialized differentiation function that comprises glycolytic and anabolic metabolism, glycosylation, calcium and lipid metabolism, and protein folding. This is achieved by either direct binding or proximity effects on neighboring genes, leading to the transformed prostate observed in localized PCa. AR activity is frequently sustained as PCa progresses and the repertoire of binding sites and target genes evolves. Consequently, androgen deprivation therapy (ADT) with agents such as enzalutamide and abiraterone has been exceptionally effective in 80% of PCa cases .Predicting the differentiation potential of PCa tumors is important because some PCa cells survive and display plasticity, which is the ability of cells to adapt to extrinsic and intrinsic perturbations and differentiate from a stem-like to a differentiated-like phenotype permanently or be in constant flux between the two (de-differentiation). Indeed, it has been shown through the transcriptional profiling of epithelial tissue that aggressive PCa is enriched for a prostate basal stem cell signature, particularly for E2F cell cycle target genes . Such drGiven the central role of the AR in prostate cancer tumorigenesis, extensive effort has been put into developing activity gene signatures for this factor. However, the AR does not act alone. p53 and HIF1α are believed to have a significant impact on prostate cancer prognosis ,18. MoreAs lethal types of PCa become more prevalent, the search for TFs alongside the AR responsible for PCa progression has intensified, and there is no shortage of candidates as nuclear hormone receptors include the glucocorticoid, estrogen, and progesterone receptors, the latter being also of paramount importance in breast cancer and other metabolically active malignancies. The glucocorticoid receptor (GR) for example, was shown to bypass the AR blockade and confer treatment resistance in the LNCaP/AR Resistant to Enzalutamide Xenograft derived (LREX’) in vivo model . GATA2 rAs discussed earlier the signatures with most potential would be ones covering a pleiotropic, multi-phenotype biology. Developing such signatures requires appropriately sized clinical sample groups for statistical power (reverse translation) and to accommodate disease heterogeneity. Resolving heterogeneity and achieving statistical power is very challenging, particularly when molecular data are generated from bulk samples. An alternative strategy is to initiate signature development using pre-clinical models. In this scenario, signature derivation is comparatively simple (forward translation), but the translation of such signatures into the clinic is much harder due to the complexity of clinical samples. This discrepancy is due to the fact that most pre-clinical models tend to be reductionist. Progress is being made to bridge this gap with recent advances in tissue explant and organoid culture, retaining some clinical complexity in a pre-clinical setting ,32. SignTo derive robust activity signatures, one cannot ignore the breadth of information provided by the chromatin landscape; rather, a multi-layered approach is needed. Such an approach needs to be highly sensitive and applicable to profiling chromatin landscapes in low cell numbers to discern in more granularity the different roles that TFs play in heterogeneous populations in situ. Exploring the chromatin architecture post treatment could uncover a wealth of information that could guide decisions as to whether changes seen at the transcriptome level through RNA sequencing are regulated by TFs—information that ChiP sequencing can provide. The pharmacological modulation of the AR may alter chromatin opening and, as such, the downstream effects seen as a result of corresponding therapeutic regimes. New versions of prognostic and diagnostic activity signatures should accommodate for the dynamic change of chromatin architecture upon pharmacological modulation and harvest the potential of combinatorial omics analysis, combining different types of sequencing outputs. Assays for Transposase-Accessible Chromatin using sequencing (ATAC) for example, offers an added component to an integrated epigenomic analysis to identify signatures of active chromatin. In addition, ATAC is amenable to single cell integration, making spatial- and cell-specific analyses possible.There are several approaches to identify the TFs that are the most relevant regulators of a gene signature. This often entails the integration ChIP-seq and gene expression datasets to identify true regulatory (TREG) TF–gene interactions in terms of interaction probability aiding in the generation of complex disease transcriptional profiles . ProstatConsequently, integrating the chromatin landscape architecture and transcriptomic landscape may prove critical in inferring regulatory relationships in clinical studies. This will occur because the accessibility of the chromatin will be variable in certain conditions, and transcript expression may provide useful information as to the TFs responsible for driving PCa progression. Changes in chromatin accessibility alter the binding ability of TFs playing a significant role in CRPC because AR-regulated, bromodomain-containing proteins such as BRD2 mediate chromatin relaxation, thus leading to the identification of a bromodomain-related gene stratification signature, BROMO-10 . IntegraThe emergence of AR-indifferent CRPC has been partly attributed to the prevalence of constitutively active AR variants that lack the canonical ligand binding domain. As such, the presence of these variants, which include AR-V7, provides a barrier to effective androgen deprivation therapy . The knoThe further integrative analytics of transcriptomic and clinical data has shown a tumor-suppressive activity of a microphthalmia-associated transcription factor (MITF), which directly affects the heat shock protein crystallin alpha B that is downregulated in PCa. This is of particular interest as an example of a TF–target combination study that leads to a PCa prognostic signature . Such siAs defined earlier, neither forward nor reverse translation in isolation seem likely to be the optimal way of deriving an activity signature. Rather, a pertinent question is: where should these two approaches intersect? The simplest example of forward and reverse translation coming together in PCa is the Prolaris signature. This was inspired by the development of activity signatures in breast carcinoma and was based on an initial 126 cell cycle progression (CCP) genes (GEO database) that were tested against 96 FFPE tumor sections, leading to a final signature of 31 CCP genes with an adjusted CCP score converging on E2F TFs . Other sThe idea that signatures derived through alternative processes may converge to common prioritized pathways is certainly an attractive one. Two sets of signatures (SIG-HES6 and SIG-DENT) were used to link overlapping hormone signaling (HES6-AR) , cell cyWith the growing number of activity signatures being published there has been a drive to develop tools that predict signature utility across heterogeneous datasets and easily map the increasing documentation of generated signatures.p-values through addCLT to produce a framework, gene signature using meta-analysis (GSMA), that analyses multiple independent studies of the same condition and provides a global list of differentially expressed genes along with meta-p-values upon leave-one-out (LOO) analysis [Meta-analytic approaches on big data collections been attempted to specifically derive TF biological information. A pipeline deriving TF activity signatures from 1056 cell lines and 9250 primary tumors combined consensus TF regulons and gene-wise expression data with unsupervised enrichment algorithms . These fanalysis .The derivation of activity gene signatures requires the application of analytical methods to omics data which include artificial neural networks (ANNs), Bayesian networks (BNs), support vector machines (SVMs), and decision trees (DTs) . These mAs it stands, there seems to be a lack of standard analytical frameworks for signature generation methods. Reproducibility is a major hurdle to forward translation, especially since most signatures born out of such methods may share some common basic tools but vary greatly in methodology and are subject to scrutiny for novelty to reach publication. As such, further work is needed to integrate the need for new ways to generate signatures  and the need for standardization necessary to bring an activity signature to the clinic.Ultimately, current AR activity signatures are limited to classical AR targets because the models used to derive such consensus signatures emerged from data prior to the administration of an AR antagonist. Though such signatures have proven exceptionally useful, they offer little in prognosticating the response of individual patients to AR inhibition (ADT). As such, exploring how the TF landscape responds to treatment is preferential to exploring what the steady state of the disease is. That is not to say that information at the point of diagnosis is not important. Indeed, exploring heterogeneity at the levels of chromatin architecture, and gene expression can support the selection of patients for radical treatment and serve as a platform for more fine-tuned investigations monitoring disease progression and treatment efficacy in biological fluids . These would involve circulating tumor cells (CTCs), exosomes and circulating free DNA (cfDNA). This approach adds the key component that diagnostic and prognostic tools and signatures lack at the moment: dynamic evolution. This, of course, will also lead to a more nuanced approach to heterogeneity because no single marker, TF, or activity signature will be appropriate for any patient for the duration of the disease. One would, however, expect to see oscillations between states captured by admixtures of activity profiles associated with classical TFs involved with PCa, such as Myc, HIF1α, and the AR.Perhaps the most optimal way of developing such synergistic activity signatures would be through a combination of functional studies and clinical data. Indeed, it has been exceptionally interesting to see the emergence of co-activation signatures in prostate cancer that not only focus on one of the classical TFs associated with PCa progression  but rather on a combination of an established TF with a biological master regulator or an established TF with another oncogene. One such example is the META-16 gene signature that aims to prognosticate treatment response and time to metastasis in patients with prostate osteometastatic clinical presentations . The co-An alternative manner of crossing biological pathways is through the crossing of generalized biochemical responses rather than single TF signatures  via both in vitro and in vivo data. Mining both hypoxia- and PCa-related signatures is how a 28-gene hypoxia-related prognostic signature was developed for localized PCa  Figure . To illuHypoxia and genomic instability may be common indices that pinpoint chances of biochemical relapse, as identified by multivariate analyses that combined DNA-based and intra-prostatic hypoxia indices to predict patient stratification into treatment intensification trials . This suCo-activation signatures pose an interesting question regarding the interplay between differential biochemical pathways that may determine the clinical relevance of basic biological research and the utility of TF activity signatures in PCa clinical practice. Contributing factors to be taken into consideration include driver mutational events linked to PCa pathogenesis. The caveat here is that the more signatures are generated on the back of further intersecting biologies, the more complicated the picture becomes, thus adding potentially valuable information for the clinic. For instance, one would expect a proportion of any signatures generated in PCa to reflect metabolic changes given that we know that metabolic change is a significant underpinning hallmark of cancer. In turn, we would expect those changes to reflect the stress response status of the tumor and, therefore, another feature of its evolution: the ability to accumulate DNA damage. This ability to ride the wave of DNA damage through an enhanced DNA damage response (DDR) and stress adaptation enables the tumor to survive and acquire somatic driver mutations. Such mutations include alterations in PTEN, p53, and RB-1. Targeting the DDR alongside the AR has recapitulated the potential of synthetic lethality in cancer treatment  Table 2Table 2. The identification of driver mutations has also enabled driver-centric transcriptional investigations into the effect of non-coding RNA in PCa progression, a field that opens new possibilities in studying the regulation of tumorigenesis, as TFs may regulate miRNA expression that, in turn, regulates the transcriptional repertoire of tumor growth through epigenetic regulation. For example, a network analysis of TP53-regulated transcriptional events has revealed that a tumor suppressor signature of long non-coding RNAs affects genes such as CDKN1A, BAX and BBC3 . The rolThis context provides a great opportunity to combine mutational data, activity signatures, non-coding RNA, and metabolomics proteomic data to produce a biomarker discovery pipeline that covers all bases of cancer evolution from the driver mutations to the adaptive mechanisms that enable cancer cells to thrive in such environments. The NMF of mutations has been used to model mutational signatures from mutational catalogues . FurtherDepending on the aggressiveness of these primary tumors, one can glean information as to what prognostic outcome of each PCa comparative tumor will be. Moreover, the analysis of primary tumors originating in likely sites of CRPC metastasis can provide clues to the molecular changes that need to occur for metastatic PCa to migrate to these sites. In addition, it may offer possibilities of combining work on key TF and stress signaling pathways to combine activity signatures to cover multiple key biochemical pathways in PCa progression. For example, a 38 hub gene signature was generated to stratify glioblastoma patients according to their IRE1/XBP1 status . XBP1s iActivity signatures are the result of collaborative research bringing together pre-clinical in vitro, in vivo, and clinical data to intertwine genetic and epigenetic activity to confront biological reality and assess clinical translation. As their primary purpose is to be a clinical tool of prognostication and stratification, they are inherently limited by inter-patient and inter-tumoral heterogeneity. As such, a major problem with pre-clinical data-derived TF activity signatures would be the overextrapolation of translational value, which could be overcome by developing patient-derived models such as organoids or explants. In addition, there is a need for optimal conditions to assess if a signature is statistically clinically relevant. Consequently, the sample size  and the condition these patients are in play significant roles in determining how representative and translational each signature is from a population and public health perspective. The explosion of the genomic era has led to many landmark discoveries in oncology and other fields. At the same time, though, it has flooded the field with information and gene signatures that not always represent a particular subset of patients but rather biological mechanisms that are found to be key in pre-clinical models. Moreover, the mechanisms that signatures are derived by may yield false positive results and poor specificity, particularly in complex biological processes, as described in a study comparing data-derived gene sets (signatures) against GO terms and literature-derived gene sets in immunological biological processes . In paraIt thus logically follows that we not only need combinatorial approaches to activity signatures but also models of disease that allow us to study the temporal dynamics of disease progression , as well as diagnostic tests that can be acquired and bio-banked throughout the duration of disease evolution. After all, a significant issue with publicly available PCa patient databases is that they are reliant on survival as a primary determinant of disease progression. As such, it is difficult to determine whether an activity signature is representative and predictive of disease evolution if there are not enough survival data to support differential survival events . Biochemical recurrence (BCR) is currently mainly predicted based on clinical observations. However, BCR-predictive signatures that include genes directly affecting TF activity (TRIM14 and SSTR1) have recently been generated using LASSO following high throughput screening combined with a risk and predictive value evaluation through a multivariate Cox regression analysis and time-dependent receiver operating characteristics . An inteRegarding the combinatorial approaches, it may well be pertinent to ask the question: of all the numerous signatures produced via different regulatory approaches in PCa and similar malignancies, how many actually converge to a common mixture of biochemical pathways and markers? Furthermore, what would the utility of a combined “master” signature be? In the end, how do we determine which signature is most clinically relevant? Currently, clonal selection is used as a way of determining if a marker is a driver of PCa. Consequently, the mutations identified through clonal selection are the ones selected for in research. However, if the observed biology is tied to a particular therapy and not a genetic driver, from a clinical perspective, it is less pertinent to know that the observed response is linked to a driver biology. Therefore, although underpinning driver mutations should be taken into consideration for prognostic reasons, determining the therapeutic regime from a pharmacological response activity signature does not have to be relevant to a driver biology. Indeed, pre-clinical models may become complementary to signature research but focus more on therapy selection rather than constitute the core pathway for signature development. This may be supported by the increasing availability of patient samples, which will reduce the dependency on cell lines. This, of course, may lead to translational activity becoming dependent on resources, with fewer centers being able to benefit and carry out such research. Of course, as discussed earlier in this review, mutational signatures can point to both driver events and micro-environmental changes. This is a direct reflection of a “concrete” mutational landscape against the dynamic character of cancer evolution in the context of its surroundings. The increasing emergence of mutational signatures may provide more translation into clinical practice, as mutational signatures provide cell-specific or environment-specific components. As such, spatial information may well form the next generation of techniques of validating activity signatures for specific populations within a tumor because it will be able to distinguish between cell-specific, tissue-specific, and lineage-specific events .Combining pharmacologically-driven and genetically-driven signatures may provide more insight into the adaptation versus selection mechanisms responsible for disease relapse and evolution in treatment-resistant forms such as the PCa to CRPC conversion. This was exemplified by the Dunning R-3327-H adenocarcinoma that showed that disease progression was down to original tumor heterogeneity rather than an original pool of identical cancer cells that managed to adapt to treatment and eventually proliferate . This, oRegarding the dynamics of disease progression, it would be pertinent to look at sample availability for metastatic disease and study both the primary and metastatic samples for DNA mutations. The presence of somatic mutations in both primary and metastatic tumours from the same patient may be regarded as a evidence of clonal selection and provide insights into how PCa tumors evolve to survive treatment and disseminate. Anchoring activity signatures back to a genetic context may be an important way of determining what is valuable or not. The backdrop of this context may be tissue specificity. Crossing information from relevant comparisons, such as primary tumors vs metastatic tumors, prostatic tissue vs urine, and cancer cells vs immune cells vs fibroblasts, will give a “whole organism” picture of what constitutes the framework of disease progression. From this cross referencing, one could determine consensus signatures that are identifiable in easily accessible biological samples  rather than samples that require invasive procedures and consequent trauma (punch biopsies) or invasive procedures that do not offer any information about the local dynamic evolution of the disease (complete prostatectomies). Such an attempt to infer temporal sequence of events in carcinogenesis has been realized through the integration of somatic mutation and gene expression data using a mixed integer linear program in breast cancer. This formulation uses both simulated and TCGA data to pinpoint mutational events that produce gene expression events over time .One may have to also consider the definition of an activity signature, like whether it is only the differential expression of prioritized genes within a dataset or that differential expression is normalized according to biological weight. A weighted signature will look completely different to one based solely on gene expression levels. Furthermore, if only gene expression levels are considered, then the signature’s applicability on a heterogenous clinical sample group is less likely to be valid. The rate limiting step in such an approach would be the threshold set that determines the weight of each gene  within the signature. The threshold itself would also determine the number of patients that the signature would be applicable to. The less precise or constrained the threshold, the less selective the signature would be for patients potentially susceptible to the biologies (and hence treatments) that the signature is describing. On the other hand, if the threshold is over-constrained, it might miss patients that could be responsive to the represented biologies and treatments.This points to the likely realization that PCa and other clinical entities will most benefit from signatures that are parts of gradients of tumoral activity. Crucially, the weighted component of each signature will be an identifier and measure of activity of where it is in the gradient, as each gene element would be weighed very precisely to measure susceptibility in a very constraint number of patients (in order not to miss patients). To this effect, bio-banking is a crucial and rate limiting component of such investigations.The creation and maintenance of biobanks is a laborious and expensive process. This unfortunately does not make it possible for most research institutes and clinics across the globe. Ideally, the maintenance of biobanks would be under multi-institutional or national control with multiple stakeholders including public bodies  and private companies . ExampleMoreover, stochastic data accumulation from biobanks would require a dedicated funding stream to maintain and curate data libraries based on samples routinely collected from multiple clinical presentations and patients without a particular research question in mind other than accumulating data for potential future promising research. Of course, this once again poses a barrier to true data sharing because it asks the question of ownership. If only a handful of institutions can finance such ventures, then the risk/benefit ratio of investing in one to produce clinically relevant signatures becomes a lot more complicated because the utility of signatures in the clinic is not as regulated as the utilization of pharmaceuticals. Indeed, it may well be down to individual clinicians based on their research expertise and collaborations to determine if signatures would be a valid diagnostic/prognostic support tool in the analysis of the genomic/transcriptomic data of their patients to design individualized treatments. This poses the very real prospect of a whole new generation of clinicians needing to be trained in the art of interpreting omics data, as exemplified by EU/ERASMUS+-driven projects such as BioS that aim to train health professionals in the utility of bioinformatics and data analytics .


Metacognition – the ability to represent, monitor and control ongoing cognitive processes – helps us perform many tasks, both when acting alone and when working with others. While metacognition is adaptive, and found in other animals, we should not assume that all human forms of metacognition are gene-based adaptations. Instead, some forms may have a social origin, including the discrimination, interpretation, and broadcasting of metacognitive representations. There is evidence that each of these abilities depends on cultural learning and therefore that cultural selection might shape human metacognition. The cultural origins hypothesis is a plausible and testable alternative that directs us towards a substantial new programme of research. Human metacognition involves discrimination, interpretation, and broadcasting of subtle cues indicating the rightness of ongoing thought and behaviour.We propose that human metacognition is made fit for purpose by cultural evolution rather than genetic evolution.In particular, we present evidence that the effective discrimination, interpretation, and broadcasting of metacognitive cues depends on cultural learning.The cultural origins hypothesis advances a programme of research on the development of metacognition, cultural variation, individual differences, and cross-species comparisons. How do cognitive mechanisms become fit for purpose? They are all complex products of nature and nurture, but who or what designs the features that enable cognitive processes to do their jobs? How come visual systems can see, learning mechanisms can learn, and reasoning processes can reason?gene-based selection , have been shaped by a cultural selection process analogous to gene-based selection ’Mindfulness  is central to Buddhist practice and has been closely associated with metacognition by scholars of Buddhism  and by wExperimental evidence that mindfulness-based meditation improves metacognitive sensitivity comes from both expertise and training studies. People who have chosen to practice mindfulness show greater metacognitive sensitivity in a tactile perception task than nonmeditators, and number of hours of practice (1–15 000) predict metacognitive sensitivity . SimilarAlt-text: Box 2Further evidence of cultural training of metacognition comes from research in the humanities, social sciences and natural sciences showing that Buddhist practices are designed to, and are effective in, promoting metacognitive sensitivity .Box 2MedThere is marked cultural variation both in metacognitive bias  and kindendophenotype. For example, children who are genetically predisposed to attend more closely to other agents may be better able to acquire metacognitive skill through cultural learning.While twin studies have indicated a genetic component in the development of metacognitive bias , metacogMore broadly, the cultural origins hypothesis calls for research relating individual differences in metacognitive sensitivity to social skills, social experience, and education. Emulating a recent study relating metacognition to mental health , one appGiven that non-human animals lack language and other cognitive resources involved in cultural learning, such as mindreading  and teacIn this section we have surveyed evidence that, in humans, metacognition is shaped by a specific kind of social learning – cultural learning. Social interaction provides more than motivation for the development of metacognitive skills, and information about the circumstances in which they should be used ,120,121.We have suggested that cultural learning enables human agents to discriminate, interpret, and broadcast metacognitive representations. It is possible that cultural learning also plays a yet deeper role in making metacognition fit for purpose. It may bring together decision and evaluation processes to create an architecture capable of assessing the rightness of cognitive representations . AlternaOutstanding QuestionsWhat is the nature and extent of cultural variation in metacognitive sensitivity? Is there cultural variation in the way confidence and error signals are computed from first order cues?To what degree is metacognitive sensitivity genetically heritable? Is this underwritten by a social–cognitive endophenotype, such as social attention?Are people with high metacognitive sensitivity better teachers of metacognition than people with lower metacognitive sensitivity?What kinds of games and routines support the development of metacognition?Do games and conversation have differential effects? For example, are games especially effective in promoting discrimination, while conversation enhances interpretation and broadcasting?What kind of experience or training would improve metacognitive sensitivity in adulthood? Does this kind of training enhance the quality of group decision-making?Alt-text: Outstanding QuestionsMetacognition is important in education, mental health, and public life. It improves learning in schools , 89.,127


Aristotle believed that happiness and success result from cultivating virtue at the mean between deficiency and excess - the golden mean. Some evidence suggests there is a golden mean of hours spent volunteering, where well-being benefits are maximized. Our study examined potential linear and nonlinear functions in the amount of time spent in prosocial work (PSW) in a day on the outcomes of eudaimonia, high arousal negative affect, and high arousal positive affect in a sample of high-commitment volunteers and prosocial leaders. In addition to nonlinear functions, interactions were explored. For example, we found that those who spend less time per week in PSW experience less negative affect when they spend more hours per day in PSW; however, those who spend more time per week in PSW experience more negative affect when they spend more hours per day in PSW. Additional findings for positive affect and eudaimonia will be discussed.


EClinicalMedicineAdolescence is a critical and challenging period during human development and globally every third death due to suicide occurs in young people EClinicalMedicine which reported protective effects of parental understanding and monitoring and negative effects of parental control on anxiety and suicidal ideation in adolescents The impact of parents-adolescent relationship on adolescent suicidal behavior is still an understudied area of research and much of the evidence comes from high-income countries. In this context, the focus of this study on parent-adolescent relationship as a primary explanatory variable for adolescent suicidal behavior is a novel and awaited step. The findings of the study are in agreement with a previous study published in Suicide and suicidal behavior are complex phenomena, and a range of individual, population, and environmental factors are known to increase suicide risk Cultural, religious, and community level factors influence parenting practices and parent-adolescent relationship. The study highlights important regional differences, especially those related to ‘respecting privacy’ in a South Asian context and no association of any parental factors with adolescent suicidal behavior in African countries. It is quite likely that adolescents across different cultures understand and interpret concepts like ‘privacy’ and ‘monitoring’ differently and that socioeconomic factors may influence suicide risk in adolescents, particularly in low-resource settings.Despite large variations across regions and countries, this study underlines the importance of parental factors in influencing mental health and suicide risk in adolescents. Emerging evidence from literature suggests that family-focused interventions have beneficial effects in improving mental health of children and adolescents Rahul Shidhaye: Conceptualization, literature search, writing-original draft, finalization of the draft.Dr. Shidhaye has nothing to disclose.