The analgesic management of dogs with spinal disease is important, as insufficient pain relief may result in chronic neuropathic pain. The retrolaminar block is a relatively new local anaesthetic technique, first developed in humans, in which the local anaesthetic is deposited into the musculofascial plane on the lamina of the target thoracic vertebra, thus providing analgesia to the thoracic region. The aim of this study was to evaluate the retrolaminar technique as a peri-operative analgesic option for dogs with spinal disease, by determining the distribution of two volumes of contrast-dye mixture and the potential complications after anatomical landmark-guided thoracolumbar retrolaminar injections in canine cadavers. Eight canine (greyhound) cadavers were randomised into two groups that received either a 10 or a 20 mL injection into the retrolaminar space at the level of the twelfth thoracic vertebra, after which they underwent computed tomography and anatomical dissection to determine the spread of the injectate. The injectate was detected in the surrounding intervertebral foramina and the paravertebral and retrolaminar fascial planes, which could suggest promising analgesic potential for spinal pain. Epidural and retroperitoneal spread was also noted, and the spread was not volume-dependent. Further studies will help to determine the exact indications for this technique.The retrolaminar block is a regional anaesthetic technique, first developed in humans, in which the local anaesthetic is deposited directly onto the dorsal aspect of the thoracic or lumbar vertebral lamina. This study aims to evaluate the distribution of landmark-guided thoracolumbar retrolaminar injections in greyhound cadavers. Thirteen injections of contrast-dye solution were performed in eight cadavers at the level of the twelfth thoracic vertebra (T12), with either 20 mL  or 10 mL  per site. The spread of the injectate was evaluated through computed tomography and transverse anatomical dissection. The groups were compared using the Mann–Whitney U test. The median (range) of the extent of the spread was 4 (2–5) and 3 (2–4) intervertebral foramina in the LV and HV groups, respectively. The median (range) of the spread along the retrolaminar space was 3 (2–3) retrolaminar segments in the LV and 3 (2–4) in the HV group. Epidural and retroperitoneal spread was identified in seven cadavers. Following landmark-guided retrolaminar injections, the injectate spread both in the retrolaminar and paravertebral spaces, without any obvious association between the volume of injectate and the extent of the spread. Further studies are warranted to determine the clinical efficacy of the technique. Multimodal analgesia regimens commonly include, where possible, a regional anaesthetic technique that is, ideally, minimally invasive, technically easy, and has a very-low-to-zero complication rate . FurtherFascial plane blocks are novel regional anaesthetic techniques that rely on the spread of local anaesthetic within the fascial plane, where a peripheral branch of the target nerve runs . A fasciSince the first description of the retrolaminar block, several other researchers have explored the technique, with or without the aid of ultrasound, either via anatomical investigation ,10,11,12The performance of the landmark-guided retrolaminar block begins with the identification of the spinous process of the vertebra of interest, followed by the insertion of the needle through the surrounding paraspinal muscles, in a parasagittal plane, until it comes into contact with the dorsal aspect of the vertebral lamina, which is where the local anaesthetic is injected . RecentlThe objectives of this study were to evaluate the distribution pattern of two different injectate volumes following a thoracolumbar retrolaminar injection, with the help of computed tomography and transverse anatomical dissection. The hypothesis was that the spread of the injectate to and within the paravertebral space, and within the retrolaminar space, would depend on the volume injected, with higher volume spreading to a greater extent along both spaces. This study was granted an exemption from full ethical review by the Animal Research Ethics Committee of University College Dublin (AREC-E-20-08-Huuskonen). ®, 300 mg I/mL, Guerbet, France), respectively. Both randomisations were achieved via the removal of a ballot from an opaque envelope containing eight and thirteen ballots, respectively. All thirteen retrolaminar injections were performed at the level of the 12th thoracic spinal vertebra (T12). The distribution of the injectate was evaluated using computed tomography in all cadavers and transverse anatomical dissection in two cadavers. Eight frozen adult greyhound cadavers, five males and three females, euthanised for reasons unrelated to this study, were used after being thawed at ambient temperature for 72–96 h. The number of cadavers was chosen according to previous, similar studies. Any cadavers with spinal abnormalities that could have affected the identification of the anatomical landmarks were excluded from the study. Three cadavers were randomly allocated to receive a unilateral right or left retrolaminar injection to evaluate the potential spread into the contralateral paravertebral space (two right and one left injection), while the remaining five cadavers received bilateral injections. The thirteen injections were further randomised into either 10 mL  or 20 mL  of a 1:20 mixture of methylene blue  and iodinated contrast (ioversol) . An 18-gauge, 1-inch needle , connected to a 20 mL syringe  containing the iodinated contrast and methylene blue dye mixture, was introduced into the epaxial muscles, in a parasagittal plane, approximately 1–1.5 cm lateral to the spinous process, at an angle of 45° to the skin. The needle was then orientated caudoventrally, and advanced until contact with the vertebral lamina was achieved. The advancement of the needle was then stopped and, following a negative aspiration for air and blood, the retrolaminar injections were performed by the same investigator (JP). The depth of the needle was approximately 1.5–2 cm, depending on the body condition score of the cadaver . A computed tomographic (CT) examination of the thoracolumbar area was performed approximately twenty minutes after the injection of the contrast-dye mixture, using a 16-slice CT scanner . The cadavers were positioned in sternal recumbency for the scan. The acquisition parameters used to obtain the scans were 0.75 mm slice thickness at 220 mAs and 130 KVp. The computed tomographic images were then viewed using image analysis software . The two EVCDI-board-certified veterinary radiologists who evaluated the CT images  were blinded to the technique used, but not to each other. The CT features assessed for each retrolaminar injection were the confirmation of injection at the T12 retrolaminar space (yes/no), the presence of contrast in the underlying ipsilateral paravertebral space (yes/no), the identification of the ipsilateral intervertebral foramina affected , the extent of the contrast distribution in the ipsilateral retrolaminar space, the contrast distribution pattern , an epidural contrast spread (yes/no), the extent of the epidural contrast spread if any, the presence of contrast in the pleural space (yes/no), the presence of contrast in the retroperitoneal space (yes/no), the presence of contrast in the peritoneal space (yes/no), the presence of pneumothorax (yes/no), intravascular contrast (yes/no), and severe abnormalities of the vertebral column that could have hindered correct needle placement. The confirmation of injection at the T12 retrolaminar space was based on image analysis and the evaluation of the maximum volume of injectate at the point of injection. We considered a craniocaudal spread of the injectate along the paraspinous muscles as a linear contrast distribution pattern, and a more lateral spread towards the intercostal muscles as an intercostal distribution pattern.Following the CT scan, two randomly selected cadavers were dissected using a band saw . On both occasions, the parietal pleura and pleural cavity were carefully inspected for any evidence of needle penetration and contrast spread following the removal of the thoracic and abdominal cavity contents. Transverse plane sections of 2.5 cm width were performed on the first cadaver, while the width was adjusted to 1.5 cm on the second cadaver to enhance the detail. The sections were performed on the thoracolumbar area of the cadavers, from the level of the tenth thoracic vertebra (T10) to the level of the third lumbar vertebra (L3), with the target retrolaminar space identified. Photographs were taken, and the presence of methylene blue dye in the retrolaminar space of interest was recorded to assist in further evaluation.Statistical analysis was performed using IBM SPSS 27  for macOS. The normality of the data was assessed using the Shapiro–Wilk test. Each hemithoracic retrolaminar injection was analysed as an independent variable. A comparison of the number of intervertebral foramina stained and retrolaminar spread with HV or LV was performed using a Mann–Whitney U test. All the statistical test results were interpreted using a 5% level of significance. The results are summarised as the median (range). Categorical data are presented as frequencies. The median (range) weight of the canine cadavers was 29.55 kg (26.3–36.4 kg), with a median (range) body condition score of 4.5 (3.5–5) out of 9 . The medp = 0.524)  occasions, regardless of the volume used. The contrast distribution pattern was always both linear and intercostal . From th= 0.524) . In 7 out of 8 cadavers, epidural contrast spread of a varying extent was noted. While, on six occasions, it was limited locally to the injection site, in one cadaver (with 10 mL injectate volume) the epidural migration was quite extensive (T4–L4). The median (range) spread of the epidural injectate involved 3 (0–14) segmental levels. Focal presence of contrast in the retroperitoneal space was also noted in 12 out of 13 injections, but no specific path could be identified. There was no contrast present in the pleural space or the peritoneal space in any of the cadavers. Pneumothorax was identified in all eight cadavers. Intravascular contrast contamination was noted in 8 out of 13 injections, and it involved vessels away from the needle’s pathway, such as the azygos vein. No injectate was identified in the contralateral retrolaminar or paravertebral spaces in the three retrolaminar injections that were performed unilaterally. No abnormality of the vertebral column was identified in any of the cadavers .Transverse anatomical dissection of two cadavers corroborated the CT study findings regarding correct T12 identification during the retrolaminar injection, the spread of the injectate into the retrolaminar spaces of interest, and the presence of dye in the underlying paravertebral space . No evidThis experimental cadaveric study describes a landmark-guided thoracolumbar retrolaminar technique in canine cadavers and evaluates the extent of the injectate spread into the paravertebral and retrolaminar spaces, while comparing two different volumes of injectate.Contrary to our hypothesis, the spread of the injectate to and within the paravertebral space was not directly proportional to the volume used. Conversely, a previous study in pig cadavers reported that lower volumes of injectate did not spread to the paravertebral space, as assessed via anatomical dissection . Our uneThe contrast spread noted in the ipsilateral retrolaminar space and the underlying ipsilateral paravertebral space supports both proposed theories about the analgesic mechanism of the retrolaminar block. The first theory suggests that the retrolaminar block is paravertebral by proxy. In previous human studies, it was hypothesised that the local anaesthetic diffused into the paravertebral space either through the porous costo-transverse ligament ,13 or thThe vast majority of the retrolaminar techniques described in the human medical literature are performed at the level of the fourth thoracic vertebra. As over 50% of thoracolumbar disc lesions in dogs are found either between the T12 and T13 or the T13 and L1  interverEpidural spread of a varied extent was also noted in seven cadavers. The previously published findings in humans are not consistent regarding the epidural migration of the injectate; some studies presenting it as a consistent occurrence and part of the exerted analgesic action of the retrolaminar technique , while oThis study also revealed the presence of injectate in the retroperitoneal space of the cadavers. The retroperitoneal injectate was noted only focally, and no path leading to the retrolaminar space could be specified. Recently, a retrolaminar block with multiple injection points was performed to provide postoperative analgesia in humans who had undergone a retroperitoneal laparoscopic nephrectomy . The bloIntervertebral disc disease is a prevalent spinal disorder in dogs that necessitates careful analgesic management , as it iThe thoracic paravertebral block (TPVB) is a regional anaesthetic technique that is well-described in veterinary literature ,48 as anRecently, a fascial plane block called erector spinal plane (ESP) block ,50 has bAn inherent limitation of this study is its cadaveric nature and the potential loss of tissue integrity that accompanies the post-mortem state, as decomposition has a detrimental effect on the physical properties of tissues . This coAll the cadavers used in this study had similar anatomical conformation, and none of them had a body condition score higher than 5 out of 9. A high body condition score could potentially obscure the anatomical landmark identification . FurtherWe also noted injectate in the intravascular space, in vessels such as the azygos vein, in 8 out of 13 injections. Direct intravascular injection into the azygos vein during the performance of the retrolaminar technique is anatomically impossible, so this finding is either a consequence of the cadaveric loss of tissue integrity or, less likely, due to the high injection pressures as a result of the high viscosity of the contrast-dye mixture. However, we used large-bore hypodermic needles to deliver the contrast-dye mixture, which reduced the pressure during injection. Similar to our findings, a CT study of a paravertebral brachial plexus block in canine cadavers identified intravascular contamination in 33% of the subjects . This haAnother limitation of the current study involves our use of iodinated contrast and methylene blue mixture, and the potential difference in the spread of this injectate compared to a local anaesthetic. The injectate distribution findings arising from any cadaveric study do not necessarily correlate with the clinical effects when the same technique is performed in live subjects , but cadSimilar studies have mostly used anatomical dissection to assess the extent of nerve staining . The extFollowing the performance of a novel landmark-guided thoracolumbar retrolaminar technique in canine cadavers at the level of T12, a proportional association between the volume injected and the spread of the injectate was not noted. The injectate was detected in the surrounding intervertebral foramina and the paravertebral and retrolaminar fascial planes, which suggests promising potential for spinal analgesia. The retrolaminar technique could perhaps be used in the future as part of a multimodal analgesic regime for canine thoracolumbar pain, provided that further studies confirm the safety and efficacy of this technique.


We analyse the impact of the three following categories of Health Policies (HP) carried out by the Greek SYRIZA-ANEL governments on the efficiency of Greek public general hospitals. These governments have implemented policies intended to change the rate of contributions to publicly funded healthcare (PCnH), policies to affect the volume and quality of publicly funded health care (PVQH) and those intended to affect the costs of publicly funded healthcare (PCH). A literary review of the PCnH. PVQH and PCH policies of the Greek SYRIZA-ANEL governments was carried out and an efficiency window-DEA study was executed using data from the Ministry of Health (MoH) and the Greek Statistical Authority (ELSTAT).The study was designed to assess the impact of PCnH. PVQH and PCH policies by the Greek SYRIZA-ANEL governments on the efficiency of Greek general hospitals. The data was collected from HEAL-Link scientific journals. Information on HPs was extracted from the work collected. The values of inputs and outputs used for the efficiency study were obtained from ELSTAT and Greek MoH databases.HPs of the Greek SYRIZA-ANEL governments extend to all three HP categories of the sample used. These policies have a dual effect on both the inputs and outputs used in efficiency. Efficiency values exhibit fluctuations with good and bad years. The SYRIZA-ANEL governments seek to ensure more equality in access to health services. Some of the policies reduce costs and have a positive impact on efficiency, while others have the opposite effect. The increase in outputs achieved as a result of health policies is counter balanced by an increase in inputs.The PCnH, PVQH and PCH policies of the SYRIZA-ANEL governance seem to have a dual orientation: some policies reduce the cost of a category and contain the total cost, thus positively contributing to an increase in efficiency. Certain policies are aimed more at fulfilling the criterion of equality in the provision of health services and thus the cost inevitably increases. From the window-DEA study, three relatively “good” years emerge  and two “bad years” . This analysis will be useful for further research on the effect of health policies on hospital efficiency in other countries and periods. HPs exercised by governments affect the inputs or outputs of health units and thus efficiency –3. The aPCnHs such as health budget protection, financial restructuring of hospital sectors and reduction of coverage by introducing or increasing user charges can reduce state health funding  and thusEach of the PCnH, PVQH and PCH policies exercised by governments affect the inputs or outputs of health units and consequently their efficiency. Economic funding policies with measures such as performance volume limit financing, or the application of costing through DRG, were observed to reduce hospitalisation days and achieve a large cost saving .The exteData Envelopment Analysis (DEA) is the prevailing method for assessing the efficiency of the healthcare system , 21. TheMeasurement of efficiency using the DEA method is carried out using the inputs and outputs involved in the conversion process in the Decision Making Unit (DMU). TE is calculated on the basis of a complex calculation between the change in outputs of each decision making unit versus its inputs . LimitinDEA efficiency studies use DMUs inputs and outputs to measure efficiency. DEA efficiency studies in Greek hospitals were reviewed and the inputs and outputs used in these were extracted. The results of this analysis are shown in Table HP decision makers are primarily the Greek Government and the Minister of Health. The managers of Health Regions (YPE) and those of general hospitals are authorised to decide on the application of a HP. Interventions apply to the secondary general hospitals of the whole country without exception. The HP interventions of the SYRIZA-ANEL government start in 2015 and are implemented over the duration of their governance.The motivation for the research stems from the desire to study the effect of health policies on efficiency for a given period. This effect has not been studied by other researchers in the past. The results of the research will contribute to postdoctoral research, and they will prove useful in shaping an integrated way of thinking about the impact of HP on efficiency.The purpose of this paper is to analyse the impact of PCnH, PVQH and PCH policies of the Greek SYRIZA-ANEL government on the efficiency of Greek public general hospitals.More specifically, through our research, we aim to demonstrate how the PCnH, PVQH and PCH policies of the afore-mentioned governments alter the efficiency of Greek general hospitals in the period under study. We suppose that PCnH, PVQH and PCH policies increase the efficiency of the general hospitals. This supposition arises from the fact that the bibliography refers to governmental policies which aim at increasing hospital performance. In fact, this increase has been calculated in certain instances , 37. TheWhat is the problem this paper attempts to answer? The problem is how PCnH, PVQH and PCH policies implemented by the SYRIZA-ANEL governments affect the efficiency of Greek public general hospitals.How do the PCnH, PVQH and PCH policies implemented by the SYRIZA-ANEL governments affect the efficiency of Greek public general hospitals?How do PCnH, PVQH and PCH policies influence the inputs and outputs selected for the Greek general hospitals efficiency study?How do the efficiency values of Greek hospitals change?PCnH, PVQH and PCH policies implemented by the SYRIZA-ANEL governments positively influence the efficiency of Greek public hospitals by reducing inputs and increasing outputs.PCnH, PVQH and PCH policies of the SYRIZA-ANEL governments reduce inputs and increase outputs.TE changes constantly in a positive way over the period considered.Ιf the main hypothesis is verified, this will mean that PCnH, PVQH and PCH policies implemented by the SYRIZA-ANEL governments positively influence the efficiency of Greek public hospitals by reducing inputs and increasing outputs.If the main hypothesis is not verified, this will mean that PCnH, PVQH and PCH policies implemented by the SYRIZA-ANEL governments do not positively influence the efficiency of Greek public hospitals by reducing inputs and increasing outputs.The HEAL-Link Database, which refers to international scientific journals as well as other databases (SCOPUS), was employed to identify the work used in this research. The literary review concerned 6275 papers referring to HPs and health unit efficiency studies. In terms of PCnH, PVQH and PCH policies, 61 papers were used, while in terms of health efficiency, 28 papers were used.The key words that we used to study HPs were Health Policies, SYRIZA-ANEL government.Papers which contained an inadequate description of a HP, or those unable to clearly classify policy measures into model categories, were excluded from our study.Τhe model of the Center of Reviews and Dissemination (2009) PICOS is used .This study is a combination of a quantitative one (using the window-DEA method) and a qualitative one . Requirements for qualitative and quantitative research are covered by the scope and quality of these studies. PCnH, PVQH and PCH policies during the SYRIZA-ANEL governance consist of the interventions made which influence the inputs and outputs of this research. We also observe that those elements influence efficiency in the long-term. The implementation of each PCnH, PVQH and PCH policy activates a mechanism which influences each input or output of this research. Thus, there is a logical connection between individual policies and their effect on each of the inputs and outputs.https://www.moh.gov.gr/).Data (inputs and outputs) generated by ELSTAT and the Greek MoH is used for the measuring of efficiency. The data is organised into groups in the 7 YPE  of the country, so that they can be adequately compared. A researcher can find more information and supplementary material in the databases of the Greek Ministry of Health (MoH)  show a minimal increase. The rest of the inputs show a significant increase . However, the increase in inputs has an unfavourable impact on efficiency.From the percentage change of the study’s outputs, it appears that hospitalisation days show almost no change, while the patients examined, and the number of hospitalised patients show an increase. The increase in outputs predisposes a positive change in efficiency. Interventions that have contributed to the change of efficiency, were the result of specific HPs. The government SYRIZA-ANE designed the HP, the Greek MoH determined their measures. HP was implemented based on the laws of the SYRIZA-ANEL governments.From these diagrams, the change in the study’s inputs and outputs is confirmed in a detailed and graphical way, in order to be useful for the interpretation of the change in efficiency.Based on the literature review and Introduction analysis, the HPs of the SYRIZA-ANEL governments can be classified into the following categories and their effect on the inputs and outputs of the efficiency study can be estimated:A)Policies that reduce inputs (and tend to increase efficiency):Changing the mode of funding through a new social security institution , tends tThe ESY human resources are reorganised during this period. As a result, staff are moved from hospitals where they were in surplus to hospitals where there was a shortage. This policy addresses the needs of previously redundant personnel and has a direct impact on the change in efficiency. Governments during this period follow previous e-health development policies . The furAn effort is made to allocate staff correctly during this period. The publication of the Health Atlas for all regions of the country  leads toThe following implemented policies of the SYRIZA-ANEL governments increase inputs. This increase is made in order to offer more health services and to meet the criterion of equal access to health services, albeit that this policy tends to reduce efficiency. The policies are as follows:The policy of increasing health funding  has a slThe policy of state and fund financing  increaseThe policy of providing extended services to beneficiaries  tends toPCnH and PVQH with an impact on inputs:B)Policies that increase outputs (and tend to increase efficiency)The SYRIZA-ANEL governments, in accordance with their electoral commitments, seek to offer more public and free health services. Although the effect of the memoranda prevents budgets from increasing, expenditure finally increases. A policy of increasing health funding  increaseThe policy of expanding beneficiaries  causes aThe abolition of tickets encourages recipients of health services to seek health services in a public general hospital. This reduces inputs but, on the other hand, definitely increases the number of outgoing patients and the number of hospitalisation days. The modernisation policy of Mental Health (MH) services increases the presence of health service recipients in public health units.Policies that reduce outputs (and tend to reduce efficiency)Development of a dual-level PFY System  causes aHPs affecting outputs:HPs of the SYRIZA-ANEL governments affect inputs and outputs and the change in hospital efficiency values as follows:A)PCHs which reduce inputsPharmaceutical cost control policy with annual budgets for classification levels of therapeutic chemical substances  reduces Health Technology Assessment (HTA) pharmaceutical expenditure policy following reconstitution of the Pharmaceutical Evaluation Committee , 51 causSustainable environmental health care with simultaneous limitation of cost  causes ePCHs that increase inputs are: the extended access policy (negative cost impact)  causes cThe depiction of the positive or negative impact of the HPs of the SYRIZA-ANEL governments on the inputs and outputs of efficiency shown in the table is useful for the discussion stage that follows.The PCHs exercised by the Syriza-ANEL governments which affect efficiency are:A)According to the Window-DEA study on the effect of the HPs of the Greek SYRIZA-ANEL governments on efficiency and on costs (costs being of significant importance to governments), the following can be noted: Regarding the change in the efficiency of Greek general hospitals, the health policies implemented during the SYRIZA-ANEL governmental period reduce the efficiency of Greek public general hospitals with ups and downs. In any case, changes in efficiency also depend on the model used, which in our case is particularly detailed, taking into account a wide range of inputs and outputs. Importantly, at this point, the following must be noted: The SYRIZA-ANEL government remained in power until July 2019 . The latThe health policies of governments during the period under study, which for the most part falls within the context of the Greek economic crisis, aim to reduce costs only in part. Cost reduction is intended to be implemented through policies that apply technocratic measures following on from previous PCHs (such as cost constraints through further implementation of ePrescription). In this respect, health policies contribute to increased efficiency . PCHs suThe SYRIZA-ANEL government reduced costs through PCnHs and PVQHs by changing the mode of funding, developing a dual-level PFY system , reorganCosts, on the other hand, increase due to certain PCnHs and PVQHs. Examples of these policies are an increase in health funding, an increase in individual categories and in total hospital costs, the policy of covering funding from the state and, the policy of expanding beneficiaries, and the policy of promoting the rights of health service recipients . Costs also rise with some of the PCHs, such as extended access policy and the health service policy during the refugee crisis .It is noted that policies are divided in terms of their impact on costs, and this concerns all three major policy categories. Policies that reduce costs (and therefore attempt to increase efficiency) and policies that increase costs (and therefore result in reduced efficiency) emerge.A reduction in the values of efficiency mean that the health policies failed to increase the hospital’s efficiency . The redB)Regarding the detailed impact of the health policies of the SYRIZA-ANEL Governments on the inputs and outputs used for the study and on the efficiency of Greek general hospitals, the following can be observed:Government health policies aim to reduce inputs in order to increase efficiency. The study found that some of the PCnHs and PVQHs tend to reduce inputs, others to increase them, the latter having a negative effect on efficiency. The reason for the detrimental effect of inputs is that the policies mentioned are aimed at achieving equality and justice. Thus, from the study of inputs, there is a large increase in economic terms, but stability in the number of beds (given that the number of beds is the most reliable data in an attempted change). However, the picture of inputs is that of an overall increase, which explains the negative impact of health policies in terms of efficiency . It seemGovernments’ policies generally aim to increase outputs in order to increase efficiency . Indeed,The study raises a number of issues that deserve to be discussed further:Survey inputs remain stable or increase:Some of the inputs (markets) increase significantly, others  increase considerably, and some  remain stable. This is detrimental to the rate of efficiency.Survey outputs remain stable or increase:One of the outputs  remains stable, one (patients examined) increases with ups and downs, and one  increases greatly. This is conducive to an increase in efficiency .HPs increase inputs:The health policies of the SYRIZA-ANEL governments are not exclusively oriented towards reducing inputs (and thus increasing efficiency). Undoubtedly, some of these policies attempt to reduce costs and inputs . However, other policies directly increase costs, leaving the possibility of increased purchases and an increase in the number of staff, as in the case of other countries . The reaHPs increase outputs:Policies that ensure equality and fairness in access to health services for all citizens and the development of the public health system, which was affected by the previous economic crisis, increase outputs .The increase in outputs is followed by the increase in inputs:It is noted that the government’s effort to increase outputs , is accompanied by an increase in inputs. Although an increase in inputs may to some extent be a desirable option for a government in order to avoid the brain-drain effect, to create jobs, or to deal with the adverse consequences of job losses in the economic crisis, it nevertheless contributes adversely to efficiency. This phenomenon (increase of outputs with simultaneous increase of inputs) as well as the reverse (decrease of inputs with simultaneous decrease of outputs) requires further investigation in order to become a general rule that can be taken into account for HPs and change in efficiency.The relation of the formation of the output value to the input value leads to a change in efficiency.The change in efficiency values can be interpreted in a one and only way: that of the change in inputs and outputs over the years and the effect of HPs on this change. We can observe the following 6 points on which the discussion takes place:The HPs of the SYRIZA-ANEL Governments extend to the three major HP categories that are applied during an economic crisis. Unlike pre-existing HPs aimed at cost reduction, these policies seem to have a dual orientation: some policies reduce the cost of a category and contain the total cost, thus positively contributing to efficiency increase. Some policies are aimed more at fulfilling the criterion of equality in the provision of health services and thus the cost inevitably increases. The overall picture of inputs shows an increase. Regarding outputs, a significant increase is achieved. It is the relative change in outputs as compared to inputs that ultimately determines the change in efficiency. From the window-DEA study, three relatively “good” years emerge  and two “bad years” . The overall picture of values is downward, and this also depends on the nature of the model used which in our case is a very detailed one. The inputs and outputs used in DEA studies are instrumental in measuring efficiency. However, it is the relative change in outputs as compared to inputs that determines the change in efficiency. The years when outputs increase significantly as compared to the increase of inputs are observed as being more efficient and vice versa. Also, a more general pattern is confirmed: an increase in outputs requires an increase in inputs.


Enhanced signaling through RAS and the mitogen‐associated protein kinase (MAPK) cascade underlies the RASopathies, a family of clinically related disorders affecting development and growth. In RASopathies, increased RAS‐MAPK signaling can result from the upregulated activity of various RAS GTPases, enhanced function of proteins positively controlling RAS function or favoring the efficient transmission of RAS signaling to downstream transducers, functional upregulation of RAS effectors belonging to the MAPK cascade, or inefficient signaling switch‐off operated by feedback mechanisms acting at different levels. The massive effort in RASopathy gene discovery performed in the last 20 years has identified more than 20 genes implicated in these disorders. It has also facilitated the characterization of several molecular activating mechanisms that had remained unappreciated due to their minor impact in oncogenesis. Here, we provide an overview on the discoveries collected during the last 5 years that have delivered unexpected insights  and allowed to profile new RASopathies, novel disease genes and new molecular circuits contributing to the control of RAS‐MAPK signaling. In this review, we briefly outline the molecular mechanisms affecting RAS‐MAPK signaling in RASopathies and provide an overview on the novel disease genes and new RASopathies emerged in the last few years.During the last 10 years, the use of hypothesis‐free disease gene discovery approaches based on genome‐wide sequencing has facilitated the identification of additional RASopathy genes /guanosine diphosphate (GDP)‐binding GTPases functioning as molecular switches that control diverse cellular processes  by modulating a multifaceted signaling network , functionally converge to favor the dephosphorylated status of RAF1 at Ser259, which is a crucial step in RAF1 catalytic activation  provides evidence of multiple circuits implicated in the negative control of RAS function as a recurrent theme of RAS‐MAPK signaling upregulation in RASopathies , also known as extracellular signal‐regulated protein kinase 2 (ERK2), encoding the terminal tier of the MAPK cascade, joined the group of the signal transducers mutated in the RASopathies.Two years ago, nearly 20 years from the discovery of 185 and Tyr187, which are located within an “activation” segment, favoring a conformation of the catalytic domain stabilizing the active site  and is a required step for the inactivation of the kinase. The reversible phosphorylated state of these two residues controls the magnitude and duration of activation as well as the nuclear localization of the kinase, which, in turn, determines the nature of the cellular response to stimuli. Specificity in substrate binding is mainly attained by interactions mediated by two “docking sites,” named D‐recruitment site (DRS) and F‐recruitment site  and reduced growth are suggestive of NS. Congenital heart defects  were reported in a relatively high proportion of patients, although hypertrophic cardiomyopathy (HCM) has not been reported. Minor skeletal defects were also a common finding. None of the subjects has been diagnosed with cancer.De novo MAPK1 variants have a non‐random distribution and promote an increased phosphorylation of the kinase, which in turn enhances the translocation of the protein to the nucleus and boosts MAPK signaling  or perturbing the regulatory mechanism controlling the activation of the kinase (changes at residues Ile74 and Ala174). Functional studies showed that the defective negative regulation exerted by MKP3 , a dual‐specificity protein phosphatase negatively controlling ERK function, represents a generalizable mechanism for the gain‐of‐function (GoF) behavior of these pathogenic variants. Of note, previous work demonstrated that amino acid substitutions in or close to the DRS can lead to either GoF or loss of function (LoF) by impairing proper MAPK1 binding to regulators and substrates, respectively  is a common finding in cancer, MAPK1 mutations define a new RASopathy. Although two individuals of the original cohort had features suggestive of NS, systematic screening of the entire MAPK1 coding sequence in large cohorts of subjects with clinical diagnosis of RASopathy did not identify other pathogenic variants. This negative result suggests that MAPK1 mutations are a rare event in subjects with NS‐related features and could be more commonly identified in patients with unclassified syndromic DD/ID.Overall, de novo 3.2LZTR1 (MIM: 600574) was identified as a gene predisposing to schwannomatosis  is a widely expressed protein that localizes to the cytoplasmic surface of the Golgi network and is characterized by six tandemly arranged Kelch motifs at the N‐terminus and two BTB/POZ  domains at the C‐terminus  are negative regulators of signaling elicited by cell‐surface receptor tyrosine kinases. Specifically, they negatively control MAPK signaling by favoring neurofibromin binding to activated GTP‐bound RAS  have recently been identified as underlying a disorder clinically resembling NS . Shared features of the originally reported patients include DD/ID, facial dysmorphism , low posterior hairline with a webbed/short neck, and cardiac involvement (HCM and/or PVS). Short stature with relative macrocephaly was documented in three individuals, and typical NS chest anomalies and other skeletal defects (spine) were also common findings. No lymphedema or skin/ectodermal anomalies were noted except for the occurrence of deep palmar creases . A number of oncogenic variants have been reported in a variety of solid tumors, including carcinomas of the endometrium, prostate, lung and liver. These somatic mutations are missense and affect residues homologous to the cancer‐associated ones mutated in HRAS, KRAS and NRAS. Of note, the p.Gln72Leu change  was identified as driver event in isolated JMML , being originally cloned from a human teratocarcinoma cDNA library , identified prenatally due to macrosomia, hydrocephalus, Dandy Walker malformation, and suspected arrhythmia, was more recently reported , this subject showed a severe and rapid lethal course of NS. He also was diagnosed with JMML. These findings, which provide the first evidence of the occurrence of genotype–phenotype correlations for this gene, indicate that mutations in RRAS2 are generally associated with a classic NS phenotype , which encodes a member of the RRAS subfamily (see above). This GTPase is implicated in a wide array of cellular processes  by modulating multiple signaling pathways, including the MAPK and PI3K‐AKT cascades , a narrow spectrum of de novo variants has recently been reported in six patients with clinical diagnosis of NS  are well known hotspots for cancer‐associated and/or RASopathy‐causing mutations in other RAS proteins , similar to what has been reported for mutations in RIT1, PTPN11 and RAF1 (limited to specific classes), and LZTR1 . Two large cohorts of patients were systematically assessed for MRAS mutations  and are not reminiscent of Mazzanti syndrome suggests that the impact of dysregulated MRAS function on intracellular signaling is not equivalent to the dysregulation driven by upregulated SHOC2 and PPP1CB function. Another aspect requiring further investigation is the specific link between MRAS hyperactivation and HCM. Accumulated evidence indicates that both the MAPK and PI3K‐AKT–mTOR pathways contribute to HCM in RASopathies . This indicates that other genes are implicated in this family of disorders or that genocopies  account for these patients. We anticipate that the routine application of genomic sequencing in the clinical practice will enable answering this open question in the next few years and is expected to provide further insights on other molecular mechanisms and circuits functionally linked to RAS signaling.None.


Systemic lupus erythematosus (SLE) is an autoimmune connective tissue disease affecting multiple organs in the human body, including the central nervous system. Recently, an artificial intelligence method called BrainAGE (Brain Age Gap Estimation), defined as predicted age minus chronological age, has been developed to measure the deviation of brain aging from a healthy population using MRI. Our aim was to evaluate brain aging in SLE patients using a deep-learning BrainAGE model.Seventy female patients with a clinical diagnosis of SLE and 24 healthy age-matched control females, were included in this post-hoc analysis of prospectively acquired data. All subjects had previously undergone a 3 T MRI acquisition, a neuropsychological evaluation and a measurement of neurofilament light protein in plasma (NfL). A BrainAGE model with a 3D convolutional neural network architecture, pre-trained on the 3D-T1 images of 1,295 healthy female subjects to predict their chronological age, was applied on the images of SLE patients and controls in order to compute the BrainAGE. SLE patients were divided into 2 groups according to the BrainAGE distribution (high vs. low BrainAGE).z-score was significantly higher in SLE patients than in controls . In SLE patients, high BrainAGE was associated with longer reaction times (p = 0.02), lower psychomotor speed (p = 0.001) and cognitive flexibility (p = 0.04), as well as with higher NfL after adjusting for age (p = 0.001).BrainAGE Using a deep-learning BrainAGE model, we provide evidence of increased brain aging in SLE patients, which reflected neuronal damage and cognitive impairment. Neuropsychiatric symptoms are commonly observed in individuals with SLE, with reported frequencies ranging from 20 to 95%, depending on the classification criteria used . These sBrain abnormalities observed in SLE patients are frequent and heterogeneous. Beyond the vascular and/or inflammatory lesions identified through MRI  and histNonetheless, these lesions lack specificity, exhibiting a continuum across patients with and without neuropsychiatric symptoms. In approximately 60% of cases, symptoms may be independent of brain damage, and instead attributed to chronic pain, altered sleep quality or corticosteroid therapy . The broRecently, an artificial intelligence method, called BrainAGE (Brain Age Gap Estimation), has been developed to measure the deviation in brain aging within a cohort of patients experiencing cognitive decline, in comparison to a healthy population without cognitive or psychiatric disorders . The appOur main objective was to evaluate brain aging in SLE by using a deep-learning BrainAGE model within an established cohort of extensively characterized SLE patients and matched healthy controls . Our sec2.2.1.The present study is a cross-sectional post-hoc MRI analysis of a well described single-center prospective SLE cohort . The stuBetween 2013 and 2016, consecutive prevalent patients with a diagnosis of SLE, attending the Department of Rheumatology, were prospectively enrolled. Inclusion criteria were as follows: diagnostic of SLE meeting at least 4 criteria of the American College of Rheumatology (ACR) classification , female n = 1; cystic lesion, n = 1).During the same time period, female control subjects within the same age range, free of autoimmune, neurological or psychiatric disorders were recruited in a control group among health care workers and university employees at our institution. Seventy-one patients and 25 age-matched controls constituted the cohort population. Two participants  were secondarily excluded from the present analysis because of they had a focal brain lesion . The concentrations of protein S100A8/A9 in serum were measured with the Bühlmann MRP8/14 ELISA kit, Switzerland, according to the manufacturers’ instructions . Plasma 2.4.2.4.1.3 isotropic, matrix = 256 × 256 × 176, TE = 2.54 ms, TR = 1900 ms, TI = 900 ms, flip angle = 9°) and a FLAIR sequence .MRI acquisitions were performed on a 3T MRI , using a 20-channel head coil. The protocol included a 3D-T1 MPRAGE (magnetization-prepared rapid gradient-echo) 2.4.4.n = 1,295, validation n = 208).For the prediction of chronological age based on brain images, our model was based on a 3D convolutional neural network architecture, as previously published . For theThe training dataset was constituted of data compiled from several publicly available sources: IXI (Information eXtraction from Images),The model input was preprocessed 3D-T1 images with dimensions of 182 × 218 × 182 voxels. The model was based on a 3D convolutional neural network using an architecture previously described . The weiAs recommended in BrainAGE studies, we removed any common variance with chronological age before submitting the residualized version of BrainAGE, using linear regression :α is the regression coefficient associated with the chronological age, and in our study α = 0.13.z-score taking the control group as reference  and brain volumes were compared between controls and SLE patients using a student-t test. WMH volumes were compared using a Mann–Whitney U test. Then, in order to estimate the risk factors for increased BrainAGE, SLE patients were divided into 2 groups (high vs. low BrainAGE). To define the cut-off value between High and Low BrainAGE, we used the “reaction time” performance. Increased reaction time is one of the main neuropsychological landmarks of brain aging. We performed a ROC-analysis and calculated the Youden Index. The optimal threshold to identify patients with low “reaction time performance” (standardized score < 80) was BrainAGE z-score = 0.9. We therefore used this threshold to separate high and low BrainAGE groups. Group comparisons for biomarkers of neurodegeneration between high and low BrainAGE patients and healthy controls were done using a ANOVA . As plasma concentrations of NfL increase with age, group comparisons were adjusted for age using an ANCOVA procedure. We also performed correlation analyses and calculated Pearson correlation coefficient  in patients and healthy controls. Finally, to identify the clinical/biological predictors of high BrainAGE, group comparisons between high and low BrainAGE patients were done using a student t-test for continuous variables and a chi-square test for categorical variables. Parameters associated with BrainAGE with a value of p <0.2 in univariate analysis were included in a multivariate logistic regression model with BrainAGE as a binary depend variable (high vs. low). To avoid multicollinearity, correlations between variables were checked and variables with r > 0.6 were excluded (disease duration and ongoing prednisolone treatment). A threshold of p < 0.05 was used for all statistical significance.Statistical analyses were performed using SPSS version 26 (RRID:SCR_002865). Continuous variables are presented as mean (± standard deviation) and categorical variables are presented as numbers (percentage). The normality of the distribution of quantitative variables was assessed visually. Plasma NfL concentrations were log-transformed to satisfy the normality assumption. First, BrainAGE . BrainAGE z-score was significantly higher in SLE patients, revealing an increased brain aging (0.6 [±1.1] vs. 0 [±1]) (p = 0.045)  and 37.0 (±9.4) years, respectively ( 0 [±1]) . SLE pat= 0.045) . The volAttention maps demonstrated that BrainAGE prediction was mainly based on the following brain areas: right lenticular nucleus, left and right medial prefrontal cortex, left dorso-lateral prefrontal cortex and inferior frontal cortex, posterior part of the body of the corpus callosum, pons, left and right cerebellum hemispheres .3.2.z-score > 0.9) and well-established biomarkers of neuronal damage (p = 0.001). White matter volume was also lower in high BrainAGE vs. low BrainAGE patients (p = 0.02). High BrainAGE patients had higher plasma NfL concentrations after adjusting for age, compared to low BrainAGE patients and healthy controls (p < 0.001) (p = 0.03), but there was no significant difference between high and low BrainAGE patients.To validate that BrainAGE was reflecting neurodegeneration in SLE patients, we analyzed the association between high BrainAGE (BrainAGE l damage . Compare< 0.001) . WMH volThen, we explored the neuropsychological correlates of increased BrainAGE.p = 0.76 and “SLICC B”: 8/24 [33.3%] vs. 14/46 [30.4%], 0.80). Nevertheless, SLE patients with high BrainAGE had poorer performance compared to low BrainAGE (BrainAGE z-score ≤ 0.9) and/or healthy controls in several cognitive domains (p = 0.001), reaction time (p = 0.02) and cognitive flexibility (p = 0.04). Illustrative cases are presented in Neuropsychiatric involvement according to the SLICC attribution models were not more prevalent in high BrainAGE patients , had shorter disease duration  and were more likely to have a non-malarial disease-modifying antirheumatic drug (DMARD) . High BrainAGE tended to be associated with higher disease activity , higher prednisolone daily dose  and low complement levels . We did not find any association between BrainAGE and the presence of anti-phospholipids antibody. In multivariate analysis .Finally, we evaluated the association between BrainAGE and the clinical and biological characteristics of disease, to identify the main determinants of increased brain aging . In univanalysis , age at 4.In this retrospective cross-sectional analysis, we provided evidence of increased brain aging in SLE by applying a deep-learning model to brain MR images. We demonstrated that high BrainAGE was associated with established markers of neurodegeneration and with worse performance in several cognitive domains. Interestingly, higher BrainAGE scores were observed in younger patients and patients with ongoing non-malarial DMARD, suggesting a more active SLE phenotype.One of the main strengths of our study lies in the use of a deep-learning model to compute a fully automated and time-efficient measurement of brain aging. This approach enables an individualized and non-invasive assessment, which could be implemented into clinical practice. Unlike conventional machine learning models based on brain parcellation and quantification of regional grey matter and white matter volumes, the use of a 3D CNN architecture allowed us to work directly on MR images registered in the MNI space, without introducing any prior assumptions. Processing steps were deliberately limited minimized, using a rigid co-registration method to preserve anatomical details. Notably, our model’s performance to predict the age of healthy controls on the validation sample was in the range of other established models . This apIn this study, the utilization of a deep-learning model allowed us to capture brain changes indicative of neurodegeneration in SLE, even within the context of an average low disease activity and subtle differences in brain volumetry between patients and controls. We found that increased brain aging in SLE patients predominantly correlated with a reduction in white matter volume. This observation is consistent with standard MRI analyses showing white matter lesions in 60% of SLE patients with neuropsychiatric symptoms . Beyond In our population, increased brain aging in SLE patients resulted in poorer cognitive performance. While SLE patients displaying low BrainAGE had cognitive test scores within the normal range, those with high BrainAGE performed significantly worse in several cognitive domains, reaction time and psychomotor speed being mostly affected. Processing speed is one of the main cognitive domains affected by normal ageing, which has been attributed to a decreased efficiency of interregional communication within the brain . Indeed,Finally, we aimed at identifying the main risk factors of increased brain aging. We found that the clinical phenotype of SLE patients with high BrainAGE was a young subject, with short disease duration, high disease activity and low complement level, treated by non-malarial DMARD and high dose of prednisolone. Drawing from these exploratory results, we can speculate that increased brain age was mainly driven by persistent inflammatory activity under medication. Typically, disease activity in SLE tends to peak early in the disease course and subsequently decline over time . PeschkeConversely, we did not find any association between increased brain age and antiphospholipid antibodies or WMH volume. A higher risk of ischemic and hemorrhagic stroke is attributed to SLE with a twofold increase compared to the general population . InteresThe main limitation of our study was its relatively small sample size, which currently curtails the applicability of BrainAGE on an individualized basis and precludes a comprehensive analysis of the intricate interplay between distinct neuropsychiatric manifestations and brain aging. We also acknowledge that the association with disease activity scores was close but did not reach statistical significance. This could be explained by the overall low disease activity observed in our cohort and it warrants validation across populations with a more severe phenotype. Future longitudinal studies will also provide additional insight into the association between age, disease duration and brain aging, as well as the role of glucocorticoids and immunosuppressive treatment.In conclusion, using a deep-learning BrainAGE model, we provide evidence of increased brain aging in SLE patients, which reflected neuronal damage and cognitive impairment. BrainAGE could be evaluated as an adjunctive diagnostic tool for assessing brain involvement in SLE.pia.sundgren@med.lu.se.The datasets presented in this article are not readily available because the data presented in this study are available from the corresponding author upon reasonable request and approval of the Institutional Review Board. Requests to access the datasets should be directed to The studies involving humans were approved by regional ethics committee in Lund . The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.GK: Conceptualization, Formal analysis, Funding acquisition, Methodology, Visualization, Writing – original draft. TR: Data curation, Investigation, Writing – review & editing. KZ: Data curation, Investigation, Writing – review & editing. RL: Formal analysis, Methodology, Software, Writing – review & editing. MG: Methodology, Software, Writing – review & editing. J-PP: Resources, Supervision, Writing – review & editing. AB: Resources, Supervision, Writing – review & editing. OH: Funding acquisition, Resources, Writing – review & editing. AJ: Funding acquisition, Resources, Supervision, Writing – review & editing. PS: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing – review & editing.


New developments in the field have provided exciting insights into how cAMP utilizes compartmentalization to ensure specificity when the message conveyed to the cell by an extracellular stimulus is translated into the appropriate functional outcome. cAMP compartmentalization relies on the formation of local signaling domains where the subset of cAMP signaling effectors, regulators and targets involved in a specific cellular response cluster together. These domains are dynamic in nature and underpin the exacting spatiotemporal regulation of cAMP signaling. In this review, we focus on how the proteomics toolbox can be utilized to identify the molecular components of these domains and to define the dynamic cellular cAMP signaling landscape. From a therapeutic perspective, compiling data on compartmentalized cAMP signaling in physiological and pathological conditions will help define the signaling events underlying disease and may reveal domain-specific targets for the development of precision medicine interventions. In eukaryotes, cAMP production is triggered by the activation of G protein-coupled receptors (GPCRs), a large and diverse protein family with around 800 members  that traThe most extensively studied target of cAMP is PKA. The inactive PKA holoenzyme consists of two regulatory (R) and two catalytic (C) subunits. There are four versions of R subunits, further classified as type I  and type II , and three versions of C: Cα, Cβ, and Cγ. cAMP binds to R in the inactive tetrameric holoenzyme R2C2 inducing a conformational change that removes the inhibitory action of R on C subunits, leading to phosphorylation of nearby substrates. PKA is an extremely promiscuous kinase with the ability to phosphorylate many targets within the same cell, thus contributing to the ability of cAMP to mediate a multiplicity of cellular effects. It is now widely accepted that cAMP signaling achieves specificity of response via compartmentalization. Given the large number and variety of activating stimuli, receptors and PKA substrates, cAMP/PKA signaling is organized within the cell as a collection of multiple pathways controlled by a precise spatial and temporal coordination of signal transduction. Such organization enables the correct translation of the message conveyed by a given external stimulus into the appropriate phosphorylation events and cellular responses .The spatial constraint on cAMP signaling became obvious with the observation, almost 40 years ago, that stimulations of cardiac myocytes with isoproterenol or prostaglandin E1 led to different cellular responses despite the fact that a similar increase in cAMP was generated in response to the two stimuli . EvidencOne example of cAMP signaling compartmentation is the organization of signaling domains at the plasma membrane, where signaling domains are organized by scaffolding proteins that may bring together GPCRs, cAMP effectors, cAMP producing (ACs) and degrading (PDEs) enzymes, signaling regulators and targets into functional domains that are few tens of nanometers in size ,10,11 F. The proAlthough GPCRs are one of the most successful therapeutic target families, current drug discovery programs remain associated with very high attrition rates due to the complexity of the signaling associated with these receptors. The realization that cAMP signaling is organized in discrete subcellular domains offers a new opportunity for the development of drugs that selectively target individual domains for therapeutic interventions that can achieve subcellular precision. Establishing the blueprint of individual cAMP signaling compartments across cell types, activation states and disease states would provide invaluable insight for targeted drug design. Quantitative proteomics methodologies that uncover protein phosphorylation, interaction and proximity provide powerful tools for this type of analysis and are increasingly being applied to study cAMP compartmentalization. Basic principles of the methods and specific examples of the applications to address particular questions are described in detail below.cAMP signaling uses different mechanisms, including protein–protein interactions (PPIs) and protein phosphorylation, to relay, process, and translate signals into cellular responses. cAMP signaling compartmentalization heavily relies on the formation of local signaling domains where cAMP signaling components involved in a specific cellular response cluster together. Compartmentalization is critically dependent on the assembly of multiple signaling components that come together via protein–protein interactions to become functional signaling units. Within such domains, cAMP signals are translated into specific cellular responses via the phosphorylation of target proteins. As such, mapping the domain interaction landscape and defining the downstream phosphorylation events are the key aspects of compartmentalized signaling studies. Typically, the proteomics analysis of PPIs and protein phosphorylation is usually performed in a quantitative manner, so the changes in phosphorylation events across experimental conditions can be compared, and true signalosome components can be distinguished from the background. To guide the reader through the applications discussed in this review paper, in Box 1Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the main workhorse driving proteomics studies. In bottom-up proteomics, proteins are first extracted and proteolyzed with a protease, such as trypsin, to yield smaller peptides . To reduTo enable protein quantification, (i) label-based and (ii) label-free methods can be additionally coupled to LC-MS/MS. In label-based methods, stable isotopes are usually introduced in a protein/peptide via (i) metabolic (in vivo) labeling or (ii) chemical (in vitro) labeling ,21,22,23It is noteworthy that not all biological samples are amenable to metabolic labeling, notably clinical samples. For these samples, chemical labeling, which involves in vitro reaction between labeling reagents and proteins/peptides, is applicable to any biological samples with the caveat of higher experimental variations since labeled samples are mixed at a later step. Chemical labeling reagents contain different heavy isotopes to produce mass shifts in the MS1 spectrum  or MFor label-free strategies there is no limitation with respect to the number and origin of samples. This approach is therefore, cost-effective and convenient, as no labeling is required. Since samples are separately collected, processed, and mass-analyzed, such experiments need very careful execution to minimize experimental or analytical variations. Label-free quantification can be as simple as spectral counting, where the number of peptide-spectrum matches (PSM) of a protein is used as a proxy for protein abundance. However, spectral counting is biased towards larger proteins, which generate more peptides than small proteins . RecentlUntil now, most discovery-based quantitative proteomics studies have been performed with mass spectrometers that operate in a data-dependent acquisition (DDA) mode. In DDA, the top N  most intense peptide precursors in a survey MS1 scan are sequentially selected for fragmentation and MS2 detection. The downside of this approach is that DDA is biased towards measuring highly abundant proteins. Additionally, due to the stochastic nature of precursor selection, if the number of precursor ions exceeds the number of top N, peptides may not be consistently detected in all LC-MS/MS runs, resulting in many missing values ,33. As aIn MS-based interaction proteomics, the constituents of protein signaling complexes are systematically mapped. Several MS-based interactomics strategies, including affinity purification-MS (AP-MS), proximity labeling-MS (PL-MS), crosslinking-MS and coFractionation-MS (coFrac-MS), have been comprehensively reviewed recently . Among tIn recent years, PL-MS has been developed to overcome these limitations. Since its introduction in 2012 , PL-MS hIf transient interactions have ceased and the binding partners have moved away from the bait protein at the time of extraction, that is not a problem since interacting, and other relevant proteins have already been “marked”. The denaturing condition can then be applied to solubilize the whole proteome without the need to preserve protein interactions. This characteristic of PL approaches is a great advantage for spatiotemporal proteomics. Biotinylated proteins are then purified using avidin/streptavidin-coated beads and are identified by MS. As the biotin-avidin association is the strongest known non-covalent interaction, multiple stringent washing steps can be performed to minimize background contaminants. Similar to AP-MS, to distinguish bona fide proximal proteins from irrelevant labeled proteins, the PL experiment is usually combined with quantitative proteomics approaches, either based on isotopic labeling or label-free quantification.2O2 treatment in the presence of biotin-phenol to produce the radicals, and labeling can be achieved in as short a time as 1 min [2O2 availability. This becomes very practical for studies that require cell incubation with ligands for a period of time before initiating the labeling. Additionally, the short half-life of the radicals (<1 ms) and their inability to cross membranes ensures that labeling occurs only within an estimated radius of 20 nm and is contained only within the candidate space [There are two main enzyme systems used in PL-MS, which are promiscuous biotin ligases, or BioID, in the case of proximity-dependent biotin identification and peroxidases, or APEX, in the case of peroxidase-catalyzed proximity labeling . The proas 1 min , meaningte space . These ate space , TurboIDte space , miniTurte space , UltraIDte space  and Micrte space  biotin lte space   and are described elsewhere . For a lTo translate a list of differentially regulated phosphorylation events into biological insight, Gene Ontology (GO) enrichment analysis can be performed to deconvolute the biological processes in which the list of proteins harboring the phosphopeptides are involved . AnotherIt is widely appreciated that not only can different GPCRs trigger different signals in the same cell, but the same GPCR activated by different agonists can transduce distinct cellular responses through receptor compartmentation . An addiGPCR signaling was initially believed to happen exclusively at the plasma membrane. In the case of GPCR signaling to ACs, it was demonstrated that the cAMP pool generated by the receptor at the plasma membrane could reach only a nanoscale distance and is short-lived due to local PDE activity  and rapi2+ handling and excitation-contraction coupling, while β2AR signaling induces modest chronotropic and no lusitropic responses. β1AR signaling drives the expression of pro-apoptotic genes while β2AR signaling promotes antiapoptotic signaling and cardiomyocyte survival, possibly through Gαi coupling or activation of β-arrestin-mediated pathways  and the highly homologous β2-adrenergic receptors (β2ARs) bind to norepinephrine and activate G protein/cAMP/PKA signaling. β2ARs are highly enriched in specialized membrane structures, including T-Tubules and caveolae, whereas the homologous β1ARs are distributed across the entire cell membrane . While βiewed in ). In theFrom the examples above, it is clear that receptor compartmentalization provides well-defined signaling domains that contribute to the specificity of the downstream response. How exactly this is achieved, however, is far from clear. For example, as the trafficking route of a given receptor appears to depend on the specific ligand bound to it, it is conceivable that the unique conformation the receptor adopts after binding could facilitate the recruitment of distinct compartment-specific sorting proteins that are not presently known. How do cAMP signals generated at internal membrane sites reach the nucleus and selectively drive ligand-specific cAMP-dependent transcription events? The identification of proteins recruited to the internal membrane domains could provide critical cues.Despite the open questions, recent findings indicate that targeting disease-specific signaling at defined subcellular locations may be a valid alternative to the prevalent and rather blunt approach that targets the receptor at the plasma membrane ,88. SuchAPEX-based proximity labeling has been exploited to capture both location- and time-dependency of ligand-specific GPCR signaling dynamics in simplified cell models ,90,91,92As a proof-of-concept, Lobingier et al. investigated the signaling networks associated with the well-studied β2AR and the less explored delta-opioid receptor (DOR) in the HEK293 model system stably expressing fusions of each receptor with APEX2 . SeveralThe same strategy was then utilized to study the underexplored DOR signaling and trafficking network, with a focus on the characterization of DOR-engaged endosomal ubiquitin network components that target the active receptor to lysosomes. Briefly, DOR-APEX2-expressing cells were exposed to the opioid agonist for varying periods of time. Biotinylated proteins identified from each time point were quantified against proteins identified from spatial references using label-free quantification. This experiment led to the identification of 29 specific interacting proteins, which were classified as being biotinylated at an early, middle, or late phase following receptor activation. WWP2 and TOM1, two ubiquitin-linked proteins that showed the strongest labeling in the late phase, were further validated as network components that mediate endosomal sorting of the DOR to the lysosome .APEX has also been utilized to probe biased signaling. One example is the angiotensin II type 1 receptor (AT1R), which responds to the peptide hormone angiotensin II (Ang II) and has a critical role in cardiovascular physiology. AT1Rs are targeted for the treatment of cardiovascular diseases, and G-protein- and β-arrestin-biased AT1R ligands have been developed . HEK293 To our knowledge, APEX-MS is currently the only platform that allows the identification and quantification of GPCR signaling components within a native condition in an unbiased, medium- to high-throughput fashion. However, the GPCR APEX is still in its infancy as, in all studies reported so far, the experiments were performed in a cell model system, with exogenous expression of the APEX-tagged receptor and treatment of the cells with high doses of agonists. This may result in protein interactions or signaling events that may not happen in more physiologically relevant settings. The next step is for such an experiment to be performed in primary cells and with tagged proteins expressed at the endogenous level. For instance, β2AR APEX could be performed in iPSC-derived cardiomyocytes expressing APEX-tagged receptors where the receptor function is of importance, and its interactome could be studied in parallel with β1AR APEX in normal and disease conditions. Moreover, the platform can also be utilized to study organelle-resident GPCRs. For instance, more than 40 functional nuclear GPCRs (nGPCRs) have been identified in several cell types, and there is a growing body of evidence supporting a pathological role for these receptors in the cardiovascular and nervous systems ,93. HoweOne of the key aspects of cAMP signaling compartmentation is the anchoring of PKA holoenzymes in proximity to the source of cAMP production and/or to their substrates, which is achieved via PKA binding to AKAPs . ProteinOwing to the AKAPs’ diverse subcellular localization and ability to assemble multiple signaling components, customized cAMP signaling units can then be arranged at specific local sites. The functional importance of AKAPs in organizing cAMP local domains is supported by several meta-analysis studies that identify a link between SNPs and mutations in AKAPs with increased risk of diseases ,97,98,99Over the past decade, studies have evaluated selective targeting of individual AKAP signaling complexes as a strategy to manipulate cAMP-mediated cellular events in diseases , with thThe cAMP-centric chemical proteomics screening approach can provide insight into which PKA-AKAP scaffolds might underlie the physiological process or pathological condition of interest. To further characterize the molecular composition of a relevant cAMP signaling domain, interaction proteomics can be applied by using the scaffold protein as bait. For example, SPHKAP was among the AKAPs that showed a dramatic increase in association with PKA in HF , suggest2+ pump SERCA-PLN complex to regulate adrenergic effects on Ca2+ re-uptake [AKAPs contain targeting domains to anchor PKA to distinct subcellular compartments and in proximity to specific substrates. In different cell types, the same AKAP can be found at different cellular locations, engaging with different sets of proteins to serve different functions. For instance, in cardiomyocytes, AKAP18γ/δ localizes to the sarcoplasmic reticulum, where it scaffolds PKA with the Cae-uptake , while ie-uptake . Endogene-uptake . Howevere-uptake .Even though the identification of components of AKAP signaling scaffolds in cell models can yield insights into the potential cellular function regulated by that complex, one should bear in mind that this role may be cell-type and/or cellular-state specific. Thus, experiments performed using physiologically relevant cells, and tissues are recommended. For AKAPs where a specific antibody is available, the signaling complex can then be studied directly in an endogenous context. For instance, AKAP12 is required to promote endothelial cell migration through unknown mechanisms. In order to identify components of the AKAP12-associated cAMP signaling domain involved in the regulation of cell migration, immunoprecipitation was performed by incubating an AKAP12-specific antibody or control IgG with the lysate from human endothelial cells where migration had been triggered by treatment with vascular endothelial growth factor (VEGF). Enriched proteins were analyzed by LC-MS/MS, and comparison was performed using the LFQ approach. The study revealed that, in migrating endothelial cells, endogenous AKAP12 was strongly associated with multiple key regulators of actin dynamics and actin filament-based movement and the VEGF stimulation was translated, via PKA-mediated phosphorylation events, into actin cytoskeleton remodeling and cell movement .2 enrichment method and identified by LC/MS-MS in a label-free fashion. Compared to WT mice, the study identified 216 phosphopeptides differentially expressed in the Cypher-KO tissue, about half of which were down-regulated. In this case, the hypophosphorylated peptides that harbor a PKA consensus motif are likely to represent PKA-dependent phosphorylation events regulated by Cypher. These include β-catenin (Ser675), vimentin (Ser72), and the known PKA substrate Troponin I (Ser23/24) [As mentioned earlier, AKAPs scaffold PKA holoenzymes with their substrates. This has led to the concept of AKAP-centric phosphoproteomics profiling to identify compartmentalized PKA phosphorylation events orchestrated by a specific AKAP. The RII-specific AKAP Cypher/ Zasp was shown to be strongly associated with dilated cardiomyopathy (DCM), and the cardiac L-type CaV1.2 calcium channel was the only known PKA effector regulated by this AKAP upon β-adrenergic activation . To screer23/24) . The stuer23/24) . This dier23/24) .The principal intracellular target for cAMP is PKA. PKA activation requires cooperative binding of cAMP to two sites on each R subunit. Upon binding of four molecules of cAMP, the catalytic subunits are freed from the inhibitory action of R subunits and phosphorylate nearby substrates at serine/threonine residues located within the consensus amino acid sequence (R/K)-(R/K)-x-(pS/pT), where x is any amino acid ,124.Besides serving as a cAMP binding site, the R subunit can be considered as an adaptor that tethers bound C subunits to distinct subcellular sites near their protein targets. cAMP binding to R subunits is thought to release active C subunits, raising the question of how specificity is maintained when the active C subunit is no longer attached to the AKAP scaffold. A study in living neurons may provide cues to this conundrum . Upon ac2/HCO3 [As a major target of cAMP, it is safe to assume that PKA resides in a large proportion of functional cAMP signaling units. In a subcellular or suborganellar compartment where the cAMP interactome is not completely known, PKA can thus be used as a “proximity bait” to discover the landscape of cAMP signaling domains in that compartment. For instance, it is apparent that there are two separate cAMP signaling systems that operate on the surface of and inside mitochondria. The signaling cascades hosted at the outer mitochondria membrane are well established and are involved in the regulation of apoptosis and mitochondrial dynamics ,128. In 2/HCO3 . Althoug2/HCO3 , studies2/HCO3 . First, 2/HCO3 . These pA recent study from Scott’s laboratory demonstrated the use of this strategy to identify the local landscape of cAMP signaling events relevant to Cushing’s syndrome . SomaticIn a different paradigm, a comprehensive analysis of phosphorylation changes upon inhibition of PKA activity can uncover specific cellular processes selectively regulated by PKA. System-level phosphoproteomic profiling of a kinase-specific phosphorylation activity can be considered a large-scale proximity analysis where the kinase comes into physical contact with its substrates . As a pr2 and Fe-IMAC sequential enrichment method. As expected, the two kinases were shown to have a substantially different set of phosphorylation targets, with most of the PKA sites being found to be decreased in Cα-null cells. Interestingly, several differentially-regulated sites of PKA-RI and RII, AKAPs, and PDEs were identified in Cα-null cells, while only one site in AKAP12 was identified in Cβ null cells, suggesting that Cβ is less associated with PKA-AKAP complexes than Cα is. Cα targets were mainly associated with cell membranes and vesicles, whereas Cβ targets were related to the actin cytoskeleton and cell junctions [The authors further investigated the PKA-dependent signaling landscape specific to Cα and Cβ. In native CD and mpkCCD cells, the two catalytic subunits are expressed at a comparable level, in keeping with the notion that they are functionally non-redundant . To dissunctions .If these findings are taken together, they indicate that the two catalytic subunits operate at discrete locations, engaging with distinct sets of local targets. Cα might be associated with a cAMP signaling domain in the AQP2 storage vesicles or at the apical membrane, while Cβ may operate at actin barrier domains. One can then utilize these two catalytic subunits as bait to dissect a specific signaling pathway in response to vasopressin. For example, a major element of vasopressin action in CD cells is the dramatic rearrangement of the actin barrier for proper trafficking of AQP2-bearing vesicles to the apical membrane . The locThe level of cAMP is determined by the rate of synthesis by ACs and the rate of degradation by the cyclic nucleotide-hydrolyzing enzyme PDEs. There are eight different families of cAMP-degrading PDEs . PDE families include multiple genes and several splice variants, giving rise to multiple isoforms. PDE isoforms differ greatly in their tissue distribution, and multiple PDE isoforms can be expressed in an individual cell type . DifferePDE inhibitors are currently in use to treat conditions that associate with the dysregulation of cAMP signaling, owing to the role of PDEs as signal terminators. For example, PDE3 inhibitors are used clinically for the treatment of acute, refractory heart failure. However, these drugs are associated with significant side effects ,147, posPDE-selective inhibitors have been exploited to study the roles of different PDEs in the regulation of cyclic nucleotide signaling. A collection of inhibitors highly selective for each PDE family is commercially available . These iThis platform was used to profile cAMP-mediated phosphorylation events involved in steroidogenesis in the mouse cell line MA-10 . An incrIn another study, this quantitative phosphoproteomics platform was used to characterize, in label-free experiments, the PDE-regulated phosphoproteomes in the T lymphocyte cell line Jurkat treated with various combinations of PDE inhibitors and a low concentration of prostaglandin 2 . TreatmeOne limitation of the PDE family-centric phosphoproteomics profiling described above is that it does not provide insight into the specific role of individual PDE isoforms, as there are no available isoform-selective PDE inhibitors. The analysis of PDE isoform-selective interactomes, however, can overcome this shortcoming, and by cross-referencing the PDE family–dependent phosphoproteome with the PDE isoform-specific interactome, a more detailed view of the full cellular landscape of cAMP signaling events in response to specific receptor activation should be attainable. This information could then be used as a starting point to design single-domain targeting interventions, e.g., based on individual PDE isoforms displacement, to raise cAMP only at specific locations, resulting in highly targeted functional effects.Surprisingly, data on a PDE isoform-selective interactome are very scarce. The analysis of the interactions in which a PDE isoform is involved is critical to link a specific enzyme with specific cellular responses. For example, treatment with an AC agonist, ectopic expression of PKA Cα, or pharmacological inhibition of endogenous PDE2A activity were shown to impair translocation of the ubiquitin ligase Parkin, a key effector of mitophagy to depolarized mitochondria, hence inhibiting mitophagy ,150. Of As more information becomes available on how cAMP/PKA signaling exploits compartmentalization to translate individual signal inputs into the appropriate functional outputs and how such organization is disrupted in disease, it also becomes apparent that the highly structured architecture of this signaling pathway provides extraordinary potential for the development of targeted interventions to normalize signaling only where required, without perturbing the entire network. The ability of PL-MS techniques to capture both stable, weak and/or transient PPIs in combination with state-of-the-art phosphoproteomics profiling technologies offers a powerful toolbox for resolving in detail the map of the cellular cAMP signaling landscape, both in physiological and pathological conditions. For example, PKA-R and/or PKA-C PL-MS can be performed to uncover the full repertoire of PKA signaling domains in the cell overall A. For a The map of the cAMP signaling landscape generated in this way can then be used to reveal potential protein targets or protein interactions that can be targeted to manipulate local signaling via domain-specific intervention. Several strategies have been proposed to intervene in discrete cAMP domains. Synthetic peptides mimicking the A-kinase binding domain of AKAPs have been developed to displace PKA from the AKAP scaffold ,151,152.Future studies will no doubt provide further details on the molecular composition, functional role and regulation of the multiple cAMP signaling domains in specific cell types and on how the cAMP signaling landscape is remodeled in disease. The already rich proteomics toolbox will see new developments and will significantly contribute to progress in this field, ultimately offering rational ground for the design of therapeutics that target molecular mechanisms with subcellular precision.


HTLV-1 is associated with other inflammatory disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), pulmonary disorders, infective dermatitis, and uveitis. Prevalence studies are lacking, but limited data suggest that at least 10 million people are infected, and the actual number is likely closer to 15–20 million infected individuals. Japan and the Caribbean are two areas globally where prevalence rates are significantly higher. HTLV transmission occurs by vertical transmission (predominantly via breastmilk) or horizontally through sexual intercourse, blood transfusions, or shared syringes.Human T-lymphotropic viruses (HTLVs) are oncogenic human retroviruses. HTLV-1, as the etiological agent of adult T-cell leukemia/lymphoma (ATL), a rare but highly aggressive CD4The current therapeutics for HTLV-associated diseases has very limited benefits. Therefore, the development of new therapies is of crucial importance. More basic, applied, and clinical research is needed in order to develop HTLV vaccines and novel treatment options against ATL and HAM/TSP. This Research Topic aims to present the recent discoveries related to potential novel drugs for HTLV-1-related diseases, new research on the link between antisense proteins and diseases, as well as clinical updates on how HTLV-1 infection affects pregnancy outcomes.Komatsu et al. examined the clinical data of HTLV-1-infected and seroconverted pregnant mothers who enlisted in the database of a prefecture-wide antenatal adult T-cell leukemia prevention program from 2011 to 2018 in Nagasaki, Japan. They produced the first report with chronological data on HTLV-1 proviral load on this population. While infection had no adverse effects on pregnancy outcomes, a positive correlation between the increase of proviral load and the number of parities in HTLV-1 positive women was observed. This report underscores the importance of thorough monitoring for HTLV-1 positive mothers and infants.Ernzen et al. evaluated the potential of an inhibitor of the protein arginine methyltransferase 5 (PRMT5) to hinder ATL induction. PRMT5 is involved in epigenetics regulation and is overexpressed in HTLV-1-transformed cell lines. They demonstrated that the PRMT5-inhibitor EPZ015666 could prevent the HTLV-1-driven immortalization of cells and exhibited selective toxicity in HTLV-1 transformed cell lines as well as in HTLV-1-infected primary T-cells in a dose- and time-dependent manner. Moreover, the treatment employing the PRMT5-inhibitor significantly reduced the mortality rate in murine models. The authors conclude that PRMT5 is implicated in the HTLV-1-mediated T-cell transformation and disease pathogenesis in vivo and that EPZ015666 may have therapeutic value for the treatment of ATL, highlighting the importance of PRMT5 as a potential new target to the development of anti-ATL drugs.Koseki et al. tested if EZH1/EZH2 (enhancer of zeste homolog 1/2) inhibitors could reduce cell proliferation and HTLV-1 proviral load and modulate cytokine production. They concluded that EZH1/2 dual inhibitors can reduce the proliferation of HTLV-1-infected cells and HAM-derived peripheral blood mononuclear cells (PBMC) in a dose-dependent fashion. Additionally, the drugs could suppress the exacerbated immune response in infected cells, as observed by the increase of IL-10, although the levels of IFN-gamma and TNF-alfa were not reduced. These data indicates that the EZH1/2 dual inhibitors may be effective therapeutic agents for HAM. To further confirm these promising data, the authors suggest the conduction of proof-of-concept clinical trials in patients with HAM.Another study aimed to address the therapeutical potential of histone methyl transferase inhibitors in HTLV-1-associated myelopathy (HAM). Lin et al. summarized the main recent findings regarding the antisense transcripts of HTLV-1, HTLV-2, and HIV-1, shedding light on the role of these transcripts and proteins on viral lifecycle, and also on the cellular factors that regulate their transcription. They highlighted that antisense mRNAs of HTLV-1, HTLV-2, and HIV-1 are transcribed from 3' LTR and have multiple transcription start sites. All of them have their expression driven by TATA-less promoters and these antisense transcripts can give rise to proteins. This review also called attention to the role of myocyte enhancer factor-2 (MEF-2) protein family on the regulation of HBZ (HTLV-1 basic leucine zipper factor) transcription. The authors also provided an overview of the role of HBZ in ATL disorder: HBZ has several host interacting partners involved in the promotion of T-cell proliferation, transition of G1/S phase, transcriptional factors, and coactivators (such as p300/CPB) and it is considered an inhibitor of tax-mediated transcription, which is a function also played by its HTLV-2 antisense counterpart, the protein APH-2 (HTLV-2 antisense protein). The authors indicate the importance of further research to elucidate the mechanism of MEF-2 family in HBZ regulation and neoplasia. They also suggest antisense genes may be potential targets for therapeutic interventions. To date, CRISPR silencing of HBZ was deleterious for ATL cell lines, xenografts, and patient samples, encouraging further studies in vivo models.Several retroviruses share an intriguing feature: they have an open reading frame on the negative strand of their genomes, which results in natural antisense transcripts. Liu et al. investigated the potential interaction between HBZ, the nucleolar proteins nucleophosmin (NPM1/B23) and nucleolin (C23), and their implications on the pathogenesis of ATL. They have proven the interaction between NPM1/B23 with HBZ in the nucleolar region in different cell lines. The authors also observed C23 interaction with HBZ. Interestingly, an isoform of HBZ, sHBZ, can interact with its own shbz mRNA. As both proteins are found in nucleolar regions and interact with each other, they hypothesize that NPM1/B23 may form a complex with HBZ and C23. Additionally, downregulation of NPM1/B23 led to a reduction of sHBZ expression along with an increase of Tax expression. In this work, the authors presented novel interaction partners for HBZ, and suggested that these interactions may be involved in HBZ functions, contributing to ATL development and calling attention to the importance of further studies to understand the outcome of these interactions in the pathogenesis of ATL.In summary, this Research Topic offers valuable insights into potential targets and promising drugs that could broaden the treatment options for diseases caused by HTLV-1. Additionally, it presents recent discoveries related to seroconversion's effects on pregnancy outcomes, explores new partners of the antisense protein HBZ, and sheds light on its role in ATL pathogenesis. These findings collectively enhance our understanding of the mechanisms underlying HTLV-1-related disorders.MC: Writing—original draft, Writing–review & editing. WZ: Writing—review & editing. LoM: Writing—review & editing. LuM: Writing—original draft, Writing—review & editing.