Statins conclusively decrease mortality in atherosclerotic cardiovascular disease (ASCVD), the leading cause of death worldwide, and are strongly recommended by guidelines. However, real-world statin utilization and persistence are low, resulting in excess mortality. Identifying reasons for statin nonuse at scale across health systems is crucial to developing targeted interventions to improve statin use.We developed and validated deep learning-based natural language processing (NLP) approaches  to classify statin nonuse and reasons for statin nonuse using unstructured electronic health records (EHRs) from a diverse healthcare system.We present data from a cohort of 56,530 ASCVD patients, among whom 21,508 (38%) lack guideline-directed statin prescriptions and statins listed as allergies in structured EHR portions. Of these 21,508 patients without prescriptions, only 3,929 (18%) have any discussion of statin use or nonuse in EHR documentation. The NLP classifiers identify statin nonuse with an area under the curve (AUC) of 0.94 (95% CI 0.93–0.96) and reasons for nonuse with a weighted-average AUC of 0.88 (95% CI 0.86–0.91) when evaluated against manual expert chart review in a held-out test set. Clinical BERT identifies key patient-level reasons  and clinician-level reasons (guideline-discordant practices) for statin nonuse, including differences by type of ASCVD and patient race/ethnicity.Our deep learning NLP classifiers can identify crucial gaps in statin nonuse and reasons for nonuse in high-risk populations to support education, clinical decision support, and potential pathways for health systems to address ASCVD treatment gaps. Cardiovascular disease (CVD) is the leading cause of death worldwide. Statins are cholesterol-lowering drugs that are recommended by major guidelines to reduce the risk of heart attacks and death. However, despite being effective and generally well-tolerated, statins are concerningly underused, and reasons for such statin nonuse are not well-understood. Using artificial intelligence (AI) approaches that can analyze complex language data including written medical terminology, we identified statin nonuse and reasons for statin nonuse from clinical notes from electronic health records of CVD patients at a large health system. We found that nearly 2 in 5 patients lacked guideline-directed statin prescriptions. We then identified the key reasons for statin nonuse including patient  and clinician reasons (not practicing according to guidelines). Our AI approaches may help target interventions to address statin nonuse and thus, improve CVD treatment. Sarraju, Coquet et al. utilise a natural language processing approach to identify reasons for statin nonuse in the health records of patients with atherosclerotic cardiovascular disease (ASCVD). Their approach identifies patient-level and clinician-level reasons for statin nonuse, with differences by ASCVD type and patient race/ethnicity. In ASCVD, statins conclusively reduce adverse events including myocardial infarctions, strokes, and mortality4. Thus, by major guidelines including the American College of Cardiology/American Heart Association (ACC/AHA) multisociety guidelines, statins carry the strongest treatment recommendation in ASCVD in the absence of contraindications5. However, statin use in ASCVD populations is alarmingly low, with discontinuation rates approaching 50% at one year following a myocardial infarction7. Racial/ethnic minorities, women, and elderly patients are more likely to prematurely discontinue statins12. Statin nonuse is independently associated with adverse outcomes including all-cause mortality and represents an important public health gap13.Atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of morbidity and mortality worldwide despite the availability of numerous therapies18. Characterizing statin nonuse directly from electronic health records (EHRs) represents a unique opportunity to capture the local epidemiology of statin nonuse in a health system, which in turn may help develop targeted interventions to improve statin use. In clinical practice, reasons for statin nonuse are documented in narrative, free-text notes in the EHR, rather than in structured fields. Identifying them requires detailed characterization of large-scale unstructured EHR data11.Reasons for statin nonuse can be multifactorial and complex, including patient, clinician, and system factors. These have typically been studied from surveys with potential selection bias and generalizability issues23. We developed a deep learning-based NLP approach  to characterize reasons for statin nonuse directly from EHR data of a multiethnic, real-world, ASCVD cohort24. The idea of this approach was to create a highly flexible deep learning model that can be incorporated into a clinical tool to accurately identify patients not meeting clinical guideline recommendations for statin use and then identify reasons for statin nonuse.Artificial intelligence (AI) technologies, including natural language processing (NLP), demonstrate promise in studying routine clinical data at scale, including for cardiovascular outcome predictionIn this study of a multisite, multiethnic EHR-based health system, we found that approximately 40% of ASCVD patients lacked structured statin prescriptions. Using Clinical BERT, we accurately identified statin nonuse and key reasons for statin nonuse from unstructured clinical notes, including the prevalence of patient-level  and clinical-level (guideline-discordant practice) reasons. We observed differences in reasons for statin nonuse by patient race/ethnicity. By guiding targeted interventions to address statin nonuse, such a tool can bridge guideline-directed statin utilization gaps in diverse, real-world settings.This retrospective study identified patients at Stanford Health Care Alliance (SHA) using EHR data from 1 January 2014 to 31 July 2019. SHA is an integrated health system, which includes an academic hospital , a community hospital , and a community practice network . The study was approved by the Stanford University Institutional Review Board (Protocol 47644). Informed consent was waived under exemption 4: research on existing data.The study included patients diagnosed with ASCVD between the ages of 18 and 89 years between 1 January 2014 and 31 July 2019 . The category of “perceived lipid control” was defined as documentation that statin use was avoided either based on available lipid levels or deferred in favor of obtaining future lipid tests to determine the need for statin therapy despite the presence of an ASCVD diagnosis. As contemporary ASCVD guidelines recommend the initiation of statin therapy as tolerated independent of baseline lipids, this was considered a guideline-discordant practice24. The Clinical BERT model was pretrained on notes from MIMIC III, a database containing EHRs from ICU patients at the Beth Israel Hospital in Boston, MA24.For the deep learning NLP dataset, as outlined above, clinical notes were obtained from a 30 day period after the index ASCVD diagnosis. For each patient, all sentences which contained mentions of statins based on a predefined statin term dictionary based on drug names  was randomly selected from the deep learning NLP dataset. Four co-authors  manually annotated this dataset to determine whether clinical notes contained documentation of active statin use or nonuse, and subsequently extracted reasons for statin nonuse from notes according to categories. To assess reviewer concordance, the overlapping review was performed in a set of 50 patients and a set of 100 patients. Discrepancies between reviewers in these overlapping notes were resolved by repeat review by a clinician expert (AS or FR). Non-muscle side effect categories were defined more granularly prior to review , but were collapsed into a single category to comply with privacy regulations that prevent the reporting of groups with less than 10 participants. Categories were not considered mutually exclusive. The categories of reasons for statin nonuse were finalized upon completion of manual review.To create a manually annotated ground-truth dataset for NLP model training and evaluation, a sample of patients  and without documented statin use (negative cases).25. Second, we used a random forest model that took the probabilities of each reason from the first step as inputs to predict the final reason for nonuse. We also separately implemented a multilabel Clinical BERT classification model to predict the reason for statin nonuse.In patients without documentation of statin use in clinical notes, we developed another model to determine documented reasons for statin nonuse, classified by the categories as previously described. We fine-tuned the Clinical BERT pretrained model to perform a multiclass classification with our dataset. For this task, we implemented a two-step pipeline. First, we trained five different models to classify one class (positive cases) against the four other classes (negative cases) in a one-versus-rest strategyFor both NLP models to classify statin nonuse and reasons for nonuse, we split the manually annotated patients into two data sets: a training set with 80% of patients and a test set with 20% of patients. We used 10-fold cross-validation to validate the model and tune the hyperparameters . To evaluate the performance of models, we assessed precision, recall, Area Under Curve (AUC) score, and F1 score. Precision  measures the fraction of correct positive predictions divided by all documents predicted as positive. Recall (or true positive rate) measures the fraction of correct positive predictions divided by the number of results that should have been predicted as positive. AUC score corresponds to the probability that a classifier will rank a positive document higher than a negative one. Finally, F1 score is defined as the harmonic mean of precision and recall. For the multiclass classifiers for the reasons for statin nonuse, we reported the weighted macro-averages of these metrics. After training and evaluation, the binary NLP model and highest performing multiclass NLP model were applied across the full deep learning NLP dataset. The distribution of reasons for statin nonuse was stratified by type of ASCVD  and race/ethnicity .t test for parametric data and chi-square/Fisher’s exact tests for categorical variables. Cohen’s Kappa coefficients were calculated to assess the concordance of the manual annotations between overlapping reviewers. All statistical tests were two-sided with a threshold of p ≤ 0.05 for statistical significance. We calculated 95% confidence intervals for NLP model performance by performing a bootstrap resampling of 1000 iterations. We also compared unadjusted and adjusted Odds Ratios (OR) of receiving high-intensity statin prescriptions (versus other statin intensities) and of receiving any statin prescriptions (versus no prescriptions) through multivariable logistic regression. Analyses were performed using Python software, version 3.7, with transformers and scikit-learn packages.For comparisons of baseline characteristics between patients with and without statin prescription, we performed an unpaired There were 56,530 patients with documented ASCVD who had at least two separate visits to the healthcare system during the study period Fig. . Of thesOf 22,296 patients without statin prescriptions, a total of 788 patients (3.5%) had documented statin allergies in the structured portion of the EHR. A manual review of 20 random patients with structured allergies confirmed the presence of allergies and lack of active statin use. Among the remaining 21,508 patients, a total of 17,579 (81.7%) had no mention of statin terms in clinical notes. The remaining 3929 patients who had any mention of statin terms in their clinical notes formed the deep learning NLP dataset for Clinical BERT model development and evaluation , community hospital patients , and patients with cerebrovascular disease, peripheral arterial disease, or polyvascular disease (compared with coronary artery disease) were less likely to receive any statin prescriptions (Supplementary Table N = 174), muscle side-effects , and patient preference   Table . Among sAfter NLP model development and evaluation, the binary statin nonuse model and the hurdle multiclass model for reasons for statin nonuse were applied to the full dataset of 3929 patients . Patients with cerebrovascular disease had a higher representation in the perceived lipid control group when compared with other groups (p < 0.001). Excerpts denoting each reason for statin nonuse are outlined in Table Reasons for statin nonuse were further stratified by type of ASCVD and by race/ethnicity Table . Non-HisAmong patients without structured statin prescriptions, individuals who were statin users according to NLP were slightly more likely to be male and have coronary artery disease compared with statin nonusers according to NLP  reliably classified statin nonuse and reasons for statin nonuse from large-scale unstructured notes including patient-level  and clinical-level reasons (guideline-discordant practice).1; yet, there remain major gaps in statin utilization7. Our study adds to this literature by highlighting a high proportion of ASCVD patients without documented statin prescriptions in a large health system, including disparities in statin prescriptions by gender, race/ethnicity, and practice setting (academic versus community-based). There is a strong need to develop interventions to bridge statin utilization gaps. Previous work studying reasons for statin nonuse has often relied on surveys that are potentially limited by selection bias, generalizability, and scalability16. A prior EHR-based study addressed statin discontinuation among patients with existing prescriptions and used a rules-based approach to analyze notes11. To our knowledge, our study is the first to employ a deep learning-based NLP approach to comprehensively track statin nonuse and reasons for statin nonuse across structured and unstructured EHR data. We capture statin nonuse in a multiethnic multisite health system in detail, including documentation gaps, clinician- and patient-level reasons for nonuse, and differences by characteristics such as ASCVD type and race/ethnicity.The benefits of statin use in ASCVD are well-established26 For example, we found clinical free-text documentation of active statin use in patients without statin prescriptions, suggesting that statin prescriptions were an unreliable surrogate for statin use in our cohort. Statin allergies in structured data were infrequently documented, but limiting side-effects were documented for patients without structured statin allergies, suggesting that using structured allergy data to infer statin intolerance would have been inaccurate. These results highlight the need for a standardized approach to document medication use in EHRs, a recognized issue for real-world data27. Importantly, research efforts that study real-world medication utilization may need to mitigate the limitations of structured data by incorporating unstructured data at scale, as in our study.EHR-based studies of medication utilization often rely on structured data such as prescriptions. However, we found substantial discordance between structured and unstructured EHR documentation of statin use. Inconsistent documentation of external prescriptions and medical care may contribute to this finding28.Our study highlights poor clinical documentation of statin nonuse, with only 18% of patients without statin prescriptions demonstrating any mention of statins in clinical notes. These findings suggest important opportunities to improve statin nonuse documentation, for example, through smart text phrases or similarly integrated user-friendly tools that are guided by NLP. Ensuring adequate documentation is mandatory in learning health systems for quality improvement (QI) and research efforts that rely on large-scale records. A deep learning NLP pipeline like ours may be well-suited to study and bridge documentation gaps by leveraging unstructured EHR data29. Differences in statin nonuse by factors such as race/ethnicity or type of ASCVD may help guide equitable statin implementation strategies30. Future work should prospectively explore the role of NLP-guided interventions to promote statin utilization.Accurately characterizing reasons for nonuse is critical to closing statin utilization gaps. An approach like ours may enhance efforts to improve statin utilization by (1) identifying reasons for nonuse to guide targeted clinical decision support tools, (2) identifying disparities by relevant clinical factors such as type of ASCVD, and (3) providing a pathway for EHR-driven statin utilization QI or research efforts. For example, patients with documented muscle-based side-effects could be flagged for further study using validated tools to confirm statin-related symptoms and to develop targeted interventions to re-challenge statins or address nocebo effectsOverall, the goal of our study was to build an accurate AI-based model to classify statin nonuse and reasons for nonuse among patients diagnosed with ASCVD. Using data derived from real-time EHRs, we trained our models to identify a patient that does not have any documentation of statin use for their ASCVD, and to identify reasons for statin nonuse. Such models are well-positioned to help overcome the challenges of real-world statin utilization by providing a better understanding of statin nonuse at scale. Such information will be a step forward towards improved statin use in routine care that is urgently needed to help reduce ASCVD disparities and gaps across diverse populations.The study should be interpreted in the context of its limitations. Our cohort was a population from Northern California that may not reflect other ASCVD populations across the United States. However, our cohort is comprised of diverse practice sites including an academic hospital (SHC), a community hospital , and a community practice network (UHA). We grouped non-muscle-based side-effects into a single category for the NLP model due to their low frequency and because we were unable to report outcomes with 10 or fewer participants due to HIPAA privacy regulations. It is possible that patients received additional care from outside health systems, which is a limitation of a single health system analysis. However, to mitigate the effects of care fragmentation and ensure regular care in our health system, we included patients with a new ASCVD diagnosis recorded in our system and at least 2 clinic visits associated with that diagnosis. Our EHR medication reconciliation section also includes medications prescribed outside of our health system and medications that are self-reported by patients. Future work should consider characterizing the frequency of more discrete categories of side-effects. We were unable to disaggregate racial/ethnic groups further or include socioeconomic information due to data limitations. Due to low overall documentation, the sample sizes of stratified groups  were small, and these results should therefore be interpreted with caution and considered hypothesis-generating. While Clinical BERT was pretrained on external notes and we report model performance from a held-out test set, external validation, as well as a prospective study of NLP-guided interventions, is needed to ensure generalizability, outcome benefit, and cost-effectiveness prior to wide deployment.In conclusion, in multiethnic ASCVD patients in a multisite health system, we observed suboptimal statin prescription rates and limited clinical documentation of statin nonuse. A deep learning NLP approach  reliably identified statin nonuse and reasons for nonuse from unstructured EHRs, including patient-level factors  and clinician-level factors (guideline-discordant practice). Advanced NLP approaches may help to learn health systems to leverage clinical documentation and characterize reasons for statin nonuse at scale from EHRs, thus potentially providing a pathway to address important ASCVD treatment gaps.Further information on research design is available in the Supplementary InformationReporting SummarySupplementary Data 1Description of Additional Supplementary Files


The Ramsay-Hunt syndrome (RHS) is characterized by the association of herpes-zoster oticus with acute peripheral facial paralysisDifferent surgical procedures have been described in the literature as triggers for virus reactivationBelow, we describe what we believe to be the first case reported of RHS happening after otoplasty, focusing on its diagnosis, treatment and evolution.A twelve year old boy, previously healthy, was submitted to bilateral otoplasty in order to correct protruding ears. There were no initial complications and the suture was removed 14 days after the procedure (PO).On the 25th day of post-op, he started having a little difficulty in shutting his left eye. The patient reported having noticed vesicles in his left ear pinna, which had been there for three days, together with ear pain and ipsilateral hearing loss. Two days later, he reported difficulties in moving his face on the left side. He had had varicella infection at 4 years of age, without complications. Upon physical exam, he had House Brackmann level III left-side facial palsy and crusty lesions on his left ear pinna . Upon otWe started treating his RHS with Dexamethasone 4mg/day per os, during 7 days, with progressive weaning (28 days of steroid treatment according to our protocol), Acyclovir 2g/day (7 days) and eye lubrication drops on the left side. The external otitis was treated with ear drops (Ciprofloxacin 0.2% + hydrocortisone 1% - 10 days). We ordered a CBC and serology tests. One week later, he had complete symptom resolution in his left ear pinna as well as his ipsilateral external otitis. Tonal audiometry showed normal hearing thresholds in both ears. CBC showed leukocytosis  without deviations.Serology tests were positive for IgG and negative for IgM for Herpes I and II, mononucleosis and cytomegalovirus; negative serology for HIV. He progressively recovered facial movement and had complete palsy resolution upon the fourth week after the diagnosis. Herpes-zoster serology (enzyme immune assay) was carried out only one month after symptom onset, which revealed negative IgM and positive IgG in high titers .The RHS is rare in childhood, with an incidence of approximately 5 cases/100,000 inhabitants/year. Nonetheless, it is the second most common cause of non-traumatic facial paralysis.Today it is believed that virus reactivation is an important cause of facial paralysis in late post-opWe may hypothesize that the otoplasty procedure triggered the viral reactivation through direct stimulation of the VII cranial nerve (ear pinna)Diagnosis is fundamentally clinicalWe described here a case of a patient who developed RHS in the late postoperative of bilateral otoplasty, having complete symptom remission after clinical treatment. The close follow up of patients submitted to otorhinolaryngological and neck/facial surgeries is highly important in order to make an early diagnosis and to provide proper treatment aiming at optimizing prognosis.


Background: Hemodynamic instability (HDI) is common during liver transplantation (LT); veno-venous bypass (VVB) is a tool used in selected cases to ensure hemodynamic stability and for surgical needs. Transesophageal echocardiography (TEE) allows the transplant team to identify the causes of HDI and to guide therapies. We present a case series of four patients showing the valuable role of TEE during LT in VVB. Methods: We report four explicative cases of TEE use in LT with VVB performed at IRCCS Azienda Ospedaliero–Universitaria di Bologna. Four transplants were performed between 2016 and 2022. Results: Many authors have highlighted the diagnostic value of TEE during LT in the case of HDI. However, its specific role during LT with VVB is poorly described. This paper illustrates multiple potential uses of TEE in LT with VVB: TEE as a guide for catheterization and optimal cannula positioning, TEE as a tool for intraoperative Patent Foramen Ovale management, TEE as help for anticoagulation therapy and finally, TEE as support when evaluating bypass efficiency and correcting hypovolemia. Conclusion: TEE is a useful instrument during LT with VVB. However, further studies are needed to assess the suitable applications of TEE during LT in patients with HDI requiring VVB. TEE should be part of the anesthetist’s cultural background. In the last two decades, the use of intraoperative transesophageal echocardiography (TEE) as a monitoring tool has constantly increased in non-cardiac surgery settings, such as orthotopic liver transplantation (LT) . IntraopVVB consists of extracorporeal circulation from the femoral vein (drainage cannulae) to the internal jugular vein (return cannulae), allowing better hemodynamic stability. An additional cannula for the portal vein (drainage cannulae) also allows venous decompression of the splanchnic circulation with reduced venostasis and intraoperative bleeding and facilitates surgical activity . HoweverThere is growing evidence that TEE can influence the anesthetic management during LT: numerous clinical conditions such as ventricular dysfunction, valvular regurgitation, air embolization, intracardiac defects and pericardial tamponade have been reported to be detected by intraoperative TEE that would not have been noticed with other monitoring methods, including pulmonary artery catheter (PAC) . TEE assIn our center all liver transplants in VVB are monitored with a Swan–Ganz pulmonary catheter. Approximately less than half of these patients are additionally monitored with TEE by the transplant anesthetist. However, not all cases undergo TEE monitoring due to the limited number of adequately trained anesthesiologists, the presence of contraindications  or the temporary unavailability of the transesophageal probe .Herein we present four clinical cases showing how TEE intraoperative monitoring improved the anesthetic management of the patients at risk of HI during LT in VVB.We retrospectively collected the clinical data of four peculiar cases who underwent LT at IRCCS Azienda Ospedaliero–Universitaria di Bologna, Italy, between January 2016 and November 2022, in whom TEE was used during the surgery and extracorporeal circulation via VVB. A GE LOGIQ 7 ultrasound machine with a GE 6T transesophageal probe was used by a trained anesthetist. In patients in whom TEE was not performed, cannulae were placed under fluoroscopy. This study was conducted in accordance with the ethical guidelines of the World Medical Association’s Declaration of Helsinki and guidelines for Good Clinical Practice ,9. FinalFrom January 2016 to November 2022, 687 LTs were performed at IRCCS Azienda Ospedaliero–Universitaria of Bologna, Italy. Of these, 58 were conducted with VVB assistance. Twenty-three LT with VVB patients received TEE during the surgical phases. A 49-year-old female patient with polycystic liver disease and severe abdominal distension was studied for LT in our center in 2016. The preoperative echocardiography showed the absence of heart disease. Due to the surgical complexity with the need to perform total caval clamping, we assisted the patient via a VVB. After the induction of general anesthesia, a right femoral vein (21 Fr cannula) and right internal jugular vein (17 Fr cannula) cannulation were performed. Cannula placement was subsequently verified with a TEE examination that revealed the jugular cannula tip positioned in the upper portion of the right atrium. During surgery, advanced hemodynamic monitoring consisted of PAC and TEE. Throughout the surgery the bypass flow was maintained between 1.5 and 3 L/min with a standard circuit range of pressures. A long and laborious dissection phase with substantial hemodynamic stability characterized the surgery. Following portal reperfusion, we observed a sudden hemodynamic change with hypotension, tachycardia, an increase in central venous pressure (CVP) from 7 mm Hg to 20 mm Hg and desaturation. The HDI onset and PAC data were suggestive of reperfusion syndrome, so fluids and norepinephrine were administered. Subsequently intraoperative TEE showed the presence of a new-onset circumferential pericardial effusion with reduced right ventricle diastolic filling, and marked changes in transmitral blood flow velocities (>25%). An emergency sternotomy was performed, and a right atrial perforation was identified and repaired, obtaining hemodynamic stability and a favourable surgery and patient outcome.3), impaired International Normalized Ratio (INR 2.2) and expected difficult liver dissection. We placed a 21Fr femoral cannula and a 21Fr jugular cannula without complications. VVB flow rate was maintained between 1.5–3 L/min. Hemodynamic monitoring consisted of the Swan–Ganz catheter and TEE, while coagulation assessment was monitored with activated clotting time (ACT) and thromboelastography (TEG). The patient received four concentrated red blood cell units for surgical bleeding during liver dissection. In this phase, during the total caval clamping, a pulmonary microembolism was identified thanks to the visualization of small floating hypoechoic images in the right heart, coming from the outflow cannula and directed towards the pulmonary circulation. The pulmonary microembolism was not causing HDI or hypoxia, and ACT was 215 s. However, a dose of heparin (1000 UI i.v.) was administered to prevent the mobilization of other thrombi during the subsequent graft reperfusion. No further thromboses were visualized and there were no bleeding complications. The post-operative course was regular.A 65-year-old male patient with Hepatitis-C-related cirrhosis complicated by hepatocellular carcinoma and severe portal hypertension was admitted for LT in our center in 2021. Preoperative transthoracic echocardiography (TTE) showed no pathological features. An intraoperative femoro-jugular VVB was required during the surgery for expected surgical complexity: in the history, the patient had performed various locoregional treatments of hepatocellular carcinoma in addition to the placement of a transjugular intrahepatic portosystemic stent-shunt complicated by stent thrombosis. Before starting the extracorporeal circulation, the heparin bolus was not administered due to severe thrombocytopenia  of 15 cm H2O, as described by Koroneos et al. [The TEE probe was placed after the induction of general anesthesia and tracheal intubation. A moderate-grade Patent Foramen Ovale (PFO) was identified (3 mm). A right-to-left shunt was documented with colour-Doppler and bubble test by applying a positive airway pressure of 20 cm Hs et al. .After starting the VVB flow, the outflow cannula (17Fr) was retracted  approxim2O and the maximum positive airway pressure was 14 cm H2O. Depending on the surgical phase, the bypass flow ranged between 1.5 and 2.5 L/min. Pulmonary and central venous pressure (CVP) increased moderately after graft reperfusion (CVP from 2 to 10 mm Hg). During surgery, the absence of a right-to-left shunt was constantly verified despite the increase in right atrium pressure due to mechanical ventilation, VVB outflow positive pressure and the onset of a moderate reperfusion syndrome. There were no intra- or postoperative cardioembolic complications.During surgery, PEEP was set between 0 and 5 cm H2O for 5 s) to rule out the occurrence of a right-to-left shunt. The shunt was never displayed during the LT, and the devices remained correctly positioned. A 41-year-old male patient with Alagille syndrome complicated by severe cholestatic liver disease, PFO and retinopathy was scheduled for LT in 2022. At age 35, he underwent percutaneous closure of the PFO (AGA PFO OCCLUDER 25 mm) for evidence of a severe right-to-left shunt during the Valsalva maneuver. Pre-LT echocardiography documented the correct placement of the atrial devices and the absence of residual shunt. The LT was performed with the VVB technique due to the surgical complexity (previous biliary tract surgery). After the induction of general anesthesia and tracheal intubation, the TEE ultrasound probe was positioned in the ME bicaval view. The central venous catheter and the PAC were then introduced using an ultrasound-guided approach. The Seldinger guide and the catheters were constantly visualized in the superior vena cava and the right atrium to avoid injury or detachment of the atrial occluder. The ultrasound-guided approach was later used for the placement of the 17Fr jugular bypass cannula. Thanks to the ultrasound guidance and the passive patient’s head rotation we initially introduced the Super Stiff Guidewire from the right internal jugular vein to the inferior vena cava without hitting the atrial occluder. The 17F jugular cannula was then introduced with ultrasound guidance up to approximately 5 mm from the atrial devices. Next, we retracted the cannula by a further 5 mm using the colour-Doppler guide to avoid the bypass flow being directed towards the occluder. After cannulation, we checked the integrity of the occluder using B-mode ultrasound in the ME-bicaval view and ME-short axis aortic valve view. During the surgery we also repeatedly performed bubble tests with the Valsalva maneuver  and, consequently, the adequate training of the entire anesthesiology team.Evidence of monitoring strategies’ superiority with PAC and TEE during LT is growing. TEE allows us to quickly identify the causes of HDI and guide the therapies to support volume status, biventricular function and vascular resistances. It has also been helpful in particular situations, such as LT with VVB. It can guide patient management and be a diagnostic tool in these circumstances. However, further studies are needed. TEE knowledge and practice should be part of the anesthesiologist’s cultural background.


Synthetic cannabinoid receptor agonists (SCRAs) are compounds that mimic the pharmacology of the psychoactive components in cannabis. These compounds are structurally diverse, inexpensive, commercially available, and difficult to identify with modern analytical methods, making them highly accessible for recreational use. Suspected SCRA toxicity, which can present with a breadth of cardiovascular, gastrointestinal, and neurological disturbances, is currently addressed through symptom management followed by a toxicological screening that often occurs long after patient discharge. Here, we report the development of four cross-reactive anti-SCRA bioconjugate vaccines as a platform for developing improved diagnostic and therapeutic interventions against SCRA intoxication, using SCRA-resembling small molecule haptens that combine common subregional motifs occurring within and across different generations of SCRA molecules. Using a combination of multiplexed competitive ELISA screening and chemoinformatic analyses, it was found that the antibodies resulting from vaccination with these bioconjugates demonstrated their ability to detect multiple SCRAs with a Tanimoto minimum common structure score of 0.6 or greater, at concentrations below 8 ng/mL. The scope of SCRAs detectable using these haptens was found to include both bioisosteric and non-bioisosteric variants within the core and tail subregions, as well as SCRAs bearing valine-like head subregions, which are not addressed by commercially available ELISA screening approaches. Vaccination with these bioconjugates was also found to prevent the changes in locomotion and body temperature that were induced by a panel of SCRAs at doses of 1 and 3 mg/kg. Further refinement of this genericized hapten design and cross-reactivity-prioritizing approach may enable the rapid detection of otherwise cryptic SCRAs that arise during overdose outbreaks, and could ultimately lead to identification of monoclonal antibody species applicable for overdose reversal. Perfect discrimination for the 0.6 MSC threshold was observed in our dataset for all concentrations above 0.625 μM. Even at the lowest tested concentration of 20 nM, the AUC for cross-reactivity detection was 0.81 C, which 1, 8, 3 and 5, respectively. Twenty-five minutes after administration, the animals were assessed for reductions in overall locomotion and core body temperatures (1). Immunization with haptens 3 and 4 also elicited significant rescue of MMB-PICA (3) and MMB-PINACA (5) induced hypothermia, respectively. Immunization with hapten 2 showed no ability to rescue hypothermia or hypolocomotion induced by NAPIC (8) at either drug concentration. This observation, in tandem with the capacity of 8 to induce physiological alterations in mice indicative of cannabinoid receptor activation, is consistent with the interpretation that structures like hapten 2 are subjected to degradation during the vaccination process [1), even at this elevated 3 mg/kg dose. The otherwise limited differences between vaccination groups at this elevated dose suggests saturation of sera antibodies with the remaining free drug being sufficient to elicit significant physiological effects. No significant interactions arising from vaccination were seen at either drug concentration for the hotplate and catalepsy tests in all four vaccination groups, due in part to high individual variability in SCRA effects on these measures. Nevertheless, the ability of our chemically stable hapten vaccines to demonstrate notable reversal of SCRA-induced locomotion and body temperature deficits at 1 mg/kg, and up to 3 mg/kg in some cases, suggests promise for future optimization of high-affinity anti-SCRA monoclonal antibodies as overdose reversal agents.Next, since reversal of overdose is a unique potential benefit of antibody-based technologies, in comparison with LC-MS approaches, in vivo vaccine efficacy was also assessed in the presence of 1 and 3 mg/kg doses of SCRAs using the cannabinoid tetrad battery, which measures four physiological or behavioral responses indicative of cannabinoid intoxication ,40. The eratures  as well eratures  with Two process . While vWith these findings, we can identify several areas for future study and technological improvement. The poor performance of hapten 2 demonstrates the importance of prioritizing hapten stability, as hydrolysis of naphthyl esters in mouse sera has been observed within days; indole-3-carboxylate thus likely served as the presented antigen for a majority of the vaccination schedule with this hapten. Future syntheses of bioisosteric haptens with metabolically stable amide or acyl-methylene linkers should be undertaken to develop antibodies capable of recognizing related diaryl ester/amide SCRAs.The bioconjugation approach for SCRAs presents another area for further optimization, which may involve methods to control rather than maximize copy number. This is highlighted by the discrepancy between the high copy number and relatively low titer of hapten 1, and the fact that the bioconjugates with the two lowest copy numbers, haptens 3 and 4, led to the greatest production of anti-SCRA antibodies while demonstrating the most sensitive detection by competitive ELISA. One possible explanation for this phenomenon could be the occurrence of overlapping haptens with certain bioconjugate conformations at higher copy numbers, causing antibodies to recognize portions of two different haptens as their epitopes. Notably, the relationship between hapten copy number and functional efficacy may also be distinct for differing carrier-proteins .50 values of both antibody populations were against their cognate SCRA , with greater IC50 increase following 5-fluoronation of the alkyl tails compared to the indole versus indazole core switches. This contrasts with what is observed for hapten 1 in which 5-fluoronation improved SCRA detection, despite the lack data for an indazole version of this hapten. Antibodies against haptens 3 and 4 also displayed binding to 7, which contains a non-bioisosteric p-fluorobenzyl tail. This result suggests that presentation of the core, linker and head subregions is sufficient to elicit an immune response in mice that creates anti-cognate SCRA antibodies. Furthermore, it is possible that most of the hexanoic tails of the haptens are sterically occluded during immune system presentation following vaccination, making this subregion inconsequential during antibody binding. Future experiments using SCRAs with other structural tail changes  could be undertaken to test this hypothesis.Further elaboration of the relationship between hapten structure and SCRA cross reactivity is also an important priority, as direct structural mimics of specific SCRAs were not always found to be the most effective at producing high-affinity species. The lowest ICDespite breakthroughs in the methods of NPS detection, identification of cryptic or novel structures remains a challenge in regular clinical practice, making this another priority area for future improvements. Many of these methods require knowledge of the NPS/metabolite structure, which is particularly difficult for illicit drug products that often contain a multitude of unknown SCRAs and other contaminants. Therefore, assessment of cross-reactivity limits for a larger pool of hapten/SCRA pairs, especially those that include recent structural variations such as tert-leucine head regions, azaindole cores, or cumyl linkers, will be critical to test the generalizability of the chemoinformatic-informed approach reported here. Such advancements in understanding of cross-reactivity immunopharmacological approaches for detecting and mitigating the effects of novel SCRAs could also translate to more broadly improved understanding of the relationship between haptenic structure and antibody function for additional clinically relevant NPS classesFinally, although the results with pre-immunization for blocking SCRA effects in vivo are a promising proof of concept, this prophylactic approach is not likely to be a realistic approach for clinical application. Therefore, the generation of optimized monoclonal anti-SCRA antibodies is imperative to maximize both their diagnostic and therapeutic potential.As NPS compounds continue to evolve through clandestine medicinal chemistry efforts, the methods of identifying these compounds during outbreaks must evolve as well. This is especially true for SCRAs, which share many bioisosteric similarities among the compounds, making the prioritization of reliable cross-reactivity essential for the utility of any immune-based approach employed for diagnostic or therapeutic purposes. This work describes the synthesis of four haptens, three of which were able to generate high titers of anti-SCRA antibodies with micromolar to sub-micromolar affinity against multiple SCRA compounds with an MCS structural similarity score of 0.6 or above, in comparison to the hapten itself. This resulted in broad detection of multiple structurally related SCRAs at concentrations below 10 ng/mL, including newer generations of SCRAs that evade detection by current commercial ELISA kits. The polyclonal sera developed in this study were also sufficient to demonstrate functional blockade of SCRA-induced toxicologic effects in mice at doses of 1–3 mg/kg, indicating the potential to use antibody-based strategies for symptom mitigation in the context of SCRA intoxication.Overall, these results indicate that anti-SCRA haptens can yield functionally relevant antibodies against multiple related compounds within and across SCRA generations, and that it may be possible to define general regions of likely antibody cross-reactivity within chemical spaces using readily accessible chemoinformatic information. These findings are especially relevant in the face of increasing numbers of otherwise unknown or cryptic SCRA analogues, as they provide a basis for further development of multiplexed SCRA ELISA screening panels that can provide actionable information about SCRA generational and structural identity within minutes to hours, rather than days to weeks. Based on the ability of the anti-SCRA antibodies reported here to blunt negative effects of SCRAs, further value may be found by combining this multiplexed screening approach with the development of monoclonal antibodies for rapid SCRA overdose reversal to yield an integrated rapid detection and intervention platform to address future SCRA intoxication outbreaks.


Phrenic nerve stimulation is a well-recognized complication related to cardiac implantable electronic devices, in particular with left ventricular coronary sinus pacing leads for cardiac resynchronization therapy.We report an unusual case of symptomatic phrenic nerve stimulation due to inadvertent placement of a right ventricular defibrillation lead in coronary sinus posterior branch in a patient with heart failure with reduced ejection fraction with a recently implanted single-chamber cardioverter defibrillator.Phrenic nerve stimulation is a relatively common complication of left ventricular pacing. Inadvertent placement of a right ventricular lead in a coronary sinus branch is a rare but possible cause of phrenic nerve stimulation. Careful evaluation of intraprocedural fluoroscopic and electrocardiographic appearance of pacing and defibrillation leads during implantation may prevent inadvertent placement of a right ventricular lead in the coronary sinus. Phrenic nerve stimulation (PNS) may complicate up to 30% of cardiac implantable electronic devices (CIEDs) implantation procedures, mainly cardiac resynchronization therapy (CRT) with left ventricular pacing, due to the anatomic contiguity of phrenic nerve to left ventricle lateral wall . AlthougA 68-year-old Caucasian male was referred to our Arrhythmology and Electrophysiology Unit for lead revision of a single-chamber ICD implanted ten months earlier in another center. Two months before ICD implantation, the patient was hospitalized for symptomatic heart failure, NYHA class 3. A 12-lead ECG showed atrial fibrillation (AF) with rapid ventricular response. A transthoracic echocardiography (TTE) demonstrated reduced left ventricular ejection fraction  and severe left atrial enlargement without significant valvular disease. An invasive coronary angiography (ICA) showed multivessel coronary artery disease (MVCAD) with a low Syntax score. Myocardial revascularization with percutaneous coronary intervention (PCI) and two drug-eluting stents (DES) implantation in the left anterior descendent (LAD) coronary artery and the obtuse marginal branch of the left circumflex was performed without complication. The patient was discharged on heart failure and rate-control therapy with a beta-blocker (bisoprolol 5 mg bid), an angiotensin receptor-neprilysin inhibitor , a mineralocorticoid receptor antagonist (MRA), a loop diuretic, and digoxin (0.125 mg qd). After two months, because of persistent severe left ventricular systolic dysfunction, a single-chamber ICD with a single-coil passive fixation lead was implanted for primary prevention of sudden cardiac death (SCD). During subsequent follow-up, the patient was referred to our center for symptomatic PNS. At admission, the patient presented with mild dyspnea and mild ankle swelling. Blood tests were in the range of normality except for increased levels of N-terminal pro-B-type natriuretic peptide . Device control showed an excessive ventricular pacing percentage (about 15%) although ICD programming in VVI mode with a lower rate of 30 beats per minute, indicating phases of slow ventricular response with the necessity of ventricular pacing. PNS at very low energy output was also confirmed. Several symptomatic episodes of high ventricular rate (HVR) indicated an unsatisfactory pharmacological rate-control despite maximum tolerated drug dosage. Lead parameters were in the range of normality with a normal trend, thus excluding lead failure due to fracture or insulation defect. A 12-lead ECG showed AF with rapid ventricular response and normal QRS duration (90 ms) . UnfortuPhrenic nerve stimulation is a potential CIEDs complication, particularly in case of CRT with left ventricular pacing, due to the anatomic contiguity of the phrenic nerve to the lateral wall of the left ventricle. Direct diaphragmatic stimulation has also been hypothesized for LV pacing leads. PNS was more familiar with unipolar and bipolar coronary sinus leads due to the limited number of pacing configurations with such leads , 4. ContThe present case highlights the importance of early recognition of inadvertent placement of a right ventricular lead in a coronary sinus branch, mainly the middle cardiac vein or a posterior branch.Such complication may be avoided with a careful fluoroscopic and electrocardiographic procedural examination. Both cardiologists and radiologists should be trained to interpret CIEDs’ fluoroscopic appearance. Inadvertent placement of a right ventricular lead in a coronary sinus branch should always be considered in the case of PNS after single or dual chamber pacemaker or ICD implantation.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Written informed consent was obtained from all participants for their participation in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.CD drafted the manuscript. PA, AB, AS, FP, and MS revised it critically for important intellectual content and given final approval of the version to be published. All authors have been directly involved in patient care, have undertaken patient investigations, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.


Using genetic strategies and viral-based directional tracers, we investigated the topological location and output networks of claustrum (CLA) neuron populations projecting to either the retrosplenial cortex, primary motor cortex, or basolateral amygdala. We found that all three CLA neuron populations clearly reside in distinct topological locations within the CLA complex and project broadly to multiple downstream targets. Each neuron population projects to different targets, suggesting that each CLA subzone coordinates a unique set of brain-wide functions. Our findings establish that the claustrum complex encompasses at least three minimally overlapping networks that are compartmentalized into different topological subzones. Such modularity is likely to be important for CLA function. Almost all hypotheses regarding the function of the claustrum (CLA) are based on the extensive brain-wide connectivity of this poorly-understood brain region , retrosplenial cortex (RSC), and the basolateral amygdala (BLA). Next, we identified co-projection targets of CLA neurons projecting to these three initial targets and, finally, we compared the degree of overlap between their projection networks. We found that neurons projecting to M1, RSC and BLA reside in distinct topological zones within the CLA and participate in vastly different projection networks. These distinct projection networks reveal that the CLA can be divided into a minimum of three topologically-distinct functional modules that may provide concerted influence on different sets of brain regions. The existence of distinct functional networks within the CLA complex reveals how the rodent CLA is organized and will guide future experiments that determine whether these specific CLA neuron populations subserve different brain functions.To reconcile these somewhat divergent views of CLA organization, we hypothesize that topologically-distinct populations of CLA neurons may also project to A total of 11 C56/BL6J wild-type male mice were used for the experiments: three mice for M1 and RSC initial targets, and five mice for experiments where the BLA was the initial target. Mice were 2–3 months old at the time of stereotaxic surgery for tracing experiments. All animal procedures were performed according to the guidelines of the NTU Institutional Animal Care and Use Committee.Mice were deeply anesthetized using isoflurane and placed in a stereotaxic frame equipped with a heating pad. Animals were monitored for breathing and their body temperature was checked throughout the surgery. For pre-surgery analgesic relief, buprenorphine and lidocaine were injected subcutaneously above the back and incision site, respectively. A skin incision was made, followed by craniotomy and dura removal above the target coordinates. Precise viral injections were made using glass pipettes with tip diameters of 30–50 μm and a pressure injector . Both the glass pipettes and Hamilton syringe  were first filled with mineral oil before front filling with viruses. Viruses were injected at 1–2 nl/s into each target region. Coordinates are defined based on the following convention: Medial-lateral (ML), anterior-posterior (AP), depth based on dura (DV) and angle represents deflection from midline with pipette tip pointing toward the center where applicable. The coordinates used for different brain regions are as follows: RSC , M1 , BLA , CLA . Injection pressure was allowed to equalize for 5 min before retraction of the glass pipette, followed by stitching up of the incision and recovery on a warming pad. Mice were monitored and supplied with Meloxicam and Baytril in their drinking water for 1-week post-surgery.via two AAV injections per animal .Because the insula projects strongly to the BLA, additional precautions were taken when the BLA was the initial target. In these experiments, Cre was also expressed in many deep layer insula neurons that are located close to the CLA (see RRID:AB_2315235), chicken-anti-GFP  and rat-anti-mcherry  from Thermo Fisher Scientific, RRID:AB_2536611) primary antibodies overnight at RT, or for 2 days at 4°C. These sections were then washed and treated with secondary antibodies  at RT for 2–3 h. Stained sections were also stained with DAPI, to visualize cell nuclei.To allow adequate time for fluorophore expression, animals were perfused and sacrificed 2–4 weeks post-surgery. Transcardial perfusion was performed using saline, followed by 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). Dissected brains were post-fixed in 4% PFA overnight, followed by 30% sucrose solution in PBS for 2 days, or until brains sank. Brain sections (60 μm thickness) were first washed, blocked and stained with rabbit-anti-PV  in the EDF mode (Extended Depth of Focus), which combines sharp images from each z-slice to reconstruct a flat projection. Tiled images were subsequently flat-field corrected and stitched using the WholeBrain program  to the anterior commissure, axons traveling through the anterior commissure were often quantified as axon densities in L6b of various cortices. The narrowness of L6b fibers in the atlas could combine with slight deviations in atlas registration to yield substantial misrepresentation of axon densities for L6b of many cortices. Hence the axon densities assigned to L6b of all cortical regions were excluded from our analysis. In addition, regions were deemed to be innervated only if axonal density was more than a threshold (0.002 pixels/area) that was empirically determined based on the distribution of axonal fluorescence. Threshold-identified regions were additionally verified for axonal expression of mCherry in the raw images. To account for variability in mCherry expression between animals and variability in absolute fluorescence intensity across animals, axon density from each brain region (axon pixels/pixels in brain region) was quantified as a fraction of the total axonal density across all innervated regions.n = 3 mice for each condition), an ANOVA test was performed per region, using the axon density from each experiment to compare innervation across the three experimental conditions. Post-hoc Tukey’s HSD was performed following ANOVA to obtain significance values for comparisons across experimental pairs for each brain region.Statistical comparisons were performed using custom R scripts. To compare differences in the axon network of the three experimental conditions  immunohistochemistry was used to define the ventral (vCLA) core and layer 5b of the agranular insula , the basolateral amygdala (BLA), and the primary motor cortex (M1). Among the many regions innervated by the CLA, these were selected to reflect different cortical hierarchies: higher/association cortex (RSC), sub-cortex (BLA), and sensorimotor cortex (M1). A viral strategy, based on Cre-lox, allowed us to visualize the axon arborizations of projection-defined CLA neurons by restricting expression of mCherry to CLA neurons that project to one of the three initial targets ..2020; ZiTo observe potential co-projection networks from CLA neurons going to different initial targets, we relied on the expression of Cre to label projection-defined CLA neurons with mCherry. The CLA-specific Cre expression from the RSC and M1 initial targets allowed us to easily limit mCherry expression to the CLA neurons , mCherryUsing the above targeting strategies, we successfully limited mCherry expression to CLA neurons, and demonstrated that the somata of CLA neurons projecting to the RSC (retRSC), BLA (retBLA), or M1 (retM1) are clearly located in distinct topological zones within the CLA complex . These rWe also examined the distribution of Cre and mCherry expression in the CLA across the rostral-caudal axis to determine whether these topological zones are restricted to particular rostral-caudal segments . PV staiThe distinct projection-based segregation of CLA topology is potentially in conflict with earlier reports of CLA neurons that project to multiple regions , postsubiculum (PoS), area prostriata (aPrS), and presubiculum Pre; . retRSC At least one region belonging to each sensorimotor module  received innervation from retRSC neurons. This sensorimotor innervation again was mostly ipsilateral, with only sparse innervation observed in some contralateral olfactory and secondary motor areas .Higher-order sensorimotor regions receiving moderate input  were innervated throughout all their layers, while other sensorimotor regions with moderate input  were mainly innervated in the deeper layers . This suIn summary, retRSC neurons reside in the vCLA core and project not only to the retrosplenial cortex but also to numerous association, sensorimotor and subcortical regions. Consistent with previous reports  are the least investigated and most controversial. The CLA-to-BLA connection has been proposed to be an important component of CLA function, potentially mediating the exchange of salience-related information between the two structures . Innervation of association regions from retBLA neurons was mainly ipsilateral, with only light contralateral projections to the mPFC, ORB, ACC, RSC, and AI . SimilarBeyond innervation of the AI, retBLA neurons also heavily innervated several ipsilateral association regions: the ORB, mPFC , ACC, Ect, and Prh . ModeratIn addition to innervating association areas, retBLA neuron axons were also identified in several sensorimotor regions. Such sensorimotor innervation was largely ipsilateral, with only weak innervation of the contralateral S1, secondary visual cortex (V2), Gu, and Vis . Of the Similar to the innervation of sensorimotor areas, axonal projections to areas within the subcortical category accounted for approximately 20% of the total distribution of retBLA neuron axons. The majority of subcortical innervation was ipsilateral, with only weak innervation of the contralateral EC, fundus of striatum, and En. Besides the ipsilateral BLA—where retrograde Cre was injected—particularly heavy ipsilateral innervation was observed in the EC and En . The heaOur results from axonal mapping of retBLA neurons establish for the first time that vCLA shell neurons project to multiple targets across the brain. Similar to the other CLA projection neurons (retM1 and retRSC), retBLA neurons also project heavily to association regions. However, retBLA outputs were more concentrated in limbic-related regions, suggesting that modality preferences do differ between the three projection-defined CLA populations.Having quantified the axonal distributions of all three projection-defined CLA networks, we next compared these topology-separated CLA networks to each other. As output networks from all three CLA neuron populations were largely ipsilateral, our analyses focused on ipsilateral connections. 3D reconstruction of axonal networks from each CLA neuron type exemplified differences in their projection preferences . Fiber dBoth the network span , and subsequently totaled for each broad region category , ORB, midline cortex , PPC and lateral cortices . Preference for association-related functional groups could be divided into three main trends . These preferences suggest that the dCLA population may primarily be involved in coordinating somatic-and-visceral-related regions that might be connected to insula function: autonomic control, interoception, pain or somatic processing (Lu et al., These differences in axonal network preferences provide strong evidence for a modular CLA, where topological organization reflects a functional segregation of different axonal networks/distribution channels. We established that the CLA can be divided into at least three topologically distinct zones that have unique network preferences which possibly allow parallel or competing processing of different global functions. It will be interesting to specifically perturb these CLA subpopulations during behavior and observe potential differences in function: it is likely that each CLA subzone is specialized to implement different behavioral functions that require coordination of multiple brain networks.Advances in CLA targeting techniques have enabled a plethora of studies that have begun to uncover the poorly-understood functions of the CLA. However, it has proven difficult to reconcile differences in the outcome of these studies because of the wide range of anatomical and genetic targeting methods that have been used. Each targeting method has interrogated different, but likely overlapping, populations of CLA neurons that reside in multiple CLA subzones. The topology-based functional segregation in the CLA that we have uncovered reveals the difficulty in comparing results that include CLA neurons from different topological subzones. In the future it will be imperative to keep in mind the zonal structure of the CLA, specifically to discern possible differences in the function of different CLA neuron subpopulations within different subzones.Our experiments have established a fundamental organizational property of claustral topology. Retrograde tracing and axonal mapping of M1-, RSC- and BLA-projecting claustral neurons revealed distinct localization of these neurons into separate claustral subregions and uncovered unique axon projection networks with distinct innervation trends. The distinctive differences in axonal networks suggest that CLA neurons residing in different topological zones are involved in drastically different forms of computation and potentially process and disseminate different types of information. Our discoveries, in conjunction with reports of topologically organized inputs to the CLA (Atlan et al., https://doi.org/10.21979/N9/FCPGSA.The raw data supporting the conclusions of this article are freely accessible at the NTU Data Repository The animal study was reviewed and approved by the NTU Institutional Animal Care and Use Committee.GH designed and performed the experiments, analyzed and interpreted the data, and wrote the article. GA designed the experiments, interpreted the data, wrote the article, and provided funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Herniation of a gravid uterus through an abdominal wall incisional hernia with overlying skin necrosis is exceptionally rare. A 29-year-old multiparous K30 + 4/40 pregnant female presented with a 1-month history of worsening abdominal wall skin changes. Magnetic resonance imaging of the abdomen and pelvis confirmed herniation of the gravid uterus into the hernia sac. A lower uterine segment caesarean section and hernia repair were performed by the general surgical and obstetrics team in view of the potential maternofoetal complications. This case highlights the importance of early recognition and the difficulties in managing gravid uterus herniation. Herniation of a gravid uterus through an anterior abdominal wall hernia is an uncommon complication of caesarean section with potential incarceration and strangulation of the gravid uterus . PlannedA 29-year-old multiparous (G5P4) K30 + 4/40 pregnant female was transferred from a peripheral centre with an irreducible incisional hernia containing her gravid uterus. Her previous four deliveries had been via lower segment caesarean section (LSCS) and aside from hernia formation were uncomplicated. The patient’s only other medical history was asthma.The patient initially presented to her local Obstetrics unit at K26 with abdominal wall discolouration. Clinically, there was suspicion that the uterus had herniated through the abdominal wall and magnetic resonance imaging (MRI) confirmed this.Upon presentation to our centre, the gravid uterus remained irreducible, but there was now ulceration and dry necrosis of the skin without evidence of infection . ObservaWith the progression of the pregnancy, there were concerns to both skin and uterine viability and ultimately the foetus. Accordingly, a semi-emergent case was planned at K30 + 6/40 conjointly between the General Surgical and Obstetrics teams.Pre-operative steroid loading was given in view of foetal prematurity. At operation, the foetus was delivered successfully via LSCS, the uterus was reduced into the abdominal cavity and the hernia sac was then excised completely. The abdominal wall defect was primarily closed in a vertical fashion with a continuous 0-PDS loop suture . Such a The patient had an uneventful recovery and was discharged four days post-operatively. She was scheduled for surgical outpatient follow-up for consideration of formal hernia repair and abdominoplasty.Gravid uterus herniation is uncommon due to the size of the gravid uterus, which is often too large to enter the hernia sac when it reaches the level of the hernia neck , 3. A reRisk factors of incisional hernias include multiparity, obesity, post-operative pneumonia, poor surgical technique, inappropriate suture choice for rectus sheath closure, additional surgical procedures, wound infection and wound dehiscence , 3, 7. IGravid uterus herniation may lead to severe maternal and foetal complications including intrauterine growth restrictions, preterm labour, abortion, lower uterine segment rupture and poor placental perfusion causing foetal death , 7. IncrManagement of herniation of the gravid uterus usually depends on the gestational age . The sucThe current management of gravid uterus herniation is guided by the patient’s clinical presentation, severity of complications and gestational age. As the hernia in our case was managed with a suture repair, a mesh repair may be required in the future if a recurrence were to occur. The number of cases of gravid uterus herniation may increase due to the increasing popularity of caesarean section; hence, its complications need to be discussed to facilitate early intervention.


Aging is an inevitable consequence of life, characterized by a progressive decline in tissue and organ function and an increased risk of death. There is growing evidence that aging is closely related to tumor development and immune regulation. However, in hepatocellular carcinoma, the relationship between cellular senescence and immune infiltration, energy metabolism, chemokines, and immunotherapeutic response is unclear and needs further study. We first analyzed 274 cellular senescence-associated genes by the NMF algorithm and identified two cellular senescence-associated clusters. Subsequently, we compared the differences between the two clusters, in terms of immune infiltration, energy metabolism, chemokines, and immunotherapeutic response to treatment. We further constructed risk models using cellular senescence-associated signature genes that could effectively identify the two subpopulations. Finally, we validated the validity and robustness of the risk model using an external dataset. We found significant differences in survival prognosis between two cellular senescence-associated clusters. In addition, we found significant differences in immune cell infiltration, expression of energy metabolism-related genes, expression of chemokine-related genes, expression of immune checkpoint-related genes, Tumor Immune Dysfunction and Exclusion between the two clusters. Also, a scoring system associated with cellular senescence was developed and validated as an independent prognostic indicator. It was validated as an independent prognostic factor and immunotherapeutic predictor for HCC. It was validated as an independent prognostic factor and immunotherapeutic predictor for HCC. The cellular senescence-related scoring system was validated as an independent prognostic factor and immunotherapy predictor for HCC, and patients with low CSS were characterized by prolonged survival time. Our study confirmed the relationship between cellular senescence and immune cell infiltration, energy metabolism, chemokines, expression of immune checkpoint-related genes, and response to immunotherapy. This enhances our understanding of cellular senescence and tumor immune microenvironment, energy metabolism, chemokines, and provides new insights to improve immunotherapy outcomes in HCC patients. It provides new insights to improve the outcome of immunotherapy in HCC patients. Despite the increasing use of surgical and local treatments worldwide3, it is estimated that about 50–60% of HCC patients will eventually undergo systemic therapy4. Since 2017, immunotherapy has been another major breakthrough in advanced HCC with encouraging results. In recent years, with the development of single-cell and second-generation sequencing, multi-omics research, bioinformatics, and tumor microenvironment (TME) research, biomarkers including gene alterations and pathway activations are of great significance for designing proper treatment regimens, and biomarker-driven therapies have shown gratifying benefits5. Targeted therapy, immune checkpoint blockers (ICB) therapy, and their combination therapy have shown superior efficacy for HCC in clinical trials. Although understanding of HCC genomics and breakthroughs in targeted therapies and immunotherapy have greatly expanded the therapeutic paradigm, challenges associated with HCC remain elusive. For example, most HCC patients have a low response rate to immunotherapy of 15–25%6, which falls far short of clinical needs. Currently, even though the objective response rate of immune checkpoint blockers has almost doubled in combination with targeted drug therapy, more than half of the patients still do not respond7. In addition, ICB can cause serious immune-related adverse events (irAEs) and hyperprogression disease (HPD) during anti-PD-1 or anti-PD-L1 therapies in about 8% of HCC cases9. Biomarker-based patient selection can help maximize efficacy and reduce the number of patients who may not benefit or even be harmed by ICB. Therefore, better immunotherapy prediction tools and biomarkers that accurately predict tumor characteristics are urgently needed to stratify patients and personalize treatment for HCC.Hepatocellular carcinoma (HCC) is a global health problem with an increasing incidence and mortality10. Regardless of the stimuli, senescent cells share some common behaviors, among which the main ones are growth arrest, apoptosis resistance, sustained DNA damage signaling, and heterochromatin modifications11. A growing body of research suggests that aging accompanies cellular senescence and that manipulating the biological process of cellular senescence can slow or retard many diseases associated with aging, such as cancer13. Recent studies have confirmed that aging and cellular senescence affect the immune microenvironment (TME) by promoting the accumulation of multiple types of immunosuppressive cells and activating various risk-related signaling molecules and cytokines, which have a broad impact on the TME and tumor growth15. At the same time, cellular senescence changes the adaptability of immune cells in the TME, thus altering the efficacy of tumor immunotherapy to some extent17. Therefore, an improved understanding of the impact of senescence on tumor immunity associated with the invasion and development of HCC is required to frame novel treatment paradigms for tumors.Cellular senescence is a complex stress response that affects cellular function and organismal health. Multiple developmental and environmental factors, such as radiation, oxidative stress, oncogenes, and protein accumulation, can activate genes and pathways leading to senescenceTo provide a comprehensive and systematic understanding of the relationship between cellular senescence and immune infiltration, energy metabolism, chemokines, and immunotherapy response in HCC, we established a cellular senescence scoring system and explored their potential importance as predictive biomarkers for prognosis and immunotherapy response. We further explored the mechanisms by which tumor cell senescence-related genes affect the TME. Subsequently, we validated the robustness and validity of CSS in multiple independent external data sets. This study provided new insights into the regulatory mechanisms of cellular senescence associated with the TME and strategies for HCC immunotherapy.https://portal.gdc.cancer.gov/), which contains 365 tumor tissues and 59 normal tissue samples, as a training set. To validate the robustness of the signature, we downloaded the sample information of tumor tissues from 231 HCC patients from ICGC (https://dcc.icgc.org/) as the validation set. Similarly, bulk RNA sequencing information and clinical information from 220 HCC patients in the GSE14520 dataset were downloaded from GEO as a validation set. Two external independent immunotherapy data were obtained from the IMvigor210 and GSE91061 datasets, respectively. The clinical features of the 365 HCC patients in the training set are summarized in Table https://genomics.senescence.info/cells/), which contains manually managed human gene data associated with cellular senescence.We collected gene expression data and complete clinical information data from 424 HCC patients using The Cancer Genome Atlas  clustering algorithm of the NMF package of R software algorithm to cluster 365 HCC samples based on the expression levels of 279 cellular senescence-related genes to identify distinct cellular senescence clusters. The “brunet” option was selected and 100 iterations were performed for the NMF. We determined the optimal number of clusters based on the apparent coefficients, dispersion coefficients, and silhouette coefficients to determine the optimal number of clusters for the 365 HCC samples.We further investigated the relationship between the clusters obtained by NMF clustering and clinical characteristics to determine the effects of cellular senescence-related genes on clinical characteristics.p-value < 0.05 and logFC > 2 or logFC < − 2. To further investigate the differences in molecular mechanisms between different clusters, we used the "clusterProfiler" package of R software to perform functional enrichment analysis of up-regulated and down-regulated genes separately, with p-value < 0.05 considered statistically significant.We used the “limma” package of the R software to perform differential analysis of the different clusters with the screening criteria of http://www.broad.mit.edu/gsea/msigdb/) contains two energy metabolism-related genes set (energy-requiring parts of metabolism and reactome energy metabolism) was used to identify the energy metabolism-related genes. We further downloaded the chemokine-related gene set from TISIDB (http://cis.hku.hk/TISIDB/). To further investigate the relationship between cellular senescence and chemokines and energy metabolism, we compared the expression of chemokines and energy metabolism-related genes among different clusters.The Molecular Signature Database  was implemented to estimate immune cell abundance of each sample in TCGA cohort based on a gene panel marking 28 immune cell types. We further analyzed the RNA expression information of 365 HCC cases using the ESTIMATE function of R software to evaluate the differences between different clusters in StromalScore, ImmuneScore, ESTIMATEScore, and TumorPurity.6). More and more studies have confirmed that MSI can not only predict the chemotherapy efficacy of early colon cancer, gastric cancer, and small bowel cancer but also be a predictor of immunotherapy efficacy of advanced solid tumors21. Richness and the Shannon Diversity Index were used to describe the diversity of the TCR repertoire. Richness measures the number of unique TCRs in the sample, while the Shannon diversity index reflects the relative abundance of different TCRs. BCR is a B lymphocyte receptor and BCR abundance is a combination of the various BCR isoforms produced in an individual. There is growing evidence that the diversity of peripheral TCR and BCR profiles can serve as a predictive and prognostic biomarker of the immune response in cancer patients. The cancer testicular antigens (CTA) score is used to evaluate tumor immunogenicity, which indirectly reflects the strength of the immunotherapy response. The richness values and Shannon diversity index valves of TCR in the TCGA LIHC patients were obtained from the Pan-Cancer Atlas study22. To determine the sensitivity of the two clusters to immunotherapy, we compared the differences in TMB, MSI, TCR richness, BCR richness, and CTA score between the clusters. We further compared the differences in the expression of immune checkpoint-related genes, such as PD-L1, PD1, PD-L2, TIM-3, and TIGIT, among different clusters. The Tumor Immune Dysfunction and Exclusion , considered to be the best predictor of response to immunotherapy, was used to predict response to immunotherapy among distinct cellular senescence clusters.We downloaded somatic mutation data from TCGA for all HCC samples and used the maftools package of R software to analyze different clusters and thus compare differences between clusters in terms of gene mutation and tumor mutation load (TMB).  * 10p-value < 0.05 and logFC > 1 or logFC < − 1, to obtain differential expressed genes. Univariate Cox regression analysis was performed to identify cellular senescence-related prognostic genes (p < 0.05). Next, the DEGs and prognostic genes were investigated using the R package “veen” to acquire prognostic cellular senescence-related DEGs, and correlations were visualized by the R package “circlize”. To eliminate overfitting, a least absolute shrinkage and selection operator (LASSO) Cox regression analysis was used in conjunction with the "glmnet" package. Finally, we utilized Cox multivariable analysis on the screened variables to find independent prognostic risk factors and construct a cellular senescence-related scoring system. Risk score = We further constructed the cellular senescence scoring system to better represent cellular senescence clusters. We used the "limma" package of R (version 4.1.2) to perform differential analysis among clusters with the screening criteria of To further examine the relationship between cellular senescence and immunotherapy response, we used two external independent immunotherapy data for validation. We calculated risk scores for each patient in both datasets using the same CSS as in the training set and divided all immunotherapy patients into high-risk and low-risk groups based on the median risk score. The proportion of patients who achieved CR/PR  or SD/PD  following immunotherapy was compared between the two groups to identify the differential in response to immunotherapy between risk groups.To verify the mRNA expression levels of two chemokine-related genes and four cellular senescence-related genes in HCC tissue samples, we collected tumor tissue samples and paraneoplastic tissue samples from 12 patients with HCC confirmed by postoperative pathology. We further compared the differences in mRNA expression of two chemokine-related genes and four cellular senescence-related genes in HCC tissue samples and paraneoplastic tissues using qRT-PCR.P < 0.05 was selected. Benjamini–Hochberg was implemented using the R function p.adjust to alter the P-value for multiple testing. R software was used to do all statistical analyses in this study (version 4.1.2).Wilcoxon t-test was used to compare differences between categorical variables. Univariate and multivariate Cox regression analyses were used to screen for independent risk factors affecting the prognosis of HCC patients. lASSO regression analysis was used to screen for signature genes for prognosis. A significance criterion of P < 0.05), while patients with Tumor Grade G3 and G4 were significantly more represented in C2 than C1 . Interestingly, we found a higher proportion of old patients (age > 60) in the C2 group  , 132 cases in Cluster 2 (C2) by a nonnegative matrix factorization (NMF) algorithm   Fig. A–D. Overp > 0.05), but the median MSI value of the C2 was higher than that of the C1 cluster  between clusters. Interestingly, C1 had the CTNNB1 gene as the major mutated gene, while C2 had the TP53 gene as the major mutated gene  and ICGC . In two independent external validation sets, ROC curves confirmed that risk scores can effectively predict 1-, 3-, and 5-year survival in HCC patients . We calculated risk scores for patients receiving immunotherapy in both immunotherapy datasets using the same formula as in the training set, and divided patients receiving immunotherapy into high-risk and low-risk groups based on the median value of the risk scores. We found a significant correlation between risk score and immunotherapy response Fig. D–E, withTo validate the robustness of the CSS, we collected tumor tissue samples and paraneoplastic tissue samples from 12 patients with HCC confirmed by postoperative pathological pathology from the clinic and verified the mRNA expression levels of four cellular senescence-related genes and two chemokine-related genes using qRT-PCR. The qRT-PCR results demonstrated that four cellular senescence-related genes  and two chemokine-related genes (CCL26 and CXCL5) were overexpressed in tumor tissues from HCC patients compared with paraneoplastic tissues Fig. . These r25. The introduction of immune checkpoint inhibitors in recent years has dramatically changed the treatment of hematologic and solid tumors, including HCC27. However, the immunotherapy of HCC has proven a challenge for the era of personalized therapy due to inter- and intra-tumoral heterogeneity. At the same time, significant problems such as low response rates to immunotherapy still exist in clinical practice, so it is crucial to use biomarkers to predict prognosis and immunotherapy response, and thus to implement personalized therapy for individuals.HCC is the sixth most common cancer in the world, and it is the fourth most lethal28. A growing number of studies have confirmed that senescent cells can be removed by SASP to stimulate the immune response and thus achieve clearance23. Thus, SASPs are positive for the body in the short term. However, in the long term, these functions could become detrimental in the context of immunosuppression of cancer to promote tumor development30. The association and value between cellular senescence, energy metabolism, chemokines, and tumor immune infiltration have not been reported, especially in HCC. Therefore, studies on the biological mechanisms and prognostic biomarkers of HCC concerning cellular senescence-related genes may offer an opportunity to identify HCC subtypes, improving the future application of precision focused treatments for HCC. In addition, the existing immunotherapeutic drugs suffer from both low response rate to immunotherapy and drug tolerance, and failed to meet clinical needs consequently. So revealing the immune microenvironment from the level of cellular senescence will provide new perspectives for the development of related drugs.Senescence is an injury-induced stress program that activates a series of pleiotropic cytokines, chemokines, growth factors, and proteases of the senescence-associated secretory phenotype (SASP), ultimately leading to persistent suppression of tumor cells and remodeling of the tumor immune microenvironmentWith the development of second-generation sequencing technologies, transcriptome analysis has paved the way for the identification of new prognostic and predictive biomarkers to discern the heterogeneity and complexity of tumors to develop new, individualized treatment strategies. Based on the expression of cellular senescence-related genes, we randomly divided the 365 HCC samples in the TCGA database into C1 and C2. Interestingly, we found significant differences in immune cell infiltration, energy metabolism, and chemokines between C1 and C2 The findings suggest that cellular senescence-related genes may contribute to different biological processes and immune phenotypes, while cellular senescence-related genes are somehow intrinsically linked to the expression of energy metabolism and chemokine-related genes. These genes have been associated with different tumorigenesis and anti-cancer immunity of individual tumors. Cellular senescence C2 is significantly enriched in cell division, and tumor-related signaling pathways, including Oocyte meiosis, Cell cycle; also associated with cellular interactions, including Neuroactive ligand-receptor interaction, Cytokine-cytokine receptor interaction. Interestingly, immune infiltrating cell analysis showed a higher percentage of MDSC, and Tregs cells in C2, suggesting that patients in C2 may be associated with an immunosuppressed state. Accordingly, immune checkpoint-related genes were significantly more highly expressed in C2, further confirming the existence of an immunosuppressive microenvironment in C2. Also, we found a lower TIDE score in C2, suggesting that patients in C2 are more likely to benefit from immunotherapy. Taken together, the newly identified cellular senescence clusters might provide novel insights into classification system of HCC.We constructed a novel prognostic signature based on the expression of four cellular senescence-related genes  by the stochastic survival forest method. In addition, we merged prognostic signatures with clinical characteristic factors and constructed Nomograms as a way to quantify risk scores and survival probabilities. Compared with other traditional predictors, the prognostic signature based on cellular senescence-related genes showed better accuracy and discriminatory performance in survival prediction. Notably, our results imply that CSS is an independent prognostic factor for HCC patients and also a predictor of immunotherapy. It has been suggested that a high-risk score may be associated with a favorable response to ICI treatment. Risk scores coupled with specific immune checkpoints may serve as predictive biomarkers of ICI response and prognosis.31. Shabangu's study showed that persistent high levels of HCV can induce altered cell adhesion and migration-promoting gene expression through the CXCL8-SRC signaling pathway32. A study by Bae et al.33. concluded that overexpression of EZH2 was significantly associated with poorer prognosis in HCC patients and that EZH2 could be a potential target for HCC immunotherapy. Lu et al.34. found that G6PD induces epithelial-mesenchymal transition through activation of signal transducer and activator of transcription 3 (STAT3) pathway, which contributes to HCC migration and invasion of hepatocellular carcinoma cells.All four related genes of our cellular senescence-related gene signature have been demonstrated in a variety of tumors. For example, a report by Li et al. found that CENPA overexpression was associated with advanced histological grade, positive serum HBsAg status, Ki-67 expression, and p53 immunopositivity35. Previous reports have suggested that microenvironmental glucose competition may be a driver of immunosuppression by depleting necessities required to maintain effector T cell function, whereas blockade of glycolysis in malignant cells can boost the efficiency of immunotherapy and metabolic remodeling between tumor cells and T cells36. Therefore, reflecting the cellular senescence status from the perspective of tumor energy metabolism and inflammatory response will help us to have a deeper understanding of tumor cell senescence. Taken together, uncovering the relationship between tumor cell senescence and energy metabolism, TME, and immunotherapy responses may provide a perspective on cellular senescence from an immune, metabolic, and inflammatory perspective, allowing us to discover how to effectively reshape the immunosuppressive microenvironment by inhibiting the senescence process or destroying senescent cells, so-called anti-aging therapies.Overall, our study provides important implications for clinical research. First, we have developed and discovered a novel scoring system that can classify patients with different treatment strategies based on different risk subgroups. We found that the risk score correlated with immune cell infiltration in TME, with more immune cell infiltration in the high-risk group, but also a higher proportion of immunosuppressive cells such as MDSC, and Tregs cells, suggesting an immunosuppressive state in HCC patients with high-risk scores. In addition, we found that HCC patients in the high-risk group exhibited lower TIDE scores and higher expression of immune checkpoint-related genes, suggesting a possible higher response to immunotherapy. Our signature showed better predictive power compared to other existing biological markers. These results suggest that CSS may further stratify patient response to immunotherapy in HCC. Thus, our study of the effect of cellular senescence on TME may enhance the understanding of immunotherapy response heterogeneity. Second, we found that cellular senescence correlated with the expression of energy metabolism and chemokine-related genes, with higher expression of energy metabolism and chemokine-related genes in HCC patients with higher risk scores, suggesting more vigorous energy metabolism and stronger inflammatory response in senescent cells. Glycolysis is a feature of both cancer cell progression and T cell activationAlthough our study has value in immunotherapy and prognosis for HCC, there are certain flaws in this study. First, the four cellular senescence-related gene sets we constructed are based on public databases, and therefore, external multicenter samples are needed for validation. Second, prospective clinical trials are necessary to verify the applicability of our findings in patients with HCC receiving immunotherapy. Third, the regulatory mechanism of cellular senescence-related genes on TME needs to be further confirmed in vitro and in vivo experiments. Finally, the preliminary interpretation of mechanisms underlying the association between cellular senescence-related genes and worse response to ICIs must be further elucidated using basic experiments.Finally, the cellular senescence risk profile presented in this study can be used to predict survival, identify molecular subtypes of HCC that may respond to immunotherapy, and provide a basis for clinical treatment planning. In the future, this signature could provide a reliable predictive tool for HCC patients.Supplementary Legends.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary Figure S6.Supplementary Figure S7.Supplementary Figure S8.Supplementary Tables.