Local anesthetics are commonly administered by nuchal infiltration to provide a temporary interscalene brachial plexus block (ISB) in a surgical setting. Although less commonly reported, local anesthetics can induce central nervous system toxicity. In this case study, we present three patients with acute central nervous system toxicity induced by local anesthetics applied during ISB with emphasis on neurological symptoms, key neuroradiological findings and functional outcome. Medical history, clinical and imaging findings, and outcome of three patients with local anesthetic-induced toxic left hemisphere syndrome during left ISB were analyzed. All patients were admitted to our neurological intensive care unit between November 2016 and September 2019. All three patients presented in poor clinical condition with impaired consciousness and left hemisphere syndrome. Electroencephalography revealed slow wave activity in the affected hemisphere of all patients. Seizure activity with progression to status epilepticus was observed in one patient. In two out of three patients, cortical FLAIR hyperintensities and restricted diffusion in the territory of the left internal carotid artery were observed in magnetic resonance imaging. Assessment of neurological severity scores revealed spontaneous partial reversibility of neurological symptoms. Local anesthetic-induced CNS toxicity during ISB can lead to severe neurological impairment and anatomically variable cerebral lesions. Peripheral nerve blocks are used for regional anesthesia. They are an effective, highly standardized and routinely used method to provide supplementary analgesia in a surgical setting , particuThe interscalene brachial plexus block (ISB) as well as the placement of a perineural catheter within the interscalene space follow a standardized technical approach. These are straight-forward procedures associated with an incidence of short- and severe long-term complications of only 0.4% . Two tecLong-term neurological deficits commonly attributable to nerve injury-induced peripheral neuropathies however are very rare and were reported to occur in 0.029% of patients who received an ISB . HoweverThere are several reports of severe and often fatal toxicity induced by accidental intraspinal injection of local anesthetics during ISB causing total spinal anesthesia ,27. HoweEthical approval of the study was granted by the institutional Review Board of the Ethical Committee at the University Hospital Frankfurt (project number: 20–699). Outcome data were gathered after the patients signed an informed consent form specifically authorizing prospective data collection.In this case series, we report on three patients who were admitted to the intensive care unit (ICU) of the Department of Neurology at the tertiary care hospital of the Goethe University between November 2016 and September 2019 with initially suspected perioperative stroke following ISB for arthroscopic rotator cuff repair performed at external hospitals.2) values between 98% and 100%. For premedication, the patient received orally administered midazolam. Single-injection ISB was performed prior to induction of anesthesia and intubation. Total intravenous anesthesia (TIVA) was maintained with propofol and sufentanil. For left ISB, the patient initially received single-dose injections of 1% lidocaine (20 mL) and 0.75% ropivacaine (20 mL) followed by a second single-dose injection of 0.75% ropivacaine (10 mL) three hours after initial local anesthetic injections and 15 min prior to termination of TIVA, and extubation. Safety checks prior to injection did not show any aspiration of blood or cerebrospinal fluid. Adverse cardiac or neurologic symptoms were not recorded. Advancement and placement of the catheter was performed in PNS technique using a stimulating catheter. An effective ISB was confirmed by physical examination prior to surgery. BP was measured every 5 min with a noninvasive cuff. The patient underwent an uneventful operative rotator cuff repair under the ISB and intravenous anesthesia. Intraoperatively, recorded systolic blood pressure (SBP) levels of 100–110 mmHg were interrupted by SBP levels of ≥90 and <100 mmHg for a maximum of 90 min due to the application of controlled hypotension. Administration of antihypertensive and/or vasoconstrictor drugs was not recorded. SpO2 (98% to 100%), and end-tidal CO2 (ETCO2) values (30% to 34%) were normal throughout the case. The duration of surgery was 170 min. After termination of TIVA, and extubation in the operating room (OR), the patient remained in a severely impaired conscious state. In the postanesthesia care unit the patient showed a sustained severe impairment of consciousness, and neurological examination by a neurologist additionally revealed aphasia, multi-modal neglect to the right, left-sided gaze preference, dysphagia, severe right-sided central facial palsy, severe right-sided hemiparesis graded as 2/5 according to the Medical Research Council (MRC) scale for muscle strength, right-sided positive pyramidal tract signs, and right-sided hemihypesthesia. These symptoms were further accompanied by complex behavioural manifestations  and facial motor movements suggesting complex focal seizures. Due to vomiting with pulmonary aspiration, the patient underwent an emergency intubation. Brain imaging four hours post-surgery did not detect any cerebral lesions.Patient 1 was a 52-year-old male with a medical history of multiple sclerosis, who underwent arthroscopic rotator cuff repair due to a spontaneous complete rupture of the left supraspinatus tendon. The positioning of the patient applied during surgery was the beach chair position. Preoperative blood pressure (BP) was 135/80 mm Hg; heart rate (HR) 66 beats per minute (bpm); oxygen saturation  followed by 0.375% ropivacaine (10 mL). There was a negative aspiration test for blood and cerebrospinal fluid prior to injection. Physical examination revealed an effective interscalene block. Adverse cardiac or neurologic symptoms were not recorded. After induction of anesthesia, and intubation, TIVA was maintained with propofol and sufentanil. BP was measured every 5 min with a noninvasive cuff. The patient underwent an uneventful operative rotator cuff repair under the ISB and intravenous anesthesia. Intraoperatively, recorded SBP levels of 100–120 mmHg were interrupted by SBP levels of ≥90 and <100 mmHg for a maximum of 35 min due to the application of controlled hypotension. Administration of antihypertensive and/or vasoconstrictor drugs was not recorded. SpO2 (98–100%) and ETCO2 values (34–37%) were normal throughout the case. The duration of surgery was 56 min. After termination of TIVA, and extubation in the OR, the patient presented with impaired consciousness and aphasia, a left-sided gaze preference, dysphagia, mild-to-moderate right-sided central facial palsy, right-sided hemiplegia, and right-sided positive pyramidal tract signs. Brain imaging four hours post-surgery did not detect any cerebral lesions.Patient 2 was a previously healthy, 52-year-old male with a spontaneous complete rupture of the left supraspinatus tendon. The patient received an arthroscopic rotator cuff repair, and a single-injection ISB combined with TIVA was selected as the anesthetic strategy. Preprocedural BP was 130/60 mm Hg; HR 66 bpm; SpOPatient 3 was a 71-year-old male with partial tear of the left rotator cuff who received an arthroscopic rotator cuff repair. The patient had an unremarkable medical history. The positioning of the patient applied during surgery was the beach chair position. An ISB combined with TIVA was planned as the anesthetic strategy. Left ISB was performed by injecting a single-dose of 1% lidocaine (20 mL) and 0.75% ropivacaine (20 mL) followed by continuous interscalene infusion of 0.2% ropivacaine at 6–8 mL/h. There was a negative aspiration test for blood and cerebrospinal fluid prior to injection. Advancement and placement of the catheter was performed in PNS technique using a stimulating catheter. Physical examination prior to surgery confirmed an effective interscalene block. Adverse cardiac or neurologic symptoms were not recorded. For the entire duration of surgery, the patient remained hemodynamically stable and the case proceeded uneventfully under general anesthesia. Immediately after termination of TIVA, and extubation in the OR, the patient presented a considerable delay of waking reactions and aphasia, left-sided gaze preference, severe right-sided central facial palsy, and right-sided severe hemiparesis graded as 1/5 according to the MRC scale for muscle strength. These symptoms persisted during postoperative monitoring in the postanesthesia care unit. Brain imaging three to four hours post-surgery did not detect any cerebral lesions.Local anesthetic-mediated central nervous system (CNS) toxicity was strongly suspected in case of new postoperative CNS symptoms including impaired consciousness or focal neurological deficits . Focal nSeizure activity was defined as continuous or recurrent rhythmic focal or generalized spikes, sharp waves, spike waves, or rhythmic waves changing in amplitude, frequency, and/or spatial distribution as evidenced by EEG . Non-conUpon admission, ICU management focused on symptomatic life-supporting treatment and seizure control. Clinical examination and laboratory findings were performed in order to rule out common causes for impaired consciousness such as hyperthermia, hypo- or hyperglycemia, hypo- or hypercapnia, anemia, sepsis or other metabolic disturbances. Patients with clinical signs of status epilepticus were managed according to the guidelines for the evaluation and management of status epilepticus .In the emergency setting, one patient received magnetic resonance imaging (MRI), while two patients had already initially received a computer tomography (CT) scan. However, MRI was subsequently performed in all patients. CT or MRI were combined with CT or MR angiography in all patients to exclude ischemic stroke secondary to large vessel stenosis or occlusion as well as cerebral vein or sinus thrombosis. Cerebrospinal fluid was examined in patients to rule out an acute manifestation of autoimmune- or pathogen-mediated encephalitis when deemed appropriate by the attending physicians. Qualitative tests for toxic agents or medications associated with seizures and other symptoms of toxic encephalopathy were performed at the discretion of the attending physicians.Patient characteristics are summarized in The patients underwent cerebral imaging including CT and MRI according to protocols designed for patients with suspected stroke. However, no abnormal imaging findings were observed in standard MRI protocol  on the day of ISB and symptom onset in all patients (Two out of three patients (patient 2 and 3) developed findings on MRI on day one and/or day five post ISB. More specifically, these patients showed T2/FLAIR hyperintensities with ADC restriction of cortical grey matter and basal ganglia of the left hemisphere b. MRI ofTotal length of hospital stay ranged from 9 to 19 days . All patPatients 1 and 2 showed partial, and patient 3 full reversibility of slow wave activity in the affected hemisphere. At the time of hospital discharge, seizure activity  was not observed in any patient . AssessmFollowing acute care, all patients underwent an inpatient rehabilitation treatment. Unfortunately, patient 2 passed away during rehabilitation due to a complicative pulmonary artery embolism. The functional outcome of patients 1 and 3 improved during their rehabilitative stay of 12 weeks. Patients 1 and 3 were invited for clinical re-evaluation after 28 and 47 months, respectively, and had achieved significant improvement of neurological functions . PatientLarge prospective studies have demonstrated that peripheral nerve block is an effective method of providing supplementary analgesia in the surgical setting. The various techniques reported are associated with a low incidence of acute complications that are mostly of transient nature. Nevertheless, there are a few reports describing persistent neurological symptoms after peripheral nerve block ,41,42, iHere, we describe to the best of our knowledge for the first time in detail three cases of severe stroke-like symptoms following ISB which we attribute to toxic effects to the brain caused by lidocaine and ropivacaine. We initially suspected a perioperative ischemic stroke in all three patients. There are several reports of patients who suffered cerebral ischemia which were linked with shoulder surgery, and especially for the beach chair position, which is used as the standard position ,44,45. HThe unilaterality of brain lesions and the absence of hemispheric watershed infarction also argued against a generalized hypoxic brain damage/hypotension-induced cerebral ischemia , especiaIn all patients, the effectiveness of ISB prior to general anesthesia had been confirmed clinically, which suggests that sufficient amounts of the local anesthetics were injected to the intended location. We neither were able to detect a hematoma of the soft tissue surrounding the extracranial arterial vessels, which are in close proximity to the injection trajectories of the interscalene approach , nor couIn summary, there is strong radiological evidence for neurotoxicity of local anesthetics for which the mechanism is apoptotic cell death , implyinNoteworthy, in our cases, signs of effective ISB prior to general anesthesia were at this time point not accompanied by symptoms of CNS toxicity. This observation is surprising at first glance, as the majority of adverse events is known to occur shortly after the injection of local anesthetic. However, a review of published cases on the clinical presentation of local anesthetic systemic toxicity revealed that cardiovascular and CNS dysfunction can have a delayed onset of greater than one hour after injection . This phToxic effects of local anesthetic on the CNS have been well characterized in preclinical studies in recent years. In these in vitro studies, all local anesthetics had neurotoxic effects, while the degree of neurotoxicity of local anesthetic was demonstrated to be concentration- or dose-dependent. Moreover, all drugs induced similar rates of early apoptosis at low concentrations, whereas at high concentrations, late apoptotic or necrotic cell death predominated ,61. ThesInterestingly, in vitro determination of toxicity induced by various local anesthetics revealed high toxicity of lidocaine and ropivacaine in neuronal cell lines ,63. The Our case study has some limitations. One is the small number of cases and some variability of clinical characteristics, which prohibits statistical conclusions and therefore generalization of the results. Moreover, there is a selection bias for critical neurologic morbidity as patients were admitted to our hospital from referring hospitals. Furthermore, the blood concentrations of the local anesthetics ropivacaine and lidocaine were not assessed. However, the symptomatology of a local anesthetic-induced toxic hemisphere syndrome seen in the three cases and presented here has not previously been reported. Thus, this case series describes and highlights this hitherto unconsidered complication.To the best of our knowledge there is no case series specifically describing patients with brain lesions induced by local anesthetics after ISB. We here report three cases of a local anesthetic-induced toxic left hemisphere syndrome caused with reasonable suspicion by local anesthetics used for ISB with an only partial improvement in functional outcome after hospital discharge. The severity of neurological impairment following peripheral nerve block observed in our patients should not degrade the value of this effective method of providing supplementary analgesia in the surgical setting. It rather should stimulate discussion around the neurotoxic potency of local anesthetics and emphasize that regional anesthetic techniques require appropriate postoperative clinical monitoring. In patients with new postoperative CNS symptoms following ISB, in addition to an ischemic or ictal etiology, a toxic effect of local anesthetics should be considered.


The heterogeneous tumor microenvironment (TME) is highly complex and not entirely understood. These complex configurations lead to the generation of oxygen-deprived conditions within the tumor niche, which modulate several intrinsic TME elements to promote immunosuppressive outcomes. Decoding these communications is necessary for designing effective therapeutic strategies that can effectively reduce tumor-associated chemotherapy resistance by employing the inherent potential of the immune system.While classic two-dimensional in vitro research models reveal critical hypoxia-driven biochemical cues, three-dimensional (3D) cell culture models more accurately replicate the TME-immune manifestations. In this study, we review various 3D cell culture models currently being utilized to foster an oxygen-deprived TME, those that assess the dynamics associated with TME–immune cell penetrability within the tumor-like spatial structure, and discuss state of the art 3D systems that attempt recreating hypoxia-driven TME-immune outcomes. We also highlight the importance of integrating various hallmarks, which collectively might influence the functionality of these 3D models.This review strives to supplement perspectives to the quickly-evolving discipline that endeavors to mimic tumor hypoxia and tumor-immune interactions using 3D in vitro models. The tumor microenvironment (TME) encompasses a complex cluster of cells that are programmed to fuel initiation, progression, and metastasis of cancer . The TME+ cell cytolytic activity driven by low oxygen could be of clinical significance as a prognostic factor for various tumor types, where hypoxia-driven CD8+ cell cytolytic activity could be associated with worse outcomes. A significant process driving hypoxic TME to influence immunosuppression is the enhancement of anaerobic glycolysis [Hypoxia within the TME is a significant tumor feature that can influence tumor-immune interactions. Under physiological conditions where oxygen is abundantly available, the conserved proline residues of hypoxia-inducible factor 1-alpha (HIF-1α) subunits are ubiquitously degraded through hydroxylation of its alpha subunits by the oxygen-dependent enzyme HIF-prolyl-4-hydroxylases (PHDs) . Followiycolysis . During ycolysis .Fig. 1H+ lymphocytes possess the capability to control cancer growth by exerting cytotoxic action over the cancerous tumor cells in a sustained manner [The continually evolving TME implements several biological events that influence tumor-immune interactions . Physiold manner . Howeverd manner , 22. Thed manner . Cancer d manner , incorred manner , and altd manner . The utid manner . In thisThe complex nature of cancer models and their ability to recreate the TME varies widely, ranging from two-dimensional 2D) models to animal-based models. Although animal models are viewed as critical platforms to test anti-tumor drugs, mouse models are costly, relatively time-consuming, and the studies performed using mice are often not repeatable in human trials . Also, tD models Approaches to recreate the TME in 3D models have evolved considerably over time. From early prototype models to the current state-of-the-art systems, 3D model designs have advanced immensely with our increased understanding of TME physiology. With time, the models have increased in complexity and served to recreate even more in vivo like parameters. As illustrated in Fig. While creating 3D experimental models to study cell-tissue interactions, the importance of using a prototype that aims to study TME hypoxia has always been a top priority. In order to fully understand the numerous consequences by which a low oxygen niche within the TME can affect tumor pathogenesis, several factors need to be concurrently embedded in a single 3D system. A hypoxic microenvironment could modify gene expression patterns in tumor cells , promoteThe effect of TME hypoxia on tumor cells has been studied extensively in 2D monolayer cultures . These 2A well-explored route taken by researchers to mimic the hypoxic TME is the development of cell spheroids . These mThe spheroid size can heavily regulate the efficient development of hypoxia within its core. A recent study  recreateAnother study harnessed the power of spheroids to evaluate the role miRNAs play in driving tumor-initiating cell (TIC) or cancer stem cell activity within the oxygen-deprived tumor . By utilOrganoids are another advancement in 3D models that have been able to recreate a hypoxic TME. The use of organoids to study tumor hypoxia hallmarks have demonstrated the recreation of miniature arrays of hypoxic cell-derived, self-organizing, tissue-specific outcomes that mimic their in vivo counterparts. One such study highlights the use of an organoid model to recapitulate tumor hypoxia and stem cell heterogeneity . In thisCerebral organoids were studied to understand how injury related to oxygen deprivation can affect the health of human neuroprogenitor cell subtypes . As it iOne of the significant hurdles that researchers face while using organoids is to correctly engineer the cellular complexity in a controlled manner that linearly demonstrates a schematic assembly of organ function. Also, when studying tumor hypoxia using organoids, a major hindrance for researchers has been the limited capacity of oxygen deprivation because of inadequate surface diffusion. This results in incubated cell death over more extended culture periods. Despite the shortcomings, scientists have rigorously used the organoid platform to examine how the diseased organ can be targeted for better treatment regimens.Biotech/biomedical companies, as well as academic scientists, have joined in the march to develop a perfectly suitable 3D scaffold that, while mimicking TME oxygenation profiles, can aid researchers’ design of improved chemotherapies. One such group at Brit Life Sciences developed the Cell-Mate 3D®, composed of natural hyaluronic acid and chitosan, which is individually responsible for providing a platform that exhibits features of the hypoxic TME . AlthougMicrofluidic 3D model platforms contribute to solid tumor hypoxia studies by supplementing a much-needed fluid-flow aspect achieved through cleverly designed micro-channels. Song et al. developed a microfluidic model that can systematically evaluate individual TME molecular factors and their relative downstream pathological effects, which is otherwise challenging to study in complex in vivo tumor samples . In thisThe need to develop dynamic TME models that allow researchers to study the extent of immune cell infiltration into the tumor corresponds to the demand to improve preclinical screening of immunotherapies. As the latest advancements in immunotherapy research are focused on the use of immune checkpoint inhibitors, these models would be essential to monitor the mechanisms by which TME influences immune evasion. Lately, the 2D models used to study tumor-immune interactions are being upgraded to 3D models as they help to reveal new immunotherapeutic perspectives . ClassicBombyx mori. Silk fibroin is being investigated extensively to produce an ideal TME building block for a 3D model [A large number of tumor ECM components have been modified to construct the TME in 3D models, such as the use of biosynthetic collagen platforms  and fibr3D model . The silMany groups have been researching techniques to develop 3D immune-oncology models that reflect TME interactions with peripheral blood mononuclear cells (PBMCs). For example, a study performed by Sherman et al. fused 96-well permeable support in a low-attachment microplate system . The hypThere is another interesting study that uses a spheroid platform (3D-3 culture), for assessing the variability in macrophage plasticity and evaluating how these differences can translate to the surrounding tumor microenvironment . The 3D-Modeling the tumor-immune microenvironment within organoids is of therapeutic importance because modeling a platform based only on peripheral immune population response alone does not lead to a clinically recognized translatable outcome for understanding checkpoint therapy mechanisms. Organoids are not adequate for reflection of the immune system in its entirety unless co-cultured with lymphocytes or other immune populations. For example, a study evaluated the usefulness of developing patient-specific autologous T cell – tumor organoid co-cultures that may help study tumor-immune interactions . The autIn order to unravel the entire potential of organoid technology to support tumor-immune studies, it is essential to explore the possibility of engineering the tumor immune microenvironment within an organoid. A holistic model recreating immune infiltration within a patient-derived organoid system has been characterized . This mo+ expression, there was a reduction in observed CD3+ cells after 8 days of culture. Besides, growth variations in several populations of myeloid-derived suppressor cells, double-negative T cells, and monocytes were found to be sensitive to drug response after immune profiling patient tumor and blood using this model. Overall, the organoid system reported in this study promises to be an essential benchmark that could help reveal a broader perspective of how various immunosuppressive mechanisms regulate tumor growth and metastasis in a patient-centric manner. The model could also be used to create patient-specific therapeutic regimens that help overcome patient-to-patient drug response differences.A third approach that researchers have commonly taken to incorporate the immune-niche into organoids is by resecting tumor tissue that already contains the patient’s stromal and immune components. For example, in the study performed by Finnberg et al., the authors propose a resected platform that can support primary tumor cells and other associated tumor cells for up to forty-four days following isolation from the source . The org+ T cell infiltration was significantly impaired under pathophysiological oxygen levels, and the inhibition of HIF or PD-L1 was able to re-sensitize breast cancer cells to cytotoxic T cells [Despite the importance of connecting tumor hypoxia and reduced immune surveillance within a single model, very few studies have achieved unifying these two hallmarks. On this note, there is a significant study that highlights the advantage of 3D cell cultures in reproducing hypoxia-dependent changes observed in the TME. In particular, hypoxia was shown to negatively regulate MHC expression in a HIF-dependent manner as demonstrated by lower MHC expression in hypoxic 3D models but not 2D tumor cell cultures in vitro. Downregulation of MHC class I expression is a hallmark of cancer and leads to cancer escaping recognition and rejection by anti-tumor T cells . Further T cells . One mor T cells . This reThe development of 3D ex vivo models is an area of rapid expansion, but the integration of immune cells in these cultures remains in its infancy. Further studies will warrant a better understanding of whether creating 3D in vitro models while mimicking tumor hypoxia and tumor-immune interactions would fare advantageous and therapeutically beneficial over classical in vivo animal models. Significant advances have been made in 3D tumor engineering toward development of the ideal TME model that may adequately accommodate multiple tumor properties. An ideal 3D TME model would function effortlessly by either being entirely inert from the patient immune variations or by implementing a personalized platform that is constructed using the patient’s cells. A 3D model that would bridge the gap between tumor hypoxia, immune evasion, and recreation of TME features as close as possible to in vivo conditions, must be suitable for all the different tumor cell types, without affecting inter-cell biology or the potentiation of immunogenic reactions. Although the advances have been noteworthy, all of the synthetic or biologically derived systems that have been fabricated with or without cancer cells have unfortunately hit a roadblock at some point in the developmental pipeline. The systems that have been developed in the presence of cellular growth are promising, but only from a proof of concept perspective. Also, the 3D models that have shown cellular growth have primarily been able to generate only specific tumor cell types: mutually exclusive populations of cancer, endothelial, stromal, or immune cells. Future studies that address the development of a system that can fuse all the different cell populations simultaneously are necessary. Results from several studies over the years have shed light on the understanding of solid tumor biology, thus forming the basis of TME modeling within a 3D in vitro niche. Despite extraordinary developments, most models recapitulate only some particular aspects of the TME; thus, further research and more holistic models are needed. As we advance in the field of 3D modeling of the TME, our methodologies and designs will hopefully become more superior. The synchronous blending of cellular growth, oxygen profile, immune cell interactions, and heterogeneity within the same 3D model would be a highly rewarding therapeutic advancement.


Metastasis is the most devastating stage of cancer progression and often shows a preference for specific organs.To reveal the mechanisms underlying organ-specific metastasis, we systematically analyzed gene expression profiles for three common metastasis sites across all available primary origins. A rank-based method was used to detect differentially expressed genes between metastatic tumor tissues and corresponding control tissues. For each metastasis site, the common differentially expressed genes across all primary origins were identified as organ-specific metastasis genes.Pathways enriched by these genes reveal an interplay between the molecular characteristics of the cancer cells and those of the target organ. Specifically, the neuroactive ligand-receptor interaction pathway and HIF-1 signaling pathway were found to have prominent roles in adapting to the target organ environment in brain and liver metastases, respectively. Finally, the identified organ-specific metastasis genes and pathways were validated using a primary breast tumor dataset. Survival and cluster analysis showed that organ-specific metastasis genes and pathways tended to be expressed uniquely by a subgroup of patients having metastasis to the target organ, and were associated with the clinical outcome.Elucidating the genes and pathways underlying organ-specific metastasis may help to identify drug targets and develop treatment strategies to benefit patients. Metastasis is a fatal step in cancer progression and is the main cause of cancer-related deaths . Tumor mGene expression profiles are widely used to explore organ-specific metastasis. Several groups have developed microarray-based diagnostic tools to determine a tumor’s site of origin , 8 or toIn this study, we investigate organ-specific metastasis by examining gene expression signatures across different tumor types that metastasize to the same organ . For each metastasis site , at least two types of primary site were included in the integrated dataset. Then, a rank-based method was used to detect differentially expressed genes (DEGs) between metastatic tumor tissues and the corresponding control tissues. We focused on common DEGs and enriched pathways across all tissues of origin, and investigated these organ-specific metastasis genes and enriched pathways in breast primary tumors. Cluster analysis and survival analysis were used to test whether the organ-specific metastasis genes and pathways were expressed uniquely by a subgroup of patients with metastasis to the target organ, and whether they were associated with clinical outcomes. In conclusion, we present here an analysis to identify signatures that are specific to the common target organ rather than to diverse primary tumor tissue types. The delineation of the roles of these signatures in the interplay between cancer cells and the target organ will lead to a better understanding of organ-specific metastasis and its susceptibilities to treatment.We searched all public databases for transcriptional profiles with clinical data of primary and metastasis site. Nine metastasis microarray gene expression datasets, covering three metastasis sites  , were coAll of nine metastasis microarray gene expression datasets were collected from the Affymetrix platform with relatively consistent quality control. For the two datasets performed on the customized GPL10379 platform (Rosetta/Merck Human RSTA Custom Affymetrix 2.0 microarray), only samples with high quality control (> 50% tumor cell content) were included in the datasets. Additional, among the five datasets metastasized from breast cancer, only one dataset GSE43837 was specific for the HER2+ subtype, this dataset was not excluded from this study in order to enhance the statistical power in DEG detection.To make full use of the information available from multiple datasets, data from different datasets were integrated using the R package virtualArray . The virGenes that were detected by all platforms Tables  and 2 weRankComp method is based on the relative ordering information of gene expression within each sample. As the relative ordering of gene expression is overall stable for particular types of normal human tissues across common platforms , reversai and j such that We further estimated effect size for the identified DEGs. To estimate the effect size in unpaired data, expressions of each DEG were assumed to follow normal distributions with different variances in condition linskaya , 23:1\don = ni + nj and q = nj/n, the denominator σ could be rewritten as:Let This effect size could, therefore, be linked to the Welch t statistic as:A medium effect size threshold of 0.5 was used to further screen the DEGs that were identified by the RankComp method.In addition, Chi-square test was used to test whether there’s a prominent bias towards available clinical characteristics (such as age and stage) between metastasis tumor and control samples for each dataset in DEG detection.To identify organ-specific metastasis genes, we compared samples grouped according to the metastasis sites. As illustrated in Fig. As individual genes often act in concert and may be responsible for multiple effects, organ-specific genes should be considered in a global context. Separate enrichment analysis was performed for up- and down-regulated organ-specific metastasis genes using known biological pathways , which wbb. Another 1979 DEGs were identified between 34 metastatic brain samples originating from lung cancer and the same brain control samples, denoted as Llb. Between these two lists, 1612 DEGs overlapped, accounting for 87% of Lbb  and 81% of Llb . There was a particularly large overlap for brain metastases from breast cancer and lung cancer  biology processes enriched for up-regulated brain-specific metastasis genes. Table S3. Gene Ontology (GO) biology processes enriched for down-regulated brain-specific metastasis genes. Table S4. Gene Ontology (GO) biology processes enriched for up-regulated liver-specific metastasis genes. Table S5. Gene Ontology (GO) biology processes enriched for down-regulated liver-specific metastasis genes. Table S6. Gene Ontology (GO) biology processes enriched for up-regulated lung-specific metastasis genes. Table S7. Gene Ontology (GO) biology processes enriched for down-regulated lung-specific metastasis genes.


Adults with congenital heart disease represent a complex and growing patient population. By virtue of their variant anatomy and the complex surgical repair often required in infancy, these patients are at risk of developing unique atrial and ventricular arrhythmias throughout their lifetimes. Electrophysiologists involved in the care of these patients should have a detailed understanding of their underlying anatomy and any prior surgical procedures to guide procedural planning and should have knowledge of the range of possible arrhythmia mechanisms that may differ from patients without structural heart disease. Despite this complexity, standard mapping techniques and electrophysiologic maneuvers may still be used to elucidate arrhythmia mechanisms, map tachycardia circuits, and guide catheter ablation. We report a case of two different macroreentrant right atrial tachycardias that were successfully ablated in a patient with congenitally-corrected transposition of the great arteries. As the morphologic right ventricle (RV) supplies oxygenated blood to the systemic circulation and the morphologic left ventricle (LV) supplies deoxygenated blood to the pulmonary circulation, patients are not typically cyanotic. However, cc-TGA is frequently associated with other congenital cardiac defects, including ventricular septal defects (VSDs) (60%–80%), pulmonary stenosis (30%–50%), and conduction system abnormalities (15%–50%). was transferred to our hospital following an episode of syncope and tachycardia. On the day of her presentation, she reported showering and experiencing a sudden onset of chest pain, palpitations, shortness of breath, headaches, and vision changes. This was accompanied by syncope. Her family also reported an episode of near-syncope approximately two weeks before this event; at the time, it was attributed to summer heat and underhydration. At an outside facility, her heart rate was recorded as 238 bpm and an electrocardiogram (ECG) confirmed a diagnosis of SVT. A dose of atenolol 12.5 mg was given, which resulted in brief periods of sinus rhythm, followed by the resumption of sustained SVT. She was also given three boluses of adenosine  without tachycardia termination. She was finally given an intravenous dose of 5 mg metoprolol, after which point, she converted to sinus rhythm. She was then transferred to our pediatric intensive care unit (ICU).A 15-year-old female with a medical history of cc-TGA, perimembranous VSD status following surgical closure, severe tricuspid regurgitation status following right atriotomy and mechanical tricuspid valve replacement, and pulmonary valvar and subvalvar stenosis status following LV-to–pulmonary artery (PA) extracardiac conduit with subsequent thrombosis On initial examination, she was afebrile and hemodynamically stable with a blood pressure of 113/65 mmHg and a heart rate of 120 bpm. Her cardiac examination revealed a resting tachycardia with a regular rhythm. She had a grade 3 harsh systolic ejection murmur at the left sternal border, normal mechanical valve click, and prominent apical heave. Her physical examination was otherwise unremarkable. Laboratory data on admission including serum chemistries, complete blood count, thyroid function studies, and cardiac biomarkers were unremarkable.. A transthoracic echocardiogram showed expected postsurgical changes related to her congenital heart disease (CHD) and a normally functioning tricuspid valve prosthesis. She also demonstrated underlying moderate systemic RV enlargement with RV systolic dysfunction.A review of her initial ECG demonstrated suspected atrial tachycardia with 1:1 AV conduction and baseline right bundle branch block . Atrial and ventricular activations were dissociated during tachycardia, excluding orthodromic reciprocating tachycardia and making AV-node reentry less likely.Given her history of complex CHD and highly symptomatic SVT, she was referred for invasive electrophysiologic study. At baseline, she had no evidence of dual AV nodal physiology on either decremental atrial pacing or during the measurement of the antegrade AV-nodal effective refractory period. The location of the AV nodal complex was usually situated. Retrograde atrial activation was decremental and concentric. Burst pacing from the RA appendage induced SVT (with a tachycardia cycle length of 262 ms) ® catheter  using the Carto® 3 electroanatomic mapping system . Propagation mapping was consistent with counterclockwise cavomitral isthmus– dependent atrial flutter . A ThermoCool SmartTouch® ablation catheter  was positioned in the cavomitral isthmus and entrainment from this location yielded concealed fusion and a postpacing interval equal to the tachycardia cycle length .High-density mapping of the RA was performed during tachycardia with the PentaRayAblation was performed during tachycardia in a slightly lateral location along the cavomitral isthmus to create a continuous lesion from the mitral annulus to the inferior vena cava. It was noted during catheter manipulation along this region that the curvature of the mitral annulus was demonstrably less convex than as is typically appreciated during cavotricuspid isthmus ablation. Tachycardia terminated during radiofrequency (RF) energy delivery, but pacing from the proximal coronary was suggestive of residual conduction through the isthmus. A series of consolidation lesions were delivered, yielding widely split double potentials, and bidirectional block was demonstrated using differential pacing. Repeat mapping was also performed during proximal coronary sinus (CS) pacing, which demonstrated “high-to-low” activation, consistent with block across the isthmus.. This tachycardia was hemodynamically unstable and required synchronized direct current cardioversion. The cavomitral isthmus was remapped during proximal CS pacing, which demonstrated persistent conduction block. Tachycardia was re-induced and persisted with 2:1 AV conduction. Activation mapping was again performed with the multipolar mapping catheter, which demonstrated a macro-reentrant atrial tachycardia originating from the RA free wall .Following this, low-dose isoproterenol was initiated. Burst pacing was again performed, which induced SVT with a slightly different atrial activation sequence (tachycardia cycle length: 236 ms) and 1:1 AV conduction With manipulation of the bipolar voltage calipers, several small channels were identified that corresponded to areas of slow conduction identified during activation mapping. Entrainment was performed in this region, yielding concealed fusion with a postpacing interval minus the tachycardia cycle length equal to 21 ms. RF application here resulted in tachycardia termination. A series of consolidation lesions were placed in areas where low bipolar voltage corresponded to arrhythmogenic channels identified in the propagation map. These were mainly located in areas of putative scar border zone and diastolic activity during tachycardia along the RA free wall. Afterward, extensive atrial burst pacing both during isoproterenol infusion and washout failed to induce any further SVT. She returned to the pediatric ICU without complications, recovering well. She was started on atenolol 25 mg once daily and discharged home the following day on her chronic medical regimen. She showed no subsequent SVT at six months of follow-up.5 Furthermore, the complex anatomic arrangement of conduction systems within the cc-TGA heart may lead to normal, anterior, or dual AV nodal systems, adding to variability in arrhythmia presentation.6 Our patient’s particular subset of associated congenital defects and surgical history makes her condition exceptionally rare.Patients with cc-TGA are rare, accounting for just 0.05% of all patients with CHD.10 Of significant concern among all types of CHD is the recurrence of previously ablated or the appearance of new atrial arrhythmias. Newer mapping methods, including the use of high-density mapping, reduce the number of lesions  required in ablation, which is likely to limit the likelihood of the creation of new arrhythmia substrate. Recurrence generally occurs within the first year after ablation (88%); those who do not experience a recurrence within this time period have a 91% chance of long-term cure.11The success of catheter ablation in adolescents and adults with CHD has improved significantly over time, likely related to advances in high-density mapping technology and ablation techniques.12 Reports of tachyarrhythmia treatment in the cc-TGA patient are sporadic, making it difficult to generalize about likely arrhythmia mechanisms.17 Studies focused upon atrial tachycardia in surgically repaired CHD do not typically carry sample sizes large enough to demonstrate isolated unique findings in the cc-TGA patient. In one study examining atrial tachycardia following the surgical repair of CHD, two of two patients with cc-TGA showed intra-atrial reentrant tachycardia (IART), whereas only three of six patients with dextro-transposition of the great arteries (d-TGA) displayed IART. This result may be linked to the particular type of procedure performed; while all six d-TGA patients underwent either a Mustard or Senning procedure, cc-TGA patients in this study received excision of subvalvular pulmonary stenosis, which possibly contributed to the arrhythmogenic substrate.18The overall prevalence of atrial tachycardia in patients with a transposition complex is 28%, although less is specifically known about the cc-TGA population.20 In general, typical atrial flutter using the cavotricuspid isthmus is the most common atrial arrhythmia mechanism in adult CHD patients,20 likely due to an increased degree of conduction delay through the crista terminalis and prolongation of atrial muscle refractoriness caused by atrial dilation and remodeling.23 While our patient’s systemic venous AV valve was a mitral valve , ablation of the initial atrial tachycardia was successful by transecting the cavomitral isthmus, suggesting that the underlying arrhythmia substrate is likely similar to that of anatomically normal patients with typical atrial flutter. As such, typical differential pacing maneuvers can be adopted to confirm conduction block as was the case in our patient.In patients with biventricular physiology following surgical repair of complex congenital cardiac lesions, the two most important anatomic obstacles allowing for RA macro-reentry are the tricuspid annulus and right atriotomy.24 Importantly, the atrial activation sequence during tachycardia cannot reliably distinguish between tachycardia mechanisms unless multiple RA catheters are used. Thus, electroanatomic activation mapping and entrainment mapping are important tools for defining the arrhythmia mechanism as well as identifying critical components of the reentrant circuits that are amenable to ablation. In our patient, the initial tachycardia was confirmed to be a cavomitral isthmus–dependent flutter by activation and entrainment mapping.As seen in the present case, IART is the next most common atrial arrhythmia mechanism in patients with prior surgery for CHD.Meanwhile, the second atrial tachycardia had a similar activation sequence but showed essentially vertical activation in the CS recordings. Typically, this is suggestive of a focal left atrial tachycardia, peri-mitral reentry, or roof-dependent flutter. However, as noted above, mapping and entrainment maneuvers identified this tachycardia as IART using the lateral atriotomy. There are several possible explanations for these disparate findings. In general, left atrial activation depends upon conduction over Bachmann’s bundle, through the interatrial septum, and through the CS musculature. In normal subjects, early activation is often noted in the distal CS due to Bachmann’s bundle connections near the base of the left atrial appendage. In this patient, slow conduction through the CS muscle and interatrial septum during IART may have allowed for a relatively greater contribution from Bachmann’s bundle to left atrial activation. Additionally, the tendon of Todaro and crista terminalis create natural barriers to conduction into the lower RA and, in the setting of altered intra-atrial conduction due to prior cardiac surgery, this may have preferentially directed activation through Bachmann’s bundle, leading to the resulting CS recordings seen in this case.24 Thus, voltage mapping can also be used to identify the critical circuit isthmus. The bipolar electrogram amplitude used to define scar varies: while many studies to date have defined scar as areas with a bipolar voltage of less than 0.1 mV, Nakagawa et al. only considered true scar as areas with no electrogram recorded above background noise, which can be as low as 0.03 mV. In the small study by Nakagawa et al., nearly half of successful ablation sites (7/15) were located in areas that would otherwise have been defined as “nonconducting scar” using a definition of bipolar voltage being less than 0.1 mV.25 Of obvious importance when using this approach is the assessment of the background noise present during mapping, which can vary significantly from laboratory to laboratory and which is affected by many external factors including cabling through the laboratory, proximity to alternating current power sources, and patient warming devices. In the present case, we were able to manipulate the voltage calipers in the mapping system to identify channels of relatively lower voltage coursing through the scar, which allowed us to create a complete line of block through the scar and prevent further SVT. Notably, using this approach, the absolute bipolar voltage below which the “scar” is annotated was less important than the relative voltage traversing between the putative slow-conduction channels and normal tissue. Channels identified in this manner can be compared to the activation map of the tachycardia to determine which channels may form critical circuit elements.The arrhythmia substrate in IART likely relies on low-voltage “channels” coursing through the scar that allow for the slow conduction required for sustained reentry.We report a rare case of a cc-TGA patient with prior tricuspid valve replacement and RV outflow tract surgery in whom multiple atrial tachycardias causing syncope were successfully treated with catheter ablation. The combinatorial use of activation, entrainment, and voltage mapping may assist in the ablation of atrial arrhythmias in complex CHD, whereas high-density mapping can be used to augment the understanding of a tachycardia’s endocardial activation sequence and scar border zones. In the cc-TGA patient, an awareness of inherent anatomical nuances coupled with the potential for multiple tachycardia mechanisms is essential to periprocedural planning and successful mapping and ablation.


Deepwater Horizon Oil Spill (DWH) on northern Gulf of Mexico (nGoM) reef fish communities. We observed significant changes in community structure immediately following the DWH, with a 38% decline in species richness and 26% decline in Shannon-Weiner diversity. Initial shifts were driven by widespread declines across a range of trophic guilds, with subsequent recovery unevenly distributed among guilds and taxa. For example, densities of small demersal invertivores, small demersal browsers, generalist carnivores, and piscivores remained persistently low with little indication of recovery seven years after the DWH. Initial declines among these guilds occurred prior to the arrival of the now-widespread, invasive lionfish (Pterois spp.), but their lack of recovery suggests lionfish predation may be affecting recovery. Factors affecting persistently low densities of generalist carnivores and piscivores are not well understood but warrant further study given the myriad ecosystem services provided by nGoM reef fishes.Large-scale anthropogenic disturbances can have direct and indirect effects on marine communities, with direct effects often taking the form of widespread injury or mortality and indirect effects manifesting as changes in food web structure. Here, we report a time series that captures both direct and indirect effects of the  Although natural disturbances can be important for maintaining diverse, resilient species assemblages8, research focused on the impacts of chronic anthropogenic stressors on biodiversity has revealed that even specious communities, presumed to be resilient, can respond unpredictably to natural and anthropogenic disturbances10. This is particularly true in marine systems which have experienced impacts from human activities for centuries and are severely degraded as a result12. Numerous examples exist of long-term community shifts from estuarine13, coral reef15, and continental shelf17 systems, and it is not uncommon for communities to remain unaffected by localized or moderate disturbances5, only to exhibit a non-linear response following a series of disturbances18 or a single event of sufficient scale or intensity19.The nature, frequency, and intensity of disturbance are important drivers of community structureDeepwater Horizon Oil Spill (DWH) was the epitome of a large-scale, anthropogenic disturbance capable of producing substantial community-level impacts. Over an 87-day period, approximately 4.9 million gallons of oil20 was released into northern Gulf of Mexico (nGoM) at a depth of ~1,500 m producing a surface slick of ~40,000 km2 at its maximum extent21. Between 4 and 14% of the total discharge was transported to the benthos by contaminated marine snow24, thus exposing numerous pelagic and benthic communities to toxic polycyclic aromatic hydrocarbons (PAHs)25 as well as emulsifying dispersants26. Both lethal and sublethal effects  of oil exposure have been well documented for numerous taxa37, and negative effects at the organismal level had the clear potential to elicit effects on community structure through bottom-up38 or top-down mechanisms40.The 2010 41, are widely studied by community ecologists42, and whose proximity favored the rapid collection of critical baseline data43. Despite extensive shoreline oiling44, impacts were mostly relegated to heavily oiled, coastal sites in Louisiana where significant vegetation loss occurred along the marsh edge45. Inshore communities, particularly nekton and fish assemblages, in areas of limited exposure showed little indication of negative effects higher than the organismal level49. Multiple assessments of community-level effects on the nGoM continental shelf also indicated no impacts or a resilient recovery. For example, changes in plankton communities were relatively brief and isolated to the active spill period52; post-DWH meiofauna diversity and abundance were comparable to pre-spill estimates53; and macroinfauna abundance and diversity showed no signs of impacts54.Much of the effort to document the community-level responses to the DWH was focused on monitoring coastal habitats that provide critical nurseries for several marine taxa56 more likely to be resilient to such a distrubance57. Considerably less is known about the impact to and response of neritic fish communities, including reef fish assemblages in the nGoM. These fish communities are diverse59, including numerous short-lived, small demersal species, as well as long-lived, fisheries species likely to be resistant to additional sources of mortality61. Key to the persistence of many fisheries species is the maintenance of multiple year classes of relatively old, mature individuals  that experience low natural mortality, which minimizes biomass loss between periodically strong year classes61. Thus, compensatory population increases following DWH-induced mortality would likely be slow.The lack of discernable DWH effects on community structure have been documented repeatedly but mostly for communities dominated by short-lived taxa62 while exploiting locally abundant prey64. However, evidence of sub-lethal PAH exposure suggests negative effects reached well beyond the footprint of the surface slick65, and recent ecosystem simulations suggest mortality resulting from acute resource limitation was perhaps more severe and widespread than exposure mortality66. Indeed, changes in feeding ecology, trophic position, and condition of red snapper (Lutjanus campechanus) were documented following the DWH67 and provide empirical support for a shift in resource availability with possible negative effects on population productivity67. Similar shifts in trophic position and pathways were observed in other reef fish species concomitant with petrocarbon cycling through the nGoM food web69. However, to date few data have been presented linking declines in reef fish abundance or community shifts that followed either direct effects of the DWH or indirect effects via food web impacts70.Resistance is also conferred through their high vagility and generalist diets which would allow individuals to disperse to avoid acute mortalityThe lack of information regarding community-level responses following the DWH may stem, in part, from the lack of pre-spill baseline data for many taxa and communities. Here, we analyze a time-series of nGoM reef fish community data that span an eight-year period starting before the DWH. We test whether significant changes occurred in reef fish community structure following the DWH, and if so whether changes occurred evenly across trophic guilds or were more concentrated within specific guilds. Results are presented in the context of acute, direct and chronic, indirect effects of the DWH, as well as factors that may affect the resiliency of reef fish communities.71, encompassed a depth range of 17–72 m, and were distributed over an 8,000 km2 area of the continental shelf. Sites were representative of the morphologically variable hard bottom habitat in the region and included low relief ledges, rocky ridges, rock rubble mounds, and flat limestone block reefs72. The epibenthic communities are dominated by coralline algae, soft corals , and sponges with limited coverage by azoozanthellate, ahermatypic corals72. Although impacts to epibenthic species were observed west of our sampling region73, sedimentation of contaminated marine snow was patchy24, and we observed no signs of habitat degradation . Thus, monitoring of the epibenthic community was not undertaken.Fish communities were surveyed at 16 natural reefs  lens for real-time viewing, twin forward projecting LEDs , and rated to a depth of 170 m. An additional forward-facing, high definition (1080p at 60–120 fps) camera  was mounted at a 45° angle to the ROV’s float block above the internal camera to record high definition video of the reef fish community.In total, 250 ROV surveys were completed between 2009 and 2017. The pre-DWH portion of our time series consisted of 26 surveys across 11 sites and occurred from the summer of 2009 to the spring of 2010. All 2010 surveys classified as pre-DWH occurred prior to surface oil entering our sampling region; surveys grouped in the 2010 time bin were sampled in November. During 2011–2013, sites were typically surveyed in the spring, summer, and fall, but later in the time series funding constraints limited sampling to summer months. Each ROV survey consisted of 3 to 4 orthogonal, 25-m transects either 1 or 2 m off the bottom, depending on visibilityet al.71. Results presented below only include those taxa identified to the level of species with the exception of purple reeffish (Chromis scotti) and dusky damselfish (Stegastes fuscus), which are difficult to distinguish on video footage and therefore combined into a single group, damselfish.Videoo samples were analyzed on a high-resolution monitor; observed fishes were identified to the lowest taxonomic level possible and enumerated. Counts were summed across transects to estimate the total abundance for each site. For active, schooling species prone to double counting , the total abundance across all transects was superseded by an estimate of the minimum number of individuals within a school obtained while maneuvering between transect locations. Species abundances were converted to densities by dividing by the total area sampled following the methods of Patterson 74, which is important to consider when evaluating environmental impacts on community structure. The PERMANOVA model had a three-factor hierarchical design with site nested within month nested within year. The nested factors, site and month, were treated as random factors while year was treated as a fixed factor. Our model also included two covariates, depth and longitude, which were z-score transformed. The reasons for this approach were: (1) each covariate represents a gradient along which reef fish community structure naturally varies75; (2) cluster analysis of pre-DWH community structure did not identify groups that might justify the use of discrete categorical factors to evaluate depth or longitude effects; (3) the use of covariates as opposed to fixed factors permitted the inclusion of the entire data set; and, (4) at this scale there is not a clear relationship between straight line distance from the well head and impacts. Changes in community structure were also evaluated using common community indices of species richness (S), diversity (Shannon-Weiner H′), and evenness (Pielou’s J′). All indices were calculated using the vegan package76 in R77. Temporal changes were evaluated with linear mixed effects models (LMMs) using the lme4 package78 followed by Dunnett’s multiple comparisons using the multcomp package79.Permutational multivariate analysis of variance (PERMANOVA) models were computed in PRIMER (v7) to test for temporal changes in reef fish community structure following the DWH. To reduce the influence of abundant species, taxa-specific densities were log(x + 1) transformed. A resemblance matrix was then computed based on the Bray-Curtis dissimilarity, with the inclusion of a dummy species at a density of 1. The dummy species was included because Bray-Curtis can behave erratically if few species are shared between sitesHaemulon flavolineatum) from guild-level estimates because densities of this schooling grunt (Haemulidae) were highly variable and often an order of magnitude larger than other guild members. Their inclusion obscured the more general, guild-level pattern  and recent analyses, and densities were summed by guild for each ROV sample. In the case of small demersal invertivores, we excluded tomtate  and a second to model the density given a guild or species was observed81. The product of the two sub-models was then used as the standardized density index for each guild or taxon. GLMMs included year as a factor and the repeated measures design was specified by including a random intercept parameter for each site. Longitude and depth were also included as covariates following z-score transformation. Least-squares means were calculated for each sub-model as the annual average from a reference grid of predictions across factor levels . Monte Carlo simulations were computed to estimate an annual density index and confidence intervals following the methods in Chagaris et al.82. Briefly, the product of the least-squares mean standard error and 10,000 random normal deviates X ∼ N was added to the least-squares mean estimate of annual density. Error deviates of the log-normal model were adjusted when the log-normal and binomial least-squares mean were correlated . Values of each Monte Carlo simulation were then back-transformed into their original measurement units to obtain a distribution of density values.Temporal trends in trophic guilds and species were assessed by computing standardized density indices with generalized linear mixed effects models (GLMM) following the delta approachThe results from binomial and log-normal models were also evaluated separately to infer whether temporal differences resulted from a significant change in presence/absence or non-zero abundance. Multiple comparisons performed using Dunnett’s method, as described above. For guilds and species observed prior to the DWH, comparisons were made between the pre- and post-DWH time periods with non-zero density estimates. For species only observed during the post-DWH time period, comparisons were made between the first and subsequent years with non-zero density estimates.Rhomboplites aurorubens) was the most abundant species in a given year   reflecting the fact either tomtate or vermilion snapper , as well as total fish density, declined following the DWH  abundance, while the decline in small demersal browsers resulted from lower densities of the cocoa damselfish (Stegastes variabilis) and seaweed blenny (Parablennius marmoratus)  and cubbyu (Paraques umbrosus) and differed from that of the tomtate, which has a looser association with the reef structure  Fig. . The treure Fig. .Figure 4Holacanthus bermudensis) was the most abundant large demersal browser and drove guild-level trends  and gray triggerfish  abundances. However, each species clearly displayed disparate patterns. The initial decline in red porgy was followed by an increase in 2011 and subsequent decline , and red grouper (Epinephelus morio) declined by 69%, 85%, and 70% following the DWH, and low densities persisted through 2017. The three most abundant piscivores, scamp (Mycteroperca phenax), gag (M. microlepis), and sandbar shark (Carcharhinus plumbeus), all declined after the spill and both scamp and gag remained at densities below pre-DWH baseline values. Densities of smaller bodied generalist carnivores, [e.g., bank seabass (Centropristis ocyurus) and belted sandfish (Serranus subligarius)  which was first observed in 2011 and rapidly increased through 2017  and damselfish  home ranges87, are heavily reliant on local resource pools, and would incur the highest cost associated with emigration. These traits not only increase the probability of exposure-related mortality but also mortality associated with resource limitation66. Impacts on pelagic production89, increased trophic position69, and greater reliance on benthic resources63 suggest small demersal reef fishes experienced increased resource competition and higher predation immediately following the DWH. The effects of resource limitation were possibly exacerbated by sub-lethal exposure37 which can result in physiological stress leading to impaired predator avoidance and foraging ability90. This inference is further supported by the fact that initial declines among large-bodied reef fishes  were less severe. These species are quite mobile, exhibit varying degrees of site fidelity on natural reefs91, and their movements can be affected by large-scale disturbances92. Additionally, a large area  of the continental shelf was closed to harvest for a few months during the spill, perhaps reducing fishing-related mortality, albeit temporarily. Emigration following the DWH also may have occurred if the cost of exposure or resource limitation outweighed the benefits of staying94. For some individuals, exposure may have been sufficient to elicit movement from affected areas. For others, the response was perhaps a shift in foraging behavior68, followed by emigration as resources became more scarce. Individuals present after the DWH probably consisted of few residents that survived acute exposure and resource limitation along with new immigrants seeking more favorable conditions93. The fact that guilds comprised of mobile reef species  showed little indication of recovery suggests a large number of individuals were either permanently displaced, perished (either from starvation or exposure66), or basal resource pools remained insufficient to support pre-DWH densities.We report declines across a range of reef fish taxa regardless of vagility, trophic position, or diet, which seem improbable without DWH-induced mortality. However, a primary challenge is identifying the extent to which declines resulted from mortality or emigration. Small fishes that live in close proximity to the reef matrix  experienced the largest initial declines, which likely reflects a high incident of acute mortality. These species can have limited 99. The 1971 red tide event is the most well-documented case of the impacts on reef fish communities and subsequent recovery. Exposure resulted in the near extirpation of reef fishes across a >1,500 km2 stretch of the WFS97. Conspicuous100 and indiscriminate reef fish mortality produced clear declines in species richness, but the most pronounced effects were changes in species relative abundance97. Recovery followed a predictable pattern of succession initiated by the arrival of small demersal species that recruit directly to reefs, followed by a peak in abundance among early pioneers, and subsequent decline in abundance superior competitors arrived; increases among large mobile fishes that do not recruit directly to reefs occurred later97. Full recovery took several years, but the community that developed was nearly identical to that observed prior to the 1971 red tide. The same general pattern of community level impacts, succession, and recovery were also observed on WFS artificial reefs following a 2005 red tide event98, corroborating observations by Smith97 and others that documented successional patterns among GoM and Caribbean reef fish communities102. Although the rate of succession can vary among sites104, after seven years of post-DWH monitoring no such pattern emerged despite similarities between community members in the aforementioned studies.The effects of natural disturbances within the region  are often insufficient to produce an observable change in large, mobile reef fish abundance105. While sedimentation of contaminated marine snow was the primary vector transporting oil and dispersants to the benthos, this phenomenon was patchy and mostly concentrated off the continental shelf and west of our sampling area107. As a result, sediment concentrations of total petroleum and polycyclic aromatic hydrocarbons (PAHs) were typical of background levels within our study area in August 201054. Nonetheless, examination of reef fish tissue samples indicated elevated levels of PAH concentrations in liver and PAH metabolites in bile persisted for years following the DWH108, thus providing evidence of continual reef fish exposure to toxic petroleum compounds for some time after the spill despite uncertainties about the mechanism of exposure.A clear difference between the DWH and natural disturbances was the potential for chronic PAH exposure, which can have long-term, higher-order impacts even at sub-lethal levels66. Consumption of swarming zooplankton by red snapper declined markedly following the DWH, a possible reflection of an initial reduction in pelagic production66. The reduction in zooplankton was balanced by increased foraging on demersal fishes and invertebrates63. This increased reliance on benthic resources continued over weeks and months following DWH, as indicated by declines in δ34S68. The associated enrichment of δ15N persisted for several years following the DWH68, and similar long-term shifts in δ15N were observed for gray triggerfish, tomtate, red porgy, and vermilion snapper concurrent with declines in δ13C, indicative of petrocarbon cycling through the food web109. The 2010 shifts in trophic position and community-wide declines that extended into 2011 also pre-date the arrival and rapid expansion of the invasive lionfish and are in general agreement with previous reports that community-wide impacts following the DWH occurred prior to the nGoM lionfish invasion70.The fact that reef fish density remained low for a number of years following the DWH suggests indirect food web effects have played an important role as well111 via predation113 and competition115. Impacts are typically most evident among small demersal reef fishes that recruit directly to reef habitats70 and vulnerable to predation both as adults116 and newly-settled recruits113. The stunted recovery of small demersal invertivores and browsers, despite their capacity for rapid recolonization following mass mortality events97 and the continued declines among native predators, provides a clear indication that the success of the invasive lionfish is affecting the response among native, small demersal fishes. How lionfish may be affecting the recovery of fisheries species has not been evaluated, but competition between lionfish and native predators  may affect population productivity117. Taxonomic resolution of dietary data for fisheries species is often poor and presents challenges when attempting to capture competitive interactions in systems with diverse species assemblages117. Diet information for the majority of small demersal reef fish is also lacking, and the potential food web effects emanating from low densities of small demersal species remains unknown.Although the role of food web effects resulting from a shift in resource availability is apparent, the success of the invasive lionfish may also be an important factor suppressing community recovery. Numerous studies have documented community-level effects of lionfishThe changes in community structure, particularly the persistently low densities among certain groups, provides a clear indication of lasting, community-wide impacts. The available evidence suggests initial declines in 2010 likely reflected both mortality and emigration resulting from exposure and resource limitation. Mortality due to direct or indirect effects of the spill likely drove initial declines of small demersal species, while large-bodied consumers were more likely to be permanently displaced or to suffer delayed mortality. Community-wide declines into 2011 were indicative of protracted resource limitation. However, the lack of recovery in small demersal reef fishes from 2012 onward may, to some extent, reflect top-down pressure from lionfish. How community shifts have altered the flow of energy to higher trophic level fisheries species or impacted system resilience remains uncertain.118 but also provide numerous economic119 and cultural120 benefits. Many of the ecosystems services we ascribe to reef fish assemblages in the nGoM are inextricably linked to fisheries harvest120, and fishery-dependent data and assessment of fishery stocks should enable tracking resilience among those species. However, continued funding for fishery-independent surveys, such as the ROV work that forms that basis of the analyses presented herein, is critical to assess the long-term effects of the DWH, lionfish invasion, and the potential for resiliency in the nGoM ecosystem.A clear challenge moving forward is identifying the underlying mechanisms driving these patterns, estimating the relative impacts of individual stressors , and developing management strategies to facilitate recovery. Efforts along these lines appear particularly relevant considering the dramatic declines observed for higher trophic position consumers that not only serve important ecological rolesSupplemental Materials.Appendix A.


Patent ductus venosus (PDV) is a congenital shunt between the portal vein (PV) and inferior vena cava (IVC). However, there are few reports on symptomatic adult-onset PDV, and the appropriate management of this condition remains unknown. In particular, there are few reports on the use of endovascular therapy for the treatment of patients with symptomatic adult-onset PDV. However, the strategies, indications, long-term efficacy, and safety of this therapy remain poorly understood. Here we report a rare case of adult-onset PDV successfully treated via endovascular coil embolization using a retrievable IVC filter.A 35-year-old man with a clinical course of progressive general fatigue and ataxia for 3 months was diagnosed with depressive personality disorder in another hospital 2 months ago and then referred to our hospital for detailed examination and further treatment. Blood test results showed hyperammonemia, indicating hepatic encephalopathy. Contrast-enhanced multidetector computed tomography and transarterial portography revealed a portosystemic shunt that connected the left PV to IVC. Endovascular coil embolization was successfully performed after temporary balloon occlusion testing and the placement of a retrievable IVC filter. After the procedure, ammonia levels gradually reduced, and his symptoms improved without any postoperative complications. No clinical symptoms were observed at the 6-year clinical follow up.This report supports the findings of other studies and offers a less invasive therapeutic option, thereby aiding clinicians in making appropriate treatment decisions for these patients. Several studies have described patent ductus venosus (PDV), a congenital shunt located between the portal vein (PV) and inferior vena cava (IVC). It is one of the causes of hepatic encephalopathy (HE) secondary to hyperammonemia in adults, but it is not well understood owing to its rarity and non-specific presentation; therefore, its diagnosis is often delayed. Consequently, these cases are often misdiagnosed as mental disorders , which suggested HE. Other laboratory data revealed no significant abnormalities, except low levels of serum albumin (3.0 g/dl) and a slightly elevated level of total bilirubin 1.4 mg/dl). Hepatitis B antigen and hepatitis C virus antibody were negative. Contrast-enhanced multidetector computed tomography (MDCT) suggested a portosystemic shunt that connected the left PV to the IVC . An 8-French sheath was inserted into the right jugular vein; following catheter insertion into the target vessel, the balloon was carefully inflated. However, the catheter was found to be floating and was easily pulled out. The aneurysmal dilatation diameter was found to be wider than that determined during the pre-procedure assessment. Therefore, we used a 7-French Swan-Ganz catheter  with a maximum balloon size diameter of 35 mm via the right common femoral vein (CFV), and it could occlude the target vessel completely. Additionally, complete occlusion was confirmed by catheter angiography via tip-hole of Swan-Ganz catheter and 5-French Judkins R. Temporary balloon occlusion testing (BOT) was performed for 3 h in intensive care unit to assess the changes in portal hemodynamics after complete embolization Fig. . ImmediaDuctus venosus is naturally closed immediately after birth (Meyer and Lind The treatment options for symptomatic PDV are as follows: conservative management with a low-protein diet, surgical management including liver transplantation, and endovascular management (Barsky et al. To the best of our knowledge, this is the first report of a patient who underwent coil embolization following retrievable IVC filter placement for PDV occlusion. Coil embolization has been performed along with balloon occlusion in previous studies to avoid coil migration (Araki et al. In previous reports, more than half of the patients with PDV were complicated with absence or hypoplasia of the intrahepatic portal venous system and were indicated for liver transplantation or surgical ligation of the shunt after BOT (Wanless et al. In conclusion, the findings of this case support those of other studies and indicate the efficacy and safety of endovascular coil embolization following retrievable IVC filter placement for the treatment of patients with symptomatic adult-onset PDV. This treatment approach may offer a less invasive therapeutic option.