Heart failure is a complex clinical syndrome associated with a significant morbidity and mortality burden. Reductions in left ventricular (LV) function trigger adaptive mechanisms, leading to structural changes within the LV and the potential development of dyssynchronous ventricular activation. This is the substrate targeted during cardiac resynchronisation therapy (CRT); however, around 30–50% of patients do not experience benefit from this treatment. Non-response occurs as a result of pre-implant, peri-implant and post implant factors but the technical constraints of traditional, transvenous epicardial CRT mean they can be challenging to overcome. In an effort to improve response, novel alternative methods of CRT delivery have been developed and of these endocardial pacing, where the LV is stimulated from inside the LV cavity, appears the most promising. Heart failure is a complex clinical syndrome associated with a significant morbidity and mortality burden. Reductions in left ventricular (LV) function trigger adaptive mechanisms aimed at regulating the cardiac output; however, over time these processes cause structural changes within the LV. This remodelling can result in the development of dyssynchronous ventricular activation, typically manifested by left bundle branch block on the 12-lead ECG. This is the substrate targeted during cardiac resynchronisation therapy; however, between 30 and 50% of patients do not experience significant benefits from this treatment [In response to the progressive haemodynamic stress associated with heart failure, compensatory structural changes occur within the heart. The left ventricular myocardial mass and composition exhibit evidence of change. As this occurs, the heart becomes less elliptical and more spherical as the geometry and volume of the LV change. This process is called remodelling and initially enables the heart to increase cardiac output by increasing both contractility and stroke volume. Whilst stroke volume initially increases, this process eventually becomes deleterious. Progressive enlargement of the ventricle leads to hypertrophy, causing increased wall tension and ultimately results in the myocardium becoming fibrotic, impairing contractility. As the left ventricle dilates and remodels, it exhibits greater contractile dyssynchrony further reducing efficiency.Dyssynchronous electrical conduction can manifest as abnormal heart rate modulation, disruption to the sequence of atrio-ventricular systole and overt ventricular systolic discoordination which typically manifests as bundle branch block on the 12-lead ECG. The development of left bundle branch block is thought to occur as a result of ventricular remodelling and/or fibrogenic damage to the cardiac condition system –6. Left In the context of left bundle branch block, the right anterior septal region is initially activated via the intact right bundle in juxtaposition to the left basal posterolateral region, which slowly propagates via cell-to-cell, intra-myocardial conduction. Contraction of the anterior right septum is imbalanced due to delayed activation of the lateral free wall and occurs unopposed. This has the effect of delaying elevation in the intra-cavity pressure gradient (dP/dtmax) as septal activation merely results in pre-stretch of the inactive lateral wall. When the lateral wall finally contracts, the septum is now in diastole, generating an energy sink which further reduces the overall ejection of blood via the left ventricular outflow tract. Cardiac mechano-energetic efficiency is further exacerbated by delayed activation of the posterolateral papillary muscle, causing sub-optimal closure of the mitral valve and ultimately mitral regurgitation.Over time, left bundle branch block (LBBB) activation causes molecular and cellular remodelling leading to alterations in glucose uptake, regional perfusion and calcium transport . These fCardiac resynchronisation therapy (CRT) aims to eliminate the dyssynchrony which results from bundle branch block activation and restore the mechano-energetic efficiency of the heart. During CRT, both the left and right ventricles are stimulated in an attempt to re-coordinate cardiac electrical activation and produce a synchronous and efficient contraction. Several large randomised controlled trials of biventricular (BiV) pacing have been conducted which have established the efficacy of this therapy, which yields both reductions in morbidity and mortality, see Fig. Around 30–50% of patients fail to respond to transvenous, epicardial CRT. This group are classified as non-responders although no unifying definition of response to CRT exists. Response can be measured in a variety of different clinical, functional and structural endpoints and patients can fail to respond for a variety of different reasons.The process of defining response to CRT is challenging as no universally accepted definition of CRT response exists. Response rates tend to be higher when clinical measures, such as subjective assessments of symptoms are used but are much lower when remodelling or outcome measurements are employed, see Fig. Additionally, symptomatic improvements do not always correlate with echocardiographic or functional classification improvements. There is also no consensus as to the optimal timeframe to re-assess LV function when gauging remodelling. The most widely accepted definition of response involves an assessment of left ventricular reverse remodelling (LVRR) 6 months after implantation, with reductions in LV end-systolic volume (LVESV) the most useful measure , 13. RemThe reasons why some patients fail to adequately respond to transvenous, epicardial CRT are multifactorial and involve several pre-, peri- and post implant factors, see Fig. Pre-implant factors associated with non-response to CRT include appropriate patient selection, baseline ECG morphology and QRS duration, gender, aetiology, presence of myocardial scar and disease severity.Optimal patient selection is critical when looking to maximise response to CRT. In both European  and AmerThe presence of left bundle branch block morphology is a strong predictor of response to CRT . Whilst The evidence for CRT in patients with non-specific intraventricular conduction delay (NICD) who possess a wide QRS without the appearance of left or right bundle branch block is also sparse. This ECG abnormality is present in between 3.8 and 5.8% of patients with impaired LV function , 23 and Conflicting outcomes following the use of CRT in patients with NICD have been reported with some studies appearing to show benefits in quality of life , whilst Women have been consistently under-represented in nearly all large-scale trials of CRT and yet gender appears to play a key part in determining response to CRT –30. FemaIn roughly 50% of patients receiving CRT, the aetiology of their heart failure will be ischaemic in origin ; howeverAround 25% of patients undergoing CRT implantation are in permanent atrial fibrillation (AF) . It is cGiven the importance of maximising the percentage of BiV pacing, it has been postulated that all patients with AF should undergo atrio-ventricular (AV) junctional ablation and this approach does appear to enhance the effects of CRT with the same magnitude as those seen in patients with sinus rhythm .Patients with a bradycardia pacing system in situ who go on to develop heart failure symptoms account for around 23–28% of CRT implants. The deleterious effects of chronic RV pacing have been well established –42 and rPeri-implant factors associated with non-response to CRT include utilising appropriate LV lead technology and optimisation of the LV lead stimulation site.Transvenous, epicardial CRT was traditionally performed by implanting a bipolar pacing lead in a tributary of the coronary sinus, facilitating depolarisation of the LV. Multisite pacing (MSP) where stimulation is delivered from two or more sites within the LV would intuitively appear preferable to single-site stimulation. In early pilot work, MSP was associated with greater reverse remodelling , 45; howMSP can also be delivered via multipoint pacing (MPP). This technique utilises quadripolar LV pacing leads which have four integrated pacing cathodes along the course of the lead, allowing greater customisation from any of the of the 10–17 MPP vectors available. The increased choice for the implanting physician means it is possible to programme around frequently occurring issues including high pacing thresholds–potentially caused by areas of scar- or phrenic nerve stimulation, where LV depolarisation causes diaphragmatic twitching. The use of MPP has been shown to yield greater improvements in acute haemodynamic response  and in aThe optimal LV pacing site displays large inter and intra-patient variability amongst patients with both DCM  and ICM Post implant factors associated with non-response to CRT include remote monitoring, frequency of biventricular pacing, device programming and optimisation.Almost all modern cardiac implanted electronic devices (CIEDs) have the capability to allow remote device follow up. Large, randomised studies have consistently shown that remote device monitoring is both feasible and reduces the need for ambulatory outpatient clinic attendance . The useWhilst CIEDs can analyse fluid status by calculating thoracic impedance as part of a multiparametric assessment, monitoring-only implantable technologies have also been devised. The CardioMEMS Heart Failure system  is a wireless pulmonary artery haemodynamic monitor, which provides an accurate assessment of real time pulmonary artery (PA) pressure, allowing the physician to optimise pharmacotherapy. Use of this system was associated with a 33% reduction in hospitalisations when compared to standard of care . Such syIn order for a CRT system to function effectively, it is essential that it is able to deliver consistent biventricular pacing. Frequent ventricular ectopics and atrial tachyarrhythmias can reduce the frequency of biventricular pacing and were identified in up to a third of non-responders to CRT . There aVentricular extra systolic beats can similarly disrupt the efficient function of a CRT system, reducing the frequency of effective biventricular pacing and as such, contributing to non-response. Again, the therapeutic target is to minimise the occurrence of such events either through the use of pharmacological therapy or catheter ablation .Appropriate programming can increase the frequency of biventricular pacing but is also essential in order to ensure the optimal mechanical functioning of the heart, which facilitates greater response. Arguably, the most important settings requiring optimisation are the pacing mode, upper and lower rate limits, the LV capture voltage, the stimulation vector and A-V and V-V intervals. Programming a high upper tracking rate ensures biventricular pacing is maintained during exercise. Similarly, the LV pacing output should include an adequate safety margin to ensure continual BiV pacing, although some modern devices can automatically adjust this parameter.Iterative optimisation of the A-V and V-V intervals using doppler echocardiography is the established reference method of achieving optimal programming by ensuring optimal diastolic filling of the LV , 71. RecThe persistent rate of non-response to transvenous, epicardial CRT has led to the development of novel forms of undertaking CRT. Of these, biventricular endocardial pacing (BiV ENDO), where LV stimulation occurs from within the LV cavity, holds a great deal of promise. This technique is associated with several advantages over epicardial activation. BiV ENDO pacing can be achieved using several different approaches including the use of novel, leadless pacing.A large body of evidence exists highlighting the potential benefits of BiV ENDO CRT.Failure to implant an LV lead in a tributary of the coronary sinus during a transvenous, epicardial CRT procedure occurs in around 5–15% of cases , 75, 76.BiV ENDO pacing is not reliant on the CS anatomy and instead, operators can choose to deliver stimulation at any site within the LV cavity. This allows for customisation of the stimulation site based on the patient’s pathology and physiology. In addition, whilst lead-based pacing systems may still encounter issues such as central venous stenosis and occlusion, particularly in the upgrade population, newer wireless pacing systems which can be delivered via a retrograde aortic approach eliminate the need for central venous access .Clinically relevant phrenic nerve stimulation (PNS) can complicate LV lead deployment during transvenous, epicardial CRT implantation and this complication is encountered in over 20% of patients at implant or at follow up . UnfortuEndocardial stimulation of the LV facilitates more rapid myocardial depolarisation as wave front propagation occurs along the shorter endocardial surface of the heart. Several studies have also demonstrated enhanced conduction velocities associated with stimulation of endocardial tissue –88. One Hyde et al. set out to establish a greater understanding of fast endocardial conduction (FEC). Non-Purkinje sub-endocardial tissue conducts impulses faster than either mid-myocardial or epicardial fibres . InsteadTraditional CRT delivers electrical stimulation to the epicardial surface of the LV wall. This pattern of activation is juxtaposed to the sequence exhibited during intrinsic myocardial activation which follows an endocardial to epicardial course. Reversing the normal depolarisation sequence is thought to promote repolarisation heterogeneity, which can prolong the QTc interval in susceptible patients and result in malignant arrhythmias –97. In cIncreasing evidence supports the hypothesis that BiV ENDO pacing has the capability to achieve greater electrical resynchronisation and consequently, maximise response to CRT. Traditionally, BiV ENDO pacing has been performed in a relatively small number of patients, typically those with failure to achieve conventional CRT or those that have been non-responders. The recently published ALSYNC study was the first multicentre study of LV endocardial pacing and demonstrated the feasibility and safety of LV endocardial CRT delivered through the atrial trans-septal approach . In 138 The acute haemodynamic benefits of BiV ENDO appear superior to those achievable utilising BiV EPI, even amongst a cohort of ischaemic, non-responders to conventional epicardial CRT . When a Chronic BiV ENDO CRT has traditionally been delivered via a pacing lead delivered via either a transventricular-septal or atrial-septal puncture. Due to the risk of thrombus formation resulting in systemic embolization, lead-based LV endocardial pacing requires lifelong anti-coagulation. In a recent meta-analysis, the stroke rate was found to be 2.5 per 100 patient years [Following a trans-septal implant, the potential also exists for an adverse interaction between the mitral valve and an LV lead which passes through it. The presence of a pacing lead can worsen valvular regurgitation and this phenomenon has been acknowledged with right-sided leads. Reassuringly in the largest series of trans-septal LV endocardial pacing to date, no system-related valvular complications were recorded. Instead, echocardiography showed reductions in mitral regurgitation as a result of improved LV function . The othLead-based BiV ENDO is predominantly delivered via the atrial trans-septal approach . SuperioVentricular trans-septal techniques allow the deployment of the lead using only superior access, eliminating the need for femoral punctures . This apA hybrid surgical/percutaneous approach has also been described where the LV apex is accessed via a mini-thoracotomy facilitating the deployment of a lead at the LV apex, which is then tunnelled up to a generator, implanted in the conventional sub-clavicular position . Whilst Whilst chronic BiV ENDO delivered via a pacing lead currently requires ongoing anti-coagulants due to the risk of thrombus formation, newer wireless technology almost entirely eliminated this risk. Formal anti-coagulation is not required and this development increases the likelihood of endocardial pacing becoming more widespread.A leadless BiV ENDO CRT pacing system has been developed, which eliminates the need for any form of pacing lead to be present within the left ventricle. Instead stimulation is delivered to the endocardial surface of the heart acoustically from an ultrasound (USS) pulse generator, implanted subcutaneously in an intercostal space, see Fig. Whilst co-axial alignment of the USS pulse generator and pacing electrode is desirable, the USS transmitter uses beam-forming to direct the energy towards the pacing electrode, minimising the amount of energy required. Refinements of this generator mean it can now be implanted via a small horizontal incision and is attached to a dedicated battery, typically placed in the mid-axillary line via traditional lead tunnelling techniques. Battery life of the current system is typically quoted at 3 years, but newer battery technology currently nearing release is expected to double this longevity to over 6 years.Early clinical studies evaluating this system confirmed implantation was safe and capable of achieving consistent biventricular capture  whilst mThe most commonly encountered complication associated with the use of the WiSE CRT system is femoral arterial access complications. In addition, not every patient has suitable anatomy to allow sufficient arterial access to allow the insertion of the electrode delivery catheter. Ischaemic heart disease is common in this population and as such, concurrent peripheral vascular disease is frequently encountered.A method of transvenously accessing the LV cavity via an atrial trans-septal puncture has been described to facilitate deployment of the pacing electrode . WiSE CRNon-response to CRT is a multifactorial issue. Improving patient selection and post implant device troubleshooting remain the cornerstone of optimising patient outcomes. Whilst newer lead technology including multipoint pacing and a greater focus on the importance of site selection are likely to be beneficial, the limitations of transvenous, epicardial CRT are widely acknowledged. In patients where implanting a transvenous lead is not possible, consideration should be given to endocardial pacing. Newer leadless LV endocardial systems no longer mandate lifelong anti-coagulation. In addition, endocardial pacing appears capable of yielding more effective electromechanical resynchronisation. Given the ability to stimulate the LV from any location, this technique would appear to offer a significant benefit to patients where transvenous lead implantation is restricted to inferior sites.


Cnidaria shared no skeletome proteins with Placozoa or Ctenophora, but did share some skeletome proteins with Porifera, such as galaxin-related proteins. Within Scleractinia and Octocorallia, we expanded the distribution for several taxonomically restricted genes such as secreted acidic proteins, scleritin, and carbonic anhydrases, and propose an early, single biomineralization-recruitment event for galaxin sensu stricto. Additionally, we show that the enrichment of acidic residues within skeletogenic proteins did not occur at the Corallimorpharia–Scleractinia transition, but appears to be associated with protein secretion into the organic matrix. Finally, the distribution of octocoral calcification-related proteins appears independent of skeleton mineralogy  with no differences in the proportion of shared skeletogenic proteins between scleractinians and aragonitic or calcitic octocorals. This points to skeletome homogeneity within but not between groups of calcifying cnidarians, although some proteins such as galaxins and SCRiP-3a could represent instances of commonality.A general trend observed in animal skeletomes—the proteins occluded in animal skeletons—is the copresence of taxonomically widespread and lineage-specific proteins that actively regulate the biomineralization process. Among cnidarians, the skeletomes of scleractinian corals have been shown to follow this trend. However, distributions and phylogenetic analyses of biomineralization-related genes are often based on only a few species, with other anthozoan calcifiers such as octocorals , not being fully considered. We de novo assembled the transcriptomes of four soft-coral species characterized by different calcification strategies  and data-mined published nonbilaterian transcriptome resources to construct a taxonomically comprehensive sequence database to map the distribution of scleractinian and octocoral  Calcification apparently has evolved multiple times independently within Cnidaria     and secr(SAARPs) —both foud CARP5) . The CARd CARP5)  is of pad CARP5) . CARPs aar space .The nonacidic regions of these acidic proteins match sequences found in other nonbiomineralizing cnidarians and bivalves, making the high occurrence of acidic residues a potential secondary modification linked to biomineralization .A. millepora appears to be taxonomically restricted genes (TRGs) in corals (A. millepora) have putative homologs in noncalcifying cnidarians, such as sea anemones and/or Hydra magnipapillata , systematically overlooking octocorals and calcifying hydrozoans  between them and their noncalcifying close relatives. For this, we de novo assembled the transcriptomes of four octocoral species, namely the massive, aragonitic blue coral Heliopora coerulea, and calcite producing species Pinnigorgia flava, Sinularia cf. cruciata, and Tubipora musica, three sclerites-forming octocorals. These species cover all calcification strategies within Octocorallia. Data-mining of newly generated and publicly available sequence resources was then used to produce fine-scaled phylogenies for selected targets of interest including acidic proteins , galaxin, and carbonic anhydrases. These results contribute to our understanding of the functional diversity and evolutionary history of coral skeletomes.Here, we conducted an analysis of the distribution of putative coral ergently , and helH. coerulea, T. musica, Pinnigorgiaflava, and Sinularia cf. cruciata, were mechanically collected from colonies cultured in the aquarium facilities of the Chair for Geobiology & Paleontology of the Department of Earth- and Environmental Sciences at Ludwig-Maximilians-Universität München in Munich (Germany) and kept under control conditions  for ca. 1 month before fixation in liquid nitrogen and subsequent storage at −80 °C.To obtain reference transcriptomes for our target octocoral species, samples of g and 4 °C) to remove remaining skeletal debris. A modified TriZol protocol , and sequenced on an Illumina NextSeq 500 at the Kinderklinik und Kinderpoliklinik im Dr von Haunerschen Kinderspital.For RNA extraction the samples were homogenized in 1–2 ml TriZol (Thermofisher) using a Polytron PT Homogenizer (Kinematica), and subsequently centrifuged  and low-quality reads (Q < 28) were removed with the Filter Illumina program from the Agalma-Biolite transcriptome package  under Bioproject number PRJEB30452. Assemblies, untrimmed/trimmed alignments, and output tree files from the various analyses are available at https://gitlab.lrz.de/palmuc/concietal_octoskeletomes.Quality control of assembled reads was done with FastQC . Source details for each data set is provided in −09) to retrieve putative homologs of coral calcification-related sequences. For the Porifera, Ctenophora, and Placozoa, databases provided in A. millepora . The analysis was limited to sequences predicted as complete by TransDecoder (see above). To visually investigate the contribution of changes in acid and basic amino acids to variations in isoelectric point, we performed a principal component analysis (PCA) on sequences grouped according to their phylogeny. Additionally, relative abundance of lysine and aspartic acid for each protein was calculated for the total proteome of seven anthozoan species, for which genomic data are available. Only species with available genomic resources were included in the analysis to avoid potential biases associated with transcriptome assemblies .Amino-acid composition of skeletal acidic proteins and their nonacidic homologs was estimated with ProtParam . Host acidic proteins were identified using a custom script (available in the project repository) using 9% aspartic acid content as the cutoff value and the identified sequences were searched  against the nonredundant NCBI database. Proteins with no hit or with octocoral-only hits were retained and their distribution mapped across octocoral data sets.The newly sequenced octocoral transcriptomes were data-mined to investigate the presence of putative biomineralization-related acidic proteins. Assembled contigs from the meta-assemblies were first assigned to either the host or the symbiont using psytrans (−09)  and galaxin-related proteins  are based on all putative homologs , with the exception of those matching galaxin-like 1 and 2 (ADI50284.1 and ADI50285.1), as these are exclusively expressed during early stages of calcification (−09) against the NCBI nonredundant database to avoid including usherin homologs in the data set. Homologous sponge collagen IV sequences were searched using the type IV collagen (Q7JMZ8) identified in the homoscleromorph sponge Pseudocoriticium jarrei as query. The analysis was limited to the N-terminal NC1 domain. Sequence of each putative homolog was checked for the presence of conserved cysteines   online. fication . Predictysteines  and addeysteines  were seahttp://tree.bio.ed.ac.uk/software/tracer/).Protein sequences were aligned with MAFFT  and MUSCA. millepora was found here restricted to acroporids. No protein was exclusively found in “Cnidaria+Placozoa” or “Cnidaria+Ctenophora,” while a small set of coral SOMPs appeared to possess homologs in Cnidaria and Porifera. These include galaxin-related proteins and the uncharacterized A. millepora protein USOMP-5 (B8VIU6.1). Although absent in Homoscleromorpha and Hexactinellida, galaxin-related proteins are ubiquitous among calcareous sponges and also found in all three currently described subclasses of Demospongiae. Within Heteroscleromorpha however, differences were observed between groups as no galaxin-related protein was retrieved from the genome of Amphimedon queenslandica  appears to be characteristic of scleractinian corals (see below). Within Octocorallia, homologs of SAARPs and CARPs could be retrieved only in the precious coral uggested . HomologC. rubrum  are occupied by proteins found occluded in coral skeletons. Only scleractinians are represented within these groups. S1 contains homologs for the acidic SOMP (B3EWY7) and P27 isolated from illepora  and S. pstillata , respectstillata a CARPs aPorites sequences, together with Favia sp. 24967, Platygyra carnosus 1685, and Pseudodiploria strigosa 22901, were placed as sister group to other scleractinians (S. pistillata protein 17235 (NS1). As previously reported  and the original G. fascicularis galaxins. The unifying feature of this clade is the RXRR endoprotease target motif described in The exception to this general pattern is a scleractinian-only clade comprising both complex and robust corals. The group includes both Rhizotrochus typus sequences nesting either within Complexa (MAFFT) or Robusta (MUSCLE). When performing the analysis on galaxin sensu stricto sequences only, galaxin-2 sequences concordantly grouped together with other complex scleractinians .As for galaxin-related proteins, Both phylogenetic analysis resulted in monophyly of collagen IV for all three sponge classes in which the protein is present  online. A common feature of skeletal proteomes is the presence of both taxonomically widespread proteins with homologs in other, not necessarily calcifying, organisms and of lineage-specific innovations or TRGs . The divDistribution analysis reflected evolutionary heterogeneity, with homologs being present across phyla or restricted to selected families. Although a few coral skeletogenic proteins remain largely restricted taxonomically, increased taxon sampling resulted in the expansion of their taxonomic distribution. In these cases, the most common pattern was their presence across phyla, or limited to Cnidaria or Scleractinia, which does not support the involvement of these proteins in biomineralization across groups. SCRiP-3a and galaxin-related proteins are, however, potential targets for future  research, because of their presence pattern . The distribution of the latter within sponges is of particular interest as we show that these proteins are present in all calcifying species, regardless of their taxonomic position. Moreover, the presence of multiple potential galaxin homologs among calcifying Calcarea and their absence among homoscleromorphs and glass sponges, supports their potential involvement in calcium carbonate biomineralization.type IV collagen appears however, as previously hypothesized by As for galaxin-related proteins, collagen IV appears either ubiquitous or absent in different sponge classes, while a patchy distribution can be observed among groups of Demospongiae. Within Heteroscleromorpha presence of S. pistillata) and B3EWY7 (A. millepora)—which are best BLAST reciprocal hits—is supported by the high similarity in the location and structure of their acidic regions. Moreover, such increases in aspartic acid could not be observed within scleractinian total proteomes. This excludes the possibility of higher aspartic content representing a lineage-specific innovation, and supports it being a biomineralization-related event.Scleractinian TRGs also exhibited a wider variety of distribution patterns, ranging from being present across both robust and complex corals down to small set of scleractinian families only . The former are of particular interest for the evolution of corals. Although different time estimates have been put forward, the accepted consensus places the divergence of Complexa and Robusta in the Palaeozoic, prior to the (ca. 240 Ma) appearance of fossil modern scleractinians in the early/mid-Triassic . The disA. millepora galaxin 1 and galaxin 2 remains uncertain due to the current lack of support in phylogenetic analyses. Despite this, phylogenetic analysis allows to confidently argue that the protein is present in the family Agariciidae and Acroporidae and it should be considered a true (sensu stricto) galaxin. One aspect that remains unsolved concerns the evolutionary history of galaxin sensu stricto outside Scleractinia. Extensive divergence between scleractinians and other cnidarians could have eroded the evolutionary signal in these proteins . The presence of TRGs (such as scleritin) in species belonging to all the three major octocoral clades (Finally, in contrast to scleractinians, octocoral TRGs were found conserved across soft-coral taxa showing similar distributions. Although the number of calcification-related genes in soft corals is currently extremely limited, intra-Octocorallia analyses are of potential interest, as they might allow for the identification of differences between calcite and aragonite-deposing species, and similarities between aragonitic animals within Anthozoa (i.e., l clades , indicatl clades , the proHere, we conducted a distribution and phylogenetic analysis of coral biomineralization genes to provide a comprehensive homolog mapping and fine-scaled phylogenies of selected genes. Through a relatively broad taxon sampling, our work allowed us to detect similarities and differences between different taxonomic groups and investigate patterns of protein presence/absence associated with skeleton polymorph. This led to the postulation of a single recruitment for calcification of galaxin sensu stricto and provided a detailed phylogeny of coral acidic proteins that revealed the increase of acidic residues during cnidarian evolution. We also provide insights into the evolution of proteins likely involved in biomineralization, such as sponge collagen IV. With the inclusion of four new octocoral transcriptomes, we have closed the existing taxon bias toward certain cnidarian taxa, specifically scleractinian corals, however gaps still exist. For instance, groups like calcifying hydrozoans remain unexplored and their inclusion in future studies on biomineralization will certainly contribute to our understanding of this process in Cnidaria. Proteomic investigations of the SOM of calcifying cnidarians other than scleractinian corals and of sponges might reveal the presence of shared skeletome components adding support to the transcriptomic presence patterns described here, and will help discover lineage-specific innovations linked to calcification in these groups. Genome Biology and Evolution online.evz199_Supplementary_DataClick here for additional data file.


The absence of virus expression during the chronic stage of bovine leukemia virus (BLV) infection and its reactivation upon ex vivo culture has become a long-lived Dogma. During the chronic stage of BLV infection the immune response limits viral replication and the mitotic division of latently infected cells, carrying BLV provirus, allows viral expansion and disease progression towards a lymphoproliferative disorder. Several stressor factors have been associated with animal production and handling. As natural mediator of stress, glucocorticoids are strong immunosuppressive agents; moreover, they can bind long-terminal repeat region of retroviruses and induce viral expression. In the present study, we present a case report describing the spontaneous reactivation of BLV infection in naturally infected cattle.pol gene strongly suggests virus reactivation in these animals. Interestingly, this simultaneous virus reactivation was unrelated to dexamethasone treatment.In order to investigate if virus reactivation occurred in vivo during the course of BLV infection, we followed up for 328 days one Holstein cow (> 3 years) chronically infected with BLV which presented high-proviral loads. This animal was neither lactating nor pregnant. Furthermore, we investigated if a stressor stimulus, in this case the administration of a synthetic glucocorticoid (dexamethasone), could impact the course of BLV infection in three additional cattle. For the first time, we observed a high level of BLV transcripts in a total of four cattle chronically infected with BLV. The detection of viral transcripts corresponding to We reported for the first time spontaneous and high level of BLV transcriptional activation in cattle chronically infected with BLV. Although virus reactivation was unrelated to dexamethasone treatment, other stressor stimuli might have influenced this outcome. Future studies will be necessary to understand these observations, since the spontaneous virus reactivation presented here might have implications on BLV pathogenesis and transmission.The online version of this article (10.1186/s12917-019-1908-7) contains supplementary material, which is available to authorized users. Bovine leukemia virus (BLV) is a deltaretrovirus from the Orthoretrovirinae subfamily and Retroviridae family. BLV causes a persistent infection in cattle and in most cases this infection is asymptomatic . In one During primary BLV infection, viral spread occurs via the production of virions able to infect new target cells (replicative infection stage) . The expHere we present a case report describing the spontaneous reactivation of BLV infection in naturally infected cattle. Moreover, we performed a pilot study to investigate if a stressor stimuli, in this case the administration of a synthetic GC [dexamethasone (DEX)], had an effect inducing the reactivation of BLV chronic infection in cattle. We hypothesized that by interacting with BLV LTR and/or modulating host immunity, DEX may provoke BLV reactivation in its natural host.Spontaneous replication of BLV in chronically infected cattle. In order to investigate if virus reactivation occurred during the course of chronic infection with BLV we followed-up for 328 days, one single Holstein cow  which was chronically infected with BLV and presented high-proviral loads. Blood samples from this animal were obtained at days 0, 20, 52, 82, 110, 160, 165, 167, 174, 181, 188, 216, 244 and 328, and the detection of viral RNA in plasma was used to assess virus reactivation in this animal under natural conditions. Refer to the Supplementary Materials for a detailed description of all materials and methods.pol RNA expression in animal 184 , suggesting different times of virus reactivation ] . Although this animal presented very high-proviral load for several years (> 3 years) it did not present any clinical sign. Moreover, we did not detect BLV RNA expression in plasma beyond day 188 , had an effect inducing the reactivation of BLV chronic infection in cattle. We hypothesized that by interacting with BLV LTR and/or modulating host immunity, DEX may provoke BLV reactivation in its natural host. For this purpose we administered 0.1 mg/kg of DEX (IV) during five consecutive days to three Holstein cattle  chronically infected with BLV; of note, none of these animals had detectable levels of BLV RNA in plasma before the initiation of the study  , might provoke virus reactivation in BLV chronically infected cattle. Stress is defined as the biological response elicited when a threat to homeostasis is perceived. Several psychological and physical stressors factors are associated with animal production and handling . Animals exposed to stress stimuli present higher levels of endogenous glucocorticoids (GC)  which in turn induce the expression and/ or repression of several genes, through binding GC-receptor (GCR) , 9. In aIn summary, we reported for the first time spontaneous in vivo BLV transcriptional activation in cattle chronically infected with this virus. Transient virus reactivation might have implications on BLV pathogenesis  and transmission. Further studies are granted to study all these preliminary findings and their implications.Additional file 1:Table S1. Detection of BLV pol gene (DNA) and transcript (RNA). (DOCX 29 kb)


Crassostrea virginica), the primary faunal component of shell rings. We estimated the timing of oyster harvest at St. Catherines Shell Ring  by statistical modeling of size frequency distributions of the impressed odostome (Boonea impressa), a parasitic snail inadvertently gathered by Archaic peoples with its oyster host. The odostome samples from three archaeological excavation units were evaluated against resampling models based on monthly demographic data obtained for present-day populations of Boonea impressa. For all samples, the harvest was unlikely to start earlier than late fall and end later than late spring, indicating that shell deposits at St. Catherines Shell Ring formed seasonally with substantial harvesting activities restricted to non-summer months. For all samples, the resampling models indicated that harvesting activities likely occurred during multiple months. However, these analytical outcomes would also be expected in the case of extensively time-averaged records of short-term, non-summer harvest events. Regardless of the exact harvest duration, the results point to seasonal harvesting and suggest that Archaic populations may have opted out of consuming summer oysters to focus on other resources, avoid unpalatable food, decrease pathogen risks, or ensure sustainable harvesting.The function of Late Archaic period (5000–3000 B.P.) shell rings has been a focus of debate among archaeologists for decades. These rings have been variously interpreted as a product of seasonal feasting/ceremonial gatherings, quotidian food refuse generated by permanent dwellers, or a combination of seasonal and perennial activities. Seasonality of shell rings can be assessed by reconstructing the harvest time of oysters ( Shell rings are circular or arcuate accumulations of shell, bone, artifacts, soil and other anthropogenic sediments deposited along the coasts of Florida, Georgia, South Carolina, and Mississippi ,2. ThreeCrassostrea virginica) are the most numerous shellfish species in nearly all shell rings, suggesting that they were a key component of the practices that contributed to ring formation  are unnecessary [for more details about the resampling protocol see supplementary material, The analytical strategy proposed here is based on computer-intensive resampling that represents a machine-learning strategy where known distributions (133 R-SFDs) are used to develop predictive models (training sets) against which the evaluated datasets  are assessed numerically. Specifically, 133 R-SFDs represent reference month sets (known harvest times) that can be used to develop 133 predictive null models. Each of those null models is developed by iterative comparisons of bootstrap samples of a given R-SFD to all 133 R-SFDs using a sample size All models are based on 2000 bootstrap replicate samples carried out separately for each archaeological sample. Repeated runs indicate that estimates are reasonably stable when 2000 replicates are used. Because 133 models each involving 133 comparisons are developed for each sample, an analysis of one archaeological sample requires (133*133*2000 iterations = ~35 million iterations) and for this reason generating more than 2000 bootstrap replicates was deemed impractical. On a reasonably powerful personal laptop , and without employing parallel (multi-core) processing, a single simulation (2000 iterations) for the specific datasets used here requires approximately 1 hour.https://github.com/MJKowalewski/Boonea. The collection accession numbers  and geographic and stratigraphic information for archaeological and modern specimen lots used in this study are provided in an appendix , changed successively across seasons (n = 6). In June, large specimens observed in May samples were nearly completely absent, indicating mortality within ~12 months of spawning in both years of sampling. From June through April, R-SFDs shifted progressively toward larger size classes and remained strongly unimodal  are also distinctly unimodal and the maximum specimen size (maximum shell height = 6.20 mm) is comparable to that observed for the present-day populations (maximum shell height = 6.19 mm). The samples derived by horizontal pooling of subsamples  are visually comparable to vertical samples . In mostB. impressa were comparable to those observed for the present-day populations. The high abundance of archaeological specimens in size classes that dominate late fall, winter, and early spring samples of recent populations, suggest that archaeological SFDs appear most consistent with the present-day SFDs of mature populations sampled through multiple late fall–early spring months  best matched the null model for a harvest period of four months starting in December and ending in March . The sam2 best maWhen specimens measuring <1.6 mm were removed from the recent dataset–to determine whether season of harvest estimates are affected by disparities between sampling methods used to process archaeological and live population samples–the results remained consistent  and 14. All above results are limited to continuous multi-month models. Whereas exploring all possible discontinuous permutations is computationally prohibitive, it is instructive to consider discontinuous variants of best continuous models. This supplementary analysis  suggestsWhile the results are consistent with multi-month harvest, this may be also due to time-averaging of short-term harvest events. For example, the apparent multi-monthly harvest could easily be produced if short-term harvest events underwent time-averaging over multiple decades or centuries. Slight differences in harvest time, spawning time, growth rate  could increase dispersion of the time-averaged SFD and make samples more consistent with multi-monthly demographic training sets. Thus, the results suggest that harvesting was strongly seasonal, but the time-averaged data (and resulting model outcomes) may overestimate the harvest duration. Nevertheless, data consistently suggest that the harvest was not likely to start earlier than November or continue past May. The modeling exercise also demonstrates that archaeological samples are highly incongruent with any substantial harvest activities during summer and early fall.Boonea impressa as a proxy for archaeological season of occupation has been criticized [B. impressa is further justified here because recent samples came from oyster reefs located in close proximity to the shell ring . Moreover, the Late Archaic time interval represented by the shell ring corresponds to an earlier phase of the current interglacial when the climate regime is expected to have been comparable to current conditions. In addition, the largest archaeological specimens closely match the largest specimens from present-day populations, and archaeological size-frequency distributions demonstrate excellent matching with some of the predictive models .The use of iticized  because iticized ,18. Our Mercenaria spp.) from St. Catherines Shell Ring, which points to predominant winter/spring harvest [Previous research on St. Catherines Island has focused on seasonal settlement and mobility patterns from the Late Archaic to the historic period . However harvest . Similar harvest . Our res harvest ,32. HoweIt is noteworthy here that because of limited sample sizes, the samples had to be pooled  limiting the interpretative power of these analyses. It is reassuring that whether samples were pooled vertically  or horizontally , the resulting pooled samples produced consistent outcomes. Analyses based on more extensive sampling of single horizons could further improve the interpretative power of the proposal approach, although it should be noted that even if the data were constrained to one well controlled horizon, the within-horizon time-averaging would still likely be substantial.The seasonal pattern of oyster harvest, augmented by other lines of evidence reported in previous studies  is congrDue to the limited number of sites with oyster seasonality data, few comparisons can be made with other shell ring sites and Archaic period midden sites. Nevertheless, a similar pattern for oyster harvest is evident at the Sapelo Shell Ring complex , where stable isotope analysis of oyster shells suggests a seasonal harvesting pattern that was primarily restricted to winter months . HoweverThe pattern of oyster harvest exhibited at St. Catherines Shell Ring supports the hypothesis that oysters were harvested seasonally, during the cooler months of late fall, winter and spring and this pattern persisted through time. That Archaic people did not consume oysters during summer months aligns with the old adage that oysters should only be eaten in the months with the letter “r”, but avoided during all other months . Today, Boonea impressa may serve as a reliable proxy for the determination of oyster harvest seasons. When determining seasonal site use or occupation, the proxy can be particularly useful when correlated with other critical lines of evidence such as stable isotopic data, use patterns for other animal and plant remains, and stratigraphy.The modeling approach developed here provides numerical support for the hypothesis that shellfish were deposited seasonally, and this practice may have been the primary means by which Archaic period people formed St. Catherines Shell Ring. When appropriately evaluated and sampled, Archaic shell-ring dwellers at St. Catherines Island, harvested oysters primarily during the late fall, winter and/or early spring. This gathering pattern matches seasonal harvesting postulated for other mollusks (clams) at this and other Archaic shell rings where similar studies have been conducted. Oyster consumption was avoided during summer months, possibly reflecting poor palatability, pathogen avoidance, or perhaps one of the earliest records of sustainable harvesting. Oysters, as ecological keystone species, are important indicators of estuarine health but their numbers are currently on the decline worldwide –42. ArchS1 AppendixBoonea impressa specimen repository information.Statistical modeling approach, methodology, zooarchaeological data, and (PDF)Click here for additional data file.S1 MovieAnimation showing a sequence of 133 null models depicted in (GIF)Click here for additional data file.S1 FigComparison of the performance of best continuous models to their discontinuous variants. Comparisons performed separately for each of the three archaeological samples. X-axis labels indicate the discontinuous month combinations. Red point marks the best continuous model for each sample and the dashed line represent the similarity index value for that best continuous model. All models evaluated using Bray-Curtis similarity index.(TIF)Click here for additional data file.S2 FigBoonea impressa plotted as a function of sample size (n) of the smaller of the two compared monthly samples. Each data point represents a pairwise comparison for the same month from different years . A. Pairwise differences in median shell length between the two compared years for a given month. B. Kolmogorov-Smirnov D statistic measuring overall difference in the shape of the two compared size-frequency distributions.Difference between monthly size-frequency distributions of live-collected specimens of (TIF)Click here for additional data file.


Cancer immunotherapy, including immune checkpoint blockade and adoptive CAR T-cell therapy, has clearly established itself as an important modality to treat melanoma and other malignancies. Despite the tremendous clinical success of immunotherapy over other cancer treatments, this approach has shown substantial benefit to only some of the patients while the rest of the patients have not responded due to immune evasion. In recent years, a combination of cancer immunotherapy together with existing anticancer treatments has gained significant attention and has been extensively investigated in preclinical or clinical studies. In this review, we discuss the therapeutic potential of novel regimens combining immune checkpoint inhibitors with therapeutic interventions that (1) increase tumor immunogenicity such as chemotherapy, radiotherapy, and epigenetic therapy; (2) reverse tumor immunosuppression such as TAMs, MDSCs, and Tregs targeted therapy; and (3) reduce tumor burden and increase the immune effector response with rationally designed dual or triple inhibitory chemotypes. The ultimate aim of immunotherapy is to boost the body's immune system to destroy tumor cells and to provide a durable antitumor immune response. The strategy of using monoclonal antibodies against two distinct inhibitory receptors on T-cells, PD1, and CTLA-4 is a major breakthrough in the field of cancer immunotherapy. The efficacy of this strategy was first established in patients with metastatic melanoma based on the antitumor immune response and increased overall survival rates of patients treated with ipilimumab, a monoclonal antibody targeting human CTLA-4 , 3. NeveAnalysis of clinical trial data suggests three types of patients: (a) those that do not respond (innate resistance); (b) those that respond initially but fail to respond in later stages (acquired resistance); and (c) those that respond initially and continue to respond , 5. ExteThe dual checkpoint blockade, using anti-PD1 and anti-CTLA-4 antibodies, was considered a first combinatorial approach in cancer immunotherapy , 24. TheIn this review, we will focus on strategies that induce tumor immunogenicity and reverse tumor immunosuppression thus increasing antitumor immune responses . We alsoChemotherapy and radiotherapy have long been used as conventional methods to reduce tumor burden. It has been recently reported that these therapies activate the immune system by releasing tumor antigens and subverting immunosuppressive factors –33. ThesStandard chemotherapeutic drugs including paclitaxel, cyclophosphamide, gemcitabine, and cisplatin are reported to modulate tumor immunity at low doses . At thei2 cyclophosphamide 3 days prior to vaccination with IMA901 + GM-CSF adjuvant had longer overall survival (OS) as compared to the ones who received IMA901 only  pyran-7-one (TP scaffold) system to design novel inhibitors having improved potency towards PI-3K . SF2523 euticals . These reuticals  in a sinr models . Anotherr models . This raIn summary, the durable antitumor response generated by the application of checkpoint inhibitors has revolutionized the field of cancer immunotherapy. Very soon it will be realized that the clinical benefit obtained from the immune checkpoint blockade is limited to only a small subset of patients and that most patients do not respond to this therapy. The combination of immune checkpoint blockade with several other anticancer treatments has shown remarkable success in various cancers and provided hope for the patients who do not respond to checkpoint inhibitor monotherapy. We have summarized the therapeutic potential of checkpoint inhibitors with drugs that increase tumor immunogenicity, reduce tumor burden, and reverse tumor-mediated immunosuppression, leading to an effective and durable antitumor immune response. In the end, we provided a novel approach to rationally design dual or triple inhibitory chemotypes that can concomitantly hit several tumor-promoting pathways and increase the immune effector response by blocking myeloid cell-mediated tumor immunosuppression. This novel approach is early in its development stage, but the promising antitumor results generated so far by use of dual or triple inhibitor chemotypes in different cancer models provide a rationale to continue exploring these agents with immune checkpoint inhibitors.


Sudden visual loss and optic disc edema caused by optic neuritis (ON) is usually followed by significant visual recovery. However, little or no recovery occurs when the loss is caused by atypical ON, especially in patients with neuromyelitis optica (NMO). Optic disc drusen (ODD) is a cause of pseudo optic disc edema and may be a predisposing factor for non-arteritic anterior ischemic optic neuropathy (NAION), thereby mimicking atypical ON. In such cases, if globular concretions are seen protruding from the disc substance, ODD may be suspected. The purpose of this paper is to describe two patients with acute visual loss followed by optic disc atrophy initially labeled as atypical ON. Though not suspected on clinical examination, optical coherence tomography (OCT) revealed deeply buried ODD as a predisposing factor for NAION.Case 1: A 48-year-old woman had bilateral sequential visual loss associated with optic disc edema. Despite treatment, vision did not improve and severe disc pallor ensued. Atypical ON was suspected. Eventually, she was started on immunosuppressant therapy based on a tentative diagnosis of NMO-spectrum disorder. On examination 5 years later, only severe optic disc pallor was observed, but OCT radial B-scans showed ovoid hyporeflective areas in the retrolaminar region of both eyes, compatible with ODD; this led to a diagnosis of NAION and deeply buried ODD. Case 2. A 35-year-old woman with suspicion of ON in the left eye and a history of previous atypical ON in the right eye was referred for neuro-ophthalmic examination which revealed diffuse optic disc pallor and a dense arcuate visual field defect in the right eye. OCT B-scans passing through the disc showed large ovoid areas of reduced reflectivity in the retrolaminar region of the optic disc in the right eye. These findings helped confirm the diagnosis of NAION in one eye, with deeply buried ODD as predisposing factor.Deeply buried ODD may be associated with NAION causing irreversible visual loss and optic disc pallor, a condition easily mistaken for atypical ON. Awareness of such occurrence is important to avoid unnecessary testing and minimize the risk of mismanagement. Acute visual loss and optic disc edema in non-senile patients is generally due to optic neuritis (ON), an inflammatory/demyelinating disease which often resolves with visual improvement in a matter of weeks . AtypicaOptic disc drusen (ODD) are laminated and usually calcified acellular globular concretions protruding from the optic disc or hidden within the disc substance. ODD near the surface of the disc are clearly visible on fundus examination but, when located below the retinal nerve fiber layer (RNFL), may alter the contour of the optic disc and mimic true optic disc edema . Though Recently, however, high-resolution OCT studies have shown that ODD deeply buried in the optic disc structure easily escapes clinical detection, even by experienced examiners . We examA previously healthy 48-year-old woman developed sudden and painless visual loss and optic disc edema in the right eye (OD). A tentative diagnosis of ON was made and high-dose intravenous methylprednisolone was administered for 5 days, followed by oral prednisone therapy, but to no avail. Three weeks later a similar event occurred in the left eye (OS). Oral corticosteroid treatment was maintained for the following 4 months with only slight improvement in vision. The disc edema resolved and was followed by severe optic atrophy. Neurologic examination, brain and orbits computerized tomography and magnetic resonance imaging (MRI) scans, cerebrospinal fluid (CSF) analysis and extensive laboratory investigation including anti-aquaporin 4 antibody assay were unrevealing, except for a weakly positive antinuclear antibody test . Because of sequential involvement and the lack of significant improvement the patient was considered to have atypical ON, presumably from NMO-spectrum disease. Oral azathioprine 150 mg/day was introduced and kept for the following years. Her visual function remained stable, and significant peripapillary RNFL loss developed on sequential spectral-domain (SD) OCT examinations, directed at quantifying peripapillary RNFL. However, high-resolution OCT scanning passing through the optic nerve were not obtained.Five years later, the patient was seen for the first time by us for a second opinion regarding her condition and her current treatment. Her vision was stable and she had no new complaints. Upon examination, visual acuity (VA) was 20/20–2 in OD and 20/25 in OS. A mild relative afferent pupillary defect was present in OS. Extraocular motility, slit lamp findings and intraocular pressure measurements were normal. The fundus examination showed diffuse disc pallor in both eyes (OU). The visual field (VF) test revealed marked sensitivity loss in the upper and lower nasal quadrants, enlarged blind spot in OD, and marked lower nasal and temporal defect associated with diffuse VF depression in OS. Swept-source (SS) OCT showed severely reduced peripapillary RNFL thickness in OU. OCT radial B-scans passing through the disc showed ovoid areas of reduced reflectivity with some hyper-reflective areas in the retrolaminar region of the disc in both eyes Fig. . The finA 35-year-old woman presented with a complaint of a small dark spot laterally in OS. Taking into account her history of acute visual loss in OD 10 years earlier, left optic disc edema from ON was suspected by her ophthalmologist and the patient was referred to us for neuro-ophthalmic evaluation. Ten years previously, she had acute VF loss and optic disc swelling in OD which regressed completely after treatment with intravenous high-dose methylprednisolone for 5 days. However, since her visual loss remained unchanged she was tentatively diagnosed with atypical ON by her previous physicians. After extensive laboratory testing including anti-aquaporin-4 antibody, MRI and CSF examination were normal, close neurological follow-up was prescribed justified by the suspicion of seronegative NMO-spectrum disease.Upon ophthalmic examination, VA was 20/20 in OU and the pupils reacted to light and near stimuli with a relative afferent pupillary defect in OD. Extraocular motility, slit lamp examination and intraocular pressure measurements were normal. The fundus examination showed diffuse optic disc pallor with a peripapillary depigmented halo around the disc in OD and mildly blurred disc margins in OS. A small round vitreous opacity presumably due to vitreous detachment was found in OS. The VF examination disclosed an arcuate dense pericentral defect in OD and was within normal range in OS. Spectral-domain (SD) OCT showed severely reduced peripapillary RNFL thickness in OD and mildly increased RNFL thickness in the superior quadrant of OS. Enhanced-depth imaging (EDI) horizontal and vertical OCT B-scans passing through the disc showed large ovoid areas of reduced reflectivity with some hyper-reflective regions in the retrolaminar region of the optic disc in OD (Fig. Subtle and insidious visual loss is relatively common manifestation of ODD, usually characterized by subclinical VF defect associated with peripapillary RNFL loss . ODD areNAION associated with ODD usually occurs in middle aged or senile patients, characterized by altitudinal VF loss, optic disc edema and occasional peripapillary hemorrhages, without significant visual improvement. In such cases, ODD is considered a predisposing factor for NAION and its presence is generally detected either in the early stages of visual loss or after resolution of optic disc edema. The observation of characteristic globular concretions protruding from the optic disc or within the prelaminar region of the optic nerve head is usually enough to establish the correct diagnosis. While further testing may be necessary to confirm it , 5, ODD The pathogenesis of NAION in eyes with ODD in not completely understood but is thought to involve a decrease in the perfusion pressure at the optic nerve head that leads to infarction . Both NAOur cases are interesting because ODD was too deep to be suspected on clinical examination, even after optic disc edema resolved. Although NAION was included in the differential diagnosis of ON, the sequential involvement and a weakly positive antinuclear antibody titer in one patient and the young age of the other patient were strongly suggestive of atypical ON. On the other hand, none had ocular pain, significant VA loss or progressive visual loss that are commonly associated with ON , 2. HoweOCT is currently considered by many authors as the best method for detecting ODD –16, supeIn conclusion, our cases show that, while clinically difficult to detect, deeply buried ODD may play an important part in the etiology of acute irreversible visual loss and optic disc edema. Ophthalmologists should be aware of this mimicker of atypical ON in order to avoid unnecessary testing and minimize the risk of mismanagement. The current cases also emphasize the utility of modern OCT technology in correctly detecting ODD, even when it is deeply buried in the optic disc.


Innate and adaptive immune cells from myeloid and lymphoid lineages resolve host infection or cell stress by mounting an appropriate and durable immune response. Upon sensing of cellular insults, immune cells become activated and undergo rapid and efficient functional changes to unleash biosynthesis of macromolecules, proliferation, survival, and trafficking; unprecedented events among other mammalian cells within the host. These changes must become operational within restricted timing to rapidly control the insult and to avoid tissue damage and pathogen spread. Such changes occur at high energy cost. Recent advances have established that plasticity of immune functions occurs in distinct metabolic stress features. Evidence has accumulated to indicate that specific metabolic signatures dictate appropriate immune functions in both innate and adaptive immunity. Importantly, recent studies have shed light on whether successfully manipulating particular metabolic targets is sufficient to modulate immune function and polarization, thereby offering strong therapeutic potential for various common immune-mediated diseases, including inflammation and autoimmune-associated diseases and cancer. In this review, we detail how cellular metabolism controls immune function and phenotype within T cells and macrophages particularly, and the distinct molecular metabolic programming and targets instrumental to engage this regulation. Immune cells arise from a common pluripotent hematopoietic stem cell that upon differentiation produces two main lineages, referred as the myeloid and lymphoid lineages. While the former fully maturate in the bone marrow and generate components of the innate immune system, the latter generally reach lymphoid organs  to complete maturation and produces most cellular constituents of the adaptive immune system. Once functional, these immune cells undergo several modifications of their genetic programming in response to an encounter, either in sterile or non-sterile conditions. These genetic modifications lead to the transition between quiescent, activated, and memory states, and control immune polarization. At the intracellular level, these transitions are accompanied by major metabolic changes. Cells notably modulate bioenergetic requirements, building blocks for their proliferation and survival , and oxidative stress. Transcriptional and post-transcriptional regulation of these genes plays a key role in the function of immune cells, dependently on the availability of various nutrients and presence of oxygen. The immune response is associated with dramatic modifications in tissue metabolism, including depletion of nutrients, increased oxygen consumption, and the generation of reactive nitrogen and oxygen intermediates. These modifications are in part due to the recruitment of various inflammatory cells.Glucose is a well-known nutrient for energy supply that feeds glycolysis and is converted into pyruvate in the cytosol . Under nAnother cellular method of producing ATP is glycolysis-mediated fermentation. Under hypoxic conditions, pyruvate remains in the cytoplasm and is mostly converted into lactate rather than acetyl-CoA. This reaction is catalyzed by lactate dehydrogenase (LDH). This glycolytic pathway is more rapid but less efficient than OXPHOS for ATP synthesis. Fermentation under normoxic conditions is aerobic glycolysis. The metabolic switch from mitochondrial oxidative to aerobic glycolysis has been observed in cancer cells from solid tumors and is known as the Warburg effect . This shSimilar to cancer cells, immune cells modify their metabolic requirements to acquire appropriate immune function in response to infection or tissue stress. Such metabolic modifications allow them to mobilize all the resources required to quickly proliferate and/or get fully activated to deploy their host-defense mechanisms. In lymphoid cells, for instance, going from a naïve/quiescent to a fully active immune effector cells implies a switch from an OXPHOS-dependent catabolic condition to a highly glycolytic anabolic state. After hazard clearing, a reversed metabolic shift then should occur in the remaining memory cells, reacquiring an oxidative non-proliferating state. As for the myeloid component, metabolic changes occur upon M1 or M2 macrophages polarization, towards higher (i) glycolytic flux and (ii) OXPHOS and fatty acid oxidation (FAO), respectively. At last, dendritic cells also switch to high glycolysis upon activation for antigen presentation. All these changes rely on complex metabolic networks that comprise numerous signaling pathways and key metabolic mediators/actors, described now to specifically control immune cellular functions at various levels. The metabolic peculiarities of various immune cell subtypes that drive their immune functions in normal and diseased conditions, including inflammatory diseases or during the antitumor immune response, are discussed below.Most lymphoid cells (T and B cells) are crucial components of the adaptive immune system that provide specific recognition of “non-self” , as wellT lymphocytes are sentinel cells that trigger an antigen-specific response. They play a crucial role in controlling infection and the pathogenesis of numerous human diseases in sterile conditions, including inflammatory and autoimmune diseases and cancer. Naive T cells recognize antigens as processed peptides presented by the major histocompatibility complex (MHC) molecular machinery that is expressed by antigen presenting cells (APCs). This interaction leads to T cell activation, rapid proliferation, and production of various effector molecules that enable the control of disease-causing pathogens. After interaction with the antigen, most effector T cells die through activation-induced cell death (AICD), leaving only a small number of activated T cells to become memory T cells. Memory T cells stably persist and can rapidly and more potently respond to antigen recalls. Each of these phenotypic transitions require a fine-tuning of metabolic networks that are necessary to cope with environmental constraints , and to acquire novel immune functions. Recent progress has been made to better define the various underlying transcriptional metabolic programming, and to better understand how this programming controls immune signaling pathways and ultimately T cell function  essential for the conversion of acetyl coenzyme A to malonyl coenzyme A .Regulation of this metabolic switch occurs at two different levels through transcriptional programming (involving c-Myc and estrogen-related receptors [ERRs]) and post-transcriptional programming (that involves AMPK activation and HIF-1α stabilization) . ExpressmTOR is a master regulator of T cell metabolism, integrating signals provided by nutrient levels and energy in T cells, cell stress pathways, and TCR and growth factor signals. CD28-dependent signaling involves the PI3K/Akt/mTOR pathway to promote glucose metabolism and to prevent T cell anergy . mTOR haAdenosine Monophosphate (AMP)-activated protein kinase (AMPK) is a metabolic energy sensor that is another master regulator of T cell activation . In contHIF-1α is also crucially involved in metabolic reprogramming, enabling T cell adaptation to hypoxic conditions (such as in tissues) . Stabilid-glucose (2DG) in HIF-deficient mice leads to the inhibition of Th17 differentiation [Ifng [Specific metabolic programming has been associated with various T cell phenotypes. Th1, Th2, and Th17 cells use glycolysis via mTORC1 signaling, whereas Regulatory T cells (TRegs) engage mostly FAO  Figure . Thus, sntiation . In micentiation . Blockinntiation ,50. Signntiation ,52. Conson [Ifng .In addition to LDH-A, stabilization of IFNγ mRNA is under the control of GlycerAldehyde-3-Phosphate DeHydrogenase (GAPDH) expression, another glycolytic enzyme that binds to AU-rich elements in 3’UTR of IFNγ mRNA when the enzyme is not engaged at a high glycolytic rate . FurtherA study that examined the proliferation and survival of activated CD4+ T cells (TCR/CD28 stimulation) using mass spectrometry to quantify protein dynamics revealed rapid remodeling of the mitochondrial proteome with a distinct metabolic signature of one-carbon metabolism . Serine,After activation, the effector T cell population contracts and the majority of cells undergo apoptosis. A small number of activated T cells persist to become memory T cells and during this transition these T cells shift their metabolism to catabolism to support quiescence and long-term persistence. AMPK plays an important role in memory T cell differentiation . In thesFAO is necessary for CD8+ T cells to differentiate into the memory phenotype, but also for their long-term persistence and reactivation after antigen recall . After nl-arginine was sufficient to shift glycolysis toward mitochondrial OXPHOS in memory T cells, leading to increased survival and antitumor function [l-Arginine sensing.Importantly, memory T cells  that infiltrate tumors are involved in efficient and long-lasting antitumor immune response in vivo in various mouse models . These Tfunction   enhances protective activity of memory CD8+ T cells against influenza virus . TherefoTReg cell metabolism relies on glycolysis-driven lipogenesis with high lipid oxidation rates , with a Myeloid cells play an important role in homeostasis by stimulating lymphocytes to respond to pathogens , eliminaAfter their generation in bone marrow, monocytes are released into the blood circulation. They are rapidly directed to various tissues where they remain as immature monocyte-derived-macrophages, acting as immune sentinels. Macrophages differ from DCs, which also derivate from monocytes, by a different expression profile of surface markers. Several subpopulations of macrophages exist based on tissues where they reside, and various subsets of functional macrophages are characterized according to the environmental signals they integrate ,73. M1 pMacrophages are a major component of immune infiltrate in solid tumors and play an important role in tumor phenotype . ThroughH. polygyrus. c-Myc is also a main transcription factor involved in M2 polarization [As described in rization . Indeed,rization .HIF-1α is one of the main transcription factor that supports the metabolic features of M1 macrophages . Data haIdh) gene, and that an aspartate-arginosuccinate shunt can compensate for this break [There are distinct patterns of amino acids use, with M1 cells using arginine as a substrate for inducible nitric oxide synthase (iNOS), whereas M2 cells use arginine as a substrate of arginase-1, which produces polyamines involved in cell proliferation and tissue remodeling to favor tumor progression . A systeis break . This shThe role of this shunt was further confirmed in a subsequent study that allowed the accumulation of fumarate and in turn an inflammatory epigenetic reprogramming through histone-acetyl transferases and methylation; these modifications ultimately provided transcriptional production of inflammatory cytokines TNFα and IL-6 . In addiThese cells are traditionally described as the frontier of innate and adaptive immunity. They induce primary immune responses because of their antigen-presentation function, and they also have an important role in priming T lymphocytes. Activation and maturation of immature DCs depends on their interaction with pathogen-associated molecular pattern (PAMPs) or damage-associated molecular pattern (DAMP) molecules. This leads to the upregulation of antigen-presenting molecules, cytokines, and chemokine receptors. Once activated, mature DCs migrate to lymphoid organs where they are efficient in their ability to induce antigen-specific T cell activation.Quiescent DCs primarily use OXPHOS to produce energy and FAO . After pUnder hypoxic conditions, the activation of HIF-1α induces the expression of glycolytic genes and increases glycolysis. This induces an increase of DC maturation in mouse models , howeverAs discussed above, efforts have been made to thoroughly characterize the intricate metabolic networks engaged in immune cells, both in response to in vitro stimuli (including antigens or PAMPs) or ex vivo in various physiological settings. As a result, metabolic peculiarities have been successfully delineated for some immune cell types, though limitations of the ex vivo experimental procedure prevent it from completely reflecting the complexity of the in vivo microenvironment. In addition to mapping metabolic networks of immune cells in a given phenotype, crucial insights have been recently gleamed, establishing that manipulation of metabolic enzymes is sufficient to modulate the phenotype of immune cells. Importantly, this metabolic manipulation of immune phenotype has been demonstrated for various immune cell types, including innate macrophages and adaptive T cells (CD4+ or CD8+). Such knowledge is of particular interest for therapeutic intervention in inflammatory diseases and cancer, where it may be possible to metabolically fine-tune immune effector function from harmful toward protective based on disease context, or to even improve persistency of exhausted tumor infiltrating cytotoxic T cells in cancer. Even though this goal cannot be reached yet in vivo due to the strong heterogeneity of immune cell populations and phenotypes, the range of metabolic targets that control the fate of immune effector function is emerging.In macrophages, mitochondrial SDH appears to play a major role in controlling the switch between pro- and anti-inflammatory phenotypes . In a stRemarkable achievements have been recently reached in reconstructing highly complex and interconnected metabolic networks in immune cells, and by applying this approach to various immune cell types from both the innate and adaptive immune system. Such outcomes have become possible by implementing complementary technologies to map metabolic changes as accurately as possible. Such approaches combine the expression of metabolic genes and proteins with high-throughput “omics” technologies  at a given steady state of immune activation with fluxomics  to identify the levels of metabolite intermediates and their biochemical origin. In depth data analyses of integrated “omics” have successfully led to the precise mapping of metabolic circuity in response to various innate or adaptive immune stimuli, clearly establishing that metabolic changes not only occur in response to these stimuli, but more importantly, that a deep metabolic reprogramming is indispensable to skew immune cell functions toward particular phenotypes. Some examples discussed here have recently elucidated the metabolic-induced epigenetic modifications that underlie these modulations of immune function.Most of these techniques have been applied in in vitro or ex vivo experimental settings, and in some cases to particular enriched immune cell populations. In these in vitro approaches, inconsistencies can easily arise between studies simply due to minor differences in nutrient compositions of cell culture media . These nutrient levels can vary in in vivo tissues and shape the immune cell metabolism in various ways; this might depend on a number of parameters dependent on the tissue itself and interactions between immune cells and their microenvironment. The current limitation now resides in the assessment of immune metabolic signatures in this complex in vivo tissue microenvironment. In the need to translate metabolic studies of immune cells into in vivo settings, a promising approach in translating immune cells metabolic studies into the in vivo environment is cellular-scale tissue microdissection coupled to proteomics analysis. Such an approach can specifically target immune cells of interest within tissue sections and offers the advantage of addressing protein expression of a large panel of metabolic markers to reconstitute circuity. It may also prove useful for translating immune cell metabolism into the clinic through the identification of biomarkers and therapeutic metabolic targets in a broad spectrum of complex human diseases as diverse as metabolic syndrome, inflammatory diseases, or cancer.The full clinical impact of immune cell metabolism is still undetermined in various contexts, and some positive effects of metabolic drugs  prescribed for inflammatory diseases might actually contribute to the modulation of immune cell function to some extent. As eagerly awaited clinical trials have begun testing metabolic molecules to prevent and treat various disease indications, the rapidly expanding clinical impact of metabolic targeting for a broad spectrum of immune-mediated illnesses will be further elucidated. Determining the Achilles’ heel of the specific immune cell population for therapeutic intervention remains outstanding. Progress has been already made to establish that adoptive transfer of T cells pretreated with metabolites or isolated from genetically modified metabolic enzymes can improve memory T cell persistence, which correlates with antitumor immune function in mice models. This has translational impact on the generation of persistent memory Chimeric Antigen Receptor (CAR) T cells through the expression of 4-1BB-induced metabolic reprogramming, which increases mitochondrial biogenesis , or the


Single injection, ultrasound-guided nerve blocks have drastically changed the multimodal approach to pain management of the acutely injured patient in the emergency department (ED). Ultrasound-guided femoral nerve blocks in the ED have become standard aspects of multiple, hospital system pain management protocols, with early evidence demonstrating improved patient outcomes. Developing a multimodal pain management strategy can improve analgesia while reducing reliance on opioids in this era of opioid addiction. Acute appendicitis is commonly diagnosed in the emergency department (ED), where intravenous (IV) opioids are the primary analgesic used to control pain while patients await definitive surgical care.6Recently, several reports suggest benefit of the ESP block for the pain associated with major open abdominal surgery, bariatric surgery, ventral hernia repair, abdominal zoster, laparoscopic cholecystectomy, inguinal hernia repair, and cesarean section.9/liters (L) (4.5-11.5 x 109/L). A contrast-enhanced computed tomography of the abdomen and pelvis revealed a 10-millimeter (mm) dilated, fluid-filled appendix with appendicolith suggestive of acute appendicitis. The patient was given IV cefoxitin and admitted to the department of surgery for planned appendectomy later that day. For analgesia, the patient received 0.5 milligrams (mg) IV hydromorphone, 30 mg IV ketorolac, and 1000 mg IV acetaminophen but was still reporting 7/10 pain. Palpation of the RLQ produced 10/10 sharp localized pain with rebound and guarding. Operative management was to be delayed for four hours; thus, after discussion with the surgical team, the patient was offered a single injection, ultrasound-guided ESP (block) for pain control.A 24-year-old male presented to the ED after seven hours of sharp mid-abdominal pain migrating to the right lower quadrant (RLQ), associated with nausea and vomiting. Vital signs were within normal limits except for mild hypertension (149/68 millimeters of mercury). Laboratory results showed a leukocytosis, white blood cells 14.5 x 10After the patient gave verbal consent he was placed on a cardiac monitor and rolled into the left lateral decubitus position. The physician stood at head of the bed facing the patient’s feet with the ultrasound monitor in direct line of sight . The firWhat do we already know about this clinical entity?Traditional pain control for acute appendicitis relies on intravenous opiates.What makes this presentation of disease reportable?Regional anesthesia has not been described for analgesia in acute appendicitis.What is the major learning point?A single injection, ultrasound-guided erector spinae plane block can provide complete analgesia for appendicitis.How might this improve emergency medicine practice?Emergency physicians can now incorporate regional anesthesia for pain control in acute appendicitis.Thirty minutes after the procedure the patient reported 0/10 pain at rest. With deep palpation to the RLQ, the patient reported mild, dull, pressure-like pain (3/10); the abdomen remained soft without guarding, rebound or localized pain. Sensory testing to cold with ethyl chloride spray revealed right-sided decreased sensation in the tenth thoracic (T10) to L2 distribution (from approximately two cm inferior to the umbilicus to the mid-anterior right thigh). Two hours later, nursing documentation indicated that the patient’s pain remained well controlled. During the remaining 5.5 hours of his ED stay the patient reported his pain was well controlled and required no additional analgesic medications. He had an uncomplicated laparoscopic appendectomy, with appendicitis later confirmed by pathology.Currently, pain control for acute surgical abdominal pain is often limited to IV opioids, sometimes combined with nonsteroidal anti-inflammatory drugs or acetaminophen. With operating room crowding, abdominal surgical patients often board in the ED for many hours, relying on repeated dosing of opioid medications that risk side effects while often failing to provide adequate pain relief. In the last quarter of 2018 at our institution, patients admitted for appendicitis boarded an average of 320 minutes before leaving the ED. Ultrasound-guided regional blocks for abdominal surgeries performed by anesthesiologists have been shown to be effective in the perioperative and postoperative period.Communication with surgical consultants with regard to performing the ESP block before the procedure should be routine. Clear documentation in both the chart and on the patient (we commonly place the time and block type) should also be part of the workflow when incorporating ultrasound-guided nerve blocks in the ED. Surgical consultants should also be aware that peritoneal blocks may significantly reduce the pain from acute appendicitis; thus, using the abdominal examination as a marker of progression of illness may not be possible.Technical considerations include adhering to block safety standards to prevent local anesthetic systemic toxicity. Because this was a novel, lower-targeted ESP block, we used a shorter acting anesthetic (1% lidocaine with epinephrine) as an additional precaution in case of adverse effects. However, a longer-acting anesthetic (such as bupivacaine or ropivacaine) would likely be optimal and consistent with prior published reports of ESP block.12Given our experience managing pain for rib fractures, we consider the ESP block to be technically feasible and highly effective. By moving the location of the single injection ESP block more caudally, we can expand the range of this block to include pain control for acute appendicitis as part of an opioid-sparing, multimodal pain control strategy for patients in the ED. We suspect that further study of this novel technique may show improved pain control for patients with acute appendicitis when compared to standard opioid-based pain regimens with fewer side effects and improved patient outcomes.


Tuberculous prostatitis is a rare and often overlooked entity that may mimic prostatic adenocarcinoma on imaging exams, especially multiparametric magnetic resonance imaging (MRI) of the prostate. Detection of a prostatic abscess is a clue to the correct diagnosis. A 73-year-old man with progressive lower urinary tract symptoms for five months was referred to our service for evaluation of prostatic enlargement. During this period, he was using indwelling bladder catheter. He denied any known epidemiological history or respiratory symptoms.PSA levels were 6.54ng/mL and digital rectal examination showed a diffusely enlarged prostate without focal nodulations. He was submitted to prostatic multiparametric MRI to exclude a concomitant neoplasia that demonstrated an enlarged prostate  with incThe histopathological analysis of the fragments on the targeted area (suggestive of abscess) evidenced a chronic granulomatous inflammatory process and the specific test for acid-alcohol resistant bacilli (BAAR) confirmed mycobacterial etiology.Tuberculous prostatitis is a rare granulomatous prostate disease that predominantly occurs in immune-compromised patients and may be mistaken for prostate cancer on clinical, laboratory and imaging findings -7. GenitImaging examinations may help in diagnostic elucidation, but there is also overlap with features of prostatic adenocarcinoma. A series of cases  suggesteThe presence of a prostatic abscesses, as in the present case, is an infrequent but characteristic finding and appears on MRI as a nodular area of intense diffusion restriction and peripheral enhancement , 6.Since there may be an overlap of clinical, laboratory and imaging findings, the gold standard for diagnosis is histopathology, confirming caseous granulomas like any other site of tuberculosis infection, as well as the presence of BAARs when stained by the Ziehl-Neelsen method. Treatment usually involves the use of classic anti-tuberculosis drugs -4.


A young woman presented to the emergency department with lethargy, hemodynamic instability, and diffuse abdominal tenderness. On point-of-care ultrasound (PoCUS), she was found to have intraperitoneal free fluid and a large pelvic mass, which were discovered intraoperatively to be hemoperitoneum due to ruptured vessels of a uterine leiomyoma. Although rare, a life-threatening, ruptured leiomyoma may be treated surgically if recognized in an expedient fashion. A PoCUS can aid the emergency clinician in prompt diagnosis. While uterine leiomyomas are common among reproductive-aged women, they rarely result in acute life-threatening events. However, the vessels overlying a leiomyoma may rupture leading to hemoperitoneum.A 43-year-old, previously known to be healthy woman was brought to the ED by emergency medical services (EMS) after her husband found her to be confused. EMS provided oxygen by facemask and obtained peripheral venous access. Upon arrival to the ED, the patient demonstrated depressed mental status but when aroused, she complained of generalized weakness. Her heart rate was 80 beats per minute, blood pressure was 78/52 millimeters of mercury, respiratory rate was 14 respirations per minute, and temperature was 37.1° Celsius (98.8° Fahrenheit). The bedside glucose level was 170 grams per deciliter (g/dL). A cardiorespiratory monitor was attached to the patient, and one liter (L) crystalloid was infused with a pressure bag. The patient was somnolent but arousable, diaphoretic, and pale. Upon auscultation, the chest was clear and cardiac rhythm regular. Radial pulses were diminished in both upper extremities. There was mild, diffuse abdominal tenderness and a firm mass appreciated in the pelvis. Rectal examination revealed brown stool.The emergency physician performed point-of-care ultrasonography (PoCUS), specifically a Rapid Ultrasound for Shock and Hypotension (RUSH) examination. The PoCUS demonstrated a moderate amount of free fluid  and a weTwo hours after arrival, the urine output totaled 40 milliliters (mL), and the repeat hemoglobin fell to 5.0 g/dL. The gynecology consultant performed a bedside transvaginal sonogram and suspected the mass to be a uterine leiomyoma. The ED staff transfused the patient with three units of packed red blood cells, and collaborative gynecology and general surgery services brought the patient to the operating room (OR) for explorative laparotomy. The gynecology team noted a leiomyomatous uterus and 2.5 L of blood in the peritoneum. The source of hemorrhage was localized to multiple bleeding vessels overlying a leiomyoma, and the gynecology team performed a myomectomy. The OR staff transfused an additional five units of packed red blood cells and three units of fresh frozen plasma. The patient had an uneventful postoperative course and hospital stay. She was discharged from the hospital several days later and appeared healthy at postoperative checkups.Although uterine leiomyomas are common among reproductive-aged women, spontaneous life-threatening bleeding is exceedingly rare. Most reported cases seem to occur without insult,7What do we already know about this clinical entity?Uterine leiomyoma are common among reproductive-age women and typically presents with pelvic pain or non-life threatening vaginal bleedingWhat makes this presentation of disease reportable?Rarely, uterine leiomyoma may rupture and present with hemoperitoneum, a potentially life-threatening conditionWhat is the major learning point?A female patient who presents with hemodynamic instability and abdominal pain, tenderness, or distention may be promptly diagnosed with ruptured uterine leiomyoma using point-of-care ultrasonography (PoCUS)How might this improve emergency medicine practice?Prompt PoCUS diagnosis of ruptured uterine leiomyoma may expedite urgent resuscitation with blood products and transfer to the operating room for definitive interventionThe most frequent cause of hemoperitoneum related to uterine leiomyoma is the rupture of an overlying, superficial artery or vein.Despite uterine leiomyoma being a common tumor in young women, the rupture of associated blood vessels resulting in hemoperitoneum appears to be exceptionally rare. However, this condition may be fatal, and swift action is required for diagnosis and surgical intervention. This case report is unique in that it presents sonographic images to demonstrate the utility of PoCUS for the diagnosis of ruptured uterine leiomyoma. This diagnosis should be considered in any female patient who presents with hemodynamic instability and abdominal pain, tenderness, or distention. A quick diagnosis with PoCUS may expedite urgent resuscitation with blood products and transfer to the OR for definitive intervention.Documented patient informed consent and/or Institutional Review Board approval has been obtained and filed for publication of this case report.