BackgroundObstructive sleep apnea (OSA) is characterized by repeated episodes of either full or partial obstruction of the upper airway. OSA is an independent risk factor for acute ischemic stroke (AIS) and a contributor to other key risk factors. OSA may damage endothelial and brain tissues and worsen outcomes following AIS. We aimed to evaluate the impact of sex differences on 90-day functional outcomes following AIS in an OSA population, as measured by the modified Rankin Scale (mRS) score.MethodologyWe performed a retrospective study of patients with OSA and AIS from the Houston Methodist Hospital Outcomes-Based Prospective Endpoints in Stroke (HOPES) Registry from 2016 to 2022. Patients with charts that noted a diagnosis of OSA before AIS or within the 90 days following AIS were included. A multivariable logistic regression model was constructed adjusting for demographics, first admit National Institutes of Health Stroke Scale (NIHSS), and comorbidities on the binary outcome. The odds ratios (ORs) and 95% confidence intervals (CIs) were reported, providing likelihood estimates of a shift to higher mRS for a given comparison between females (reference category) and males. Statistical significance was defined as two-tailed p-values <0.05 for all tests.ResultsFrom the HOPES registry, 291 females and 449 males were found to have OSA. Males had a higher proportion of comorbid conditions such as atrial fibrillation  and intracranial hemorrhage compared to females . The multivariate logistic regression model showed that males were at two times higher risk for developing poor functional outcomes at 90 days , p < 0.001).ConclusionsMales were found to have two times higher risk for developing poor functional outcomes at 90 days. This may be due to more severe oxygen desaturation, increased susceptibility to oxidative stress, and greater frequency of full airway obstruction in males. Greater emphasis on early diagnosis and treatment of OSA may be necessary to reduce the disproportionate incidence of poor functional outcomes, particularly among apneic male stroke survivors. Obstructive sleep apnea (OSA) is characterized by short and repeated episodes of either full (apnea) or partial (hypopnea) obstruction of the upper airway. This episodic interruption of breathing translates into intermittent hypoxia and repeated arousals from sleep. Sleep apnea is an independent risk factor for acute ischemic stroke (AIS) and a contributor to the development and treatment resistance of other risk factors, primarily hypertension and atrial fibrillation -3. OSA mElevated blood pressure after apneic arousals may escalate to sustained hypertension or greater blood pressure variability ,9, factoCIH also triggers decreased levels of nitric oxide (an endothelium-derived relaxing factor), resulting in endothelial dysfunction and increased vasoconstriction. OSA patients exhibit impaired endothelial repair capacity . EndotheRodent and cell culture models of CIH offer insights into the molecular mechanism by which OSA may enhance chemoreflex, provoke endothelial dysfunction, and induce oxidative stress. In these models, CIH increases hypoxia-inducible factor-1α (HIF-1α) expression, corresponding to increased transcription of pro-oxidant enzymes, and decreases hypoxia-inducible factor-2α (HIF-2α) expression, corresponding to decreased transcription of antioxidant enzymes. The result is a rise in reactive oxygen species (ROS) and oxidative stress that damages cells and contributes to further sympathetic hyperactivation, hypertension, and endothelial dysfunction. The increase in endothelin due to OSA may be the result of increased ROS because increased endothelin-1 (ET-1) levels in rodents exposed to CIH were due to ROS-dependent activation of the enzyme that generates active ET-1 ,13. LipiInflammation from OSA-related cell damage triggers the secretion of proinflammatory cytokines such as tumor necrosis factor-α and interleukin-6 . ElevateThe obstructed attempts at forced inhalation during apneic episodes may create negative intrathoracic pressure. Negative intrathoracic pressure swings put stress on the atrial walls and may result in atrial remodeling that predisposes to arrhythmia and new-onset atrial fibrillation. Additionally, the sympathetic overstimulation, oxidative stress, and systemic inflammation fostered by OSA further elevate susceptibility to atrial fibrillation and poor treatment outcomes -24.atypical symptoms such as fatigue, insomnia, mood disturbances, and depression and are less likely to report snoring or witnessed apneas [While OSA has been well-established as a risk factor for poor functional outcomes following AIS , the impd apneas . Men pred apneas ,28.However, the gap in OSA prevalence and severity between men and women narrows with age. Women report greater impairment in quality of life than males with similar AHI, and OSA symptoms manifest at lower AHI in women, indicating that AHI is not always an adequate measure of clinical OSA severity for women ,30. WomeWomen and men with OSA tend to present with different comorbidities, many of which are especially relevant in the context of stroke. Women with OSA have a greater incidence of hypothyroidism and are more likely to experience depression as a result of their OSA . The conPremenopausal women seem to be less susceptible to oxidative stress, one of the central mechanisms by which OSA may elevate stroke risk and hinder recovery , as indiIn this retrospective study, we aim to evaluate the impact of sex on the extent to which OSA affects 90-day functional outcomes following ischemic stroke, as measured by the modified Rankin Scale (mRS) score. Furthermore, we aim to discern whether any sex differences in 90-day functional outcomes among OSA patients are more pronounced within a specific etiology of AIS, as defined by the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification of ischemic stroke .2) test was used to identify the differences between categorical variables. The outcome variable was categorized using the mRS, with mRS between 0 and 2 considered a good functional outcome, while mRS 3 to 6 considered a poor functional outcome at 90 days. The independent variable was patient gender. A multivariate logistic regression model was constructed by adjusting for demographics, first admit National Institutes of Health Stroke Scale (NIHSS), and comorbidities on the binary outcome. The odds ratios (ORs) and 95% confidence intervals (CIs) were reported, providing likelihood estimates of a shift to a higher mRS score for a given comparison between females (reference category) and males. Statistical significance was defined as two-tailed p-values <0.05 for all tests. All analyses were performed using R statistical software version 4.1.1 .We performed a retrospective analysis of patients with OSA and AIS from the Houston Methodist Hospital Outcomes-Based Prospective Endpoints in Stroke (HOPES) Registry from 2016 to 2022 . The HOPFrom the HOPES registry, a total number of 740 patients were diagnosed with OSA. Of these 740 patients, 291 were females and 449 were males. There was an equal distribution of age(s) in both females (mean = 66.91) and males (mean = 66.60 years) (p = 0.60). Both the female and the male cohorts were predominantly white . The female cohort, however, had a higher proportion of black individuals .Males had a higher proportion of comorbid conditions such as atrial fibrillation  and intracranial hemorrhage  compared to females. Females and males had equal proportions of comorbid conditions such as congestive heart failure , transient ischemic attack , hypertension , end-stage renal disease , diabetes , dementia , coronary artery disease , and hyperlipidemia . Incidence of previous stroke was high among both the females and males included in this study .The most prevalent stroke etiology among both females and males was cardioembolism: 95 females and 166 males were identified as having strokes of a cardioembolic origin. Females and males had equal proportions of cardioembolic strokes  and small vessel  etiologies. Males, however, had a higher proportion of large artery atherosclerotic  stroke etiology. Table A multivariate logistic regression model after adjusting for demographics, first admit NIHSS, and comorbidities showed that males were at a two times higher risk for developing poor functional outcomes at 90 days . Table In our retrospective analysis of patients presenting with OSA, males were found to have over two times higher risk of poor functional outcomes at 90 days, as measured by the mRS score. The HOPES Registry included 291 females and 449 males diagnosed with OSA within the 90 days following their AIS, accounting for 15% of females and 23% of males documented with AIS in the registry. Given the considerable prevalence of OSA among patients recovering from AIS, especially in males, the results of this study indicate that greater emphasis on early diagnosis and treatment of OSA may be necessary to reduce the disproportionate incidence of poor functional outcomes among apneic patients in general, but especially among apneic males.The sex differences revealed by this retrospective study offer insights into the most significant mechanisms by which OSA likely impedes functional recovery from stroke and factors that may reduce the negative impact of OSA on recovery. Cardioembolic and large artery atherosclerotic etiologies were the most commonly determined etiologies among both sexes in this cohort. Given that cardioembolic stroke was by far the most common etiology of stroke in this study, the negative intrathoracic pressure swings, increased cardiac oxygen demand, and oxidative stress induced by OSA may be of particular concern in the context of stroke recovery and may explain the sex gap in functional recovery that we observed. Men are more susceptible to oxidative damage than women , which mThe prevalence of the large artery atherosclerotic stroke subtype in this cohort further supports the roles of the severity of oxygen desaturations and susceptibility to oxidative damage in determining the incidence of poor outcomes following ischemic injury. A study by Schulz et al. found that the contribution of OSA to large artery atherosclerosis is related to the severity of oxygen desaturation , which iGiven that only a fraction of patients within the HOPES Registry underwent screening for OSA, there may exist a considerable population of patients with undocumented OSA in the registry which could not be included in this study. Future prospective studies that avoid this selection bias are needed. Another limitation of this study is the lack of pre-admission mRS scores for comparison. Because 24% of females and 27% of males had a prior diagnosis of stroke, pre-admission mRS scores are necessary to account for cases in which mRS scores indicating poor outcomes already exist from the prior stroke. Furthermore, the males included in this study had a higher proportion of comorbidities such as atrial fibrillation and intracranial hemorrhage compared to their female counterparts, which may have had a role in the poor 90-day outcomes noted among the males.dipping, reduces mortality, protects against new vascular events, and mitigates depressive symptoms [A particularly significant limitation of this study was that patients with both treated and untreated OSA were included. Compliance with continuous positive airway pressure (CPAP) treatment for OSA could not be definitively determined based on chart review for the apneic patients included in this study. Future studies are needed to determine if sex differences in functional recovery may be effectively reduced among stroke survivors compliant with early CPAP treatment. Polysomnography should be performed early during the post-stroke period, especially among males, to screen for OSA so that treatment may be implemented as early as possible. Treatment with CPAP should be strongly encouraged for those who present with OSA to reduce symptoms and the likelihood of poor functional outcomes. There is evidence that treatment of OSA with CPAP in stroke survivors accelerates neurological recovery, improves functional and motor outcomes, lowers blood pressure, restores nocturnal symptoms -61. Sevesymptoms ,62. TherIn our study of AIS patients with OSA, the male sex was associated with a higher risk of poor 90-day functional outcome after AIS, especially of large artery atherosclerotic etiology, after adjusting for comorbidities and demographics. Earlier discovery of OSA and adherence to CPAP treatment may help reduce the risk of poor functional outcomes among apneic male stroke survivors.


Enhancer RNAs (eRNAs) are non-coding RNAs produced from transcriptional enhancers that are highly correlated with their activities. Using capped nascent RNA sequencing (PRO-cap) dataset in human lymphoblastoid cell lines across individuals, we identified inter-individual variation of expression in over 80 thousand transcribed transcriptional regulatory elements (tTREs), in both enhancers and promoters. Co-expression analysis of eRNAs from tTREs across individuals revealed how enhancers interact with each other and with promoters. Mid-to-long range interactions showed distance-dependent decay, which was modified by TF occupancy. In particular, we found a class of ‘bivalent’ TFs, including Cohesin, which both facilitates and insulates the interaction between enhancers and/or promoters depending on the topology. In short ranges, we observed strand specific interactions between nearby eRNAs in both convergent or divergent orientations. Our finding supports a cooperative convergent eRNA model, which is compatible with eRNA remodeling neighboring enhancers rather than interfering with each other. Therefore, our approach to infer functional interactions from co-expression analyses provided novel insights into the principles of enhancer interactions depending on the distance, orientation, and the binding landscapes of TFs. Genes are expressed from promoters, where regulatory signals are integrated from proximal and distal enhancers to determine the amount of RNA product. Enhancers act as distant regulatory elements for promoters through specific TF binding, while at the same time produce bidirectional enhancer RNAs (eRNAs). Regulatory networks by enhancers are key to most cellular processes, including development, cell-type differentiation, and stress response, whereas their misregulation could cause disease. Numerous studies revealed that a large majority of disease associated genetic variations affect TREs14. Understanding TRE networks requires knowledge of how TREs interact with one another, and the mechanisms through which this regulation is achieved.Regulation of transcription is achieved mainly through the binding of transcription factors (TFs) at transcription regulatory elements (TREs), such as promoters and enhancers4. Consequently, relying on RNA products to identify TREs emerged as an efficient approach. For instance, the FANTOM5 consortium used Cap Analysis of Gene Expression (CAGE) to create an atlas of enhancer activity across numerous cell types and tissues6. While CAGE is a simple and powerful method to quantify transcription initiation at genes, it is less well suited for the quantification of transcriptional activity at enhancers, which produce particularly unstable eRNAs. Sequencing methods that capture nascent RNA, such as global Precision nuclear Run-On sequencing with 5´-capped (m7G) RNA enrichment (GRO-cap or PRO-cap), measure transcriptional activity directly and are, therefore, better suited to the quantification of TRE activity15.The bidirectional production of RNA from TREs, including both enhancers and promoters, is one of the most important hallmarks of regulatory activity in vertebrates, as transcribed enhancers are most active16. By coupling co-expression analysis to TF binding profiles, the mechanisms of the regulatory circuits can be characterized. While physical interaction between TREs, charted through chromatin conformation assays21, remains the gold standard for mapping chromatin interaction networks, such interactions are not always readily detectable. Functional interactions identified based on TRE co-expression can, therefore, complement physical interaction maps to fill gaps in the regulatory network.After identifying and quantifying active TREs, which TREs control gene expression and how they do so becomes an eminent question. Systematic analysis of expression variation can reveal these targets and mechanisms. Co-expression network uses variation in expression across different samples to elucidate regulatory circuits19. We investigate the interactions between tTREs measured by PRO-cap in lymphoblastoid cell lines (LCLs) from ~ 70 individuals. We use co-variation between tTREs as an indicator of functional interactions between sites and explore how those interactions are globally affected by various transcription factors. We identified thousands of putative interactions and found global signatures of either facilitation or inhibition of interactions for multiple TFs. We also explored interactions between the different strands of neighboring tTREs and found evidence of cooperativity at sites with converging polymerases, discovering new rules of eRNA interactions and their potential roles.This study leverages the variation in transcription initiation at transcribed TREs (tTREs), majority of which are eRNAs21, we leverage our previously published PRO-cap datasets from ~70 YRI LCLs to study functional interactions between tTREs using a tTRE co-expression analysis17. This dataset includes 87,826 tTREs, of which about 1/5 correspond to promoters and the remainder are defined as putative enhancers. These were identified based on bidirectional transcription of a pair of nascent RNAs within 300 bps of each other. Of these, about 40% are variably expressed across the individuals. As a measure of co-expression, we used Pearson’s correlation coefficients for linear regressions across individuals of the PRO-cap signals between pairs of variably transcribed tTREs in short range near SLFN5 gene promoter and in long range at BCL2 super-enhancer locus . The distribution of the coefficients reaches background levels  by 1 Mb , and the majority of the coefficients above background are for tTRE pairs within 200 kb . This distance effect is unlikely to be bias from linkage disequilibrium (LD) of the genotypes at tTRE pairs, since the YRI population is known to have LDs smaller than 5 kb22 and a similar degree of distance decay is observed using only the tTRE pairs with independent genotypes .We explored whether the co-expression reveals the principles of functional interactions between tTREs, such as the rule of distance. When we bin the correlation coefficients based on the distance between tTREs, covariation of the PRO-cap levels decreases as the distance increases . The tTREs located upstream of the TSSs have a covariation pattern that gradually decreases upon distance. Interestingly, coefficient distribution to the downstream tTREs seem to drop faster with increasing distance. This is consistent with a recent report that intragenic enhancers are less activating for their host gene, and can even attenuate expression23.We also explored how covariation between variably transcribed tTREs and mRNA levels varied based on the distance between the tTRE and gene promoters or mRNA TSSs  datasets from ENCODE’s Factorbook repository26 in a representative LCL (GM12878) to determine the effect of TF binding on the global tTRE interaction trends.Binding of transcription factors can modify interactions between TREs. Insulator proteins such as CTCF can disconnect the communication between two regions27 between them and plotted the distribution of correlation coefficients as a function of distance . The presence of CTCF sites between tTREs reduced co-expression overall and within each distance bin. We also looked at the activating effect of P300 on co-expression, comparing tTRE pairs that are occupied by P30026 to ones where P300 is not bound. As expected, P300-bound tTRE pairs tend to have higher levels of co-expression than the unoccupied pairs . These examples show that our co-expression analysis is able to detect the expected effects of TF binding, both when the TF is intersecting and when it is occupying the tTREs.To examine the insulating effect of CTCF on functional interactions, we separated tTRE pairs into “no intersection” and “intersection” categories based on the number of CTCF ChIP-seq peaks26. To generate a metric of how TF binding modifies the distance-dependent decay of co-expression, we focused on tTRE interactions in the top 5th percentile of the correlation coefficient distribution at each distance bin, and plotted the top 5th percentile as a function of the distance. We then calculated the difference in the area under the curve (ΔAUC) between “occupancy”/”no occupancy” and “intersection”/”no intersection” tTRE pairs. We compared the ΔAUC to permuted background ΔAUC distributions to evaluate the significance of the difference .We expanded our analysis to all 60 TFs for which ChIP-seq data were available from the ENCODE repositorySupplementary Fig. 2d). Insulating TFs, such as CTCF, reduce tTRE co-expression both when intersecting and occupying tTREs. The opposite is true for activating TFs, such as P300. Interestingly, the activating category contains many TFs that are immune or B-cell specific . This pattern is no longer evident for tTREs that are far enough apart to make direct read-through unlikely (> 5kb) . Also, if the enhancer pairs are interlaced by another enhancer in between, the convergent correlation pattern disappears  would show lower overall correlation than the diverging strands (upstream minus and downstream plus) . We perfsappears . These fsappears .We explored the activity, architecture, and functional interactions of eRNA transcribing tTREs using the variation in transcription initiation across human LCLs. The results reveal the principles of functional interactions between tTREs dependent on distance, TF binding, and direction of transcription.Identification of transcriptional regulatory elements (TRE) based on capped nascent RNA sequencing provides a direct measure of transcriptional activity and, therefore, shows higher sensitivity. A direct measure of transcriptional activity is important, as non-productive transcripts such as eRNAs and uaRNAs are rapidly degraded in the nucleus. Other transcription-based approaches, such as CAGE and nuclear short RNA analysis, are impeded by this instability. As we showed, CAGE performs well in identifying promoters but less efficiently detects enhancers than PRO-cap. Additionally, by focusing on bidirectional eRNA transcription start sites (TSS) from PRO-cap data enabled us to filter out spurious transcription from only one strand. Taken together, we propose that PRO-cap provides a high quality set of tTREs.17. Our model suggests a single TF binding site between the eRNA TSSs as a universal architecture, whereas other models have proposed two TF binding sites for promoter TREs, one for each strand34. Our interpretation is that two adjacent highly-directional tTREs form a larger nucleosome free region, where convergent strands are below the detection limit, and identified as a single TRE.Bidirectional transcription has been widely accepted as a general architecture of TREs. Our previous analysis of transcription initiation (tiQTL) and directional initiation quantitative trait loci (diQTL) implies that both the central TF binding and the core RNA polymerase initiating regions must be taken into consideration when assessing the potential impact of non-coding genetic variantsDrosophila that are formed by paused RNA polymerases35.Beyond understanding individual TREs, we explored how TREs interact with one another to form cis-regulatory networks. Physical interaction maps have been the standard for identifying direct interactions. However, co-expression network approaches to identify functional interactions, though indirect, provide independent evidence for interactions and a good complement to physical maps. We were able to capture mid-range interactions (within 200 kb), which were dependent on distance and insulator/co-activator binding in a manner consistent with current knowledge. Interestingly, factors associated with strong promoters were insulating, indicating that strong promoters may dominate functional interactions and repress looping across them. This may be analogous to chromosomal boundaries in 23. To compare the tTRE correlations independent of the genotype correlations in the population structure (linkage disequilibrium), we used genotypes at the SNPs nearest to the TSSs with the minor allele frequency greater than 0.05, and selected tTRE pairs within 1 Mb distance whose associated genotypes are not correlated .We used the variably expressed tTREs , and calculated correlation coefficients of the 75 individual normalized readcounts  between two tTREs within 5 Mb distance . For the distance analysis, we binned the correlation coefficients by the distance between 2 tTREs. The bins are generated based on fixed distance intervals up to 1,024 kb, or a fixed number of tTRE pairs  with variable distance intervals. With the variable interval bins, we generated plots for the 5th percentile, median and the 95th percentiles of the correlation coefficient distributions within each bin of 1000 pairs along the distance , and we obtained the z-score = +8.34 and p = 7.26 × 10−17 for CTCF intersections affecting correlation decay plots. The intersection and the occupancy scores of all other 76 factors available for GM12878 in FACTORBOOK were calculated in this way. Only factors with sufficient number of binding sites in each category were reported. These p-values and z-scores were used in hierarchical clustering and t-stochastic neighbor embedding (tSNE) analysis to cluster TFs.The top 5 percentile of the correlation coefficients in the variable interval bins was used as an indicator of tTRE co-expression. This correlation coefficient percentile serves as a function of the distance, and we refer to it as the correlation decay plot. To test whether the abundance of ENCODE FACTORBOOK binding sites - located either between the tTREs or at the tTREs - affect the correlation decay plots, we calculated the difference between the area under the correlation decay curves (ΔAUC) in the 0 – 200 kb distance range. The significance of the ΔAUC was estimated by comparing to the background distribution of ΔAUC which was generated by random shuffling of the factor binding sites. We calculated the background ΔAUC distributions by 100 permutations of 5,000 – 70,000 randomly shuffled ENCODE factor binding sites in GM12878. The background ΔAUC distributions followed a normal distribution and were dependent on the number of binding sites. We approximated the expected mean and standard deviation (sd) of the ΔAUC dependent on the actual number of the specific factor’s binding sites, and used it to generate a z-score and p-value of the ΔAUC between correlation decay curves enriched or depleted with the factor binding sites. For example, CTCF contains 41,465 ChIP-seq binding sitesFor each tTRE, the nearest neighboring tTRE downstream was identified and a linear regression between their strands was computed across the samples according to the following categories: Sense , antisense convergent (plus-minus), and antisense divergent (minus-plus). Pairs of tTREs were binned based on the distance between them and the distributions of Pearson correlation coefficients were compared between the categories. Pairs within 250 bp were not included to avoid any possibility of the same read being counted in both tTREs.


Several cases of temporal lobe epilepsy (TLE) showing false lateralization of ictal scalp electroencephalography (EEG) have been reported. However, TLE with cavernous malformation indicating false lateralization of both ictal scalp EEG and semiology as in the present case is rare. The aim of this report is to call attention to avoiding overestimation of ictal scalp EEG findings in epilepsy patients with cavernous malformation.A 25-year-old man without any medical history suffered from seizures for a year despite appropriate anti-epileptic medication. Magnetic resonance imaging (MRI) revealed cavernous malformation in the left amygdala. The seizure type was brief impaired consciousness with left dystonic posturing, preceded by a sensation of blood rushing to the head. Long-term video EEG with scalp electrodes showed periodic sharp waves beginning from the right temporal area during seizures. Although both semiology and ictal scalp EEG indicated right TLE, intracranial EEG revealed the onset of low-voltage fast activity from the left hippocampus near the cavernous malformation. This patient therefore underwent removal of cavernous malformation and left amygdala, and achieved freedom from seizures postoperatively.We reinforce the importance of performing intracranial EEG for cavernous malformation-associated epilepsy when discrepancies between scalp EEG and MRI are evident. •Scalp ictal electroencephalogram (EEG) showed false lateralization.•Semiology sometimes indicates false lateralization.•Intracranial EEG may be important in cavernous malformation-associated epilepsy. To identify the epileptic focus, a variety of assessments are performed prior to focus resections, including video electroencephalography (EEG), imaging tests such as magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography, magnetoencephalography, and neuropsychological tests. Intracranial EEG may be skipped in patients with concordant findings on all of these examinations . HoweverIctal EEG findings from long-term video scalp EEG tend to be emphasized when deciding on foci ,3, and cThis report describes an extremely rare case in which both semiology and scalp EEG falsely indicated the laterality of the seizure onset zone, despite a lack of hippocampal sclerosis or cortical atrophy. Based on this case report, we would like to suggest avoiding an overvaluation of ictal scalp EEG and semiology in cavernous malformation-associated temporal lobe epilepsy.2A right-handed, 25-year-old man without any medical history such as febrile seizures, head trauma or intellectual disability had presented with focal impaired awareness seizure (FIAS) with unnatural posturing of the left upper limb at 24 years old. Perampanel (4 mg/day) was unable to control seizures. MRI revealed a small “popcorn” lesion of mixed signal intensity with hypointense rim in the left amygdala on T2-weighted imaging , represeLong-term (48-h) video EEG with scalp electrodes was performed after discontinuing perampanel and 5 seizures were recorded. The seizure began with a “head rush” sensation, followed by impaired awareness with unusual posturing of the left upper extremity. One of the 5 seizures showed a left dystonic posture , althougDue to the discordance between imaging and scalp EEG, we conducted stereotactic electroencephalography (SEEG) with three targets, in bilateral hippocampi and the right amygdala, before focus resection surgery . In our We eventually diagnosed this patient with left cavernous malformation-associated temporal lobe epilepsy (TLE) and performed resection of the cavernous malformation and left amygdala under a transcortical approach. MRI after surgery confirmed complete removal of the cavernous malformation . The pat3The current case involved cavernous malformation-associated temporal epilepsy, with false lateralization from both scalp EEG and semiology. The results of intracranial EEG confirmed that laterality of the epileptic focus was misguided by scalp EEG, suggesting that LVFA originating from the left medial temporal area near the cavernous malformation propagated rapidly to the contralateral hippocampus and spread throughout the right temporal region earlier than the left side. Although three reports have described the side of ictal onset in scalp EEG contralateral to cavernous malformation ,4,7, to Although long-term video scalp EEG is an inevitable non-invasive examination before focal resection surgeries , severalUnilateral dystonic posturing is considered a key sign for judging lateralization of the seizure focus, since the probability that the focus of seizure onset is located contralateral to the side of dystonic posturing is reported as 92–100% . DystoniPatients with a more than 75–90% probability of having IEDs unilaterally are more likely to have the seizure foci on the side with more IEDs and achieve a seizure-free state after focus resection surgery ,19. IED There were some limitations in this case report. First, we cannot discuss magnetoencephalography or single photon emission computed tomography, as we did not use these examinations. Second, we did not perform simultaneous scalp and intracranial EEG. Proving the mechanism of the IED false lateralization described above would require simultaneous scalp and intracranial EEG.Although the ictal and interictal scalp EEG and semiology strongly suggested right TLE, the intracranial EEG confirmed left TLE in this patient. The seizure onset zone was in the vicinity of the cavernous malformation, such as the left hippocampus, but early propagation of ictal waves to the right hippocampus caused left dystonic posture and false lateralizing findings of scalp EEG in this case. Although ictal EEG findings are reportedly more credible than imaging , intracrNo ethical approval was needed because this was an anonymous case report. The patient signed a consent form for publication. We adhered to the Declaration of Helsinki.Written informed consent for publication of this case report and accompanying images was obtained from the patient. A copy of the written consent is available for review by the Editor of this journal.The data are not publicly available due to issues of patient privacy. The datasets used during the current study are available from the corresponding author on reasonable request.The authors declare that they have no competing interests.No funding was obtained for this study.All authors listed have significantly contributed to the investigation, development and writing of this article.info@forte-science.co.jp) for English language editing.We would like to thank Forte Science Communications (email: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


This case series aims to highlight the efficacy of peripheral nerve stimulation (PNS) in the treatment of phantom limb pain, as well as provide an alternative method for the treatment of this pain syndrome. In this report, we describe three amputee patients with severe phantom limb pain who obtained substantial analgesia and improvement in physical functionality after implantation of a temporary PNS device. Future studies should assess predictors of successful response or poor response to PNS therapy, such as mental health, environmental stressors, coping skills, and procedural factors. These factors may facilitate an individualized approach for each patient to ensure appropriate candidacy for PNS and better prognosis. Considering that patients in our cohort did not achieve long-lasting benefit after removal of temporary PNS, future research should assess if patients with phantom limb pain would benefit from permanent PNS, rather than temporary PNS. Nearly 200,000 extremity amputations are performed annually in the United States . DespiteA 68-year-old female with a history of left transhumeral amputation, end-stage renal disease on hemodialysis, hypertension, type 2 diabetes mellitus, and anxiety presented to the pain clinic for phantom limb pain. Her left transhumeral amputation was due to a planned surgical resection due to necrotizing fasciitis two years ago.Her main complaint was severe phantom limb pain based around the area where her thumb, dorsal hand, and wrist were previously located. She reported constant shooting and burning pain with intermittent “lightning bolt” sensations. She also described residual nociceptive limb pain which was less intense compared to the phantom limb pain. She had previously trialed gabapentin 100 mg twice a day, nortriptyline 10 mg daily, 5 mg oxycodone four times daily, lidocaine patches, ice application, and mirror therapy without success.A median nerve neuroma was identified during examination with ultrasound, and thus, the decision was made to perform PNS of the left median nerve for pain relief. A diagnostic block with 2% lidocaine at the left median nerve resulted in 80% pain relief. A temporary stimulator  was implanted under ultrasound guidance in close proximity to the left median nerve. A Sonosite linear ultrasound transducer (15-6 MHz) was used to identify the left median nerve (neuroma). A short-axis, in-plane approach was used to place the stimulating needle in close proximity to the neuroma . The intA 77-year-old female has a history of hypertension, diabetes, chronic kidney disease, depression, and a high-grade spindle cell sarcoma that resulted in a left transfemoral amputation. She reported phantom limb pain diffusely around the area of the left foot. She had previously tried gabapentin, duloxetine, tramadol, intra-articular hip steroid injections, and mirror therapy without any significant relief. The pain was described as severe 10/10 pain exacerbated by weather changes, and intermittent radiation to the residual stump.She underwent a left femoral and sciatic nerve ultrasound-guided diagnostic block with 2% lidocaine which provided significant pain relief (100%) for 4–6 hours. Two months later, she underwent a temporary PNS device placement targeting the left femoral and sciatic nerves. A Sonosite linear ultrasound transducer (15-6 MHz) was used to identify the left femoral and sciatic nerve at the inguinal and subgluteal region, respectively. A short-axis, in-plane approach was used to place the stimulating needle in close proximity to the nerves. The subcutaneous tissue layers were identified, and local anesthetic was infiltrated in the subcutaneous tissue. The introducer needle was first advanced to near the left femoral nerve and stimulation was carried out with the patient stating that she was feeling paresthesia in the left femoral nerve distribution. We then inserted the lead and re-performed stimulation and then styleted the lead. We repeated these same steps for the left sciatic nerve. After the stimulating needle exhibited optimal spread of coverage, a PNS lead was advanced through the stimulating needle. The external portion of the lead was secured to the skin with Dermabond  and Tegaderm .At the 2-week follow-up visit, the patient reported a 100% improvement for both pain intensity and physical functionality, with a sustained 100% improvement in both measures at the 6-week follow-up visit. She reported lower frequency of pain flares and greater satisfaction with PNS therapy compared to baseline. After severe recurrence of pain was noted at day 45, ultrasound evaluation revealed migration of the PNS lead. The PNS settings were re-programmed, and efficacy was achieved again with 75% improvement in pain intensity. After removal of the PNS device 60 days post-implant, she had a relapse of phantom limb pain in 2 months that was 50% less intense than pre-implant pain intensity. No other complications were reported.A 16-year-old male reported a history of chronic left lower extremity pain in the setting of refractory erythromelalgia and left transtibial amputation secondary to severe pain and recurrent infection. After amputation, he began experiencing severe phantom limb pain at the left foot about three weeks after amputation. A diagnostic nerve block at the sciatic nerve provided 80% pain relief, and therefore, a temporary PNS stimulator was implanted under ultrasound guidance to stimulate the sciatic nerve. A Sonosite linear ultrasound transducer (15-6 MHz) was used to identify the left sciatic nerve at the subgluteal region. A short-axis, in-plane approach was used to place the stimulating needle in close proximity to the nerve. The subcutaneous tissue layers were identified, and local anesthetic was infiltrated in the subcutaneous tissue. The introducer needle was advanced to near the left sciatic nerve and stimulation was carried out with the patient stating that he was feeling paresthesia in the left sciatic nerve distribution. We then inserted the lead and repeated stimulation and then styleted the lead. After the stimulating needle exhibited optimal spread of coverage, a PNS lead was advanced through the stimulating needle to the optimal site of stimulation. The external portion of the lead was secured to the skin with Dermabond  and Tegaderm .At the 2-week follow-up visit, the patient reported pain intensity was 2/10 compared to 10/10 at baseline. At the 6-week follow-up visit, pain intensity was 5/10. He reported less frequent pain flares and great satisfaction. After removal of his PNS device at 60 days, his pain intensity subsequently increased to 10/10 within one month following removal. No complications were reported.We report a case series of post-amputee patients with severe phantom limb pain who obtained substantial analgesia and improvement in physical functionality after implantation of a temporary PNS device. Reduced frequency of pain flares, good patient satisfaction, and no adverse events were reported. However, after removal of the temporary PNS device, pain intensity worsened in all patients and regressed completely back to pre-implant baseline levels in two patients. This small case series highlights that temporary PNS provides substantial short-term pain relief and improved physical functionality, although this therapeutic effect is temporary and does not extend long-term after device removal.The mechanism in which patients experience PLP is not entirely delineated but likely has both central and peripheral components. Central components include somatosensory cortical reorganization of the area representing the amputated limb, as well as spinal reorganization in the dorsal horns after deafferentation from a peripheral nerve . PeripheOur results are concordant with the literature, which highlights efficacy of PNS in treatment of post-amputation pain , 5, 10. Additional clinical studies are needed to further assess the efficacy of PNS in the treatment of phantom limb pain. The current literature suggests that PNS therapy may provide clinically significant pain relief and improvement in the quality of life of patients with chronic neuropathic post-amputation pain, which usually includes both residual limb pain and phantom limb pain , 10, 12.When evaluating post-amputee patients who are experiencing phantom limb pain, the first step should be to assess prosthetic fit and ensure proper wound healing if amputation occurred recently. If none of these are sources of pain, referral to a physical therapist would be warranted to work on multiple exercises and pain management modalities. Pharmacologic treatment with oral gabapentinoids, selective norepinephrine reuptake inhibitors (SNRIs), or tricyclic antidepressants (TCAs), followed with a formal biopsychologic evaluation, should be the next step in cases where pain is refractory to rehabilitation and alternative therapies. Neuromodulation techniques such as PNS and spinal cord stimulation may be considered for those patients who do not obtain benefit from conservative and pharmacologic treatment. Additionally, other procedural interventions such as dorsal root entry zone ablation, peripheral nerve blocks, and radiofrequency ablations can also be considered. In some cases, surgical intervention may be necessary if none of the aforementioned interventions improve the patients' pain and quality of life.Future studies should assess predictors of successful response or poor response to PNS therapy, such as mental health, environmental stressors, coping skills, and procedural factors. These factors may facilitate an individualized approach for each patient to ensure appropriate candidacy for PNS and better prognosis. Considering that patients in our cohort did not achieve long-lasting benefits after removal of temporary PNS, future research should assess if patients with phantom limb pain would benefit from permanent PNS, rather than temporary PNS.


This case report describes the successful use of Integra® , an artificial skin substitute, for the reconstruction of soft tissue defects following soft tissue sarcoma resection. We present a case of a 75-year-old female presented with a progressively enlarging lesion on her right hand. Imaging revealed tumor involvement in the extensor tendons and adjacent to the index finger tendon. A percutaneous biopsy confirmed an undifferentiated pleomorphic sarcoma. The patient underwent neoadjuvant radiotherapy followed by wide excision of the tumor. Integra® dermal regeneration matrix was utilized to cover the exposed bone during the surgical procedure. This allowed for wound closure and provided a favorable environment for tissue regeneration and subsequent split-thickness skin graft. Complete wound healing was obtained. Regular follow-up examinations showed no evidence of local recurrence or secondary lesions after one year. The successful use of Integra®, in this case, demonstrates its efficacy as a reconstructive option for complex hand sarcomas. It offers immediate wound coverage and promotes tissue regeneration, thereby avoiding the need for more extensive treatment modalities with associated donor-site morbidity. The utilization of Integra® resulted in high patient satisfaction and excellent recovery. This case highlights the importance of utilizing innovative techniques and materials in achieving optimal outcomes in challenging hand sarcoma reconstructions. Soft tissue sarcomas are rare heterogeneous groups of solid malignancies arising in connective tissues and derived from mesenchymal cell origin that can occur in various locations throughout the body, including the extremities . Soft tiThis case report presents a patient with soft tissue sarcoma located in the dorsal aspect of the hand, involving the overlying skin and resulting in exposure to the underlying bone. We describe the successful application of an artificial skin substitute, Integra® , for coverage of the exposed bone following tumor resection. The use of Integra®, in this case, facilitated wound closure, promoted tissue regeneration, and ultimately restored functionality to the hand.A 75-year-old right-handed female patient with a history of hypertension, dyslipidemia, and hypothyroidism was initially referred to our institution in February 2022 due to a gradually enlarging lesion on the dorsal aspect of her right hand, measuring 5.4 cm in the largest dimension. Magnetic resonance imaging (MRI) revealed a heterogeneous lesion invading the extensor tendons of the third, fourth, and fifth digits and close to the extensor tendon of the index finger Figure .A percutaneous biopsy confirmed the presence of an undifferentiated malignant tumor composed of spindle and epithelioid cells, consistent with a grade 2 undifferentiated pleomorphic sarcoma according to the Fédération Nationale des Centres de Lutte Contre Le Cancer (FNCLCC) grading system . After dThe patient underwent neoadjuvant radiotherapy with a total dose of 50 Gy in two fractions, completed in May 2022. The resection of the tumors involved sacrificing extensor tendons, periosteum, and a layer of dorsal interossei muscles to achieve negative margins. We attempted to preserve the second extensor tendon, but intraoperative observations indicated that conserving it would be challenging due to the tumor's close proximity. Considering our concern about not achieving negative margins, we made the decision to include it in the resection. Following the tumor resection, the metacarpal bones (second-fifth) were exposed, and Integra® was used to cover the soft tissue defect  scans every four months revealed no evidence of local recurrence or secondary lesions after one year of follow-up Figure . The patThe management of soft tissue sarcomas in hand presents several challenges, including achieving complete tumor resection with negative surgical margins while obtaining wound coverage and ensuring the deliverance of radiotherapy.Preoperative and postoperative radiotherapy is a commonly proposed treatment in the management of sarcoma along with surgery, each offering distinct benefits and risks ,8,9. PreIn cases where tumor involvement necessitates the resection of a large amount of soft tissue, the resulting bone exposure poses additional difficulties in achieving successful wound healing. After removing the periosteum, exposed bone cannot form a layer of healthy granulation tissue where skin graft can be applied directly and integrated. Additionally, it is important to consider early coverage of exposed bone to avoid bone infection and accelerate wound healing, especially in the case of sarcoma patients when adjuvant therapy is needed. Traditional reconstruction methods, such as skin grafts or local flaps, may be limited in their ability to provide durable coverage.Integra®, a bi-layered dermal regeneration matrix, has demonstrated considerable success in various reconstructive applications, including complex wounds and soft tissue defects, as well as in cancer resection -16. It aIn the present case, the use of Integra® proved to be a valuable adjunct in achieving successful wound closure and functional restoration, even with the history of neo-adjuvant radiotherapy. By covering the exposed bone, Integra® provided a protective barrier, facilitated the ingrowth of blood vessels and fibroblasts, and promoted the formation of a neodermis. This allowed for subsequent split-thickness skin grafting, resulting in a durable and aesthetically satisfactory outcome.The successful use of Integra®, in this case, highlights its potential as an effective option in reconstructing complex hand wounds following sarcoma resection. It offers several advantages, including covering exposed bone, promoting tissue regeneration, minimizing scarring, and improving patient outcomes. Moreover, patients can benefit from definitive wound coverage within a short timeframe of 2-3 weeks following Integra® placement, as demonstrated in the presented case. Further studies and long-term follow-up are warranted to assess the durability and functional outcomes of Integra® in similar cases.The case presented demonstrates the successful utilization of Integra® as an artificial skin substitute in the reconstruction of a soft tissue sarcoma involving the dorsal aspect of the hand. The application of Integra® facilitated wound healing and promoted tissue regeneration without requiring free tissue transfer or distant flaps, which would have added additional morbidity to the patient. The case highlights the importance of innovative techniques and materials in achieving optimal outcomes in challenging reconstructive procedures.


Dual immune checkpoint inhibition with ipilimumab plus nivolumab is currently the most effective, but also by far the most toxic treatment for advanced melanoma. Therefore, other combination partners that also lead to high and long-lasting responses but cause fewer adverse events were explored.Relatlimab, a LAG-3 blocking antibody, was investigated in combination with nivolumab in a phase 2/3 randomized double-blind trial (RELATIVITY-047) and could demonstrate significantly improved progression-free survival in treatment-naive advanced melanoma patients compared with nivolumab monotherapy. While the safety profile is more favorable than that of ipilimumab plus nivolumab, no significant survival benefit has yet been demonstrated with the new combination over nivolumab monotherapy.The approval of relatlimab plus nivolumab by both the Food and Drug Administration and the European Medicines Agency expands the arsenal of treatment options for melanoma but raises new questions in clinical practice and a re-evaluation of currently established treatment standards and sequences. The development of immune checkpoint inhibitors has fundamentally revolutionized the therapeutic landscape and has led to a marked improvement in survival outcomes in patients with advanced melanoma by enabling profound and durable responses . Based o+ and CD8 + T lymphocytes and inhibits the tumor immunological microenvironment by negatively affecting T cell proliferation and inducing T cell exhaustion , LAG525 [NCT03484923], MK4280 [NCT02720068], and RG6139 [NCT04140500]). In a phase 3 trial the efficacy and tolerability of fianlimab (REGN3767) in combination with cemiplimab compared to pembrolizumab in patients with previously untreated unresectable locally advanced or metastatic melanoma will be evaluated [NCT05352672]. Fianlimab is a newly developed fully human anti-LAG-3 antibody with high affinity, blocking LAG-3/MHC II driven T cell inhibition. Together with cemiplimab, a high affinity, human, hinge-stabilized IgG4 antibody to PD-1 receptor, fianlimab showed promising antitumor effects in preclinical studies. In a phase 1 dose escalation study [NCT03005782], combined therapy with fianlimab and cemiplimab demonstrated an acceptable safety profile and already indicated clinical activity in patients with advanced malignancies . PrelimiThe approval of the new combination therapy with relatlimab plus nivolumab for patients with advanced melanoma expands the arsenal of immune checkpoint inhibitors, raising new questions in clinical practice and requiring re-evaluation of currently established therapeutic standards and sequences. A key role is played by understanding factors that determine treatment response, resistance, and toxicity, in order to identify the appropriate combination and sequence of treatments for each patient and define an individually tailored therapy. Both, anti-PD-1 monotherapy or in combination with ipilimumab represent the current standard of care for patients with advanced melanoma. Although the Checkmate-067 trial was not powered enough to compare ipilimumab plus nivolumab with nivolumab alone, it demonstrated a numerical efficacy benefit • establi(unpublished data) [Although the new dual checkpoint inhibition shows positive antitumor activity and appears to be effective by prolonging PFS, a large proportion of patients will not respond to therapy or will experience disease progression after a period of response, requiring follow-up therapy. To date, we do not know whether the same patients who do not respond to relatlimab plus nivolumab also do not respond to ipilimumab plus nivolumab or vice versa and whether they differ. In a small, pooled, retrospective, multicenter analysis, anti-CTLA-4-based therapy was shown to be less effective after treatment failure on relatlimab plus nivolumab with an overall response rate of 11%, although the cohort was too small to allow robust analysis between ipilimumab mono and combined therapy with ipilimumab plus nivolumab •. In theed data) ••. Withied data) .Furthermore, it remains to be seen what value the new dual checkpoint combination will have in distinct patient groups such as brain metastases, elevated LDH and liver metastases. Therapy with ipilimumab and nivolumab shows superior efficacy with long-lasting effects compared with anti-PD-1 monotherapy while inducing both extracranial and intracranial responses. Based on the results of two phase 2 trials the combination therapy with ipilimumab and nivolumab lead to an ORR of up to 56% intracranially in patients with active brain metastases. Thus, it has become established as first-line treatment in melanoma patients with brain metastases , 62. SinThe need for oncological treatment options with an improved benefit-to-risk ratio has increased with new treatment options for patients that can significantly prolong survival and, in some cases, lead to cure. Dual immune checkpoint inhibition has become the focus of research by both prolonging duration of response and improving therapy response rates . Relatli


Penicillin (PCN) allergy is commonly reported. Yet most patients labeled allergic can tolerate beta-lactams (BLs), the antibiotics of choice for many infections. Documentation of reaction details is important to identify low risk patients who can safely receive BLs, but it is often incomplete. A May 2018 point prevalence study from the electronic medical record (EMR) using a one-week sample of Atrium Health Greater Charlotte Market (AH) PCN allergy patients showed that 52% had no reaction documented.This was a retrospective, observational study in thirteen hospitals in the AH system. To improve BL allergy assessment, a BL allergy questionnaire was developed during the launch of a new AH wide EMR. During the initial roll-out, a best practice advisory (BPA) fired for nursing when entering a BL allergy, prompting a link to a separate BL allergy questionnaire for nursing completion. After all sites were live, we assessed questionnaire completion rates. During the optimization phase, the questionnaire was incorporated directly in the BPA and simplified from ten to three questions, allowing completion in one step . The answers are then automatically sent to flowsheets where they are available for reference and feed into assessment algorithms that provide the allergy level of risk and clinician treatment guidance upon antibiotic order entry . The BPA fires daily as a reminder until questionnaire completion.Pre-optimization: Over a ten month period from December 2021 through October 2022, 4118 inpatient questionnaires were completed. Beginning in August 2022, when all facilities were live on the new EMR, the average monthly questionnaire completion was 833 . Post-optimization: In November 2022, completion increased 38.9%, resulting in an additional 4743 questionnaires over four months (average of 1186 per month). The number of questionnaires completed thereafter decreased as the number of patients with previously completed questionnaires increased.The creation of a targeted BL allergy questionnaire in the EMR improved BL allergy documentation across a large, diverse health system. The effect was enhanced through workflow optimization incorporating the questionnaire into a one-step BPA.All Authors: No reported disclosures


Kaposi sarcoma (KS) is a low-grade tumor of the vascular endothelium. The majority of individuals affected have advanced human immunodeficiency virus (HIV) or acquired immunodeficiency syndrome (AIDS). The disease typically manifests as cutaneous lesions but reports have shown that systemic disease is not uncommon. Because gastrointestinal KS is often asymptomatic, it is likely underdiagnosed. Those with symptoms can present with vague abdominal pain, nausea/vomiting or anemia. Rarely the tumors can cause bowel obstruction or perforation. We present a case of small bowel obstruction cause by KS tumors in a young transgender male to female patient with poorly controlled AIDS, supported by literature review of the clinical presentation, diagnosis and treatment recommendations. Kaposi sarcoma (KS) is the most common tumor seen in acquired immunodeficiency syndrome (AIDS) with typical findings of cutaneous lesions. Previous reports have shown that 10–66% of those patients with skin lesions also had systemic disease . We presPatient is a frail 28-year-old transgender male to female with poorly controlled human immunodeficiency virus (HIV)/AIDS  who presented to the ED with one day of abdominal pain. History was limited as the patient was uncooperative. Vitals on arrival in the ED were: Temp 98°F, Pulse 78, BP 106/55, RR 18, SpO2 97%. The physical exam was significant for cachexia (BMI 17.3), skin changes compatible with skin type Kaposi’s Sarcoma, abdominal distention, and peritoneal signs. Computed tomography (CT) abdomen and pelvis showed multiple dilated small bowel loops with swirling of mesentery  and possThe patient was taken to the operating room for exploratory laparotomy given concerns for peritonitis in the context of closed loop obstruction. Intra-operative findings consisted of three ileal tumors spanning an approximately 40-cm segment, located 45 cm proximal to cecum accompanied by significant lymphadenopathy –3.A palliative en bloc resection of the segment containing the three tumors was carried out with creation of an end ileostomy and mucous fistula. Final pathology showed malignant vascular tumor with extensive ulceration involving all layers of bowel wall, positive staining for CD-34 & HHV-8 consistent with KS.The patient was started on HAART therapy post-operatively, had an uncomplicated hospital course and was eventually discharged to rehab. Unfortunately, the patient refused chemotherapy on discharge and was lost to follow up.KS is a vascular angio-proliferative neoplasm which usually presents on mucocutaneous sites but may involve visceral organs. In the US, KS predominantly affects HIV infected patients, with the majority of individuals having advanced immunosuppression (CD4 count <150 cells per cubic millimeter) and a high viral load (>10 000 copies per millimeter) at the time of diagnosis . While gGiven the indolent nature of GI KS, it raises the question of whether routine endoscopic screening should be done. Endoscopically KS appears as red or purple patchy lesions of different sizes and shapes of polypoid/nodular morphology. These lesions are typically identified in the stomach, esophagus, and duodenum . The rolIn patients with AIDS-related KS with systemic manifestations, combined HAART and systemic chemotherapy is indicated. Liposomal doxorubicin is the agent of choice, and trials have shown that delivery via pegylated doxorubicin is effectively concentrated in lesions with greater tolerability and less toxicity than other chemotherapeutic agents .The surgical management of small bowel obstruction due to KS remains controversial– the decision to perform surgery should be made on a case-by-case basis. In general, surgery may be indicated in cases of complete or high-grade partial obstruction, perforation, or ischemia, which are associated with a high risk of morbidity and mortality . HoweverKS should be included in the differential diagnosis of small bowel obstruction in patients with AIDS. Although this extent of progression of GI KS is rare, high index of suspicion and perhaps increased endoscopic screening is warranted when dealing with this vulnerable demographic.


At follow-up, LBBAP seemed suboptimal, with a paced QRS duration of 185 ms with paced left bundle branch block (LBBB) morphology, possibly due to microdislodgment of the lead. We opted for implantation of a new LBBAP lead, with conventional BiV-CRT as bailout strategy. BiV-CRT was considered to be associated with increased risk because of a pseudoaneurysm compressing the coronary sinus as a complication of the previous NeoChord procedure. The new LBBAP lead was implanted according to the method previously described by Heckman et al.A 72-year-old man with ischemic cardiomyopathy and a previous NeoChord  and Tendyne  procedure for severe mitral regurgitation was referred to our device department. After implantation of a DDD pacemaker 2 years before referral with LBBAP because of second-degree AV block, his LV function had declined. Left ventricular ejection fraction (LVEF) decreased from 40% to 15%. The electrocardiogram recorded after initial implantation showed a paced qR morphology in lead VWith the availability of alternative pacing strategies such as LBBAP to conventional BiV-CRT in cardiac resynchronization therapy, patient selection is an important issue, even more so when both strategies can be combined with LOT-CRT.,BiV-CRT has proven to be an effective therapy in patients with heart failure and LBBB.,LOT-CRT has been described as an alternative pacing strategy to classic BiV-CRT and LBBAP when these strategies alone result in suboptimal cardiac resynchronization therapy.In this case report, we showed that stimulus-V6RWPT can be longer than expected in LBBAP with left bundle branch capture due to severe distal conduction system disease, resulting in LBBAP failing to correct dyssynchrony. In patients with nonselective LBBP and severe distal conduction system disease, LV septal myocardium and subsequent LV activation might be faster than LV activation via the (diseased) conduction system, in essence resulting in LV septal pacing only despite capture of the left bundle branch. LOT-CRT can be a good addition to LBBAP when the latter fails to correct electrical dyssynchrony.


Surgery for endocarditis of the aorto-mitral continuity can be a challenge incase of extensive tissue destruction. We report two cases of a modified monoblocreconstruction of the aortic and mitral valves and of the aorto-mitral fibrousbody. Two valve bioprostheses were sutured to each other and implanted as acomposite graft. A pericardial patch sutured to the valves was employed toreconstruct both the noncoronary sinus and the left atrial roof. This technicaladjustment allows adaptation to variable anatomical conditions in theseparticularly difficult cases. Surgery requires debridement ofall infected tissue and may require reconstruction of the aorto-mitral annulus andleft atrial roof. Previouslydescribed techniques for reconstruction include use of monobloc aorto-mitralhomograft and of handmade aorto-mitral bioprosthetic or mechanical valve compositegrafts,4,5. As a novelpossible adaptation of this technique to some cases marked by “extreme” tissuedestruction, we describe a variant of the aorto-mitral monobloc valve implantation,adding a pericardial patch to reconstruct the noncoronary sinus, the left atrialroof, and the aorto-mitral continuity. All patients provided written consent priorto surgery for use of personal data to research purposes.Infective endocarditis carries a particularly high morbidity and mortality in casesinvolving abscess and disruption of the aorto-mitral continuityCase 1 was a 51-year-old patient with bivalvular  endocarditis. Transesophageal echocardiography revealed mitral andaortic vegetations , a significant periprostheticaortic leak, and central mitral regurgitation. An aortic root abscess extendingtowards the anterior mitral leaflet and the left atrial roof was also noted.Case 2 was a 70-year-old patient with bivalvular endocarditis. Transesophageal echocardiography revealed a bicuspid aortic valve,severe aortic regurgitation, large mitral vegetation, severe mitralregurgitation, and a fistula between the left sinus of Valsalva and the leftatrium.Both cases had preserved ventricular function and normal coronary arteries.Cardiopulmonary bypass (CPB) was established using bicaval cannulation and femoralreinjection. Cold Custodiol® cardioplegia was instilled. An oblique aortotomywas performed and the mitral valve approached via a transseptal incision extendedtowards the left atrial roof.The infected aortic (bioprosthetic and native) valves, the native mitral valves, andpart of ascending aorta had to be debrided. Anterior mitral leaflet infection,aorto-mitral disruption, and abscess involving two thirds of the aortic root wereobserved in both cases. The posterior leaflet of the mitral valve was free frominfection . The sacA modified “monobloc repair” was performed. Two Medtronic Mosaic© aorticvalves  were joined to two AbbottEpic© mitral valves  , using aIntensive care unit stay was seven days in case 1 and 10 days in case 2. Both casesrequired permanent pacemaker implantation. Discharge echocardiography revealedsatisfactory bivalvular function, as well as the control at one year of follow up.Both patients were discharged home with satisfactory functional status.,4, aimed at facilitating surgical reconstruction incases with extensive destruction of the native aorto-mitral continuity, left atrialroof, and valve annuli due to infectious endocarditis.The surgical management of infectious endocarditis is based on a radical excision ofthe infected tissues. Nonetheless, reconstruction can be difficult in somecircumstances. Herein we describe a modified monobloc aorto-mitral replacementtechnique,4. This modification entails the useof a V-shaped patch to reconstruct both the left atrium and the noncoronary aorticsinus; this patch is secured directly to the composite valve graft. When thedestruction is limited to the noncoronary sinus at the level of the root, thisstrategy might help avoiding full root replacement and coronary buttonsreimplantation, andtherefore reduces the complexity of the repair as well as the CPB and aorticcross-clamping times. Also, a distinct patch can be inserted between the two valvesto provide versatility in their placement while respecting the native aortomitralangle. We also suggest performing valve sizing by inserting into the defect both theaortic and mitral valve sizers at the same time, in order to understand theprospected relationship of the two valves.The strategy presented here adjusts the previously described “monobloc”reconstruction techniques to cases with aortic root and coronary ostiapreservationWe underline the usefulness of bicaval canulation and a biatrial access to achieve anoptimal exposure of the lesions  and facilitate complex reconstructions. We suggest that bicaval cannulationshould be liberally employed in patients operated on for echocardiographicallyevident extensive abscess of the aortomitral body.This modified monobloc aorto-mitral valve implantation should be considered as partof the toolbox for surgical management of severe infectious endocarditis. Itenhances the possibilities to adapt the reconstruction to variable anatomy defectsafter debridement.


Traumatic dental injuries are extremely common in children, and trauma to developing permanent teeth can disrupt root maturation; vital pulp therapy is an appropriate treatment for these teeth. This case report describes a 9-year-old boy who suffered dental trauma while playing football, resulting in an enamel–dentin fracture with pulp exposure in the left central incisor with an open apex (Cvek's stage 3) and an enamel–dentin fracture in the right central incisor with an open apex (Cvek's stage 3). Apexogenesis with mineral trioxide aggregate was performed to preserve the neurovascular bundle, allowing normal radicular formation in the left central incisor. During a 2-year follow-up, the tooth showed no signs and symptoms, and radiographic examinations revealed no evidence of radiolucent lesions in the periapical region. This case study provides compelling evidence that the utilization of the described agent yields significant efficacy in treating traumatic fractures accompanied by pulp exposure. A complicated crown fracture refers to a dental injury where there is damage to both the enamel and dentin of a tooth, leading to exposure of the pulp. The prevalence of such fractures can vary between 2% and 13% of all dental injuries [When dealing with traumatized immature teeth with open apices and pulp exposure, vital pulp therapy is the preferred treatment option. Apexogenesis and other vital pulp therapies are gaining popularity due to several advantages, including shorter appointments and less technique sensitivity. Apexogenesis encourages natural root development, leading to apical closure and strengthening of the root structure . In thisFor many years, calcium hydroxide has been the primary pulp-capping agent. However, it has certain disadvantages, such as the formation of defects in the dentin bridge beneath the calcium hydroxide layer and increasing the risk of failure , 7. In rThe objective of this clinical case report is to describe a partial pulpotomy procedure using MTA in a complicated crown fracture of an immature permanent incisor tooth on the left side. Additionally, the report highlights the accelerated apical closure observed in the affected tooth with an open apex during a two-year follow-up period.A nine-year-old boy presented to the Department of Pediatric and Preventive Dentistry with a chief complaint of dental fractures. The patient had an Ellis III fracture in the maxillary left central incisor (tooth #21) and an Ellis II fracture in the maxillary right central incisor (tooth #11) due to a traumatic injury sustained while playing football . The parDuring the intraoral examination, pulp exposure was observed in the maxillary left central incisor tooth #21) with mild pain upon percussion. No periodontal pockets were detected, and the affected teeth showed class I mobility. Pulp vitality testing revealed a positive response to cold stimulation. The extent of pulp exposure in tooth #21 was measured to be approximately 2–3 mm. The radiographic examination did not reveal any root fractures or periradicular radiolucency in the regions of teeth #11 and #21, but it did confirm an enamel–dentin fracture in the maxillary right central incisor  affects the outcome of partial pulpotomy with calcium hydroxide dressing , 14. TheSince many years ago, vital pulpotomy has used calcium hydroxide to induce coagulation necrosis, a low-grade irritation that causes undifferentiated pulp cells to undergo differentiation. These cells produce predentine, which is then mineralized, whereas the coagulated tissue is calcified , 19. MTAMTA has been scientifically proven effective in various endodontic procedures. In this study, MTA apexogenesis treatment was completed in 24 months, resulting in a calcified barrier and no need for further treatments. This suggests MTA's suitability as a pulp-capping material. However, it is crucial to note that this conclusion is based on one study, and longer clinical studies are recommended for better long-term effectiveness and safety data. Healthcare professionals should consider patient factors and case-specific requirements, and consult current research and guidelines for treatment decisions.


It is generally recognized that the initiation of obesity-related hepatocellular carcinoma (HCC) is closely associated with hepatic inflammation. However, the paradoxical role of inflammation in the initiation and progression of HCC is highlighted by the fact that the inflammatory HCC is accompanied by significant immune effector cells infiltration compared to non-inflammatory HCC and HCC with enhanced immune response exhibits better survival. Importantly, the cancer progression has been primarily attributed to the immunosuppression, which can also be induced by obesity. Furthermore, the increased risk of viral infection and thus viral-HCC in obese individuals supports the view that obesity contributes to HCC via immunosuppression. Here, we have reviewed the various mechanisms responsible for obesity-induced tumor immune microenvironment and immunosuppression in obesity-related HCC. We highlight that the obesity-induced immunosuppression originates from lipid disorder as well as metabolic reprogramming and propose potential therapeutic strategy for HCC based on the current success of immunotherapy. Chronic and mild inflammation can induce local or systemic insulin resistance (IR), which in turn induces high levels of insulin and IGF-1, thus stimulating abnormal hepatocyte proliferation , 7. 2) C+ T cells as well as M1 macrophages, implies strong antitumor immunity, and HCC with enhanced immune response exhibits better survival  cells. However, these cells undergo function loss and numeric decrease owning to the metabolic dysfunction and lipotoxicity  to aerobic glycolysis, and thus robust proliferation  and granzyme B when compared to mice with chow diet, and altered FA partitioning could be responsible for the reduced function of CD8+ T cells  via lipid peroxidation , tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) are able to adjust to lipid-rich environment as well as TIME, and thereby can facilitate immunosuppression. FAO plays a vital role in this process.5.1+ T cells  and adoptive cell therapy have shown strong anti-tumor effects for HCC. Despite of the prevalence of obesity-related HCC, there are few studies that have focused on the potential impacts of obesity on immunotherapy of HCC.7.1Obesity appears to play a positive role in immunotherapy (mainly including ICIs therapy and adoptive cell therapy) of various cancers. ICIs therapy (such as anti-PD-1 and anti-CTLA4 antibodies) works by targeting depressed immune effector cells and enhancing their function, resulting in robust immune activation. In patients with NSCLC, high BMI was found to be independently associated with a better prognosis after atezolizumab treatment . A multi7.2Adoptive cell therapy works by modifying immune effector cells (such as T cells) to express specific immune effector marker (such as chimeric antigen receptor), resulting in enhanced anti-tumor immunity in patients. Currently, studies about the effects of obesity on adoptive cell therapy for HCC are lacking. Wenshu Tang et al. observed that obesity-induced hypercholesterolemia can effectively impair NK cell function, whereas Wenhao Qin et al. indicated that high cholesterol increased NK cell lipid accumulation thereby enhancing their function , 96. Thu8Overall, based on the general understanding, obesity-related HCC is primarily attributed to NAFLD and IR. However, there are few studies that have focused on the inhibitory effects of obesity-induced metabolic disorder on antitumor immunity. In this review, we have presented a comprehensive overview about the distinct mechanisms through which obesity facilitates HCC progression via remodeling TIME and inducing immunosuppression Figure 1Obesity-related immunosuppression is mechanistically related to the disrupted balance between adipocytes, hepatocytes, and immune cells. Excessive lipid deposition in visceral adipose tissue is the trigger for obesity-related HCC, and can directly lead to adipose tissue expansion . Thus, tConventional therapy including surgery resection and systemic therapy is widely used in HCC treatment. The impact of obesity on HCC resection remains controversial. Many studies indicated that obesity has shown no effects on lethality rate after HCC resection, whereas several studies had shown that severe obesity increased lethality rate after HCC resection , 182. BeAbove all, obesity can facilitate HCC progression via remodeling TIME and inducing immunosuppression, all of which mainly originate from the inflammatory environment but can exert pro-tumor effects once HCC has occurred. Given the tolerance to conventional therapy and the continued success of immunotherapy for HCC, more emphasis should be placed on obesity-induced immunosuppression. Investigations about obesity-related HCC progression from an immunosuppressive perspective can provide the basis for biomarker discovery and facilitate diagnostic evaluation as well as therapeutic development. However, it is unclear when the inflammatory environment might shift to immunosuppressive state, which may be an important direction for future insights into the development of obesity-related HCC.JY designed and drafted the manuscript. JH and YF revised the manuscript. MX designed and edited the manuscript. All authors contributed to the article and approved the submitted version.


One of the two X chromosomes in female mammals is epigenetically silenced in embryonic stem cells by X chromosome inactivation (XCI). This creates a mosaic of cells expressing either the maternal or the paternal X allele. The XCI ratio, the proportion of inactivated parental alleles, varies widely among individuals, representing the largest instance of epigenetic variability within mammalian populations. While various contributing factors to XCI variability are recognized, namely stochastic and/or genetic effects, their relative contributions are poorly understood. This is due in part to limited cross-species analysis, making it difficult to distinguish between generalizable or species-specific mechanisms for XCI ratio variability. To address this gap, we measured XCI ratios in nine mammalian species , ranging from rodents to primates, and compared the strength of stochastic models or genetic factors for explaining XCI variability. Our results demonstrate the embryonic stochasticity of XCI is a general explanatory model for population XCI variability in mammals, while genetic factors play a minor role. XCI evolved to balance the gene dosage between females with two X-chromosomes and males with one X-chromosome4. While the exact timing can vary across species5, XCI usually occurs during preimplantation embryonic development6. During this process, one of the two X-alleles in each female cell is independently, randomly, and permanently chosen for transcriptional silencing to match the single X-allele in male embryos9. The choice of silenced X-allele is inherited through cell divisions, propagating the random choice of allelic inactivation down each cell’s subsequent lineage. This produces whole-body mosaicism for allelic X-chromosome expression in each adult mammalian female, originating from very early embryonic development10.Every female mammalian embryo undergoes X-chromosome inactivation (XCI) as an essential step for successful development12. XCI ratios affect the phenotypes of X-linked diseases, as they can either protect or expose individuals to disease variants13. The factors that influence XCI variability are mostly studied in mice and humans, and include stochasticity12 and genetics16, but their relative roles are controversial17. Cross-species comparisons of XCI variability stand to reveal general or species-specific mechanisms of XCI. For instance, genetic determinants of XCI are well-established in lab mice20, but not in humans22, where they are harder to identify and measure. Exploring XCI variability in other mammals presents the opportunity to test models of stochasticity or genetics in the context of evolution.In humans, both X-alleles are equally likely to be inactivated, but XCI ratios vary widely among adult females, from balanced to highly skewed1, it was recognized that the inherent embryonic stochasticity and permanence of XCI is the simplest explanation for the observed variability in XCI among adults and positions this adult variability as a window into embryonic events27. For example, flipping 10 coins is more likely to result in 8 heads than flipping 100 coins is likely to result in 80 heads, meaning that the variability in heads-to-tails ratios depends on the number of coins flipped. Similarly, the variability of XCI ratios in a population of female mammalian embryos is determined by the number of cells at the time of XCI  allele33. In humans, genetic influence on XCI is mostly observed in small family studies or disease cases, with no strong evidence for the broad allelic effects seen in mice22. Another genetic influence on XCI is allelic selection, where natural or disease-causing variants favor certain X-alleles38. However, evidence for allelic selection through natural variation remains elusive in human populations. Thus, the relative contributions of stochasticity and genetics to population XCI variability in mammals remain unclear with currently limited data from mouse and human studies.In addition to stochasticity, genetic effects can influence the choice of allelic inactivation and contribute to population variability in XCI ratios. Allelic inactivation during XCI is mediated by the cis-acting long non-coding RNA XIST39 dataset. Our approach leverages natural genetic variation to sample X-linked heterozygosity and eliminates the requirement for costly phased or strain specific genetic information to assess XCI ratios across diverse mammals at population scale. We start by establishing the population-level XCI ratio distributions for all nine mammalian species and use models of embryonic stochasticity to predict the number of cells fated for embryonic lineages , resulting in a total of 19,180 initial samples , includilineages , Fig. 2.lineages , as welllineages , may impWe use bulk RNA-sequencing (RNA-seq) data to measure the X-linked allelic expression of a sampled tissue by computing allele-specific expression ratios of heterozygous single nucleotide polymorphisms (SNPs). The parental proportion of X-linked allelic reads are expected to follow a binomial distribution dependent on the number of sampled reads and the XCI ratio of the tissue see . The bin41  from the EN-TEx consortium43, achieving nearly perfect agreement in XCI ratio estimates for samples with folded XCI ratios of 0.60 or higher, demonstrating the accuracy of our approach.We fold the distribution of reference allelic-expression ratios around 0.50 to aggregate data across both alleles and enable a robust estimate of the XCI ratio magnitude for the bulk RNA-seq sample . We fit 41 . Interes41 , suggestBy calling SNPs from RNA-seq reads and employing folded distributions to model reference-aligned allelic expression, we can estimate the magnitude of XCI in any female mammalian bulk RNA-seq sample. We source female annotated bulk RNA-seq samples of 8 non-human mammalian species from the SRA database , additioTo ensure the allelic variability we report from X-linked SNPs is specific to XCI, we estimate autosomal allelic imbalances for all samples using the same pipeline and approach as for the X-chromosome analysis . CompariAfter generating population distributions of XCI ratios for the 9 mammalian species, we next explore how well models of embryonic stochasticity explain the observed adult XCI ratio variability. The initial variability in XCI ratios among mammalian embryos is dependent on the number of cells present during XCI , where aAn important consideration when estimating embryonic cell counts from XCI variability in adult tissues is the fact adult tissues only represent the embryonic lineage of the blastocyst as opposed to extra-embryonic lineages. This positions XCI variability of adult tissue samples as informative for the number of cells present within the last common lineage decision for all adult cells, i.e. the number of cells present within the epiblast of the mammalian blastocyst. If XCI occurs after epiblast specification, the variability in XCI ratios is determined by the number of epiblast cells at the time of XCI. On the other hand, if XCI occurs before epiblast specification, XCI variability within the embryonic lineage is influenced by both the initial stochasticity of XCI and the stochasticity associated with cell sampling during epiblast lineage specification. The temporal ordering of XCI among these lineage events cannot be resolved without cross-tissue sampling of both the extra-embryonic and embryonic tissues. As such, estimating cell counts solely on XCI variability in adult tissues provides an estimate of the number of cells present within the epiblast of the embryo.For the least and most variable species (macaca and dog), the estimated autosomal imbalances offer additional context for the reported XCI population variability. The reported X-linked variability in macaca is in excess to the reported autosomal allelic variability . This deModeling XCI ratio variability across numerous species allows comparisons in light of evolution for determining generalizable or species-specific characteristics in XCI. Broadly, we demonstrate XCI ratios are variable in each species we assess, revealing variability in XCI ratios itself as a conserved characteristic of XCI. The exact variance in XCI ratios varies across the species, with differences in the timing of XCI and/or embryonic/extra-embryonic lineage specification (differences in cell counts) as one putative explanation. We compare our estimated cell counts to the evolutionary relationships among the species we assess , suggestAfter determining stochastic models can explain population XCI ratio variability across mammalian species, we turn to testing whether we can identify any genetic correlates with XCI ratios. Our approach leveraging natural genetic variation to quantify XCI ratios enables us to assess a large catalog of genetic variants for associations with XCI ratios across mammalian species . One putative genetic contribution to XCI ratio variability is allelic selection during development, where increased X-linked heterozygosity , is more likely to produce selective pressures between the two X-alleles. It follows that samples with higher X-linked heterozygosity would be expected to exhibit more variability in XCI ratios.44. The trend in heterozygosity across species is as expected, with rats (likely laboratory strains) as the most inbred . For each species, we rank the samples based on their estimated XCI ratio and score the placement of samples carrying a given variant within the ordered list , IRAK148, and FLNA49. This suggests our analysis is able to recover putative examples of selection impacting XCI ratios via disease-variants, though with small effect sizes and low frequencies in our sampled population. In general, we are unable to identify strong associations between genetic variation and XCI ratios across all 9 mammalian species, both along the whole X-chromosome and for individual variants.The distribution of AUROCs for each species show striking similarities to a null comparison , indicatWe modeled tissue XCI ratios from bulk RNA-seq samples across 9 mammalian species and found population-level variation in XCI ratios, reflecting differences in developmental events such as XCI timing or lineage specification. We showed that embryonic stochasticity models fit the XCI data well and estimated epiblast cell counts at the time of XCI across species. We also searched for genetic factors influencing XCI ratios and found a pervasive lack of strong genetic associations with XCI ratios, indicating that XCI variability is mainly driven by stochasticity rather than genetic variation in mammals.33 has supported the hypothesis that a similar genetic mechanism can exist in humans and drive population XCI variability21, though evidence for XCE-alleles in human populations remains inconclusive22 and data from other mammalian species is historically absent. Although genetic influences on XCI, particularly variants affecting XIST15 or disease-associated variants37, have been identified, they do not constitute a general mechanism that can fully account for observed population-level XCI variability. Comprehensive assessment of genetic influence on XCI would require combined DNA and RNA sequencing data, which is challenging to perform at a large scale across mammalian populations. Our approach for extracting heterozygous variants from RNA-seq data28, while providing a sample of genetic variability, is still able to assess hundreds of X-linked genes per species for associations with XCI and culminated in only weak evidence for limited genetic influence on XCI ratios. In contrast, we demonstrated models of embryonic stochasticity can explain population XCI variability with exceedingly small amounts of error consistently across mammalian species, providing a much more general explanation for population XCI variability.The lack of cross-mammalian comparisons of population XCI variability has previously limited our understanding on the sources of XCI variability in mammals. The existence of XCE-alleles in laboratory mice45 and stochastic allelic drift during development20. We found a link between the variance in X-linked allelic expression and the inbreeding level of some species  accounts for the lack of evidence for broad allelic-selection or individual variants influencing XCI ratio variability in mammals, as both parental alleles are mostly equivalent. This does not apply to disease variants, but they cannot explain the widespread XCI ratio variability across mammalian species. We find genes associated with increased XCI ratios that have prior evidence for causing highly skewed XCI in disease cases, but their effect sizes and population frequencies are small in our samples. Therefore, the inherent stochasticity of XCI during embryogenesis is the main source of the observed XCI ratio variability in mammalian populations.A general model of X-linked genetic variability depletion , where only samples annotated as female were selected, using the metadata provided through SRA. Details for download and processing of the GTEx39 data can be found here28. The entire sample processing pipeline uses a standard collection of bioinformatics software tools, all available for installation via Conda . All Snakemake workflow rules, environment setup procedure, analysis commands and options, and underlying libraries are available on Github at https://github.com/gillislab/cross_mammal_xci, and https://github.com/gillislab/xskew. Briefly, a .fastq file acts as input, for either single- or pair-end sequencing experiments, and a .vcf and .wig file are produced as outputs for subsequent compiling of allele-specific read counts in R v4.3.0. The R script used for combining the .vcf and .wig information is also made available at https://github.com/gillislab/cross_mammal_xci/tree/main/R. Genome generation and alignment was performed with STAR, with the addition of the WASP58 algorithm for identifying and excluding reference biased reads. We extract chromosome-specific alignments from the .bam file (X chromosome or specific autosomes) and use GATK tools to identify heterozygous SNPs from that chromosome. The suite of GATK tools for identifying heterozygous variants from RNA-sequencing data was used following the GATK Best Practices recommendations. Specifically, the tools utilized include AddOrReplaceReadGroups -> MarkDuplicates -> SplitNCigarReads -> HaplotypeCaller -> SelectVariants -> VariantFiltration.All non-human mammalian fastq data was downloaded from the Sequencing Read Archive . In each case the latest assembly version path was used, and the genomic.fna and genomic.gtf was downloaded. Annotated and indexed genomes were generated with STAR using --runMode genomeGenerate with default parameters.Reference genomes and gene annotations (.gtf files) for each species were sourced from the NCBI Refseq database . Using the subset of skewed samples for each species, we averaged the folded allelic-expression ratios for all SNPs present in 1 mega-base (MB) bins across the X-chromosome . ChromosStarting with a single parental allele, the sampled maternal allelic-expression of a heterozygous X-linked SNP can be modeled with a binomial distribution, dependent on the ratio of active maternal X-alleles in the sample and the read depth of the SNP.60. Since SNPs vary in read-depth, we use a folded-normal model as an approximation of the underlying mixture of depth-dependent folded-binomial distributions. The probability of allelic-expression under the folded-normal model is defined as:Where The folded-normal model can also be applied to autosomal data to estimate allelic-imbalances. For each species, we extract chromosome-specific alignments from the .bam file for the two autosomes closest in size to the X-chromosome . We emplXCI is a binomial sampling event, where the number of cells choosing to inactivate the same X-allele follows a binomial distribution defined as:Embryonic XCI ratios can be modeled as: We estimate We model population XCI ratios as:We identify the normal distribution with minimum sum-squared error between its CDF and the empirical population XCI ratio CDF, minimizing error over the tails of the distributions with percentiles <= 0.40 or >= 0.60 . We compWe compute sample heterozygosity as the ratio of SNPs detected in a sample (20 read minimum) to the total number of unique SNPs identified across all samples for a given species. We quantify associations between X-linked heterozygosity and XCI ratios as the spearman correlation coefficient between the sample X-linked heterozygosity ratio and the fitted mean and variance of the maximum-likelihood folded-normal distribution of the sample . We onlyWe quantify the strength of XCI ratios as a predictor for the presence of a given variant through the AUROC metric. Given a ranked list of data (XCI ratios) and an indicator of true positives (samples with a given variant), the AUROC quantifies the probability a true positive is ranked above a true negative. An AUROC of 1 indicates all true positive samples were ranked above all true negative samples, demonstrating XCI ratios were a perfect predictor for the presence of that variant. An AUROC of 0.50 indicates random placement of true positives and negatives in the ranked list, demonstrating XCI ratios performed no better than random chance for predicting the presence of that variant. We compute the AUROC through the Mann-Whitney U-test, defined as:We estimate the power of each variant through bootstrap simulations. We randomly sample with replacement the XCI ratios of the true positive and true negative samples, those that either carry or do not carry a given variant. We match the sample size of the original true positive and negative labels. We compute a bootstrapped AUROC and p-value from the simulated data, repeating for 2000 simulations to compute a bootstrapped distribution of AUROCs. The AUROC power  is defin61 v4.3.0. All plots were generated using ggplot262 v3.4.2 functions. The phylogenetic tree in http://www.timetree.org/.All analysis was performed in RSupplement 1