Pulmonary rehabilitation is typically used for reducing respiratory symptoms and improving fitness and quality of life for patients with chronic lung disease. However, it is rarely prescribed and may be underused in pediatric conditions. Pulmonary rehabilitation can include inspiratory muscle training that improves the strength and endurance of the respiratory muscles. The purpose of this narrative review is to summarize the current literature related to inspiratory muscle rehabilitation training (IMRT) in healthy and diseased pediatric populations. This review highlights the different methods of IMRT and their effects on respiratory musculature in children. Available literature demonstrates that IMRT can improve respiratory muscle strength and endurance, perceived dyspnea and exertion, maximum voluntary ventilation, and exercise performance in the pediatric population. These mechanistic changes help explain improvements in symptomology and clinical outcomes with IMRT and highlight our evolving understanding of the role of IMRT in pediatric patients. There remains considerable heterogeneity in the literature related to the type of training utilized, training protocols, duration of the training, use of control versus placebo, and reported outcome measures. There is a need to test and refine different IMRT protocols, conduct larger randomized controlled trials, and include patient-centered clinical outcomes to help improve the evidence base and support the use of IMRT in patient care. Pulmonary rehabilitation (PR) is a class of treatments that aims to help patients suffering from chronic respiratory symptoms . PR usuaIn most cases, PR is prescribed by a physician and leverages the expertise and guidance of physical and respiratory therapists . PR has The lungs cannot fulfill their gas exchange role without a mechanical apparatus (or pump) that expands the lungs and draws air into the lower airways. This complex apparatus includes the thoracic cage , the lung pleura, and the respiratory muscles. The respiratory muscles are similar in nature to other skeletal muscles, and during quiet breathing, they work in unison to expand the thoracic cage and induce inhalation. Respiratory muscles have both voluntary and involuntary innervation and are thought to have greater resilience to fatigue than other skeletal muscles. The muscular diaphragm is the main driver of inhalation. During quiet involuntary breathing, the diaphragm mostly acts alone. As the diaphragm contracts, it flattens and descends into the abdominal cavity, leading to drops in intrapleural and alveolar pressures. Assuming airway patency, air moves from the atmosphere to the lower, subatmospheric pressures in the alveoli.Exhalation, on the other hand, is a more passive process and results from the relaxation of the respiratory muscles. The diaphragm relaxes and ascends, allowing recoil of the lungs and thoracic cage to the resting volume known as functional residual capacity (FRC). During hyperventilation or volitional exhalation, the rectus abdominal muscles  promote thoracic recoil and forced exhalation. During volitional breathing or hyperventilation induced by exercise, exhalation is facilitated mainly by the abdominal muscles  and secondarily by the internal intercostals. Contraction of these muscles together actively augment the ascent of the diaphragm, raising the pleural and alveolar pressures and leading to rapid exhalation.th and 12th ribs; it separates the thoracic from the abdominal cavity. After the diaphragm is fully developed, the descent of the diaphragm during contraction stabilizes and elevates the lowest ribs via its anterior connections. This is known as the bucket handle effect. It is thought that children have less efficient inspiratory motion due to incomplete diaphragmatic development [Key developmental issues are involved in inspiration during health and disease. All respiratory muscles originally derive from embryonic  mesoderm. The embryonic and early-life development of the diaphragm is critical to normal respiration and can exhibit developmental defects that impair breathing efficiency. The muscular diaphragm develops as a thin sheet that is both a central tendon and emanating muscle connecting anteriorly to the costal cartilages of ribs 7–10 and xiphoid and posteriorly to the lumbar vertebral bodies, transverse processes, and the 11elopment , which cSeveral factors can predispose respiratory muscles to fatigue in health and disease. Any factor that increases the energy demands of respiration impairs the capacity of the respiratory muscles to handle an increased ventilatory workload and quickly leads to respiratory muscle fatigue and dyspnea. Two common examples include restrictive lung disease  and airway collapse and hyperinflation , both of which limit the respiratory muscles from handling increased ventilatory workload. Secondly, reduced energy reserves, malnutrition, or homeostatic problems, such as circulating acidosis or reactive oxygen species, can impair respiratory muscle function leading to fatigue. Lastly, deconditioning or atrophy , anemia, low cardiac output states, and hypoxemia all predispose to diaphragmatic dysfunction and fatigue. More research is needed to understand how best to measure and follow changes in respiratory muscle functioning. The strength of the respiratory muscles can be quantified in the clinical setting easily by the maximum inspiratory pressure (MIP) at the mouth by manometry against a closed mouthpiece . These tTraining approaches for inspiratory muscles can broadly be separated into strength-or endurance-training approaches. Strength-training approaches generally involve higher external loads (intensity) with lower training volumes, while endurance-training approaches typically use lower external loads and higher training volumes . AlthougVIH training typically requires vigorous hyperventilation for up to 30 minutes to target respiratory muscle endurance. To prevent hypocapnia, incorporation of a dead space rebreathing circuit or supplemental oxygen to avoid hypoxemia and maintain isocapnia is necessary. Training sessions generally occur at a frequency of 3–5 times per week at an intensity of 60–90% of maximal voluntary ventilation (MVV). A limitation of VIH is that high motivation is required as patients may find training to be strenuous and time-consuming –21. OverFlow-resistive loading provides the respiratory muscles an external load during ventilation through a variable diameter orifice. Traditionally, this is accomplished with the use of analog devices with adjustable dials, which allow the orifice surface area to be manipulated to change the flow and resulting load. For a given flow rate, the load increases as the orifice surface area decreases, although load also varies with flow rate. Therefore, in order to provide a quantifiable load, respiratory flow or pressure must be measured, which has led to the development of devices that provide electronic flow-resistive loading. For example, the K-series devices from POWERbreathe® provide an external load via an electronically controlled orifice that varies to achieve a target load or flow rate. Similarly, the PrO2™ device  providesPressure-threshold loading provides an external load to the respiratory muscles via ventilation through an orifice that remains closed with a spring-loaded valve or other methods until a target pressure is achieved  improvements were noted in most [Although the performance was not improved after IMRT in the national-level competitive swimmers, IMRT improved 50-meter and 200-meter swim performance in short- and middle-distance swimmers training 20 hours/week , 100-met in most , 29, 30  in most , 28 studWe performed a review of the literature on IMRT in children with neuromuscular diseases. Several studies were excluded due to insufficient details regarding the specifics of their training protocol or outcomes –38. Of t1 were unchanged.Improvements in inspiratory muscle endurance were noted in all three of the studies which included endurance measures including improved MVV , the durThe population of interest in the three remaining studies were children with infantile-onset Pompe disease (IOPD) including a before-and-after trial in 9 subjects  and 2 caOverall, respiratory training appeared to improve respiratory muscle endurance and is likely to offer benefits in children with neuromuscular diseases considering the effects of respiratory muscle fatigue under stress on the morbidity and mortality in this population. Additionally, training appeared to be well-tolerated with no reported adverse events ,43,45,46β2 agonists (>1 puff/day) for breakthrough asthma symptoms. The increased inspiratory muscle strength has seen following IMRT correlated closely with a decrease in dyspnea with exercise (measured by Borg scoring) and a decrease in β2-agonist consumption [n = 81) demonstrated reduced dyspnea, respiratory symptoms, and activity impairment with IMRT [β2-agonist bronchodilator use. There were no studies describing exacerbation events that required the use of reliever medication, emergency department visits, or hospital admissions. This 2013 Cochrane review strongly recommended that studies on children be conducted in the future.A Cochrane review involving 115 adult patients was published in 2013 summarizing the results of 5 studies employing IMRT in patients with asthma . The IMRsumption . Since tith IMRT , 51. TheTo date, only two IMRT studies have been published in pediatric asthma , 53, whin = 17) or placebo IMRT at 5% of MIP . Both interventional groups received a conventional respiratory rehabilitation program. The researchers reported significant post-treatment differences between the IMRT and placebo groups in FEV1 (P = 0.003), FVC (P = 0.001), FEV1/FVC (P = 0.004), MIP (P = 0.002), and MEP (P = 0.004), adjusted to the pretreatment values, in favor of the IMRT group. In addition, IMRT was associated with significant improvement in asthma symptom scores measured by the validated Asthma Control Test (ACT) (P = 0.001). Elnaggar concluded that the incorporation of IMRT in children with asthma can improve respiratory function, enhance respiratory muscle strength, and improve children's perception of asthma symptoms.More recently in 2020, Elnaggar investigated the efficacy of IMRT on lung function, respiratory muscle strength, and asthma symptoms in asthmatic children . ElnaggaStudies that have examined the effects of IMRT in overweight and obese adults have reported improved exercise performance –57, MIP IMRT appears to represent a viable non-pharmacologic option for patients in need of improving their exercise tolerance and reducing dyspnea. The exact mechanisms by which IMRT reduces asthma symptoms remain poorly defined. For children with asthma, we speculate that IMRT improves disease control through improved lung function, reduced airway closure, and reduced respiratory muscle fatigue leading to reduced frequency of dyspnea, which previously was perceived as asthma symptoms. While there have not been any published reports on the effectiveness of IMRT in children with obesity and asthma, a special population that experiences a high symptom burden , favorabRelatively few children have been formally studied with high-quality IMRT utilizing randomization and blinding. Among studies included in this review, approximately half included both inspiratory and expiratory muscle training. It may be reasonable to hypothesize that expiratory muscle training would improve expiratory muscle strength . Although this hypothesis was supported by two randomized trials , 28 and Dyspnea and exercise intolerance are common symptoms noted in pediatric patients with neuromuscular diseases, asthma, and obesity. Only a handful of studies have investigated the effects of IMRT on clinical outcomes and underlying physiological mechanisms in these pediatric patients. These studies demonstrate that IMRT is likely effective in improving respiratory muscle strength and reducing airway closure  and potentially in improving breathing mechanics and reducing dyspnea during exercise. These mechanistic changes can help explain improvements in symptomology and clinical outcomes, and they highlight our evolving understanding of the role of IMRT in pediatric patients.


Ecological studies showed correlations between a shift toward animal-protein-rich diets and longer life-expectancy; however, only a few studies examined individual-level association of protein source and mortality risks using appropriate iso-caloric substitution models adjusted for total energy intake. We used EPIC-Heidelberg (European Prospective Investigation into Cancer and nutrition) to create iso-caloric substitution models and determined relative all-cause, cardiovascular and cancer mortality hazards associated with dietary intake of animal protein and other macronutrients, employing Cox proportional hazard models. For comparison with other studies, we also synthesized evidence from a systematic review relating animal protein intake to mortality risk from seven prospective cohort studies in the USA, Europe and Japan. Substitution of 3% of total energy from animal protein for fat  and carbohydrate  was associated with all-cause mortality (Hazard Ratios [HR] from 1.05 to 1.11), mostly driven by cardiovascular mortality (HR from 1.13 to 1.15). Independently of animal protein, substituting poly-unsaturated fat for saturated fat increased cancer-related mortality risk by 12 percent. The systematic review largely corroborated our findings. Overall, higher proportions of dietary energy from animal protein, combined with low energy intake from either carbohydrate sub-types or dietary fats, increases all-cause and cardiovascular mortality risks, but not cancer-related mortality. A long-standing question in nutrition epidemiology concerns the optimal dietary macronutrient composition in relation to an individual’s overall health. On a population level, national statistics of food availability have documented important changes in average per capita availability and consumption of food types along with the economic development of countries . OverallBetween-country comparisons have shown strong positive correlations between the average per capita availability of animal protein, sugar and total and saturated fats and incidence rates of cancer, diabetes and cardiovascular incidents . By contTo assess associations on an individual level, several recent studies have addressed the relationship between macronutrient composition of diet and all-cause and cause-specific mortality rates in prospective cohorts in the USA, Australia, Japan and several European countries, focusing analyses on energy intake in the form of animal protein as compared with energy from plant protein or other macronutrient sources ,7,8,9,10Here, we report findings from a prospective EPIC-Heidelberg cohort, using statistical models to comprehensively examine relative mortality risks  associated with different possible iso-caloric substitutions of animal protein for other possible macronutrient sources, as well as for further possible nutrient substitutions independently of animal protein. In addition, we performed a systematic review of previous iso-caloric modeling studies on the effect of animal protein substitutions, so as to compare their findings with those from our own analyses, and to generate an overall synthesis of evidence relating animal protein intake to mortality risk from prospective studies so far.EPIC-Heidelberg recruited participants and collected data between 1994 and 1998 as part of the larger European EPIC study. The study design and methods for the European EPIC study, including the Heidelberg component, have been described previously ,13. BrieInformation about habitual diet was collected using a self-administered food frequency questionnaire (FFQ), which had been extensively validated in prior studies ,16,17. BTotal energy intake was defined as the daily energy intake from total protein, total fatty acids and total carbohydrates. Since alcohol is not considered feasible with regard to nutrient substitution, alcohol was not included in total energy intake but was adjusted in the models as a covariate ,19. The In EPIC-Heidelberg, mortality outcomes were ascertained first through regular record linkages with municipal registries for vital status, and for all cases of death further information on causes of death (death certificates) was collected from regional health offices, which was coded according to the International Statistical Classification of Diseases and Related Health Problems (ICD)-10 by trained medical study personnel. The present analyses are based on complete case ascertainments from June 1994 to May 2019. n = 949) at recruitment and those in extreme 1 percentile of ‘energy intake/energy requirement’ ratio (n = 485), 24,106 remained for the analysis. Those with unknown information about smoking history (n = 341) were imputed using a fully conditional specification multiple imputation method [p-value are provided in 2), intake of alcohol  and dietary fiber (g/day). Proportional hazard assumption in the Cox model was tested using Schoenfeld residuals [p < 0.05 or 95% confidence intervals excluding the null, and all analyses were performed using SAS Version 9.4 . After the exclusion of participants with prevalent cancer diagnosis (n method . Relativn = 829) from the analytic sample. Many past studies have also suggested that the mortality association with animal protein may be largely driven by red meat consumption [To examine the robustness of ND models, we additionally performed an equivalent series of analyses using “standard” linear models (“S” models) , in whicsumption ,25. To eThe systematic review was conducted and reported based on pre-defined PRISMA guidelines  and was Among the 24,106 participants retained for the present analyses, a total of 4029 cases of death were registered until the end of the follow-up (May 2019), of whom 982 (24.3%) died of cardiovascular events, 1603 (39.7%) of cancer and the remaining 1444 (35.8%) of other, miscellaneous conditions . The medThe model with maximum macro-nutrient breakdown  was chosen for the final analysis as it had a significantly better model fit compared with models in which macronutrients were considered on a more aggregate level . Using tA basic nutrient density model (ND) minimally adjusted only for age, sex and total energy intake as confounding variables , Model AExcluding participants with diabetes diagnoses at the baseline and adjusting for red meat intake in the multivariate adjusted models did not significantly change the various associations observed between nutrient substitutions and mortality risk . In addiOverall, 563 studies were identified in the initial search. As there were no duplicates, all 563 studies were included for title screening. Forty studies were included for abstract screening and seven studies were included for a full text review. All seven studies were included in the final review based on inclusion criteria. Most studies adapted a similar breakdown of macronutrients into components of proteins  and fats , except for further breakdown into alcohol energy by Chen et al. . Only thIn this analysis of the prospective EPIC-Heidelberg cohort, we found that animal protein stood out as an important predictor of all-cause mortality, in large part driven by the association of higher animal protein intake with increased risk of cardiovascular mortality. These increased risks were observed in relation to iso-caloric substitutions of animal protein either for saturated fats or for simple or more complex carbohydrates. Independently of animal protein, our analyses also indicated an increase especially in cancer-related mortality risk in association with higher intakes of poly-unsaturated fats, notably as iso-caloric substitution for saturated fats. Our findings of increased cardiovascular and overall mortality risks in association with higher dietary intakes of animal protein are, generally, largely in line with those from previous iso-caloric modeling studies in other prospective cohorts, although all studies varied with regard to the degree of macronutrient breakdown in statistical risk models, specific macronutrient substitutions considered  and additional covariates considered as confounding factors ,7,8,9,10The reviewed studies generally reported mortality hazards ratios only for selected macronutrient substitutions by animal or plant protein, but did not provide an overall picture of all possible pairwise substitutions between sub-types of protein, fat and carbohydrates. In our analysis, breaking down each macronutrient  into their sub-types improved model fit, suggesting that improvement in model fit may not be entirely related to animal protein intake, but significant substitution of other macronutrients could have contributed to model fit. Significant substitution effects correspond to significant differences between weighted sums of positive and negative coefficients of nutrients considered, where this weighted difference is significantly different from zero. Therefore, not considering all pairwise macronutrient substitution could potentially limit the interpretation of strength and direction of associations as important, details might be lost when the macronutrients are aggregated versus broken down. Importantly, because we tested all possible pairwise substitutions, we found that—in addition to the association of animal protein with overall and cardiovascular mortality—poly-unsaturated fat substituted for saturated fat was associated with cancer mortality. The findings from our and previous iso-caloric modeling studies in prospective study cohorts stand in contrast with those from international correlation studies that have shown higher cancer incidence rates, but lower rates of overall mortality (with longer life expectancy), in countries that have higher per capita availability and percent energy intake from animal protein ,29,30. TAll things considered, the robust associations seen between higher animal protein intake and overall and cardiovascular mortality in our study and those of others suggests that factors uniquely associated with animal protein could perhaps explain these associations. In past studies it was speculated that branched-chain amino acids, largely present in animal protein sources, could lead to insulin resistance and excess weight, which are important predictors of chronic disease incidence, particularly cardiovascular disease, and can ultimately increase mortality risk ,34,35. AA first limitation of this study is that our primary exposure, nutrient intake, was assessed at only one point in time, at the baseline recruitment. Therefore, there is a chance for the participants to have changed their dietary habits or lifestyle, which could result in attenuated associations. Second, nutrient intake information derived from self-administered FFQ inherently has considerable random measurement error, also leading to attenuation of rich associations. Third, the EPIC-Heidelberg cohort had a comparatively better socioeconomic indicator than the underlying population and, as a result, the nutrient intake measures may not entirely represent the underlying population. In terms of strength, this was a large prospective cohort study with detailed information collected about lifestyle habits such as smoking, alcohol consumption and BMI, which are strong confounders for the association of nutrient intake and mortality. Thus, we were able to adjust for all important known confounders of the association. That said, the possibility of residual confounding by unmeasured factors cannot be entirely eliminated.Overall, the findings from our and other studies indicate that higher proportions of dietary energy from animal protein, combined with low energy intake from either carbohydrates or dietary fats, increases mortality risk. Crucially, animal protein was not the only macronutrient associated with increased mortality risk. Therefore, future studies should further attempt to establish a causal association—by addressing residual confounding issues in prospective cohort studies and by establishing clear biological mechanisms—between animal protein intake and overall and cause-specific mortality.


In recent years, there has been an increasing recognition of coronary computed tomographic angiography (CCTA) and gated non-contrast cardiac CT in the workup of coronary artery disease in patients with low and intermediate pretest probability, through the readjustment guidelines by medical societies. However, in routine clinical practice, these CT data sets are usually evaluated dominantly regarding relevant coronary artery stenosis and calcification. The implementation of radiomics analysis, which provides visually elusive quantitative information from digital images, has the potential to open a new era for cardiac CT that goes far beyond mere stenosis or calcification grade estimation. This review offers an overview of the results obtained from radiomics analyses in cardiac CT, including the evaluation of coronary plaques, pericoronary adipose tissue, and the myocardium itself. It also highlights the advantages and disadvantages of use in routine clinical practice. Due to the improvement in computed tomography technology, the importance of additional diagnostic tools in the field of cardiovascular imaging has been recognized by medical societies, leading to a readjustment of guidelines and recommendations for cardiac CT angiography (CCTA) . In the Nevertheless, the main focus in daily routine lies in the determination of coronary artery stenosis. However, the introduction of radiomics to the medical society, as an opportunity to analyze quantitative data by extracting numerous features from images—invisible to the human eye, offers new potential for cardiac CT imaging . These nIn the last years, radiomics analysis showed incremental value in the cardiovascular imaging field ,12. ThisRadiomics analysis uses methods from the field of artificial intelligence to quantify textural information by extracting the spatial distribution of Hounsfield or signal intensities and relationships. In contrast to previously known computer-aided diagnosis and detection systems (CAD), radiomics analysis is not a standalone system to deliver a single answer but a process for extracting numerous quantitative data from digital images and mining the data for hypothesis generating . It starRadiomics features can be divided into different subgroups. First-order features describe the distribution of voxel intensities independently of their spatial relationship. Texture features on the other hand take the spatial relationship into account and characterize the heterogeneity of the underlying lesion. Mainly five different types of texture features are used in the routine, defined by gray-level cooccurrence matrix (GLCM), gray-level run-length matrix (GLRLM), gray-level size zone matrix (GLSZM), gray-level dependence matrix (GLDM), and neighboring gray-tone difference matrix (NGTDM). Additionally, shaped-based features further quantify the spatial complexity, and finally, transform-based features convert spatial information into specific new information or filter specific information ,18,19,20p < 0.0012). The five most differentiating parameters were short-run low-gray-level emphasis, long-run low-gray-level emphasis, surface ratio of component 2 to total surface, long-run emphasis, and surface ratio of component 7 to total surface . Hence, radiomics analysis of coronary artery plaque outperformed conventional parameters in identifying NRS plaques in cardiac CT NAF-PET positivity with the presence of two out of four high-risk features in CCTA and surface of high attenuation voxels in radiomics analysis . Summarizing these results, radiomics analysis outperformed conventional parameters for the identification of plaque vulnerability significantly ..42].p-value < 0.0001). As the authors defined LV septum width as a surrogate of myocardial remodeling, they considered the EML score as a potential tool for evaluating myocardial remodeling [In addition, Cavallo et al. outlined a successful CCTA-based radiomics approach to identify left ventricular remodeling in patients with arterial hypertension. They included 83 patients with arterial hypertension and 75 control patients who underwent cardiac CT and segmented the left ventricular myocardium. In total, 377 radiomics features were extracted. The dataset was divided into two parts using a 7:3 ratio for training the classification model and afterward for testing and evaluating the model performance. Models with an accuracy higher of 60% were selected and finally, an Ensemble Machine Learning (EML) score was calculated. The EML score correlated to LV septum width . Comparing these findings with conventionally measured HU values, radiomics analysis (namely wavelet_LHL and wavelet_LLH) could differentiate better between thrombi and circulatory stasis in patients with left atrial appendage filling defects [First attempts have also been made in mass characterization on cardiac CT images. In 2019, Nam et al. investigated 39 periprosthetic masses in 34 patients suspected of periprosthetic valve obstruction clinically. All patients underwent cardiac computed tomography and were clinically suspected of prosthetic valve obstruction (PVO). The final cause of PVO was then identified using redo-surgery and follow-up imaging as a standard reference. In 20 cases pannus could be finally diagnosed, in 11 cases thrombi, and in eight cases vegetations. Visual analysis of differentiating pannus from other abnormalities was compared to a radiomics score. Radiomics analysis was able to differentiate between pannus and other tissue (AUC = 0.876), leading to the combination of visual and radiomics assessment to outperform the visual assessment alone . In line defects .However, cardiac imaging goes beyond purely investigating the heart itself. As already mentioned above, perivascular adipose tissue, surrounding blood vessels immediately, is known to be metabolically active . VariousSummarizing literature, Even though radiomics analysis shows promising results in cardiac CT imaging, the implementation of radiomics analysis in clinical routine has not been fulfilled yet. Radiomics analysis is susceptible to many technical factors and hence lacks reproducibility. Different studies describe the influence of different manufacturers as well as various image acquisition parameter settings on feature stability . Not onlDespite the challenges of radiomics analysis, the radiomics of cardiac CT will find its way into the clinical routine in one way or another. The strength of feature analysis in cardiac CT is to go beyond the pure visual assessment of image data and to add quantitative information. This quantitative information will be needed for future clinical decision-making as well as for exploring biomarkers in prevention and personalized cardiovascular medicine. Even though this is an ambitious goal, the foundation is already laid, and future work must and will overcome the limitations to implement radiomics of cardiac CT in clinical routine.


In survivors of moderate to severe traumatic brain injury (msTBI), affective disruptions often remain underdetected and undertreated, in part due to poor understanding of the underlying neural mechanisms. We hypothesized that limbic circuits are integral to affective dysregulation in msTBI. To test this, we studied 19 adolescents with msTBI 17 months post-injury  as well as 44 matched healthy controls . We leveraged two previously identified, large-scale resting-state (rsfMRI) networks of the amygdala to determine whether connectivity strength correlated with affective problems in the adolescents with msTBI. We found that distinct amygdala networks differentially predicted externalizing and internalizing behavioral problems in patients with msTBI. Specifically, patients with the highest medial amygdala connectivity were rated by parents as having greater externalizing behavioral problems measured on the BRIEF and CBCL, but not cognitive problems. The most correlated voxels in that network localize to the rostral anterior cingulate (rACC) and posterior cingulate (PCC) cortices, predicting 48% of the variance in externalizing problems. Alternatively, patients with the highest ventrolateral amygdala connectivity were rated by parents as having greater internalizing behavioral problems measured on the CBCL, but not cognitive problems. The most correlated voxels in that network localize to the ventromedial prefrontal cortex (vmPFC), predicting 57% of the variance in internalizing problems. Both findings were independent of potential confounds including ratings of TBI severity, time since injury, lesion burden based on acute imaging, demographic variables, and other non-amygdalar rsfMRI metrics , as well as macro- and microstructural measures of limbic circuitry . Supporting the clinical significance of these findings, patients with msTBI had significantly greater externalizing problem ratings than healthy control participants and all the brain-behavior findings were specific to the msTBI group in that no similar correlations were found in the healthy control participants. Taken together, frontoamygdala pathways may underlie chronic dysregulation of behavior and mood in patients with msTBI. Future work will focus on neuromodulation techniques to directly affect frontoamygdala pathways with the aim to mitigate such dysregulation problems. Traumatic brain injury (TBI) continues to be a leading cause of death and disability in youth , 2. EstiNormal affective function relies on limbic pathways that may be particularly vulnerable to the contusional and torsional forces of brain trauma, including less granular portions of the prefrontal and temporal cortices as well as their subcortical targets in the medial temporal lobe, striatum, hypothalamus, and brainstem –21. TBI-The present study leveraged a systems neuroscience framework for limbic circuitry centered on the amygdala . We prevAlthough the resting-state correlates of affective and behavioral dysregulation are understudied in adolescent TBI, prior work in other populations provide some direction for hypotheses. Specifically, studies on typical development  and condWe studied patients after a non-penetrating msTBI who were recruited from four different pediatric intensive care units in Los Angeles County. Inclusion criteria included a Glasgow Coma Scale (GCS) score between 3 and 12 , aged between 8 and 18, normal or corrected-to-normal visual acuity, and proficient English competence to understand instructions for the neuropsychological inventories. Exclusion criteria included prior TBI, neurologic deficits that would preclude them from completing any of the planned testing, or prior neurologic, developmental, or serious psychiatric diagnoses. For comparison, 46 age- and sex-matched healthy controls (HC) were recruited through flyers, magazines, and school postings. Controls met the same inclusion and exclusion criteria as the TBI group other than those pertaining to the brain injury under investigation. All patients who underwent MRI were also required to be eligible for scanning . The institutional review boards of University of California, Los Angeles (UCLA) and the other sites of recruitment approved this study.The study protocol has been previously described in detail . In the 2).All patients underwent MRI scans on a 3T Siemens Trio with a 12-channel Head Matrix Coil (Siemens AG). Patients were instructed to remain still throughout the scan and foam pads were placed around their heads to further minimize head motion. For rsfMRI, T2* weighted BOLD fMRI was acquired while Patients rested with their eyes open for 7 min and 55 s, utilizing an echo-planar imaging (EPI) pulse sequence . For anatomical reference, T1-weighted 3D MPRAGE images were acquired . For microstructural metrics, diffusion weighted imaging (DWI) was acquired  in CONN using intermediate settings (97th percentile in normative sample). After scrubbing removed volumes with framewise displacement (FD) > 0.9 mm, we removed 2 subjects from the HC group from further analyses because their scrubbed time series had an overall mean FD > 0.9 mm. All rsfMRI and T1 images were visualized for lesions that would overlap with the networks of interest; no lesions or encephalomalacia were observed in close vicinity to the amygdala network masks used. Scrubbed time series were then passed into CONN's denoising steps, including band-pass filtering (0.008–0.09 Hz) and regression of noise components generated from individually segmented white matter and cerebrospinal fluid masks (5 components for each via CompCor) as well as motion parameters and their first-order derivatives (generated from ART). From the denoised times series for each subject, we computed rFC in the a priori defined amygdala networks  and skull stripped using BET. Fractional anisotropy (FA) and mean diffusivity (MD) maps were computed using dtifit. Whole-brain HARDI tractography was performed with Camino (http://cmic.cs.ucl.ac.uk/camino/). The maximum fiber turning angle was set to 35°/voxel to limit biologically implausible results, and tracing stopped when FA dropped <0.2, as is the standard in the field. The Eve atlas was registered, linearly and then non-linearly, to each patient's FA map using Advanced Normalization Tools (ANTs) and its ROIs were correspondingly warped to extract 19 tracts of interest, of which we focused on bilateral uncinate fasciculus given it connects the frontal lobe to medial temporal lobe. Five white matter tract atlases were created from healthy controls, and each patient's FA map was linearly and nonlinearly registered to each of these in order to refine fiber extractions, the results of these five tract identifications were fused for each patient for a final, cleaned fiber clustering. Registrations at each step were visually checked for quality. MD was averaged within ROIs to generate summary measures for the medial and ventrolateral amygdala subregions, using the cluster masks previously published (DTI data were processed as described previously –53. Brieublished .http://surfer.nmr.mgh.harvard.edu) to perform a quantitative morphometric analysis of the T1 MRI scans (We used an automated segmentation and probabilistic region-of-interest (ROI) labeling technique   is a parWe were most interested in investigating associations between resting-state connectivity and ratings of mood and behavior regulation. As such, our primary behavioral outcome measures were Emotional Control and Externalizing subscales of the BRIEF and CBCL, respectively. We also assessed the Working Memory subscale of the BRIEF to test whether amygdala network connectivity was specific to mood and behavior regulation rather than more general to frontal regulation phenomena.t-tests using SPSS, accepting alpha of 5% as significant.All variables of interest were assessed for normality and outliers, removing values greater than 3 standard deviations from the mean. We first compared TBI and HC groups on potential confounding variables using independent samples For whole network analysis, we performed a series of independent first order Pearson correlational analyses in the TBI group to discern whether connectivity for the medial and ventrolateral amygdala networks predicted externalizing and internalizing problems, respectively and specifically, as hypothesized. For the externalizing behavior domain, we used two distinct measures to test for convergent validity, the Emotional Control subscale on the BRIEF and the Externalizing subscale on the CBCL. For the internalizing behavior domain, we did not have a similar measure to test for convergent validity. For both externalizing and internalizing domains, we also examined the discriminant validity of the brain-behavior findings by testing whether amygdala network connectivity correlated with a less affective domain, the Working Memory subscale of the BRIEF. We also tested the specificity of the amygdala network-behavior associations by assessing whether macro- and microstructural metrics within limbic regions and circuits, including uncinate fasciculus FA, medial amygdala MD, and amygdala volume, accounted for any of the predicted variance in the behavior domain of interest. Finally, we tested whether potential confounds accounted for any of the predicted variance, including age, sex, handedness, time since injury, and severity of injury , presence of gross injuries on hospital CT .Given that the resultant clusters from the voxelwise analysis are part of the canonical default mode network, we investigated whether their connectivity to one another – a good proxy for default mode connectivity – correlated with these behavioral ratings. Again, supporting the specificity of the main finding, we found no significant correlation between rACC to PCC connectivity and the Emotional Control or Externalizing Behavior scales , explaining 57% of the variance in internalizing problems .T = 2.26, p = 0.028; Externalizing: T = 2.34, p = 0.02), although not Internalizing . The reported brain-behavior associations were specific to the TBI population in that healthy controls did not show a correlation in amygdala connectivity with emotional control, externalizing or internalizing problems . In addition, medial and ventrolateral amygdala connectivity with the peak voxels from the voxelwise analyses did not differ across groups .Supporting the clinical significance of these brain-behavior correlations, all behavioral ratings were higher (less well-controlled) in the TBI group as compared to the HC group (Emotional Control: This study is unique in that it combined functional connectivity from rsfMRI with measures of macro- and microstructure to investigate the mechanisms of mood and behavioral dysregulation after msTBI in adolescents. Findings from this study suggest that specific functional circuits within the amygdala's broad connectome, particularly its frontoamygdala circuitry, explain significant variance in affective and behavioral problems that are hallmarks of chronic TBI. We showed that the functional connectivity predictors of externalizing and internalizing problems were differentiable at the amygdala subregional level and could not be better explained by injury severity, gross lesion burden, time since injury, patient demographics, or structural measures of amygdala circuit integrity, diffusivity, and volume.For adolescents in the chronic phase after msTBI, medial amygdala network rFC predicted problems in emotional control and externalizing behavior that were not accounted for by measures of structural aberrancy or injury severity. Medial amygdala connectivity to voxels in the rACC and PCC drove this relationship, predicting upwards of 48% of the variance in externalizing problems . This liThe regions in the medial amygdala network play a critical role in integrating memories and goals with salient information from the environment, particularly the salient actions of others, to regulate affective, approach, and affiliative behaviors . In prioConsistent with this role and the findings from the current study, prior work in typically developing adolescents found that elevated rFC between the amygdala and rACC predicted greater externalizing behaviors  ConnectiFor adolescents in the chronic phase after msTBI, ventrolateral amygdala network rFC (yellow network in Pre-clinical work demonstrates parallel and more causative insights into the mechanisms linking connectional differences after injury with internalizing problems. Specifically, prior studies have shown cellular structural differences with increased dendritic arborization in the basolateral amygdala  as well Our study had many limitations in common with other studies in TBI but also unique to this specific cohort. In general, studies of patients with TBI suffer from a heterogeneity in injury characteristics, including different severities, mechanisms, intracranial lesions, neuropsychological history, and more. We attempted to control for many of these and other obvious demographic confounds as well as measures of macro- and microstructure that could account for the observed findings in rsfMRI. None explained our main findings. While the sample size is relatively comparable to other similar studies of this population in the literature, it is quite small, and does not allow subgroup analyses by sex, age, severity, mechanism, or other potentially confounding variables. In future work, larger samples will enable investigation of more subtle effects of potential confounds, particularly sex given its role in amygdala development and function. These findings will clearly need replication as recent work demonstrates poor replicability in brain-wide association studies involving less than thousands of subjects . FurtherWhereas markers of injury severity, including GCS and pathological findings on CT, did not explain differences in affective and behavioral outcomes in our adolescents with msTBI, aberrancies in the functional connectome of limbic circuitry did account for problems in externalizing and internalizing behavior. The results were specific to circuits situated in the limbic components of the medial frontal and temporal lobes rather than other limbic or more neocortical circuits. This could reflect a specific process of recovery, compensation, or premorbid vulnerability in the TBI group that reflects recruitment of ongoing reparative processes, additional effort needed, or chronic alteration, damage, among other possibilities. Current treatments do not target the underlying etiology of affective disability in pediatric TBI. Rather, the majority target the specific symptoms of traumatic brain injury, such as antidepressants for mood problems, cognitive-behavioral therapy for anxiety, or family therapy for academic social adjustment . This ci


The influence of metabolism on signaling, epigenetic markers, and transcription is highly complex yet important for understanding cancer physiology. Despite the development of high-resolution multi-omics technologies, it is difficult to infer metabolic activity from these indirect measurements. Fortunately, genome-scale metabolic models and constraint-based modeling provide a systems biology framework to investigate the metabolic states and define the genotype-phenotype associations by integrations of multi-omics data. Constraint-Based Reconstruction and Analysis (COBRA) methods are used to build and simulate metabolic networks using mathematical representations of biochemical reactions, gene-protein reaction associations, and physiological and biochemical constraints. These methods have led to advancements in metabolic reconstruction, network analysis, perturbation studies as well as prediction of metabolic state. Most computational tools for performing these analyses are written for MATLAB, a proprietary software. In order to increase accessibility and handle more complex datasets and models, community efforts have started to develop similar open-source tools in Python. To date there is a comprehensive set of tools in Python to perform various flux analyses and visualizations; however, there are still missing algorithms in some key areas. This review summarizes the availability of Python software for several components of COBRA methods and their applications in cancer metabolism. These tools are evolving rapidly and should offer a readily accessible, versatile way to model the intricacies of cancer metabolism for identifying cancer-specific metabolic features that constitute potential drug targets. Cancer involves a complex set of dysregulations in multiple biomolecular layers including metabolism. Metabolic changes in cancer result from and lead to profound changes in the behavior of cancer cells and their surrounding environment. Although extensively studied, these metabolic changes are difficult to accurately measure and model in an unbiased manner due to the need to consider a heterogeneous tumor environment encompassing different cell types, many difficult-to-measure metabolites, and lack of standardization of models . While rGenome-scale metabolic models (GEMs) can provide a compelling approach towards understanding cellular metabolism. GEMs are curated computational descriptions of entire cellular metabolic networks. Derived from genome annotations and experimental data, GEMs are composed of mass-balanced metabolic reactions and gene-protein associations that map the relationship of genes to proteins involved in each reaction Figure 1Compared to omics analysis, cancer metabolism may be more accurately modeled by combination of GEMs and a family of methods called Constraint-Based Reconstruction and Analysis (COBRA). COBRA methods perform systems-level analyses on metabolic networks to uncover how genetic and environmental factors affect phenotype on a biomolecular basis. COBRA framework utilizes a stoichiometric matrix that transcribes mass-balanced metabolic reactions of a cellular system, including the system’s uptake and secretion rates, into a matrix that represents the change in levels of reactants and products for each reaction Figure 1COBRA methods for metabolic network analysis are now incorporated into many software packages across several programming languages like MATLAB and Python . Of thesThis review will summarize the set of packages currently available in Python for various COBRA methods. We identify the advantages and shortcomings of the Python ecosystem to guide users’ decisions on their choice of a software platform and inspire future research ideas. We focus on the application of COBRA methods to cancer metabolism. Finally, we will explore the future directions of COBRA methods development and their importance in cancer modeling.in silico perturbation. Next, we summarize various methods for adding biological constraints like multi-omics and biophysics. In addition, we review methods for unbiased pathway analysis and sampling methods. We also summarize the development of COBRA methods for models at the single-cell and population level. Finally, we touch upon packages for visualization and interactive web applications.To make COBRA open-source and accessible, multiple Python packages have been developed by the scientific community to perform the different analyses within COBRA. Here we describe the major components of COBRA and list their associated packages Table 1,via GitHub and checks for correct annotation, model components, and stoichiometry (COBRA for Python (COBRApy) uses an object-oriented programming approach to represent models, metabolites, reactions, and genes as class objects with accessible attributes. Using this design, COBRApy recapitulates functions for standard metabolic flux analyses of its MATLAB counterpart while being extendible and accessible. First, it has the capabilities to read and write models in various formats such as MAT-file (for storing MATLAB variables), JSON, YAML, and Systems Biology Markup Language (SBML) , the curhiometry . To use hiometry Figure 2The most common COBRA method is flux balance analysis (FBA), which assumes the system is at steady state, follows mass-balance described in the stoichiometric matrix, and restricts reaction fluxes by bounds. Furthermore, FBA searches for sets of steady-state reaction fluxes that maximize or minimize an objective function representing a biological function, such as using biomass production objective to model cellular growth . The objvia SOA can be implemented using COBRApy and SciPy. Alternatively, a very different approach to dynamical metabolic modeling was proposed by DMPy, which translates a GEM into a dynamic reaction equation model using Michaelis-Menten approximations and infers missing kinetic constants using Bayesian parameter estimation with thermodynamics constraints  . There astraints . HoweverFlux distributions, even under an optimal objective, are usually not unique as many alternative fluxes can yield a maximum biomass production. The most representative solution can be found using geometric FBA in COBRApy, which looks for a unique flux distribution that is central to the entire solution space . To bettQuantitative flux predictions are useful to experimentalists because of their potential to explain or even predict the effect of environmental and genetic changes. For investigating the relationship between the external environment and the modeled system, COBRApy provides tools for specifying the growth medium and exchange rates of a model. Instead of extracellular conditions, intracellular changes such as genetic mutations and gene modulation can be interrogated as well. To identify essential genes and reactions for biological functions, FBA is performed with gene knockout simulations to assess the effects of the knockouts on objective functions . Similarin silico strain design, which determines optimal genetic changes that would maximize production of desired metabolites. Straight maximization of only the desired reaction is problematic, since it ignores the drainage of cellular resources needed for cellular growth. Therefore, strain design methods couple product yields with cellular objectives to optimize for fast-growing cells that have high productivity. Such metabolic engineering tools are available in a COBRApy-derived package called cameo . For example, we take the minimum expression of required subunits, but take the sum of isozyme expression. This calculation can be performed in Python packages like CORDA (Cost Optimization Reaction Dependency Assessment)  and MEWp3E , which 3E , INIT . However, due to stochasticity and existence of alternative optima, GEM reconstruction and gap-filling of the same network can give rise to multiple GEMs that could yield different flux predictions. To account for the uncertainty in network structure, ensemble modeling compresses such a set of alternative models into an ensemble object to reduce redundancy while capturing variation. Ensemble modeling can be performed through Medusa, a Python package for generating ensembles, performing ensemble simulations, and coupling ensembles with machine learning (ML) .via MEWpy, and a continuous TRN method called probabilistic regulation of metabolism (PROM) , post-translational modifications, epigenetics, protein–protein interactions and protein stability, allostery, and signaling networks . Severalm (PROM) , 114. Otm (PROM)  integratm (PROM)  integratExtraction of context-specific GEMs requires a reference GEM that is often manually curated. To automate the laborious process of GEM reconstruction, several tools were developed to reconstruct microbial GEMs from genome sequences . Severalvia removal of thermodynamically infeasible pathways or calculations of Gibbs free energy. The vastness of solution space can also be attributed to thermodynamically infeasible loops where metabolites are cycled infinitely. COBRApy includes two implementations for removing such loops: one method ll-FBA (add_loopless) utilizes mixed-integer linear programming  and molecular weight. These protein allocation constraints are applied by Python package MEWpy using a method called GECKO , which adds many pseudo-metabolites and pseudo-reactions to represent enzymes , which assumes that minimizing overall total flux approximately finds efficient pathways that minimizes the total enzyme mass . Availab enzymes . Another enzymes . AutoPAC enzymes . Studies enzymes , 70. Ins COBRAme  and was ynamicME . All pacvia lexicographic reverse search, an implementation that significantly improves performance and memory usage  analysis finds the minimum reaction sets  that can maintain steady state. Different variations of EFM have been implemented in Python. For example, EFMlrs is a Python package that performs EFM enumeration ry usage . In addiry usage , minimalry usage , and elery usage , 116. Exry usage .Another set of unbiased methods is Markov chain Monte Carlo (MCMC) sampling methods, which can characterize the solution space by estimating the probability distribution of feasible fluxes. This could be performed with or without constraining by an objective function. Currently, COBRApy integrated MCMC methods such as artificial centering hit-and-run (ACHR)  and opti+ biosynthesis using COBRApy . We here summarize these metabolic changes and provide the rationale for using COBRA methods to analyze cancer metabolism and TME. Indeed, COBRA methods have been utilized for various applications in cancer research in the past decades. We describe how the analyses begin with building cancer-specific metabolic models, from which one can infer metabolic dysregulation through pathway and network analyses. Next, we showed how these models were used for quantitative prediction of cancer metabolic activity and drug targets. Finally, we highlight the frontiers of modeling the TME using multicellular or single-cell COBRA methods.The dramatic functional and environmental changes that occur during cancer formation and progression are accompanied by accordingly dramatic metabolic reprogramming in cancer cells Figure 3via FBA which requires an objective function. While designing an objective function for tissue-specific eukaryotic cells is usually challenging, cancer cells can be reasonably modeled by biomass objective function, because cancer is mainly characterized by cellular growth  , 184, thvia knockouts and analyzed potential gain of function mutations by adding promiscuous reactions predicted by chemoinformatics , but also new drug targets explained by protection against oxidative stress and methylglyoxal toxicity . AnotherWhile the presence of pathways is indicative of activity, analyzing the pattern of how these pathways connect could provide additional insights. For this purpose, topological analysis is a network-based analysis that characterizes metabolic models based on network properties that describes the degree and patterns of connection between metabolites, genes, and reactions. The same models from Agren et al.  were conin silico knockouts  that could reduce cancer growth  and requires more expertise than the typical omics measurements for constraining COBRA methods. Python packages for modeling label tracing data are available via FluxPyt and mfapy  and the Parker Institute for Cancer Immunotherapy (JH). YS is a Damon Runyon Quantitative Biology Fellow supported by the Damon Runyon Cancer Research Foundation (DRQ-13-22). YS was additionally supported by the Mahan Fellowship at the Herbold Computational Biology Program of Fred Hutch Cancer Research Center and the Translational Data Science Integrated Research Center New Collaboration Award at Fred Hutch Cancer Research Center and in part through a pilot fund from the NIH/NCI Cancer Center Support Grant P30 CA015704. PB acknowledges the support of 5U01AG061359-02 and 5U01AG061359-03 from NIA and 5R01HD091527-06 from NICHD. JL was funded by the Brotman Baty Institute Catalytic Collaborations Trainee Grant program.JH is a board member of PACT Pharma and Isoplexis and receives support from Gilead, Regeneron and Merck.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


TAFAZZIN gene . This debilitating disorder is characterized by cardio- and skeletal myopathy, exercise intolerance, and neutropenia. TAFAZZIN is a transacylase that catalyzes the second step in the cardiolipin (CL) remodeling pathway, preferentially converting saturated CL species into unsaturated CLs that are susceptible to oxidation. As a hallmark mitochondrial membrane lipid, CL has been shown to be essential in a myriad of pathways, including oxidative phosphorylation, the electron transport chain, intermediary metabolism, and intrinsic apoptosis. The pathological severity of BTHS varies substantially from one patient to another, even in individuals bearing the same TAFAZZIN variant. The physiological modifier(s) leading to this disparity, along with the exact molecular mechanism linking CL to the various pathologies, remain largely unknown. Elevated levels of reactive oxygen species (ROS) have been identified in numerous BTHS models, ranging from yeast to human cell lines, suggesting that cellular ROS accumulation may participate in the pathogenesis of BTHS. Although the exact mechanism of how oxidative stress leads to pathogenesis is unknown, it is likely that CL oxidation plays an important role. In this review, we outline what is known about CL oxidation and provide a new perspective linking the functional relevance of CL remodeling and oxidation to ROS mitigation in the context of BTHS.Barth syndrome  is a genetic disorder caused by variants of the  As the hallmark lipid of the mitochondrial inner membrane, cardiolipin (CL) is essential for a myriad of cellular functions. CL undergoes a unique and highly regulated remodeling process catalyzed in part by the enzyme TAFAZZIN . Variatiox) forms a complex with the intermembrane space (IMS) protein cytochrome c (cyt c), and that this complex catalyzes the oxidation of additional PUFA-CL molecules (Newly synthesized CL contains predominantly saturated fatty acid chains. The primary outcome of CL remodeling is the incorporation of polyunsaturated fatty acids (PUFAs) into CL molecules. Unlike saturated and monounsaturated fatty acids, PUFAs are susceptible to oxidation in the presence of elevated reactive oxygen species (ROS), and oxidation of PUFA-CL has been shown to underlie cellular sensitivity to apoptosis . Recent olecules .Here we describe a new perspective for understanding the evolutionarily conserved role of CL remodeling in the context of CL oxidation and BTHS. This model represents a paradigm shift for BTHS research by identifying a novel link between TAFAZZIN function and BTHS pathophysiology. Future studies should be aimed at improving our understanding of this model and evaluating the potential for treating BTHS by inhibiting this oxidation pathway.CL is a uniquely dimeric phospholipid found almost exclusively in mitochondria. It is comprised of a glycerol backbone bridging two canonical phosphatidylglycerol molecules. The variable structure of the four associated fatty acid chains, along with the negative charge carried by the two phosphate head groups, are important for the many protein-lipid interactions attributed to CL . FollowiTAFAZZIN is the primary enzyme responsible for reacylation of CL in healthy cells, but two additional acyltransferases have been described in mammals. Similar to TAFAZZIN, both of these enymzes, acyl-CoA:lysocardiolipin acyltransferase 1 (ALCAT1) and monolysocardiolipin acyltransferase 1 (MLCL AT-1), preferentially transfer oleic and linoleic acid acyl chains to CL, generating predominantly unsaturated CL species . InteresIn mammalian cells, it is likely that remodeled CL confers a fitness advantage. Intact CL remodeling has been shown to be important for various aspects of cellular and mitochondrial homeostasis, including regulation of mitochondrial dynamics, induction of mitophagy and apoptosis, protein turnover, and calcium uptake . In humaAlthough the above findings demonstrate the general importance of CL remodeling, the exact nature of how PUFA-CL benefits cells is not clear. One molecular hypothesis is that protein-lipid packing in the inner mitochondrial membrane (IMM) imposes mechanical stress that favors the accumulation of PUFA-CL, which can only be generated through remodeling. Due to its intrinsic negative curvature, membranes enriched with PUFA-CL can accommodate a higher density of electron transport chain (ETC) complexes and thereby act as more efficient sites for oxidative phosphorylation (OXPHOS) . Howevervia OXPHOS relies on the electron shuttling activity of cyt c, an IMS protein whose localization is maintained, in part, by binding with CL. The interaction between CL and cyt c is a critical determinant of cell homeostasis. Cyt c contains two CL binding sites; loose binding of CL to the “A-site” is mediated by reversible electrostatic interactions between positively charged lysine residues 54/55, 72/73, 86/87, and the negatively charged head groups of CL, whereas tighter binding to the “C-site” involves hydrophobic residues in the fatty acyl chains of CL . The reduced affinity of cyt c for CLox likely contributes to detachment of the former from the IMM, though the exact biochemistry of this process remains obscure. Subsequent translocation of cyt c into the cytosol requires permeabilization of the OMM. The mechanism(s) responsible for OMM permeabilization are the subject of much debate, but two potential explanations have received considerable attention  free fatty acids (FAox). FAox, and highly reactive aldehydes readily derived from them  suggests that it confers a fitness advantage in mammalian cells, and CL remodeling is often thought of solely as a mechanism for converting saturated CL into PUFA-CL. However, if CL remodeling only functions to generate PUFA-CL, why have cells not evolved to synthesize PUFA-CL directly, rather than depend on the additional multi-step process of converting nascent, primarily saturated CL to PUFA-CL through remodeling? Indeed, it has been shown that exogenously obtained PUFAs can be readily incorporated into newly synthesized CL, indicating that remodeling for the sole purpose of producing PUFA-CL would be redundant when PUFAs can be dietarily acquired . This suox . Furtherated ROS . Taken tox and regenerate non-oxidized CL presupposes that the phenotype of having elevated CLox is more deleterious than generating FAox and associated reactive aldehyde species. This point is supported by the fact that cells have robust mechanisms for neutralizing FAox/aldehydes, including adduction with glutathione, reduction by aldo-keto reductases or alcohol dehydrogenases, and oxidation by aldehyde dehydrogenases  . This meomplexes . The keyox production. As an extension of this framework, a major pathophysiological outcome of TAFAZZIN deficiency in BTHS is the inability of cells to recycle CLox and regenerate non-oxidized PUFA-CL. Future studies should be aimed at testing this model, as it may suggest a new avenue for treating BTHS.In summary, this novel paradigm posits that CL remodeling has been evolutionarily conserved as a means for mitigating the deleterious effects of CL


Anxiety symptoms increase during the peri-menstrual phase of the menstrual cycle in people with anxiety disorders. Whether this reflects a heightened variant of normal menstrual-related changes in psychological states experienced by healthy (i.e. non-anxious) people is unknown. Moreover, menstrual-related change in anxiety symptoms is a poorly understood phenomenon, highlighting a need for pre-clinical models to aid mechanistic discovery. Here, we review recent evidence for menstrual effects on anxiety-like features in healthy humans as a counterpart to recent reviews that have focused on clinically anxious populations. We appraise the utility of rodent models to identify mechanisms of menstrual effects on anxiety and offer suggestions to harmonise methodological practices across species to advance knowledge in this field.Consistent with reports in clinical populations, some evidence indicates anxiety symptoms increase during the peri-menstrual period in healthy people, although null results have been reported, and these effects are heterogeneous across studies and individuals. Studies in rats show robust increases in anxiety during analogous phases of the oestrous cycle.Studies in female rats are useful to identify the evolutionarily conserved biological mechanisms of menstrual-related changes in anxiety. Future experimental approaches in rats should model the heterogeneity observed in human studies to increase alignment across species and advance understanding of the individual factors that increase the propensity to experience menstrual-related changes in anxiety. The female menstrual cycle Fig. A modulatNotwithstanding notable methodological limitations in many of these studies, there is relative consensus that anxious symptoms in this subset of people worsen peri-menstrually, i.e. the weeks prior to and during menstruation •, a phenMost research on menstrual changes in psychological symptoms in healthy people have focused on mood-related symptoms  or used Despite the overall limitations of the literature outlined above, some studies support the existence of menstrual-related fluctuations in anxiety symptoms in healthy people. An early study  reportedOf the studies that have directly compared menstrual effects in anxious versus non-anxious people, some have reported no evidence for menstrual changes in self-reported anxiety symptoms, or panic symptoms, in non-anxious people , 18, 42,2 challenge (e.g. fear of losing control) were higher in the premenstrual phase relative to the follicular phase, only in people high in anxiety sensitivity. There was no impact of the menstrual cycle in either group on physical or self-reported panic symptoms. A recent study [Other studies have examined individual difference factors that moderate menstrual fluctuations of anxiety symptoms in healthy people. Nillni et al.  comparednt study  reportedCombined, evidence to date for menstrual-related fluctuations in anxiety in healthy people appears more equivocal than that in populations with anxiety disorders. However, of the studies that do report significant changes, in general (with some exceptions), it is the peri-menstrual phase that is associated with anxiety symptom exacerbation, consistent with the pattern of PME reported in clinical populations.The work by Nillni and colleagues highlights the importance of the moderating role of individual difference factors, such as anxiety sensitivity and health anxiety. These factors may be widely dispersed in a healthy population, as opposed to a clinical population which may cluster more uniformly around one point of these dimensions (e.g. high anxiety sensitivity). The wider dispersion of these factors in healthy populations could lead to greater variation in the magnitude, presence, or direction of menstrual cycle fluctuation and may partly account for null results or inconsistencies between studies in healthy people. Relatedly, menstrual fluctuation in anxiety symptoms is likely a heterogeneous phenomenon experienced by a subset of those who menstruate, and there may be distinct sub-patterns of cyclic changes in symptoms. This is exemplified in work by Kiesner •, who trOther methodological considerations include the importance of measuring for hormonal contraceptive use, like the oral contraceptive pill, which introduces high and relatively steady levels of synthetic oestradiol and progestins. The oral contraceptive pill chronically suppresses ovulation and inhibits ovarian production of oestradiol and progesterone. Several studies did not assess hormonal contraceptive use (e.g. ), which Symptoms of anxiety disorders can be modelled in rats using unlearned fear paradigms. The most common unlearned fear tasks capitalise on the animal’s innate fear of ethologically relevant stimuli  and therefore do not require training to evoke behavioural fear responses , termed ‘anxiety-like behaviour’. These approach-avoidance conflict tasks include the elevated plus maze, open field test and light–dark box. Other unlearned fear tasks that do not depend on approach-avoidance conflict include those measuring active avoidance behaviour , startle response  or social behaviour .. That is, female rats exhibit higher anxiety-like behaviour when tested during oestrous phases of low sex hormones (metestrus or diestrus) compared to oestrous phases of high sex hormones (proestrus). These effects are highly robust across various rat strains, testing paradigms, lighting conditions and experimental designs and have not diminished over time, with several recent studies reporting similar results [The impact of the oestrous cycle Fig. B on anxi results , 69•. A  results –75. The  results . For exa results , which mPotential biological mechanisms underlying hormonal-associated fluctuations in anxiety that have been examined in female rats and humans include oestradiol and the oestrogen receptors , 79, theAlthough there are broad similarities between the human and rat reproductive cycles Fig. , there a. Moreover, while some studies examine distinct oestrous phases [We suggest that translational researchers justify their selection of the oestrous phase being examined and the time of day of testing based on the research question and indicate how it aligns with the human menstrual cycle. For example, if a researcher is interested in declining oestradiol and progesterone levels as experienced during the premenstrual phase in humans, this may correspond to the oestrus phase in rats. However, this may confound the impact of declining sex hormones with the period of sexual receptivity, so testing during metestrus or diestrus may be more appropriate. Some researchers use the afternoon of diestrus in female rats as an analogue to the premenstrual phase in humans, given that during diestrus, there is a withdrawal from a mini peak in progesterone which is then proceeded by a surge in oestradiol , 83–85. s phases , 70–73, s phases •, 74, 75Whereas most rodent studies on oestrous effects adopt a between-subject design, most human studies on menstrual effects adopt a within-subject design, which, as noted, is better suited to detect menstrual-related changes in anxiety. Behaviour on unlearned fear tests in rats, such as the EPM, is commonly evaluated using a single trial to avoid behavioural changes that may arise due to non-associative learning processes (e.g. sensitization or habituation). This concern stems from the observation of a ‘one-trial tolerance’ to the anxiolytic-like effects of benzodiazepines on the EPM, wherein the anxiolytic effects are diminished upon a second exposure to the EPM , 87. Howabsolute levels of hormones, changes in hormones and/or the ratio of hormones are associated with variability in oestrous effects on anxiety-like behaviour. Studies in rats could also identify the neurobiological processes underlying heightened sensitivity to hormonal fluctuations in the absence of differential hormone/neurosteroid levels. For example, unlike in humans, studies in rats could easily examine brain changes  to typical hormonal changes across the oestrous cycle in rats identified as ‘hormone sensitive’ (i.e. rats that exhibit cyclic-dependent anxiety-like behaviour). This is important given recent demonstrations that cell cultures from people with versus without PMDD exhibit differential changes in gene expression following treatment with oestradiol and progesterone [Examining oestrous effects on anxiety-like behaviour using a within-subjects design is advantageous because it allows researchers to identify underlying mechanisms that account for individual differences in oestrous-related exacerbation in anxiety-related behaviour, with the aim of translating these findings to understand the biological source of the heterogeneity of menstrual effects on anxiety in humans. Indeed, individual differences in oestrous effects on anxiety-like behaviour have been demonstrated on a marble burying task, such that only 30% out of 85 female rats exhibited cycle-dependent burying behaviour, with higher marble burying during metestrus versus proestrus . Future esterone . These fJust as anxiety symptoms in humans are comprised of affective, behavioural, physiological, and cognitive dimensions that can be assessed using different procedures, tests of unlearned fear in rats capture different facets of anxiety-like behaviour . For exaAn advantage of using measures such as the acoustic startle response in female animals is that this test can also be examined in humans. Indeed, some studies report higher startle response during the late-luteal phase compared to the early follicular phase in humans , 95, altnormally bred rats may offer insights into menstrual-related changes in anxiety in healthy people, future studies testing oestrous effects on anxiety-like behaviour in female rodents selectively bred for high anxiety-like behaviour may be a promising animal model for examining the genetic, neurobiological, and endocrinological causes of menstrual-related anxiety fluctuations in clinically anxious women. Additionally, examining oestrous effects on anxiety-like behaviour in both normally bred and high-anxiety rodents may be a useful analogue to examine potential differences in menstrual cycle effects on anxiety between healthy and clinically anxious human populations.To the best of our knowledge, all research on oestrous effects on anxiety-like behaviour have been conducted using normally bred adult female rats, with one exception  that exaFluctuations in anxiety across the menstrual cycle are evident in both clinical and healthy human populations, although evidence to date indicates a more robust effect in clinical populations, and substantial heterogeneity is evident in both populations. Analogous fluctuations in anxiety-like behaviour across the oestrous cycle in rats point to an evolutionarily conserved biological mechanism that at least in part underlies these effects. Cross-species translational frameworks can be better leveraged to investigate and understand this effect. Future research should endeavour to improve the alignment of cross-species models through increasing the reliability and comparability of the hormonal phases that are examined, employing within-subject designs, and further investigating which individual factors may contribute to the incidence and pattern of menstrual-related fluctuations in anxiety across species.


Hypoxic-ischemic brain damage (HIBD) is the main cause of perinatal mortality and neurologic complications in neonates, but it remains difficult to cure due to scarce treatments and complex molecular mechanisms remaining incompletely explained. Recent, mounting evidence shows that endogenous neurogenesis can improve neonatal neurological dysfunction post-HIBD. However, the capacity for spontaneous endogenous neurogenesis is limited and insufficient for replacing neurons lost to brain damage. Therefore, it is of great clinical value and social significance to seek therapeutic techniques that promote endogenous neurogenesis, to reduce neonatal neurological dysfunction from HIBD. This review summarizes the known neuroprotective effects of, and treatments targeting, endogenous neurogenesis following neonatal HIBD, to provide available targets and directions and a theoretical basis for the treatment of neonatal neurological dysfunction from HIBD. Neonatal hypoxic-ischemic brain damage (HIBD), which is caused by perinatal asphyxia, is a primary etiology for acute neonatal mortality and long-term infant neurological dysfunction –3. The iEndogenous neurogenesis is the process by which neural stem cells (NSCs) undergo symmetric and asymmetric divisions, after which they proliferate and subsequently differentiate into directed progenitor cells that gradually migrate to functional brain areas while undergoing uninterrupted plastic changes and establishing synaptic connections to produce neurological functions  Figure . Under nOver recent years, our understanding of spontaneous endogenous neurogenesis shifted from its existence exclusively during embryonic and prenatal mammalian development  to be prNSCs differentiation depends mainly on the components of their microenvironment, including neuronal cells, stromal cells, and the extracellular matrix. Based on these factors, NSCs of the same origin can differentiate into different cell types, including astrocytes, oligodendrocytes, and neurons . Next, tAlthough endogenous neurogenesis can improve neurological dysfunction by replacing neurons lost to injury, this is not always the case. As endogenous neurogenesis is a highly regulated process, exposure to a hypoxic-ischemic environment may lead to abnormalities in its course. In some cases, neurodevelopmental dysplasia may be a secondary process leading to brain dysfunction , like dyIt was recently shown that post-neonatal HIBD can induce NSCs proliferation in neurogenesis sites, with subsequent migration of proliferating progenitor cells to a damaged brain region where they acquire the desired phenotype; furthermore, in the damaged brain region, new neurons differentiated from NSCs can integrate into functional neural loops and repair damaged nerves . Plane ein vivo pathophysiological responses, including angiogenesis, cell proliferation, and transcriptional induction of survival genes (Hypoxia-inducible factor (HIF-1) is a major transcriptional activator induced by hypoxia and ischemia. In hypoxic conditions, HIF-1 acts as the main mediator of a series of al genes . The maial genes . Some stal genes –34 have Erythropoietin (EPO) is the first protein that has been identified downstream of HIF-1 to activate erythropoiesis . In hypoThe sonic hedgehog (Shh) signaling pathway plays an important role in endogenous neurogenesis  and can The Notch signaling pathway is a highly conserved signaling pathway that plays a critical role in the process of endogenous neurogenesis . Under nThe Phosphatidylinositol-3-kinase (PI3K) pathway has already been identified to play an important role in cell survival of endogenous neurogenesis and many studies have focused on use of the PI3K pathway to treat brain injury after stroke . A recenWingless-type mouse mammary tumor virus integration site/β-catenin (Wnt/β-catenin) signaling is a vital pathway for endogenous neurogenesis and an essential signaling system during embryonic development and aging . Most stvia the BDNF-TrkB-CREB signaling pathway  binds to the VEGFR2 receptor and activates the Rho/Rok pathway, promoting synaptic growth and playing a crucial role in neuroprotection and neurogenesis , 53. BDN pathway .The ability of spontaneous endogenous neurogenesis after neonatal HIBD to repair damaged neurological functions is markedly limited. Therefore, developing therapeutic techniques  to promoSeveral clinical studies have shown that hypothermia initiated at less than 6 h after birth reduces death or disability for infants with HIBD at 36 weeks or later gestation  and hypoStem cell-based treatments for HIBD have shown promising therapeutic efficacy in preclinical studies . Endogenvia regulating endogenous neurogenesis.Exosomes are endosomal origin membrane-enclosed small vesicles that contain various molecular constituents including proteins, lipids, mRNAs and microRNAs . Many stvia cerebral vasoconstriction, decreasing cerebral blood flow and reducing cerebral hematoma, and simultaneously increasing oxygen supply to ischemic tissues  encompasses both social interaction and the surrounding environment, based on the principle of maximizing opportunities for multisensory stimulation, voluntary physical activity, and social stimulation. EE is an intensive intervention  that canHigh expression of the pro-apoptotic protein p53 in NSCs leads to disruption of endogenous neurogenesis after neonatal HIBD . Accordivia blocking microglia activation, which may also apply to neonatal HIBD, because neuroinflammation is also one of the important injuries mechanisms of neonatal HIBD (Neuroinflammation has been demonstrated to inhibit neurogenesis and the presence of various inflammatory components, such as immune cells, cytokines, or chemokines, plays a role in regulating the survival, proliferation, and maturation of NSCs . It has tal HIBD .Numerous growth factors can increase the proliferation of endogenous NSCs under a variety of conditions, thus promoting the endogenous neurogenesis process. For example, BDNF and its analogues GSB-106 and GSB-214 can upregulate the PI3K/Akt and ERK pathways to promote endogenous neurogenesis after brain injury and improve neurological function , 69. OthCurrently, HIBD remains the leading cause of neonatal death and long-term neurological dysfunction. There is no effective treatment for the neurological sequelae caused by HIBD, neurogenesis is a promising therapeutic target for preventing HIBD-induced neurological sequelae in neonates. All of the treatments discussed above may improve the prognosis of neonatal HIBD through the target of neurogenesis. Thus, the search for optimized combined neuroprotective treatments is urgent. Moving forward, the physiological processes of endogenous neurogenesis, their molecular pathways, and the intervention mechanisms of novel technologies should be investigated in greater depth. In these ways, the most appropriate treatments and technologies can be identified to alleviate the sequelae of neurological damage in children with HIBD, with a goal of also alleviating a heavy burden on their families and society.


Important breakthroughs have considerably improved the outcomes of the percutaneous treatment of valvular heart diseases during the last decades. However, calcium deposition and progressive calcification of the left-sided heart valves present a challenge with prognostic implications that have not been addressed until recently. In the case of native mitral stenosis with no surgical options, a compelling need for tackling heavily calcified valves has led to the development of novel debulking techniques and to the use of aortic balloon-expandable bioprosthesis in the mitral position. In this section of the special issue “Mitral Valve Disease: State of the Art”, we will review standard approaches and indications for the treatment of native mitral stenosis; summarize these two innovative solutions and their evidence, describing both procedures in a “step-by-step” fashion; and briefly comment on future directions in this field. Structural percutaneous cardiac interventions have revolutionized the treatment of valvular heart disease (VHD) over the last years. Transcatheter aortic valve replacement (TAVR) for aortic stenosis has been extensively investigated and has finally become the standard of care for thousands of patients worldwide, even for those with a low surgical risk . In the From an epidemiological standpoint, MS is the fourth type of VHD affecting left-sided heart side valves in terms of incidence and is mainly caused by rheumatic fever or degenerative processes. Chest radiation, carcinoid tumor, or atrial mass-derived MS are exceptional. Rheumatic MS is still an important healthcare problem in developing countries, and degenerative MS will increase in the following years in progressively aging developed ones.2) with symptoms should be treated (class I/level B recommendation), initially with percutaneous dilatation over surgery if no unfavorable characteristics for PMC are present. These include old age, previous commissurotomy, a New York Heart Association (NYHA) functional class IV classification, permanent atrial fibrillation, severe pulmonary hypertension, or prohibitive echocardiographic scores. However, PMC should only be attempted when commissural fusion exists. In other words, this technique is restricted to rheumatic MS (chronic inflammatory disease) and is not useful in degenerative cases  [According to the latest clinical practice guidelines, clinically significant (moderate or severe) MS  are also factors that should tip the scale towards valve surgery, but two novel procedures have emerged that may challenge this paradigm in native valves: mitral lithotripsy and “valve-in-MAC” (ViMAC). In the following sections, we will review both techniques, excluding the “valve-in-valve” (ViV) and “valve-in-ring” (ViR) approaches, since they are carried out in non-native (bioprosthetic) mitral valves and rings.Nowadays, severely calcified aortic valves are well managed with contemporary debulking and specific TAVR devices, a bioprosthesis being immediately delivered  in most cases. The mitral setting is different, especially for rheumatic MS, in which balloon dilatation is the final treatment (PMC). In cases of heavily calcified mitral valves (rheumatic or degenerative), this “uncontrolled” balloon-based dilatation could lead to undesired tears or ruptures of the calcific tissue in the leaflets (not splitting of the commissures) and severe acute MR that requires emergent surgery. The frequency of this complication ranges from 2% to 19% .Since the 1980s, sonic pressure (shock) waves have been used to fragment renal and biliary stones. After the first in vitro experience by Nowak et al. in 1989 with twelve explanted calcified rheumatic mitral valves, the technology was abandoned due to inefficacy and serious risk of calcium embolism . Thirty One year later, Sharma et al. published another case of lithotripsy-facilitated PMC but in a rheumatic patient in the NYHA functional class IV with concomitant aortic stenosis and severe left ventricular dysfunction . Just afFinally, lithotripsy-facilitated PMC has also been performed in a rheumatic setting at the same time as TAVR by our group (“one-step” approach) . A patieThe procedure of applying shockwave pulses to the mitral valve is demanding and comes with a learning curve. Given its complexity and duration, general anesthesia is advised. An echography-guided vascular puncture (large-bore sheaths) allows for safe venous and arterial access. At least two femoral veins will be needed . Transesophageal echocardiography (TEE) guidance is also recommended to attain a mid-posterior transseptal puncture. A high-support wire will retain left ventricular access with the help of a guiding catheter , which is mandatory to create an interatrial septostomy because of the size of the devices and sheaths to be advanced to the mitral valve .The next steps consist of crossing the mitral valve with three long high-support 0.014´´ wires downstream into the left ventricle (LV)  and thenMAC consist of circumferential calcific depositions around the mitral valve, especially in the posterior aspect , but somSurgical repair or replacement approaches for MAC are challenging, requiring decalcification of the annulus and showing higher rates of periprosthetic paravalvular leaks, “valvular area mismatch” with intra-annular suturing, atrioventricular disruption, stroke, or damage to the left circumflex coronary artery. Other strategies, such as the implantation of valved graft conduits between the left atrium and the LV apex, are less appealing and barely used .(a)Paravalvular leakage (PVL) since the mitral annulus is not circular but D-shaped. This complication can be avoided by prosthesis over-sizing;(b)Bioprosthesis embolization, in cases of non-complete circumferential MAC;(c)Obstruction of the LV outflow tract (LVOT) due to displacement of the anterior leaflet of the mitral valve. This event carries higher mortality rates, is more frequent in ViMAC than in ViV or ViR, can be anticipated with precise imaging-based preprocedural planning (CT), and may be solved with midline lacerations of the anterior leaflet   or by me(d)Similar surgical treatments, valvular mismatch, or the impossibility to implant any available prosthesis in very large mitral annuli.With regards to percutaneous solutions and as aforementioned, PMC (with or without lithotripsy) can be attempted in MS with MAC of rheumatic origin, but its efficacy in degenerative MAC is controversial . Therefore, new percutaneous or minimally invasive technologies are being developed, taking advantage of the calcific scaffold that lies beneath the mitral valve. These include a variety of balloon-expandable bioprostheses, which can be implanted in MAC in a transseptal, transapical, or transatrial fashion . The main limitations of this procedure include the following:Some of these complications can be solved with the transatrial (“hybrid”) approach, which requires the multidisciplinary work between cardiac surgeons, anesthesiologists, and interventional cardiologists . Once su2 in the LVOT once the valve is implanted (“neo-LVOT”) is highly predictive of this complication  must be the contrary to the one for TAVR. Once the procedure is finished, an atrial septal defect closure may be needed in 30–50% of the patients to avoid left-to-right shunts. Cerebrovascular protection devices are also advised in ViMAC.From the percutaneous point of view, the procedure is more straightforward than mitral lithotripsy. The material and hardware are very similar to those described above , but in this setting, mitral pre-dilatation is rarely required (around 10–15% of the cases). Once transseptal access is attained (preferably with TEE guidance) via the femoral vein, interatrial septostomy is needed to ease the advancement of the valve delivery systems. When the stiff wire is positioned in the LV, it can be externalized as a “loop” through a transapical access in difficult cases . Furthermore, under rapid pacing, the aortic valve will be delivered and implanted according to standard techniques. Newer generation balloon-expandable valves with smaller sheath profiles and outer skirts  may improve navigation and annular sealing, thus reducing paravalvular leaks. The valve size and type will be determined by a CT, based on mitral annular dimensions and LVOT measurements. If neo-LVOT  is < 220 mmShockwave-based lithotripsy has more applications in addition to the treatment of MS. For instance, it has also been proved safe and effective when dealing with calcified mitral valves with severe regurgitation to aid in transcatheter edge-to-edge repair procedures. In a case report by Fam et al., calcific deposits in the posterior leaflet/annulus had to be debulked with two lithotripsy balloons before PMC and finally edge-to-edge repaired with a MitraClip  . Further(a)In MITRAL II (NCT04408430), a prospective non-randomized multicenter trial, 110 patients will be treated with ViMAC (SAPIEN 3 and SAPIEN 3 Ultra), and 100 patients not suitable for ViMAC will be treated with optimal medical treatment and will serve as controls.(b)In SITRAL (NCT02830204), 30 patients at a high risk for mitral valve surgery or deemed inoperable due to the extent of calcification will be treated with surgical SAPIEN 3 valve implantation.In the MAC-derived MS setting, two studies are still in the recruiting phase to further explore the clinical outcomes of TMVR and/or mitral preparation strategies before TMVR:Finally, novel minimally invasive  valvular technologies have been designed for MAC with MR and have already showed promising results, such as the NeoChord  and the Tendyne  devices . Other vCalcific deposits stand as one of the strongest enemies for interventional cardiologists when treating coronary arteries and also valvular structures. Calcifications are linked with worse outcomes and entail a grim prognosis in different forms of cardiovascular disease. Given the unstoppable and progressive aging of populations worldwide, more patients will require calcium-modifying technologies in the future. Currently restricted to inoperable, high/extreme surgical risk, and no-option patients, these technologies will broaden their indications in the next years, as it has happened with TAVR.In the mitral valve scenario, good results have been demonstrated with shockwave lithotripsy balloons in case reports of MS with a large calcium burden. Disrupting and debulking just calcific tissue may offer better results after PMC without damaging mitral leaflets or subvalvular structures. Despite the absence of prospective studies and randomized clinical trials, this approach will expand in the following years as per clinical indications in degenerative and especially in rheumatic MS.Regarding TMVR for MS, the best results have been reported for ViV procedures, followed by ViR. ViMAC remains the most challenging setting, but with new valve developments, multimodality imaging tools, better supporting technologies, and critical analysis of the results of ongoing studies, percutaneous and hybrid treatments for MAC will improve their outcomes with lower and acceptable mortality rates in the years to come.Thus, either by mitral lithotripsy before valve dilatation or by bioprosthetic valve implantation in severely calcified native mitral annuli, we will be able to offer optimized percutaneous solutions and, eventually, a better prognosis to selected patients with MS.