Acute mechanical circulatory support (MCS) devices are widely used in cardiogenic shock (CS) despite a lack of high-quality clinical evidence to guide their use. Multiple devices exist across a spectrum from modest to complete support, and each is associated with unique risks. In this review, we summarize existing data on complications associated with the three most widely used acute MCS platforms: the intra-aortic balloon pump (IABP), Impella systems, and veno-arterial extracorporeal membrane oxygenation (VA-ECMO). We review evidence from available randomized trials and highlight challenges comparing complication rates from case series and comparative observational studies where a lack of granular data precludes appropriate matching of patients by CS severity. We further offer a series of best practices to help shock practitioners minimize the risk of MCS-associated complications and ensure the best possible outcomes for patients. Historically, CS has been managed largely with intravenous inotropes and vasopressors, medications which improve systemic perfusion at the cost of worsening myocardial supply/demand imbalance. Despite seeming to temporarily improve hemodynamic indices and traditional markers of tissue perfusion, empiric studies have shown that escalating inotropes and vasopressors fail to meaningfully reverse the downward hemodynamic spiral that occurs in CS, with very poor survival observed in patients requiring multiple agents2.Cardiogenic shock (CS) is an advanced state of hemodynamic compromise representing a convergent endpoint of cardiac decompensation resulting from acute myocardial infarction, end-stage heart failure, myocarditis, and various other conditions. In-hospital mortality in CS remains unacceptably high, with recent estimates ranging from 27–51%3. The IABP became widely used and gained a class I indication for CS until the landmark IABP-SHOCK II trial showed that it failed to improve short- or long-term mortality in acute myocardial infarction complicated by CS (AMICS)6. Over the past decade, several other acute MCS platforms capable of providing much greater support have been developed and adopted to varying degrees in clinical practice. The trans-valvular axial flow pumps such as the Impella device  directly unload the left ventricle, capable of providing between 3 and 5.5 L/minute of flow while reducing native myocardial oxygen demand7. A percutaneous right ventricular Impella device (Impella RP) is also available, which bypasses the right ventricle by displacing blood from the right atrium to pulmonary artery8.Mechanical circulatory support (MCS) devices are commonly used to augment cardiac output and decouple systemic perfusion from native myocardial energy expenditure in CS. The intra-aortic balloon pump (IABP) was first applied in CS in the late 1960s, promising to improve hemodynamics through balloon counterpulsation synchronized with the cardiac cycle7. Because of the technical complexity and complications associated with the need for trans-septal puncture for delivery of the left atrial drainage cannula, the TandemHeart device is less commonly used in contemporary practice and thus will be only briefly discussed here. Finally, veno-arterial extracorporeal membrane oxygenation (VA-ECMO), a peripheral modification of cardiopulmonary bypass, is the only device which provides full systemic circulatory and respiratory support, though at the cost of increased left ventricular afterload9. Recognizing the futility of escalating inotropes as well as the imperative to effectively intervene before impaired systemic perfusion progresses to an irreversible state of widespread metabolic failure, we and others have proposed CS management algorithms which incorporate early application of these advanced MCS platforms, with device selection tailored to the individual patient’s hemodynamic profile13.The TandemHeart device  consists of a left atrial drainage cannula connected to an extracorporeal centrifugal flow pump, which returns blood to the descending aorta at flow rates of up to 5 L/minute14. Intravascular shear and varying degrees of hemocompatibility with non-biologic surfaces can induce hemolysis, which in combination with systemic inflammation and thromboembolism can predispose to renal failure, with significant prognostic implications15. Understanding the relative risks of these various complications with different MCS devices and their implications for device selection in patients has been extremely challenging.All MCS devices are associated with risk. This risk is increased among patients in CS who are commonly treated with vasoconstrictive agents, anticoagulants, and anti-platelet drugs, and may be exposed to other devices requiring large bore access such as hemodialysis catheters, pulmonary artery catheters, and other venous or arterial sheaths for monitoring and drug delivery. Use of large-bore MCS access along with a requirement for systemic anticoagulation increases the risk for bleeding, limb ischemia, and stroke4. Two small randomized trials comparing the TandemHeart device to IABP showed a clear signal toward higher rates of bleeding and limb ischemia with TandemHeart, though these trends were significant in only one of the two studies17. Three small, underpowered randomized trials have been conducted evaluating the use of Impella in CS. The ISAR-SHOCK (Efficacy Study of LV Assist Device to Treat Patients with Cardiogenic Shock) trial was a small study (n = 25) comparing the early generation Impella 2.5 to IABP, powered for a surrogate endpoint of hemodynamic improvement 30 minutes after device insertion18. A numerically higher incidence of bleeding, hemolysis, and limb ischemia was observed among Impella patients, though the sample was too small to evaluate the significance of these trends. The IMPRESS in Severe Shock  trial was designed to compare all-cause mortality between IABP and the Impella CP in AMICS but ended up being completed as an exploratory safety trial because of miscalculation of expected event rates19. A trend toward higher rates of bleeding and hemolysis was observed with the Impella CP, though the small sample size limited any conclusive findings. Finally, the IMPELLA-STIC trial compared IABP alone to IABP plus Impella 5.0 in 12 patients with AMICS and found a significantly higher rate of major bleeding in the combined device group, though the study was too small to evaluate the significance of other complication trends20. To date, only one randomized trial has evaluated the use of VA-ECMO in CS: the Extracorporeal Life Support in cardiogenic Shock complicating acute myocardial infarction (ECLS-SHOCK) trial randomized 42 post-arrest AMICS patients to VA-ECMO or no MCS and found similar rates of complications between groups, though again the study was underpowered for clinical outcomes including safety conclusions21.Straightforward comparison of complication rates across devices is possible only by using data from randomized trials in which equivalent patients are randomized to different device strategies. Complication rates observed in randomized trials performed to date are summarized inet al. comparing rates of stroke, bleeding, and limb ischemia among AMICS patients treated with IABP, percutaneous VADs (Impella or TandemHeart), or VA-ECMO are exemplary of these trends. In this large sample, stroke was observed in 3.1% of patients with IABP, 5.6% of those with pVADs, and 9.7% of those treated with VA-ECMO. Similarly, bleeding occurred in 19.4%, 29.9%, and 54.2% of each group, respectively, while limb ischemia occurred in 0.9%, 3.6%, and 7.7%, respectively. All of these complications were associated with increased length of stay and hospitalization costs23. While it stands to reason that more invasive devices would lead to higher complication rates, it must be noted that these observational studies do not account for significant baseline differences between the real-world patients in whom the devices are deployed. Crude insight about the severity of shock across device cohorts can be gained by comparing the short-term mortality rates in89. Two patients (4%) had major vascular events related to flow-limiting dissections of the femoral artery at device removal, with no device related mortality observed.Given the paucity of sufficiently powered randomized trials, most data on complication rates come from observational case series and registries, which are summarized inet al. performed a matched analysis comparing 237 CS patients treated with Impella in 13 European centers to 237 matched patients taken from both arms  of the IABP-SHOCK II trial24. Aminet al. identified patients undergoing PCI with MCS (linked by same-day billing data), some of whom had CS, and performed a propensity matched analysis comparing those managed with Impella with those managed by IABP25. Finally, Dhruvaet al. linked two registries to identify AMICS patients managed with Impella or IABP, matched for 75 baseline variables 26. Each study reported a higher rate of complications associated with Impella compared to IABP. Even more striking, the Amin and Dhruva studies reported a significantly higher rate of in-hospital mortality in patients treated with Impella, raising the concern that the additional complications associated with Impella may translate to higher mortality. While these analyses corroborate the trends observed in observational case series as summarized in27.A final class of studies which have been used to compare relative complication rates between devices are comparative observational analyses, which attempt to adjust for the significant baseline differences observed in cases series by attempting to match patients across available clinical variables. Three such studies have recently spurred substantial debate about complications related to Impella use. SchrageIn the absence of high-quality randomized data clarifying the net risks and benefits of MCS platforms in CS, practitioners still need to move forward and manage individual patients with CS. Recognizing that there are likely higher rates of at least some complications  with Impella and VA-ECMO compared to IABP, shock practitioners should redouble their efforts to adhere to the following best practices.11.Institutions should design structured responses (such as the ‘Shock Team’) to ensure that patients with suspected CS are rapidly identified and evaluated by qualified practitioners so that necessary resources can be urgently made available and evaluations begun regarding the likelihood of recovery or candidacy for durable MCS or transplant. Depending on the suspected inciting insult and local staffing patterns, the shock team might include interventional cardiologists, heart failure specialists, (cardiac) intensivists, and cardiac/vascular surgeons along with perfusionists and critical care nurses. Some have suggested that the cath lab be used as a default staging ground where right heart catheterization, coronary angiography, and fluoroscopic MCS insertion can all be rapidly performed12. Recent data suggest that algorithms incorporating hemodynamically guided decision-making may lead to improved survival in AMICS  to guide device selection. Just as it is important to identify the crashing patient with severe biventricular congestion who is unlikely to stabilize with IABP alone, it is equally important to identify the STEMI patient with isolated left ventricular failure well-suited for left-sided Impella, sparing them the additional complications of a more invasive device like VA-ECMO. We and others have proposed algorithms for device selection based on the ventricular congestive profile and validated indices of right ventricular function such as pulmonary artery pulsatility index (PAPi), along with the presence of concurrent respiratory failuren AMICS , though 9. We would refer readers to a comprehensive review by Subramaniam and colleagues for further discussion of risk factors and strategies to reduce complications from acute MCS devices14.Many complications can be avoided or their impact minimized if recognized and managed promptly. For example, a careful vascular assessment should be performed daily to monitor for signs of limb ischemia, which may require intervention such as external bypass or addition of a distal perfusion catheter. Similarly, markers of hemolysis should be continually tracked to assess the need for device repositioning and thorough neurologic exams performed to identify signs of stroke, which can be particularly difficult to recognize in unconscious patients. In ECMO patients, right upper extremity oxygen saturation and pulmonary capillary wedge pressure should be continuously monitored for the development of Harlequin syndrome and left ventricular distension, which may require optimization of ventilator settings, upgrade to a VAV-ECMO configuration, or addition of a left ventricular vent30.Aside from complications occurring at the time of device insertion or removal, most occur as a function of time on support. Multimodal data  should be continuously re-integrated to assess for the possibility of device weaning or the need for escalation. Specific thresholds  have been proposed to guide consideration of escalation or addition of right-sided support, though specific device algorithms will depend on local availability32. Each operator must develop these technical skills to improve outcomes.Large bore access devices above 17Fr are commonly referred to vascular surgery for removal. However, with emerging techniques, devices ranging from 12Fr to 17Fr may be removed with percutaneous closure approaches. The Perclose device (Abbott Inc) can be used at the time of device implantation (pre-closure approach) or at the time of device removal (post-closure approach) to rapidly achieve hemostasis. The Manta closure device (Teleflex Inc) has recently been introduced and may represent another approach for percutaneous vascular hemostasis. The introduction of the Impella CP with a side-arm access port allows for device removal and post-closure, thereby mitigating vascular complication risk at the time of device removalCS is a complex clinical syndrome that remains a major cause of global mortality and morbidity. MCS device utilization is growing for cardiogenic shock, though each MCS platform is associated with risks. High-quality randomized controlled trials evaluating the use of MCS devices for CS are currently lacking but are in development. Recent reports utilizing administrative datasets and retrospective registries are of limited value other than to raise awareness that randomized controlled trials are needed to improve outcomes for patients. Progress in the field will be made only when high-quality randomized trials are conducted in defined populations, powered for all-cause mortality. The decision to place an advanced device should be made by an experienced shock practitioner or team using the most complete information possible. In the meantime, clinicians should educate themselves about hemodynamically driven decision-making algorithms and best practices to reduce complications associated with each MCS platform.


Colorectal cancer surgery is complex and can result in severe post-operative complications. Optimisation of surgical outcomes requires a thorough understanding of the background complexity and comorbid status of patients.The aim of this study is to determine whether certain pre-existing comorbidities are associated with high grade post-operative complications following colorectal cancer surgery. The study also aims to define the prevalence of demographic, comorbid and surgical features in a population undergoing colorectal cancer resection.A colorectal cancer database at The Prince Charles Hospital was established to capture detailed information on patient background, comorbidities and clinicopathological features. A single-centre retrospective study was undertaken to assess the effect of comorbidities on post-operative outcomes following colorectal cancer resection. Five hundred and thirty-three patients were reviewed between 2010–2018 to assess if specific comorbidities were associated with higher grade post-operative complications. A Clavien-Dindo grade of three or higher was defined as a high grade complication.Fifty-eight percent of all patients had an ASA grade of ASA III or above. The average BMI of patients undergoing resection was 28 ± 6.0. Sixteen percent of all patients experienced a high grade complications. Patients with high grade complications had a higher mean average age compared to patients with low grade or no post-operative complications . Univariate analysis revealed patients with atrial fibrillation, COPD, ischaemic heart disease and heart failure had an increased risk of high grade complications. Multivariate analysis revealed pre-existing atrial fibrillation  and COPD  were independently associated with an increased risk of high grade complications.Pre-existing atrial fibrillation and COPD are independent risk factors for high grade complications. Targeted perioperative management is necessary to optimise outcomes. Colorectal cancer (CRC) is the third most common cancer in Australia, with over 17 000 people diagnosed annually . While cCurrent larger scale colorectal databases rarely capture granular details surrounding patient comorbidities. This can make it difficult to ascertain which specific comorbidities have the greatest negative impact on post-surgical outcomes. The Charlson Comorbidity Index has widely been used to evaluate comorbid burden as a measured index to predict mortality. However, this classification system uses grouped comorbidity groups for analysis, thereby lacking disease-specific data and preventing analysis on the specific impact of certain diseases –8.As well as comorbidity analysis, the assessment of post-operative outcomes has also varied widely in literature and within colorectal cancer databases. Several factors can influence the incidence of complications. The definition of ‘post-operative complications’ and the period which is defined as ‘post-operative’ are examples of the variation in complication monitoring. The Clavien-Dindo Classifications are an objective, reproducible and standardised categorisation system, developed for comparing post-operative complications. The classifications have been validated for use in several surgical subspecialties include hepatobiliary and urological surgery , 10.While there is literature that outlines the prevalence of comorbidities in certain tumour types, there is a paucity of information on colorectal cancer. Moreover, there is a lack of consistency in grading and comparing postoperative outcomes within these studies.The few studies that observe the effect of comorbidities on post-operative outcomes were published over ten years ago and reflect older surgical techniques, standards and treatment guidelines –13.The aims of this study are to 1) Determine the prevalence of comorbidities, surgical and pathological features of patients with colorectal cancer and; 2) Describe the association between specific patient comorbidities and high grade post-operative complications following colorectal cancer surgery.The Prince Charles Hospital (TPCH) in Brisbane, Australia is a tertiary cardiothoracic centre that cares for patients with advanced and complex heart and lung diseases. The General Surgical department at TPCH provides care for colorectal cancer patients whose clinical background and comorbidities are captured in a detailed clinicopathological database. A retrospective study using The Prince Charles Hospital colorectal cancer database was undertaken to investigate the effect of patient comorbidities on post-operative outcomes. Patient charts were individually reviewed and data extracted by trained medical personnel. Data was obtained from previous and current admissions as well as correspondence letters, follow up documentation and outpatient/readmission notes.Patients who underwent surgical resection of colorectal cancer at The Prince Charles Hospital between January 2010 and December 2018 were included in the study.Patients were excluded if they had endoscopic resection of the malignant lesion without surgical intervention. Patients below the age of 12 were also excluded from the study.Basic demographic data such as age, gender, date of birth, height, weight, body mass index (BMI) and American Society of Anaesthesiologists (ASA) grade were documented. Specific pre-operative conditions were grouped into comorbid categories: cardiovascular, respiratory, metabolic, autoimmune and renal disease.Cardiovascular diseases recorded included ischemic heart disease, previous coronary artery bypass grafting, previous percutaneous coronary intervention, pacemaker insertion, defibrillator insertion (AICD), previous valve repair, heart failure (any type), cardiomyopathy, hypertension, pulmonary hypertension and atrial fibrillation.Respiratory diseases recorded included asthma, COPD, bronchiectasis, cystic fibrosis and obstructive sleep apnoea.Metabolic diseases recorded included type I diabetes, type II diabetes and hyperlipidaemia. Autoimmune conditions recorded included rheumatoid arthritis, psoriasis, polymyalgia rheumatica, and systemic lupus erythematosus (SLE). Baseline renal function was noted in each patient.Surgical data included the operation performed, urgency of surgery, operative approach, discharge date and length of stay. Pathological data included tumour histopathology, histological grade and TNM stage of disease.The database and patient charts were reviewed for specific post-operative complications and their treatment was documented. Complications were Graded from I to V according to the Clavien-Dindo Classification of Surgical Complications . ComplicDifferences in demographic features, comorbidities and surgical/pathological features between the two complication groups were assessed using t-tests and Fisher’s exact tests as appropriate. Statistically significant results were defined as those with p ≤ 0.05.The association between comorbidities and high grade post-operative complications was assessed using uni- and multivariate cox regressional analysis to assess odds ratios for each comorbidity. All comorbidities were assessed using univariate analysis, while only those that were found to be statistically significant were analysed in the multivariate model. Statistical review was performed by a qualified biomedical statistician. Data was analysed with Stata v14 software (StataCorp).Ethics approval for this database was granted by the Prince Charles Hospital Human Research Ethics Committee (Reference: HREC/17/QPCH/295). A waiver of consent was approved to allow access to confidential patient information without consent. Patients were not anonymized prior to data collection. However, patient names and certain other identifying data was not recorded in the database to help guard against confidentiality breaches. Patient data was accessed between January 2018 and June 2019.Five hundred and thirty-three patients who underwent colorectal cancer resection between January 2010 and December 2018 were reviewed.The mean age of patients undergoing colorectal cancer surgery was 67.4 years ± 15.6 SD. The mean BMI was 27 ± 6.0 SD. There was a 50:50 even distribution of males and females within the cohort and the majority of patients  had an ASA of ≥3 (indicating the patient is living with a severe disease process).Fifty-nine percent of all patients who underwent colorectal cancer resection had a cardiac comorbidity, with hypertension  being the most common condition followed by ischemic heart disease  and atrial fibrillation .Twenty-eight percent of patients had respiratory disease with asthma  and COPD  being the most common lung conditions. Fifty-four percent of all patients had metabolic disease (n = 288) with hyperlipidaemia being the most common  followed by type II diabetes . Only 2% of patients (n = 11) had an autoimmune disease. The majority of patients had a preoperative estimated glomerular filtration rate (eGFR) >90 ml/min/1.73m2  which is indicative of no underlying renal disease. Thirty percent of the cohort had an eGFR between 60–89 while 9% had an eGFR between 45–59. Forty-three percent of patients had a degree of renal impairment prior to surgery. Fourteen percent of patients were current smokers at the time of surgery (n = 76). The majority of cancers  were located within the right side of the colon (from the caecum to the hepatic flexure). Left sided colon cancers (from the splenic flexure to the sigmoid) made up 30% of cases (n = 161) while rectal cancers made up 17% (n = 88).Right hemicolectomies, extended right hemicolectomies, appendicectomies and caecectomies  made up the majority of surgical cases and reflects the dominance of right sided colon cancers. The majority of surgical cases were performed laparoscopically   while open cases (including laparoscopic converted to open) made up 23% of all operations (n = 121).The most common histopathology was adenocarcinoma of no special type . Most patients in both the nil/low and high grade complications groups had an early stage of caner (Stage I and IIA). The majority of our patients had stage I disease, with no difference in distribution of disease between the two groups (p = 0.73). The surgical and pathological features of the cohort are outlined in Complications were divided into surgical complications and medical complications.One hundred and twenty-nine patients (24%) developed surgical complications.The most common surgical complications were prolonged ileus , superficial wound infection  and an abdominopelvic collection . Anastomotic leaks accounted for 2.6% of complications (n = 14). One hundred and twenty patients (23%) had a medical complication. The most common medical complications were cardiac arrhythmias , respiratory infections  and impaired renal function . The median length of stay (LOS) for patients following surgery was 10 days with a 30-day mortality rate of 2% (n = 9). The surgical and medical complications of the patients are outlined in Patients who had Clavien-Dindo Complication Classifications graded III-V were classified as having high grade post-operative complications. The demographic features of high-grade post-operative complications are outlined in Out of the entire study cohort, 85 patients (16%) had high grade post-operative complications. Patients who had high grade post-operative complications were older than those with nil/low grade complications .Those with high grade complications had a higher proportion of patients with ASA III-V when compared to those with nil/low grade complications .Patients with high grade complications were found to have higher rates of ischemic heart disease , coronary artery bypass grafting , heart failure , cardiomyopathy  and atrial fibrillation .Patients with high grade complications were also found to have double the incidence of COPD  . There wPatient background data, comorbidities and their association with high grade complications was analysed by univariate analysis and presented in Patients with pre-existing atrial fibrillation , heart failure  and an ASA IV grade  had a threefold higher risk of high grade complications when compared to patients who did not have these comorbidities. Patients with chronic obstructive pulmonary disease  and ischaemic heart disease  had approximately double the risk of developing high grade complications .Coronary artery bypass grafting was found to be just above the threshold for statistical significance . Types II diabetes, obstructive sleep apnoea, hyperlipidaemia, asthma and percutaneous coronary intervention were not found to be associated with high grade complications.Multivariable logistic analysis  was perfOf the six comorbidities found to be associated with high grade complications, pre-existing atrial fibrillation  and COPD  were the only comorbidities independently associated with high grade post-operative complications.We performed a retrospective study to determine the effect of comorbidities on surgical outcomes following colorectal cancer resection.Out study demonstrated that patients with pre-existing atrial fibrillation were almost three times more likely to have severe post-operative complications while patients with COPD were found to be twice as likely to have similar complications.The independent association between atrial fibrillation and poorer surgical outcomes has never before been described in literature.Our results demonstrate that patients with high grade complications following colorectal cancer surgery were more likely to be older, have significantly higher rates of ischaemic heart disease, previous coronary artery bypass grafting, atrial fibrillation, heart failure and COPD. This is of particular interest as previous studies have associated ‘cardiac disease’ with post-operative complications without delineating the effect of specific comorbidities. While ischaemic heart disease, heart failure and increased age were found to be associated with the development of high-grade complications on univariate regression analysis they were not independently associated with high grade complications on multivariate analysis.Atrial fibrillation (AF) is a common cardiovascular comorbidity that frequently occurs within a similar demographic to that of CRC. Approximately 5% of Australia’s population over the age of fifty-five have atrial fibrillation . AlthougSeveral studies have demonstrated an association between cardiovascular disease and an increase in post-operative complications , 19, howA study from Walsh et al. demonstrated poorer all-cause mortality for CRC patients affected by AF . HoweverCOPD was also found to be independently associated with high grade complications. COPD is the second leading cause of disease burden in Australia in patients aged 65–74 . In a 20One third of all patients with pre-existing atrial fibrillation and 29% of patients with COPD developed high grade post-operative complications. Amongst these patients, there was a higher proportion of medical complications compared to surgical complications. Patients with COPD who developed high grade complications had higher incidences of post-operative respiratory infections, ischaemic cardiac events and had an increased requirement for respiratory and ventilatory support. Patients with AF who developed complications were more likely to develop cardiac arrhythmias and respiratory complications.Complications arising in those with AF and COPD seem to largely be due to an exacerbation of their underlying cardiorespiratory disease rather than a specific post-operative surgical aetiology. Further to this, atrial fibrillation and COPD also appear to be markers of poorer overall health and a higher comorbid burden in patients.It is pertinent to note that across all patients within the study, there was a high burden of comorbid disease. The association between pre-operative atrial fibrillation and high grade complications following colorectal cancer resection is a novel finding. The association between COPD and poorer surgical outcomes is equally important in order to help optimise patients perioperatively and manage prospective outcomes. Medical optimisation of patients and their comorbidities in the perioperative period is also important in minimising high grade post-operative complications. Collaborating with physicians, anaesthetists, peri-operative medical teams and allied health is crucial in minimising the risk of high grade post-operative complications and improving patient outcomes.This study highlights the importance of pre-existing atrial fibrillation and COPD as specific diseases which require careful perioperative optimisation, planning and management in order to minimise their impact on the incidence of high grade complications.S1 Appendix(PDF)Click here for additional data file.


Mycobacterium tuberculosis from patient sputum samples for clinical diagnosis of drug-resistant tuberculosis (DR-TB). However, the complexity of sequencing platforms has limited their uptake in low-resource settings. The goal of this study was to evaluate the use of the tNGS-based DST solution Genoscreen Deeplex Myc-TB, for use on the compact, low-cost Oxford Nanopore Technologies MinION sequencer.Targeted next-generation sequencing (tNGS) has emerged as a comprehensive alternative to existing methods for drug susceptibility testing (DST) of  Mycobacterium tuberculosis from patient sputum samples for clinical diagnosis of drug-resistant tuberculosis (DR-TB). However, the complexity of sequencing platforms has limited their uptake in low-resource settings. The goal of this study was to evaluate the use of the tNGS-based DST solution Genoscreen Deeplex Myc-TB, for use on the compact, low-cost Oxford Nanopore Technologies MinION sequencer. One hundred four DNA samples extracted from smear-positive sputum sediments, previously sequenced using the Deeplex assay on an Illumina MiniSeq, were resequenced on MinION after applying a custom library preparation. MinION read quality, mapping statistics, and variant calling were computed using an in-house pipeline and compared to the reference MiniSeq data. The average percentage of MinION reads mapped to an H37RV reference genome was 90.8%, versus 99.5% on MiniSeq. The mean depths of coverage were 4,151× and 4,177× on MinION and MiniSeq, respectively, with heterogeneous distribution across targeted genes. Composite reference coverage breadth was >99% for both platforms. We observed full concordance between technologies in reporting the clinically relevant drug-resistant markers, including full gene deletions. In conclusion, we demonstrated that the workflow and sequencing data obtained from Deeplex on MinION are comparable to those for the MiniSeq, despite the higher raw error rates on MinION, with the added advantage of MinION’s portability, versatility, and low capital costs. Targeted NGS on MinION is a promising DST solution for rapidly providing clinically relevant data to manage complex DR-TB cases.Targeted next-generation sequencing (tNGS) has emerged as a comprehensive alternative to existing methods for drug susceptibility testing (DST) of  Despitesistance . The lonsistance .r TB cases detected and has reduced diagnostic and treatment delays (–The uptake of WHO-recommended rapid diagnostics (WRDs), Xpert MTB/RIF Ultra , and line probe assays for use on direct clinical samples has contributed significantly to the global increase in number of RIFt delays –6. Howev delays –, and can delays –.Mycobacterium tuberculosis complex (MTBC) can provide deep and comprehensive data on all clinically relevant resistance mutations and is the leading alternative to existing phenotypic and molecular DST methods (Next-generation sequencing (NGS) of  methods  currentl methods .In contrast, targeted NGS (tNGS) can be performed on direct clinical samples and has emerged as a feasible option for comprehensive, fast, and clinically relevant sequencing from patient sputum samples –15. Howe–The aim of this study was to evaluate the adaptability of an existing TB tNGS assay kit and DST analytics solution , originally developed for application on Illumina  sequencing instruments, for use on the portable ONT MinION sequencer in order to demonstrate the feasibility and potential of rapid tNGS on a compact, low-cost sequencer.For this study, 104 DNA samples from randomly selected smear-positive, Xpert MTB/RIF-positive sputum specimens, previously characterized by Deeplex Myc-TB on the Illumina MiniSeq platform, were resequenced using MinION. Samples were retrieved from the archived collection available at Ospedale San Raffaele, Milan, Italy. No patient data were used, and the study was done without any modification of clinical and laboratory management.We performed ONT MinION sequencing on the Deeplex Myc-TB amplicons produced from clinical samples previously sequenced on the Illumina MiniSeq instrument, in order to compare and contrast results and performance. Sequencing of Deeplex Myc-TB amplicons on MiniSeq was considered the reference technology for this study. Laboratory procedures, including sputum sample processing, DNA extraction and purification, and PCR amplification by the Deeplex Myc-TB kit, are described in detail in the supplemental material.While the sample preparation steps were common to both sequencing instruments, the library preparation steps for sequencing on the MinION and MiniSeq were different based on the manufacturers’ instructions. Each previously produced Deeplex Myc-TB hi-plex amplicon was used for MinION library preparation, as described in detail in the supplemental material.The Deeplex Myc-TB kit contains a sequencing analysis and clinical interpretation software solution originally developed for Illumina raw sequence data. This solution is currently not optimized for sequence data from the MinION. For the purposes of this study, we therefore computed read quality and mapping statistics for MinION data using an in-house, proprietary pipeline we developed, while for the MiniSeq results we used the software solution provided by the manufacturer as described in the supplemental material and complemented the analysis with the MTBseq pipeline . The reaAll 104 sediment samples amplified by Deeplex Myc-TB and sequenced using MiniSeq were also successfully sequenced on MinION.The average percentage of MiniSeq reads identity mapping to the reference genome was 99.5% . The aveTwo sequencing runs of 48 and 56 samples were performed on the MinION device using two flow cells to sequence the whole study collection of 104 isolates. The average identity percentage of MinION reads mapping to the H37RV targeted genome was 90.8%, with a MAPQ  value around 60 .rrs and rrl). Mean composite reference coverage breadth was over 99%, indicating complete coverage of the targeted genome regions (Data Sets S6 and S7).The average depth of sequencing coverage relative to reference genome obtained on MinION was 4,151×. As with MiniSeq, the MinION coverage depth was stratified by targeted genomic region for all study samples (Data Sets S4 and S5). We compared the total variant calls between the MinION and MiniSeq, setting different minimum coverage thresholds to explore variant calling with MinION, as described in the supplemental material. Overall, we observed good overlap of variant calls between sequences generated from MinION and MiniSeq (rrs gene (linked to streptomycin resistance at genomic coordinates 1472337 and 1572359) were detected by MiniSeq and missed by MinION at the 40× threshold adopted, but these were, however, identified also by MinION with a lower number of reads . The MinION method correctly identified the resistance patterns determined by MiniSeq, including 3 RIFr, isoniazid-resistant (INHr) strains with additional pyrazinamide resistance (PZAr) and streptomycin resistance (SMr), PZAr and ethambutol resistance (EMBr), and PZAr only; 1 RIFr, INH-susceptible (INHs) sample with additional capreomycin resistance (CAPr); 6 RIFs, INHr cases, with additional SMr in 3 cases; and 5 strains showing SMr only (Data Set S19). In two samples we observed a full pncA gene deletion on MiniSeq, which was correctly identified by MinION as well.Regarding the presence of confidence-graded mutations associated with resistance , 23 to tReplacing or complementing phenotypic DST with tNGS-based resistance prediction could avoid or significantly decrease our reliance on culture-based DST methods and has the potential to transform the management of DR-TB globally. The recent development of compact, portable NGS instruments for field deployment will accelerate the decentralization of NGS testing and make near-patient tNGS for clinical diagnosis of drug-resistant TB a near-term reality, with remarkable time and cost savings compared to conventional sequencing instruments , 18. TheIn this study, we demonstrated that the existing tNGS Deeplex Myc-TB assay, optimized primarily for larger Illumina sequencing platforms, could be successfully implemented on a portable MinION sequencing device. With only minor protocol modifications, we were able to adapt the library preparation of amplicons (average size of 600 bp) for optimal, long-read sequencing direct from patient clinical samples. This approach resulted in excellent coverage across the targeted MTBC genomic regions, but with higher error rates than for MiniSeq sequences from the same sample amplicons . CriticaThe raw base call error rate was found to be high in MinION sequencing, at around 9%, in comparison to the negligible error rates in MiniSeq data . Even acrrs and rrl).The variable MinION depth of coverage for the Deeplex Myc-TB-targeted regions  was anotWith regard to variant calling, we have demonstrated that by applying appropriate minimum call thresholds to the coverage depth at a given target region  and 4, irrs, tlyA, gidB, and gyrA (rrs and tlyA).The majority of discrepant variant calls between MiniSeq and MinION were found when the stringent 400× minimum cutoff to call a variant , and batch size for a single run was higher on MiniSeq than on MinION . In this study, we assessed two different batch sizes for MinION (48 and 56) obtaining linear cumulative yield in gigabases, suggesting that MinION throughput may be still increased over 56, without significant loss in coverage depth.Turnaround times to prepare samples for MinION and MiniSeq sequencing reactions from DNA extraction were also similar (3 working days). Future customization of Deeplex Myc-TB primers for MinION chemistry, avoiding the need of library preparation modifications to the protocol, would enable shorter library preparation time and is something that should be explored. Importantly, the length of the MinION run could also be optimized to reduce time and cost. In the two study runs, the cumulative yield of reads after 12 h accounted for about the 60% of the total (Data set S20), with read lengths constant up to the end of the MinION sequencing. Therefore, the two runs could have been stopped after 12 h with a >2,000× average depth of sequencing coverage from the samples included in the run, likely enabling one to reliably analyze all Deeplex Myc-TB targets. Batching fewer samples on a MinION amplicon run, likely the pragmatic solution for a routine laboratory with limited daily samples, would enable one to generate optimal sequencing reads for clinical interpretation within a few hours. Additionally, there is the potential to reuse flow cells on MinION, which is not possible on MiniSeq and could significantly reduce the time and cost per sample sequenced. The versatility and physical robustness of the MinION compared to MiniSeq, accompanied by lower capital and maintenance costs and the demonstrated accuracy for identifying clinically relevant mutations in MTBC, make this tNGS platform a promising solution for decentralized, culture-free clinical sequencing in laboratories with fewer daily samples.This study has some limitations. The Deeplex Myc-TB assay was applied only on smear-positive sputum samples, and prior complete sequencing results were obtained from all selected samples on the Illumina platform. The sensitivity and specificity of this tNGS solution (Deeplex Myc-TB and MinION sequencing) still need to be evaluated using appropriate analytical and clinical studies. Mycobacterial species identification and MTBC genotyping were not assessed with MinION data, as the current study was meant primarily to compare the total variant calls and drug resistance interpretation of the two sequencing technologies.The high-frequency-variant threshold adopted for MinION  was set to avoid the introduction of additional variables in this first mutation analysis comparison between the two platforms. Furthermore, the Deeplex web application used to analyze the MiniSeq data did not detect the presence of any relevant low-frequency variant (at 3% threshold); therefore, the overall comparative SNP analysis should not be biased.In this study, we demonstrated that the workflow and sequencing data obtained from Deeplex Myc-TB amplicons from direct clinical specimens, sequenced on a MinION instrument, are comparable to the Illumina MiniSeq process and outputs, with the added advantage of the MinION device’s portability and low capital costs, making this platform more suitable for use in clinical mycobacteriology and to a wider extent in clinical microbiology laboratories where the same platform can be used to identify MDR bacterial pathogens . The app


The lateral hypothalamus encompasses neural circuits that are highly active during feeding behavior and rapid eye movement sleep. Yet, it remains unclear whether these mutually exclusive behaviors share common neural populations. Here, we recorded and perturbed the activity of inhibitory neurons of the lateral hypothalamus (LH) across feeding behavior and sleep in freely behaving mice. We found that feeding is reliably encoded by specific patterns of neuron activity and that these patterns are reactivated during rapid eye movement sleep. Disrupting the activity of these inhibitory neurons specifically during rapid eye movement sleep decreased subsequent feeding behavior. These results suggest that rapid eye movement sleep stabilizes the hypothalamic representation of feeding behavior and modulates future food intake. vgat) show unique activity patterns during feeding that are reactivated during REM, but not non-REM, sleep. REM sleep-specific optogenetic silencing of LHvgat cells induced a reorganization of these activity patterns during subsequent feeding behaviors accompanied by decreased food intake. Our findings provide evidence for a role for REM sleep in the maintenance of cellular representations of feeding behavior.During rapid eye movement (REM) sleep, behavioral unresponsiveness contrasts strongly with intense brain-wide neural network dynamics. Yet, the physiological functions of this cellular activation remain unclear. Using in vivo calcium imaging in freely behaving mice, we found that inhibitory neurons in the lateral hypothalamus (LH Rapid eye movement (REM) sleep  is the most puzzling brain state in vertebrates. It is associated with sensory-motor development and learning , 2, memo311vgat) show high activity during wakefulness or spontaneous REM sleep  carrying a Cre-dependent expression cassette of GCaMP6s (Syn-Flex-GCaMP6s) into the LH of vgat-IRES-Cre mice (Lower Left). We observed long-lasting expression of GCaMP6s in the LH area -positive neurons , another REM sleep-active neuron population in the LH . We longitudinally recorded the calcium transients from GCaMP6s-expressing LHvgat neurons, concomitant to EEG/EMG signals and behavioral video-tracking during a free-feeding task that did not involve instrumental learning or behavioral conditioning (Movie S1).We first recorded the activity profiles of LHn the LH , 36. Tran the LH , respectvgat neuron populations reliably encode feeding , food approach , or nonfeeding behaviors using a cross-correlation analysis. Population vectors were computed from the fluorescence intensity values of single LHvgat neurons for each recorded frame of an imaging session and correlated with each other .We first investigated whether LHch other H. Each oo frames G. Hereafehaviors I. The rovgat neuron population activity , whereas the one for food approach decreased linearly .To assess the stability of this representation, we computed an index of similarity by correlating the activity patterns of single frames occurring during individual feeding and food approach bouts to the corresponding map . We found that the feeding representation was stable over successive feeding bouts A and indlinearly B. Note tvgat cells based on their activity profiles during food approach and feeding to reflect their contribution to the appetitive or consummatory aspects of feeding behavior, respectively , food approach , or when animals were outside the food quadrant and not eating . Interestingly, the activity of feeding-max and unspecific cells linearly decreased over successive feeding bouts, while the activity of nonfeeding-max neurons increased  and a redistribution of LHvgat neuron clusters .We then classified single LHectively C. We founcreased E. Furthencreased F. These vgat neurons to either wakefulness or REM sleep, we next performed simultaneous recordings of cellular activity and EEG/EMG signals from spontaneously sleeping animals. We observed that the activity of LHvgat neurons was strongly modulated across sleep–wake states , wakefulness , both wakefulness and REM sleep  or non-REM (NREM) sleep , and no selectivity for either of the sleep states or wakefulness .To assess the contribution of LHe states A, with se states C. Unsupevgat neuron activity patterns during REM sleep similar to those occurring during awake feeding? To first test whether LH circuits contributing to awake feeding behaviors and sleep stages share common cellular substrates, we computed a coclassification matrix of the different LHvgat neuron clusters associated with the feeding task and sleep–wake states . We found a significant modulation of the average activity of the neurons in the different feeding clusters across sleep–wake states. Whereas activity of all clusters was lowest during NREM sleep, it was highest during REM sleep for all functional clusters except the nonfeeding-max group .At the population level, we next looked at the activity patterns during the different sleep–wake states. We found that the activity patterns of LHach maps C. While ach maps C. The siach maps D and E. vgat neurons during REM sleep contributes to the maintenance of appropriate feeding behavior during wakefulness. To achieve this, we targeted the expression of the light-sensitive proton pump ArchT3.0 to LHvgat neurons by stereotactically injecting an adeno-associated virus (AAV2) carrying a Cre-dependent expression cassette of ArchT  or YFP  into the LH of vgat-IRES-Cre mice (Lower Right). We tested for the biological function of the opsin by recording the electrophysiological response to optogenetic silencing in mice that were chronically implanted with tetrodes inside a microdrive and an optical fiber in the LH (Left). Of 18 recorded neurons, 8 showed a significant decrease in spike rate upon continuous illumination delivered selectively during REM sleep episodes  and 10 did not change their spike rates in response to the light delivery . Consistent with our imaging results . An additional cohort of mice were implanted with cortical EEG electrodes, EMG wires, and bilateral optical fibers above the LH. These animals were fed ad libitum and habituated to the free-feeding task for three consecutive days. On the fourth day, LHvgat neurons were optogenetically silenced selectively during REM sleep episodes prior to the free-feeding paradigm in both ArchT3.0- and YFP-transduced animals .We then tested whether activation of LHn the LH A, Left.  results , we obse animals D and E. vgat neurons coexpressed ArchT-tdTomato . Mice were then instrumented with a GRIN lens for consecutive REM sleep-specific optogenetic silencing followed by imaging concomitant to EEG/EMG recordings during the free-feeding task . We found that optogenetic silencing induced a significant change of the feeding map in comparison to their baseline representation. The change persisted for at least 4 d . This change was not due to daily variation of the feeding map  or changes in overall neuronal activity  since both remained stable over days. Note that also the food approach map was significantly changed , consistent with its variation within recording session .To investigate whether REM sleep provides a window for activity tuning of neuronal network dynamics, we targeted the expression of GCaMP6s and ArchT to the right LH by stereotactic coinfusion of AAV5-Syn-Flex-GCaMP6s and AAV2-CAG-Flex-ArchT-tdTomato in vgat-IRES-Cre mice . This decrease was not due to changes in the cellular recruitment because the number of LHvgat neurons per cluster remained stable over days, and after optogenetic silencing , despite daily variation of the cellular composition of the clusters . These effects were not due to photobleaching of GCaMP6s fluorescence upon laser light stimulation .At the single-cell level, the activity of feeding-max neurons was significantly decreased during postsilencing feeding behavior but not during food approach or nonfeeding behavior  that are stable within, and between, behavioral sessions, but distinct from those observed during nonfeeding behavior. Thus, this map captures both active and inactive LHvgat neurons that may ultimately represent an optimal state of LHvgat neurons to control feeding through their downstream synaptic targets. Indeed, LHvgat neurons that modulate circuits implicated in attention . We recorded male vgat-Cre mice  aged betTo assess the feeding behaviors, animals were exposed to a square, 29.7 by 29.7 cm, open-field arena. In three corners of the arena either food, the animals’ bedding, or their nesting material were presented. One corner was left empty. Animals were video tracked, and their behavior and position were visually scored. Sleep recordings were performed in the open top home cages (18 × 29 cm) of the mice. Sleep stages were scored offline based on EEG and EMG measurements.F/F) was obtained by first subtracting an estimate of the baseline fluorescence from the signal and then dividing it by its noise envelope. Using this approach, the time series captured when neurons are inactive is approximately zero-mean and has unit standard deviation and cellular activation is characterized by transient activity peaks > 2 normalized ΔF/F.To extract the calcium transients for the individual neurons, the calcium movies from all of the acquired sessions were motion-corrected and aligned to a reference image and finally concatenated. Single neurons and their calcium signals for each mouse were extracted using the CNMF-E toolbox  from theThe similarity between activity patterns was assessed using Pearson’s correlation coefficient between population vectors. To test whether pattern similarity was higher than chance, the signal was shuffled in the time domain (referred to as permutation test), retaining the respective identities of individual neurons. For comparisons of the activity levels between cells and cell groups, we used the average fluorescence and performed ANOVA.SI Appendix, Materials and Methods.For more details on surgical procedures, behavioral paradigms, and signal acquisition and processing, refer to Supplementary FileSupplementary File


The imbalance between BCL-2 homologues and pro-death counterparts frequently noted in cancer cells endows them with a cell autonomous survival advantage. To eradicate ectopic cells, inhibitors of these homologues (BH3 mimetics) were developed to trigger, during anticancer treatment, full activation of the canonical mitochondrial apoptotic pathway and related caspases. Despite efficiency in some clinical settings, these compounds do not completely fulfill their initial promise. We herein put forth that a growing body of evidence indicates that mitochondrial integrity, controlled by BCL-2 family proteins, and downstream caspases regulate other cell death modes and influence extracellular signaling by committed cells. Moreover, intercellular communications play a key role in spreading therapeutic response across cancer cell populations and in engaging an immune response. We thus advocate that BH3 mimetics administration would be more efficient in the long term if it did not induce apoptosis in all sensitive cells at the same time, but if it could instead allow (or trigger) death signal production by non-terminally committed dying cell populations. The development of such a trade-off strategy requires to unravel the effects of BH3 mimetics not only on each individual cancer cell but also on homotypic and heterotypic cell interactions in dynamic tumor ecosystems. Mitochondria are essential for life due to their position at the core of cellular metabolism and respiration. At the same time, they constitute the most actionable intracellular organelle to execute active cell death and ensure cell and tissue homeostasis. BCL-2 family members play a key executioner role by integrating various exogenous or intracellular signals into loss or maintenance of mitochondrial outer membrane (MOM) integrity. Upon overwhelming stress conditions, cells with an altered network of BCL-2 proteins, favoring BAX/BAK pore forming activity engage into a process of complete mitochondrial outer membrane permeabilization (MOMP), leading to massive activation of caspases. The latter proteases cleave many substrates actively preparing cells to die by apoptosis.Apoptosis is crucial during development and for tissue homeostasis, but is often impaired in cancer. Adaptation to apoptosis is now understood to be a fundamental step not only during tumor natural course (for initiation or progression), but also for tumor resistance to anticancer treatments including chemo- and radiotherapies. Resistance to treatment occurs through pre-existing or acquired mechanisms relying on genetic modification and/or rewiring of intracellular signaling pathways. It is a major factor driving tumor relapse with frequent metastasis and ultimately cancer-related deaths. The contribution of the mitochondrial apoptotic pathway, and of BCL-2 family members that regulate it, to cancer cell resistance justifies the development of agents targeting MOMP. Among these, BH3 mimetics are small molecules developed to inhibit pro-survival proteins , unleashing BAX/BAK to promote MOMP and apoptosis onset in otherwise aberrantly surviving cancer cells. Their success in treating hematological malignancies opens new therapeutic opportunities for a wider use in oncology. The dose-limiting secondary effects of these molecules nevertheless imply that more efficient therapeutic strategies need to be designed to fully exploit their therapeutic potential. This involves the development of predictors of efficiency in cancer cells, but also better understanding of the cross talks between apoptotic cell death and other modes of cell death whose triggering may lead to biological variations involved in cancer progression. The influence of these cross talks on the tumor microenvironment and the immune system also need to be deciphered. As a matter of fact, BAX/BAK dependent MOMP leads not only to cytochrome-c (cyto-c), SMAC cytosolic translocation and caspase activation to execute cell death, but also trigger viral mimicking inflammation. Moreover, caspases downstream of MOMP also influence proinflammatory signaling which contributes to tumor response or resistance to treatment and shape antitumoral immune response. We herein review how MOMP, and BH3 mimetics may modulate other cell death modalities and intracellular communications in the context of cancer treatment and discuss how it could reshape tumor ecosystem dynamics upon cytotoxic treatment, to better control tumor response and improve survival.Many studies have reported the contribution of BCL-2 family to tumor initiation, progression, or resistance to therapy ,2,3. AltEach member of BCL-2 family contains one or more BCL-2 homology (BH) domains (BH1 to BH4), discriminating multi-BH domain proteins (either pro- or anti-apoptotic) from BH3-only (activators or sensitizers) members. The multi-BH domain proteins BAX and BAK are the central pro-apoptotic members that execute MOMP. In addition, BOK an unconventional BAX/BAK-like BCL-2 effector, can also induce MOMP (even in the absence of BAX/BAK), but in contrast to BAX/BAK its activity on MOMP is constitutive and not regulated by BCL-2 family members but by proteasomal degradation . In normBCL-2 homologues prevent initiation and execution of MOMP by binding to BH3-only activators and to activated BAX/BAK molecules, respectively. Both types of interactions occur via the same binding of the hydrophobic face of the BH3 domain on the proapoptotic member into the hydrophobic groove formed by the BH1-BH3 domains in prosurvival proteins. This explains why binding to this interface  by BH3-only sensitizer proteins NOXA or BAD alleviate BAX or BAK from their apoptotic load and promote their activation. Sensitizers essentially function as competitive inhibitors of interactions between anti-apoptotic members and activators (or active BAX/BAK). Yet, proteins such as BCL-xL may be part of higher-order complexes engaging multiple BH3-only proteins that can be allosterically regulated by BH3-sensitizer in a non-competitive mode . Thus, MThe biochemical consequences of MOMP driven by BAX and BAK is in great part due to the release of soluble proteins from the mitochondrial intermembrane space, such as, as mentioned above, cyto-c, but also SMAC/DIABLO or Endonuclease G. Cyto-c in addition to its contribution to the electron transport respiratory chain in the intermembrane space, allows the formation of the apoptosome together with the adaptator molecule APAF-1 and the downstream activation of the caspase cascade when in the cytosol. The apoptotic executioner caspases are responsible for the cleavage of several hundreds of substrates and the characteristic morphological changes of apoptosis that include membrane blebbing, cell shrinkage, the formation of ‘‘apoptotic bodies,’’ and chromosomal DNA fragmentation.Many tissue specific transcriptional regulations and protein degradation processes, by the ubiquitin-proteasome system in particular, govern BCL-2 family proteins’ levels in cells . Cancer How quantity, stability, activity, and subcellular localization of BCL-2 family proteins tightly control MOMP onset in connection with the cellular context and regulation of BCL-2 proteins on the mitochondrial surface, is still subject to intensive research ,27,28. PThe fact that cancer cells strongly rely on BCL-2 homologues for survival maintenance is particularly blatant in hematological malignancies  but singGreat efforts were dedicated to the development of molecules that by inhibiting prosurvival protein functions, promote apoptosis in cancer cells. Small molecules called BH3 mimetics, that bind into the BH3 binding pocket of the prosurvival proteins BCL-2, BCL-xL, or MCL-1 (as BH3 domains of the proapoptotic ones do), favor mitochondrial apoptosis in cancer cells and have already useful clinical applications. In freeing the pro-apoptotic proteins sequestered by the prosurvival proteins, these molecules increase a proapoptotic load on BAX and BAK and promote subsequent MOMP in cells that are highly dependent on these proteins for their survival . HoweverThe first BH3 mimetic ABT-737 was discovered by Oltersdorf et al. in 2005 using nuclear magnetic resonance (NMR)-based screening of a chemical library to identify small molecules . ABT-737Exploiting the subtle differences between the binding interfaces of BCL-2 versus BCL-xL led to the identification of the BCL-2 specific (selective) BH3 mimetic ABT-199 or venetoclax. In 2016, US Food and Drug Administration (FDA) approved venetoclax for treating chromosomal 17p-deleted chronic lymphocytic leukemia (CLL) used as single agent ,37. RemaTargeting BCL-2 is most advanced clinically and has shown great potential in hematological malignancy but less efficacy in solid cancers. While hematopoietic malignancies appear addicted to a single pro-survival protein, the survival of cancer cells in carcinoma is often safeguarded by multiple pro-survival BCL-2 family proteins . For insSelective BCL-xL inhibition could be especially useful to treat tumors that often regulate BCL-xL as a mechanism of adaptation . ImportaTargeting MCL-1 by small molecules was more challenging because of its more complex structure, and several MCL-1 inhibitors have been produced using different strategies showing modest selectivity. However, the novel molecule S63845 exhibits high selectivity for MCL-1 over BCL-2 or BCL-xL and shows promising results with good tolerance in preclinical studies . MCL-1 mOverall, combining BH3 mimetics with chemotherapy is under intense investigation, in particular in solid cancers that exhibit more heterogeneous and evolutive survival dependencies towards BCL-2 family proteins . TargetiThe potential for BH3 mimetics to selectively kill cancer cells over normal cells is based on the concept of their higher apoptotic priming due to apoptotic oncogenic signaling and/or microenvironmental apoptotic pressure ,20. In fDespite complete response rates of up to 50% in CLL treatment by venetoclax, secondary resistance is the most frequent cause of treatment failure. Resistance mechanisms observed in CLL patients treated with venetoclax include the acquisition of BCL-2 mutations such as the Gly101Val mutation that reduce venetoclax binding to BCL-2, or compensatory overexpression of other pro-survival proteins BCL-xL and MCL-1 ,68,69,70In addition to the intrinsic resistance to tumor cells, a growing body of evidence indicates that support of tumor cell survival by cancer-associated fibroblasts  critically favors cancer progression and regrowth post therapy . We indeFailures of BH3 mimetics may rely on incomplete MOMP, on an unprimed cellular state or on survival addiction of cancer cells to other/several antiapoptotic proteins.MOMP is often rapid and complete, enrolling the majority of mitochondria in a given cell after apoptotic stress. Under these circumstances extensive MOMP appears as an all-or-nothing and as a point of no return , possiblIn some instances, at the level of a single cell, mitochondria may in fact undergo permeabilization in a less coordinated manner as usually understood, and this might contribute to resistance. An incomplete MOMP allowing cell survival was evidenced, with intact mitochondria repopulating a viable mitochondrial network . IncomplInsufficient BH3-only proteins in cells also results in resistance to MOMP and cyto-c release after mitochondria exposure to sensitizer BH3 peptides . ResistaAt cellular level, resistance to BH3 mimetics may also be influenced by dynamic changes in apoptotic signaling induced by anticancer treatments. Montero and colleagues interrogated the dependence of cancer cells on the BCL-2 family proteins after targeted cancer therapies and observed that multiple oncogenic driver-targeting therapies, including BRAF or EGFR inhibitors, eventually downregulate NOXA expression because the MAPK-dependent pathway favors NOXA mRNA stabilization. This generates a dependence on MCL-1 in treated cancer cells, that can be therapeutically overcome by sequential inhibition of BRAF and MCL-1 in preclinical studies . The effAnother limitation to the use of BH3 mimetics is that they may inhibit neither fully the canonical function of their targets nor their non-canonical functions. Prosurvival BCL-2 proteins may interact with their pro-apoptotic counterparts through their BH3-binding site but also through other interfaces, as described by Andrews and colleagues for interactions between BIM and either BCL-2 or BCL-xL, that engage into ABT-737 resistant complexes . AntiapoWith the initial view of MOMP as an action switch button in the apoptotic process, BH3 mimetics offered the promise of triggering massive and complete apoptosis in cancer cell populations. However, as mentioned above, extensive exploration of BH3 mimetics effects and resistant clinical cases underline that their action is disappointing on that aspect. Indeed, these compounds induce fractional killing at best (like many other cytotoxic agents), occasionally sparing mitochondria subsets and possibly failing to induce full caspase activation. This may cast doubt on their usefulness as anticancer agents in the long term in solid tumors. As discussed below, however, the use of BH3 mimetics as laboratory tools to explore the biological effects of mitochondrial integrity alterations has, at the same time, helped redefine the biological role of MOMP and highlight the abundant intracellular crosstalks between distinct cell death pathways. In part because it corresponds to a rupture of the symbiotic relationship between mitochondria and the host cell, MOMP is involved in cell death modes that are more inflammatory than apoptosis stricto sensu and caspases regulate this process. Thus, in addition to target cancer cells, BH3 mimetics may be useful to recruit and amplify the effects of anti-tumoral immunity.Mitochondrial apoptosis is classically regarded as immunologically silent. This feature is achieved by limited plasma membrane breakdown, selective release of cellular contents, and display of ‘‘find me’’ and ‘‘eat me” signals by apoptotic cells to trigger the rapid phagocytic clearance of apoptotic bodies. Chekeni and colleagues showed that apoptotic cells are not passive during this process of removal; they actively maintain certain metabolic pathways and only selected metabolites are released through caspase-mediated opening of pannexin 1 channel in the plasma membrane. These function as tissue messengers triggering in neighboring cells proliferation, wound healing, and suppression of inflammation . HoweverParadoxically, with regard to its major role in apoptotic cell death, MOMP is inflammatory. In 2014, two studies (using BH3 mimetics) showed that during BAX-BAK dependent MOMP, mitochondrial DNA (mtDNA) is released in the cytosol where it activates the cytosolic DNA sensor pathway cGAS/STING to promote, as does viral infection, an inflammatory reaction with type I IFN expression ,96. MechIn the case of cGAS activating  MOMP, induction of type I interferon (type I-IFN) pathway is blunted by downstream caspases and only unmasked when caspases are inhibited or genetically ablated . This neThe antagonistic effects of MOMP and of downstream caspases on the anti-tumoral secretory phenotype of cells committed to viral mimicry and/or necroptosis advocates that caspase inhibition may be beneficial for anticancer treatment and that a contrario, caspase activation by apoptosis inducing agents (which BH3 mimetics are by design) is not. Previous studies have already reported that caspase inhibitors can have antitumor effects . Most rePyroptosis is a form of cell death most commonly occurring as an innate immune response, depending on the cleavage of caspase-1 by inflammasome complexes leading to the activating cleavage of the proinflammatory cytokine IL-1β (a pleiotropic mediator of inflammation) and the cytosolic protein gasdermin D (GSDMD) promoting its capacity to form membrane pores leading to cell lysis ,115. InfThese ambiguous connections raise the question of whether caspase activity in a whole tumor is linked in any way to its immune status. A universal role for apoptotic caspase activity in antagonizing inflammatory processes would predict that high levels of active caspases should negatively correlate with inflammation markers and immune infiltration. Exploration of public databases comprising matched proteomic and RNA expression data indicate that this is not always observed . It is Notably, cell death mode in response to MOMP may not only be determined by downstream caspase activation but also by additional mitochondrial modifications. During MOMP, progressive mitochondrial dysfunction linked to electron transport chain decoupling from ATP synthesis, inducing ATP loss and ROS accumulation, mediates a metabolic catastrophe that contributes to cell demise. This underlies the success of ROS-targeting therapeutic strategies (such as ionizing radiation or oxidant-producing chemotherapeutics) in the treatment of cancers. ROS foster the mitochondrial permeability transition linking mitochondrial metabolic dysfunction to apoptosis . UnderscMOMP may also support ferroptosis which is characterized by iron-dependent accumulation of lipid hydroperoxides to lethal levels in close relation with ROS accumulation, that occurs through progressive mitochondria dysfunction. In some circumstances, both cell death pathways ferroptosis and autophagy-dependent cell death may also crosstalk to apoptosis through ROS production related to mitochondria dysfunction or through Beclin interaction with BCL-2 antiapoptotic proteins, respectively . How theWith the almost systematical, yet pleiotropic, influence of mitochondrial apoptotic stress on inflammation, chemosensitivity, immune response, and tumor progression following  therapy, arguably relies on how treatments remodel the tumor microenvironment and adaptative immunity. In particular their influence on intercellular communications between malignant clones and non-malignant cells is critical: committed or dying cells can generate active signals that drive tumor response to anticancer treatments.It is established that exposure to chemotherapeutic agents can change the types and abundance of components in the tumor secretome. For example, IL-6 and IL-8 are frequently induced, and their expression strongly correlates with tumor recurrence and poor responsiveness to therapy in various cancers . They prIn addition to soluble messengers, committed cells also provide extracellular vesicles that can transport a variety of cellular contents opening an intercellular communication that emerges as an important mechanism in the development of chemoresistance  Figure . As an eAltogether, these observations indicate that tumor cells, including those dying upon anticancer treatments, intensively communicate with the tumor environment through many routes and that these communications critically shape the global therapeutic response. The numerous connections between MOMP and “immunogenic” cell death modes imply that BH3 mimetics, even if they target limited cell subsets, may nevertheless be useful if they exploit the anti-tumoral effects of these communications (spreading therapeutic response) and/or if they can target cells that produce pro-tumoral signals. Our recent data argue that efficient strategies will have to find a compromise between the biochemical pathways leading to the production of death signals and the own vulnerabilities of cells elaborating them. Indeed, we found that cGAS active cancer cells following anti-mitotic treatments produce a TNF/type I-IFN pro-apoptotic secretome that induces NOXA expression in neighboring cells, rendering them dependent on BCL-xL . These dProgress made in the past decades in understanding how the BCL-2 family of proteins controls mitochondrial integrity and survival in malignant and non-malignant cells has provided an unprecedented opportunity to target this pathway using BH3 mimetics for efficacious cancer treatment. BH3 mimetics provide great opportunities to treat hematological malignancies either as single agent or in combined regimen with low toxicity. In solid tumors survival dependencies are less evident maybe due to the higher level of heterogeneity or phenotypical plasticity. Combining chemotherapies with BH3 mimetics often provides a better tumor response in numerous preclinical studies but remains to be confirmed in clinical trials.BH3 mimetics allowed to explore the full spectrum of MOMP biological effects. Related studies have shown that mitochondrial apoptosis and alternative cell death pathways are entwined. In retrospect, an alternative interpretation of the seminal observation made by Letai and colleagues in 2011 (that predisposition to MOMP prior treatment correlates with chemotherapeutic efficiency ) may be A growing body of evidence indicates that during treatments committed cells can actively contribute to the expansion and prolongation of therapy-induced effects through apoptotic spreading within the tumor as well as immune response activation and maintenance over time. Therapeutic induction of MOMP should therefore not be evaluated on the sole basis of its effect on each individual cancer cell apoptotic onset, but also on its ecosystemic effect. Arguably, caspase activation may provide unsuitable secondary effects by preventing inflammation and mask BH3 mimetic efficiency. This should be considered by designing therapeutic strategies that trigger death onset in the appropriate cell types but also rigorously influences the mode of cell death executed. To reach this objective, the role of the BCL-2 proteins and of MOMP in shaping progression and response to treatment in tumor ecosystems need further investigation to better unravel the intricate and possibly actionable communications involved, including during treatment where adaptative resistance may install.


Across groups, CoM displacement during perturbation exposure increased when directed towards the impaired or non-dominant limb with no influence of timing knowledge. In addition, peak CoM displacement was less with known timing irrespective of the perturbation direction. Generally, the CoM displacement during perturbation exposure correlated well with the CoM medial-lateral velocity during unperturbed walking, supporting evidence that human response dynamics to lateral perturbations are influenced by the instantaneous state of the body’s momentum.Previous research found that below-knee prosthesis users proactively increase their lateral margin-of-stability on their impaired side in anticipation of an impending perturbation when the timing is predictable and potentially directed toward the impaired limb. While knowledge of perturbation timing and direction influences proactive strategies, the consequences of such knowledge and anticipatory behavior on recovery from perturbations is unclear. This study characterized center-of-mass (CoM) dynamics of below-knee prosthesis users and non-impaired controls following a lateral perturbation when the perturbation direction is known but  Maintaining frontal-plane locomotor stability is a known challenge for below-knee prosthesis users (BKPUs) , 2. Conta priori knowledge of an impending discrete lateral perturbation [BKPUs and non-impaired individuals use a combination of control mechanisms during walking to maintain the periodic frontal-plane oscillations of their center of mass (CoM) within their base of support , 14–16. urbation , BKPUs wurbation . This ina priori but timing knowledge is varied. In light of our previous research finding that knowledge of perturbation timing and direction influenced BKPUs anticipatory strategies [known or unknown and directed toward their impaired limb that prosthesis users would exhibit a greater lateral CoM displacement when compared to the effects of perturbations directed towards their sound limb. We also hypothesized that BKPUs’ strategy of increasing their lateral MoS when perturbation timing is both known and possibly directed toward the impaired limb [It is difficult to evaluate how the cumulative effect of these control mechanisms impact BKPUs’ capacity to respond to discrete lateral perturbations. Thus, the primary aim of this study was to characterize the resulting CoM dynamics of BKPUs wearing passive ankle-foot prostheses and non-impaired controls following discrete lateral perturbations during walking when the direction of the perturbations were known rategies , we hyporategies –10, whenred limb  would rered limb  and limbred limb , a seconThe study protocol was approved by the Northwestern University Institutional Review Board (IRB # STU00200872) and all participants provided informed written consent prior to study involvement. The individual pictured in this manuscript has given written informed consent (as outlined in PLOS consent form) to publish these case details. We recruited both BKPUs and non-impaired participants. Inclusion criteria for all participants included: 18 to 65 years of age, normal/corrected vision, and able to walk continuously for 10 minutes without undue fatigue or health risks. BKPUs were also required to possess a unilateral below-knee amputation, be daily users of their clinically prescribed prosthesis for ambulation, walk in the community without a mobility aid, prosthesis usage for at least one year, and a residual limb in good condition . Exclusion criteria for all participants included: self-identified musculoskeletal (apart from amputation) and/or vestibular pathologies impairing balance and/or stability, taking medications affecting proprioception and/or balance, and cognitive deficits that preclude understanding of testing instructions.Participants walked on a treadmill  with an oversized belt (1.39m width) that provided space for responding to lateral perturbations. For safety, participants wore a passive overhead harness  adjusted so it did not restrict lateral movement. A cable-driven robotic device , 30 deliA ten-camera motion capture system  recorded 3-D positions (100 Hz) of retro-reflective markers attached to the pelvis  and bilaterally on the greater trochanter, lateral knee, lateral malleolus, calcaneus, and second and fifth metatarsals. Prosthetic foot marker positions approximately matched the sound foot. The motion capture system also collected synchronized kinetic data from the robotic device’s load cells at 1000 Hz.Participant demographics and additional health history from BKPUs on prosthesis use (type and experience), time since amputation, amputation etiology, functional balance, and socket comfort were collected first. The Berg Balance Scale, a valid and reliable outcome measure for lower limb prosthesis users , and thea priori knowledge of the lateral perturbation:Participants performed a series of walking trials at their preferred speed that were grouped into blocks based on participants' Baseline–no perturbations were appliedUnknown Time and Unknown DirectionUnknown Time and Known DirectionKnown Time and Known DirectionKnown Time and Unknown DirectionBold conditions were analyzed for this experiment. We have previously analyzed this dataset and reported on anticipatory adaptations for all the above conditions [* Only nditions .Each block contained six trials. The order of the blocks were randomly presented to minimize order bias. All blocks, except for baseline, were repeated twice to collect equal numbers of right- and left-directed perturbations. Each trial included 20 seconds of continuous walking. Participants received a single discrete lateral perturbation during each non-Baseline trial. Results analyzing the anticipatory locomotor strategies for all five conditions have been published separately . Here weA large arrow pointing to the right or left was displayed on the monitor to provide knowledge of the perturbation direction . A visuaParticipants were instructed to walk along the center of the treadmill, defined by a distinct yellow chalk line, and to return to the treadmill center as quickly as possible after each perturbation. Participants were specifically instructed not to consider the chalk line as a “tight-rope.” To minimize fatigue bias participants were allowed to rest between individual trials and test blocks for as long as requested. Although participants regularly rested between blocks, none chose to rest between trials and so each block was tested during continuous walking. However, subsequent trials were started only after the participant was observed to have fully regained steady-state walking (approximately 20 steps). The treadmill was gradually stopped after each block.Using Visual 3D software , marker position trajectories were low-pass filtered , and the CoM position was estimated as the center of a pelvis model built from the pelvis markers. Evidence suggests that this simple approximation of CoM position is comparable to more complex multi-segment models when estimating instantaneous medial-lateral CoM position .Visual 3D was also used to estimate initial foot contact and toe-off events for each limb as the maximum anterior distance between the calcaneus and pelvis center, and maximum posterior distance between the fifth metatarsal and pelvis center, respectively. Accuracy of these event times were manually  confirmed and adjusted as appropriate.Response dynamics to each perturbation were characterized through three features of the temporal medial-lateral CoM position across a given trial :ΔCoMexposureΔCoMexposure= CoM position at perturbation end–CoM position at perturbation onset);CoM displacement in the same direction as the perturbation during the perturbation exposure period of 400 msec (ΔCoMpeakPeak CoM displacement in the same direction as the perturbation following the perturbation onsetCoMpeak= Maximum CoM position following the perturbation onset–CoM position at perturbation onset);. that the displacement was slightly in the direction opposite to the perturbation. As relevant to the primary set of hypotheses, the CoM displacements and time to reversal were used to characterize the spatial and temporal response components, respectively. While the temporal component did not directly address our hypotheses, it was considered an important metric to help interpret reactive control.Custom MATLAB  software was used to calculate each of these parameters for a given trial and the average values were calculated for each test condition across the six trials. The two CoM displacement metrics were normalized to body height and replaced with a value of zero in the rare occasion  or non-dominant (control) limb. Plots were created with data separated by perturbation direction, group, and timing knowledge, and zero values corresponding to no displacement in the perturbation direction were removed.To address the secondary aim, identifying the influence of gait phase on acute response dynamics, ΔCoMexposure, ΔCoMpeak, and Treversal. Specifically, analyses of ΔCoMexposure and ΔCoMpeak addressed the first and second hypotheses, respectively. The absence of violations of normality that would affect the results given robustness of mixed ANOVA was confirmed using the Shapiro-Wilk test and observation of Q-Q plots of the model residuals. Assumptions of homogeneity of variance and covariance were confirmed with the Levene’s test and Box’s test, respectively. Effect sizes were estimated using the partial η2 values.To address the primary aim of assessing the direction and timing knowledge effects on CoM response dynamics, a three-way mixed ANOVA  was used to test the main and interaction effects of timing knowledge , direction , and group  on ΔCoMexposure). Graphical observation of ΔCoMexposure versus perturbation onset time suggested that for both groups the CoM displacement profile: 1) resembled that of the baseline (unperturbed condition) temporal CoM medial-lateral velocity across the gait cycle, and 2) was not considerably influenced by timing knowledge. Consequently, the data were reduced to four sets separated by group and perturbation direction, and for each dataset a repeated-measures correlation analysis was conducted between ΔCoMexposure and instantaneous baseline CoM medial-lateral velocity to determine the strength and significance of their relationship across participants while accounting for paired data within participants [ΔCoMexposuredata point was temporally advanced by the percentage of the average baseline stride time corresponding to 200 msec (16% for BKPUs and 19% for controls) representing the temporal midpoint of the perturbation.To address the secondary aim of assessing how perturbation response was affected by gait-phase dependent factors that theoretically influence CoM dynamics, we focused on evaluating the relationship between instantaneous lateral CoM velocity and CoM displacement during the exposure period  and R statistical software , respectively, using a critical alpha value of 0.05.Participants included 13 non-impaired controls  and 6 BKPUs  using their customary prostheses. The BKPUs  for controls and BKPUs were 1.3±0.1 m/s and 0.8±0.3 m/s, respectively. The perturbations noticeably destabilized all participants, but none experienced a failed recovery . Recovery was achieved through either side or cross-over stepping strategies . Plots ΔCoMexposure, ΔCoMpeak, and Treversal are displayed in Figs ΔCoMexposure versus gait cycle for controls and BKPUs are presented in Average ΔCoMexposure, (2 = 0.240), with displacement greater with perturbations towards the impaired/non-dominant side, but the main effect of group, main effect of timing, and interaction effects were not significant ≤1.541, p≥0.231, partial η2≤0.083).For xposure,  the mainΔCoMpeak, (2 = 0.331), with less displacement when perturbation timing was known, but the main effect of group, main effect of direction, and interaction effects were not significant ≤2.933, p≥0.105, partial η2≤0.147).For CoMpeak,  the mainTreversal, (2 = 0.219), with more time when perturbation timing was known, but the main effect of group, main effect of direction, and interaction effects were not significant ≤1.912, p≥0.185, partial η2≤0.150).For eversal,  the mainΔCoMexposure and ΔCoMpeak, respectively, without significant interaction effects, but found but no significant main or interaction effects on time to reversal. While key statistical values have been presented, findings should be interpreted given these collective results.To note, secondary three-way ANOVAs were performed on these data with a randomly selected subset of six participants from the able-bodied sample to create a balanced design. The results from this secondary analysis confirmed the significant main effects of perturbation direction and timing knowledge on ΔCoMexposure and unperturbed CoM medial-lateral velocity (The repeated-measures correlation coefficients (r) evaluating the relationship between velocity  at correa priori knowledge of onset timing of a lateral perturbation on the CoM response dynamics of BKPUs and non-impaired controls. We hypothesized that BKPUs would exhibit a greater lateral CoM displacement when perturbations directed toward their impaired limb occurred, irrespective of timing knowledge, when compared to the effects of perturbations directed toward their sound limb. Further, we hypothesized that due to the additional anticipatory control strategies observed in BKPUs when perturbations of known timing were possibly directed toward their impaired limb, there would be smaller CoM displacements resulting from known timing perturbations than unknown time perturbations. These hypotheses were generally supported when considering both groups and depended on the CoM displacement metric, but no group or interaction effects were significant to suggest that responses were not specific to BKPUs.This study explored the effects of direction and ΔCoMexposure, the acute displacement of the CoM during the period of perturbation exposure, was greater when BKPUs and control participants were perturbed towards the impaired/non-dominant limb respectively  was 400 msec. While this perturbation period was used to generate an acute disturbance, this feature also made it difficult to isolate the effects of the perturbation to a specific instant in the gait cycle. Second, the study design involved random delivery of perturbations during the gait cycle and so the timing of the disturbance relative to cycle was not controlled. Consequently, the secondary aim of evaluating the relationship between gait-cycle dependent COM velocity and perturbation response could only be achieved through the available data and not a systematic sweep of cycle time. Fourth, the relatively small BKPU sample size limited the statistical power of this study, and therefore may have reduced ability to detect group differences. Moreover, the response behavior findings in this study resulted from analyses across groups and were not specific to BKPUs to directly support or reject the stated hypotheses. However, these results revealing significant main effects of perturbation direction and knowledge of timing but non-significant group effects suggest directions for future investigation on this topic but with larger BKPU sample sizes for greater statistical power. Furthermore, the tested sample of community-dwelling BKPUs demonstrated high levels of baseline balance function, and the results are therefore limited by the representativeness of cohort. However, fall risk is relevant for prosthesis users at all stages in the rehabilitation journey , includiThe results of this work have particular clinical implications. The greater COM displacement on the impaired and non-dominant limb side during perturbation exposure might suggest greater potential to fall towards that side. For BKPUs particularly, a greater excursion of the COM when ankle strategies are lacking would require some alternative motor strategy to maintain balance and possible reliance on hip musculature . Furthera priori timing knowledge of a medial-lateral perturbation on response behavior of BKPUs and non-impaired controls. The results suggest that the displacement of the COM during the short exposure period of the perturbation was greater on the impaired or non-dominant limb for BKPUs and controls, respectively. However, peak COM displacement was reduced for both cohorts when the timing of the perturbation is known, which also increased the timing delay between perturbation onset and redirection of the COM back towards center. Importantly, medial-lateral COM displacement during perturbation exposure appears to be coupled with instantaneous medial-lateral COM velocity during the gait cycle. Knowing how these factors (a priori knowledge and gait cycle timing) affect response behavior may facilitate the design of perturbation paradigms to train for improved locomotor stability.This study observed the effects of direction and