Almost 140 years after its discovery, tuberculosis remains the leading infectious cause of death globally. For half a century, patients with drug-sensitive and drug-resistant tuberculosis have undergone long, arduous, and complex treatment processes with several antimicrobials that primarily function through direct bactericidal activity. Long-term utilization of these antimicrobials has been well-characterized and associated with numerous toxic side-effects. With the prevalence of drug-resistant strains on the rise and new therapies for tuberculosis urgently required, a more thorough understanding of these antimicrobials is a necessity. In order to progress from the “one size fits all” treatment approach, understanding how these antimicrobials affect mitochondrial function and bioenergetics may provide further insight into how these drugs affect the overall functions of host immune cells during tuberculosis infection. Such insights may help to inform future studies, instigate discussion, and help toward establishing personalized approaches to using such antimicrobials which could help to pave the way for more tailored treatment regimens. While recent research has highlighted the important role mitochondria and bioenergetics play in infected host cells, only a small number of studies have examined how these antimicrobials affect mitochondrial function and immunometabolic processes within these immune cells. This short review highlights how these antimicrobials affect key elements of mitochondrial function, leading to further discussion on how they affect bioenergetic processes, such as glycolysis and oxidative phosphorylation, and how antimicrobial-induced alterations in these processes can be linked to downstream changes in inflammation, autophagy, and altered bactericidal activity. Mycobacterium tuberculosis (Mtb) and remains the leading cause of death by a single infectious agent in the world. In 2018, TB disease resulted in ~1.5 million deaths and still presents a major clinical challenge globally  is caused by infection with lly WHO, . Treatmelly WHO, . The reglly WHO, . Treatmelly WHO, . Table 1lly WHO, .Mtb. Although many TB antimicrobials have yet to be investigated in terms of host immune modulation, this review aims to demonstrate the importance of such future studies. As The use of these antimicrobials has been the mainstay for TB treatment for almost half a century now. Despite the multitude of readily available antimicrobials, TB still persists as a major global infectious hazard due to the increasing prevalence of drug-resistant strains, as well as other factors such as immunosuppression, the prevalence of latent TB infection and its mode of transmission. This necessitates the need to identify better therapies to help simplify and shorten treatment times. Recently, much attention has turned to the use of host-directed therapy (HDT) to help improve TB treatment through the enhancement of immune responses in infected host immune cells  which profoundly changes how these cells respond to Mtb infection  and the damage associated molecular patterns (DAMPs) that are engaged.The mitochondria play key protective roles during TB infection   increases ROS production and decreases expression of liver fatty acid binding protein (L-FABP) as well as its target gene, PPAR-α, in zebrafish larvae  and levofloxacin (LVX) inhibit The ribosomal inhibitor, linezolid (LZD), elicits a range of toxic side effects after long-term treatment, with disturbances in mitochondrial function thought to be the main cause of the majority of these adverse effects  , an inhibitor of bacterial F1/FO ATP-synthase , and the citric acid (TCA) cycle play in the host response to Mtb infection  directly disrupts the ETC, targeting copper chelation and inhibits complex IV activity in human fibroblasts, resulting in a decrease in OXPHOS efficiency, as measured by a reduction in oxygen consumption  and CIP also increase lactate production in primary human cells and cell lines, however, at high concentrations, these drugs have the opposite effect, decreasing glycolytic activity, as measured by reduced extracellular lactate production , could also help infected immune cells to fight a variety of both bacterial and viral infections.In conclusion, future studies are required in elucidating how TB antimicrobials affect mitochondrial function and bioenergetic profiles JP: conceptualization and software. JK and JP: funding acquisition and administration. JP and CC: visualization and writing-original draft. All authors: validation, writing-review, and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dear Readers,The coronavirus disease 2019 (COVID-19) pandemic has adversely affected the field of cardiac electrophysiology in a significant way. Most tragic has been the loss of colleagues, friends, and relatives. Many hospitals in heavily affected areas have halted some or all elective procedures. Finally, scientific conferences were either canceled or changed to a virtual format and in-person patient visits have been modified to the same.2 Also, recent advances in technology have allowed the safe discharge of patients after atrial fibrillation ablation; these include the use of vascular ultrasound to guide vascular access, intracardiac echocardiography to reduce the risk of cardiac perforation and assess the pericardial space at the end of the procedure, uninterrupted oral anticoagulation, improved ablation technologies allowing for faster pulmonary vein isolation, and venous closure devices to facilitate patient mobilization just two hours after the procedure. There is significant evidence supporting this practice and research suggests that same-day discharge after atrial fibrillation ablation is safe and not associated with higher hospital readmission or complication rates after discharge.3However, the COVID-19 pandemic has also accelerated the adoption of some beneficial trends, one of which is same-day discharge after electrophysiology procedures, which has served to free up hospital beds and reduce the risk of viral infection. Early mobilization and same-day discharge after cardiac implantable electronic device placement have been practiced in some centers for many years, albeit excluding high-risk patients such those at risk for bleeding or on oral anticoagulation, those with complete heart block, those with secondary-prevention implantable cardioverter-defibrillator indications, and those experiencing immediate procedural complications. Studies have shown that this approach may lead to greater patient satisfaction and cost savings without adversely affecting readmission rates.As the COVID-19 pandemic continues, pursuing same-day discharge where appropriate may help us to limit the effects of this public health crisis on the field of cardiac electrophysiology and continue to provide our patients with safe and timely care.Best wishes for a happy holiday season.Sincerely,MD, FHRS, FACCMoussa Mansour, Editor in ChiefThe Journal of Innovations in Cardiac Rhythm ManagementMMansour@InnovationsInCRM.comDirector, Atrial Fibrillation ProgramJeremy Ruskin and Dan Starks Endowed Chair in CardiologyMassachusetts General HospitalBoston, MA 02114


Heparin-induced thrombocytopenia is an immune-mediated reaction to heparin and heparin analogs, which results in an acquired hypercoagulability syndrome resulting in paradoxical arterial and venous thrombosis leading to thrombocytopenia. Organs with high vascularity, such as the adrenal glands, are at an increased risk of injury in heparin-induced thrombocytopenia due to thrombus formation in the adrenal vein causing adrenal insufficiency. The standard of treatment remains discontinuation of heparin and heparin analogs and starting corticosteroids and non-heparin antithrombotic therapy such as argatroban. Patients on heparin therapy for either venous thromboembolism treatment or prophylaxis have an increased thrombotic risk due to heparin-induced thrombocytopenia. Heparin-induced thrombocytopenia is seen in patients either on unfractionated heparin or on low molecular weight heparin. It is an immune-mediated reaction to heparin causing a prothrombotic state which causes paradoxical arterial and venous thrombosis leading to thrombocytopenia. Adrenal glands are susceptible to injury as they are highly vascular structures. Acute adrenal insufficiency due to heparin-induced thrombocytopenia should be high on differentials for patients on heparin therapy, if the patient develops pain in the abdomen, hypotension, and fever with thrombocytopenia. We report a case of heparin-induced thrombocytopenia leading to acute adrenal insufficiency in a critically ill patient and was treated with discontinuation of heparin and starting corticosteroids with improvement in the clinical outcome for the patient.A 64-year-old female with a past medical history of end-stage chronic obstructive pulmonary disease, active recreational heroin use, and previous deep vein thrombosis of lower extremities was admitted to the medical intensive care unit for acute on chronic hypercarbic respiratory failure secondary to opioid overdose and exacerbation of end-stage chronic obstructive pulmonary disease requiring intubation and mechanical ventilation. At the time of admission, the patient was hypothermic at 93.7 Fahrenheit, tachycardic to 116 bpm with elevated white blood cells (WBC) count, and was started on empiric antibiotics. Blood work showed troponin of 0.8 ng/mL, platelet at 216,000/µL, and hemoglobin at 9.6 g/dL. The patient was also found to have non-ST segment elevated myocardial infarction, at the time of admission, for which the patient was started on heparin drip for anticoagulation  for heparin-induced thrombocytopenia and heparin drip was discontinued. The patient was also found to be hypotensive with mean arterial pressure ranging between 58-63 mmHg and had a low-grade fever. When asked about pain in the abdomen, the patient complained of diffuse body pain due to opioid withdrawal. The direct Coombs test was negative. Other laboratory values were remarkable for prolonged prothrombin time (PT) and partial thromboplastin time (PTT), elevated d-dimer, low fibrinogen, suggestive of disseminated intravascular coagulopathy, secondary to adrenal insufficiency. The patient also developed microangiopathic hemolytic anemia with low hemoglobin, high lactic dehydrogenase (LDH), low fibrinogen, and low haptoglobin and peripheral blood showing schistocytes secondary to disseminated intravascular coagulopathy.Computed tomography of the head was negative for hemorrhage and computed tomography abdomen/pelvis, and computed tomography angiogram of abdomen/pelvis were negative for retroperitoneal bleeding. The adrenal gland could not visualized be on a computed tomography angiogram of the abdomen and pelvis.The patient’s hemodynamic instability was most likely due to adrenal insufficiency. The patient was transfused multiple units of red blood cells and fresh frozen plasma and was started on stress dose steroids with hydrocortisone, ascorbic acid, and thiamine (HAT therapy) after which her blood pressure improved with resolution of thrombocytopenia, and the patient was transferred from medical intensive care unit to the general medical floor. The patient, unfortunately, died due to complications related to the end-stage chronic obstructive pulmonary disease.Heparin-induced thrombocytopenia occurs in patients who are undergoing heparin treatment, resulting in an acquired hypercoagulability syndrome. It is defined as an immune-mediated reaction to heparin products resulting in a paradoxical arterial and venous thrombosis leading to thrombocytopenia .Heparin-induced thrombocytopenia is an autoimmune phenomenon involving immunoglobulin G (IgG) antibodies that attach to the heparin-platelet factor-4 protein complex leading to platelet activation. The induced platelet activation leads to systemic thrombosis and platelet consumption, which eventually presents as thrombocytopenia. The adrenal glands in particular are at a higher risk of injury due to heparin-induced thrombocytopenia, due to their vascularity. The vascular supply to each adrenal gland is drained by a single central adrenal vein, which is highly susceptible to outflow obstruction. The systemic thrombosis caused by heparin-induced thrombocytopenia can cause adrenal vein thrombosis which subsequently leads to arterial adrenal gland hemorrhage and eventual life-threatening adrenal crisis . The immHeparin-induced thrombocytopenia, which is an immune-mediated disorder, can cause adrenal insufficiency and should be suspected if patients have abdominal pain and fever with hypotension. Our case underlines the importance of maintaining a high index of awareness for the occurrence and impact of the recognition of heparin-induced adrenal insufficiency and its implications on the clinical course of critically ill patients and treating this subset of patients with discontinuation of heparin and starting corticosteroids.


Alcohol use disorder is a pervasive healthcare issue with significant socioeconomic consequences. There is a plethora of neural imaging techniques available at the clinical and preclinical level, including magnetic resonance imaging and three-dimensional (3D) tissue imaging techniques. Network-based approaches can be applied to imaging data to create neural networks that model the functional and structural connectivity of the brain. These networks can be used to changes to brain-wide neural signaling caused by brain states associated with alcohol use. Neural networks can be further used to identify key brain regions or neural “hubs” involved in alcohol drinking. Here, we briefly review the current imaging and neurocircuit manipulation methods. Then, we discuss clinical and preclinical studies using network-based approaches related to substance use disorders and alcohol drinking. Finally, we discuss how preclinical 3D imaging in combination with network approaches can be applied alone and in combination with other approaches to better understand alcohol drinking. Alcohol abuse is a pervasive societal problem with substantial socioeconomic and medical consequences . AlcoholIn humans, the emotional and physical signs of withdrawal from alcohol can manifest in several ways such as increased anxiety, increased irritability, increased frustration, increased aggression, mood swings, insomnia, tremors, convulsions, higher blood pressure, accelerated pulse, accelerated breathing, dehydration, and delirium tremens ,12,13,14Preclinical animal models, especially in rats and mice, are ideal for studying alcohol drinking and alcohol addiction due to the ability to adequately model negative affect and excessive drinking behavior. Some of the most prominent animal models to study excessive and binge-like drinking are the intermittent access to ethanol (IAE) model, using two-bottle choice ,58,59,60Rats and mice have also been selectively bred to express excessive drinking behavior, allowing for greater focus on genetic aspects associated with increased drinking. In rats, the alcohol-preferring strain, the high alcohol-drinking strain, and the Marchigian Sardinian alcohol-preferring rats have been examined ,77,78,79To study excessive and compulsive-like drinking due to alcohol dependence, the intermittent ethanol (CIE) vapor exposure model has been heavily used in both rats and mice ,104,105.Although our knowledge of the neurocircuitry that underlies excessive alcohol drinking and alcohol dependence has improved dramatically over the last decade ,106, theThe development of site-specific recombinase systems ,108,109 The optogenetic method takes advantage of the genetic incorporation of light-gated cation channel channelrhdopsin-2 (ChR2) and chloride and proton pumps  into neural tissue ,112,113.Optogenetic approaches in preclinical models have been used to identify numerous circuits throughout the brain involved in alcohol drinking. Several studies have used optogenetic manipulation to identify projections from the central amygdala (CEA) that are critical for addiction-like behaviors ,117,118.Designer receptors exclusively activated by a designer drug (DREADDs) are a class of chemogenetically engineered receptors activated by small molecules . DREADDsSeveral studies have taken advantage of chemogenetics to explore mechanisms of alcohol drinking. For example, a reduction of alcohol consumption in mice was found by a suppression of activity in the NAc core with hM4Di-mediated inhibition . PharmacTwo-photon calcium imaging is a method for monitoring the in vivo activity of distinct neurons in brain tissue . This alOne of the most commonly used neuroimaging techniques is magnetic resonance imaging (MRI), which is relatively fast, non-invasive, and can be done in vivo. MRI takes advantage of the ability of atomic nuclei to absorb radio frequency (RF) in the presence of an external magnetic field . Then, tRecent developments in tissue-clearing methods allow for three-dimensional (3D) imaging of intact tissues, such as the whole brain. Hydrophobic and hydrophilic tissue-clearing methods are both solvent-based and generally remove lipids, pigments, and calcium phosphate to reach an appropriate refractive index for imaging. The 3D imaging of tetrahydrofuran (THF) cleared organs (3DISCO) was developed by Ertürk et al. The use of THF, a dehydrating and de-lipidating agent, instead of alcohol enhances the refractive index homogeneity of the samples . Renier Network-based approaches are highly applicable to neuroscience and have been used to analyze large sets of data. In neuroscience, networks can be examined at the structural or functional connectivity level. Structural networks are mapped by measuring the physical inter-neuronal connectivity or inter-regional connectivity, whereas functional networks examine the effective connectivity of neural activity between regions . MethodsGraph theory has been used to identify specific features of neural networks in more detail. Graph theory is a branch of mathematics that is used to analyze complex networks ,178,179.In terms of brain mapping, numerous studies have examined the human brain . Many ofModularity is defined as the division of a network into modules. A network expressing high modularity consists of nodes with high intramodule connectivity and sparse intermodule connectivity . MathemaIn modeling neural networks, hub brain regions will emerge that are characteristic of the specific brain state being examined. A hub is defined as a node with high connectivity to other nodes in the same module  or to other modules (connector hubs) ,192. HubThere have been attempts to identify modules and neurocircuitry involved in SUDs from large-scale analysis of prior literature ,195. HowAlcohol-dependent individuals and binge drinkers have been shown to have major alterations to functional connectivity in several studies. Resting-state functional connectivity in executive control brain regions has been found to be reduced in subjects suffering from AUD ,208. AcuFos protein begins to be produced at detectable levels approximately 30 min after neural activity, and peaks at 60 to 90 min, and returns to baseline at approximately 180 min; thus, the data from Fos protein represents neural activity across minutes to hours. This method allows immunostaining for multiple proteins and combination with other approaches such as optogenetics, chemogenetics, or pharmacology. This approach allows for data to represent normal awake-behaving neural activity, allowing for multiple experimental paradigms to be explored. Some limitations to single-cell whole-brain imaging are that it is ex vivo and only samples a single timepoint. In contrast, fMRI can capture a time course within the same subjects in vivo with data representing neural activity across seconds per measurement. Some limitations to fMRI are lack of brain region specificity in the data and that the animals are either immobilized or anesthetized during sampling, which prevents assessing many experimental paradigms . aradigms .Functional MRI has been used in preclinical models of neurological disease and SUD to identify general trends in functional connectivity, as well as key brain regions and neural pathways involved in these states. Compared to controls, the thalamus was found to be heavily involved in the neural network activity of rats in acute abstinence from cocaine self-administration. Interestingly, changes in the neural network seen by fMRI measurement disappeared after 2 weeks . A longiFos mRNA expression during auditory fear conditioning to reveal patterns of Fos induction that are similar among shock-only and tone-only fear conditioning and fear recall conditions, suggesting simple associative learning ensembles that are activated by arousal rather than by a specific sensory cue [An unbiased approach to examine whole-brain neural activity by light-sheet fluorescent imaging of immunostained and optically cleared tissue has recently been developed (iDISCO) and used to uncover brain regions differentially activated during parenting behavior ,146. Anosory cue . Fos in combination with functional connectivity and network analysis. The quantification of 81 brain regions for Fos protein, using traditional immunohistochemistry, after fear conditioning led to the identification of a fear network [Fos protein approach, using the single-cell whole-brain imaging (iDISCO+) method, has been combined with functional connectivity and graph theory to identify changes to neural network structure and function caused by withdrawal from alcohol and psychostimulants [Recent studies have begun to use protein detection of the immediate early gene  network . This fe network . Similarimulants ,217.Neural networks associated with withdrawal from individual psychostimulants  were identified in mice by using single-cell whole-brain imaging of neural activity . While wSimilarly, the unbiased single-cell whole-brain imaging and network analysis approach identified a massive increase in coactivation among brain regions and reduced modular structuring of the brain during abstinence from alcohol dependence when compared to control networks . In the The network-based approach using single-cell whole-brain imaging provides a unique opportunity to assess different aspects of alcohol drinking and AUD through various preclinical models  and states of drinking/dependence depending on the question of interest. The different preclinical behavioral paradigms models  of alcohol drinking and alcohol-preferring rodent strains can be used to assess neural networks across severities and conditions of alcohol drinking . Similarities and differences in brain-wide networks can be compared across alcohol drinking paradigms, and the unique components of a given network can be identified.T2 that is expressed in an activity-dependent manner from the loci of the immediate early genes Arc and Fos. This allows for the labeling of activated neurons at a given time/behavioral state  while maintaining the ability for further testing before collection of the brain to examine neural networks [Fos labeling from a previous timepoint of neural activity  can be combined with traditional Fos immunostaining to mark for natural immediate early gene protein production, using iDISCO, which is associated with neural activity from a time window shortly prior to euthanasia . Then, the neural activity from these two timepoints could be used together to assess neurons that are activated at both timepoints and potential similarities and differences in neural network activity between the two timepoints.Perhaps most exciting of all is the possibility to combine single-cell whole-brain imaging and network analysis with other recently developed preclinical neuroscience technology. For example, the Targeted Recombination in Active Populations (TRAP) technique, in which mice contain a tamoxifen-dependent recombinase CreERnetworks ,224. ThePreclinical network analysis using immediate early gene immunofluorescence can also be combined with immunostaining or endogenous fluorescence that signals specific neuronal cell types. This approach will provide information on a wide scale about what types of neurons and receptors contribute to overall network activity. Furthermore, viral tracing between brain regions can determine structural connections and then be used to compare structural to functional connectivity within a given network.Optogenetics or DREADDs can be combined with single-cell whole-brain imaging to explore the effect of manipulation of specific circuits or cells on the neural connectivity of the whole brain. This approach would be ideal for delineating regions that play a role in a given behavior associated with the specific circuit but that is at the tertiary, quaternary, or further level of separation from the stimulation/inhibition. Additionally, using optogenetics or DREADDs to disrupt the activity of brain regions identified by network analysis combined with whole-brain imaging as “hub” regions associated with a behavioral state can verify their functional importance for the behavior. Calcium imaging can also be used to uncover more specific neuron-to-neuron local networks within a given brain region alongside an examination of brain-wide networks in the same animals. There is also the potential to use multi-brain region fiber photometry to collect neural activity data from neurons across a large number of brain regions , either Traditional pharmacology with drug candidates that are potential medications to treat alcohol drinking can be explored as well using neural network approaches. It would be interesting to know how efficacious a given drug is at restoring the network function of the brain to a more normal state after a history of drinking. Perhaps current medications for the treatment of AUD, such as the FDA-pproved naltrexone, acamprostate, and disulfiram, only relieve changes to brain-wide network activity partially, or for a short period of time, which may explain to some degree the limited/moderate efficacy . InteresNetwork-based approaches in preclinical studies have the potential to make significant contributions to our understanding of alcohol drinking. The combination with other relevant neuroscience approaches provides a unique opportunity to enhance our systems-based understanding of the brain in a way that was not previously available. Further, network analysis may help improve medication development and lead to better therapeutic options for AUD.


Haematobia irritans exigua) (BF) and closely related horn flies (Haematobia irritans irritans) (HF) are invasive haematophagous parasites with significant economic and welfare impacts on cattle production. Wolbachia are intracellular bacteria found widely in insects and currently of much interest for use in novel strategies for the area wide control of insect pests and insect-vectored diseases. In this paper, we report the transinfection of BF towards the development of area-wide controls.Buffalo flies . The success of transinfection and infection dynamics was compared by real-time PCR and FISH and fitness effects were assessed in transinfected flies.Three stages of BF; embryos, pupae and adult female flies, were injected with different Wolbachia infection was recorded in flies successfully reared from injected eggs. Adult and pupal injection resulted in higher survival rates and somatic and germinal tissue infections, with transmission to the succeeding generations on some occasions. Investigations of infection dynamics in flies from injected pupae confirmed that Wolbachia were actively multiplying in somatic tissues. Ovarian infections were confirmed with wMel and wMelPop in a number of instances, though not with wAlbB. Measurement of fitness traits indicated reduced longevity, decreased and delayed adult emergence, and reduced fecundity in Wolbachia-infected flies compared to mock-injected flies. Effects varied with the Wolbachia strain injected with most marked changes seen in the wMelPop-injected flies and least severe effects seen with wAlbB.BF eggs were not easily injected because of their tough outer chorion and embryos were frequently damaged with less than 1% hatch rate of microinjected eggs. No Wolbachia successfully replicated in somatic tissues. The Wolbachia-induced fitness effects seen in transinfected BF suggest potential for use of the wMel or wMelPop strains in Wolbachia-based biocontrol programmes for BF.Adult and pupal injection were the most suitable methods for transinfecting BF and all three strains of  Haematobia irritans exigua are obligate hematophagous ectoparasites of cattle    and wMelPop-injected flies (27.98 ± 3.92)  compared to the control injected flies (46.95 ± 4.15), but no significant difference was observed with the wAlbB-injected flies (Tukey’s multiple comparison test: P = 0.77) , wMel (P = 0.0052) and wMelPop (P = 0.0051) in comparison with the mock-injected flies , not previously adapted in Haematobia cells, infection rates and persistence in the injected flies were high . In a few batches, transmission to the next-generation was confirmed.Although embryonic microinjection has been the primary method used to develop transinfected insects, adult microinjection can be advantageous in that females can be selected for injection . Furtherresponse . Our resWolbachia distribution and dynamics in injected females was critical for us to identify the optimal timing for collecting infected eggs for the establishment of an infected colony (11–15 days). Wolbachia density significantly decreased to day five possibly due to host immune response [wMelPop and wAlbB were injected into Anopheles gambiae adult mosquitoes [wAlbB, wMel and wMelPop Wolbachia in Haematobia cells were also observed to initially decrease, possibly due to an innate immune response mediated by the Imd pathway [Wolbachia to be present in all the vital somatic tissues , except for the ovarial tissues, suggesting that Wolbachia might need extra time to infect the ovaries. However, injection with wAlbB, wMel and wMelPop Wolbachia caused > 40% death in flies by day seven post-injection, further reducing the likelihood of collecting infected eggs. Therefore, we hypothesised that microinjecting 1–2 h-old pupae would give more time than with adult microinjection for Wolbachia to multiply, spread and establish in the ovaries. Pupal injection has previously been conducted with Trichogramma wasps and resulted in successful ovarian infections and persistence of Wolbachia in the wasp colony for 26 generations [As oviposition by BF may begin as early as three days after eclosion from the pupae and continue until death, knowledge of response  but recosquitoes . The ini pathway . Real-tierations ., wMel and wMelPop overcame host immune responses and established in both somatic and germline tissues. Further, in two instances, next-generation (G1) BF from wAlbB and wMel injected pupae were positive for Wolbachia, indicating next-generation transmission as a result of pupal injection. The main disadvantages of pupal injection in comparison with adult injection were limitation on the volume of Wolbachia that could be injected and inability to distinguish female from male pupae for injection.With BFwMelPop strain is a virulent type of Wolbachia, and its over replication in somatic tissues and brain cells, known in other infected insects [Wolbachia kinetics we found a higher density of wMelPop than with the other two strains following both adult and pupal injection. Reduction in the longevity of infected Ae. aegypti mosquitoes caused by infection with wMelPop, decreasing the potential extrinsic incubation time for the dengue virus, was one of the characteristics that led to the hypothesis that wMelPop infection would reduce dengue spread [wMelPop could also markedly reduce BF life span and their ability to transmit Stephanofilaria sp. nematodes. These nematodes have been implicated in the development of buffalo fly lesions, a significant production and welfare issue in north-Australian cattle [Stephanofilaria has an extrinsic incubation period of up to 3 weeks in Haematobia spp. [Wolbachia shown in our study could markedly reduce the vector competency of infected flies. There is also the possibility the Wolbachia infection could more directly compromise the vector competency of BF for Stephanofilaria sp., as has been seen in the case another filarial nematode, Brugia pahangi transmitted by mosquitoes and in the case transmission of the dengue virus by Ae. aegypti [The  insects , 62, maye spread . Infectin cattle . Stephanbia spp.  and the  aegypti , 67.Wolbachia-infected males are released CI, vertical transmission, and a relatively higher fertile egg production by infected females in comparison with uninfected flies may increase the chances of successful establishment of Wolbachia in a new host population [Wolbachia have been found to enhance and reduce egg production depending upon both the strain of the Wolbachia and the host [wAlbB, wMel and wMelPop significantly reduced total egg production in pupal injected flies. Also, Wolbachia infection caused delayed and decreased adult emergence of BF post-pupal injection. Wolbachia being an endosymbiont lacks nutritional biosynthetic pathways and depends on its host for wide range of nutrition [Wolbachia and BF for nutritional resources such as amino acids and lipids [Wolbachia was found in all of the critical tissues involved in the endocrine cascades for egg production and maturation in insects , it interfered with egg production by this means [wMelPop infection has been documented in mosquitoes on a number of occasions [Wolbachia infection in BF, they could have a significant impact in altering population dynamics or even crashing BF populations [Wolbachia-infected BF, together with adult life span reduction might decrease overwintering and survival of BF, particularly during periods of unfavourable fly conditions and at the edge of the BF range.Fecundity of insects has a significant influence on population dynamics of insect populations  and whenpulation . Wolbachthe host , 69–74. utrition . Hence, d lipids , 76. Anois means . In addiccasions , 25. If ulations , 78. Forulations . ProlongwMel, wMelPop and wAlbB Wolbachia strains and infection can induce a number of fitness effects in injected flies. However, embryonic injection proved challenging with BF and it was not possible to establish an infected isofemale line using this technique. Pupal and adult microinjection gave much higher fly survival rates, high titres of Wolbachia in somatic tissues and ovarian infection and transmission to the next-generation in a number of instances. Despite relatively limited testing, this gives hope for the establishment of sustainably Wolbachia-infected strains of BF and the future development of a Wolbachia-based area-wide control programme.BF are competent hosts for the growth of


Induced pluripotent stem cells (iPSCs) represent an unlimited source of pluripotent cells capable of differentiating into any cell type of the body. Several studies have demonstrated the valuable use of iPSCs as a tool for studying the molecular and cellular mechanisms underlying disorders affecting bone, cartilage and muscle, as well as their potential for tissue repair. Musculoskeletal diseases are one of the major causes of disability worldwide and impose an important socio-economic burden. To date there is neither cure nor proven approach for effectively treating most of these conditions and therefore new strategies involving the use of cells have been increasingly investigated in the recent years. Nevertheless, some limitations related to the safety and differentiation protocols among others remain, which humpers the translational application of these strategies. Nonetheless, the potential is indisputable and iPSCs are likely to be a source of different types of cells useful in the musculoskeletal field, for either disease modeling or regenerative medicine. In this review, we aim to illustrate the great potential of iPSCs by summarizing and discussing the in vitro tissue regeneration preclinical studies that have been carried out in the musculoskeletal field by using iPSCs. Musculoskeletal conditions such as osteoporosis, osteoarthritis, fractures, muscular dystrophy and skeletal malformations are the second-greatest cause of disability worldwide . AccordiIn order to develop effective treatments, it is of uttermost importance to elucidate the cellular and molecular bases that underlie human diseases through the understanding of critical biological processes . A betteHuman primary cell cultures and cell lines have substantially improved our understanding of the mechanisms responsible for many rare and common diseases and have driven the development of novel therapeutic strategies . AlthougiPSCs were generated over a decade ago by Takahashi and Yamanaka, who succeeded in reprogramming mouse tail-tip fibroblasts by means of retroviral introduction of only four transcription factors: octamer-binding protein 3/4 (Oct3/4), SRY (sex-determining region Y)-box 2 (Sox2), Krüppel-like factor (Klf4) and c-Myc ,33. iPSCAlthough the potential of iPSCs for tissue regeneration, disease modeling and drug screening has been largely recognized, the findings of iPSC research to date are mostly focused on the neurology, cardiology and hematology fields . NonetheAs previously mentioned, the use of primary and immortalized cell lines as in vitro models for musculoskeletal diseases presents intrinsic drawbacks. Although not exempt from limitations, the use of iPSCs has proven to be the best alternative to overcome the problems associated with these conventional cell-based in vitro models . ReprogrThe first models of disease employing iPSCs were reported in 2008, when Park and colleagues produced the first large repository of disease-specific iPSCs . Later oOn the other hand, the more widespread availability of gene editing techniques makes it easier to generate in vitro iPSC-based models, which allow not only to better study the pathology of musculoskeletal disorders and their associated phenotypes but also to correct pathogenic mutations in patient-derived iPSCs. Common technologies for genome-editing are meganucleases , zinc fiBone disorders have been defined as a group of varied acute and chronic traumatic, degenerative, malignant or congenital conditions affecting the musculoskeletal system . ResearcBone is a dynamic tissue that is continually remodeling through coordinated osteoclast-mediated destruction followed by osteoblast-mediated reconstruction ,76. This2+ influx assay. Furthermore, using TALEN-mediated correction of the mutation in MFS-iPSCs, they demonstrated that it was possible to rescue the compromised osteogenenic differentiation [MFS is an autosomal dominant genetic disorder of the connective tissue caused by mutations in the FIBRILLIN-1 (FBN1) gene and associated with skeletal deformities , among other symptoms ,64,65. Kntiation  reprograntiation .Panicker et al.  using iPFOP, characterized by progressive ossification in soft tissues, is another rare genetic disorder that has been ‘modeled in a dish’ employing patient-derived iPSCs . MatsumoThe generation of iPSCs from patients with osteopetrosis ,75, an aOsteoporosis still represents a significant public health problem affecting a broad spectrum of the elderly population. To our knowledge, there are no studies modeling osteoporosis per se but rather modeling rare diseases where osteoporosis is part of their clinical manifestation. That is the case of the iPSCs generated from patients with Turner syndrome (TS) , a rare Li-Fraumeni syndrome (LFS) is another rare genetic condition associated with bone malignancy, osteosarcoma being one of the main cancer types seen in LFS patients. Lee et al.  generateInterestingly, in vitro iPSC-based models of bone-affecting diseases are scarce and are usually reported by only one single research group, which makes it difficult to compare protocols and phenotypes. AS  and GD ,114 modeApart from bone diseases, patient-specific iPSCs have been created in order to model cartilage diseases. Refined protocols for chondrogenically differentiating iPSCs have paved the way to developing in vitro models for cartilage diseases, as well as new screening platforms for testing new drugs based on their use. Among previously published studies, several groups have focused on modeling cartilage diseases produced by monogenic mutations but studies modeling complex cartilage diseases affected by several factors are also starting to arise.Xu et al.  employedThe understanding of the pathophysiology of neonatal-onset multisystem inflammatory disease (NOMID) arthropathy has also improved thanks to iPSC technology. NOMID arthropathy is an auto-inflammatory disease caused by NACHT, LRR and PYD domains-containing protein 3 (NALP3) gene mutations. Yokoyama et al. generated iPSCs derived from NOMID patients and produced chondrocyte-like tissues with mutant and wild-type NLRP3. Interestingly, they revealed a previously unidentified connection between the inflammasome-associated molecule NLRP3 and the master regulator of chondrocyte differentiation SOX9. Furthermore, they showed that SOX9 was overexpressed via the cAMP/PKA/CREB signaling pathway in chondrocytes with disease-causing mutations in NLRP3 . SurprisFGFR3 chondrodysplasia is another monogenic cartilage disorder that was modeled using iPSC lines derived from patients ,85, whicIn addition to being used for genetic diseases, patient-specific iPSCs have been also generated from patients with complex diseases such as rheumatoid arthritis (RA)  and OA ,88,89. IOverall, these recent advances set the basis for the generation of a new tool for the study of cartilage disorders. The possibility to model ‘disease in a dish’ by means of iPSC technology opens the door to the development of novel therapeutic compounds and, more importantly, to an improved understanding of cartilage diseases.In addition to bone and cartilage diseases, patient-derived iPSCs, which possess the ability to differentiate into myogenic progenitor cells followed by myotubes, can be a useful tool for drug screening and modeling of skeletal muscle diseases . For theSo far, different groups have demonstrated the suitability of iPSCs to derive large quantities of myogenic precursors  and to mMyotonic dystrophy 1 (DM1) is a multisystem disorder primarily affecting the central nervous system, heart and skeletal muscle. Recently, iPSC-based DM1 models were established using the PAX7 conditional expression system. These cells were differentiated into myogenic progenitors and, subsequently, terminally differentiated into myotubes. Interestingly, DM1 iPSCs differentiated into the myogenic lineage recapitulated the molecular events associated with the DM1 phenotype, such as the splicing disruption of MBNL1 target genes . DastidaThe term “laminopathies” comprehend 16 rare disorders that have mutations in LMNA as a common characteristic. Four out of 16 disorders affect skeletal muscle as well as other tissues: dilated cardiomyopathy (DCM), Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy (LGMD) type 1B (LGMD1B) and LMNA-related congenital muscular dystrophy (L-CMD) . The exaMM is a congenital distal myopathy caused by defective muscle membrane repair as a result of mutations in the DYSFERLIN gene. iPSC-based MM disease models developed by Tanaka and colleagues  were ablIn addition to MDs, there are many other diseases affecting the muscular system that have been modeled by means of iPSCs, such as carnitine palmitoyltransferase II deficiency , valosinTo our knowledge, there is only one study that generated iPSCs from patients with degenerative disc disease . In thisBone, intervertebral disc and muscle all have the inherent ability to regenerate after injury, unlike cartilage, tendon and ligaments, which either have almost no intrinsic repair potential or heal with inferior properties. However, while bone and skeletal muscle can easily regenerate, they usually fail to do it when a large volume of diseased tissue is involved . MoreoveProgenitor cells, responsible for the renewal of most normal tissues in the body, have largely been proposed as the alternative to adult mature cells in therapeutic strategies to treat musculoskeletal disorders . The useNew therapeutic strategies have increasingly involved the use of stem cells in order to replace/repair damaged tissues and improve homeostasis ,27. iPSCBelow, we provide examples of iPSC-based repair therapies for bone, cartilage and muscle .MSCs derived from iPSCs were found to be comparable to adult MSCs in terms of multipotency even when derived using different protocols ,138,145.Differentiation of iPSCs into chondrocytes showed better repair capacity of osteochondral lesions than iPSCs or iPSC-MSCs in independent studies ,138. UtoOther works did not compare iPSCs with adult MSCs or autologous grafts for osteochondral repair. Xu et al.  implanteIn bone regeneration, it is also usual to differentiate iPSCs into osteoblasts prior to transplantation. iPSCs obtained from patients with craneodysplasia were differentiated into osteoblasts before transplantation in bone defects in rats . The autStudies about skeletal muscle regenerative medicine using pluripotent stem cells have been mainly performed using ESCs . Some stIn the case of degenerative disc disease, several studies have focused on the resettlement of cells that had been lost in the intervertebral disc by injecting iPSCs  differenIn general, all these studies suggest that MSCs derived from iPSCs show similar repair results than those obtained using adult MSCs. Moreover, further differentiation into mature cells showed better outcomes than intermediate progenitor-derived cells from iPSCs. However, improved differentiation protocols seem to be necessary in order to obtain better regeneration results in preclinical models.Despite the valuable potential of iPSCs, there are some limitations that need to be accounted for when pursuing applications for disease modeling and regenerative medicine. Some of the drawbacks affecting the research on iPSCs are mainly related to cell differentiation, both in terms of differentiation potential varying among cells  and of oThe current scarcity of robust and efficient differentiation protocols cannot be underestimated either . ImmaturSafety concerns and new advances in the study of stromal and stem cell secretome have opened the door to new cell-free tools for regenerative medicine, particularly the therapeutic potential of extracellular vesicles (EVs) ,169. IniEVs, including exosomes and microvesicles, are cell-derived membrane-enclosed signaling organelles that mediate intercellular communication . EVs areFew studies have proposed the therapeutic effect of exosomes isolated from iPSCs on musculoskeletal diseases. For OA treatment, the effects of exosomes obtained from iPSC-MSCs and from synovial MSCs were compared . ExosomeThese results, while encouraging, need further confirmation. Moreover, the mechanisms underlying the reparative effect need to be elucidated for further clinical application.The enormous potential expected from iPSCs since they were generated more than a decade ago has been confirmed by recent studies showing their regenerative potential, as well as their ability to recapitulate the characteristics of musculoskeletal diseases in vitro. Nowadays the generation of genetically corrected iPSCs is feasible and that it is possible to identify the cellular and molecular mechanisms underlying musculoskeletal diseases using iPSCs. Despite the many remaining challenges and the long way ahead until iPSC-based therapies become clinical treatments, the use of these cells in the orthopedic field is only expected to increase as our ability to generate more accurate and specific cells improves. The concomitant development of phenotypically and physiologically relevant in vitro models of bone, cartilage, muscle and tendon offers exciting prospects for disease modeling, drug discovery and regenerative medicine applications.


Despite considerable appeal, the growing appreciation of biosignals complexity reflects that system complexity needs additional support. A dynamically coordinated network of neurovisceral integration has been described that links prefrontal-subcortical inhibitory circuits to vagally-mediated heart rate variability. Chronic stress is known to alter network interactions by impairing amygdala functional connectivity. HRV-biofeedback training can counteract stress defects. We hypothesized the great value of an entropy-based approach of beat-to-beat biosignals to illustrate how HRVB training restores neurovisceral complexity, which should be reflected in signal complexity. In thirteen moderately-stressed participants, we obtained vagal tone markers and psychological indexes  before and after five-weeks of daily HRVB training, at rest and during stressful cognitive tasking. Refined Composite Multiscale Entropy (RCMSE) was computed over short time scales as a marker of signal complexity. Heightened vagal tone at rest and during stressful tasking illustrates training benefits in the brain-to-heart circuitry. The entropy index reached the highest significance levels in both variance and ROC curves analyses. Restored vagal activity at rest correlated with gain in entropy. We conclude that HRVB training is efficient in restoring healthy neurovisceral complexity and stress defense, which is reflected in HRV signal complexity. The very mechanisms that are involved in system complexity remain to be elucidated, despite abundant literature existing on the role played by amygdala in brain interconnections. Although it has become increasingly evident that physiological systems are complex, in the sense that many interdependent components interact at different hierarchical levels and simultaneously operate at different time scales, there can be no direct quantification of complexity in living systems. Rather, an intuitive approach with considerable appeal has been that physiological/biomedical signals that are generated by such systems may carry information on the system complexity, its self-organization, and potential adaptability, so pointing to signal complexity analysis is a reliable way to examine coordinated interactions in neurophysiological networks. Prior knowledge of system organization might allow for anticipating, to some extent, system responses through a dynamical organization as well as long-term (persistent) adaptations. Accordingly, in controlled conditions, a logical expected system behavior should help in strengthening the link between system complexity if one can demonstrate that signal complexity change concurrently . UltimatIt has been known for years that the brain and the heart exhibit permanent top-down and bottom-up interactions that are critical beyond cardiovascular health, for behavioral, cognitive, and emotion regulations . As a liStudies encompassing psychology, cardiovascular, and neuroimaging domains provide converging evidence of a link between short-range (HF) HRV dynamics and the prefrontal subcortical circuits through an intricate network ,16,17,18It follows from above that, reasonably enough, one might expect a causal link between amygdala functional connectivity, a coordinated neurovisceral integration, and complexity in the healthy unconstrained CAN.., stress management. While the response to stress in humans is a healthy adaptive function in situations of acute challenge, a prolonged exposure to stressors might cause persistent dysregulations [Researchers have recently more closely associated mood and cognition to complexity markers in HRV dynamics ,21,22. Iulations , which aulations ,25. Heigulations ,27,28. Aulations . The HRVulations , which eulations ,31, or iulations ,33. To dThe aim of the present study was to provide a novel application of a complexity-based method to evaluate coordination in a neurophysiological network, the CAN, through complexity in its output signal, HRV dynamics.For that, Refined Composite Multiscale Entropy (RCMSE) in heartbeat time-series was assessed during stress-cognition interactions in self-reported moderately stressed participants, before and after HRVB training to trigger system adaptations. We hypothesized that improved stress defense is associated with greater signal complexity, which could reflect better neurovisceral coordination.The procedures are in agreement with the French law that allows for performing experiments on humans and publishing the obtained results without requiring approbation and ID by an IRB or ethical committee, because the experiments are part of the research training that has been approved by the faculty steering committee, which has full responsibility on the training program. The experimental group (‘Heart Rate Variability Biofeedback’: HRVB group) consisted of 13 healthy participants  performing administrative work at the faculty. They all reported being somewhat stressed (see stress quantification below) and they have difficulty for balancing work, family, and lifestyle. Six unstressed people  served as the control group (CTRL group).None of the participants were receiving medical treatment before enrollment. They were required to abstain from food or drink for two hours before the HRVB training procedure, scheduled on early morning and early evening before breakfast and lunch. The participants abstained from caffeine ingestion on the experimental days. After five-weeks of HRVB training, three participants of the HRVB group dropped-out of follow up. They argued for too high constraints being linked to the day-to-day HRVB training. Thus, the final sample undergoing both assessments encompassed 10 subjects .The experimental protocol consisted of two 10 min sequences that were separated by a few minutes that were dedicated to fill psychological questionnaires. The same sequences were repeated before and after HRV biofeedback training. During each sequence, the subjects stayed quietly seated in front of a computer, breathing at spontaneous rate, while the heartbeat time series were recorded, as described below. The resting conditions corresponded to the first 10 min of watching a calm and soothing documentary. During the second 10 min sequence, the participants performed cognitive tasks in a controlled stressful environment. They had to respond to 31 questions that were displayed on a computer screen, which needed the mental processing of logic, memorization, and calculation in a balanced proportion. Questions were created with the E-Prime software , so that the participants answered by pushing dedicated keys on a keyboard. The added stressors had the form of predetermined response time, visual feedbacks for false responses, and an attentive and evaluative audience (two people standing near the participant and taking notes). Flashing lights crowd noises, car honks, and sirens completed the set of added stressors. The number of questions, the type of logic memorization and mental calculation questions, the negative feedback, and the two people for evaluative audience were different before and after HRVB training to avoid undesired consequences of habituation.® . As feedback, heart rate was detected non-invasively by infrared finger photoplethysmograph and processed to detect respiratory sinus arrhythmia (RSA) and the presence of a unique mode (frequency) in HRV, which characterizes cardiac coherence, thereby, suitable conditions for afferent cortical-subcortical stimulation through vagal afferent traffic.HRVB training was assigned to the experimental group for five weeks. The participants had to control their breathing rate at ~ 6 cycles per min without changing their natural tidal volume, in quiet conditions for 5-min periods twice a day (morning and evening). A connected device that was developed by URGOTECH linked by Bluetooth to a smartphone application, URGOfeel, guided the controlled breathing rateThe participants filled out a series of questionnaires. The Spielberger’s State-Trait Anxiety Inventory (STAI ) consistCardiac interbeat intervals  were recorded while using a Polar H10 chest belt that was linked by Bluetooth to a smartphone. Polar chest belts demonstrated great accuracy in assessing RR intervals when compared to ECG recordings ,38. The Spectral power was computed in the low frequency band  and the high frequency band , and then interpreted as pure sympathetic and dominantly vagal activities, respectively. LF power/HF power was computed as an indicator of sympatho-vagal balance.Complexity in the RR time series was captured by computing Refined Composite Multiscale Entropy (RCMSE), an improved method for obtaining sample entropy ,42 at seThe quantitative variables were expressed as mean, standard deviation (SD), and coefficient of variation (CV %).t-tests with Bonferroni corrections for multiple comparisons were applied to observe the effects of HRVB training on the psychological and physiological indices. The effect Size with Hedge’s g was calculated. Values above 0.80 were adopted with high magnitude (‘large’), above 0.5 with medium (‘med.’) and 0.2 with small  magnitude. The Pearson’s correlation coefficient was computed for analyzing the relationship between two variables. The Receiver Operating Characteristic (ROC) curve through Sensitivity, Specificity, Area Under Curve defined the efficacy of the HRV indices in time (RMSSD), frequency , and non-linear (entropy) domains. The respective p values were used between the pre- and post-HRVB training set by Youden Index.The normality of each dataset was determined using the d’Agostino–Pearson normality test. One-way analyses of variance followed by paired or unpaired All of the statistical calculations were performed using GraphPad  and XLSTAT .p = 0.0026 and p = 0.0165, respectively), whereas the perceived cognitive load (NASA TLX score) remained unchanged (p = 0.4258). This observation is not trivial, because it supports the idea that a lower stress/anxiety is not the consequence of less attention being paid to the cognitive task (since the cognitive load is intact), but a pure HRVB training beneficial effect on anxiety and perceived stress when facing our stressful controlled conditions. The absence of changes in the participants of the control group confirmed the pure effect of training. Overall, psychological markers indicate that HRVB training helped participants to prevent a rise in anxiety/stress while facing the stressful cognitive task.The main effects of HRVB training on HRV at rest and during stressful cognitive conditions are indicated in The RMSSD and HF-HRV values indicate a small effect size of training on vagal activity at rest and a moderate effect during stressful cognitive tasking. The sensitivity analysis demonstrated that the main effects of HRVB training were effective during stressful cognitive tasking 2 = 0.789, F = 29.97, p = 0.0006, We highlighted a link between training benefits at rest and those that were observed during stressful cognitive tasking Taken together, the above adaptations in vagal activity after training indicate that enhanced vagal tone at rest could help in reaching higher vagal control during a stressful task.Remarkably, autonomic adaptations to training were more consistent and clear-cut when assessed with a complexity marker, RCMSE. First, entropy exhibited a small coefficient of variation (~20%), which contrasts with CV in other markers , to identify changes in the coordinated interconnectivity of the central autonomic network (CAN). It was hypothesized that a coherent profile in entropy changes during stress-cognition interactions provides a meaningful approach of CAN complexity and neurovisceral adaptability to HRVB training. The main finding in this sense was that entropy in the output signal was heightened despite stress, thanks to HRVB training. This was accompanied with training benefits on vagal activity, which is known to prevent disruption in amygdala functional connectivity ,18. We sTo obtain a realistic interpretation of a link between system complexity and signal complexity in our conditions, a pre-requisite is that autonomic responses and long-term adaptations match with previous observations that consistently report on the link between vagal activity, anxiety, and interconnectivity in the neurovisceral circuitry when the brain has to respond emotionally and cognitively. A low resting vagal HRV and/or an excessive vagal tone withdrawal when one faces an acute challenge has been associated with poor health and poor effectiveness in coping with a variety of stimuli and challenges . These dThe capacity to maintain high vagal activity at rest as well as during a cognitive task is critical in stress defense , and it Interestingly enough, we show a correlation between the training-induced gain in vagal activity, which confers better psychophysiological status to a participant , and theWhile we use RCMSE here, a large panel of complexity-based methods for analyzing interbeat time series can be drawn. To evoke a few representative examples, sample entropy has been applied to wavelet-based decomposition in very-low (VLF), low (LF), and high-frequencies (HF) at different ages ; multiscUsing a multiscale entropy approach for that is not without limitations. Mainly, the significance of sample entropy at given scale strongly depends on the length of the analyzed time series ,46. We iIn brief, here we suggest that a complexity-based approach of cardiac interbeat time series during stress-cognition interactions is helpful in understanding complexity changes in an intricate central-autonomic neurovisceral circuitry. This statement finds strong support in the combined markers of cognitive load, state anxiety, perceived stress, vagal activity, and entropy, which collectively offered a coherent vision of cooperative mechanisms. Although advanced knowledge on the role of amygdala has recently been provided, an obvious limitation in the present study is the absence of any metrics regarding amygdala functional connectivity or direct evidence of changes in brain networks complexity. Therefore, we conclude that, although HRV biofeedback training appears to be an effective means to preserve a healthy complexity, and that this very property is reflected in HRV entropy, the very mechanisms that link neurovisceral coordination to signal complexity remain to be established.


Independent of total body fat mass, predominant upper body fat mass distribution is strongly associated with cardio-metabolic comorbidities. However, the mechanisms underlying fat mass localization are not fully understood. Although a large body of evidence indicates sex-specific fat mass distribution, women are still excluded from many physiological studies and their specific features have been investigated only in few studies. Moreover, endurance exercise is an effective strategy for improving fat oxidation, suggesting that regular endurance exercise could contribute to the management of body composition and metabolic health. However, no firm conclusion has been reached on the effect of fat mass localization on fat oxidation during endurance exercise. By analyzing the available literature, this review wants to determine the effect of fat mass localization on fat oxidation rate during endurance exercise in women, and to identify future research directions to advance our knowledge on this topic. Despite a relatively limited level of evidence, the analyzed studies indicate that fat oxidation during endurance exercise is higher in women with lower upper-to-lower-body fat mass ratio than in women with higher upper-to-lower-body fat mass ratio. Interestingly, obesity may blunt the specific effect of upper and lower body fat mass distribution on fat oxidation observed in women with normal weight during endurance exercise. Studying and understanding the physiological responses of women to exercise are essential to develop appropriate physical activity strategies and ultimately to improve the prevention and treatment of cardio-metabolic diseases. The current global obesity epidemic and related cardio-metabolic comorbidities have resulted in a growing interest in adipose tissue features . BesidesIt is recognized that in resting conditions, the upper and lower body fat masses have specific effects on metabolism . Moreover, the higher lipolysis activity of the upper body fat mass, compared with the lower body fat mass, may differentially influence substrate metabolism  . While eRegular endurance exercise promotes weight control and is an effective way to counteract cardio-metabolic alterations, particularly by increasing fat oxidation . Indeed,This review focused on studies that compared endurance exercise–induced fat oxidation in women with upper or lower body fat depot phenotypes and an actual lack of consensus is observed. Two studies , 2014 by2max and for 40 to 60 min). Haufe and collaborators found that exercise-induced fat oxidation is not associated with fat mass localization   . Women w 45 min) . We sugg 45 min) . Catecho 45 min) .In resting condition, growth hormone concentrations were not different between groups and the minimal growth hormone-induced lipid mobilization during exercise suggested a negligible effect. Interestingly, while glucose concentrations were not different between groups, women with higher upper-to-lower-body fat mass ratio exhibited higher post-prandial insulin levels, indicating an insulin resistance risk. The significant difference in ANP concentrations at rest and during exercise suggests a specific regulation of ANP in function of body shape  Figure . It appeIn addition, the lower metabolic flexibility in women with higher upper-to-lower-body fat mass ratio increases their risk of cardio-metabolic alterations, particularly insulin resistance . SimilarEndurance exercise has many health benefits, including on body weight and composition . Enduran2max for about 60 min, 3 times per week for 12 weeks) increased the relative fat oxidation rates during exercise only in women with upper body obesity , no difference in fat oxidation rates during exercise was observed compared with pre-training in women with obesity (n = 5/each) were pooled, and this did not allow investigating the impact of fat mass localization on exercise training-induced fat oxidation. In addition, training intensity (70% of VO2max) might have been too high for assessing optimal fat oxidation. To our knowledge, no information is available on the impact of exercise training on substrate oxidation in relation with fat mass localization in women with normal weight.Interestingly, while no difference was observed between women with upper and lower body obesity at baseline, low-intensity exercise training  after the 12 weeks of endurance training. These results question the influence on body composition of the increased fat oxidation in response to endurance training in this population. Indeed, endurance exercise increases the capacity to use fat at rest and during exercise, suggesting an effect on body weight and fat mass loss via greater fat oxidation . HoweverNevertheless, even if increased fat oxidation may not be associated with a decrease in fat mass in response to endurance training, the exercise-mediated improvement in fat oxidation is important not only for body composition and weight management, but also for cardio-metabolic health. Indeed, the capacity to oxidize fat during exercise is inversely related to cardio-metabolic comorbidities  and to the progression of metabolic diseases . TherefoIt is recognized that aging is associated with increased fat mass accumulation and menopause leads to a shift toward upper body fat mass deposition. However, and surprisingly, little is known about the effect of fat mass localization on substrate oxidation during endurance exercise in post-menopausal women. Some studies investigated the influence of menopause and the related body composition modifications on substrate metabolism at rest and during exercise , but nonIt has been reported that in women with normal weight, whole-body lipolysis is not affected by menopause in post-absorptive and also in hyperinsulinemic conditions . Lipolys2max for 40 min) in men with obesity and post-menopausal women with obesity showed that while upper body fat mass was not significantly different between men and women, VAT was lower and abdominal SAT and fat oxidation rates were higher in women than in men. This suggests the importance of upper adipose tissue quality for substrate metabolism. The authors noted that sexual dimorphism in substrate oxidation during exercise was unlikely to be explained by plasma estrogen concentrations, which were comparable between groups. They hypothesized that intramyocellular triglyceride content and muscle morphology may play a role (2max for 30 min) is comparable in post-menopausal women undergoing or not hormone replacement therapy; however, hormone replacement therapy is associated with greater reliance on fat proportional with exercise duration  might explain the reduced weight loss rate and the predisposition to weight regain when individuals return to a normocaloric diet . In posty a role . Interesduration . ProlongOverall, more randomized clinical trials are needed to investigate the effect of fat mass localization on fat oxidation during acute and chronic endurance exercise in women with normal weight and obesity, before and after menopause. In addition, due to the lack of experimental studies, the present review only focused on fat oxidation during endurance exercise and it is not known whether these findings might also apply to other exercise modalities , also relevant for the prevention and treatment of cardio-metabolic diseases .Over the years, many studies have described, mainly at rest, the deleterious effects of upper body adiposity and its association with the risk of cardio-metabolic alterations. Although it is acknowledged that regular physical activity plays a pivotal role in fat metabolism regulation, and thus in body composition management and cardio-metabolic health, data on the impact of fat mass localization on substrate utilization during exercise in women are scarce.Interestingly, although few studies are available on this topic, the weight status appears to be a confounder in the relationship between fat mass localization and fat oxidation during acute endurance exercise in premenopausal women . Higher LI and NB had the idea for the review article. LI and GE performed the literature search and data analysis and drafted the review article. NB critically revised it. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cardiac arrhythmias are responsible for many cardiovascular disease-related deaths worldwide. While arrhythmia pathogenesis is complex, there is increasing evidence for metabolic causes. Obesity, diabetes, and chronically consuming high-fat foods significantly increase the likelihood of developing arrhythmias. Although these correlations are well established, mechanistic explanations connecting a high-fat diet (HFD) to arrhythmogenesis are incomplete, although oxidative stress appears to be critical. This review investigates the metabolic changes that occur in obesity and after HFD. Potential therapies to prevent or treat arrhythmias are discussed, including antioxidants. Cardiovascular disease (CVD), including coronary heart disease, heart failure, stroke, and hypertension, persists as the leading cause of death worldwide despite advances in medical treatment ,2,3,4. I2 or more) over the past few decades, concerning trends in dietary patterns, and the increased prevalence of CVD and arrhythmias motivate a closer examination of the metabolic impacts of a chronic high-fat diet (HFD), both concurrent with and independent of comorbidities such as obesity. With a global increase in the consumption of high-fat foods and prevalence of obesity, antioxidant therapy to combat these metabolic conditions has potential for cardioprotection. The aim of this review is to examine the potential mechanisms that mediate the relationship between obesity or the long-term consumption of high-fat foods and the development of pro-arrhythmogenic metabolic conditions, as well as novel antioxidant approaches to pharmacological treatment. For a more clinically oriented review, the interested reader is referred to this recent publication: [Public health concerns regarding the surge in obesity , it is trivial to state “metabolism is involved in disease X.” Of course, it is. How could metabolism not be involved? Nonetheless, we accept that some diseases are more metabolic than others and figuring out the detailed mechanisms is not a trivial undertaking. Given the intense metabolic demands on the heart, abnormal metabolism is important for heart disease as a broad category.In order to understand myocardial metabolism, we begin with a brief review of mitochondria. Mitochondria account for approximately one-third of cardiac myocytes’ cell volume—the most densely packed mitochondria of any tissue . Approxiβ-oxidation of fatty acids provides a constant supply of energy to the heart since lipids contain more than twice the energy per gram compared to carbohydrates and protein. In fact, conditions such as heart failure (HF), arrhythmias, and ventricular hypertrophy are, in part, due to mitochondrial remodeling that results in reduced capacity for fatty acid oxidation (FAO) . With suIn order to protect itself against oxidative stress, the heart can express proteins that result in increased antioxidant activity, for example forkhead box O3a (FOXO3a), catalase, and metallothionein 2 (MT2) . This maSince the effects of saturated fat of cardiac myocytes seem like an important topic, one might think that there would be an extensive body of literature examining this, but, in fact, there are very few publications in this area. In a set of reductionist experiments, we exposed isolated cardiac myocytes from wild-type mice to physiologic levels of fatty acids . The satThough the term “high-fat diet” is commonly used in research publications without specifying the source of fat, it is important to consider the different effects of various classifications of fatty acids: Saturated, monounsaturated, polyunsaturated, and trans fats. Importantly, naturally occurring vitamins—such as vitamins A and E—are fat-soluble and can only be absorbed with fatty foods; thus having at least some form of fat in the diet is essential .There is clinical evidence that consuming foods predominately containing polyunsaturated fatty acids (PUFAs) as the source of fat leads to cardioprotective and anti-inflammatory outcomes ,25,26. PAt present, people are significantly more likely to become obese and develop diabetes than people living 50 years ago, underscoring the importance of identifying protective therapies and lifestyle recommendations for an environment enriched with calorie-dense foods . ObesityDIO rodents also have morphologic changes in that they develop cardiac hypertrophy ,36. The Obese humans have abnormal cardiac electrophysiology, most commonly measured by electrocardiography (ECG). Specifically, obese humans have increased frequency of ventricular ectopy (premature ventricular complexes: PVCs) and abnormal cardiac repolarization, measured as long QT (LQT) ,43,44. LWeight-loss surgery decreases both QT and QT dispersion, indicating that this is a causal effect rather than an association . Obese hOur prior work has shown that wild-type mice develop heart rhythm abnormalities when they are made obese by eating an HFD for about three months . We wereDysfunctional mitochondria may also have reduced ability to use fatty acids as a fuel source, which could result in myocardial lipid accumulation, worsening the effects of lipid overload in a feed-forward manner ,61. The Dietary fat is not the only dietary compound that leads to the macro- and microscopic accumulation of fat. Excessive long-term carbohydrate intake can also cause obesity. Fructose, while an isomer of glucose, is metabolized quite differently compared to other simple sugars . Both huEven in the absence of dietary fat and fructose consumption, the human body still maintains a mechanism for de novo lipogenesis. Glucose can be converted into fructose by ketohexokinase (KHK) via the polyol pathway. Mice lacking KHK, and thus unable to convert glucose into fructose, are protected from the insulin resistance, dyslipidemia, and weight gain observed in wild-type mice fed a glucose-rich diet in the absence of fructose . This no3R), also respond to cellular cues to release calcium from the SR [Abnormal calcium handling within the cardiac myocytes is central for many types of arrhythmias. The contraction of cardiac myocytes begins with the introduction of external calcium into the cytosol of the cell through the L-type calcium channels (LTCCs), signaling the SR to release more calcium into the cytosol via ryanodine receptor channels (RyRs)—specifically the RyR2 isoform in cardiac tissue ,74. Thesm the SR .Whereas the release of calcium from the SR marks the initiation of contraction in cardiac myocytes, the removal of calcium from the cytosol following systole is equally vital. The translocation of cytosolic calcium back into the SR by the sarco/endoplasmic reticulum calcium ATPase (SERCA) is essential in diastole and represents a sensitive window of time in which improper calcium clearance can have significant arrhythmogenic consequences . MutatioA potential explanation for the increased risk of developing arrhythmias in individuals with obesity is in the altered expression of various ion channels and gap junction proteins within the myocardium, which has been shown in mice fed a high-sugar diet and with HFD-induced obesity ,81,82,83This, in addition to fibrotic remodeling of myocardial tissue, offers an electrophysiological mechanism for obesity-related arrhythmias, though the precise details remain unclear. It is possible that these ion channel modifications and fibrosis could be prevented with mitochondria-targeted antioxidant treatment, once again highlighting the impact of mitochondrial ROS in the development of arrhythmogenesis .Arrhythmias require not only a trigger for initiation but also a subsequent mechanism or substrate for perpetuation. Chronic HFD consumption and downstream excess FAO and metabolic stress, along with chronic low-grade inflammation associated with obesity, can trigger the onset of an arrhythmia by cellular modifications that result in calcium leak within the cardiac myocyte. This substrate can lead to RIRR, as mentioned earlier, and may further cause heterogeneous repolarization of cardiac myocytes, a mechanism of arrhythmic propagation. In fact, we have previously shown via optical mapping that rodents fed a high-saturated-fat diet have significantly greater repolarization heterogeneity and increased action potential duration (APD) than mice fed standard chow . This coAF is the most frequent sustained arrhythmia seen in clinical practice . AlthougAlthough human genetics have shown modest power to predict AF, it is largely a disease of age with a contribution from diet and lifestyle . Diabetes is a well-recognized risk factor for AF . ObesityAdditional evidence for the role of cardiac metabolism in promoting AF comes from human atrial tissue samples taken from patients undergoing cardiac surgery. One study found abnormalities in atrial tissue metabolism comparing samples from patients with permanent AF to patients who had never had AF . SpecifiOther studies using mice with mutated RyR2 channels, causing increased calcium leak, indicate that mitochondrial oxidative stress may have a role in atrial arrhythmia pathophysiology . Large aRecent work has shown that DIO mice have more inducible AF than control mice . InvestiAlthough it is widely reported that the long-term consumption of a diet high in saturated fat results in increased oxidative stress in various tissues, we do not have a good understanding of how this relates to ventricular arrhythmias. Cardiac lipid overload caused by transgenic over-expression of peroxisome proliferator activated receptor gamma (PPARγ), without obesity or diabetes, causes spontaneous ventricular tachycardia and SCD with prolonged repolarization due to a decrease in Kv channel expression . SeveralBoth ROS production and mitochondrial function are important in evaluating the arrhythmogenic effects of an HFD. The pathology likely involves oxidation and phosphorylation of the RyR2 calcium-release channels. Whereas ROS production affects calcium handling through protein oxidation and activating kinases’ pathways leading to ectopic beats, impaired mitochondrial biogenesis and quality control could result in inefficient ATP production from FAO, which could also contribute to abnormal heart rhythm . Though As mentioned earlier, the process of arrhythmogenesis following a high-fructose diet is mechanistically similar to that seen following an HFD, resulting in near identical cellular modifications: Oxidation of CaMKII, phosphorylation of RyR2, and increased production of ROS to name a few ,67,72. PSimilar to the effects of a chronic HFD in individuals without comorbidities, abnormal lipid accumulation and systemic low-grade inflammation are observed in those with obesity and diabetes and may contribute to the development of arrhythmias . Levels There is also evidence the insulin receptor pathway can modulate ion channel function. One group showed that decreased phosphoinositide 3-kinase (PI3K) activity, which is downstream of the insulin receptor, contributes to increased action potential duration in both type 1 and type 2 diabetics’ cardiomyocytes, and this was mediated by increased late sodium current . AnotherIn a genetically engineered mouse model with accelerated accumulation of mitochondrial DNA damage, older mice exhibited a mosaic pattern of mitochondrial damage in the heart. This heterogeneity resulted in a significant increase in ventricular arrhythmias at both 12 and 18 months . AlthougAdenosine monophosphate-activated protein kinase (AMPK) is a conserved sensor of cellular energy state. The biology of AMPK has been investigated extensively in the context of ischemia, heart failure, and hypertrophy, but much less is known about the role of AMPK in arrhythmias. AMPK signaling is complex, with both transcriptional and nontranscriptional functions. Activation of AMPK increases glucose utilization, increases mitochondrial biogenesis, and reduces oxidative stress . AMPK isAdiponectin is an insulin-sensitizing adipocytokine and is thought to have cardioprotective effects . Serum lThere are conflicting data regarding insulin resistance in adiponectin KO mice ,127. WhoOther adipokines may also have electrophysiologic effects. Leptin (a hormone stored and secreted from adipocytes) has also been shown to be important in the development of HFD-dependent atrial fibrosis and AF, which may relate to the importance of fat loss for arrhythmia protection in overweight individuals .It seems likely that the most impactful dietary/lifestyle modifications for reducing the risk of arrhythmias include consuming foods low in saturated and trans fats and maintaining a healthy BMI. Cellular alterations resulting from obesity—such as tissue fibrosis, atrial remodeling, and diminished conduction velocity—are reversible with weight loss . The conA significant potential benefit in the utilization of antioxidants as anti-arrhythmic therapy is the lack of pro-arrhythmic side effects sometimes observed in traditional pharmacological interventions, particularly due to altered APD . AlthougAnother therapeutic target is the mitochondrially targeted antioxidant mitoTEMPO. The specificity of this mechanism allows for the prevention of mitochondrial ROS-induced proteomic alterations that follow an HFD, providing a feasible option for reducing risk of HF and SCD ,140. In As mentioned previously, activation of PKD and CaMK appears to be important for the pathophysiology of the obese and/or HFD heart. Chemical inhibitors of these kinases could be effective; however, off-target effects could result in unanticipated consequences. Activation of AMPK could be beneficial for heart rhythm in the context of obesity and/or HFD but, to the best of our knowledge, this has not been tested. Though this discussion of potential pharmacological targets is certainly not exhaustive, a final therapeutic intervention involves c-Jun N-terminal kinase, or JNK. This kinase, responsible for the activation of CaMKII, is a factor in the development of atrial arrhythmias . Left unCardiac arrhythmias are responsible for many cardiovascular disease-related deaths worldwide. While arrhythmia pathogenesis is complex and multi-factorial, there is increasing evidence for metabolic causes. Obesity, diabetes, and chronically consuming high-fat foods significantly increase the likelihood of developing arrhythmias. Although these correlations are well established, mechanistic explanations connecting an HFD to arrhythmogenesis are incomplete. Animal models have been used to better understand the molecular mechanisms. A working model is presented as a schematic in More research is needed to better gain an understanding of the mechanisms that connect abnormal metabolism and suboptimal nutrition to arrhythmias. Mitochondrial dysfunction appears to be critical and more information is emerging. As it stands, there is very little work investigating the role of mitophagy in arrhythmias. There is some debate as to the role of mitochondrial calcium in arrhythmias, which is regulated (in part) by the mitochondrial calcium uniporter (MCU) . The mec