Lower incidences of positive sensitivity response were identified for the electrical  I2 = 51%) and cold tests  I2 = 68%). The calculated levels of REP success were similar to those reported for immature teeth. With a very low certainty of evidence, the meta-analysis showed a high incidence of REP’s success for mature teeth with necrotic pulp evidenced by periapical lesion reduction and moderate positive responses to sensitivity tests.This work aimed to investigate the use of Regenerative Endodontic Procedures (REP) on the treatment of pulp necrosis in mature teeth through systematic review and meta-analysis of evidence on clinical and radiographic parameters before and after REP. A search was performed in different databases on 9 September 2020, including seven clinical studies and randomized controlled trials (RCT). The methodological quality was assessed using Revised Cochrane risk-of-bias (RoB 2) and Before-and-After tools. Meta-analyses were performed to evaluate the success incidences regarding the reduction of periapical lesion and recovery of sensitivity. The certainty of the evidence was assessed using GRADE. Meta-analysis showed a high overall success of 0.95  I Pulp necrosis is caused by the death of pulp connective tissue, including the deterioration of blood and lymphatic vascularization and existing nerve fibers , affectiOstby 1961) carried out studies involving biological agents such as blood clots, leading to what is now known as Regenerative Endodontics 1 carried defines Regenerative Endodontics employs the tissue engineering triad  in the root canal space to regenerate the pulp tissue ,10. RegaIn bioengineering, a favorable microenvironment of interaction between stem cells and a biocompatible scaffold is a crucial factor for tissue neoformation. Therefore, it is particularly important to establish a specific biological niche capable of offering adhesive, attractant, and proliferative conditions for stem cells, guiding and facilitating tissue repair ,14. ReceConsistent data regarding the use of Regenerative Endodontic Procedures (REP) in immature teeth with necrotic pulp have been produced ,21, and Therefore, the body of knowledge supporting the benefits of Regenerative Endodontics, mainly produced with immature teeth studies, may not directly apply to mature teeth with necrotic pulp. Although contemporary conventional endodontics is assertive in the recovery of mature teeth, there is interest in investigating the challenges and strategies to perform regenerative treatments in adults ,23. A reHowever, the comparison between REP and conventional endodontic treatment related to clinical parameters becomes questionable since pulp sensitivity tests would not be applicable to a group with filled canals. An analysis comparing teeth before and after REP, therefore, could contribute to a better understanding of REP with mature teeth with necrotic pulp. In this context, the present study aimed to evaluate clinical and radiographic parameters of mature teeth with necrotic pulp before and after REP through systematic review and meta-analysis. A meta-analysis was performed with the available evidence for REP regarding bone healing and the achievement of a positive response to sensitivity testing that, according to the AAE 2018) [18 [20] pThis systematic review was conducted and reported following the PRISMA statement . The proSince no comparison was expected with conventional endodontic therapy, this review based the eligibility criteria on a PIO variant of the PICO framework , which iEven though scientifically relevant, study designs and types of publication that did not produce the type of primary data pertinent to the present analysis were also excluded: in vitro studies, animal studies, studies on immature or open apex teeth, case reports, and literature reviews.Electronic searches were performed on 9 September 2020 in PubMed, Cochrane Library, Scopus, Web of Science, and Latin American and Caribbean Health Sciences Literature database (LILACS)/Brazilian Library in Dentistry (BBO). The grey literature, defined as documents produced on all levels of government, academics, business, and industry in print and electronic formats but not controlled by commercial publishers, was explored using the databases Bielefeld Academic Search Engine (BASE), Science.gov , Global ETD search , and OAlster .Specific search strategies were developed for each database . No restTwo reviewers (P.S. and F.P.G.) independently read the retrieved studies’ titles and abstracts, removed the duplicates, and selected the studies following the eligibility criteria. Full texts were examined to confirm their eligibility. Any disagreement on the suitability of the selected studies was solved through discussion and consensus with a third reviewer (I.P.C.).For data extraction, scientific and technological information items were tabulated and analyzed in Microsoft Office Excel 2013 . Two reviewers (F.N.S and E.S.L.) performed the analyses independently, with a high level of tested agreement. The extracted data of the selected studies included: author(s) and the year of publication, number of participants, number of teeth, type of teeth, presence of control, regenerative material used, irrigation solution, intracanal medication, size K-file apical to induce bleeding near the apical foramen, internal sealing, external sealing, follow-up period, the type of anesthetic used, the time elapsed from the first visit, and outcomes regarding tooth survival assessment and clinical and image evaluations.The assessment of the methodological quality of all included studies was performed by I.C. and M.Z.S. The methodological quality of the randomized controlled trials (RCT) was assessed using the Revised Cochrane risk-of-bias tool (RoB 2) . For priIn addition, the methodological quality of the regenerative intervention groups of all studies, including the non-randomized clinical trials, was assessed by the Before-and-After tool  , consideSuccess was measured according to the attainment of elimination of symptoms, with evidence of bone healing and positive response to sensitivity testing, among the main objectives of REP . To eval2 index, and the predictor intervals were calculated in analyses where a random effect was applied.In all analyses, the number of teeth that presented positive results and the total number of teeth evaluated were included to assess the incidence of therapy success and its 95% confidence interval (CI). A meta-analysis fixed-effect model was applied when a small number of studies were included (three or fewer studies), and a random-effect model was applied when four or more studies were included in the meta-analysis . HeterogSeeking harmonization of outcomes across reviews and aiming to generate evidence , the authttps://gradepro.org/ accessed on 9 November 2020) [The Grading Recommendations Assessment, Development and Evaluation framework , available at er 2020)  was useder 2020) . The Inder 2020)  and Dioger 2020) , describThree hundred and one records were obtained from the search. From those, 149 duplicates were removed, and 152 records remained . ConsideThe primary data for each selected paper are summarized in Regarding the methodological quality of the four RCT studies, the study conducted by Arslan et al. (2019)  had a loThe Before-And-After quality assessment was applied to all selected papers, analyzing 11 items regarding the REP groups. A single study was classified as having poor methodological quality , two werA meta-analysis was performed by evaluating the prognosis of REP in mature teeth with necrotic pulp. The incidence of events  was evaluated after REP, compared to before treatment. Therefore, it was possible to include both RCTs and non-randomized studies with parallel or single-arm (before and after) designs in the same meta-analysis since only the comparable REP groups were included in the present quantitative analysis.Data exclusively related to the REP intervention in all included studies were statistically analyzed and interpreted to integrate the reported information, considering the overall success incidence of mature teeth treated with REP, reduction of periapical lesion obtained after REP, and electrical and cold test responses after REP, as described below. The summary of the certainty of the evidence for each comparison, evaluated using the GRADE approach, is presented in 2 = 6%)   with a p2 = 6%) .2 = 13%)  a, with a2 = 37%), in comparison to the estimated results for REP in immature teeth [Considering a 12-month follow-up of 79 mature teeth with necrotic pulp treated with regenerative endodontics, 73 treatments were considered successful  Ire teeth    c. The GR2 = 51%) .2 = 68%)  d. The ce2 = 68%) .The present study selected primary articles for a systematic review whose outcomes led to a meta-analysis. In the following sessions, the main characteristics of the studies will be discussed with regard to important methodological aspects of the REP. Subsequently, the evidence for the success of these treatments used for mature teeth with necrotic pulp will be discussed in light of the meta-analysis results.The studies included in the present review showed a wide variation in their age groups, which did not allow the assessment of correlations between age and the success of REP on mature teeth with necrotic pulps. A previous study on immature teeth reported that younger patients (9–13 years) obtained better results in the REP than older participants (14–18 years old) . RegardiThe use of apical enlargement in REPs seeks to form a vascularized tissue similar to the pulp tissue through the blood influx from the periapical tissues. Therefore, it allows for the structuring of an autologous scaffold containing growth factors, contributing to the migration of mesenchymal cells . Such prSimilarly, Chrepa et al. (2015)  showed tBleeding can be optimized with an intentional over-instrumentation 2–3 mm past the apex into the periapical region, using K-files #20–#40 to induce it near the apical foramen ,39,40,43Blood derivatives such as platelet aggregates have been widely used in dentistry for regenerative procedures in soft and hard tissues due to their fibrin composition capable of sustained release of growth factors and inflammatory cytokines . The maiTwo selected studies employed autologous blood processing protocols for REP. Brizuela et al. (2020)  employedIt is noteworthy that, in this case, these materials were of heterologous origin, with cells originated from a cell bank  and PPP fractions from the irradiated plasma of healthy donors. Although the use of heterologous materials is considered a viable alternative, the cost of the procedure and the risk of cross-contamination are both increased .g for 10 min) of blood aliquots without the addition of anticoagulants or any other substance, in a low-cost method. This protocol allows the production of an autologous biomaterial through the entrapment of platelets, immune cells, and growth factors in its fibrin network, providing structural stability and long-term production/release of growth factors [The second study, by Zhou et al. (2017) , compare factors . Neverth factors .Another point to be highlighted in the included studies was the recurrent use of ethylenediaminetetraacetic acid (EDTA) ,40,41,43Regarding the intracanal medications used in the REP protocols of the included studies, diverse substances were employed. While both antimicrobial materials for irrigation and intracanal medications have limitations , a signiHowever, some relevant studies have shown that concentrations equal to or greater than 1 mg/mL of antibiotic pastes can produce adverse effects on stem cells of the apical papilla (SCAPs). On the other hand, calcium hydroxide promotes the proliferation of SCAPs, possibly through the release of bioactive growth factors from dentin . TherefoRandomized controlled trials are considered a reference of excellence or a gold standard among all methods of clinical investigation. They provide direct scientific evidence with less probability of error to clarify a cause–effect relationship between two events. In this sense, this review included four RCT studies, even though three of them presented some methodological concerns ,41,43, aThe limitations observed in Jha et al. (2019)  referredSince the present systematic review did not intend to compare REP with conventional endodontic treatments but to assess the outcomes of REP interventions, non-RCT before-and-after studies were also included. Considering the outcomes of interest  as the most relevant for REP, all the studies were also evaluated by Before-And-After criteria, regardless of study design ,64. InteRegarding the outcomes and assessing methods, some authors considered that the treatment success was restricted to the absence of symptoms and the reduction or resolution of the periapical radiolucency ,37,40. OMoreover, the analysis of the results of cold ,43 and eThe overall results of this systematic review identified evidence indicating REP as successful in the treatment of mature teeth with necrotic pulp, suggesting the clinical relevance of REP as a paradigm shift in conventional endodontics. It is noteworthy that the use of biological approaches (blood clot and derivatives) offers the opportunity to regain the sensitivity to thermal and electrical tests, even in mature teeth. A regenerated dental pulp restores the flow of immunological cells to the innate immunity within the canal root, reducing potential reinfections, increasing teeth hydration, and increasing the mechanical resistance to fractures, as compared to endodontically treated teeth .Among the present review’s limitations, the small number of articles is included, as the search was restricted to mature teeth with closed apex, and the impossibility of performing some statistical comparisons due to the high methodological heterogeneity of the selected studies. These restrictions limit the conclusions of the present review as rather preliminary, and it is patent that more high-quality clinical studies are still needed to support the statement that REP is related to a good prognosis in mature teeth, mainly due to the overall low certainty of evidence available to date. The identified factors that most influenced the downgrade of the certainty of the evidence were the risk of bias, the heterogeneity, and the indirectness . Considering the outcomes of interest, the periapical tissues repair could be clearly assessed and compared by the radiographic parameters, observing periapical lesion reduction. However, difficulties regarding the evaluation of the pulp health status were observed, as this remains a challenging subject . The theIn this sense, the authors recommend the proposal of a standardized REP clinical protocol designed to mature teeth with necrotic pulp, based on the best evidence available, that could support future studies with good methodological quality. The best level of evidence could come from randomized controlled clinical trials whose controls were vital homologous teeth and not conventional endodontically treated teeth, as these would not respond to thermal and electrical tests. Further clinical studies should also consider larger sample sizes and extended follow-up periods to increase validity, and special care on randomization, measurement of outcomes , and selection of reported results to reduce risk of bias and achieve high certainty of the evidence on the success of REP for mature teeth.There is a high incidence of success of REP on the healing of mature teeth with a necrotic pulp, evidenced by reduction on the periapical lesion and positive responses to sensitivity tests. However, the low certainty of the available evidence indicates the need for more clinical studies to determine the clinical relevance of Regenerative Endodontic Procedures as a paradigm shift in conventional endodontics.


Atherosclerosis, as a civilization disease, is a serious epidemiological problem. Significant carotid disease and significant coronary artery disease result in acute consequences, such as ischemic stroke and myocardial infarction, which are the major causes of cardiovascular mortality. Typically, atherosclerosis of the aortic arch branches involves the bulbs of the common carotid arteries and the proximal segments of the internal carotid arteries, and can be effectively assessed by ultrasonography. Computed tomography angiography enables the identification of patients with less typical clinical manifestations of atherosclerosis, e.g., brachiocephalic trunk stenosis with symptoms of the steal syndrome and moderate stenosis in the coronary arteries. We present examples of computed tomography angiography images of this type of changes. A 74-year-old male was referred to a cardiology outpatient clinic for further evaluation of a transient loss of consciousness.The medical history revealed arterial hypertension, type two diabetes, previous rheumatic disease (14–15 years), and revascularization of the right lower limb arteries. The patient reported an episode of syncope during housework. The syncope episode occurred when the patient raised his arms upward during the work. The patient suffered from balance disorders, dizziness, and numbness in the upper limbs.Physical examination results: good circulatory and respiratory function, constant HR at 70 bpm, right brachial BP 125/65 mmHg, left brachial BP 130/70 mmHg, and small varicose veins in the left shin.Ultrasound examination of the carotid arteries revealed bilateral atherosclerotic plaques in the visualized carotid arteries sections. A Doppler examination revealed significantly lower flow velocities in the carotid arteries on the right side compared to the contralateral side.Due to the reported non-specific cardiovascular complaints and the ultrasound image of the carotid arteries, the patient was referred to the computed tomography (CT) laboratory for computed tomography angiography (CTA) of the coronary arteries and computed tomography of the carotid and vertebral arteries.The CTA of the coronary arteries, performed using a 384-slice Siemens Force CT, revealed a coronary artery calcium score of 412.5 (left main (LM): 185, left anterior descending (LAD): 16.7, and left circumflex (LCx): 210.8), the value of which indicated a high risk of a significant coronary artery disease A; the exThe CTA of the carotid and vertebral arteries performed using a 384-slice Siemens Force CT revealed a typical origin system of the aortic arch branch, numerous atherosclerotic plaques of various morphotic types in the aortic arch and its branches, significant ostial stenosis of the brachiocephalic trunk by 70–90% of the vessel lumen D, recessThe patient was qualified for further diagnostics and treatment. Further cardiological diagnostics were planned to objectively evaluate the stenosis in the left circumflex, in addition to a consultation with a vascular surgeon to consider invasive treatment of the significant stenosis in the brachiocephalic trunk.The complaints reported by the patient could be most likely related to the steal syndrome, which intensified when the patient raised his right arm. Such an explanation of the syncope episode is indicated by the summary of all data from the anamnesis , physical examination , and imaging examinations . Blood pressure values were only slightly lower on the right arm than on the left arm. However, it should be remembered that, physiologically, blood pressure values measured on the right arm should be higher; the measurements taken were resting measurements made in the standard position of the upper limbs. Moreover, the patient was treated with antihypertensive drugs due to the diagnosed arterial hypertension.The steal syndrome resulting from flow disorders in the brachiocephalic trunk is a relatively rare type of this type of pathology. Most often, the steal syndrome affects the left subclavian artery and results from the narrowing of its proximal part . In the The relationship between the degree of coronary stenosis and myocardial ischemia is one of the key diagnostic problems of coronary artery disease . Current


Biomolecular phase separation denotes the demixing of a specific set of intracellular components without membrane encapsulation. Recent studies have found that biomolecular phase separation is involved in a wide range of cellular processes. In particular, phase separation is involved in the formation and regulation of chromosome structures at various levels. Here, we review the current understanding of biomolecular phase separation related to chromosomes. First, we discuss the fundamental principles of phase separation and introduce several examples of nuclear/chromosomal biomolecular assemblies formed by phase separation. We also briefly explain the experimental and computational methods used to study phase separation in chromosomes. Finally, we discuss a recent phase separation model, termed bridging-induced phase separation (BIPS), which can explain the formation of local chromosome structures. The various components of cells  are organized both spatially and temporally for efficient functioning; membrane-bound organelles are examples of spatiotemporal compartmentalization. However, other types of organelles exist that lack a membrane structure, known as membraneless organelles , and incC. elegans. P granules exchange their components with the cytoplasm and exhibit fusion, dripping, and wetting behaviors. The authors also estimated the viscosity and surface tension of the granules. Subsequently, the material properties and biological implications of membraneless organelles have attracted significant interest ..20]. In ower law F. The crear drop . The freAlthough BIPS and SIPS seem to be opposing concepts, they can work together to induce efficient phase separation. As discussed, in the BIPS model, a bridged loop can act as a nucleation point B. The loin-silico and in-vitro examples have been identified, it needs more examples of phase separation driven by proteins bridging two different DNA segments using multiple DNA-binding sites. Moreover, the in-vivo relevance of the BIPS model needs to be clearly demonstrated.In this review, we discuss the fundamental principles of biomolecular phase separation, including the stickers-and-spacers model, and the current understanding of phase separation involved in DNA-related processes in chromosomes. The stickers-and-spacers model is a simple conceptual framework inspired by polymer theories and can be applied to the phase separation of biopolymers with various architectures. Microphase separation is another important concept adopted from polymer physics, which can explain the segregation of euchromatin and heterochromatin. Various nuclear/chromosomal biomolecular assemblies formed by phase separation are involved in chromosome organization or many genome-related biologically important functions. Notably, in chromosomes, very long DNA molecules are involved in phase separation. The BIPS model illustrates the role of DNA in phase separation and utilizes multivalent DNA interactions of a protein to drive phase separation. However, although a few Currently, a complete conceptual framework to understand the phase separation involved in chromosome organization is lacking, as the inside of a cellular nucleus is filled with many molecules, forming a heterogeneous and disordered mass of biopolymers, which is far from being in equilibrium. To cut the Gordian knot of the chromosome, a fuller understanding of the local and global topologies of chromosomes is required. In particular, the static and dynamic actions of phase separation on DNA topology are key factors for determining the structure and properties of chromosomes, as illustrated by the BIPS model. We need to observe how topological changes in DNA induce phase separation in real time; however, the spatiotemporal resolution of current microscopes is limited. Monitoring the full dynamics of DNA topology and protein assembly may show this process clearly. Regarding simulations, although it is not yet possible to simulate the full nucleus on the atomic level, theoretical and numerical methods can provide valuable information that the experiment cannot access. In this complicated system, multi-scale simulations are promising for accessing a wide range of length and time scales. User-friendly and open-source packages will greatly benefit the community.In the future, we expect that these approaches will provide a clear understanding of the role of phase separation in the chromosome, such as how the chromosome is segregated into two compartments, and how other bimolecular condensates involved in DNA-related functions are condensed with DNA. This understanding will help to explain multiple phenomena in the nucleus throughout the cell cycle.


This book covers basics for facial cosmetic surgery, hard and soft tissue surgical technique, current advancements in cleft and orthognathic surgeries, and various updates of technical and principles for facial cosmetic surgery Fig. . From thVarious field of facial cosmetic surgeries are fully discussed and contemporary surgical techniques are explained with readily visible illustrations and photographs. At the same time, four chapters are accompanied with videos that is quite useful for clinicians. Interestingly, the book contains lifting procedures as an important part of the cosmetic surgery, which reflects current needs and trends of cosmetic facial procedures. This book also contains the office-based procedures such as filler, botulinum toxin, laser, hair restoration, and various non-surgical rejuvenation procedures. It is quite interesting to note that at the end of each part arch as facial contouring (Part III & Part V), rhinoplasty (Part VI), office-based procedures (Part VII), blepharoplasty (Part IX), lifting procedures (Part X), orthognathic surgery (Part XI), and intra-oral plastic surgeries (Part XII), the Q & A discussion or commentaries are accompanied. These sections enables in-depth explanation of a techniques and highlight the important points of the surgical techniques. At the same time, the editors tried to give a chance to think about the critical aspects of each surgical principles to the readers.This comprehensive text composed of 73 chapters provides practical guides for facial cosmetic surgery for not only of the training doctors but also for the specialists. The authors of the book tried to reflect the current enthusiasm and demands of clinical practitioners. And the texts are complemented by tables, figures, and various schematic illustrations; anatomy, surgical technique, and surgical concepts. This is an excellent resource and reference for surgeons tries to perform facial cosmetic procedures as well as for specialists who want to expand the knowledge and skills of cosmetic surgery. This book fully covers the current advanced techniques in cosmetic surgery and would be able to contribute the advancement of this surgical field.


Nerium oleander (Apocynaceae) and is used as a traditional herbal medicine due to its excellent pharmacological properties. It is widely applied for various disease treatments, such as congestive heart failure. Recently, oleandrin has attracted widespread attention due to its extensive anti-cancer and novel anti-viral effects. However, oleandrin has a narrow therapeutic window and exhibits various toxicities, especially typical cardiotoxicity, which is often fatal. This severe toxicity and low polarity have significantly hindered its application in the clinic. This review describes natural sources, structural properties, and detection methods of oleandrin. Based on reported poisoning cases and sporadic animal experiments, the pharmacokinetic characteristics of oleandrin are summarized, so as to infer some possible phenomena, such as enterohepatic circulation. Moreover, the relevant factors affecting the pharmacokinetics of oleandrin are analyzed, and some research approaches that may ameliorate the pharmacokinetic behavior of oleandrin are proposed. With the toxicology of oleandrin being thoroughly reviewed, the development of safe clinical applications of oleandrin may be possible given potential research strategies to decrease toxicity.Oleandrin is a highly lipid-soluble cardiac glycoside isolated from the plant  Nerium oleander, a commonly cultivated ornamental shrub of the Dogbane family, Apocynaceae  pandemic caused by SARS-CoV-2 worldwide. There is currently a lack of systematic pharmacokinetic reports of oleandrin. Additional studies on the pharmacokinetic profile of oleandrin is necessary for the development of safe clinical applications. To date, only poisoning cases and sporadic animal experiments provide a basis for an in-depth understanding of the pharmacokinetic properties of oleandrin leandrin . ThroughAll available information on oleander or oleandrin was collected from Science Direct, China National Knowledge Infrastructure (CNKI), PubMed, Google Scholar, Baidu Scholar, the Web of Science, and related books. In this review, we summarized the sources, structural features, detection methods, pharmacokinetics , and toxicology of oleandrin in order to provide a theoretical basis for further research and development of the glycoside.N. oleander by Lukowski in 1861 -3-[oxy]-14-hydroxycard-20(22)-enolide (CAS Registry No.559-83-1), was the first CG and main active component isolated from  in 1861 . N. olea flowers   is a white crystalline powder with a melting point of 250°C and a molecular weight of 576.727 Da, and it is insoluble in water and soluble in methanol, ethanol, and chloroform. Oleandrin, digoxin, and digitoxin are typical CGs; their structural formulas are shown in Oleandrin  with an oral bioavailability of approximately 30%  dose of oleander extract, whereas humans reached the Cmax within 3 h after an intramuscular dose of oleander extract  and efflux transporters (ETs), which lead to different efficacies and toxicities among patients . For exain vivo metabolism of oleandrin require further extensive characterization, as do their pharmacological contributions. The biotransformation of oleandrin by commonly used microbial strains should be systematically studied, related products obtained, and the enzyme systems involved in the transformation process understood. It may be inferred from the number of metabolites whether there are differences in the metabolic pathways of oleandrin metabolism in the body under the action of the intestinal flora of different species such as humans and rats. In vitro metabolism studies, the structure of oleandrin can be modified to study microbial metabolic processes. In the future, we can use the summary of the reported research studies and the WinNonlin pharmacokinetic software to construct pharmacokinetic models of oleandrin to aid in systematic research.The pH also has a significant effect on the metabolism of CGs. Gault in 1981 showed that an increase in gastric acid secretion caused digoxin hydrolysis in the stomach, producing a large amount of digoxin aglycone, and an increase in metabolites excreted in the urine . Wang inExcretion is the process of transporting the parent drug or its metabolites out of the body through excretory or secretory organs, either under acidic, alkaline, or neutral conditions . There aOleandrin, oleandrigenin, oleandrin conjugates, and other metabolites are excreted in feces, with a small quantity excreted in urine . Gastroiin vivo. Administration of radio-labeled drugs to animals provide the most definitive information on the routes of drug clearance. The administered radiolabeled formulation is usually prepared by mixing the non-radioactively labeled drug with the radiolabeled ones so that the total concentration of the drug and its metabolites could be quantified by determining the radioactivity of the radioactive tracer , and toxicology of oleandrin. Oleandrin is a highly fat-soluble CG with broad pharmacological activities, mainly derived from Seeking attenuation programs and improving the water solubility are key factors to improve the therapeutic capacity of oleandrin. The pharmacokinetic behavior and toxicity of oleandrin are reviewed, which can provide a systematic reference for the expansion of its clinical applications. A shorter pathway to clinical trials can be provided and the development of new drugs and clinically-safe medication can be facilitated if the toxicology and pharmacokinetics of oleandrin are well established. To date, oleandrin, as the main ingredient of several drugs, has been approved and used in clinical Phase I and Phase II trials. In the future, the possible requirement of personalized medicine-directed use of oleandrin for drug safety may be necessary and practical to achieve an ideal therapeutic efficacy with minimal toxicity in clinical practice. Currently, the world’s population is still plagued by COVID-19, generating much interest in antiviral drugs. The discovery of the novel antiviral activity of oleandrin will also accelerate future research, which may lead to the development and application of the glycoside in the treatment of various diseases.


The World Health Organization (WHO) recommends 2 alcohol-based formulations to lessen the spread and infectivity of the novelty virus. Kratzel et al. investigated the efficacy of both the WHO recommended formulations and found them equivalent in terms of efficacy for COVID-19.3Currently, there is no specific treatment for coronavirus disease (COVID-19), and the availability of the newly developed vaccine is limited. Precautionary measures, such as wearing masks, social distancing, handwashing, surface disinfecting, and using hand sanitizers, have remained the only effective measures during the ongoing pandemic.4 According to the CNN news reported in New Mexico, out of 7 people who ingested methanol-based hand sanitizers, 3 died, 3 were in critical condition, and 1 experienced permanent blindness.5 The Food and Drug Administration (FDA) has issued a warning for the general public to avoid sanitizers from unauthentic companies  due to the risk of toxic effects among users.6Unfortunately, to date, 7593 cases of hand sanitizer exposure in children under the age of 12 years have been reported.7 A similar surge was observed in other developing and developed countries. It is pertinent to mention that the announcement by the WHO on the effectiveness of sanitizers caused stock shortages and price hikes. To overcome the shortage and to meet the increasing demand, many pharmaceuticals and chemical, perfume, and beverage industries have started the production of hand sanitizers.Following the WHO recommendations on the use of hand sanitizers as a preventive measure for COVID-19, the demand for these products surged around the globe, resulting in their shortage and unavailability. Pakistan has reported an increased demand of hand sanitizers up to 3000 tons from its regular demand of 1200 tons.4 We believe that health authorities should run parallel maneuvers to ensure the quality and standards of hand sanitizers as a component of a vigorous COVID-19 containment campaign.The increased demand for hand sanitizers resulted in a mass production, which subsequently led to the availability of substandard products with varying concentrations of alcohol in the consumer market. It must be noted that the use of substandard hand sanitizers can be linked to numerous adverse events involving permanent blindness, gastrointestinal irritations, portal vein embolism, mild mucosal irritation, eczema, skin irritation, respiratory problem, resistance to antibiotics, hormonal problems, depression, hypothermia, hypertension, ketoacidosis, alcohol poisoning, and in severe cases – death.To overcome or fulfill the demand for hand sanitizers, controlling the price hike, and lessening the hazards of these products, we are inclined to share a few suggestions to curb the situation of COVID-19. The industrial pharmacist can play the role in providing standard production of hand sanitizers by maximizing the production, quality control, assurance, and compliance with official guidelines as provided by the WHO, FDA, and pharmacopeias. Drug regulatory and health authorities should ensure the standard production and the availability of hand sanitizers in hospitals, pharmacies, health care centers, and high-risk vicinities at affordable prices. Moreover, implementation monitoring strategies and ensuring the regulatory practices are of utmost importance during the current circumstances. In cases of shortage, priority should be given to the high-risk areas, and supply should be maintained. In addition, community pharmacists can ensure the safe and effective use of these products and can optimize the preparation of hand sanitizers within the pharmacy premises by taking advantage of small-scale manufacturing. Moreover, they are much aware on the importance of complying with the recommended specifications and guidelines. These practices would not only be translated into a drastic reduction of the cost to the public, but also will aid in the provision of standard and quality products to the consumers. Furthermore, a community pharmacist is an accessible professional to the community and can educate the public on the safe use of hand sanitizers, hereby ensuring the safety on the community level. Community pharmacists counsel people to avoid touching the facial area after using hand sanitizers, as exposure can cause skin irritation. They can also counsel the public regarding the storage of hand sanitizer at room temperature, preferably at cool places, and keeping the hand sanitizer out of the reach of children. The pharmacist can also play the role in providing post-ingestion management of hand sanitizers. Pharmacists can assist in the case management of adverse events following ingestion of hand sanitizers either directly or indirectly by providing the swift information on treatment protocols. Pharmacists as health care professionals can play a major role in current crises by providing awareness, education, and guidance related to appropriate selection, buying and use of hand sanitizer, monitoring any substandard hand sanitizer brands, which can provide a threat to public health instead of helping curb the COVID-19. Taken together, collaborative efforts of pharmacists with health authorities will mitigate the harms and burden of sanitization associated adverse events, for which none of the countries is readily prepared amid an overwhelming health care system.


Coronary artery ectasia (CAE) is defined as a diffuse or focal dilation of an epicardial coronary artery, which diameter exceeds by at least 1. 5 times the normal adjacent segment. The term ectasia refers to a diffuse dilation, involving more than 50% of the length of the vessel, while the term aneurysm defines a focal vessel dilation. CAE is a relatively uncommon angiographic finding and its prevalence ranges between 0.3 and 5% of patients undergoing coronary angiography. Although its pathophysiology is still unclear, atherosclerosis seems to be the underlying mechanism in most cases. The prognostic role of CAE is also controversial, but previous studies reported a high risk of cardiovascular events and mortality in these patients after percutaneous coronary intervention. Despite the availability of different options for the interventional management of patients with CAE, including covered stent implantation and stent-assisted coil embolization, there is no one standard approach, as therapy is tailored to the individual patient. The abnormal coronary dilation, often associated with high thrombus burden in the setting of acute coronary syndromes, makes the interventional treatment of CAE patients challenging and often complicated by distal thrombus embolization and stent malapposition. Moreover, the optimal antithrombotic therapy is debated and includes dual antiplatelet therapy, anticoagulation, or a combination of them. In this review we aimed to provide an overview of the pathophysiology, classification, clinical presentation, natural history, and management of patients with CAE, with a focus on the challenges for both clinical and interventional cardiologists in daily clinical practice. Coronary artery ectasia (CAE) is defined as a diffuse or focal dilation of an epicardial coronary artery, with a diameter that exceeds of at least 1.5 times the normal adjacent segment. It is described in up to 5% of all comers' patients undergoing coronary angiography, but with a considerable variability in relation to the patients' clinical presentation and the definition adopted for CAE . While aThe aim of the present review is to outline the classification, etiopathogenesis, clinical presentation and diagnostic assessment of CAE, with a specific focus on management strategies and long-term outcome.To date the definition of CAE remains uncertain: the lexical variability prevents to accumulate the data for robust scientific evidence on this issue. The mainstream of the literature directs the nomenclature on the basic distinction between the terms ectasia and aneurysm that, although sometimes used as synonyms, represent two different phenotypes of the disease: while the first one identifies a diffuse dilatation that involves > of 50% of the length of the vessel, the term coronary artery aneurysm (CAA) refers a focal dilatation . CAAs arCAE is a relatively uncommon angiographic finding, with a prevalence ranging from 0.3 to 5% of all comers' patients undergoing coronary angiography , 2, 17. Several reports showed a gender predominance with a high prevalence of CAE in men , 18, 19.Right coronary artery (RCA) is the most frequently involved, followed by left anterior descending (LAD), left circumflex (LCx), and less frequently left main (LM) .CAE has been frequently reported in patients with aneurysms in other vascular beds, such as ascending and abdominal aorta and pulmonary arteries . MoreoveAlthough the etiopathogenesis of CAE is not fully understood, atherosclerosis seems to be the most frequent cause in adults . This hyHowever, CAE shows some other histopathological features that are not usually observed in patients with CAD, like the relative preservation of the intima and the extensive loss of musculoelastic components of the media, which is thought to be one of the central mechanisms in the pathogenesis of CAE , 33. IndThe coexistence with other conditions, such as BAV and aortic aneurysms, corroborates the conception of CAE as the manifestation of a systemic disease, in which individual genetical susceptibility might be one of the contributing factors , 28.Systemic inflammatory diseases, infections and connective tissue disorders are other possible causes of CAE , 40, 41.CAE has also been reported among cocaine users and its pathogenesis may be related to severe hypertension episodes and to the direct endothelial damage caused by drug-induced vasoconstriction .CAAs or pseudoaneurysms are rare but potential complications of PCI, especially following brachytherapy, atherectomy, or drug-eluting stent (DES) implantation –48. ResiA distinct setting of aneurysmatic disease is represented by aortocoronary saphenous vein graft aneurysms (SVGAs). SVGAs are a rare and late complication of coronary artery bypass grafting (CABG), usually occurring after ~15 years from surgery. As for CAE, etiopathogenesis is not fully understood and several mechanisms have been proposed, including structural deterioration caused by graft atherosclerosis, technical issues related to surgical manipulation and adaptation of vein graft to higher arterial pressure with vessel wall weakening and dilatation. SVGAs usually progress over time, often reaching large diameter, and have a high incidence of life-threatening mechanical complications, such as compression of adjacent structures, fistulous communications, and rupture , 57.The clinical manifestations of CAE are heterogeneous, and often it is occasionally recognized during coronary angiography or computed tomography (CT). However, CAE may become clinically overt through different possible scenarios, including ACS, effort angina, exercise-induced ischemia, microvascular dysfunction, compression of adjacent cardiac or non-cardiac structures and the tragical, albeit rare, complication of rupture with acute cardiac tamponade , 58–60. The gold standard for the diagnostic assessment of CAE remains coronary angiography: the typical angiographical features are delayed antegrade contrast filling, segmental back flow, and local deposition of dye in the dilated coronary segment, all of them able to depict the severe flow disturbances of CAE. However, slow flow and blood stasis might complicate the angiographic assessment of CAE, especially when evaluating the true size of the vessel or the presence of thrombotic material. IVUS is useful to better assess vessel wall architecture and to distinguish between true aneurysm, pseudoaneurysm, normal segments with adjacent stenosis or complex plaques angiographically mimicking CAAs : this diOptical coherence tomography (OCT) is another possible tool in the invasive assessment of CAE. Compared to IVUS, OCT has greater axial and spatial resolution that might be helpful in CAE to assess atherosclerotic plaque features, thrombus burden and mechanisms of PCI failure. However, its application in large vessel is limited by the low penetration depth, with loss of image definition as the distance of the anatomical structures from the lens increases .Other technologies are available for the interventional cardiologist in the toolbox of CAE investigation, but they play a minor role. CT may be helpful in the non-invasive assessment of CAE, increasing the prevalence of incidentally found CAE, which was described as a rare finding on coronary angiography in previous decades , 63. CT Magnetic resonance angiography (MRA) is another possible tool in the assessment of CAE, especially in patients with contraindication for other diagnostic techniques. MRA provides important anatomical and functional information, especially in large proximal vessels, while its resolution decreases in smaller and distal segments , 68.Transthoracic and transesophageal echocardiography have a role in the evaluation of patients with CAE, especially in children with KD and large CAAs of the proximal LM and RCA. Echocardiography is useful to assess the location of the CAA and the presence of intraluminal thrombi. Given its non-invasive nature, it is an ideal method for long-term follow up in adulthood of patients with history of KD .The long-term outcome of patients with CAE has been poorly investigated. Although flow disturbances, enhanced thrombogenicity and high risk of procedural drawbacks during PCI would be perceived as associated to unfavorable prognosis, literature does not provide conclusive evidence on the clinical outcome of patients with CAE. Krüger et al. demonstrated the occurrence of angina and exercise-induced ischemia in patients with isolated CAE and non-obstructive CAD, suggesting that flow turbulences might potentially impair myocardial perfusion even in absence of coexisting significant stenoses . Howeverper se the procedural success of PCI and the long-term clinical outcome of these patients. Bogana Shanmugam et al. compared 25 STEMI patients with an EIRA to a cohort of 80 non-EIRA STEMI patients and found that the EIRA group, despite similar in-hospital outcomes, had a higher incidence of long-term cardiovascular events, particularly driven by recurrent MI, unstable angina (UA), and need for surgical revascularization , often associated with high complexity of the lesion and large thrombus burden, might affect rization . Conversrization .Similarly, Ipek et al. showed no difference in terms of in-hospital and 1 year mortality and revascularization between STEMI patients with and without EIRA undergoing primary PCI, despite a higher rate of no-reflow in the EIRA group . HoweverIn accordance with these findings, Gunasekaran et al. assessed the prognostic significance of CAE anatomical extension in a retrospective study on 317 patients and found that a higher grade of CAE (Markis type I and II) was associated with a significantly higher risk of ACS at long term compared to lower grade of CAE (Markis III and IV), despite similar severity of underlying CAD. Moreover, patients with Thrombolysis In Myocardial Infarction (TIMI) flow <3 showed a higher occurrence of ACS at follow up compared to patients with normal epicardial flow, with further adverse impact on outcomes when associated with multivessel CAE . A cohorTaking all these findings together, it is reasonable to consider CAE as an aggressive phenotype of coronary disease associated with a multifactorial risk of cardiovascular events at follow up: technical issues of PCI and the intrinsic complexity of the disease lead to the imbalance of coronary hemodynamics, susceptibility to local thrombotic disorders and adverse clinical outcome.The main studies evaluating clinical outcome of patients with CAE are summarized in The optimal management of patients with CAE is largely unknown. The uncertainties regarding the natural history of CAE and the absence of robust randomized and large-scale data complicate the resolution of this clinical conundrum. Moreover, the most relevant amount of the current evidence on the treatment of CAE has been extracted from cohorts of symptomatic patients, often with ACS as clinical presentation, but conversely a lack of data on asymptomatic patients with incidentally found CAE and no evidence of significant CAD exists. Possible treatment options include medical therapy, PCI, and surgery, but each of these strategies reserves technical and clinical challenges. In absence of specific recommendations, management strategies are still individually tailored according to clinical presentation, anatomical features, and procedural complexity.Percutaneous treatment of CAE is a valuable option in patients with suitable anatomical and clinical features. However, PCI of ectatic and aneurysmatic lesions presents several challenges, starting from lack of specific indications. First, it is uncertain whether conservative or interventional strategy is to be preferred in CAE patients without obstructive CAD, and there is no supportive evidence on PCI outcomes in this setting. Nonetheless, in patients with CAE and significant coronary stenoses and/or ACS, PCI has several technical challenges that must be carefully balanced into decision-making process when considering the optimal revascularization strategy. In the setting of an ACS with an EIRA, high thrombus burden with distal embolization and microvascular damage substantially increases the technical complexity of PCI, with a high risk of procedural failure and adverse events at long term . Given tBesides procedural complications, previous data showed that the presence of a CAA is a risk factor for stent thrombosis (ST) after PCI in patients with ACS . IannopoIn consideration of these technical challenges, if PCI is clinically indicated, different percutaneous techniques should be adapted to the anatomical scenario. Exclusion with covered stents might be indicated in saccular CAAs or small pseudoaneurysms not involving major side branches . Figure Beyond procedural technicalities, another limitation of covered stents is the high risk of ISR and ST at long-term follow up, with a substantial higher risk of target lesion revascularization (TLR) compared to new-generation DES –98. The In cases where covered stent implantation is not possible due to severe tortuosity, calcifications or fear of side branch loss, stent-assisted coil embolization can be used, especially in large CAAs with wide neck. With this technique, commonly used in the treatment of cerebral aneurysms, a floppy guidewire supported by a microcatheter is directed inside the CAA before stent implantation. A coronary stent covering the entire neck of the CAA is then deployed at low pressure, jailing the microcatheter into the CAA; coils can then be passed through the microcatheter to wrap around the stent. Post-dilation of the stent is securely performed, after the microcatheter removal. Additional coils can be advanced through the stent struts if needed . There aPercutaneous interventions are a valuable option for the treatment of SVGAs, especially in patients with high surgical risk and/or with preserved myocardial perfusion. These techniques have been increasingly used in recent years and include covered stent implantation, percutaneous closure with Amplatzer occluders and coil embolization with or without PCI of the native grafted vessel .There is lack of robust evidence on the surgical treatment of CAE. Possible surgical techniques include CAA resection, ligation, or marsupialization with bypass grafting –106. HowThe optimal medical therapy of patients with CAE is an area of ongoing debate. Considering that atherosclerosis is involved in the pathogenesis of CAE in most cases, an aggressive modification of cardiovascular risk factors should be mandatory in these patients.However, antithrombotic therapy is the most controversial issue on the pharmacological treatment of CAE. Severe coronary dilatation is associated with flow disturbances and blood stasis, thus predisposing to the activation of coagulation cascade with increased risk of local thrombosis and distal embolization , 107. MoHowever, because of the observational design and the relatively small sample size of these studies, these data should be interpreted with caution and should be confirmed by dedicated randomized trials or large multicentre registries.In patients with KD OAC is recommended in selected patients with large and/or rapidly expanding CAAs . MoreoveGiven the role of atherosclerosis, inflammation and endothelial disfunction in the pathogenesis of CAE, statins and ACE-inhibitors have been proposed as possible therapeutic options due to their pleiotropic effects on the endothelium . Fan et Several anti-ischemic drugs have been proposed as possible therapeutic options in patients with CAE. Krüger et al. showed that the development of inducible ischemia in CAE depended on heart rate . TherefoUnlike β-B, calcium channel blockers (CCB) have anti-spastic effects and might have a beneficial role by reducing the risk of coronary spasm and thrombus formation . Other pTrimetazidine can also improve coronary flow and myocardial preconditioning by increasing adenosine levels, and showed to reduce exercise-induced myocardial ischemia in patients with CAE .Unlike patients with CAD, nitrates should be avoided in patients with CAE because of their promotion of flow disturbances and exacerbation of myocardial ischemia .Despite growing evidence on CAE in recent years, several controversies remain about its natural history and treatment. Although several studies showed a poor outcome in patients with ACS and angiographic evidence of CAE, the risk of future adverse events in asymptomatic patients with isolated CAE and non-obstructive CAD is unknown. Due to the absence of data from large multicenter studies and of specific recommendations, there is great uncertainty on what is the best medical and interventional treatment of isolated CAE.In patients with obstructive CAD and indication for PCI, there are several procedural issues: (1) The use of intravascular imaging during PCI might be helpful for stent sizing and landing zone identification, but the large caliber may affect the quality of images, particularly for OCT; (2) The high thrombus burden in the setting of an ACS is associated with a higher risk of no-reflow, but the optimal periprocedural antithrombotic therapy has not been clarified yet; (3) The technology advances in interventional cardiology have provided several options for the percutaneous treatment of patients with CAE ; however, the long-term performance of these devices needs to be investigated; (4) Optimal antithrombotic therapy is another missing piece in the complex framework of CAE; OAC and long-term DAPT have been proposed as possible therapeutic options, but the evidence is still controversial and limited to small number of patients.Moreover, given atherosclerosis as the most prevalent underlying condition, it is unknown whether aggressive lipid lowering therapies with PCSK-9 inhibitors might provide a prognostic benefit in CAE patients with ACS. Lipoprotein (a) [Lp(a)], despite being associated with aortic and cerebral aneurysms, has an unclear role in the pathogenesis of CAE –119. In The renovated interest in CAE in the recent years emphasizes the crucial role of some required improvements in its knowledge pathway: the importance of a unique shared definition, of a deeper characterization of CAE phenotypes in real-world registries, and of effective therapies in randomized trials.CAE, defined as a diffuse or focal dilatation of an epicardial coronary artery, is reported in up to 5% of patients undergoing coronary angiography. The clinical manifestations of CAE are heterogeneous, ranging from asymptomatic cases to high-risk patients with ACS . There iLE, MD, FC, and MB: drafting of the manuscript. AS, TA, FT, and GE: revising critically the manuscript. CB, CV, and GG: final approval of the manuscript. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The adrenocortical stress-response is extraordinarily conserved across mammals, birds, fish, reptiles, and amphibians, suggesting that it has been present during the hundreds of millions of years of vertebrate existence. Given that antiquity, it is relatively recent that primate social complexity has evolved to the point that, uniquely, life can be dominated by chronic psychosocial stress. This paper first reviews adrenocortical evolution during vertebrate history. This produces a consistent theme of there being an evolutionary tradeoff between the protective effects of glucocorticoids during an ongoing physical stressor, versus the adverse long-term consequences of excessive glucocorticoid secretion; how this tradeoff is resolved depends on particular life history strategies of populations, species and vertebrate taxa. This contrasts with adrenocortical evolution in socially complex primates, who mal-adaptively activate the classic vertebrate stress-response during chronic psychosocial stress. This emphasizes the rather unique and ongoing selective forces sculpting the stress-response in primates, including humans. This is meant to contrast with the present, whether that means since the invention of agriculture and unequal material culture, since the Industrial Revolution, or since the invention of Facebook or deep-fried double stuf Oreos. The take-home message is always the same – the transition to the particular contemporary lifestyle has been too rapid for hominin evolution to catch up, and pathology arises from that mismatch. The archetypal example of this is the current obesity epidemic: during that 99% of hominin history, food was often sparse and unpredictable, and it was a rarity for hunger to be regularly satiated. As a result, a history with little evolutionary pressure to develop strong physiological signals of satiety must now contend with the novelty of Westernized diets.The present paper concerns a time span one-to two-orders of magnitude longer than hominin history, in considering the blink-of-an-eye existence of complex primate sociality in the context of hundreds of millions of years of vertebrate evolution. The rationale for this contrast is the remarkable conservation of the endocrine stress-response across vertebrates. The organization of the hypothalamic-pituitary-adrenocortical axis, the reliance upon a handful of types of glucocorticoid molecules (GCs), the rapid increase in GC secretion in the face of challenge are all conserved across mammals, birds, fish, reptiles and amphibians. As one considers the subtleties and complexities of, say, the interactions of socioeconomic status, familial support, social capital and the psychology of attributional style on stress-related health, it is startling to realize that a vast length of time ago, while evading a predator or pursuing a prey, a dinosaur secreted GCs.Despite this conservatism and antiquity of the adrenocortical stress-response a point must be made, however – no dinosaur ever worried itself sick from perseverating on the absurd idea that an asteroid might strike earth. For 99% of vertebrate history, the stress-response evolved amid selective pressures related to the likes of grassland predator/prey relations, bird migrations or fish schooling. What is one to make of the relatively recent evolution of the stress-response in social primates that can chronically worry? Much as with Oreo cookies in the context of hominin history, chronic psychosocial stress in primates in the context of vertebrate history produces a picture of evolutionary mismatch.2--Since the days of Selye, there is the inevitable lack of clarity as to whether “stress” refers to the external perturbation or the internal response . Through--An increase in GC secretion is not synonymous with stress, as shown by the daily circadian rhythm of GC concentrations. The non-stress roles of GC secretion have been aptly termed “predictive homeostasis”  and as w--Stressor-induced GC secretion is not synonymous with the entirety of the stress-response, as would be testily emphasized by any autonomic nervous system expert. The heavy emphasis in the stress literature on GCs (and in this review as well), in part reflects the extraordinary range of GC effects on metabolism, immunity, reproductive physiology, brain function and behavior; however, it also reflects the far greater difficulties in studying the secretion of sympathetic catecholamines, especially in naturalistic settings.3--Stressors are less about the absolute intensity of a challenge than about its degree of discrepancy from the norm. For a bird species migrating between the arctic and the tropics, dealing with intensely cold temperatures that, nevertheless, are precisely what would be expected constitutes a normal workday in the arctic for the adrenocortical axis, whereas an unexpectedly chilly day in the tropics can be a life-threatening allostatic challenge ; b.--Different pathologies can arise from abnormalities in basal GC secretion, versus stress-induced secretion, versus secretion during the recovery period after a stressor abates. Not surprisingly then, evolutionary forces have acted independently on these different components of GC secretion .--An array of GC actions involve the familiar direct effects on target tissues in response to an ongoing stressor, and these effects have long dominated thinking about stress. However, it must be remembered that GC effects are often, a) indirect, permissively augmenting the direct actions of other signaling systems ; b) preparative, in anticipation of a stressor ; this harks back to preparative homeostasis, and c) suppressive, reversing the effects of early mediators of the stress-response during the recovery period (such as the stimulatory effects of GCs on appetite) .--As appreciated since the golden age of steroid receptor-ology some decades back, variation in levels of receptors for GCs (as well as in levels of receptor cofactors and transport binding globulins), are at least as important as variation in circulating levels of GCs themselves. Framed in an evolutionary context, there can be instances where selective evolutionary pressure on GC receptors has been greater than on GC levels .This review first considers the evolutionary forces that have sculpted the adrenocortical stress response throughout vertebrate history, before then turning to the forces pertinent to the recent emergence of complex primate sociality. One theme will be the remarkable differences in the types of selective forces in these two realms; another will be the need in both domains to question whether individual differences in aspects of the stress-response translate into meaningful differences in Darwinian fitness.4protective effects of GCs during any given stressor and those favoring protection from GCs and their cumulative adverse effects over time. A variety of factors come into play in resolving these trade-offs .4.1A major source of variability is the severity of an ongoing environmental stressor in the context of the long-term costs of chronic GC overexposure. For example, among American redstarts, a migratory bird, elevated basal GC levels predict survival of birds who overwinter in a harsh environment , but not those who overwinter in a more benign one (mangroves) . In nume4.2). The need for trade-offs regarding GC actions are dramatic in the context of reproduction. On one hand, there is the typical stressfulness of reproduction, reflecting its metabolic costs, the demands of courtship displays that are intrinsically competitive and often agonistic, or the necessities of migrations to mating or spawning grounds. On the other are the well-known anti-reproductive effects of the stress-response, particularly via GCs, meaning that from a reproductive perspective, increased GC secretion during a mating period would be at precisely the wrong time. As such, across a range of vertebrates, higher average GC levels  during a breeding season predict lower Reproductive Success, while higher levels before a breeding season predict the opposite  . As anot4.3The evolutionary trade-off between ongoing protective effects of stress-induced GC secretion and potential future pathologies of GC excess is strongly influenced by species lifespan. Being relatively long-lived is, in effect, a prerequisite for chronicity of any sort, including chronic GC hypersecretion and its eventual cost. Thus, the longer the future there typically is for a species, the greater the likelihood of paying a price for cumulative GC exposure. Commensurate with that, across bird species, there has been selection for lower basal and stress levels of GCs in species that are longer-lived . Within 4.4Considerable amounts of research have focused on uncovering the mechanisms, epigenetic or otherwise, by which early life experience can cause persistent changes in physiology and behavior, changes that can be life-long or even multigenerational. The particular ability of early life stress to persistently alter features of the future stress-response has been framed in at least two contrasting ways cf .Developmental Constraint models focus on how such life-long GC overexposure is deleterious, but is an unavoidable price for the protective effects of GCs at the time of a stressor . As another example of this point, there are consistent booms and busts in population size among snowshoe hares, driven by cycles of predation risk; despite this, periods of maximal risk do not constitute pathophysiological stressors . And as The other tradition revolves around animals in natural settings, exposed to naturalistic stressors (as opposed to the increasingly common anthropogenic stressors). As reviewed below, there is ample evidence to suggest that chronic social stress can decrease fitness in other primates; nonetheless, some authors have emphasized how little actual evidence there is for naturalistic stressors lessening fitness in wild populations . This isTo summarize the evolutionary forces that have acted upon the adrenocortical axis during vertebrate history: a) The tradeoff between the protective effects of GCs in the face of stressors versus the adverse long-term consequences of GC excess varies by species, populations and individuals as a function of predictability of environmental demands, the frequency and nature of mating opportunities, lifespan, body size and metabolism. B) Throughout the life of some species (and particularly during development), there may not actually be a tradeoff involved, in that the protective effects of GCs during a stressor have evolved to also adaptively prepare organisms for future stressors. C) Whether sustained stress has been decreasing fitness for hundreds of millions of years of vertebrate evolution depends, in a sense, on the ethological appropriateness of labeling an event as a stressor; importantly, what might seem self-evidently stressful to us may merely be the predictable and normal exigencies of everyday life in other species.This summary positions us to now appreciate just how different evolutionary pressures have played out in social primates.5Humans share with other primates a number of deeply fundamental similarities, including long lives, living in stable social groups with individuated relationships, where the strength of social bonds can have at least as much impact on fitness as does social status, and where there is the complexity that today's source of social stress may be an individual who must be cooperated with tomorrow .Among other things, these shared traits produce a continuum between humans and other primates as to the social circumstances that can alter GC secretion. Some of these similarities can be remarkably subtle. One concerns the strong, stable personality differences observed in other primates. For example, among wild baboons and independent of rank, higher basal GC levels are observed among the males whose ongoing grooming is most likely to be disrupted by the mere non-threatening presence of a higher-ranking male. Moreover, this personality/endocrine profile is stable over years, and is demonstrable on a day in which there has not been an incidence of ongoing grooming being disrupted under these circumstances. Thus, this is the endocrine picture of a constitutional tendency to see social threat that others do not .As another example, most dominance interactions among male baboons occur with individuals of adjacent ranks ; even among baboons, politics is local. Furthermore, such adjacent interactions are typically the most tense and fraught with the potential for aggression and injury, and are most sensitive to male testosterone levels . OccasioAnother demonstration of this continuity comes with the power of female sociality and support. An adult male baboon will occasionally transfer into a troop; such males are often initially aggressive and destabilizing, displacing aggression on females after tense dominance interactions with resident males. At such times, females respond to this social stressor with increased intensity of grooming interactions with other females, and those who already have the most stable and focused grooming relationships show the smallest increases in GC levels .As a final example, if wild baboons are injected with beta-carboline antagonists of benzodiazepine receptors, they transiently burst with anxiety-like behaviors -- frenzies of repetitive self-grooming and scratching, teeth grinding, nose wiping, slapping at the ground or purposeless breaking branches or pulling up grass. This disinhibition of anxiety is most pronounced in subordinate animals, who have the most reasons to have elevated endogenous benzodiazepine signaling. However, that is only seen in troops in which subordinates are subject to high rates of displacement aggression by dominant individuals .Thus, stress-related physiology in other primates can reflect personality differences, the social meaning of a behavior independent of its actual performance, the protective effects of social support networks, or cultural differences in different populations. The continuity with our species should be abundantly clear. (In discussing these studies, it is important to note two things. First, these psychoendocrine correlates among wild primates cannot reveal anything about the direction of causality, if any. However, studies of captive primates, in which social settings can be experimentally manipulated, indicate that social circumstance drives the endocrine changes, rather than the reverse cf . Second,These examples underline the different evolutionary pressures on social primates, as compared to the rest of vertebrates. As discussed, for other vertebrates, there is the perpetual tradeoff between the protective effects of GCs in the face of a present threat balanced with whether there is a long-term cost to chronic GC hypersecretion. But the crucial point is that there is no such tradeoff in these primate instances; in the example of tonically elevated basal GC levels in baboons who consistently perceive events to be threatening when others don't, the point is that there is no actual present-tense threat.The same is observed in humans, in that depression and human anxiety disorders are pathologies of chronic psychosocial stress, built upon perceived lack of control and predictability, lack of coping outlets, and lack of social support. Importantly, both have long been known to be psychopathologies of chronic overactivation of components of the stress-response, with major depression typically associated with elevated basal GC levels or resistance to GC negative feedback , and anxInvoking evolution in the context of primate psychosocial malaise must raise the question of whether these states actually have fitness consequences. As discussed above, the brutality of an arctic winter is merely quotidian if you are an arctic ground squirrel. Does chronic psychosocial stress actually exact a fitness cost in primates? This question has been raised , whether6A standard trope is that there has been insufficient time for evolution to have caught up with the costs of a Westernized diet. Similarly, there has been insufficient time for evolution to have caught up with primate social complexity and psychosocial stress.An optimal profile of the adrenocortical stress-response would consist of low basal GC levels, rapid and substantial stress-induced GC secretion but only in the face of “real” vertebrate stressors, and rapid recovery once stressors abate. As such, an easy prediction is that ongoing evolutionary forces are slowly selecting for this sort of profile, selecting against baboons who see threats that other baboons do not and humans crippled by depression or anxiety. Potentially, we may only be a few million years away from primates and, say, marine iguanas having far more different adrenocortical axes than is presently the case.However, there is a countervailing force that is relevant. It is maladaptive to be incapacitated by constant anxiety about non-existent threats. In contrast, it can be life-saving for say, someone Jewish, Roma or LGBTQ in Berlin in the 1930s to be among the first to perceive substantial threat and leave. Much research and theorizing have focused on context-dependent evolutionary selection, where the same trait can have diametrically opposite fitness consequences in different settings, and on evolutionary bottle-necks, where some unique circumstance removes nearly all of a population, leaving only those with a previously obscure genetic trait. A combination of these evolutionary forces may be occurring now among we primates, producing a scalloped pattern of long periods of gradual selection against those most prone towards psychosocial stress, punctuated with periods of rapid and severe selection where only the canaries in the mine-shafts of psychosocial stress come out the other end. Context-dependent selection and selective bottlenecks mean that primates will be dealing with the evolutionary consequences of malignant sadness and corrosive worry for epochs to come.Robert M. Sapolsky: I, as the author, am solely responsible, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, and all aspects of writing of paper 210308-017180..I have no conflicts of interest.


Mitochondria are key players of aerobic respiration and the production of adenosine triphosphate and constitute the energetic core of eukaryotic cells. Furthermore, cells rely upon mitochondria homeostasis, the disruption of which is reported in pathological processes such as liver hepatotoxicity, cancer, muscular dystrophy, chronic inflammation, as well as in neurological conditions including Alzheimer’s disease, schizophrenia, depression, ischemia and glaucoma. In addition to the well-known spontaneous cell-to-cell transfer of mitochondria, a therapeutic potential of the transplant of isolated, metabolically active mitochondria has been demonstrated in several in vitro and in vivo experimental models of disease. This review explores the striking outcomes achieved by mitotherapy thus far, and the most relevant underlying data regarding isolated mitochondria transplantation, including mechanisms of mitochondria intake, the balance between administration and therapy effectiveness, the relevance of mitochondrial source and purity and the mechanisms by which mitotherapy is gaining ground as a promising therapeutic approach. Despite the early recognized relevance of mitochondria for cell energetics and homeostasis, there was little or no information regarding mitochondria’s dynamism and transfer feasibility between cells. It was only in 1982 that Clark and Shay demonstrated that isolated mitochondria carrying a mutation for chloramphenicol and efrapeptin resistance in its genome, naturally transferred the antibiotic resistances to sensitive cells by simple incubation . These w0 cells, successfully restoring mitochondrial respiration [Still, it took over two decades since Clark and Shay findings for a follow up demonstration of mitochondrial spontaneous transfer. In 2006, Spees and colleagues reported the first evidence supporting the horizontal (cell-to-cell) transfer of mitochondria between mammalian cells. The co-culturing of A549 mutated and depleted cells with human mesenchymal stem cells (MSCs) successfully rescued the mitochondrial function in the former, as shown by the increase in ATP content, membrane potential and oxygen consumption [In the last two decades, several studies showed an in vitro relocation of mitochondria, either by horizontal transfer  or an iBased on the above findings, the first study of the potential of mitotherapy in an animal model was published by McCully and colleagues in 2009 . RespiraStudies of the in vivo models of liver insults indicate that transplanted mitochondria may also contribute to metabolic recovery of hepatocytes. For example, an intrasplenic infusion of isolated mitochondria attenuated oxidative stress, cell death and the serum levels of liver enzymes in a rat liver model ischemia-reperfusion . The sysIn the field of respiratory diseases, an intratracheal injection of mitochondria reversed airway hyperresponsiveness to acetylcholine in rats . The traIn parallel with the studies above, evidence was gathered that mitotherapy may also be protective in the central nervous system (CNS), as shown in the next section.Diseases of the CNS are often terminal due to the intrinsic inability of central neurons to regenerate and its non-permissive microenvironment. Among new therapeutic approaches to overcome these impediments, several studies examined the potential of mitochondria transplantation in neurological disorders .In the first such study, the mitochondria derived from hamsters attenuated the area of brain infarction and neuronal death, and restored motor performance in a stroke model of middle cerebral artery occlusion (MCAO) in rats, either via local intracerebral or systemic intraocular-arterial administration. In addition, the transient proliferation of microglia and astrocytes suggested a role of immune response to mitotherapy . In subsA few studies also tested the efficacy of mitotherapy in models of Parkinson’s disease (PD), a prevalent and disabling neurodegenerative condition characterized by progressive motor as well as eventual cognitive dysfunction. There is strong evidence of mitochondrial dysfunction in PD, manifested by a reduced bioenergetic potential, the disruption of redox homeostasis and an increased susceptibility to excitotoxic neuronal death. In a well-established rat model of PD produced by the administration of the neurotoxin 6-hydroxydopamine, an injection of mitochondria in the medial forebrain bundle successfully attenuated the oxidative damage and degeneration of dopaminergic neurons and improved locomotion . In anotMitotherapy also showed promising results in experimental models of schizophrenia (SZ) and Alzheimer’s disease (AD). Several mitochondrial dysfunctions were reported in SZ, including alterations of proteome, disruption in the cellular respiration and dissipation of membrane potential (Δψm) ,26. In aThe impairment in mitochondria homeostasis is observed in psychiatric disorders as well. In a lipopolysaccharide (LPS)-induced model of depression, an intravenous administration of mitochondria reduced the period of immobility in the forced swimming and tail suspension tests, improved sucrose preference and exploratory behavior in the open field test, attenuated astrogliosis and microglia activation, restored ATP production and promoted neurogenesis . These dC. elegans, mitochondria density increases and correlates with regeneration [Unlike the peripheral nervous system, central neurons lack the ability to successfully regenerate axons toward their targets after injury. Despite several efforts to overcome the inhibitory microenvironment and limited intrinsic growth capacity, there are no definitive therapeutic approaches available. In the pursuit of new strategies, mitochondria dynamics have been constantly related to retinal ganglion cell (RGC) viability and axonal regeneration in several models ,33,34. Fneration , which rneration . Mitochoneration ,37. Thusneration . Furtherneration . In contneration . There aOur own group addressed the regenerative potential of mitochondria transplantation in an experimental model of glaucoma based on optic nerve crush in rats . An intrAn important matter for mitotherapy is the route of delivery. Most studies have either administered directly into the target tissue or at the respective artery, proximal to the area of interest, while others have used a systemic route.Systemic administration was consistently performed in several studies ,19,24,31Other groups have chosen a more direct way to deliver mitochondria to the tissue of interest. The group of James McCully, who mainly focus on cardiac tissue, have provided important advances for the in loco administration of mitochondria. In their studies, they either injected mitochondria directly into the cardiac tissue ,8,9,10 oSome sort of tropism appears to regulate the internalization of mitochondria, since experiments with no tissue injury displayed exogenous mitochondria dispersed in the organism ,16,24, wThe source of mitochondria for mitotherapy is critical and depends on the ease to obtain the organelles, the age of the tissue, the donor’s health conditions, the metabolic characteristics of the organ of origin, as well as histocompatibility. Our group chose the liver as the donor of mitochondria based on the high density of the organelle, easy access to the tissue, high regenerative potential and relatively high oxidative phosphorylation coupling index .The variations of the bioenergetic characteristics of mitochondria among organs have already been reported such as the high membrane potential observed in muscle mitochondria, although muscle, brain, brown and white adipose tissue showed no significant differences in mitochondria number ,45. NotaMus spretus, induced transformation in M. m. domesticus mice zygotes after microinjection [0 cells [The relevance of histocompatibility for the transplantation of mitochondria is still uncertain. In vitro analysis indicated that resistance from the host cells did not happen following the insertion of mouse mitochondria in a human cell line, suggesting that there are strong limits for cells to incorporate exogenous mitochondria . In contnjection . Consist[0 cells .In the first study in adult animals, the mitochondria isolated from a hamster cell line successfully provided neural protection against ischemic stress in rats . FurtherTaken together, the data indicate that the source of mitochondria is relevant in certain scenarios. However, since tissue-matching, unmatching, autologous, allogeneic and xenogeneic transplants induce cell/tissue protection in various models, the choice in each case may be mainly based on the ease of mitochondria isolation.Another important issue is the quality of the isolated mitochondria, which we define based on its functionality: a functional mitochondrion phosphorylates ADP in vitro and displays high oxidative coupling factor, parameters that can be easily checked with the use of a high-definition oximeter .Furthermore, a protocol is recommended to control organelle integrity either using cytochrome C (CytC) on a respiration analysis or performing activity assays of specific enzymes of the mitochondrial matrix. A high-quality mitochondrion must not have its respiration highly induced with CytC; therefore, a threshold must be adopted: we recommend no higher than a 15% increase in oxygen consumption. In addition, no activity of matrix enzymes should be detectable in the supernatant of the organelles. Other experiments can give precise information of the quality and purity of the organelle, but those described above are fast, have low space requirements and can be easily applied to the clinic.In a series of experiments with either intact or disrupted organelles and with purified components from mitochondria as ATP and DNA, McCully and colleagues showed evidence that the therapeutic effects of mitochondria depend on the functional organelle . CorroboThe data above support the relevance of the isolation procedure. However, depending on the donor tissue, the procedure can be too costing to allow its therapeutic use. Soft tissues, such as the brain and liver, are easily homogenized and do not require digestive or rough disruptive processes. On the other hand, tissues such as skeletal muscle require more steps and the isolation may take longer. To overcome this issue, the group of McCully developed an optimized protocol for the purification of skeletal muscle mitochondria ,47.Overall, the procedures must be oriented to the acquisition of the highest possible number of mitochondria as quickly as possible to guarantee the quality, purity and sterility of the organelles.The precise mechanism of organelle internalization in mitotherapy is still controversial. Despite the lack of scientific data regarding free mitochondria internalization, it is likely that some of the known mechanisms described for horizontal transfer may occur in mitotherapy as well.Human MSCs containing healthy mitochondria restored the bioenergetics of A549 cells with deleted or depleted mitochondrial DNA, by transferring healthy mitochondria . CorroboInterestingly, the mitochondria with damaged DNA were unable to be transferred between cells, which suggests a selective process to protect the cell from receiving pathogenic or dysfunctional mitochondria . Yet, an+-cADPR-Ca+2 signaling pathway [Regarding specific mechanisms of the internalization of mitochondria, back in 1982, it was already suggested by Clark and Shay that endocytosis was the mechanism involved in the incorporation of free mitochondria . Transmi pathway . In addi pathway , which w pathway . Heparan pathway .As for clinical applications, several techniques have been used to improve mitochondria transfer, such as the mediation of liposomes , membranTherefore, evidence is strong that the endocytic mechanism may drive free mitochondria internalization by tissues at large, although tissue-specific factors cannot be excluded, and, as discussed in the beginning of this section, the well-established data regarding the horizontal mitochondrial transfer may be relevant for new strategies of the internalization process in mitotherapy. Still, the mechanisms of free mitochondria internalization must be better understood to further improve mitotherapy’s application.A compelling question regarding mitotherapy is how the organelles can induce modification in the target tissue to promote the reported results. Despite the promise of mitotherapy, the mechanisms of mitochondrial effects remain elusive. In this session we addressed both this issue and the clinical advances of mitotherapy.Of prime concern is whether mitochondria must enter the target cell. Despite the initial evidence from photomicrographs that the internalization of exogenous mitochondria was required for the in vitro gain of antibiotic resistance  and respIn contrast, a subsequent publication showed fluorescent-labeled mitochondria within myocytes, even though most transplanted organelles remained in the interfibrillar space . Notably+, on acetaminophen-treated hepatocytes and on hippocampal neurons upon injury [An additional issue is the dose–response of mitochondria for therapy efficiency. Here, the concentrations of mitochondria showed a positive dose-responsive effect on the SH-SY5Y cells treated with the neurotoxin MPPn injury ,17,24,69n injury  and injun injury . These rAn additional matter refers to the course of mitotherapy. Most publications thus far report a single administration of mitochondria leading to positive results. However, mice with nonalcoholic liver disease chronically treated with mitochondria showed an improvement in markers of damage and a reversal of the injury phenotype , suggestThe mechanisms of the beneficial effects of mitotherapy are yet to be fully understood, inclusive of its eventual specificity. Since many medical conditions involve mitochondrial distress as either cause or consequence, it is usually assumed that adding new mitochondria to the system would rescue the organelle’s related mechanisms. Such a premise explains the prevalence in the literature of targeting the modulation of bioenergetic parameters such as mitochondrial matrix enzymes, NADH, ATP, membrane potential, content of ROS, oxidative stress markers and antioxidant enzymes ,31,40,71Notwithstanding, other studies reported results less obviously linked to mitochondrial health. Reduced markers of apoptosis are a common effect of mitochondrial transplantation ,22,39,71To better understand the role of immunity in the effects of mitochondrial transplantation, either syngeneic or allogeneic mitochondria were intraperitoneally injected to test for reactivity and tissue recognition using skin grafts and found neither an inflammatory response nor an induction of IgM production, which favors both the safety and usability of mitochondria transplantation . OverallIn line with the cellular rescuing effect of mitotherapy, several studies reported the functional recovery of damaged tissues. In the lung, mitotherapy successfully recovered organ performance to sham levels after ischemia  and promIn addition to the advances in several experimental models, mitotherapy has also advanced toward clinical application. Pediatric patients who required central extracorporeal membrane oxygenation support for ischemia-reperfusion-associated myocardial dysfunction after a cardiac surgical procedure were transplanted with autologous isolated mitochondria. Importantly, patients did not develop short-term complications, such as arrhythmia, and four of the five subjects had an improved ventricular function . DespiteIn summary, despite the limited understanding of the mechanisms of mitotherapy, evidence is strong that exogenous mitochondria can help prevent damage, promote functional tissue recovery in several in vivo models of biological disorders, and may, therefore, be applicable in future clinical trials.Mitotherapy holds promise from a therapeutic point of view, since most of the studies of diverse diseases and conditions showed positive results. However, important limitations still need to be overcome, such as those raised by the group of Brian O’Rourke, who claimed that mitochondria might not survive the toxic levels of the extracellular calcium concentration ,74. AlthAs pointed out, since mitochondria can be easily harvested from cells in culture, clinicians may benefit from dedicated cell culture centers capable of providing fresh mitochondria isolated by a uniform protocol when autologous transplantation is not possible. Further studies are required to address the possibility of chronic treatment, and clinical trials may be on the horizon, pending every ethical and security issue. Overall, mitotherapy still remains a promising treatment, in need of more information to reach its full potential.