Cannabis and its variants with little to no somaclonal variation or disease for pharmaceutical and for other industrial use has been an emerging area of research. A limited number of protocols have been developed around the world, obtained through a detailed literature search using web-based database searches, e.g., Scopus, Web of Science (WoS) and Google Scholar. This article reviews the advances made in relation to Cannabis tissue culture and micropropagation, such as explant choice and decontamination of explants, direct and indirect organogenesis, rooting, acclimatisation and a few aspects of genetic engineering. Since Cannabis micropropagation systems are fairly new fields, combinations of plant growth regulator experiments are needed to gain insight into the development of direct and indirect organogenesis protocols that are able to undergo the acclimation stage and maintain healthy plants desirable to the Cannabis industry. A post-culture analysis of Cannabis phytochemistry after the acclimatisation stage is lacking in a majority of the reviewed studies, and for in vitro propagation protocols to be accepted by the pharmaceutical industries, phytochemical and possibly pharmacological research need to be undertaken in order to ascertain the integrity of the generated plant material. It is rather difficult to obtain industrially acceptable micropropagation regimes as recalcitrance to the regeneration of in vitro cultured plants remains a major concern and this impedes progress in the application of genetic modification technologies and gene editing tools to be used routinely for the improvement of Cannabis genotypes that are used in various industries globally. In the future, with more reliable plant tissue culture-based propagation that generates true-to-type plants that have known genetic and metabolomic integrity, the use of genetic engineering systems including “omics” technologies such as next-generation sequencing and fast-evolving gene editing tools could be implemented to speed up the identification of novel genes and mechanisms involved in the biosynthesis of Cannabis phytochemicals for large-scale production.The development of a protocol for the large-scale production of  Cannabis sativa L. (Hemp) (Cannabaceae) is a fast-growing herbaceous species that originated from Central Asia ; 3) 0.6 M sucrose, 3.8 M glycerol, and 20% ethylene glycol in liquid MS—were tested by Uchendu et al. [Cannabis lines. More recently, u et al. . Of thesGermplasm preservation has been enabled by encapsulation of plant cells and tissues in artificial seeds and various encapsulation methods are available, allowing for not only the preservation of germplasm as part of conservation plant management but also the national and international exchange of rare, endangered and/or high-value commercial plant genetic resources . Some ofCannabis, where many legal restrictions prevail in different regions and countries. The low cost in preserving high-quality genotypes that may be rare and/or economically important, at a large scale, is an added benefit [Cannabis and other hemp products have acknowledged the advantage of using artificial seeds as the synthetic seeds can be transplanted directly after transport into growing medium for a single genetic clone, saving time and manual labour associated with the generation and rooting of cuttings whilst circumventing the self-crossing problem associated with Cannabis agriculture [Artificial seed technologies are highly relevant for plant species whose in vitro regeneration capacities remain unpredictable using conventional micropropagation. The advantages of this technology are many as this allows for better handling of plant material when it is being transported as part of national and international plant resource exchange programs. This is particularly relevant for a medicinal crop such as  benefit . Commerciculture . Cannabis, the present exploration of this technology is at its infancy and reliable protocols are thus largely lacking. However, axillary buds and nodal segments have been used to produce an artificial seed [2.2H2O proved to be the most viable route successful synseed production [For ial seed ,91, respoduction . In the oduction  the inclIn an effort to optimise in vitro conditions for the production of undifferentiated callus and somatic embryogenic callus, the study by Hesami et al.  utilisedCannabis cultivation and genetic transformation and editing, in general. The application of synthetic seed technology at the moment is currently hampered by a paucity of available scientifically validated protocols and this is an avenue of research that may see more interest in the future due to its potential in assisting with clonal lines of superior fidelity that may fit well in a commercial pipeline for Agrobacterium-mediated transformation has been explored by Feeney and Punja [Cannabis and for their application in industrialised production of Cannabis-derived metabolites. Experimentation with the Agrobacterium co-cultivation strategies to enable for successful production of transgenics is an important consideration [A. rhizogenes MSU440 strain was the most efficient in generating various rol-transformed transgene lines. Successful genetic transformation using Agrobacterium-mediated technology is often challenged by different responses associated with using a variety of different strains and the study by Sorokin et al. [A. tumefaciens (EHA105) harbouring the pCAMBIA130-uidA GUS reporter gene. Although many of these studies aim to ultimately increase metabolite yields in plant systems, other researchers and biotechnology companies intend to focus on using microbial systems for the heterologous manufacture of desired compounds for en masse production of cannabinoids that occur in nature in trace quantities [Cannabis. The establishment of micropropagation protocols for a particular species, cultivar or strain often precedes the utilisation of genetic engineering and gene modification technologies as they offer alternative routes to produce interesting metabolites that are of industrial value. With this in mind, several different strategies are available for genetic manipulation despite the plant showing recalcitrance to in vitro regeneration  and genend Punja  and the nd Punja . Such cuderation . Of the n et al.  showed pantities . With thC. sativa and C. indica and with future applications being imminent as CRISPR Cas 9-mediated editing has several advantages for Cannabis metabolite engineering. As detailed by Deguchi et al. [Cannabis genome also offers superior methods that will likely have a higher public acceptance than conventional genetic modification strategies for the production of elite chemotypes of C. sativa [Cannabis strains but also to impart better disease and pest resistance, polyploidy manipulation plus the alterations to general patterns of plant and growth development of Cannabis plants [The application of CRISPR Cas9 gene editing techniques also holds promise for use in both i et al. , gene kn. sativa . Such tes plants ,98. Cannabis has met with some stumbling blocks despite major advancements in determining the regulatory and biosynthesis genes that control specialised metabolic pathways of key medicinal metabolites uniquely produced by Cannabis that could be used for pathway engineering [Cannabis. The most comprehensive study thus far in solving these problems is that of Zhang et al. [Cannabis are limited in their application across many commercial varieties. Broadening the scope of using gene manipulation techniques in ineering . The maig et al. , where tAgrobacterium-derived genetic transformation with several genes  that promote somatic embryogenesis and act as gene regulators of pathways that control organogenetic plant responses in other model plant species were explored therein. Although the hemp strain referred to as YUNMA7 is a commercial variant that is most important in China was included in the experiments, the best Cannabis strain was determined to be the DMG278 type. With the aim of genetic transforming hemp, this was then chosen as the main experimental model as it was more amenable to Agrobacterium-mediated genetic transformation using an AGL1 strain. Transgenics produced in this way were ultimately grown as F2-generation transgenic crops in the field. Together with the production of transgenics, gene editing experiments used a protoplast-based method that was tested initially using six different guides for the CsPDS1 target locus. Although the production of viable protoplasts was at times inefficient, a cocktail of enzymes, in particular, 12 g/L pectinase and 5 g/L cellulose R10 enzymes, allowed for a greater pool of protoplasts that were transformed with the pAT-GFP plasmid. The explant choice, transformation with developmental regulators and the use of gene editing tools as a combined set of processes enabled for the regeneration of hemp transgenics to be successful, setting a new paradigm for exploitation in functional genomics research and for using synthetic biology approaches for improving Cannabis plants [Arabidopsis auxin-responsive element, eliciting IAA-related changes in transgenic protoplasts [CRISPR-Cas9 mutagenesis and s plants . In anottoplasts . Agrobacterium-based gene modifications. In addition to this, nanoparticle-assisted transformation may utilise passive infiltration of plant tissues, making it easier to introduce multigene constructs into target tissues that would otherwise require sequential transformation steps with Agrobacterium. The latter method may thus become labour intensive and be plagued by poor transformation frequencies when the purpose is to introduce multiple genes into target tissues. As a proof of concept, Ahmed et al. [Cannabis of two soybean transcription GmMYB29A2 and GmNAC42, cloned into the pGWB6 plasmid, that control the synthesis of glyceollin, which functions as a defence metabolite. Lower leaf surfaces of the Tygra variety were passively infiltrated with poly-ethylenimine cationic polymer-modified silicon dioxide-coated gold (PEI-Au@SiO2) nanoparticles using a multigene delivery system for the MYB transcription factors that were linked to a GFP expression marker. Attempts to use Agrobacterium-mediated transformation are ongoing as researchers try to expand the varieties that are being tested with this method. Concomitantly to assessing the effectiveness of agrobacterial co-cultivations with seedlings of the short-day C. sativa agricultural lines  and the day-neutral FINOLA type with the intention of generating transgenic plants, an in vitro regeneration protocol was assessed when plants were placed on 0.2 mg/L NAA: 4 mg/L TDZ MS medium [Agrobacterium tumefaciens LBA4404 (pBIN19), as expected, not all these explants had similar transformation rates. Hypocotyls were most amenable to Agrobacterium-mediated gene modification, recorded at 53.3%. Some regenerants had an albino phenotype on the 100 mg/L kanamycin selection medium but spontaneous in vitro regeneration and rooting were evident in certain transformed lines. This study by Galán-Ávila et al. [Cannabis. According to Ahmed et al. , plant gd et al.  showed tS medium . Althouga et al.  also indCannabis. Because the unique enzymes that are responsible for cannabinoid synthesis have been elucidated using genome sequencing tools, regulation of this pathway is highly interesting for biotechnologists and natural product researchers alike. Protoplast transformation appears to be a more dependable and viable pathway for greater number of strains. With the intention to use this method for CRISPR/Cas9 gene editing, to study protein–protein interactions and other genetic-biochemical regulatory mechanisms that control cannabinoid metabolism, Matchett-Oates et al. [C. sativa genotype to examine PEG-mediated protoplast transformation from leaf mesophyll cells. The transformation was confirmed to be correlated to the plasmid and PEG concentrations to elicit transient GFP expression. Even though PEG-assisted protoplast transformation was optimised for a high THC-containing strain, two other strains (Cannbio2 and a high CBD strain) responded adequately with protoplasts that could also be used for future gene editing manipulations and other molecular-based scientific investigations. The difficulties in obtaining transgenic plants of this plant continue to hinder scientific progress in studying its genes using functional screening assays, making it impossible to gain deep insights into the regulatory mechanisms that control specialised metabolite production in s et al. , in a reCannabis strains. Such protocols, that can be routinely applied across many different laboratories, are most likely imminent as this research area has gained considerable attention from biotechnologists with a vested interest in turning Cannabis into a model species to study its metabolism. It is thus clear that the possibilities that genetic engineering and gene editing present for furthering our general understanding of cannabinoid metabolism and its regulation provide strong incentives to continue researching simple, less labour intensive, viable protocols for producing a broad-spectrum of genetically modified Cannabis and a tissue culture step is often a prerequisite for the generation of plants with different chromosome composition than the mother stock [Cannabis polyploids with higher CBD content and sesquiterpene accumulation via tissue culture albeit rooting was lowered in the tetraploids developed using oryzalin, a compound that alters microtubule formation during mitosis. Another example is illustrated by the work of Kurtz et al. [Non-transgenic methods using chemical mutagens are also suitable for polyploidy generation in er stock . As an eer stock  generatez et al.  using coCannabis farming, with feminised seed being highly sought after. Polyploids are useful for plant breeding as more desired phenological traits and quality assured phytochemical traits may be expressed in bred plant lines [Cannabis variants. 5. Conclusions and Future ProspectsFemale plants are favoured in nt lines —such manCannabis sativa for mass production of the crop yields with high vigour whilst space requirements are minimised for mass amounts of plant clones. Direct organogenesis allows for genetic preservation of the plants, producing genetically identical clones that are vital for industry use. Micropropagation of Cannabis is a fairly new field that is, however, rapidly developing with the opening of new industrial markets globally that have been spurred on by the legalisation of Cannabis derived products. Although there have been protocols developed, many more trials and combinations of PGRs experiments are needed to gain further insight into the development of an appropriate species-specific micropropagation regimen that elicits highly prolific organogenesis in culture of morphologically normal and healthy plants. The exploration of different strains of Cannabis is also still at its infancy and available protocols are not necessarily highly efficient as they are plagued by low regeneration capacity. To reiterate, more strain-specific investigations are thus also urgently needed to provide a better understanding of strain-related differences with respect to in vitro culture. There is thus room to investigate methods that will allow for efficient, reliable and easy-to-reproduce regimes that are characterised by high multiplication rates and rapid ex situ plant establishment under greenhouse and/or field conditions. Many of the micropropagation regimes discussed in this review do not necessarily include a post-culture analysis of the phytochemistry once plants have been acclimated. This is important for quality control purposes if the application of in vitro cultivation techniques is deemed to maintain the chemical integrity of desired commercial chemotypes. Several different approaches may be useful in this regard including microsatellite analyses to determine genetic-associated somaclonal variation resulting from microculture conditions. Epigenetic effects that illicit unprecedented genetic changes have the potential to alter chemical fingerprints of cultivated Cannabis and, together with high-throughput metabolite fingerprinting, this may be useful in monitoring such changes as these can become heritable in subsequent generations. This is particularly important as most Cannabis is used for an industry dependent on phytochemical consistency. In the future, it is likely that cryopreservation as a germplasm conservation strategy may be applied for long-term preservation of unique chemotype variants of C. sativa. The preservation of heirloom seeds using synthetic seed technologies may be complimentary in the augmentation of breeding activities that solely focus on Cannabis and its relatives. Micropropagation via direct organogenesis or indirect organogenesis may be a useful tool in propagation of Cannabis in vitro are no longer an impediment to the generation of healthy and true-to-type clonal propagules, many other genetic engineering applications including the use of fast-evolving gene editing tools will come into routine application in many different laboratories and biotechnology start-ups. This will lead to a revolution in the innovations associated with both medicinal marijuana and hemp. In addition, when the issues associated with difficulties in propagating


Acute hemorrhage in small animals results from traumatic and non-traumatic causes. This review seeks to describe current understanding of the resuscitation of the acutely hemorrhaging small animal (dog and cat) veterinary patient through evaluation of pre-clinical canine models of hemorrhage and resuscitation, clinical research in dogs and cats, and selected extrapolation from human medicine. The physiologic dose and response to whole blood loss in the canine patient is repeatable both in anesthetized and awake animals and is primarily characterized clinically by increased heart rate, decreased systolic blood pressure, and increased shock index and biochemically by increased lactate and lower base excess. Previously, initial resuscitation in these patients included immediate volume support with crystalloid and/or colloid, regardless of total volume, with a target to replace lost vascular volume and bring blood pressure back to normal. Newer research now supports prioritizing hemorrhage control in conjunction with judicious crystalloid administration followed by early consideration for administration of platelets, plasma and red blood during the resuscitation phase. This approach minimizes blood loss, ameliorates coagulopathy, restores oxygen delivery and correct changes in the glycocalyx. There are many hurdles in the application of this approach in clinical veterinary medicine including the speed with which the bleeding source is controlled and the rapid availability of blood component therapy. Recommendations regarding the clinical approach to volume resuscitation in the acutely hemorrhaging veterinary patient are made based on the canine pre-clinical, veterinary clinical and human literature reviewed. Acute hemorrhage in small animals results from traumatic and non-traumatic causes. While there are differences in overall patient management depending on the cause of the acute blood loss, systemic effects associated with volume of blood loss are repeatable. Co-morbidities associated with acute loss of intravascular volume and oxygen carry capacity are related to the proportion of blood volume lost and compensatory reserve of the individual patient , 2. WhilIn human medicine, acute hemorrhage and its sequelae are the leading cause of preventable death in military and civilian settings . Over thThis review seeks to describe current understanding of the resuscitation of the acutely hemorrhaging small animal veterinary patient through evaluation of pre-clinical canine models of hemorrhage and resuscitation, clinical research in dogs and cats, and selected extrapolation from human medicine. It is recognized that mechanism-based reasoning for interventions in veterinary medicine predominate clinical decision making in the absence of large randomized clinical trials and systematic reviews of the clinical literature . AdditioFor the purposes of this review article, patients experiencing acute blood loss are divided into 5 categories based on the cause of bleeding and the patient's hemostatic status upon arrival to a veterinary care facility .These are patients that have sustained injury from an external force (blunt and/or penetrating) leading to blood loss that can vary from minor to catastrophic. Source of blood loss can occur externally , intracavitary , or into tissues . Primary intervention goals in these patients are to attenuate any ongoing bleeding not stopped by endogenous hemostasis , 8, apprTrauma Induced Coagulopathy (TIC) is defined as the culmination of endogenous responses to hemorrhagic shock plus tissue injury . At presThese are patients that are experiencing blood loss as a result of vascular defects caused by neoplasia  or underlying disease . During primary survey, these patients can be a bit more challenging to identify as hemorrhagic shock until initial diagnostics are obtained. As such, in a patient showing signs of hypovolemic shock at presentation, initial intervention is typically volume expansion while preliminary diagnostics are performed to determine cause. If present, once shock is identified as hemorrhagic, intervention goals are similar to that of traumatic injury non-coagulopathic patients.Similar to TIC, these are patients that have progressed to coagulopathy as a result of blood loss (without concurrent tissue injury). However, in contrast to TIC, these patients are identified as being in Disseminated Intravascular Coagulopathy (DIC) as a result of the consumptive processes associated with endogenous hemostatic efforts plus or minus contributions of endothelial damage due to underlying disease . SimilarThese are patients that are bleeding spontaneously from normal vascular “wear and tear” and have an inadequate ability for the endogenous hemostatic system to stop bleeding. Examples include thrombocytopenia , thrombocytopathia , clotting factor deficiencies , or inactivity . Primary intervention goals in these patients are to attenuate any ongoing bleeding which may require supplementation of endogenous hemostatic components that are deficient , and based on patient's clinical status and degree of blood loss , approprIn the continuum of advancing clinical care from bench to bedside, mechanistic models serve to describe the biological processes and physiology of a disease and/or the mechanism of action of an intervention. Utilization of the dog in preclinical and mechanistic models of hemorrhage is uncommon, but a number of published studies do exist –20. ModeThe physiologic dose and response to whole blood loss in the canine patient is repeatable both in anesthetized –20 and aDecreased arterial oxygen content resulting from loss of hemoglobin is countered by mobilization of abdominal organ blood reserves into splanchnic circulation. In both dogs and cats, splanchnic circulation contributions to counteract blood volume loss is similar in total . In dogs, splenic contraction is the biggest contributor whereas in cats, the contributions from liver, intestine and spleen are similar . In spleOther systemic consequences and effects are dependent on the proportion of volume lost, duration of ongoing bleeding, and individual patient factors regarding compensatory reserve . While iWhile an increase in heart rate (HR) and decrease in systolic blood pressure (SBP) are noted in canine models of hemorrhagic shock, compensatory mechanisms may result in these values remaining in the clinically normal reference interval until ~15–20% of total blood volume is lost . HoweverAs blood volume is lost, compensatory mechanisms including buffering systems and ventilation adjust in an effort to maintain normal blood pH in the face of systemic metabolic acidosis characterized by significantly lower base excess and increases in lactate. The acidosis associated with acute hemorrhage can compound coagulopathy and result in continued bleeding . Volume Hemostatic abnormalities are a major factor in the treatment of acutely bleeding patients, both in traumatic and non-traumatic hemorrhage. While there was a period of research that suggested that acute large volume hemorrhage in trauma was primarily associated with hypocoagulability and hyperfibrinolysis, growing evidence supports a continuum dependent on not only duration, amount of blood loss and clinician interventions, but also patient factors . This spCross reference Frontier articles: “The effects of resuscitative fluid therapy on the endothelial surface layer,” Smart/Hughes AND “Article 7 Colloids, Yes or No? Pros and Cons of Colloids?” (Adamik/Yosova). Large clinical trials evaluating alternative initial resuscitation fluid based on severity of disease and time to definitive care are underway in human medicine , resources available to the practitioner and pet owner influence the clinical approach to the acutely hemorrhaging veterinary patient. Despite a varied range of manifestations of the acutely hemorrhaging patient, a systemic approach as outlined below is recommended.There has been a substantial change in human medicine regarding resuscitation for hemorrhagic shock from massive fluid administration to minimal fluid resuscitation while maintaining permissive hypotension and utilization of transfusion with a balanced ratio of blood products and goal directed correction of coagulopathy , 4. In sFor years, aggressive crystalloid therapy was utilized to treat hemorrhage shock. In fact, Shoemaker et al. recommended supra-normal resuscitation with crystalloids during shock. He found that this mode resulted in increased oxygen delivery to tissues and improved survival in critically ill patients . UnfortuHypertonic saline is an attractive low volume resuscitation crystalloid. Its benefits include rapid increases in cardiac output and blood pressure , improvecross reference Frontiers article: Colloids…“Article 7 Colloids, Yes or No? Pros and Cons of Colloids?” (Adamik/Yosova)).Synthetic colloids such as hydroxyethyl starch (HES) have historically been used as a resuscitative fluid in hemorrhagic shock patients in veterinary medicine due to their rapid volume expansion, availability and cost , 58. RecSimilar to the philosophical approach to damage control surgery (DCS) where there is an attempt to stop or minimize hemorrhage as soon as possible in the surgical suite, anecdotal clinical observations of increasing blood pressure with fluid resuscitation may create vascular pressure exacerbated bleeding. To this end, studies were developed to investigate the effects of minimizing increases in blood pressure to allow for vital organ perfusion but limited excessive pressures beyond that. Many of the initial studies were conducted in animal models and a summary of the results was published in a meta-analysis of these studies . All resThere is not strong evidence to support an ideal pressure goal with hypotensive resuscitation and how long it can be maintained. In addition, most of these studies have used crystalloid solutions as part of their resuscitation and there have been no definitive studies using hypotensive resuscitation with blood component therapy. None of the studies cited above were performed in patients with head trauma and hypotensive resuscitation in patients with head trauma and increased intracranial pressure is not recommended , 64.Early studies on animal blood transfusion demonstrated the effectiveness of whole blood transfusion on resuscitation in hemorrhagic shock but it was fraught with infections, clotting and other complications. With refinement in collection techniques and anti-coagulants, these hurdles were overcome and whole blood transfusion became a useful therapy in hemorrhagic shock. The scarcity of blood products eventually led to the more efficient use of component therapy.Early standard resuscitation component therapy included sequential resuscitation with crystalloids, artificial colloids, and packed red blood cells . With thThe underlying benefits using component therapy compared to crystalloid resuscitation are not completely elucidated but several reported advantages include but are not limited to mitigation of hyperfibrinolysis and platelet function and repair of endothelial integrity , 72. In Given the improvement in outcomes using plasma and blood component therapy studies have been performed to investigate the use of platelets in blood component therapy. Similar to plasma, high ratios of platelet concentrates to pRBC have demonstrated decreased risk of death in massively transfused patients , 73, 74.Given the success of component therapy, why not just use fresh whole blood? The use of fresh whole blood in war casualties has an extensive history particularly from the conflicts in Afghanistan and Iraq . StudiesComponent therapy has become the preferred method in resuscitation of severe hemorrhage in humans but there are hurdles to the use of it in veterinary medicine. Many of the studies cited in this section above indicated that this therapy is most effective within the first few hours of hemorrhage and its effect on outcome diminishes after this period. Anecdotally, it is recognized that the speed with which hemorrhaging veterinary patients are taken to surgery for definitive hemorrhage control is much more delayed compared to human medicine (with some exceptions). This delay may negate the positive results with component therapy. Similarly, component therapy may not be readily available to the many veterinary hospitals. Additionally, plasma concentrates are also not consistently available. While there remains much to learn in clinical veterinary patients, experimental studies in animals show that replacement of lost blood, sometimes as components, administered early has improved outcomes , 20, 23.Consensus on the best approach to volume resuscitation in the acutely hemorrhaging patient does not exist in veterinary medicine due to multiple factors including, but not limited to, lack of prospective clinical trials, limited similarly designed projects amenable to meta-analyses, and challenges extrapolating human and pre-clinical model findings to clinical canine and feline patients with similar, but not identical, physiologic responses to hemorrhage and resuscitation. However, this challenge is not unique to veterinary medicine, and continued research efforts to individualize resuscitation in the hemorrhaging patient based on patient phenotype, response to interventions, degree of disease and environment remain a significant pursuit , 12, 78.Initial triage of the acutely ill patient has many approaches with different assessment algorithms taught and applied. The British military has added Catastrophic bleeding (< C>) to the American College of Surgeons Airway (A), Breathing (B), Circulation (C), Disability (D), Environment/Exposure (E) approach to the primary survey of the acutely injured patient methodology that it advocates utilizing . RegardlDuring the intravascular resuscitation phase of the acutely hemorrhaging patient, reassessment parameters include physical exam findings , biochemical measurements , and macro-perfusion measurements (blood pressure). Once circulatory stabilization is underway, continued determination of underlying cause  and consWhile there continues to be a shift in first choice resuscitation in the recently hemorrhaged patient in shock, current literature (and logistics) supports a 10–20 ml/kg dog (5–10 ml/kg cat) intravascular dose of a balanced electrolyte solution . In the subset of hemorrhaging patient's whose clinical status has not stabilized, the clinician's choices from there vary based on patient status, response to interventions and blood product availability. Consideration for blood product administration in the poorly responding hemorrhaging patient early in resuscitation is recommended .A series of veterinary papers evaluating various triage values and subsequent blood transfusion in canine trauma patients found an association between abdominal fluid score (AFS), base excess (BE) and ionized calcium (iCa), respectively, as independent predictors of patients receiving a blood transfusion. These findings suggest consideration for readying blood product for administration when initial triage diagnostics include an AFS ≥ 3, BE < − 6.6 mmol/L, and ionized iCa < 1.24 mmol/L or < 2.50 mEq/L , 81, 82.Veterinary and human literature demonstrate a spectrum of coagulation and fibrinolytic abnormalities in acutely hemorrhaging patients –11, 34. In summary, current evidence supports judicious use of a balanced electrolyte solution, and early movement to “replacing what is lost” in the acutely hemorrhaging patient by utilization of blood product, when available . Future All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Internal or common carotid artery encasement (CAE) is observed in almost 2-7% of head and neck cancers (HNC) and designates the tumor with the T4b category. This clinical scenario is associated with a dismal prognosis, owing to the risk for thrombosis and bleeding that usually characterizes such an advanced cancer. Standardized radiological criteria to infer invasion of the carotid artery are lacking. Complete surgical resection in the context of a multimodality treatment is supposed to offer the greatest chances of cure. Surgery can either be carotid-sparing or include carotidectomy. Data on probability of cerebrovascular and non-cerebrovascular complications, risk of carotid blowout, poor oncologic outcomes, and less-than-certain efficacy of diagnostic and interventional preventive procedures against cerebral infarction make it difficult to define surgery as the recommended option among other therapeutic strategies. Non-surgical therapies based on radiation therapy possibly combined with chemotherapy are more frequently employed in HNC with CAE. In this context, carotid blowout is the most feared complication, and its probability increases with tumor stage and cumulative radiation dose received by the vessel. The use of highly conformal radiotherapies such as intensity-modulated particle therapy might substantially improve the manageability of HNC with CAE by possibly reducing the risk of late sequalae. Despite evidence is frail, it appears logical that a case-by-case evaluation through multidisciplinary decision making between head and neck surgeons, radiation oncologists, medical oncologists, diagnostic and interventional radiologists, and vascular surgeons are of paramount value to offer the best therapeutic solution to patients affected by HNC with CAE. Internal or common carotid artery encasement (CAE) by head and neck cancers (HNCs), including salivary gland and sinonasal cancers, designates the tumor with the T4b category . CarotidCriteria to define CAE at imaging, mastering various therapeutic approaches for locally advanced disease with CAE, management options to prevent CA blowout (CB), as well as knowledge of potential cerebrovascular risks related to permanent CA occlusion are all contemporary aspects that should raise the interest of different physicians who deal with advanced HNCs. The present perspective will emphasize on these aspects based on the most relevant current evidence.So far, there are no standardized radiological criteria to distinguish simple CAE from frank vessel involvement. Preoperative magnetic resonance imaging (MRI) has been demonstrated capable in predicting involvement of the CA in cases with near-circumferential encasement . In contvia an interpositional vascular graft. Moreover, prior to indicate CA resection without cerebral revascularization in a patient tolerating a temporary balloon occlusion test, one should consider that the rate of delayed cerebrovascular events in patients with negative occlusion test accounts for 15-22% , although a trend was found in favor of CRT, possibly because of the treatment-naïve nature of these patients. Of note, imaging was assessed in 7/8 patients treated with radical CRT, and all had near-total circumferential CAE , delivered either alone or in combination with chemotherapy (CRT), are aimed at avoiding complications of CA resection. An HNC may be labelled as “unresectable” either because genuinely unsuitable for a wide-margin resection  or due to the estimated unfavorable balance between risks of surgery and its potential benefits from an oncologic standpoint. Over one-third of these patients are usually treated by neoadjuvant chemotherapy followed by CRT –29. Howetial CAE .No data on CB events were reported in non-surgically treated patients in these two latter series , 30. HowPatients with naïve or recurrent advanced HNC with CAE may still have a chance to be cured if treated with modern surgical and RT techniques. In particular, this should be taken into account in young patients with a relatively indolent disease, who may potentially have a relatively long life expectancy. In this clinical scenario, a careful evaluation of the available management strategies to secure CA should be put in place to achieve the best oncological results while minimizing the risk of cerebrovascular and non-cerebrovascular sequelae. Precise analysis of tumor extension, adequate treatment planning, and proper counseling should save patients an ineffective invasive treatment, such as an unintentional R2 surgery, and the risk of dangerous and potentially life-threatening complications, such as CB. These prerequisites are best fulfilled in tertiary referral centers, where the multidisciplinary team can handle very advanced cancers with CAE by exploiting the available strategies and customizing treatment based on special characteristics of the single case. It is authors’ opinion that prospective studies are needed to objectively assess the risk-benefit ratio of CA securing strategies  that are adopted to deliver the locoregional treatment of HNC with CAE.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.Authors contributed as follows: conception . Perspective design (EO and MF). Paper draft (EO and MF). Draft correction . Supervision (LL and PN). 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.


Cervical cancer rates in Sub-Saharan Africa (SSA) are amongst the highest worldwide. All three of the Human Papillomavirus (HPV) vaccines  provide primary protection against the most common cancer-causing strains of HPV (types 16 and 18) that are known to cause 70% of cervical cancers. Over the last five years, there has been an increase in Sub-Saharan African countries that have introduced the HPV vaccine. The majority of research has been conducted on supply-side barriers and facilitators to HPV vaccination uptake in SSA, yet little research has been conducted on demand-side or end-user perspectives of, and decisions around, HPV vaccination. In order to complement existing research, and inform current and future HPV vaccination implementation approaches, this qualitative systematic review explored Stakeholders’ understandings of HPV vaccination in SSA. This review searched the following databases: Embase (via Scopus), Scopus, MEDLINE (via PubMed), PubMed, EBSCOhost, Academic Search Premier, Africa-Wide Information, CINAHL, PsycARTICLES, PsycINFO, SocINDEX, Web of Science, and the Cochrane Controlled Register of Trials (CENTRAL) and found a total of 259 articles. Thirty-one studies were found eligible for inclusion and were analyzed thematically using Braun and Clarke’s methods for conducting a thematic analysis. The quality of included studies was assessed using the Critical Appraisal Skills Programme (CASP) checklist. Three major themes emerged from this analysis; knowledge of HPV vaccination and cervical cancer is intertwined with misinformation; fear has shaped contradictory perceptions about HPV vaccination and gender dynamics are relevant in how stakeholders understand HPV vaccination in SSA. Human papillomavirus (HPV) is a central causative agent of cervical cancer, the second most common cancer among women in Africa, where approximately 372.2 million women aged 15 years and older are at risk of developing cervical cancer . Such stThe quadrivalent HPV vaccine was first approved by the U.S Food and Drug Administration (FDA) in 2006, offering primary protection against the most common cancer-causing strains of HPV  followed by the bivalent vaccine approved in 2009 (protection against type 16 and 18) (both are which are primarily used in SSA), and finally the 9-valent vaccine approved in 2014 . In SSA, late presentation to care is an established trend associated with health inequities, gender disparities, socioeconomic and cultural factors that have ultimately increased cervical cancer mortality rates … could be prevented, so I was very happy because it meant that if I got vaccinated I would not die of cancer”  will not go to the clinic unless she is sick…So expecting healthy people to voluntarily come for vaccination is difficult”  on television all the time talking about cervical cancer and we had an overwhelming response…The [patients] who were coming told us that [they] heard about it from the first lady” […a child does not doubt [a teacher]. They can go home and convince the parent ‘this is what the teacher said”   because there is AIDS and HIV out there and we all are aware of it… My child can be raped, and I will feel bad about it, but I am at peace [knowing] that she is participating in the Kganya Motsha [HPV vaccine trial] and will be protected against [this] sexually transmitted disease”  the cultural background that a woman should seek permission from her husband, whether she should take her daughter for the vaccine… so those are cultural issues that will always be there”  they also play an important role” [In general, male and female stakeholders across studies urged men to be more included in education around cervical cancer and HPV vaccination. In addition to men being carriers of HPV and being susceptible to cancer outcomes such as anal, penile and throat cancer ,24, greant role” .Three major themes emerged from this review. Firstly, stakeholders’ understandings and perceptions of cervical cancer, HPV and HPV vaccination are shaped by a mix of knowledge and misinformation, both of which are influenced by social processes and relationships and both of which act as both barriers and facilitators to HPV vaccination. Within the broader literature, this review supports Abdullah and colleagues that there is sub-optimal knowledge about vaccine preventable diseases and associated vaccines in Africa, albeit moderate levels of positive attitudes towards HPV vaccination . The stuA second central theme that emerged from this review was how beliefs around infertility have created a significant divide in understandings of HPV vaccination in SSA. Stakeholders tended to hold one of two opposing views of the causes of infertility. Some attributed infertility to cervical cancer thus encouraging support for the HPV vaccine for prevention of common cancer-causing strains of HPV and by extension, infertility. Conversely, others attributed infertility to the HPV vaccine itself, which fueled HPV vaccination hesitancy and refusal amongst stakeholders who harbor such beliefs. This supports broader findings that infertility amongst African women can have serious psychological and social implications for women, along with being a major factor for marital instability, social pressure and ostracization, stigmatization and abuse ,57,58,59A final theme that emerged from this review was the significant role that gender dynamics play in how stakeholders perceive HPV vaccination. Gendered dynamics were present in stakeholder perceptions and understandings of HPV vaccination in the following ways; the notion that HPV vaccination can be a viable harm reduction strategy against female gender-based violence, significant differences across countries in SSA in regard to whom makes decisions within households about health seeking behaviors (such as HPV vaccination), and finally, that female-only HPV vaccination campaigns encourages the perception of HPV as a female specific issue which perpetuates misinformation. This is aligned with the broader research examining the feminization of HPV due to the focus on cervical cancer screening, which has not only made females the primary group responsible for HPV prevention, but has also resulted insufficient protection from HPV-related illnesses in males and females alike ,56,57,60The findings from this review have various practical implications for how demand for HPV vaccination might be enhanced. The dynamics of trust and drivers of distrust between communal stakeholders and the government, medical and public health community need to first be recognized and then be addressed through wide spread meaningful engagement. Prior to rollout, education and social mobilization around the HPV vaccination Programs should sufficiently engage communities in order to thoroughly gauge local understandings, conceptions and misconceptions, language nuances, baseline perceptions and fears around cervical cancer, HPV and HPV vaccination. Utilizing local language nuances of disease terminology and recognized symptomology may be an important consideration for HPV vaccination roll out strategies across SSA when shaping content and focus of education and awareness-raising interventions ,33,37,43Although some studies used focus group discussions and interviews to assess preliminary perceptions of cervical cancer, HPV, and HPV vaccination, often following up with an educational component at the completion of the study to address misinformation, it is evident that efforts to address misinformation require first understanding the social context and the social dynamics within that context, as identified in this review. This supports previous research that community acceptability towards HPV vaccination is a slow process that requires first acknowledging rumors, fears, and concerns, prior to expressing the benefits and safety of HPV vaccination . This reThe lead researcher has a background in public health and acknowledges that this has shaped positive attitudes towards vaccination more generally. As cervical cancer is largely preventable, the lead researcher undertook this analysis in order to better understand extreme rates of morbidity and mortality from HPV and cervical cancer in SSA .A strength of this review is the consideration and acknowledgement of divergent findings within the dataset and what such conflicts reveal about understandings of HPV vaccination in SSA. This review provides a ‘big picture’ demand-side perspective to contribute to improvements in current and future HPV vaccination strategies in SSA, an under-researched area overall.This was a rapid qualitative review and thus has various limitations common to this approach. Limitations include: a single review author for all stages of the search strategy . A ‘Confidence in Evidence from Reviews of Qualitative Research’ assessment was not conducted . The datStakeholders who create a demand for the HPV vaccine are arguably the most vital to its uptake and continued necessity, especially when the momentum of HPV vaccination in SSA has only recently been initiated. How stakeholders understand cervical cancer, HPV, and HPV vaccination will be vital to the short- and long-term success of HPV vaccination Programs in SSA. This review found that stakeholder understandings of HPV vaccination are shaped by a complex relationship between knowledge and misinformation, a significant fear of infertility associated with both cervical cancer and the HPV vaccine, and social and gender dynamics in SSA. This review iterates the importance of first working with communities to gauge local and context-specific understandings, before trying to implement change through one-size-fits all education, sensitization and behavior change strategies.


FUS gene cause amyotrophic lateral sclerosis (ALS-FUS). However, the exact pathogenic mechanism of mutant fused in sarcoma (FUS) protein is not completely understood. FUS is an RNA binding protein (RBP) localized predominantly in the nucleus, but ALS-linked FUS mutations can affect its nuclear localization signal impairing its import into the nucleus. This mislocalization to the cytoplasm facilitates FUS aggregation in cytoplasmic inclusions. Therapies targeting post translational modifications are rising as new treatments for ALS, in particular acetylation which could have a role in the dynamics of RBPs. Research using histone deacetylase (HDAC) inhibitors in FUS-ALS models showed that HDACs can influence cytoplasmic FUS localization. Inhibition of HDACs could promote acetylation of the FUS RNA binding domain (RRM) and altering its RNA interactions resulting in FUS maintenance in the nucleus. In addition, acetylation of FUS RRMs might also favor or disfavor its incorporation into pathological inclusions. In this review, we summarize and discuss the evidence for the potential role of HDACs in the context of FUS-ALS and we propose a new hypothesis based on this overview.Mutations in the  Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease . PTMs refer to the covalent enzymatic modifications of proteins following biosynthesis which can affect their function in health and disease. Many lines of evidence suggest that PTMs of proteins linked to motor neuron diseases are powerful modifiers of their activity. For example, in TDP-43 it is well-established that acetylation can act as a strong modulator of ALS pathogenicity, affecting both TDP-43 aggregation propensity and cytoplasmic mislocalization   granules, such as stress granules, which are transient regulatory structures formed by RNA complexes stalled in translation  domain  can increase FUS mislocalization as it reduces FUS-Transportin interactions. In this way, FUS mutants with acetylation mimetics in the K510 residue showed higher mislocalization and an increase also in cytoplasmic inclusions and colocalization with stress granules  might prevent RNA binding leading to FUS nuclear localization. Indeed, studies using HDAC inhibitors have shown an increased localization in the nucleus of FUS (Kuta et al., As of today, it has not yet been proven that RRM acetylation produces both a reduction in FUS mislocalization and its associated cytoplasmic toxicity. Acetylation mimetics in K315/K316 did not prevent mislocalization but reduced FUS cytoplasmic inclusions and colocalization with stress granules (Arenas et al., FUS or TARDBP-43 mutations, so it is not clear whether this therapy would also be effective in sporadic ALS cases.It is worth pointing out that HDAC inhibitors have been shown to acetylate not just the K315/K316 but also the K510 residue (Arenas et al., In conclusion, FUS acetylation through HDAC inhibitors is presented as potential therapeutic strategy for FUS-ALS , althougCT wrote the article. This was checked by DP and LV. 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 aim of this study was to map evidence of how physical activity (PA) strategies are integrated and explored in research of interdisciplinary, time-limited reablement for community dwelling older adults and to identify knowledge gaps. Following an apriori protocol, we searched eight databases for eligible studies, in addition to citation and reference searches. Study selection and data-extraction was made independently by two reviewers. Fifty-one studies were included, showing that exercises and practice of daily activities were included in the majority of intervention studies, but in most cases little information about exercise components or strategies for increasing PA were provided. There was insufficient evidence for any synthesis of how reablement affects older adults’ PA levels, their physical fitness or how PA is experienced in reablement. There is a need to further investigate how the promotion of PA can be adequately implemented in reablement and how it may affect older adults’ function.


Understanding the resources needed to achieve desired implementation and effectiveness outcomes is essential to implementing and sustaining evidence-based practices (EBPs). Despite this frequent observation, cost and economic measurement and reporting are rare, but becoming more frequent in implementation science, and when present is seldom reported from the perspective of multiple stakeholders , including those who will ultimately implement and sustain EBPs.Incorporating a multi-level framework is useful for understanding and integrating the perspectives and priorities of the diverse set of stakeholders involved in implementation. Stakeholders across levels, from patients to delivery staff to health systems, experience different economic impacts  related to EBP implementation and have different perspectives on these issues. Economic theory can aid in understanding multi-level perspectives and approaches to addressing potential conflict across perspectives.This paper provides examples of key cost components especially important to different types of stakeholders. It provides specific guidance and recommendations for cost assessment activities that address the concerns of various stakeholder groups, identifies areas of agreement and conflict in priorities, and outlines theoretically informed approaches to understanding conflicts among stakeholder groups and processes to address them. Involving stakeholders throughout the implementation process and presenting economic information in ways that are clear and meaningful to different stakeholder groups can aid in maximizing benefits within the context of limited resources. We posit that such approaches are vital to advancing economic evaluation in implementation science. Finally, we identify directions for future research and application.Considering a range of stakeholders is critical to informing economic evaluation that will support appropriate decisions about resource allocation across contexts to inform decisions about successful adoption, implementation, and sustainment. Not all perspectives need to be addressed in a given project but identifying and understanding perspectives of multiple groups of key stakeholders including patients and direct implementation staff not often explicitly considered in traditional economic evaluation are needed in implementation research. Contribution to the literatureAssessment of cost and implementation time required to implement EBPs is centrally relevant to implementation science.Costs vary from the perspective of different stakeholder types .Key cost-related issues from the perspectives of different stakeholders are summarized, including those such as patients and delivery staff that are often not explicitly identified and included in economic evaluation.This article summarizes the application of economic theory to inform multi-level stakeholder perspectives.Recommendations are made for future pragmatic research, application of cost assessment, and its use for decision-making with different stakeholder types.An integral component of assessing value for implementation efforts is capturing the costs and benefits of implementing evidence-based practices (EBPs) relevant to various groups of stakeholders . EstimatLocal economic considerations are a critical factor influencing whether or not individuals and organizations, versus larger healthcare systems and society, adopt and sustain EBPs . The burImplementation costs and effects accrue at different levels and are variable across different parties. Costs across these different levels have a notable impact on implementation success and sustainment. A recent systematic review of implementation costing methods by Bowser et al. reflects this reality; researchers found a wide range of costing perspectives represented in implementation research, including organizational, facility, provider, and societal perspectives . Yet, trThe purposes of this paper are to (1) apply a multi-level framework to identify key types of stakeholders in implementation efforts and what cost information (broadly defined) they need for decision making; (2) discuss how we consider simultaneously multiple perspectives and options for applying economic theory to advance coordination across perspectives; (3) address the role of stakeholder partnerships and engagement in determining relevant implementation cost data, pragmatic costing tools, and reporting guides to facilitate this; and (4) present examples of multi-level cost application and provide recommendations and future directions in the field.Incorporating a multi-level framework is useful for integrating the perspectives and priorities of the diverse set of stakeholders involved in implementation research. Ferlie and Shortell’s  multi-leTable The purpose of economic evaluation in healthcare is to maximize outcomes related to health that are subject to a set of constraints . This puApplying economic theory in the context of implementation research can aid in identifying and understanding multi-level perspectives and approaches to integrating these perspectives. Applying principles of economic theory can also help increase the relevance of implementation cost information used in decision-making across different stakeholder groups to ultimately enhance uptake, implementation, and sustainment of EBPs. We provide an example of applying economic theory to implementation issues using multiple stakeholder perspectives. These examples illustrate important first steps in recognizing potential areas of conflict or unintended economic effects across stakeholder groups and developing plans for resolution. Economic theory can be useful in providing guidance and support around decision-making in the implementation of health services , but theUnderstanding the distribution of costs and benefits across stakeholders is critical to designing implementation strategies that facilitate economic cooperation among stakeholders. There are numerous examples in implementation science when stakeholders can reduce total joint costs and realize savings by pooling resources and cooperating. Healthcare delivery organizations entering into accountable care organizations entering into bundled payment models must allocate the total costs and savings from episodes of care . AlternaTheories from related fields such as decision sciences  and behavioral economics  can also aid in understanding multiple stakeholder perspectives when considering costs and benefits of specific courses of action , 21. A fStakeholder engagement is vital for closing the gap between research and practice. The implementation science field increasingly recognizes that participatory approaches are foundational to successful implementation and sustainment, including from an economic perspective . CommuniUnderstanding cost and benefit implications and perceived value from various stakeholder perspectives, from end-users to delivery staff to the larger system/environment, and what implementation cost information is most beneficial for each of these groups is critical in advancing economic analyses in implementation research. Stakeholder engagement across levels is especially critical when organizations face overwhelming competing demands and scarce resources and/or stakeholders experience disproportionate costs related to implementation, such as opportunity costs for frontline staff engaging in implementation efforts in lieu of billable patient care . AssessiMulti-level perspectives are especially important when considering implementation efforts in low-resource contexts. For example, if the organizational leadership wishes to implement an EBP to reduce system costs  but the resources required to effectively adopt, implement, and sustain it, such as substantial time from highly paid professional staff (provider team), is not aligned with resource capacity within a setting, the likelihood of implementation success is low. Such misalignment can exacerbate rather than mitigate health disparities . SettingFirst, while it may still be possible to implement an EBP in a low-resource setting, substantial, and potentially costly, adaptations to the intervention and/or implementation strategy are often needed. Identifying and conducting initial rapid cost estimates of lower-cost implementation strategies for an EBP is vital to addressing equity issues in implementation and sustainment. Conducting this type of rapid cost estimate requires considering the organizational/community capacity and resources from multiple perspectives . Discussion of how to tailor EBPs and implementation strategies to low resource settings and changing context is a separate topic addressed in detail elsewhere , 28, butSecond, both researchers and stakeholders may consider adopting innovative approaches that substantially reduce costs commonly seen in implementation efforts in low- and middle-income countries. In some instances, resource-challenged settings have demonstrated high levels of success in implementation and health outcomes at a dramatically reduced cost. This is sometimes achieved by the use of “task-shifting” in which delivery of an EBP is accomplished by much lower level—and thus lower cost—staff members such as community health workers or peer coaches . UtiliziWhile reporting guidelines exist for economic evaluation, including the Consolidated Health Economic Evaluation Reporting Standards (CHEERS), these guides focus primarily on healthcare payer or societal perspectives . While cDeveloping reporting guides can aid in identifying areas of cooperation and conflict and in applying theories from economics, behavioral economics, and decision sciences, to address economic barriers within and across stakeholder groups. This is consistent with conclusions from a recent systematic review whereby researchers recommended standardized guidelines around costing perspectives, instrumentation, and reporting as critical next steps to advance the field . At the Another important consideration is the reporting of uncertainties in cost estimates and cost-effectiveness/benefit analyses. Each cost and benefit input (parameter) includes specific model/structural, and methodological assumptions  that can influence estimates and, ultimately, conclusions from economic evaluations. Various types of sensitivity analysis can be performed to investigate the influence of these uncertainties on the economic output. For instance, will small changes in an input parameter, such as provider or staff type or time, lead to a big change in cost and/or the relative benefit of adopting an innovation? These sensitivity analyses, from simple  to complex , can provide stakeholders with some estimates of the model parameter’s impact on the economic output. For example, increasing the labor cost of front-line staff from the base-case assumption may change an implementation plan from being cost-effective to not cost-effective. An example of sensitivity analysis and the information it can provide to stakeholders is included in our case example. Another option may be to build user-friendly model tools, such as described previously, so that different organizations could easily change some of the input assumptions to fit their specific needs; this is, again supporting the concept of “costing in context” . For exaThis illustration above demonstrates some of the complexities involved in data collection and reporting the results of economic analyses in implementation research. These issues are especially consequential when needing to summarize findings for either non-economist audiences or organizational decision-makers. In our experience, most decision-makers in potential adopting sites are not concerned with either the costs in a randomized research study or detailed economic issues such as marginal costs and economies of scale. Rather, they are concerned with what it would cost them to implement a program in question in their setting—e.g., replication costs. The underlying economic factors such as marginal costs would come into play when calculating and presenting options and potential adaptations , but decision-makers are often frustrated by many details, qualifications, or jargon from either economics or implementation science. Rather they want to know “what is the bottom line for me in this setting under our conditions.”Frontline providers are often the ones who are most impacted daily by the implementation of new evidence-based practices. Taken as a group, front-line providers are generally interested in patient care and safety and the resources required to achieve those goals. For instance, frontline hospital staff involved in a patient safety intervention identified patient safety as a priority; but they cited a lack of proper equipment/supplies and facility issues as major operational barriers that their institutions did not prioritize when implementing a new policy . AdditioIn contrast, the implementation of policies and practices are usually decided at an upper administration level  based on financial incentives. For instance, reducing fall-related injuries is a financial incentive for hospitals so an EBP to address this may be a priority at the policy and/or organizational level . In theoThis example highlights where a solution  resulted in both the hospital administrators and frontline nursing staff benefiting from the fall-prevention program that was implemented at their facilities despite potential conflicts—hospital administrators achieved net savings while frontline staff had more time to devote to other duties.We provide specific guidelines and recommendations for cost assessment activities involving multiple stakeholders, guided by steps as outlined in Rapid-Cycle Research described by Johnson et al. in Table As the field of implementation science moves forward with building the evidence base on estimating costs and conducting comparative economic evaluation of implementation, we must specify the perspectives, and cost considerations, including the specific costs relevant to each stakeholder group as a vital first step. There can be considerable variation in costs and cost-effectiveness based on perspective and costing methodology , and recTable Working with stakeholders during pre-implementation phases to “model” approximate costs of an intervention and its associated implementation strategy (or strategy bundles as often used), and to consider their potential impact on resource demands and allocation (as well as benefits) across stakeholder levels, may ultimately save time, money, and enhance cooperation on economic issues. Adapting existing tools, such as the economic impact inventory template posited by Neumann et al., can advance the field by organizing, and presenting various types of costs and consequences from the perspectives in which they occur . FacilitThis article has several limitations. First, there is not enough literature to conduct a systematic review and there have not, to our knowledge, been comparative studies of different methods to address the issues above. Stakeholders often do not agree and engaging multiple parties in open discussions and problem-solving to come to a consensus is at present more art than science. More research is needed on the specific types and formats of cost feedback to provide to different stakeholders. Cidav et al.  provide Other potentially useful theories for advancing multiple stakeholder perspectives in economic evaluation in implementation science, such as those from behavioral economics and decision sciences, were not fully addressed here. Zimmerman et al., for example, have leveraged the concept of bounded rationality as a foundation on which they have built comprehensive simulation models to aid healthcare stakeholders in providing treatment services for veterans with posttraumatic stress disorder . These aThere has been a recent surge of publications related to the collection of costs in implementation science , 31, 36,We conclude that there are compelling reasons to collect, report, and understand costs from the perspectives of different stakeholders. Failure to do so may result in (1) lack of program adoption, especially if organizational and policy perspectives are not considered; (2) poor reach to individual participants, especially those at high risk and with few resources; (3) poor quality implementation, especially if not considering the perspective of both supervisory and delivery staff; and (4) poor sustainment when not considering all these perspectives.Our recommendations are based upon the synthesis of existing literature, input from the diverse disciplines represented on our authorship team, various economic theories, applied experience collecting cost data, and implementation science frameworks, but not on a systematic review or meta-analyses.The recommendations we offer above and in Table