Essentially all HIV transmission is from people living with HIV who are not virally suppressed. An HIV micro-epidemic control approach that differentiates treatment support and prevention services for people living with HIV and their network members according to viral burden could optimize the impact of epidemic control efforts. Essentially all HIV transmission is from people living with HIV (PLHIV) who do not know their infection status or have not yet achieved viral suppression, making support for these individuals and their risk contacts a priority for treatment and prevention efforts.Proven approaches exist to reduce viral burden and interrupt HIV transmission from PLHIV who are not yet virally suppressed, but these approaches must be implemented with enhanced focus and scale to maximize benefit.Improved diagnostic approaches offer new opportunities to increase public health impact by prioritizing support for unserved or underserved individuals with the greatest viral burdens and among members of their risk networks.Policy makers should pursue the implementation and evaluation of diagnostic approaches that can focus services among individuals and networks with the greatest viral burdens.Program managers should treat viral burden as a primary consideration in the provision of differentiated HIV services, applying an HIV micro-epidemic control framework to prioritize and tailor services for PLHIV and their risk contacts along a continuum of progression to viral suppression. Acute infection is characterized by a 2–4-week period of exceptionally high viremia as HIV replicates rapidly in the body before a person’s immune system mounts a response and reduces the level of circulating virus to much lower—but typically not undetectable—levels for a period of months to years.,,–In key populations engaged in frequent behavioral risks, up to an estimated 50% of all HIV transmission occurs from individuals during AHI when viremia is very high prior to the development of an immune response including anti-HIV antibodies (Ab) that yield reactivity on third-generation Ab assays.Approaches that differentiate service delivery to better address the preferences and needs of unserved and underserved individuals have been identified as a priority to close outstanding gaps in access to HIV prevention and treatment.Nevertheless, the resources and technologies needed to activate a more comprehensive differentiation of support based on viral burden historically have been limited. With the advent of expanded access to viral load testing and options to screen for AHI, opportunities now exist to prioritize support for individuals and in risk networks with the greatest viral burdens. This prioritization can help interrupt epidemic HIV transmission associated with AHI through early diagnosis, HIV treatment, and provision of PrEP and other proven prevention approaches to risk contacts. Because HIV morbidity, mortality, and transmission risk are most closely associated with viral burden, this enhanced focus can guide the allocation of limited resources to maximize the impact of prevention and treatment efforts.Prioritizing viral load testing and screening options for individuals with the greatest viral burdens can help interrupt epidemic HIV transmission.We propose an HIV micro-epidemic control framework to characterize these opportunities to accelerate impact, with a primary focus on addressing the differentiated service preferences and needs of individuals who are not yet virally suppressed, as well as the members of their risk networks. This framework aims to organize and integrate both new and existing approaches to tailor support for PLHIV and their risk contacts based on progression to sustained viral suppression. By profiling the characteristics of those who face challenges in achieving viral suppression—such as barriers to diagnosis, to treatment initiation and retention post-diagnosis, and to viral load testing and suppression—programs can introduce solutions that both help these individuals and that remove barriers for others.By profiling the characteristics of those who face challenges in achieving viral suppression, programs can introduce solutions that both help these individuals and remove barriers for others.The approach features variable treatment and prevention services and service intensity grouped according to 4 different stages along this continuum of progression to viral suppression . The modAlthough progress varies by region, an estimated 59% of PLHIV globally are already receiving HIV treatment and are virally suppressed.Partner notification services, also known as index testing, are recommended by the World Health Organization as a safe, effective strategy to accelerate HIV epidemic control by asking PLHIV to list and refer their sexual and injecting partners and biological children to HIV testing services on a voluntary basis.A substantial proportion of individuals who have previously received an HIV diagnosis have either not yet initiated ART or have not achieved viral suppression.Immediate support should be provided to PLHIV who are diagnosed but not virally suppressed to help address root causes of adherence or treatment failure.In the process of providing personalized treatment support to PLHIV, a range of voluntary index testing options can be offered to encourage referrals of their risk contacts to prevention services such as PrEP and condoms until they have achieved viral suppression, or to HIV treatment services, as needed.,,PLHIV who have not yet received a diagnosis can be similarly offered tailored support to maximize individual treatment and population-level prevention benefits. An expanded range of options for accessing HIV testing services, including HIV self-testing options with dispensing through pharmacies and peer networks, can help to close gaps in diagnosis among PLHIV who might otherwise not otherwise access diagnostic or other services.Expanding screening for AHI among key populations and other individuals facing elevated HIV infection risks can help realize the largely untapped treatment and prevention benefits of identifying, treating, and index testing individuals with AHI. To maximize efficiency, AHI screening can be prioritized for the risk network members of individuals identified with AHI, those with recent HIV infections, and newly identified PLHIV. Screening can also be focused on key populations reporting recent behavioral risks, as well as among those with other sexually transmitted infections. Upon diagnosis, all PLHIV can be offered an accelerated path to viral suppression with same-day treatment initiation. Programs can assess the differentiated characteristics of newly diagnosed, recently infected, and acutely infected PLHIV to further optimize the relevance and focus of HIV testing efforts.,While the treatment and treatment-as-prevention benefits of prioritizing support according to viral burden may have evident advantages, the HIV micro-epidemic control approach also aims to enhance prevention benefit by focusing services in the risk networks in which active HIV transmission is occurring. The majority of risk contacts of PLHIV who are undiagnosed will be uninfected but at high risk of acquiring HIV infection, making linkages of these individuals to prevention services a priority. These focused prevention efforts should employ a combination of evidence-based prevention strategies relevant to the specific preferences and needs of the populations being served.The HIV micro-epidemic control approach aims to enhance prevention benefit by focusing interventions in the risk networks in which active HIV transmission is occurring.For all risk contacts of PLHIV who are not yet virally suppressed, PrEP is a critical, evidence-based, and likely short-duration priority.,Stigma, discrimination, violence, and other structural factors impose considerable barriers to service uptake, particularly among PLHIV and key populations living in criminalizing environments.The proposed micro-epidemic control approach emphasizes diagnosis of and intervention during AHI in light of the substantial role that AHI plays in epidemic transmission of HIV. Most current national HIV testing algorithms rely on antibody-based serological testing that cannot detect AHI. As a result, these approaches misdiagnose potential core transmitters as HIV-uninfected and miss critical opportunities to maximize the prevention benefits of HIV treatment. Affordable, accurate, and scalable solutions to diagnose AHI have remained elusive.,The brief duration of AHI poses a major challenge to diagnosis.The brief duration of acute infection and cost of testing poses barriers to diagnosis.Cost is also an issue. Point-of-care platforms for detection of HIV RNA such as Alere Q (Abbott Laboratories) and GeneXpert (Cepheid) are now available but are generally perceived as expensive (US$17–24). Fourth-generation point-of-care rapid HIV tests, such as the Alere HIV Combo kit (Abbott Laboratories), detect both p24 antigen and HIV antibodies within 20 minutes at a lower cost (US$2–4) and can be substituted as the first, sensitive screening test in a national HIV testing algorithm for diagnosis.,Nevertheless, a clinical trial of PrEP in Uganda, South Africa, and Zimbabwe found that 28% of infections missed by current third-generation rapid diagnostic tests would have been identified with the use of Alere HIV Combo, suggesting some advantages of using a fourth-generation test over standard antibody testing.,–AHI is sometimes accompanied by transient clinical flu-like and other symptoms, including rash, fever, sore throat, fatigue, muscle/joint aches, oral and genital ulcers, diarrhea, and swollen lymph nodes.To overcome some of the logistical and resource-related barriers to the expansion of AHI screening, diagnosis, and intervention, we propose 2 potential solutions to encourage further consideration of both these and other context-relevant approaches: (1) conducting pooled HIV-1 RNA polymerase chain reaction (PCR) on dried blood spot samples; and (2) expanding the use of fourth-generation point-of-care rapid HIV testing, leveraging recency testing data where possible to help focus AHI screening in networks in which ongoing HIV transmission is occurring.The “gold standard” for detection of AHI is molecular testing, specifically HIV-1 RNA PCR. This approach is considered the standard of care to facilitate early infant diagnosis among children born to HIV-infected mothers. However, PCR is relatively expensive.,In resource-limited settings, collecting dried blood spot samples for pooled PCR is a promising approach to circumvent some of the cost barriers associated with point-of-care platforms and the logistical challenges associated with separation, storing, and transfer of plasma samples. Whole blood spots can be collected with a finger prick and can be stored and shipped with relative ease. Recalibration of the PCR is necessary because of a degradation of viral RNA in dried blood that may result in a 2-log reduction in assay sensitivity and because of the potential presence of viral DNA, which may partially compensate for this loss in sensitivity.In resource-limited settings, collecting dried blood spot samples for pooled PCR is a promising approach to circumvent some logistical and cost barriers to detecting acute HIV infection.To maximize the benefits of screening for AHI, the time from sample collection to case identification and intervention must be minimized. A pooling approach is more practical in high volume settings in which batches can be run every day. While the time needed to process samples will vary according to the proximity and availability of laboratory infrastructure, we anticipate many programs being able to provide results in 1–2 days given the predominantly urban concentration of laboratory resources and of HIV key population risks in many country settings. The expansion of point-of-care viral load testing can further reduce turnaround times, facilitating earlier action.The routine collection of dried blood spot samples to detect and intervene during AHI also presents some additional value-added opportunities to improve HIV service delivery that are worthy of consideration. Drug-resistance genotyping can be performed on samples from all individuals who are confirmed through either molecular or serological testing to have HIV infection. In addition, these samples could be used to conduct phylogenetic analyses of potential HIV transmission clusters to enhance the focus of targeted testing and index testing implementation.,,,Rapid HIV recency assays, such as the Asanté HIV-1 Rapid Recency® Assay (Sedia Biosciences) and the Maxim Swift HIV Recent Infection Assay , were developed to help identify individuals who have become HIV infected within the past year—on average in the past 6 months—to help estimate HIV incidence and improve the focus of programming in settings, populations, and networks in which incident infections continue to occur.Rapid recency assays only measure antibody avidity after HIV seroconversion; they do not detect HIV RNA or p24 antigen and therefore are unable to detect AHI. In typical use, they are only offered to individuals who have been confirmed to have HIV infection with a standard antibody-based national HIV testing algorithm. Recency assays are also pending review for diagnostic purposes by the World Health Organization and are currently only approved for research use by the U.S. Food and Drug Administration. The World Health Organization has endorsed the use of recency assays for surveillance purposes but has not yet made a determination regarding program or individual-level benefits pending further evidence.That said, the rapid recency algorithm has demonstrated capacity to identify individuals who became HIV infected within the past year, and the U.S. President’s Emergency Plan for AIDS Relief has identified the scale up of recency testing as a “minimum standard” for HIV program implementation in an expanding set of countries receiving U.S. government support.Testing the contacts of recently infected individuals could improve a program’s capacity to detect and treat undiagnosed individuals.To leverage recency testing data to help focus AHI screening as part of an HIV micro-epidemic control model, programs would need to adopt strategies to: (1) integrate AHI screening into practice; (2) secure client informed consent for recency testing and the confidential use of those results; (3) support confidential profiling of individuals with recent and acute infections; (4) target testing with AHI screening in populations, settings, and networks aligned to these profiles; and (5) prioritize index testing with AHI screening among the contacts of recently and acutely infected individuals.An illustrative workflow for AHI screening supplemented by recency testing to improve focus is shown in Prioritizing AHI screening as part of index testing for the risk network contacts of recently infected individuals, as well as eventually for the contacts of individuals with AHI as these are identified, could then help programs increase the likelihood of detecting people with AHI. Case profiles can be developed to describe actionable characteristics of recently and acutely infected individuals to guide the prioritization of targeted, differentiated HIV testing, AHI screening, and prevention strategies for these individuals and members of their risk networks. Individuals who are recently or acutely infected can also be offered opportunities to serve as peer mobilizers and/or to distribute HIV self-test kits to help accelerate linkages of their network contacts to testing, treatment, and prevention services, as relevant.As individuals are screened as potentially having AHI, these individuals can be offered immediate confirmatory HIV RNA testing and HIV treatment. On the rare occasion that HIV RNA confirmatory testing is not immediately available, presumptive HIV treatment could be provided pending confirmation in a manner analogous to the provision of HIV postexposure prophylaxis, which is generally considered safe and effective.,,Current concerns about the potential impact of providing acute or recency test results to clients include increased risk of criminalization of key populations, as well as criminalization of HIV transmission and increased risk of gender-based or intimate partner violence.Viral burden is the primary predictor of HIV-related morbidity, mortality, and ongoing transmission. Although a majority of PLHIV globally have achieved viral suppression through sustained access to HIV treatment, achieving an end to the HIV pandemic is contingent on addressing the preferences and needs of virally unsuppressed and persistently unserved and underserved PLHIV and members of their risk networks. Proven solutions exist to prevent and treat HIV, but the approaches and technologies needed to differentiate and focus support based on viral burden historically have been limited. Now, with the expansion of viral load testing and an expanded set of options to screen for and treat AHI, we may be better equipped to improve both the impact and efficiency of efforts to accelerate epidemic control.An HIV micro-epidemic control approach that prioritizes personalized treatment support for PLHIV who are not virally suppressed—and in the process focuses HIV testing and relevant HIV prevention and treatment support among their network members—offers a framework to integrate these advances into current practice to maximize client benefits and overall impact. In particular, such an approach offers a path to integrate the detection and treatment of AHI into routine programming, potentially curbing a substantial proportion of ongoing HIV transmission that occurs during this period and has historically continued apace beyond the reach of efforts to leverage HIV treatment as prevention at scale. However, the ultimate advantages of such an approach remain largely undocumented. Additional investments in the development, implementation, and evaluation of practical strategies to differentiate support based on viral burden are needed to assess the real-world benefit of the proposed HIV micro-epidemic control approach.


Ibotenate produced spherical, perikaryal-specific lesions regardless of the site of injection, and experiments with specific glutamate receptor antagonists showed that its effects were mediated by the N-methyl-D-aspartate receptor. Because of this uniform neurotoxicity and near ubiquitous efficacy, ibotenic acid became the excitotoxic lesioning agent of choice. The discovery of the excitotoxic properties of the tryptophan metabolite quinolinic acid and of the anti-excitotoxic, neuroprotective effects of the related metabolite kynurenic acid in the Schwarcz laboratory then gave rise to the concept that these endogenous compounds may play causative roles in the neuropathology of a wide range of neurological and psychiatric disorders.The neurotoxic action of glutamic acid was first described by Lucas and Newhouse, who demonstrated neural degeneration in the inner layers of the neonatal mouse retina after systemic treatment with L-glutamate. Olney extended these findings by showing that neuronal degeneration affected other brain structures including neurons within the arcuate nucleus of the hypothalamus and the area postrema, that the lesion spared axons passing through these areas, and that the neurotoxic potency of glutamate analogs correlated with their excitatory potency, resulting in the designation “excitotoxins.” As this method affected only a small number of brain regions, it was not suitable for targeted brain lesions. The Coyle laboratory showed that direct injection of the potent glutamate receptor agonist, kainic acid, into the rat striatum caused a rapid degeneration of intrinsic neurons while sparing axons of passage or termination including the unmyelinated dopaminergic terminals. Kainic acid also exhibited this perikaryal-specific and axon-sparing profile when injected into the cerebellum, hippocampus and eye. However, neuronal vulnerability was highly variable, with hippocampal CA3, pyriform cortex and amygdala neurons exhibiting great sensitivity due to kainate’s high convulsive activity. In a comparison study, ibotenic acid, a potent glutamatergic agonist isolated from the  Lesioning neurons of interest has long been a tool in neuroscience research to define their physiologic and behavioral roles as well as their synaptic connections. These lesions may be accomplished by electrolytic  probes or transection of axon pathways. Degenerating axons become argentophilic, permitting the tracing of their pathways in brain sections to their fields of synaptic termination . Brain lAt the time, another challenge in neuroscience research was to recreate in experimental animals the neuropathology characteristic of neurodegenerative disorders. These disorders typically involve a very selective degeneration of a population of neurons while sparing of adjacent neurons and axons of passage – unlike penetrating lesions or strokes. For example, Parkinson’s disease affects the dopaminergic neurons in the substantia nigra ; Huntingin situ excitotoxin lesion method has been widely used in basic and translational neuroscience research for over 40 years  (In order to understand the basis for developing the em (CNS) . These aem (CNS) . The disem (CNS)  led to tem (CNS) , which iem (CNS) .In a review of these studies, 2 +  led to the proposal that more than one receptor might mediate the action of these glutamate analogs . Precise2 + . Another2 + .in situ injection of an excitotoxin to produce a perikaryal-specific lesion was motivated by an interest in reproducing the pathology of Huntington’s disease (HD). The prominent symptoms of HD, a choreo-athetotic movement disorder complicated by progressive dementia, may result from degeneration of neurons primarily in basal ganglia  hypothesized that the apparent “axon-sparing” striatal lesions in HD may be excitotoxic in nature. Direct injection of a potent glutamate receptor agonist could therefore induce perikaryal-specific neurodegeneration similar to HD in experimental animals. To test this idea, we chose kainic acid, reputed to be among the most potent glutamate receptor agonists , as an ejunction . An init tissues . In cont tissues .in situ kainic acid lesion, which offered “a new technique for making selective brain lesions that will be useful for examining neuronal connectivity” . Glutamate, which is ~50-fold less potent than kainate in electrophysiologic studies in vitro  compound kynurenic acid are now regarded as major players in the pathophysiology of a wide range of neurological and psychiatric disorders.The concept of excitotoxicity emerged over 60 years ago from the observation that systemic treatment with glutamate causes degeneration of neurons in the inner layers of the retina. Subsequent studies showed that glutamate-induced neuronal degeneration also occurred in a few discrete brain regions with deficient blood-brain barriers. Since the toxicity correlated with excitatory potency of glutamate analogs, Olney coined the term “excitotoxins.” To harness the lesion method so that it could be targeted at neurons intrinsic to any brain region, Both authors wrote and edited the review.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.


Samples of thyroid cancer and healthy thyroid tissue were collected from patients undergoing thyroidectomy and analyzed with 1H-NMR spectroscopy for a wide array of metabolites. We found a significant increase in lactate content in thyroid cancer tissue compared to healthy tissue. Metabolomic analysis demonstrated significant differences between cancer tissue and healthy tissue, including an increase in aromatic amino acids, and an average decrease in citrate in thyroid cancer tissue. We hypothesize that these changes in metabolism may be due to an oxidative stress-related decrease in activity of the Krebs cycle, and a shift towards glycolysis in cancer tissue. Thus, thyroid cancer cells are able to reprogram their metabolic activity to survive in conditions of high oxidative stress and with a compromised antioxidant system. Our findings, for the first time, suggested a connection between oxidative stress and the alteration of the metabolic profile in thyroid tumors.Thyroid cancer cells demonstrate an increase in oxidative stress and decreased antioxidant action, but the effects of this increased oxidative stress on cell function remain unknown. We aimed to identify changes in the metabolism of thyroid cancer cells caused by oxidative stress, using proton nuclear magnetic resonance ( The metabolomic analysis performed (and the type of instrumentation used) did not allow us to discriminate between reduced or oxidized glutathione, only showing an increase in total glutathione. Our findings, of course, only partially clarify the complex mechanisms underlying thyroid cancer. Other studies are needed to better understand the role of other metabolites and enzymes involved in thyroid cancer, as well as the mechanisms involved in the overproduction of free radicals. However, the data is quite clear that thyroid cancer cells are able to reprogram their metabolic activity to survive in conditions of high oxidative stress and with a compromised antioxidant system. A recent study by Yanyun Li et al  on 16 paThyroid cancer cells show evidence of increased oxidative stress and impaired antioxidant defenses. In this study, using NMR spectroscopy to explore differences in the metabolites of thyroid cancer tissue compared to healthy tissue, we demonstrate evidence of metabolic “reprogramming” in cancer cells, with changes in the metabolites involved in mitochondrial activity and in glucose/pyruvate metabolism. Our findings suggest that thyroid cancer cells have metabolic plasticity that allows them to survive in extreme environmental conditions, such as those with high oxidative stress levels. The metabolic pathways involved in this adaptation may provide additional targets for cancer-fighting therapeutic agents. Lactate, for example, may be considered not only as a cancer marker but also as an oncometabolite potentially capable of increasing cellular resistance to harmful stimuli. The metabolic pathways involved in lactate synthesis may represent a novel molecular target to counteract thyroid cancer.


New species arise as the genomes of populations diverge. The developmental ‘alarm clock’ of speciation sounds off when sufficient divergence in genetic control of development leads hybrid individuals to infertility or inviability, the world awoken to the dawn of new species with intrinsic post-zygotic reproductive isolation. Some developmental stages will be more prone to hybrid dysfunction due to how molecular evolution interacts with the ontogenetic timing of gene expression. Considering the ontogeny of hybrid incompatibilities provides a profitable connection between ‘evo-devo’ and speciation genetics to better link macroevolutionary pattern, microevolutionary process, and molecular mechanisms. Here, we explore speciation alongside development, emphasizing their mutual dependence on genetic network features, fitness landscapes, and developmental system drift. We assess models for how ontogenetic timing of reproductive isolation can be predictable. Experiments and theory within this synthetic perspective can help identify new rules of speciation as well as rules in the molecular evolution of development. Are certain developmental processes especially likely to be disrupted in hybrids? This question has been surprisingly neglected given that hybrid defects provide a rare window on those developmental processes and pathways that diverge rapidly between taxa.’ – “Distinct species are those separate collections of genomes that, if you were to put them together in the same cells, the mixture would create a broken organism. Development in the hybrid individuals would go awry in such a way as to cause inviability, infertility, or a phenotypic mismatch to ecology or mating interactions that compromises further reproductive success. Given common descent from an ancestor, how could evolution produce such disastrous phenotypic consequences of genetic change? Darwin recognized this dilemma (Darwin 1859), in that natural selection will oppose changes that confer a net fitness cost. But evolution can circumvent this problem through interactions between multiple genetic factors, as intuited by W. Bateson, T. Dobzhansky and H.J. Muller and made explicit in the genetic mechanism of Dobzhansky-Muller incompatibility (DMI) for post-zygotic disruption in hybrids . Put simBox 1.Visualizing, conceptualizing, and modeling Dobzhansky-Muller incompatibilities.Dobzhansky-Muller Incompatibilities (DMIs) can be viewed as disrupted gene networks and as valleys (or holes) in fitness landscapes . DistincIn this way, the mutational substitutions that accumulate in one population are indifferent to the substitutions that accumulate in other populations, but only so long as the two populations do not share alleles with one another through gene flow. When the populations of incipient species do intermingle genetically, then developmental programs in hybrid individuals must confront whether alleles derived from such ‘out-of-sight, out-of-mind’ evolution interact in a way that allows ontogeny to proceed normally . Incompacis- and trans-acting regulators of gene expression . In the present state of the field, we find that few general answers emerge from these factors considered in isolation, motivating deeper attention from theory and empirical study.To address this goal, here we first consider how molecular evolution is influenced by the properties of genes and genetic networks, such as pleiotropy, modularity, robustness, and As we explore how the evolution of development intersects with speciation, it is valuable to consider some key aspects of genetic architecture from the perspective of multi-gene networks that, in turn, control organismal development . Here, wThe mapping of genotype on phenotype and fitness leads us to predict that evolution will proceed along genetic lines of least resistance . GeneticThe extent of pleiotropic effects induced by mutation to a gene is a major determinant of the likelihood of evolutionary change to that gene . This locis-regulatory elements, in contrast, generally influence only a subset of a gene’s expression, and so will exert the fewest pleiotropic effects relative to coding sequence and trans-regulatory changes. Consequently, trans-regulatory evolution and coding sequence divergence ought generally to be slower for genes with high centrality and connectivity in genetic networks. We should expect such genes to have especially high ratios of cis:trans regulatory divergence, provided that those genes also have reasonably complex arrays of cis-regulatory elements controlling their expression that can facilitate divergence. Because trans-acting regulation tends to show greater condition-dependence than does cis-regulation  for genes expressed differentially across development provide a means of assessing selection on the encoded protein sequences used in genetic networks. A simple way of testing for trends in purifying selection and positive selection across ontogeny is to compare the average dN/dS value for the set of genes with peak expression at a given developmental timepoint , other factors may also contribute to Haldane’s rule . Genes tvolution . Sex-limcis-regulatory regions, however, correlate only weakly  may actually be less likely to create DMIs than changes with opposing cis-trans effects (thought to result from stabilizing selection within species) , as well as to the likelihood of contributing to a DMI. For example, genes showing evidence of vergence . And, DMpression . Specifispecies)  (but seespecies) . ConsequMight the functional role of genes also represent an axis of predictability to factors that instigate DMIs in inter-species hybrids? The relatively few known ‘speciation genes’ in animals give little clue to whether particular molecular functions may be predisposed to involvement in inter-species incompatibilities . At a coGene duplication is a powerful factor in the evolution of phenotypic novelty . MoreoveGene expression profiles and genetic network architecture are dynamic over the course of ontogeny. This dynamism suggests that molecular evolution could differ for the distinct subsets of genes associated with different stages of development . ImportaThe early conservation model (‘von Baer’s third law’) derives from the fact that time is unidirectional, so changes early in development may cascade catastrophically as cell division and differentiation proceeds. Consequently, genes expressed early in development would experience stronger purifying selection, with slower molecular and phenotypic evolution early in ontogeny . Some peHmr in Drosophila [A caveat about inferring the developmental timing of hybrid dysfunction is that early-acting incompatibilities may preclude detection of DMIs that would otherwise be revealed later . Disproportionate observation of early-acting developmental defects in hybrids thus may not imply that molecular evolution disproportionately accrues for genes in early-acting developmental programs. This ‘pull of the early’ represents a general challenge in characterizing the profile across ontogeny of disrupted genetic networks that confer post-zygotic reproductive isolation. There are at least four ways to potentially address this issue empirically: (i) assess distinct species comparisons from different phylogenetic depths, (ii) exploit partial penetrance of F1 hybrid dysfunction , (iii) uosophila ), or (ivnp with n DMIs each with probability p of not occurring in the earliest stage). This null model therefore predicts a negative relationship of the genetic distance between species and the terminal stage to which hybrids develop, and predicts that later stages will have more tissue types exhibiting dysfunction in hybrids. Note that these predictions overlap with several other models that we describe, but do not depend on differential selection pressures, pleiotropy, or network features for genes expressed at different times in development. This model, however, predicts no association of molecular evolutionary rates for genes (or the mode of selection) with the developmental timing of their expression.We suggest that the ‘uniform chance’ model represents a null model for the manifestation of hybrid incompatibilities over the course of ontogeny. If molecular evolution is unbiased with respect to the timing of expression of genes that have diverged between species, then every point in development can be considered to have an equal chance of expressing a DMI to yield hybrid disruption of a tissue type or termination of development . This coThe mutation accumulation model of aging and senescence predicts that genes and genetic networks expressed earlier in development will evolve more slowly . Here, tcis and trans, with trans-acting changes being more likely to exert pleiotropic effects and arising at a higher rate   to frame these issues about how ontogenetic features link to molecular evolution and hybrid dysfunction. Surprisingly, few broad conclusions can be drawn from even these well-studied systems, and general principles await concerted research efforts. Nevertheless, these study systems present clear promise for future research to disentangle the constellation of causal contributing factors that link microevolutionary mechanisms to developmental programs and macroevolutionary patterns.To decipher how predictable molecular evolution and post-zygotic reproductive isolation might be, it is important to consider the ontogenetic context of organisms to identify trends in the tempo of gene expression, cell division, and differentiation . The kinmammals) . While hmammals) . Here, wC. elegans development as a point of reference, embryonic cell numbers grow approximately exponentially until ~520 cells upon which cell count increases relatively slowly to the 959 somatic and ~2000 germline cells that comprise the adult hermaphrodite animal . We still have only a rudimentary understanding of how the genetic clockwork might set the ontogenetic timing of this developmental alarm clock in the dawn of new species. Consequently, it is a challenge to extract consensus on patterns and predictions about sequence evolution and DMI incidence over the course of ontogeny.Key features of an ontogenetic view of molecular evolution include the pleiotropic roles of genes and the pleiotropic effects of genetic perturbation, as well as the dynamism of genetic network modularity over development, to influence the evolvability of genes and the robustness of phenotypic outputs. A growing body of empirical literature is documenting the dynamics of gene expression and molecular evolution over developmental time. What is missing in the nascent state of the field is an integrated set of theoretical expectations for how these features can produce emergent trends of genome evolution and of dysfunctional genetic networks in the divergent set of genomes of hybrid individuals. Empirical tests of existing theoretical predictions, from models that focus primarily on only a subset of ontogeny, will benefit from exploring the relative influence of adaptive molecular evolution and purifying selection on genes that vary in expression over all ontogeny. Answering the neglected question -- ‘Are certain developmental processes especially likely to be disrupted in hybrids?’ -- offers the promise to identify new rules of speciation as well as rules in the molecular evolution of development.


Fibromyalgia is a chronic health condition characterized by widespread, severe musculoskeletal pain that affects an estimated 5–7% of the global population. Due to the highly comorbid nature of fibromyalgia, patients with the disorder often respond poorly to traditional pain treatments. Recent studies suggest that patient response may be more favorable to alternative analgesics, such as cannabis. However, the therapeutic potential of cannabis-based pain treatment for fibromyalgia remains unclear. The present study examined the most recent cannabis literature (2015–2019) and provides a critical review of current research on the safety and efficacy of medical cannabis treatments for fibromyalgia.We followed Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines in searching the PubMed and Medline databases using the search terms “cannabis + fibromyalgia” and then “cannabinoids + fibromyalgia.” Inclusion criteria were a) English language, b) published in peer review journals, c) published from 2015 to 2019, d) all study designs except for systematic reviews and meta-analyses, and e) all cannabis preparations.The search identified five applicable studies involving 827 participants that used six different treatments. Review suggested several methodological problems pertaining to generalizability and validity.Although the critically reviewed studies superficially suggest that medical cannabis is a safe and effective treatment for fibromyalgia pain, serious methodological limitations prevent a definitive conclusion regarding the use of cannabinoids for pain management in fibromyalgia patients at this time. Fibromyalgia is associated with widespread musculoskeletal pain that is commonly accompanied by additional symptoms such as fatigue, cognitive problems, mood disturbances, and problems with sleep Clauw . In the Pathophysiological symptoms of fibromyalgia include a sensitized or hyperactive central nervous system that is associated with an increased gain in pain and sensory processing Clauw . FibromyMoreover, results have shown that, in some cases, fibromyalgia patients with multiple comorbid conditions that lead to pain respond well to centrally acting pharmacological therapies, such as cannabis  was used for this review  also varied across studies and included delivery methods of smoking, inhalation via vaporization, and oral administration (oil drops). Dosage amount, treatment strategy and duration, diagnostic criteria, inclusions criteria, and baseline considerations were also inconsistent across selected studies. The inconsistencies in both methodological design and results across existing studies make the establishment of a solid foundation of empirical evidence challenging.Additionally, since cultural considerations are not as relevant when determining the biological effectiveness of cannabinoids for pain management within the human species, the lack of broad cultural diversity among study participants was not considered a limitation in the selected studies. Moreover, since studies have shown that fibromyalgia has a high female:male ratio, it was not surprising that the majority of study participants across all selected studies were female. However, research has shown that there are significant biological differences between males and females regarding all areas of cannabis use, including addiction potential and outcomes  and cannabidiol (CBD) represent two principal components Madras . THC, thTherefore, a key aspect in determining the efficacy and safety of cannabis agents needs to involve not only tracking the precise agent used with patient outcomes but also noting the ratio of THC to CBD in each dosage. However, identification and isolation of cannabinoids across products is challenging due to the lack of available information in this area. Moreover, as previously noted, research has shown that the mechanism of entry into the human body of different agents plays a role in efficacy and safety. Future research is needed to ascertain the most appropriate ROA for each agent, which is currently difficult due to rapidly changing cannabis-related technology.Evidence highlighting the efficacy of cannabis in the treatment of chronic pain for fibromyalgia patients will not have acceptable validity if the type, strain, and dosage is not carefully tracked. Further, correlations between assessed outcomes and specific types of cannabis cannot be accurately determined if dosage and strain are not carefully tracked alongside outcomes. In the selected studies, Sagy et al.  utilizedHabib and Avisar  did not There was a high level of demographic variety across all five selected studies. However, minimal effort was made to minimize bias by ensuring that groups were appropriately comparable at baseline for demographic and other key factors. While diversity would generally lead to higher generalizability, when assessing cannabis use for fibromyalgia, variability across participants is not as desirable. For example, cannabis use and efficacy are highly affected by gender (Calakos et al. While gender-specific studies on cannabis use itself are few, those available have shown that gender differences do exist, most notably that males are more likely to use cannabis medicinally and that females are quicker to become addicted (Cuttler et al. As previously noted, the majority of participants across the selected studies were female; 85% (Habib and Avisar Studies have indicated that past and concurrent cannabis use influences the efficacy, safety, and tolerance of any form of concurrent analgesic use in chronic pain patients across diverse diagnoses (Salottolo et al. Yassin et al.  did not Studies have indicated that cross-drug tolerance is an important factor when assessing the efficacy of analgesics for pain management as cross-drug tolerance varies widely between individuals and substance interactions (Askay et al. While 100% of the selected studies focused on participants with a fibromyalgia diagnosis, only two studies (Van de Donk et al. For example, Yassin et al.  only incIn addition, the selected studies generally failed to clearly record participant symptoms at the start of each study protocol. Symptoms such as constipation, dizziness, dry mouth, and dry eyes consistent with symptoms of fibromyalgia may also be attributable to cannabis use (Van de Donk et al. There is a high level of agreement in the extant literature that fibromyalgia is a comorbid disorder, rarely occurring in isolation (Fitzcharles et al. For example, the prevalence of depression in the fibromyalgia population is 25–60%, and research has shown that fibromyalgia patients with comorbid long-term or preexisting depression are less responsive to certain pain medications than fibromyalgia patients with short-term depression (Silverman et al. Several different methods were used to assess outcome across the selected studies. Van de Donk et al.  assessedThe use of such a wide variety of assessments for determining treatment outcome, particularly concerning measuring chronic pain levels, decreases the generalizability of results across studies and the overall broader fibromyalgia patient population. Further, chronic pain as a construct was not operationalized in any of the five selected studies. Continuity regarding pain as a construct will help researchers to determine appropriate assessment measures. Operationalizing the specific type of pain that is being targeted in fibromyalgia patients in relation to cannabis as an analgesic and selecting appropriate outcome measures will be an important aspect of future studies.The authors of the selected studies collectively suggest that medical cannabis is a safe and effective treatment option for patients with fibromyalgia, with reports of significant improvements in pain intensity/severity (Habib and Artul Additional weaknesses in methodological design occurred in varying configurations across all selected studies. These weaknesses affected the overall generalizability of outcomes and included problems with inclusion and recruitment criteria, lack of control groups or appropriate reference groups, short treatment duration, and small sample sizes. Randomized clinical trials with proven methodological design are a critical need in the field of cannabis research as it pertains to assessing chronic pain management in fibromyalgia patients. The growing legalization and increasing use of cannabis across all populations indicates that cannabis use, for any reason, is a significant public health concern, and empirically-based information is urgently needed.Although the five critically reviewed studies would seem to suggest that medical cannabis is a safe and effective treatment option for patients with fibromyalgia, the serious methodological limitations of this research preclude drawing any strong conclusions about efficacy. Instead, we advise that the reviewed body of literature lends very little evidence in support of medical cannabis as an efficacious treatment modality for chronic pain management in fibromyalgia patients. We conclude that no studies to date have established a compelling relationship between any form of medical cannabis treatment and symptom improvement in fibromyalgia patients suffering from chronic pain.Moreover, the studies reviewed in this paper indicate a high prevalence of adverse side effects associated with cannabis use in fibromyalgia patients. The majority of reviewed studies utilized or indicated smoking as the ROA for cannabis treatment despite empirical evidence indicating that smoking cannabis has adverse health effects and is not recommended for treating chronic conditions. Randomized clinical trials using ROAs specifically indicated for increased safety, tolerance, and efficacy are needed before cannabis can be safely recommended as a treatment modality for managing chronic pain in fibromyalgia patients. It should be noted, however, that the validity of randomized clinical trials of cannabis use may be compromised by challenges to adequate participant blinding. Participants’ awareness of the lack of psychoactive effects of placebo cannabis may result in the inadvertent overestimation of the effectiveness of medical cannabis Casarett .Additionally, further research is needed in order to ascertain the clinical benefits as well as the safety and tolerability profiles of all strains and compositions of cannabis used for symptom relief. Studies are also needed to identify the effects of short- and long-term drug interactions with cannabis in fibromyalgia patients who concurrently use conventional analgesics or unauthorized forms of cannabis for chronic pain. Furthermore, fibromyalgia patients often have multiple comorbidities, making the effects of medical cannabis on specific symptoms hard to parse out across the varying symptoms brought by diverse and often overlapping medical conditions. Future study design should also include post hoc analysis to assess the effect of baseline characteristics on the mean pain scores across patients with varying baseline characteristics such as demographic specifications, comorbidities, past cannabis use, concurrent drug use, symptoms, pain levels, and other such relevant factors. Separating fibromyalgia patients into groups based on baseline indications and comparing them to reference groups is a crucial aspect of sound methodological design that was not sufficiently implemented across the selected studies in this review.Due to the current legal climate regarding cannabis use in the United States, health professionals are often confronted with the need to educate patients regarding safe cannabinoid use. Standardization of treatment compounds and regimens is needed so that health practitioners can offer safe, evidence-based information to patients. However, given the inconsistent results across studies, reaching a definitive conclusion regarding the use of cannabinoids for chronic pain management in fibromyalgia patients is not possible at this time.


Current parallel sequencing technologies generate biological sequence data explosively and enable omics studies that analyze collective biological features. The more omics data that is accumulated, the more they show the regulatory complexity of biological phenotypes. This high order regulatory complexity needs systems-level approaches, including network analysis, to understand it. There are a series of layers in the omics field that are closely connected to each other as described in ‘central dogma.’ We, therefore, have to not only interpret each single omics layer but also to integrate multi-omics layers systematically to get a full picture of the regulatory landscape of the biological phenotype. Especially, individual omics data has their own adequate biological network to apply systematic analysis appropriately. A full regulatory landscape can only be obtained when multi-omics data are incorporated within adequate networks. In this review, we discuss how to interpret and integrate multi-omics data systematically using recent studies. We also propose an analysis framework for systematic multi-omics interpretation by centering on the transcriptional core regulator, which can be incorporated in all omics networks. Nowadays, an enormous amount of sequencing data has been generated by increasing levels of size exponentially. Next-generation sequencing (NGS) technology is a key breakthrough that performs parallel sequencing with fewer specimen than traditional sequencing methods. The old gene studies focused on a set of genes within a couple of samples, while current studies profile whole genome from a myriad of samples. We call this genomics, which indicates the study of collective features of whole genomes as described by its suffix ‘-ome,’ meaning ‘totality.’ Recent genomics studies have unraveled mechanisms underlying complex phenotypes or diseases such as cancer, which had not yet been well understood. Especially, large-scale genomics have revealed that most complex traits have polygenic loci on human genomes in regard to complex diseases , chromatin immunoprecipitation (ChIP), and mass spectrometry (MS). These experimental technologies have been incorporated into NGS accordingly by developing transcriptomics, epigenomics, and proteomics, although proteomics requires a particular process   has been used based on pairwise correlations between gene expression variables  directly . PTM is a series of protein modification processes, such as the addition or removal of chemical moieties of amino acids, changes in protein properties caused by proteolytic processing, and the formation of disulfide bridges between cysteine residues  based on proteomic analysis can offer another key regulatory explanation. The transcription factor, chromatin modifier, and other epigenomic regulators are core regulators in transcriptional regulatory networks, which are composed of proteins. This implies that variations in these regulators at the protein level are also important to understanding the regulatory mechanisms of regulatory networks. Not only is profiling DEPs themselves important, but analyzing PTM will give us additional information for multi-omics systems analysis. Moreover, PPI networks can supplement and enhance transcriptional regulatory networks by attaining gene-level signatures from profiling results of proteins and PTMs to construct integrative regulatory networks. For example, when we discovered that transcription factor A regulates a set of genes with DEGs or DEPs, which have functional enrichment relevant to an interesting phenotype such as a specific disease, the alteration of transcription factor A can be crucial in explaining the marker for the development of the disease. However, there may be no alteration for transcription factor A at the (epi)genomic, transcriptomic, and proteomic levels in another cohort, although it ranks highest among candidates for markers of the disease. In this case, protein interactome analysis specifically can help to determine another factor that affects transcription factor A in interaction regulation. This is because alterations of other co-regulating factors that interact with transcription factor A might influence transcriptional regulation. By integrating this interactome network information with the constructed network, we can get a full regulatory map from transcription to translation for a specific phenotype.To understand regulatory mechanisms in a complex biological system, all layers of omics that include genomics, epigenomics, transcriptomics, and proteomics are necessary as they play independent but related roles in relation to one another. In addition, considerable expressions of mRNA do not always result in protein expressions, although large portions of mRNA production are correlated with protein levels  (Dettmer et al. Taken together, a series of omics data using the relevant network construction will enhance our understanding of complex mechanisms that underly biological phenotypes when the multi-omics data are incorporated within an adequate framework. The framework should be constructed based on ‘central dogma,’ which indicates information flow from transcription to translation. Each step of making mRNAs and proteins is composed by complex networks, and they interact with each other. Therefore, systems approaches that use transcriptional regulatory network, protein interactomes, and functional networks have to be incorporated properly to the relevant omics data. In addition, we propose analysis that identifies a core regulator of transcriptional regulatory networks. This transcriptional core regulator-centered approach has the advantage in multi-omics studies because the perturbation of the core regulator can be affected by or affect not only transcription but also translation. Most of the core regulators may be transcription factors so that they actively participate in transcriptional regulation. Furthermore, they are associated with translation as they are composed of proteins. Therefore, core regulators can be incorporated in both of transcriptional regulatory networks and protein interactomes. Systematic multi-omics interpretation in the context of perturbation of transcriptional core regulators will successfully give us clues to unravel mechanisms about complex biological phenotypes.


ADHD is defined by behavioral symptoms that are not well characterized in relation to ADHD’s neurobiological mechanisms. This approach has limited our ability to define ADHD nosology and predict outcomes because it does not systematically examine facets of the disorder such as the inability to maintain cognitively effortful activities, as promoted in the NIMH RDoC approach. Existing data indicate ADHD is associated with differences in reward valuation and processing, but we do not know whether ADHD is also associated with higher levels of aversion to exerting cognitive effort and/or altered reward x effort interactions. Our ongoing study addresses this knowledge gap by examining individuals’ preferences between rewards associated with minimal effort and reward alternatives with a higher payoff but higher effort costs (“effort discounting”); thereby permitting us to characterize differences in biases and tradeoffs during effort-related decision-making in ADHD. The study takes advantage of a well-defined sample of ADHD-diagnosed and healthy control individuals to address three aims. First, we determine whether ADHD is associated with steeper discounting of larger, more effortful rewards. Second, we examine the subjective perception of effort in youth diagnosed with ADHD and healthy controls using tasks requiring varying levels of cognitive effort. Third, we explore relationships amongst indices of effort discounting, theoretically-related traits , biomarkers of effort-related decision-making (eye movements and pupil size), and various cognitive measures. Successful completion of the aims will permit us to better characterize ADHD-healthy control differences and lay a foundation for more computational approaches to ADHD diagnostic criteria. The inability or reluctance to engage in cognitive  effort is a cross-cutting feature of numerous psychopathologies and related to the Research Domain Criteria (RDoC) construct: Reward Valuation; subconstruct: Effort . One sucIn the last few years, interest in the neurobiological mechanisms underlying effort-related decision-making has been growing, but most of these studies have examined physical effort, rather than cognitive effort, e.g., squeezing handgrips –5, typinIn terms of component cognitive mechanisms, previous research in ADHD has focused on reward valuation and reward processing ,11, on cN-back memory task, then made decisions between small and large rewards associated with a low or high N-back requirement. Data from these studies, and research using hypothetical tasks  effort has been previously discussed in connection with psychopathology and is recognized in the RDoC, very little research has attempted to move this concept from the qualitative to the quantitative realm, as proposed in this study. The methodology under-development in this project is highly innovative with the intention of elucidating the mechanisms underlying the concerns faced by these clinical populations in a novel way. By systematically examining the effect of changes in the ISI and Stimulus Duration parameters on performance on the cognitive effort tasks, as well as the subjective ratings of effort, we hope to provide a methodology to permit responses to sustained attention and working memory loads to be quantified under Third, successful completion of The study design has a number of potential limitations. First, as with any quasi-experimental study, the ADHD-diagnosed and healthy control individuals may differ in ways that are related to their group membership that affect cognitive effort discounting, for example, differences in the types and severity of co-morbid conditions like depression and co-occurring use of substances/medications. The Health Questionnaire will provide some data about these sources of group differences and permit some exploratory analyses of the robustness of effects when participants are omitted. However, such analyses would only provide a foundation to justify future research, rather than provide a definite answer to the role of these comorbidities in differences in effort discounting. A second study limitation is that if group differences in effort discounting are primarily due to differences in perceived difficulty, our plan to control for perceived difficulty level by matching groups on these ratings may eliminate group differences in cognitive effort discounting. However, such a lack of differences is still informative and suggest that the neuropsychological processes involved in devaluation of effortful rewards are not different between groups; rather differences reported outside the laboratory are driven by ADHD-diagnosed individuals perceiving the cognitive effort costs as higher.A “dislike of mental effort” is a diagnostic criteria for ADHD, a trait closely associated to attention and working memory . Howeve


Attempts to achieve digital transformation across the health service have stimulated increasingly large-scale and more complex change programmes. These encompass a growing range of functions in multiple locations across the system and may take place over extended timeframes. This calls for new approaches to evaluate these programmes.Drawing on over a decade of conducting formative and summative evaluations of health information technologies, we here build on previous work detailing evaluation challenges and ways to tackle these. Important considerations include changing organisational, economic, political, vendor and markets necessitating tracing of evolving networks, relationships, and processes; exploring mechanisms of spread; and studying selected settings in depth to understand local tensions and priorities.Decision-makers need to recognise that formative evaluations, if built on solid theoretical and methodological foundations, can help to mitigate risks and help to ensure that programmes have maximum chances of success. Many countries worldwide see large-scale system-wide health information technology (HIT) programmes as a means to tackle existing health and care challenges –3. For eSummative evaluations that seek to capture the eventual outcomes of large national programmes appear to answer questions about the effectiveness of public investments. However, funders and administrators are under pressure to demonstrate outcomes quickly - often within the lifetime of programmes, whilst the full benefits of major change programmes can take a long time to materialise. Premature summative evaluation can generate unwarranted narratives of “failure” with damaging political consequences .The success or failure of HIT projects involves many different dimensions and at times incommensurable factors , 9. The Our team has conducted several formative evaluations of large HIT programmes and developed significant expertise over the years –16. In dWe here offer an extension of this work to explore not only implementations of specific functionality ), but their programmatic integration with ancillary systems . This can help to gain insights into the emergence and evolution of information infrastructures (systems of systems) that are increasingly salient as we see functional integration within hospitals and across care settings. We also consider mechanisms of spread, evolving networks/processes, and vendor markets .The first challenge concerns the difficulty of attributing outcomes (i.e. exploring what caused a specific outcome) for major changes in HIT. Although often required to justify investments, the direct effects of complex HIT such as EHRs are difficult to track and measure . This isTheoretically informed formative evaluations that draw on science and technology studies and acknowledge the interrelationship between social and technological factors can help to address this issue . A partiDecision-makers cannot simply roll out standard solutions across the health service as sites vary in terms of clinical practices, existing information systems and data structures, size and organisational structures, contexts and local demographics. A key challenge for evaluation of large programmes is reconciling tensions between bringing specific sites up to international best practice, and levelling up the local ecosystem . OrganisThere is a tension between local and national priorities – and there is no stable way to reconcile these. Instead, strategies constantly shift between these poles, never standing still, pulled by a network of stakeholder groups with conflicting interests in a process that has been conceptualised as a swinging pendulum Fig.  14]. Fo. Fo14]. To explore this process and associated tensions, evaluators need to study evolving networks, relationships, and processes to understand how various stakeholders are mobilised nationally and locally as part of the change programme and what the perceived effects of these mobilisations are. This may involve working closely with national Programme Leads to identify current policy directions and intended national strategy, whilst also exploring local experiences of this strategy. From our experience, it can be helpful to move from arms-length critical analyst to constructive engagement with different stakeholders groups. Establishing long-term personal relationships with senior decision-makers whilst retaining independence is important is this respect. These need to be characterised by mutual trust and frank discussion where evaluators play the role of a ‘critical friend’ at times delivering painful truths.A recurring example of tensions in our work relates to progress measures. National measures of progress designed to provide justification for programme resources are liable to clash with local priorities and circumstances. Participating local organisations may negatively perceive achieving these as requiring large amounts of resources in comparison to limited local benefits and driven by the need to satisfy reporting demands. Some agreement over a limited core set of measures to satisfy both local and national demands may be helpful.Takian and colleagues noted how the policy context changed in the course of a single long-term change programme . These fAlthough important, shifting socio-political environments only constitute part of the picture. A long-term view of nurturing evolving infrastructures highlights that visions of best practices will inevitably change over time . They alChanges in medical techniques and diagnosis, models for care delivery, and vendor offerings affect available technologies (and vice versa). The market may not immediately be able to respond to new policy-driven models, and therefore evaluations and policies need to consider these dimensions . This maLarge HIT change programmes are often concerned with not only stimulating local changes but also with promoting ongoing change ensuring that efforts are sustained and scaled beyond the life of the programme . But thiStudies of the emergence and evolution of information infrastructures have in turn helped articulate new strategies for promoting/sustaining such change –37. HoweEvaluators can explore success factors and barriers to scaling qualitatively and formatively feed these back to decision-makers who can then adjust their strategies accordingly. Evaluation needs to address local change in tandem with evolving networks at ecosystem level. By studying a range of adopter sites and their relationships with each other, as well as other stakeholders that are part of the developing ecosystem, evaluators can identify mechanisms that promote digital transformation and spread. Understanding these dynamics can also help decision makers focus strategy on achieving programme objectives. By addressing networks and relationships, evaluators can, for example, explore how knowledge spreads throughout the wider health and care ecosystem in which the change programme is embedded, and how stakeholders were motivated to exchange and trade knowledge .We are now entering an era that emphasises patient-centred care and data integration across primary, secondary and social care. This is linked to a shift from discrete technological changes to systemic long-term infrastructural change associated with large national/regional HIT change programmes. There are some attempts to characterise and study these changes including our own . HoweverWe now need new methods of programme management geared towards developing learning in ecosystems of adopters and vendors. These evolutionary perspectives also call for broader approaches to complex formative evaluations that can support the success of programmes and help to mitigate potential risks.Although there is no prescriptive way to conduct such work, we hope that this paper helps decision-makers to commission work that is well suited to the subject of study, and implementers embarking on the evaluative journey to navigate this complex landscape.


Volumetric Muscle Loss (VML) is associated with muscle loss function and often untreated and considered part of the natural sequelae of trauma. Various types of biomaterials with different physical and properties have been developed to treat VML. However, much work remains yet to be done before the scaffolds can pass from the bench to the bedside. The present review aims to provide a comprehensive summary of the latest developments in the construction and application of natural polymers-based tissue scaffolding for volumetric muscle injury. Here, the tissue engineering approaches for treating volumetric muscle loss injury are highlighted and recent advances in cell-based therapies using various sources of stem cells are elaborated in detail. An overview of different strategies of tissue scaffolding and their efficacy on skeletal muscle cells regeneration and migration are presented. Furthermore, the present paper discusses a wide range of natural polymers with a special focus on proteins and polysaccharides that are major components of the extracellular matrices. The natural polymers are biologically active and excellently promote cell adhesion and growth. These bio-characteristics justify natural polymers as one of the most attractive options for developing scaffolds for muscle cell regeneration. Skeletal muscle dysfunctions can be caused by acquired disorders of muscle , genetic , cachexia ), sarcopenia or injury ,2. SkeleAdvances in material engineering and known chemistry of synthetic materials including greater accessibility to control chemical modification have triggered the development of engineered polymer-based scaffolds for the use in various biomedical fields including treatment of VML. Synthetic polymers are an attractive class of materials in the biomedical fields, especially in tissue scaffolds, due to their useful traits and can be easily tailored to meet specific mechanical properties, porosity and degradation time according to their application requirements. In recent years, major classes of biodegradable polymers such as polyesters, polyurethane and polyamides are among the commonly used polymers in tissue engineering, due to the predictable and reproducible mechanical and physical properties such as tensile strength, elastic modulus and degradation rate. Moreover, synthetic polymers are well known for their enormous availability and can be easily mass-produced with uniform quality at a low cost. However, despite the excellent qualities offered by the synthetic polymers, concerns about the toxicity always hindered their employability as implants in the human body. The toxicity from implants degradation is always associated with the release of polymer degradation products and by-products in a local acidic environment at the implant site . The smaCompared with its synthetic counterparts, natural polymers are often components in the extracellular matrix (ECM), a macromolecular network present within cells. This makes natural polymer is mostly biocompatible with less inflammatory response, hence providing a conducive environment for cells proliferation. Attempts have been made to use scaffolds composed of natural polymers to promote the repair of VML injury by providing structural and biochemical frameworks. The natural polymer-based scaffold properties are expected to closely resemble those of the ECM macromolecular network that offers mechanical stability and structural integrity to tissue and organ. The inherent capacity for cell binding is exerted by the presence of the following molecular sequences: Polypeptide base—carrying specific protein structural motif such as RGD (arginine/Glycine/aspartic acid)  and polyThe present review provides a short overview of skeletal muscle structure and organization, volumetric muscle loss along with recent advances in tissue engineering scaffolds to treat the VML. The cell source and critical design criteria for developing scaffolds for In vitro cell expansion are highlighted. Finally, future direction and challenges in the field of severe skeletal muscle injury are examined.Skeletal muscle also known as voluntary muscle is a biological machine that converts the adenosine triphosphate (ATP), the source of energy used for force production and mechanical work . The humMajor traumatic injuries involved severe damage of skeletal muscle and peripheral nerves often impaired the function of myoneural junctions, chemical communication between a nerve fiber and a muscle cell . Upon skThe current gold standard of care for VML is typically based on surgical intervention with autologous muscle graft. This procedure involves a healthy muscle transplant from an unaffected site to restore the loss of impairment functions. When no adjacent muscle is available, autologous transplantation and neurorrhaphy in the form of free functional muscle transfer would be applied . HoweverCell based-therapies represent the most exciting and promising strategies to restore normal functions of damaged and injured tissues and organs . The strEmbryonic stem cells (ESCs) are stem cells derived from the undifferentiated inner cell mass of human embryos . ESCs arEthical tensions related to ESCs clinical translation have limited the use of embryonic stem cells as a cell-based therapy in regenerative medicine. This makes the mesenchymal stem cells (MSCs) an appealing choice in clinical translation as the MSCs are self-renewal, well-tolerated and have no ethical concerns . MesenchThe earliest work on the employment of MSCs isolated from bone marrow to treat transgenic mice’s injured muscle was first reported by Ferrari et al. . They foConsidering all the challenges posed by ESCs and adult MSCs, it is worth exploring the potential of fetal-derived stem cells obtained from the human amniotic fluid as an alternative for cell-based therapy. Amniotic fluid stem cells (AFSCs) can be isolated using immunoselection with antibodies specific for c-Kit . AFSCs r5 of iPSCs into mice models with muscle-induced-injury to study the efficacy of iPSCs migration in vivo. They had found that the iPSCs successfully migrated and differentiated into myofiber after intramuscular engraftment in immunodeficient mice in vivo. Moreover, upon optimizing the differentiation process, they have further discovered that the cells expressed fast major histocompatibility complex and skeletal muscle proteins and formed functional sarcomeres . As an artificial matrix, a scaffold act as a temporary guide or template for cell adhesion, growth and function. The desired engineered scaffolding should offer the following characteristics as summarized by Hoque et al. .BiocompIn this section, recent advances in skeletal muscle tissue scaffolding with the use of electrospun and hydrogels from natural polymers are highlighted.Over the past decade, advances in tissue engineering have sparked interest in biodegradable and biocompatible natural polymers for fabricating tissue scaffolding that mimics the functions of native ECM macromolecules both structure and functions. In this regard, collagen always the best choice as it is a major structural constituent of many tissues such as skin, bone, tendon, ligament and other connective tissues, and accounts for roughly one-third of total body protein . The majConsiderable effort has been made to produce electrospun collagen. Earlier work on collagen electrospinnability was first reported by Matthews et al. . They haAnother widely used natural polymer is gelatin, a natural biopolymer derived from collagen and has almost identical composition and biological properties as those of collagens . GelatinGelatin has many distinctive features that confer an advantage in tissue regeneration. However, without proper treatment the gelatin exhibits poor mechanical properties. Post-spinning treatment, such as crosslinking proved to improve water-resistant ability, thermo-mechanical properties and tensile strength. In crosslinking, the aqueous glutaraldehyde reacts to the hydroxyl and sulfide group from amino residues to form strong covalent bonds through intra and intermolecular bonding . Zhang eBesides collagen, fibrin is another fibrillar protein that is naturally involved in wound healing and tissue repair. For years, fibrin has been widely used as hemostatic agents and tissue sealants in various clinical applications including neuro, cardiac and liver surgery. Due to its angiogenic potential, fibrin has been the subject of many studies in the past few years, particularly in volumetric muscle loss injury repair. Recently, Guo et al.  have sucCellulose is a renewable polymer available abundantly in plant-based materials. It consists of a linear chain of glucose molecules with a degree polymerization up to ten thousand. The two monomers of anhydroglucose rings joined together by a covalent bond called β 1-4 glucosidic bond to form a linear configuration of the cellulose chain . The chaβ-(1-4)-N-acetyl-glucosamine) is the principal structural polysaccharide of arthropods (such as crabs and insects) and cell walls of fungi and yeast. Considering the amount of chitin produced annually in the world, it is the most abundant polymer after cellulose [Chitin, poly  and DMF. This system reached a pH of 11, compared to 13 of the NaOH/DMF system. The average nanofiber diameter of 39 ± 12 nm was obtained when 1.5% hyaluronic acid dissolved in 2:1 (v/v) NH4OH: DMF solutions. Besides basic solvents, an alternative acidic solvent system could also generate electrospun hyaluronic acid nanofibers. A system consists of formic acid, water and DMF was studied [Similarly, hyaluronic acid has also been reported to enhance repair in damaged nerve and skeletal muscle. The hyaluronic acid promotes the high release of myogenic regulatory factors that facilitate the functional repair of mammalian skeletal muscle in vivo. However, their potential in nanofiber electrospun has not been fully exploited. This is because hyaluronic acid shows poor processability during electrospinning due to polyelectrolyte nature that caused high solution viscosity and surface tension at very low polymer concentrations. To reduce the polyelectrolyte effect, Li et al.  have ele studied . As suggOver the past decades, hydrogels have revolutionized the biomedical field and have been employed in a wide variety of applications including in muscle regeneration especially after volumetric muscle loss. The hydrogel is a 3D network polymer that is made of crosslinked natural or synthetic materials that can swell yet maintained its mechanical integrity. The ability to swell under biological conditions and good mechanical properties from the crosslinking makes them an ideal candidate in tissue engineering and drug delivery. Hitherto, two techniques are commonly applied to muscle regeneration using hydrogels including injectable hydrogels and pre-formed hydrogels-based scaffolds. The latter technique is widely used to guide in vitro muscle tissue formation or to orchestrate in situ skeletal muscle regeneration . NaturalRecently, increasing efforts have been made to design and fabricate natural polymeric based hydrogels due to their similar structure to components in the body. Moreover, they elicit a limited inflammatory response and exhibit adequate biocompatibility for tissue regeneration . Among tOther than fibrin, gelatin is one of the most attractive protein-based candidates for consideration as biomaterial scaffolds in muscle tissue regeneration. Recently, Gattazzo et al.  has succThe exploration of self-healing hydrogel has gained immense attention especially by the exploitation of dynamic covalent bonds present in polysaccharides, such as imine bonds that can be prepared between amino and aldehyde groups under physiological conditions . ChitosaBiological scaffolds derived from de-cellularized tissues and organs are progressively applied in regenerative medicine and tissue engineering . The tecIn another work, Urciuolo et al.  have higA study by Svystonyuk et al.  revealedIn recent years, researchers have focused on mimicking the ultrastructure of native muscle tissue composed of highly oriented myofibers . This isKim et al.  present The field of volumetric muscle loss injury and repair is an exciting one. Many research groups are attempting to design new and successful strategies to repair and reconstruct the artificial muscle. The natural based scaffold has proven to be an extremely useful approach to regenerate muscle loss by promoting myoblast migration . DespiteIn vivo energy generation and internal charging by the natural body activity and the physiological environment has played a vital role in cell division, intracellular communication, neural activities, epithelial healing and mechanotransduction . Electro