The heterogeneity of sarcomas with regard to molecular genesis, histology, clinical characteristics, and response to treatment makes management of these rare yet diverse neoplasms particularly challenging. This review encompasses recent developments in sarcoma diagnostics and treatment, including cytotoxic, targeted, epigenetic, and immune therapy agents. In the past year, groups internationally explored the impact of adding mandatory molecular testing to histological diagnosis, reporting some changes in diagnosis and/or management; however, the impact on outcomes could not be adequately assessed. Transcriptome sequencing techniques have brought forward new diagnostic tools for identifying fusions and/or characterizing unclassified entities. Next-generation sequencing and advanced molecular techniques were also applied to identify potential targets for directed and epigenetic therapy, where preclinical studies reported results for agents active within the receptor tyrosine kinase, mTOR, Notch, Wnt, Hedgehog, Hsp90, and MDM2 signaling networks. At the level of clinical practice, modest developments were seen for some sarcoma subtypes in conventional chemotherapy and in therapies targeting the pathways activated by various receptor tyrosine kinases. In the burgeoning field of immune therapy, sarcoma work is in its infancy; however, elaborate protocols for immune stimulation are being explored, and checkpoint blockade agents advance from preclinical models to clinical studies. Sarcomas are a broad family of cancers that arise from cells of mesenchymal origin in virtually every tissue of the body, and they can differentiate along a number of tissue lineages, such as adipose, muscle, fibrous, cartilage, or bone. As such, the pathology of these neoplasms is extremely diverse, with over seventy described subtypes . HistoriThis histological and molecular heterogeneity makes sarcomas particularly difficult to diagnose, leading to debate surrounding the sufficiency of histological diagnosis versus the need for ancillary molecular diagnostics. Treatment has proven equally challenging, and research findings in one subtype often do not translate to others. These limitations are magnified within the context that sarcomas are among the rarest of cancer diagnoses, making research and trials more difficult. In the US, sarcomas represent 1% of new cancer diagnoses and of cancer-related deaths , though Sarcomas, as a group, are resistant to conventional cytotoxic chemotherapy, save for some successes with anthracycline-based therapy for rhabdomyosarcoma, Ewing sarcoma, and osteosarcoma . Late reNonetheless, the past year has seen advances in genomics-based sarcoma science and the publication in major journals of significant positive results from clinical trials. In this review, we aim to summarize recent developments in both diagnostics and treatment, including translational science and clinical trials in chemotherapy, targeted therapy, epigenetic therapy, and the burgeoning field of immune therapy. The scope of this review includes works published from late 2014 to early 2016.Multi-platform “omics” approaches were undertaken to elucidate comprehensive mutational landscapes for liposarcomas, epithelioid sarcoma, and rhabdomyosarcomas.Kanojia et al  used a cJamshidi et al  publisheShern et al  characteTo interrogate the need for and value of ancillary molecular diagnostics for sarcomas, Italiano et al  designedRegardless of the necessity for molecular testing to support certain diagnoses, detailed genetic analysis of a patient's tumor tissue can have important implications for targeted therapy. Studies at many institutions are applying next-generation sequencing strategies in an attempt to identify “actionable” mutations with some clinical significance to cancer, sarcoma centres included. Peds-MiOncoSeq  employedOutside of the clinic, several more studies searched for actionable/targetable and recurrent/driver mutations across sarcomas. Andersson et al  conducteReturning to the question of the necessity of comprehensive molecular analysis for the diagnosis and management of sarcomas, we conclude that the evidence at this point is not strong enough to support mandatory use of these expensive and work-intensive diagnostic tools. As of yet, there has been no significant clinical impact reported in terms of treatment response or patient survival. While comprehensive genomics may certainly prove useful in complex cases, it may be most practical for the use of these tools to remain at the discretion of the sarcoma subspecialty pathologist. In the setting of routine clinical practice, traditional morphological assessment  is likely sufficient for most sarcoma diagnoses.Next-generation sequencing of RNA opens the door to identify both known and novel fusion transcripts. Hofvander et al  applied This type of approach may be useful to assist in identifying molecular events that can be useful for the differential diagnosis of some challenging sarcoma categories, such as vascular tumors of bone. Epithelioid hemangioma of bone can be difficult to differentiate from other vascular neoplasms due to its highly variable histological presentation. Ijzendoorn et al  identifiRNA sequencing has also proven useful in defining new entities among otherwise unclassifiable sarcomas. Le Loarer et al  conducteWhile basic and translational research delve deeper into tumor biology to identify new strategies to target these multifaceted malignancies, conventional chemotherapy remains a mainstay in the treatment of a number of sarcomas. Indeed, this was the subject of many major findings over the past year.For patients with primary soft tissue sarcomas, surgery with or without radiotherapy can offer a cure, but nearly half of patients recur and eventually die, with an estimated median survival of 12 to 15 months . As a reEribulin is an analogue of the marine-derived compound halichondrin B that acts as an inhibitor of microtubule dynamics through binding to a single site on tubulin, thereby suppressing the stability and growth of microtubules ,33. EribThe role of eribulin was evaluated in a phase III trial that randomized patients with advanced leiomyosarcomas or liposarcomas to receive either eribulin or dacarbazine . EligiblTrabectedin is a marine-derived drug that has several anti-cancer mechanisms of action . It actsA phase III trial assessed the role of trabectedin versus dacarbazine in a similar cohort to that included in the aforementioned eribulin trial, again including metastatic leiomyosarcoma or liposarcoma patients who had received at least one prior systemic therapy in addition to anthracyclines . This trThe role of trabectedin as a modulator of the transcription of oncogenic fusion proteins was demonstrated in a phase II trial assessing the efficacy and safety of trabectedin as a second line (or later) therapy for patients with advanced translocation-associated sarcomas . PatientBeyond the established activity of trabectedin as second-line therapy for sarcomas, its role in the first-line setting was examined in a phase II single-arm clinical trial using a combination of trabectedin and doxorubicin . Among 1Aldoxorubicin is a novel pro-drug of doxorubicin, where a pH-sensitive linker conjugates doxorubicin to albumin. The exposure of the albumin-drug conjugate to the acidic tumor microenvironment releases doxorubicin, which preferentially localizes in tumor cells that are pinocytotically-active ,45. DrugSuperior clinical activity of aldoxorubicin was recently demonstrated in a phase IIb study that showed a significantly greater PFS (the primary endpoint), in patients with advanced soft tissue sarcoma randomized to receive aldoxorubicin or doxorubicin . HoweverGemcitabine and docetaxel combinations have previously displayed a proven activity, particularly in advanced leiomyosarcoma . These rBeyond its role in the metastatic setting, the gemcitabine-docetaxel combination was recently assessed in early-stage soft tissue sarcomas. A phase II trial compared the combination of gemcitabine-docetaxel against conventional therapy with doxorubicin-ifosfamide in patients with localized, resectable, high-risk sarcoma. While this trial showed that the gemcitabine-docetaxel combination was tolerable, it did not show a significant superiority of gemcitabine-docetaxel over doxorubicin-ifosfamide in hospitalization rate, which was  defined as the primary endpoint of this study .Further phase II trials have also investigated whether the activity of gemcitabine and docetaxel is enhanced by the addition of anti-angiogenic agents such as bevacizumab , particuBasic science studies have identified a number of targetable oncogenic pathways activated in specific sarcomas. Figure Evidence behind receptor tyrosine kinase inhibitors in sarcomas is comparatively advanced, with a number of agents in clinical trials. Aside from gastrointestinal stromal tumors, there is little known about biomarkers predictive for response. A phase II study of imatinib in 33 progressive, unresectable desmoid tumors  assessedIn pre-clinical studies of osteosarcoma, Liu et al  show synMalignant peripheral nerve sheath tumors (MPNSTs) have been shown to have receptor tyrosine kinase gene amplifications of PDGFR and cKIT ; howeverShang et al  observedIn a rhabdomyosarcoma study aiming to identify targetable proteins associated with alveolar rhabdomyosarcoma fusion oncoprotein , microarHedgehog pathway inhibitors show promise in osteosarcoma, through targeting of SMO  or GLI fHsp90 is a chaperone protein that assists in the folding of many of the signalling molecules mentioned above. In a study of undifferentiated pleomorphic sarcoma , Hsp90 wMDM2 is a suppressor of genome guardian p53, and its genetic amplification is a hallmark of well- and de-differentiated liposarcoma. Bill et al  comparedFLI1, the most common translocation partner of EWSR1 in the oncogenic fusion protein driving Ewing sarcoma, requires binding to transcriptional cofactor RNA Helicase A for full activity. YK-4-279, a small molecule that blocks the interaction between FLI1 and RNA helicase A, was tested in a Ewing sarcoma cell line xenograft  and induFOXM1 is a transcription factor - sometimes activated as part of the Wnt or Hedgehog pathways - that contributes to proliferation and cell cycle progression. It has been shown to be overexpressed in numerous sarcomas ,78, whicWhile significant advancements have been reported in clinical trials of cytotoxic chemotherapeutic agents, metastatic soft tissue sarcomas still have poor outcomes and few effective therapeutic options. Table Imatinib is a tyrosine kinase inhibitor that inhibits several oncogenic pathways. Its impressive activity in sarcomas has been primarily shown in GIST, by interrupting the constitutive activation of KIT-mediated signal transduction characteristic of that sarcoma –81. ImatThe role of imatinib as a PDGFR-β inhibitor was also tested among metastatic chordoma patients, a disease displaying PDGFR-β protein expression but not amplification or other activating mutation. In a recent retrospective analysis that included 46 PDGFR-β-positive metastatic chordoma cases, median PFS was 9.9 months. Within a median follow up of 24.5 months, 34 of 46 of patients had stable disease by RECIST 1.0, with no partial or complete responses observed . These rSorafenib, a multi-kinase inhibitor, has been shown to act through different signal transduction pathways, including via inhibition of pro-angiogenic vascular endothelial growth factor receptors (VEGFR and PDGFR-β). In phase II clinical trials, sorafenib has been shown to have activity in metastatic soft tissue sarcoma , specifiA recently published phase II trial evaluated the role of sorafenib as a PDGFR-β inhibitor in locally advanced and metastatic chordoma patients (n = 27) . In thisDasatinib is a multi-kinase inhibitor that targets several oncogenes. Its main activity in sarcoma is thought to be through inhibiting the c-SRC kinase pathway. Gene expression profiling has reported the c-SRC pathway to be highly expressed in leiomyosarcoma  and chonPazopanib is a multi-kinase inhibitor that has been shown to have activity in metastatic soft tissue sarcoma, primarily through the phase III PALETTE trial published in 2012, which randomized metastatic soft tissue sarcoma patients to receive either pazopanib or placebo. This trial demonstrated a significantly prolonged PFS by 3 months in pazopanib arm, although no significant differences were observed in OS . In a rePazopanib has also been reported to be active against desmoid-type fibromatosis , which iThe role of pazopanib in the neoadjuvant setting was also investigated in several studies, based on recent evidence suggesting a synergistic effect when combining radiotherapy with angiogenesis-targeted therapies that act through inhibiting the supplying vasculature for sarcoma cells . A recenDenosumab is a monoclonal antibody that targets the receptor activator of nuclear factor-kappa b ligand (RANKL), which is normally expressed on osteoblasts and functions to activate osteoclasts to control bone regeneration and remodeling. This agent has exhibited a particularly significant clinical benefit in giant cell tumors, which highly express RANKL –111. WhiEpigenetic and epigenomic modifications are emerging as key mechanisms in the pathogenesis of many sarcomas. Some subtypes could be described as exhibiting an “epigenomic mutator phenotype,” wherein a single missense mutation at an epigenetic modification target site, generates an “oncohistone,” resulting in aberrant repression and de-repression of genes at the global transcriptome level. An example of one such mutation, investigated by Lu et al , is seenIn 2014, MPNST saw major breakthroughs in understanding the epigenetic mechanisms by which somatic mutations bring about malignant transformation, which have since led to practical diagnostic advances. Lee et al  identifiIn rhabdomyosarcoma, the DNA methylome was characterized for 37 tumors and 10 cell lines , and PAXBromodomain and extra terminal domain (BET) proteins are “readers” of histone acetylation marks, facilitating the transcription of genes in marked areas. In cancer, BET proteins are key translators of aberrant acetylomes, and as such, BET inhibitors are emerging as promising treatments for some cancers, including bone sarcomas.In osteosarcoma, Lee et al  and BakeIn Ewing sarcoma, BET inhibition was shown to directly block transcription of the fusion oncoprotein in two independent studies ,127, whiEZH2 is the catalytic subunit of the PRC2 transcriptionally repressive chromatin remodeling complex. Lv et al  found thLaporte et al  employedIn pre-clinical studies, histone deacetylase (HDAC) inhibitors have been shown to be particularly active in translocation-associated sarcomas such as synovial sarcoma and Ewing sarcoma, through reversing aberrant transcriptional repression induced by the underlying fusion proteins in these sarcomas ,138,139.The immune microenvironment of sarcomas is poorly characterized to date, leaving open the question of which sarcoma subtypes are immunogenic. D’Angelo et al  conducteSmaller studies focusing on specific subtypes revealed broadly similar results. A study of 35 well- and de- differentiated liposarcomas  found TIHot tumors are those that are immunogenic, associated with high numbers of TILs and tumor associated macrophages, but that are actively modulating the immune response to survive, for example by expressing immune checkpoint ligands that suppress anti-tumor immune responses. Hot tumors are the most likely to benefit from immunomodulatory therapies such as checkpoint inhibitors.Lussier et al  showed tIpilimumab, an anti-CTLA-4 therapeutic monoclonal antibody, was tested in a phase I trial on 33 pediatric patients with refractory solid tumors, including 17 sarcomas  . At 3 weIn an abstract recently published for ASCO, Tawbi et al  presenteCold tumors are those that appear not to have been recognized by the immune system much - if at all - and theoretically would benefit best from stimulatory immune therapy such as cytokine treatment, immune cell engineering, and/or cancer vaccines.Yang et al  investigDendritic cell strategies involve removal of a patient's own immune cells, dendritic cell training with or without tumor antigen exposure/engineering, and re-introduction of the trained dendritic cells. In osteosarcoma, dendritic cells from an osteosarcoma rat model were electrically fused with an osteosarcoma cell line to generate a fusion tumor vaccine . This fuThe innate immune system's natural killer (NK) cells are newer targets for immune therapy interventions. NKG2D is an activating immune receptor on NK and cytotoxic T cells whose ligands are frequently present on tumor cell surfaces but rarely detectable on normal cells. Fernández et al  found moA phase II cancer vaccine trial for 29 sarcomas by Merchant et al  employedIn a phase I trial for alveolar soft part sarcoma and clear cell sarcoma by Goldberg et al , tumor cImmune therapy against specific tumor antigens may prove a particularly useful strategy in sarcomas, due to their comparatively low genomic complexity (and presumed low burden of tumor neoantigens). Chimeric antigen receptor T-cells (CAR-T-cells) are T-cells, usually autologous cells extracted from the patient receiving therapy, that have been engineered to target a specific tumor antigen. Ahmed et al  conducteDendritic cell vaccines can also serve to target specific tumor antigens, as seen in a phase I trial by Krishnadas et al  in childRecent publications have shown that significant, albeit incremental progress can still be made using cytotoxic chemotherapy agents, including eribulin, trabectedin, and aldoxorubicin. Efforts to conduct larger international studies have been somewhat successful, and there has been a partial move toward more histology-specific studies rather than the lumping together of completely disparate entities. Targeted therapies have shown fewer advances at the level of clinical practice, despite numerous trials involving various receptor tyrosine kinase inhibitors; however, advances in basic science, driven by spectacular technological advances in genomics, highlight targetable pathways, including mTOR, Notch, Wnt, Hedgehog, and MDM2, and other targetable proteins, such as Hsp90, RNA helicase, and FOXM1. Epigenetic approaches have had some preclinical success with BET and EZH2 inhibitors, and immuno-oncology approaches are advancing from pre-clinical to phase I- and II- level studies in both immunostimulatory and immune checkpoint therapies.


All living organisms need to adapt to ever changing adverse conditions in orderto survive. The phenomenon termed hormesis describes an evolutionarily conservedprocess by which a cell or an entire organism can be preconditioned, meaningthat previous exposure to low doses of an insult protects against a higher,normally harmful or lethal dose of the same stressor. Growing evidence suggeststhat hormesis is directly linked to an organism’s (or cell’s) capability to copewith pathological conditions such as aging and age-related diseases. Here, wecondense the conceptual and potentially therapeutic importance of hormesis byproviding a short overview of current evidence in favor of the cytoprotectiveimpact of hormesis, as well as of its underlying molecular mechanisms. In his masterpiece "The Twilight of the Idols", the German philosopherFriedrich Nietzsche stated: "What does not kill me, makes me stronger".Indeed, at least in specific cases, exposure to low amounts of a toxic substance orstressor may render an organism more resistant to higher doses of the same trigger. This adaptive response is known as hormesis (from the Greek"to set in motion"): the exposure to mild levels of harmful factorspreconditions a cell or an organism in that it stimulates the activation of stressresistance mechanisms, thus fostering the cellular capacity of maintenance and repair.Accordingly, in toxicology, hormesis defines a two-phase dose-response relationship, inwhich a low and a high dose trigger a stimulatory  and an inhibitory (toxic)effect, respectively 2O2, rendering cells more resistant tosubsequent exposure to higher H2O2 doses 2O2 concentrationsdepends on the formation of low levels of superoxide anions, which presumably drive anadaptive program The universal and deep-seated concept of hormesis can be exemplified in a variety ofdifferent experimental models. For example, treatment of bacteria with sub-lethalconcentrations of antibiotics not only provokes the surge of resistant populations, butalso induces a general stress response, such as increased biofilm formation thatcontributes to elevated adaptability C. elegans, for instance, moderate mitochondrial stress 13The involvement of mild oxidative stress in the hormetic response has spotlightedmitochondria as central control levers for hormesis, coining the term"mitohormesis" Mechanistically, hormesis seems to be executed by a variety of physiological cellularprocesses which (probably cooperatively) converge on enhanced stress resistance andlongevity . For example, mild heat stress in flies leads to increasedexpression of stress response proteins, including heat shock proteins that aredetectable after more than a week post-treatment 2021The relevance of hormesis for both human pathophysiology and specific disease treatmentis being increasingly recognized. As already mentioned, many anti-aging interventionsmay follow hormetic features, suggesting that preconditioning might have a preventivemedical character. Accordingly, mild dietary stress, i.e. calorie restriction withoutmalnutrition, as it can be achieved through different fasting regimens Both dietary factors and moderate exercise have been linked to improved brain healththrough hormesis 2728Besides these preventive features, hormesis has a direct clinical relevance. Forinstance, the ischemic preconditioning frequently used before heart surgery, where shortand mild cycles of ischemia are applied, protects the heart and brain against thesubsequent, more prolonged deprivation of oxygen and nutrients To sum up, hormesis defines a biphasic dose-response relationship that has a directimpact on preventive and clinical medicine. While the main molecular trait determiningthe beneficial character of low-dose stimulation for a cell or organism seems to berooted in the activation of stress resistance, the detailed mechanisms underlying thisphenomenon still need to be elucidated. In the same lines, the pleiotropic impact ofhormesis on the human body must be explored and evaluated in further detail.Irrespectively, accumulating evidence concedes a point to Nietzsche’s thoughts: a littlestress for some more stamina.


International migration to middle-income countries is increasing and its health consequences, in particular increasing transmission rates of tuberculosis (TB), deserve consideration. Migration and TB are a matter of concern in high-income countries and targeted screening of migrants for active and latent TB infection is a main strategy to manage risk and minimize transmission. In this paper, we discuss some aspects of TB control and migration in the context of middle-income countries, together with the prospect of responding with equitable and comprehensive policies.TB rates in middle-income countries remain disproportionally high among the poorest and most vulnerable groups in large cities where most migrant populations are concentrated. Policies that tackle migrant TB in high-income countries may be inadequate for middle-income countries because of their different socio-economic and cultural scenarios. Strategies to control TB in these settings must take into account the characteristics of middle-income countries and the complexity of TB as a disease of poverty. Intersectoral policies of social protection such as cash-transfer programs help reducing poverty and improving health in vulnerable populations. We address the development of new approaches to improve well-established strategies including contact tracing and active and latent TB screening as an ‘add on’ to the existing health care guidelines of conditional cash transfer programs. In addition, we discuss how it might improve health and welfare among both poor migrants and locally-born populations. Authorities from middle-income countries should recognise that migrants are a vulnerable social group and promote cooperation efforts between sending and receiving countries for mitigation of poverty and prevention of disease in this group.Middle-income countries have long sent migrants overseas. However, the influx of large migrant populations into their societies is relatively new and a growing phenomenon and it is time to set comprehensive goals to improve health among these communities. Conditional cash transfer policies with TB screening and strengthening of DOTS are some strategies that deserve attention. Reduction of social and health inequality among migrants should be incorporated into concerted actions to meet TB control targets. Tuberculosis (TB) rates have declined significantly in the 20th century worldwide, but HIV and multidrug-resistant tuberculosis (MDR-TB) have had drastic effects in some of the poorest countries , 2. TB bTB and TB/HIV coinfection partially reflects the income and development level of a country, with medium incidence rates at least 20 times higher in low-income countries (LIC) than in high-income countries (HIC) Fig.  1, 4]. , 4. 1, 4In HIC, migrants from high TB burden countries typically maintain higher active TB prevalences compared to the local population and, TB prevalence can reach at 3.3/1000 among migrants . It may Over the last 15 years, the economic development of Upper-middle income countries (UMIC), especially the BRICS  . The recMigration in middle-income countries is generally directed to major cities , 18. MigIn addition to strengthening Direct Observed Treatment, Short-Course (DOTS) strategy and Bacillus Calmette–Guérin (BCG) vaccination among migrants since the 1980s, many high-income countries with low TB burden have also incorporated active and latent TB screening. TB screening strategies directed to migrants are aimed to reduce TB burden and the risk of TB transmission: migrant screening for LTBI is estimated to prevent many new TB cases , potentiWhile some HIC have chosen compulsory screening for some types of migrants, others do not consider screening . The decThe strategies for TB control in migrant populations in middle-income countries are poorly discussed in the literature. TB control in middle-income countries is more complex due to multifaceted problems such as poverty, inequality and higher infectious disease rates in locally born populations. It is known that the prevalence of LTBI is likely to be higher in low- and middle-income countries than in high-income countries. In Brazil, active and latent TB screening is only suggested for contacts of TB patients, for HIV-infected individuals, the homeless and prisoners  . The estAnother issue is that targeting migrants may have a discriminatory effect. Moreover, irregular migrants may not respond to these policies for fear of detection and criminal conviction or expulsion . In ordeTB-related Millennium Development Goals have brought substantial improvements in implementation of free diagnosis and TB treatment . In BrazAlthough the proportion of cases of TB among migrants in middle-income countries is unknown, we estimate they account for a small proportion of cases. Few studies estimated the burden of TB among migrants in MIC, although migration has been increasingly important for disease control , 29–31, Free access to TB diagnosis and treatment has been implemented among migrants in many high-income countries, but access to health care can be especially difficult. Migrants face many social and cultural barriers to accessing treatment and sometimes they experience prejudice in health care settings . PolicieMDR-TB is currently a major challenge for TB control . MigratiIt is crucial for TB control to consider the characteristics of migrants in MIC, their interaction and social impact on host societies. Heterogeneity in TB incidence across different risk groups has been suggested as playing an important role in maintaining disease transmission, indicating that universal control measures are doomed to have limited impact if social inequalities are maintained , 34.Conditional cash transfer programs have been successfully implemented in several high-, middle- and low-income countries in the past decade. Although inclusion criteria and benefits vary, these programs are usually integrated into education and health care policies. For example, the Brazilian conditional cash transfer (CCT) program “Bolsa Família”  providesFew studies have focused on the impact of social protection policies on infectious disease burden, especially TB burden. Experimental provision of food baskets was associated to increased TB treatment completion and cure rates in Brazil . There iThere is lack of data about social protection policies among migrants, but we suggest that poor migrants should be offered what is offered to poor locally born people. In MercosulIn middle-income countries with marked social inequalities such as Brazil, control strategies would be more successful if they take into account the intricacies of TB, placing control policies within the broader context of health care and social equity. Despite differences between public policies to promote social and health equity in Brazil and the circumstances in other middle-income countries, we believe that social protection policies must incorporate TB control strategies and a migration-related component. Migrants’ sociodemographic aspects, TB morbidity and mortality and migratory patterns specific to each country must be taken into account.The United Nations post-2015 agenda established more ambitious health targets and called for intersectoral policies. Since TB is a disease of poverty, predominantly affecting people living in large cities, and recognizing the wide heterogeneity existing between middle-income countries and source countries of migrants, from labour migrants to refugees, it is key to establish TB control programs alongside intersectoral public policies that promote social equity. Conditional cash transfer programs integrated into active TB screening and strong contact tracing could have a positive impact on TB control among the poorest and most vulnerable populations.Cooperation and technology transfer among the BRICS and other low- and middle-income countries would also advance the control of neglected diseases . These cThese notable initiatives could be expanded to include more specific targets for TB control, as improving health care may not be sufficient to guarantee equity in access for migrants. Old and new TB control strategies may not have the desired impact unless there are initiatives for poverty reduction and early disease diagnosis among migrants. Moreover, because middle-income countries have limited health care resources, established health policies may be compromised if there is an increased demand due to migration and intersectoral policies are not planned.Middle-income countries and especially UMIC have been facing the same phenomena as seen in high-income countries in past decades: labour migration to large urban cities. Because of marked inequalities, big cities from middle-income countries must prepare to provide health care to incoming migrants, building on the reverse experience of being primarily a source of migration to more developed regions. In this context, intersectoral TB control strategies are needed to address social and health inequalities taking into account both migrants and locally born vulnerable groups in these countries. Conditional cash transfer policies may benefit poor migrants, especially during the first years after migration, and improve access to health care. Strengthening of DOTS and TB screening must also include migrants, especially irregular migrants that might not be supported by CCT programs. Finally, since migration flows largely take place within or between neighbouring regions , innovat


Innovative engineered nanomaterials are at the leading edge of rapidly emerging fields of nanobiotechnology and nanomedicine. Meticulous synthesis, unique physicochemical properties, manifestation of chemical or biological moieties on the surface of materials make engineered nanostructures suitable for a variety of biomedical applications. Besides, tailored nanomaterials exhibit entirely novel therapeutic applications with better functionality, sensitivity, efficiency and specificity due to their customized unique physicochemical and surface properties. Additionally, such designer made nanomaterials has potential to generate series of interactions with various biological entities including DNA, proteins, membranes, cells and organelles at nano-bio interface. These nano-bio interactions are driven by colloidal forces and predominantly depend on the dynamic physicochemical and surface properties of nanomaterials. Nevertheless, recent development and atomic scale tailoring of various physical, chemical and surface properties of nanomaterials is promising to dictate their interaction in anticipated manner with biological entities for biomedical applications. As a result, rationally designed nanomaterials are in extensive demand for bio-molecular detection and diagnostics, therapeutics, drug and gene delivery, fluorescent labelling, tissue engineering, biochemical sensing and other pharmaceuticals applications. However, toxicity and risk associated with engineered nanomaterials is rather unclear or not well understood; which is gaining considerable attention and the field of nanotoxicology is evolving promptly. Therefore, this review explores current knowledge of articulate engineering of nanomaterials for biomedical applications with special attention on potential toxicological perspectives. Nanostructures are engineered assemblies of materials with at least one dimension equivalent to 100 nm or less as defined by the National Nanotechnology Initiative (NNI). These nano scale materials are significantly important and increasingly being employed for commercial purposes in various sectors, wherein some of the advanced nanomaterials are at the leading edge of nascent fields of nanobiotechnology and nanomedicine . MateriaDue to the above stated unusual physicochemical belongings, nanomaterials differ considerably from the bulk material of the alike composition, permitting them to execute remarkable feats of better functionality, sensitivity, efficiency and specificity in terms of their therapeutic or biomedical applications , 8, 9. FIn the context of nanomedicine, a variety of materials have been utilized for their potential medical applications, wherein metallic nanoparticles have been proven the most convenient and suitable due to their unique optical, physical and electrical properties. These materials have found noteworthy applications in imaging, sensing, drug delivery and gene targeting. Numerous studies related to metallic particles such as gold and silver nanoparticles have been discussed in different sections of this review in terms of their application and toxicity , 23–31. 20–PPO69–PEO20) [poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide)] for their utility as a non-viral DNA delivery vector in cellular environment using Escherichia coli DH5α as a model microorganism. In this research, optimum weight ratio of 1:10 of plasmid DNA to copolymer P-123 was screened to achieve higher transformation efficiency. The schematic mechanism by which pDNA and copolymer P-123 nano-constructs release pDNA into the bacterium has been illustrated in Fig. Quantum dots (QDs) have also emerged as a novel class of fluorescent probe for in vivo biomolecular and cellular imaging due to their size-tuneable light emission, improved signal brightness, resistance toward photo-bleaching and simultaneous excitation of multiple fluorescence colours. Moreover, current research has led fabrication of multifunctional nano-probes that are highly bright and stable under different in vivo conditions. Additionally, polymer-encapsulated QDs have been prepared by encapsulating luminescent QDs with amphiphilic block copolymers and linking the polymer coating to tumor-targeting ligands and drug delivery functionalities. Interestingly, these materials have been found to be nontoxic to the cells and such conjugated QDs have raised new possibilities for ultrasensitive and multiplexed imaging of molecular targets in living cells, animal models and possibly in humans; however, their long-term in vivo toxicity and degradation need to be more carefully evaluated . In addiDespite of numerous potential biomedical applications, toxicological perspective of engineered nanomaterials is poorly understood or rather unclear, which is gaining considerable attention in terms of nanotoxicology. Although, nanotoxicology is in embryonic stage of its development; it is a vital part of nanomedicine and discusses interactions of engineered nanomaterials with biological systems or environment; wherein, particular emphasis is given on the correlations between the physicochemical and surface properties of nanomaterials with induction of toxic or adversarial biological responses. In addition to this, nanotoxicology aims to discover favourable physicochemical characteristics of various nanomaterials, which may render them more responsive toward inner biological environment for therapeutic benefits , 44. TheIn the context of biomedical applications of nanomaterials, it is vital to recognize that the concomitance of nanomaterials and biological entity may exert detrimental effects on biological systems , 51. TheIn terms of nanomaterials toxicity, three principles have been elucidated which are referred as transport principle, surface principle and materials principle. All these fundamental principles of materials toxicity need to be considered pragmatically for dictating specific interactions between nano objects and biological systems. Moreover, these three basic principles provide insight of each nanomaterial separately for their specific physicochemical property, which are imperative in creating adversarial biological effects .Therefore, to contrive nanomaterials for biomedical applications, it is imperative to rationally engineer nanomaterials with controlled physicochemical properties to dictate nano-bio interface toward desired interactions to achieve highest level of safety with better functionality, sensitivity, efficiency and specificity. Moreover, basic understanding of nano-bio interfacial interactions between engineered nanomaterials and biological objects will allow predictive relationships at the nano-bio interface. Such predictive interactions are essential for the perspective of further development of designing strategies and safe usage of nanomaterials –55. FromIn this perspective, a variety of nanomaterials have been rationally designed including engineered metallic nanoparticles, their alloys and oxides, super-paramagnetic oxide crystals, quantum dots, semiconductors, dendrimers, polymeric micelles, liposomes, aquasomes (carbohydrate-ceramic nanoparticles) and polyplexes/lipopolyplexes for their vast potential towards biomedicine application from diagnostics to treatment of disease , 56–60. Nanomaterials display exceptional physicochemical properties and can be exploited for various biomedical applications as illustrated in Fig. Moreover, different physicochemical belongings of nanomaterials have possibility to produce specific chemical atmosphere within the cells to encourage a pro-oxidant environment, initiating an imbalanced cellular energy system reliant on redox potential, thus leading to hostile biological consequences. Such hostile biological effects may range from the commencement of inflammatory pathways through to ultimate cell death . TherefoIn the context of biomedical applications of engineered nanomaterials, the foremost distinctive feature is their size, which fall in-between individual atoms or molecules and corresponding bulk material. The reduced size of nanomaterials will not only provide an opportunity for increased uptake but also will build chances to interact with biological tissues to a greater magnitude to achieve desired type of selective biological action for therapeutic purposes . FurtherAdditionally, nanomaterials are anticipated to cross biological obstacles, gaining entrance to the body and subsequently nano size may govern their kinetics, absorption, distribution, metabolism and excretion that would not be possible otherwise with the bulk material of akin composition , 66. WelLikewise, theoretical traits propose that reduced particles size will have higher surface area, which may possibly promote interactions between the nanomaterials and the surface of biological entities, which may influence living organism adversely. In this perspective, size dependent toxicological consequences of various nanomaterials have been established by employing silver nanoparticles, palladium nanoparticles, single-walled carbon nanotubes and multi-walled carbon nanotubes toward bacterial cells. These studies show that smaller sized particles directly interact with bacterial cells leading to antagonistic effects confirming size-dependent toxicity –71. MoreIn addition to size and type of material (composition), shape or morphology of a nanomaterial is another important characteristic feature at nanoscale level. However, only few investigations are focusing on the biomedical or toxicological relationship associated with this parameter alone. Nevertheless, in metallic nanomaterials various properties including electromagnetic, optical and catalytic properties are strongly influenced by their shapes –75; consEscherichia coli. Furthermore, this study proposed that nano range size and the existence of (111) lattice plane combine to encourage antibacterial potential and nanoparticles commenced shape-dependent interaction with bacterial cells intracellular by increasing Ca2+ influx and inducing release of intracellular Ca2+ stores via endoplasmic reticulum through IP3 receptor channels. All these variations can result in higher apoptosis and lower cell proliferation, subjected to cell type. Added modulation of cell apoptosis and proliferation may involve direct nanoparticle effects on intracellular signalling mechanisms [Moreover, critical role of surface charge of gold nanoparticles in modulating membrane potential of different malignant and non-malignant cell types followed by their downstream intracellular events was established in recent times; which revealed a novel mechanism for cell-nanoparticle interactions and gold nanoparticles uptake. Positively charged gold nanoparticles were taken-up intracellularly based on the membrane potential and generate membrane depolarization. This action improved [Cachanisms . All sucIn addition to various inherent physicochemical characteristics as discussed in previous sections, external influences may also have noteworthy consequence on biomedical or toxicological belongings of nanomaterials. For example, in the presence of light, photosensitive nanomaterials will be capable of producing higher amount of reactive oxygen species (ROS) which will have considerable toxicological effects. In this context, three photosensitive nanomaterials, titanium dioxide, silicon dioxide and zinc oxide have been assessed to possess toxicity with varying degrees toward Gram positive and Gram negative bacterial strains in water suspensions by particle concentration dependent manner. This research claims that the stimulus of light had significant influence under most of the examined experimental conditions which is possibly related with its role in motivating production of ROS .Furthermore, purity of any nanomaterial is one of the most important characteristic, which need to be considered for its biomedical or toxicological role. However, it has often been neglected, which needs to be considered for its active role in therapeutics or otherwise. Existence of residual contaminating foreign metals, unreduced metal ions, chemicals or other agents  may actually be responsible for noxious actions rather than the actual nanomaterials itself and the quantity of contaminating materials are fully dependent upon the synthesis method used. Currently, numerous post-production nanomaterials processing methods are known to remove most of these precursor metal catalysts and chemical agents from nanomaterials; however, such purified nanomaterials may still have some amount of remaining substances. Therefore, the effects of such chemical impurities, residual metals and presence of counter ions on both potential biomedical and deleterious effects cannot be overlooked. Hydrophobicity/hydrophilicity, electron transfer capability, surface smoothness/roughness/defects, oxidizability of nanomaterials in physiological conditions and counter ion effects are other essential physicochemical parameters of diverse nano objects that need to be considered to control their toxic potential while engineering nanomaterials for their biomedical applications. For instance, one of the recent studies used multiparametric methodology to understand high-content imaging coupled with gene expression analysis on fundamental pathways for evaluating cell-nanomaterial interactions. By employing this approach the effect of the surface charge and hydrophobicity of gold nanoparticles on cell-material interactions were parametrically evaluated followed by their validation through biochemical assays. Interestingly, the data evidently divulge that while surface hydrophobicity of nanomaterial does not essentially affect cellular uptake levels, nevertheless increased surface hydrophobicity was found to be associated with higher cell membrane damage and induction of autophagy, which had greater influence than the effect of surface charge ranging between −50 and +20 mV . In anotNanotechnology has significant potential to influence field of biology and medicine due to nanoscale size of basic biological entities and it is gaining considerable attention in terms of nanomedicine. However, toxicological perspectives of engineered nanomaterials are poorly understood or rather unclear, which is limiting full potential of nanomedicine. Therefore, the often ignored toxicological concerns of engineered nanomaterials need urgent attention and it is essential to carry out fundamental research to address these issues. Moreover, the future of nanomedicine will depend on rational engineering of various nanomaterials with controlled physicochemical properties to dictate their interactions in anticipated manner with biological systems for biomedical applications. Additionally, detailed and thorough understanding of nano-bio interactions will be required to discover favourable physicochemical characteristics of various nanomaterials, which may render them more responsive toward inner biological environment for therapeutic benefits without any toxic impact.


Neurological diseases, including dementias such as Alzheimer's disease (AD) and fronto-temporal dementia (FTD) and degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS), are responsible for an increasing fraction of worldwide fatalities. Researching these heterogeneous diseases requires models that endogenously express the full array of genetic and epigenetic factors which may influence disease development in both familial and sporadic patients. Here, we discuss the two primary methods of developing patient-derived neurons and glia to model neurodegenerative disease: reprogramming somatic cells into induced pluripotent stem cells (iPSCs), which are differentiated into neurons or glial cells, or directly converting (DC) somatic cells into neurons (iNeurons) or glial cells. Distinct differentiation techniques for both models result in a variety of neuronal and glial cell types, which have been successful in displaying unique hallmarks of a variety of neurological diseases. Yield, length of differentiation, ease of genetic manipulation, expression of cell-specific markers, and recapitulation of disease pathogenesis are presented as determining factors in how these methods may be used separately or together to ascertain mechanisms of disease and identify therapeutics for distinct patient populations or for specific individuals in personalized medicine projects. Despite recent, rapid advancement in our understanding of biology and genetics, neurological disease remains a persistent and fatal threat to human health. The World Health Organization (WHO) estimates that the fraction of worldwide deaths attributable to neurological conditions increased 89% from 2000 to 2015 WHO, . Alzheimin vitro models.Staining of post-mortem tissue remains the only means of officially diagnosing several neurological diseases, and studying the mechanisms behind defining proteinopathies is difficult in this static state. The postmitotic nature of neurons and difficulty of tissue access make biopsies of the CNS infeasible, and the blood-brain barrier isolates many biochemical signals of disease progression from peripheral biofluids. Thus, in order to properly study pre-symptomatic disease or disease progression mechanisms in patients, we must rely on human For genetically inherited disease, where a specific causal mutation has been identified, cellular models of disease can be created by genetic manipulations  of cells lines or primary rodent cell cultures. Alternatively, primary rodent cell cultures can be generated from existing animal models of disease . These approaches do come with limitations. For example, overexpression models can simultaneously mask subtle disease phenotypes while falsely exaggerating or creating entirely non-biological pathology. While these models may be used for early studies into disease mechanisms, true biological representation, especially for potential therapeutic development, requires endogenous expression of known genetic mutations and their genetic interactors, which emphasizes the need for patient-derived models.Patient-derived models become even more necessary for studying sporadic disease, as there is no way of definitively recapitulating the disease in a non-diseased organism. The genetic and epigenetic heterogeneity of sporadic patients may make specific causative mutations statistically undetectable, or a confluence of small changes may be required to cause disease phenotypes. Such complexity would be impossible to recapitulate through genetic manipulation of a non-diseased cell, even if the full spectrum of contributory expression was identified. Since sporadic patients make up the majority of patients in diseases such as ALS, Frontotemporal Dementia (FTD), and AD  and then differentiated into the desired cell type or directly converted (DC) into the desired cell type Figure . Each mein vitro models from adult patients. Experiments which showed that ESC fusion could confer pluripotency to fibroblasts suggested that certain cellular products might induce pluripotency even in an adult somatic cell   capable of converting adult mouse  translated dipeptide repeat proteins (DPRs) in C9 ALS . These have been used primarily for the development of therapeutic neuron replacement  are generated by treatment with retinoic acid and sonic hedgehog (SHH), which is required for floor-plate differentiation  in therapeutic transplantation, mature iPSC-derived oligodendrocytes have been generated. Early reports suggested that iPSCs might contain some inherent factor which prevented oligodendrocyte maturation   , has been examined extensively in patient-derived iPSCs, and these studies been thoroughly reviewed  to understand whether, even at a pre-neuronal stage of development, human cells show AD phenotypes -E693delta mutation displayed ER and oxidative stress, which was alleviated with treatment of docosahexaenoic acid (DHA)  human cell culture systems   has been extensively modeled using patient-derived iPSCs , focuses on differentiating healthy oligodendrocyte precursors for transplantation replacement therapy , DLX1, DLX2, and MYT1L or ISL1 and LHX3, respectively  inhibiting the polypyrimidine-tract binding (PTB) protein has been shown to be sufficient for transdifferentiation . Small molecules have been shown to increase efficiency of conversions when combined with transcription factors  can be generated via expression of pluripotency factors in possible combination with small molecules  , a causative ALS gene  or PSEN1  showed higher extracellular levels of Aβ40 and Aβ42 when compared to controls. iNs from AD patients also showed an increase in total tau and phosphorylated tau  patient fibroblasts converted into hiMNs saw reduction in neurite growth rate and, after 60 days Direct conversion cells have demonstrated the ability to mimic disease pathology and physiology in a number of neurological disorders Figure . What haPatient-derived CNS models have expanded rapidly in the past decade. A variety of adult somatic cells can be reprogrammed into iPSC's and differentiated or directly converted into the cells necessary to model disease, test novel drug candidates, and be used for cell transplantations. As researchers today design new studies of familial and sporadic neurological conditions, they must carefully consider which model and method most efficiently answers their scientific question Figure .Patient-derived CNS models are time intensive. While direct conversion decreases the time to generate neurons from somatic sample collection due to the lack of reprogramming, the time it takes to differentiate and mature functional neurons from fibroblasts or iPSCs is similar. iPSC-based models offer more efficient production of cells than direct conversion because cells are infinitely expandable in both the iPSC stage and the NPC stage. While the yield of the desired cell type  is high, sub type  yield can vary widely between different small molecule and growth factor-based differentiation methods. Virally-expressed transcription factors may increase specific cell subtype yield in iPSC differentiations. While fibroblasts used for direct conversion are infinitely expandable, the lack of an intermediate progenitor stage creates a maximum one-to-one ratio of starting fibroblasts to desired cell type. In addition, there are reports of desired neuronal subtype yields of fewer than 10%  in iNeurons and iPSNs have demonstrated the loss of aging signatures in reprogramming (Mertens et al., The clonal nature of iPSCs allows for the generation of CRISPR-Cas9 correction of disease-causing mutations, which can help establish whether certain pathologies observed in the model are attributable to the mutation and provides an isogenic control. This technique is not currently available in direct conversion; however, there may be some advantage to generating model neurons from a genetically and epigenetically heterogeneous population of somatic cells rather than from copies of a single iPSC clone, especially given the heterogeneity of disease pathology between cells in patient tissue.Both direct conversion and iPSC differentiation have produced glia and several neuronal subtypes. Specific differentiation methods, including transcription factor choice, small molecule and growth factor choice, and plating methods have been and will continue to be adjusted to create a higher yield of desired cell types in less time. While, due to its earlier discovery, iPSC differentiation technology is currently better explored and developed, it is likely that iNeurons and other direct conversion cell types will be equally fine-tuned in the near future. With either method, care should be taken in regards to the true identity of generated cell types, given the extensive cellular conversion these cells undergo. Genetic and proteomic analyses of differentiated or converted cell types, along with comparisons to postmortem cell-type specific analyses, can address some of these concerns.in vitro human cell culture models, they are capable of recreating disease-specific pathologies. In addition, new disease pathways have been discovered using patient-derived cell culture models which were then validated in postmortem patient tissue, such as the presence of nucleocytoplasmic transport deficits and nuclear pore dysfunction in ALS, FTD, HD and, likely, in other neurodegenerative diseases. It is therefore likely that other novel disease mechanisms will be revealed using these human cell culture models.The extensive reports of disease pathologies and pathogenesis summarized in this review suggest that, despite the limitations of Patient-derived cells offer hope for an endogenous, human model of neurological disease, especially for sporadic cases which cannot be modeled any other way, and provide a platform for rapid therapeutic discoveries that are both personalized and clinically translatable.LG, AS, AN, and RS: conceptualized and wrote the manuscript.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.


GARS, encoding the ubiquitous enzyme glycyl-tRNA synthetase (GlyRS), cause peripheral nerve degeneration and Charcot-Marie-Tooth disease type 2D (CMT2D). This genetic disorder exemplifies a recurring paradigm in neurodegeneration, in which mutations in essential genes cause selective degeneration of the nervous system. Recent evidence suggests that the mechanism underlying CMT2D involves extracellular neomorphic binding of mutant GlyRS to neuronally-expressed proteins. Consistent with this, our previous studies indicate a non-cell autonomous mechanism, whereby mutant GlyRS is secreted and interacts with the neuromuscular junction (NMJ). In this Drosophila model for CMT2D, we have previously shown that mutant gars expression decreases viability and larval motor function, and causes a concurrent build-up of mutant GlyRS at the larval neuromuscular presynapse. Here, we report additional phenotypes that closely mimic the axonal branching defects of Drosophila plexin transmembrane receptor mutants, implying interference of plexin signaling in gars mutants. Individual dosage reduction of two Drosophila Plexins, plexin A (plexA) and B (plexB) enhances and represses the viability and larval motor defects caused by mutant GlyRS, respectively. However, we find plexB levels, but not plexA levels, modify mutant GlyRS association with the presynaptic membrane. Furthermore, increasing availability of the plexB ligand, Semaphorin-2a (Sema2a), alleviates the pathology and the build-up of mutant GlyRS, suggesting competition for plexB binding may be occurring between these two ligands. This toxic gain-of-function and subversion of neurodevelopmental processes indicate that signaling pathways governing axonal guidance could be integral to neuropathology and may underlie the non-cell autonomous CMT2D mechanism.Dominant mutations in  GARS, cause Charcot-Marie-Tooth disease type 2D  genes linked to CMT  gene, GARS have been shown to cause a similar conformational change to GlyRS, leading to a structure that can bind to erroneous targets  including neurotransmission , suppresses mutant GlyRS synaptic binding and associated neuropathology. This reveals an important role for plexins in mutant GlyRS pathology and suggests a possible mechanism by which mutant GlyRS can exert non-cell autonomous effects.As mentioned above, we now report that mutant GlyRS expression mimics the axonal branching defects generated by loss-of-function mutations in the two fly plexins, Drosophila stocks were cultured on standard molasses/maize meal and agar medium in plastic vials or bottles at 20 or 25°C. The garsP234KY and garswt stocks have been previously described  agar plate and counting the forward body wall contractions exhibited in 2 min. Twenty flies per genotype were scored.Adult viability assays were conducted by crossing Larvae were reared, dissected and processed as previously described with analysis of hemisegment A2 , as per the manufacturer’s instructions, and reverse transcribed as published previously , ectopic synaptic contacts from both the TN , nor when wild-type gars is ubiquitously expressed using the same driver , suggesting that these defects are not general to all Drosophila models of motor neuron degeneration.We now report a novel CMT2D phenotype of defective synaptic wiring in the r Figure . This inplexA , phenocopies the wiring defects of both plexA and plexB loss-of-function mutants  and plexB (plexBKG00878) to flies ubiquitously expressing garsP234KY, and re-scored the number of ectopic contacts occurring between the TN and muscles 6 and 7. We found that heterozygous reduction of both plexA and plexB enhanced the ectopic branching defects observed in mutant GlyRS flies , suggesting that the enhancements are specific to the CMT2D model.A recent study has shown that mutated GlyRS binds to the membrane-bound protein Nrp1 . The RNA of both genes was detected in the larval central nervous system, and the adult brain and ventral nerve cord , suggesting that both plexins function throughout life.Mutant GlyRS causes both larval and adult phenotypes in the fly . Ubiquitous expression of garsP234KY significantly reduced adult viability, with only 7% surviving to the adult stage. We found plexA and plexB reduction had opposing effects on the adult viability defect of garsP234KY flies: reduction of plexA compounded the reduced viability , whilst diminishing plexB levels significantly improved survival . Alternatively, increasing plexA had no significant effect, while plexB overexpression reciprocally exacerbated the garsP234KY viability phenotype, with no flies (0%) making it to the late pupal stage  decrease in number (~20 contractions/min are observed in the mutant compared to ~39 mean in controls). The defects in muscle function of mutant gars-expressing flies were worsened in plexA heterozygotes (~10 contractions/min) and marginally improved in plexA overexpressors (~27 contractions/min). PlexB reduction improved contraction frequency (~28 contractions/min) and plexB overexpression exacerbated it (~4 contractions/min). It should be noted that reduced PlexB levels enhanced the wiring defect . Concordant with the differential effects on viability , while plexB overexpression enhanced Brp accumulation , suggestive of a reciprocal pattern associated with down- and up-regulation of Plexin B. No significant alterations were observed with plexA reduction or overexpression, suggesting that the synaptic accumulation of mutant GlyRS is observably modified by Plexin B alone. The expression patterns of plexins, are not well understood, due in part to a lack of reliable antibodies. We therefore overexpressed myc-tagged Plexin B and found that it localized to the presynaptic neuron , while PlexB is the main receptor for the secreted semaphorin Sema2a . It is therefore possible that plexA modification of the mutant GlyRS phenotype, and therefore the amelioration caused by Sema2A overexpression, is at least partially mediated through increased PlexB levels.In stark contrast, Sema2a overexpression ameliorated the GlyRSt Figure  and abert Figure . Moreovet Figure . SurprisDrosophila model of CMT2D can be modified by alterations in plexin-semaphorin signaling. Drosophila possess two plexins, PlexA and PlexB, which differentially bind semaphorins to provide cell-cell communication that drives axon repulsive and attractive cues. We show that mutant GlyRS expression leads to ectopic axonal branches in the peripheral nervous system, and this phenotype is modifiable by altering the levels of the Drosophila plexins, PlexA and PlexB. We then show that heterozygous reduction of PlexB ameliorates the lethality and neuropathology caused by garsP234KY expression, while PlexA reduction exacerbates the same phenotypes. In addition, PlexB, but not PlexA, appears to be important for the association of mutant GlyRS with the neuromuscular synaptic membrane. Furthermore, we found that increasing expression of the secreted PlexB ligand, Sema2a, but not the PlexA ligand, Sema1a, can also supress the mutant GlyRS binding and motor defects. Finally, we show that the fluctuations in mutant GlyRS build-up at the NMJ, caused by manipulation of plexin-semaphorin components, correlates significantly with the pathological features identified in the CMT2D model (Figures We have shown that key pathologies in a  Figures . Taken tDrosophila indicate that mutant GlyRS interferes with the fidelity of axon guidance pathways that are partly governed by both PlexA and PlexB signaling. As mutant GlyRS expression both phenocopies and modifies the plexA and plexB axonal branching defects, we suggest that the ectopic branching phenotype arises from mutant GlyRS interaction and interference with canonical plexin signaling. Importantly, however, whilst we found opposing effects of PlexA and PlexB on toxicity phenotypes and associated pathology, the same neurodevelopment defects were exacerbated with reduction of either plexin. Hence, the data suggest that axonal guidance defects are de-coupled from the neurodegenerative pathology, mirroring the developmental switching in sensory neuron identity observed in Gars mice that appears to be independent from the progressive peripheral nerve degeneration (Sleigh et al., In both invertebrates and vertebrates, plexin signaling drives the attractive and repulsive signals required for high-fidelity nerve-nerve, nerve-muscle and nerve-glia interactions (Winberg et al., Drosophila PlexA has been shown to drive repulsive axon guidance cues in the embryo, whilst PlexB can initiate either repulsive or attractive cell-cell interactions depending on the cellular environment (Winberg et al., Sema2a in larval muscle suppresses the motor and neuronal phenotypes observed when mutant GlyRS is expressed. In contrast, there is no change in the phenotypic readouts when the class 1 semaphorin, Sema1A, is overexpressed. Accordingly, PlexB reduction, unlike PlexA, was able to partly rescue the mutant GlyRS lethality. We thus propose that PlexB is required for the build-up of mutant GlyRS at the NMJ, and ultimately the toxicity caused by expression of garsP234KY. Mutant GlyRS could be competing with Sema2A binding at the larval NMJ, and interfere with other membrane-bound or secreted factors associated with different neuronal cell types, or at different developmental time points. It is likely that the plexB heterozygote fly did not produce a complete rescue of mutant GlyRS because plexB is still expressed, albeit at a reduced level. Consistent with a role for PlexB-mutant GlyRS binding in synaptic accumulation, the overexpression of Sema2A, particularly in the reduced plexB fly, led to further reduction in GlyRS build-up and an additive improvement in disease phenotypes, presumably by out-competing mutant GlyRS.In this study, we use the larval NMJ to examine the interaction between the plexin-semaphorin components and mutant GlyRS toxicity. However, unravelling the complete contribution of plexin-semaphorin signaling to CMT2D, in both the peripheral and central nervous systems, is made difficult by the complexity of the temporally and spatially diverse functions mediated through both plexin types (Neufeld et al., Within the cell, GlyRS charges glycine to its cognate tRNA; however, in a non-canonical tumur-defense capacity, GlyRS also binds to the extracellular receptor protein K-cadherin (Park et al., gars phenotype amelioration conferred by Sema2A overexpression, suggesting that PlexA may act independently on mutant GlyRS toxicity at the NMJ, and downstream of PlexB. Beyond this, as multiple semophorin-plexin interactions control nerve-nerve, nerve-muscle, and nerve-glia communication (Ayoob et al., We have suggested that Plexin B may either be an anomalous target for mutant GlyRS, or an indirect modifier of the interactions that drive the association of mutant GlyRS with the presynaptic membrane. Our data also suggest that PlexA may further modify the phenotypes indirectly through its physiological antagonism of PlexB-driven processes. Interactions of this nature have been noted previously; for example, PlexA has been shown to partially rescue the PlexB mutant, but PlexB did not reciprocally rescue PlexA mutants (Ayoob et al., In summary, we have shown that mutants GlyRS expression interferes with plexin-mediated axonal branching, and that the alteration of plexin-semaphorin signaling can modify mutant GlyRS lethality, neuromuscular dysfunction, and neuropathology. This toxic gain-of-function, and alteration of neurodevelopmental pathways, highlights mechanisms analogous to a mammalian model of the disease. Our findings provide additional evidence of a new disease paradigm in CMT2D, which provides further explanation for the selective effect on the nervous system of mutations in a widely expressed protein.SJG, JNS and MZC designed the experiments and wrote the article. SJG and JNS performed the experiments and analyzed the data. All authors have approved submission of this work.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.


Critically ill patients frequently experience severe agitation placing them at risk of harm. Physical restraint is common in intensive care units (ICUs) for clinician concerns about safety. However, physical restraint may not prevent medical device removal and has been associated with negative physical and psychological consequences. While professional society guidelines, legislation, and accreditation standards recommend physical restraint minimization, guidelines for critically ill patients are over a decade old, with recommendations that are non-specific. Our systematic review will synthesize evidence on physical restraint in critically ill adults with the primary objective of identifying effective minimization strategies.Two authors will independently search from inception to July 2016 the following: Ovid MEDLINE, CINAHL, Embase, Web of Science, Cochrane Library, PROSPERO, Joanna Briggs Institute, grey literature, professional society websites, and the International Clinical Trials Registry Platform. We will include quantitative and qualitative study designs, clinical practice guidelines, policy documents, and professional society recommendations relevant to physical restraint of critically ill adults. Authors will independently perform data extraction in duplicate and complete risk of bias and quality assessment using recommended tools. We will assess evidence quality for quantitative studies using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach and for qualitative studies using the Confidence in the Evidence from Reviews of Qualitative Research  guidelines. Outcomes of interest include (1) efficacy/effectiveness of physical restraint minimization strategies; (2) adverse events  and associated benefits including harm prevention; (3) ICU outcomes ; (4) prevalence, incidence, patterns of use including patient and treatment characteristics and chemical restraint; (5) barriers and facilitators to minimization; (6) patient, family, and healthcare professional perspectives; (7) professional society-endorsed recommendations; and (8) evidence gaps and research priorities.We will use our systematic review findings to produce updated guidelines on physical restraint use for critically ill adults and to develop a professional society-endorsed position statement. This will foster patient and clinician safety by providing clinicians, administrators, and policy makers with a tool to promote minimal and safe use of physical restraint for critically ill adults.CRD42015027860PROSPERO The online version of this article (doi:10.1186/s13643-016-0372-8) contains supplementary material, which is available to authorized users. Physical restraint is highly prevalent in the critically ill, with up to 75% of mechanically ventilated adults having physical restraints applied at least once during their intensive care unit (ICU) admission . AnnuallIn the ICU, physical restraint is frequently used for clinician concerns for patient and/or clinician safety including prevention of accidental removal of medical devices  . HoweverDespite the intention to promote patient and clinician safety, physical restraint can adversely impact patient safety resulting in detrimental physical and psychological consequences –22. Negaall advocate for minimization of physical restraint across all healthcare settings [Given the recognized adverse physical and psychological consequences of physical restraint and the lack of efficacy for preventing device removal, government legislation, hospital accreditation standards, and professional society guidelines settings , 25, 26.The identification of effective physical restraint minimization strategies is an immediate clinical and research imperative and has implications in terms of patient- and family-reported experience and outcome measures. To address this need, we will conduct a systematic review with the overall objective of producing an updated practice guideline on physical restraint for critically ill adults and developing a professional society-endorsed position statement. Our objectives include synthesizing quantitative studies of the (1) efficacy and effectiveness of physical restraint minimization strategies; (2) adverse events  and associated benefits including harm prevention; (3) ICU outcomes ; and (4) prevalence, incidence, and patterns of use including patient and treatment characteristics and chemical restraint. Additionally, we will synthesize quantitative and qualitative evidence on barriers and facilitators to minimization and patient, family member, and ICU healthcare professional perspectives and identify professional society-endorsed recommendations relevant to ICU patients and evidence gaps and research priorities.This review protocol was prepared using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocol (PRISMA-P) guidelines . We comphttp://apps.who.int/trialsearch website. We will search major guideline sites  and websites of relevant professional societies and conduct general searches using tools such as Google Scholar, ScienceWatch, and MSN. We will examine reference lists of relevant studies and will contact corresponding authors for additional published or unpublished work.We created a preliminary search strategy  and keywords . Vocabulary and syntax will be adjusted across databases. We will remove animal-only studies and opinion pieces . We will not impose language restrictions in our database searches but will only seek to obtain policy documents written in English. As we are including a range of study designs, we will not apply study-specific filters.We will include a range of study designs, including qualitative studies describing perspectives/restraint experience as well as clinical practice guidelines, policy documents, and professional society recommendations relevant to critically ill adults. To determine the efficacy/effectiveness and safety of physical restraint minimization strategies, we will include parallel group randomized controlled trials including crossover trials, quasi-randomized trials , and non-randomized studies including non-randomized controlled trials; controlled before and after studies; historically controlled studies; and retrospective or prospective cohort studies that include a control group. Inclusion of evidence from qualitative studies that explore the experience of patients and family members receiving interventions and healthcare providers delivering the intervention, as well as studies that evaluate barriers and facilitators to implementation of interventions, have an important role in maximizing the value of a systematic review to inform clinical practice, policy, and decision-making –32.Our population of interest is adults admitted to a high intensity care setting including ICUs, specialized weaning centres, and high-dependency units. We will include all high intensity care setting patient populations including those admitted for medical, surgical, neurological, cardiac, and trauma diagnoses. We will exclude studies of physical restraint use in patients admitted to other acute care settings such as the emergency department, postoperative recovery units, hospital floors/wards, psychiatry, and in long-term care settings.For studies examining the efficacy and safety of physical restraint minimization, interventions will include any strategy used for the purpose of decreasing the frequency and duration of physical restraint. Such strategies may comprise pharmacological and non-pharmacological interventions used to alleviate anxiety and optimize analgesia and alternatives to ensure patient safety such as sitters or observers. Patients may receive chemical restraint in the form of sedatives and neuromuscular blockers. For non-interventional and qualitative studies, we will include data from patients that are not physically restrained during admission to a high care intensity setting.For studies examining the efficacy and safety of physical restraint minimization and non-interventional and qualitative studies, comparator groups will receive usual care that comprises physical restraint methods such as wrist restraints, ankle restraints, chest belt, bed rails, vest restraints, mitts or mittens, four-point restraints, and five-point restraints.Our primary outcome of interest is the efficacy/effectiveness of physical restraint minimization strategies defined as the proportion of patients physically restrained. Secondary outcomes include (1) adverse events including unintentional device removal ; delirium; severe agitation; psychological impact including anxiety, depression, and post-traumatic stress disorder ; physical injury resulting from restraint; and associated benefits including harm prevention; (2) ICU outcomes including duration of mechanical ventilation, ICU length of stay, and mortality; (3) prevalence, incidence, and patterns of physical restraint use including patient and treatment characteristics and chemical restraint; (4) barriers and facilitators to minimization; and (5) patient, family member, and healthcare professional perspectives on physical restraint. Additionally, we will identify recommendations for restraint minimization endorsed by professional societies and accreditation and regulatory agencies and evidence gaps and priorities for research.Two authors  will independently screen search results against eligibility criteria. Full-text publications of all potentially relevant articles, selected by either author, will be retrieved and examined for eligibility. We will resolve any disagreements though discussion and refer to an independent arbiter (CD) if required. We will use the reference management software EndNote X7  to removTeams of two authors will independently extract data from selected studies on key components addressing our research questions including features of study design, patient characteristics, prevalence and incidence, study outcomes, benefits and adverse events, perceptions, and recommendations using designed data extraction forms, iteratively refined based on pilot data extraction. We have developed three data extraction forms for (1) studies with a control group, (2) studies without a control group, and (3) studies with a qualitative design. Due to inherent difficulties with determining what constitute findings in a qualitative research , we willWe will critically appraise included papers in duplicate and independently for risk of bias. For randomized and quasi-randomized trials, we will use the domain-based evaluation recommended by The Cochrane Handbook.  These doFor qualitative studies, we will assess study quality using the multi-dimensional concept of quality recommended by the Cochrane Quality and Intervention Methods Group  that incSearch results will be summarized in a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) study flow diagram . We willIf we identify sufficient studies evaluating interventions to minimize physical restraint, we will perform meta-analyses for the following outcomes: proportion of patients physically restrained, duration of mechanical ventilation, ICU length of stay, mortality, and adverse events. If we identify fewer than three randomized or quasi-randomized trials, we will include non-randomized studies in this meta-analysis.P value <0.05 to be significant. We will use RevMan 5.3 software to conduct meta-analyses. If we identify insufficient studies for meta-analyses, we will provide a descriptive qualitative synthesis.For binary outcomes, we will calculate risk ratios (RRs) with 95% confidence intervals (CIs) using a random effects model. For continuous variables, we will calculate a pooled difference of means with 95% confidence intervals using a DerSimonian Laird random effects model. If the mean  is not reported or unavailable from the study authors, we will use the median (IQR) to approximate the mean using the method described by Hozo and colleagues  and willIf we identify sufficient studies of interventions to minimize physical restraint, we will perform subgroup analyses considering intervention type and patient characteristics such as age and admission category . We will conduct two sensitivity analyses, one excluding non-randomized studies and the second excluding studies rated as high risk of bias.We will use individual study participants as the unit of analysis. If we identify multi-arm studies, we will combine groups to create a single pairwise comparison as recommended by the Cochrane Handbook for Systematic Reviews of Interventions . If combWe (LR) will attempt to contact study authors for unreported data or clarification of study methods using a maximum of three e-mails. If data remains unavailable, we will analyse the available data and report the potential impact of missing data in the discussion section.P < 0.05 represents significant heterogeneity) and using the I2 statistic, which represents the percentage of variability across studies attributable to heterogeneity rather than chance [I2 statistic is >50% statistic, indicating moderate to substantial heterogeneity, we will assess the type and sources of heterogeneity  such as ICU patient type and physical restraint minimization intervention used.If we identify sufficient studies evaluating interventions to minimize physical restraint, we will assess clinical and methodological heterogeneity with forest plots and chi-square tests  approach  to asseshighly likely, moderate confidence if likely, low confidence if it is possible, and very low confidence if it is not clear that the review finding is a reasonable representation of the phenomenon of interest [We will use the Confidence in the Evidence from Reviews of Qualitative Research  approach to assess confidence in qualitative evidence synthesis . This reinterest . We willTabulated data, quality of evidence, and confidence in evidence assessment findings will be provided to an interprofessional and international guideline development group to develop recommendations for inclusion in an updated practice guideline on physical restraint for critically ill adults.While nursing homes and psychiatric units have seen a significant reduction in physical restraint use over the last two decades, use amongst the critically ill remains extremely common and may have detrimental physical and psychological consequences , 17–22. We will use our mixed-methods systematic review findings to develop a professional society-endorsed position statement and to update the 2003 guidelines  on physi


Adult patients with an esophageal cancer can potentially be compromised with postoperative leaks or fistulae if patients’ nutritional status is in a vulnerable stage. Currently in Australia, there is a growing need for clinicians to know whether use of immune-enhancing formulas (IEFs) containing Arg, omega-3, and RNA are a cost-effective approach compared with isonitrogenous-isocaloric formulas to reduce postoperative infectious complications in esophagectomy patients. Since IEFs may carry higher costs, this has led to inconsistencies in practice among clinicians and hospitals.Our aim is to compile and present the most up-to-date nutrition evidence available regarding the provision of IEFs containing Arg, omega-3, and RNA to help clinicians develop an evidence-based nutrition care plan; identify available evidence of whether an esophagectomy patient should receive IEF; determine the cost-effectiveness and safety of such nutrition; and determine appropriate administration quantity and timing .This review will include RCTs involving the use of IEFs enriched with Arg, omega-3 polyunsaturated fatty acids, and RNA in the pre-, peri-, or postoperative period (for at least 5-7 days) given orally or via enteral feeding tube, in adult cancer patients undergoing esophageal resection. Lower gastrointestinal, gastric, or head cancer surgery with parenteral nutrition or non-IEF or use of isolated immunonutrient (Arg vs omega-3 vs RNA) will be excluded. Primary outcome comprises postoperative infectious complications. Secondary outcomes (pre/postoperatively) consist of cost-effectiveness, length of stay, survival/mortality, quality of life, nutritional status, percentage of weight loss, and biochemical changes. The risk of bias will be independently assessed by the reviewers, using a domain-based evaluation tool. Blinding will be assessed for subjective and objective outcome measures. Publication bias will be visually assessed by funnel plots. A meta-analysis will be generated by the Review Manager 5.3 software and represented in forest plots.The first results are expected in 2018. Outlining the protocol will ensure transparency for the completed review.This protocol for a systematic review and meta-analysis will enable a comprehensive appraisal of the literature to help determine whether overall institutional savings are associated with this approach. Findings will form a knowledge base relevant to stakeholders across the health system and researchers who are involved in decision making on evidence-based nutrition care plan pathways for patients undergoing esophagectomy, as well as the use of IEF, timing, and administration quantity.PROSPERO Registration Number: CRD42017056908; http://www.crd.york.ac.uk/PROSPERO/ display_record.asp?ID=CRD42017056908 (Archived by WebCite at http://www.webcitation.org/6rLyeqaD6) Adult patients with an esophageal cancer or intractable strictures often require resection of the esophagus with complex anastomoses that can potentially be compromised with postoperative leaks or fistulae if patients’ nutritional status is in a vulnerable stage ,2. AdditAlthough it is challenging to define and measure nutritional status in oncology, cancer-associated malnutrition seems to affect this population even in those overweight or well-nourished patients with a recent diagnosis . Up to 8Currently in Australia, there is a growing need for clinicians to know whether the use of immune-enhancing formulas (IEFs) containing arginine (Arg), omega-3, and ribonucleic acid (RNA) is a cost-effective approach compared with isonitrogenous- isocaloric formula to reduce postoperative infectious complications (POIC) in esophagectomy patients . Since tThus, this systematic review will enable a comprehensive appraisal of the literature to assist in determining whether overall institutional savings are associated with this approach. This review also seeks to compile and present the most up-to-date available evidence on the provision of IEFs containing Arg, omega-3, and RNA, to help clinicians develop an evidence-based nutrition care plan. Furthermore, we aim to identify available evidence of whether an esophagectomy patient would be the ideal case to receive IN, and a cost-benefit analysis to determine appropriate administration quantity and timing .We aim to address the following research questions. In cancer patients undergoing esophageal resection and requiring postoperative nutritional support: (1) Is there sufficient quality evidence on perioperative IEF enriched with omega-3, Arg, and RNA to recommend it as routine practice among clinicians?, (2) Do IEFs enriched with omega-3, Arg, and RNA confer additional clinical benefits such as reducing the risk of POIC and improving patients’ health care outcomes compared to standard enteral nutrition?, and (3) Are IEFs enriched with omega-3, Arg, and RNA a cost-effective strategy to be considered by clinicians?This review will include randomized controlled trials (RCTs) on National Health and Medical Research Council level II intervention and use of IEFs in esophagectomy patients. The trials selected should contain IEFs  compared with standard enteral formulas (SEFs) either pre-, post-, or perioperative. Non-RCT intervention studies such as cohort studies will be excluded, since the potential risk of bias is higher. Likewise, we will exclude observational studies, abstracts, review papers, conference proceedings, and studies that are not English, Spanish, or Portuguese language.Ethical approval is not required for the proposed systematic review and meta-analysis because the data used in this review will not involve the privacy of individual patients. Findings will be reported in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . As wellAll patients 18 years or over undergoing surgical procedure for esophageal cancer will be included. Also, those included should be receiving IEF  pre-, peri-, or postoperatively for at least 5-7 days. Moreover, we will include all inpatient and outpatient/ambulatory patients in the health care facility where patients are having surgery .The intervention will be the use of IEFs containing the immunonutrients Arg, omega-3 polyunsaturated fatty acid, and RNA provided either orally or via an enteral feeding tube. To be included, studies will have IEF given pre-, peri-, and/or postoperatively as an intervention. Co-intervention with other oral or parenteral substances will not be included. The control group should be receiving the traditional isonitrogenous-isocaloric SEF or polymeric nutritional supplements either orally or via enteral feeding tube.The primary outcome will be POICs including wound infections or ﬁstulae formation, bacteremia, sepsis, anastomotic leakage, abscess and pulmonary complications, especially pneumonia or bronchopneumonia within the first 2 weeks of surgery and during the whole stay in hospital . The meaInclusion criteria:Adult patients (≥18 years) undergoing an elective surgical procedure for esophageal cancer; perioperative patientsAdult patients in various stages of the disease including I, II, III, and IVAll patients (inpatient and outpatient) undergoing surgeryReceiving perioperative IEF Exclusion criteria:Lower GI cancer surgery, gastric, or head cancer surgeryPatients with cancer having parenteral nutrition or enteral feeding with non-IEF or IEF with only one isolated nutrient (Arg vs omega-3 vs RNA)We will consider the following secondary outcomes measured pre- and/or postoperatively:Health-related costs/cost-effectiveness/cost-benefit: defined by the authorsHealth care use: length of stay (LOS) measured in number of days; readmissions to acute care, subacute care or intensive care unit; re-operations measured in number of casesSurvival/mortality: number/proportion of deaths related to treatment ˂18 months  ; number/European Organisation for Research and Treatment of Cealth-related quality of life questionnaires  and 1 week and/or 3 weeks postoperativelyBiochemical changes: as per trial authors, including C-reactive protein levels (from ≤7 days prior surgery and first 2 weeks postoperatively)Searches for RCTs will be conducted systematically by the reviewers, with no publication year restriction. Non-English/Spanish/Portuguese studies might be excluded unless translations were provided or arranged. Also, original authors may be contacted for clariﬁcation and request of further data if trial reports seem unclear. Computerized searches will be performed for relevant published studies on the following databases from their inception until July 2017: the Cochrane Central Register of Controlled Trials (CENTRAL-The Cochrane Library), PubMed, EMBASE, CINAHL, LILACS, ClinicalTrials.gov, and the Trip (Turning Research into Practice) database. Subject strategies for databases in the search strategy will be modeled and designed for CENTRAL, CINAHL, and PubMed , which cThe reference lists of identiﬁed publications will be scanned for further trials, and some trial authors may be contacted if required. Additional searches with Google Scholar, Google, and Bond University Library online will be conducted to retrieve remaining systematic reviews and meta-analyses pertinent to this review, in order to scan their reference lists for additional existing trials. A search of gray literature may be performed using the and .The results of the searches will be combined as indicated in Data such as study design and setting, number of participants, outcome measure, and timing of IN initiation, route feeding, and total duration of IN will be autonomously extracted and organized on a matrix table . The metCochrane Handbook for Systematic Reviews of Interventions [The risk of bias in the eligible studies will be independently assessed by the reviewers. Any disagreements will be resolved through discussion with an external arbitrator if required. Risk of bias assessment will be performed using the domain-based evaluation tool described by the  ventions .Every RCT will be appraised according to the quality of 6 domains: (1) random sequence generation and allocation concealment (selection bias), (2) blinding of participants and personnel (performance bias), (3) blinding of outcome assessment (detection bias), (4) incomplete outcome data (attrition bias), (5) selective reporting (reporting bias), and (6) any other potential concerns to validity . BlindinBased on study reports, preliminary information will be collected in matrix tables to inform the risk of bias assessments . Trial aA low risk of bias RCT will be considered if all or the majority of domains were gauged as adequate. RCTs will be considered as having a high risk of bias when one or more of the assessed domains within that trial were unclear or inadequate . The “risk of bias or internal validity” will be reported as part of the “characteristics and outcomes of identified studies” matrix  as well P value of <.05 for both continuous and dichotomous variables will be considered statistically significant , which will be calculated/extracted for the analysis of most of the outcome measures apart from health care use  and biochemical/immunological changes that are exclusively represented as continuous variables . Also, nnificant .Heterogeneity must be considered from both a clinical and statistical perspective. On one hand, clinical expertise will be used to decide whether it would be meaningful to combine the studies based on the potential sources of heterogeneity for each outcome measure. The potential sources where the RCTs may not show the exact same result could depend on factors such as the patients’ demographics , nutritional status baseline, type of surgery (invasive or less invasive), different doses and timing of pre- versus peri- versus postoperative IEF, definition of the outcome measures, as well as the timing for measuring those outcomes . Hence, 2 test [On the other hand, statistical heterogeneity, which quantifies the variation due to heterogeneity and not due to chance across the RCTs, will be also checked through the statistical calculation of an IAfter that process, if the degree of clinical and statistical heterogeneity of studies are not excessive, a quantitative summary measures  will be generated by the Review Manager (RevMan) 5.3 software and represented in forest plots . Thus, tWhere statistical pooling is not possible, the findings will be presented in narrative form or a table to describe the outcomes, different tools used to assess the outcomes listed for this review, or continuous data that can be pooled for meta-analysis.Grading of Recommendations Assessment, Development and Evaluation working group methodology. Therefore, the quality of evidence will be assessed across 5 domains: risk of bias, consistency, directness, precision, and publication bias. Additional domains may be considered where appropriate. High-quality evidence will be adjudicated when further research might be very unlikely to change the reviewer’s confidence in the estimate of effect; moderate quality if further research is likely to have a significant impact on the confidence and may change the estimate; low quality if further research is needed and is very likely to have an important impact and likely to change the estimate; and very low quality if there is considerable uncertainty about the estimate of effect.The quality of evidence for all outcomes will be judged using the The first results are expected in 2018. Outlining the protocol will ensure transparency for the completed review.Given the nature of esophageal cancer and the significant potential for nutritional deficiencies prior to resection as compared with other gastrointestinal (GI) pathologies, it seems appropriate that there should be a specific focus on the role of perioperative nutritional supplementation . ResearcIEFs enriched with a combination of immunonutrients  are well known to Australian clinicians. However, IN is not consistently used with cancer patients undergoing esophageal resection as standard practice . Since tTo date, there have been several systematic reviews and meta-analyses of RCTs undertaken to determine the effectiveness of IN in elective upper and lower GI surgical practice ,16,21,30