Drosophila, and from those of other systems, that mitonuclear interactions—either conflicting or cooperative—are common features of GxE and GxG. We argue that mitonuclear interactions are an important model for how to better understand the pervasive context-dependent effects underlying the architecture of complex phenotypes. Future research in this area should focus on the quantitative genetic concept of effect size to place mitochondrial links to phenotype in a proper context.The mitonuclear genome is the most successful co-evolved mutualism in the history of life on Earth. The cross-talk between the mitochondrial and nuclear genomes has been shaped by conflict and cooperation for more than 1.5 billion years, yet this system has adapted to countless genomic reorganizations by each partner, and done so under changing environments that have placed dramatic biochemical and physiological pressures on evolving lineages. From putative anaerobic origins, mitochondria emerged as the defining aerobic organelle. During this transition, the two genomes resolved rules for sex determination and transmission that made uniparental inheritance the dominant, but not a universal pattern. Mitochondria are much more than energy-producing organelles and play crucial roles in nutrient and stress signalling that can alter how nuclear genes are expressed as phenotypes. All of these interactions are examples of genotype-by-environment (GxE) interactions, gene-by-gene (GxG) interactions (epistasis) or more generally context-dependent effects on the link between genotype and phenotype. We provide evidence from our own studies in This article is part of the theme issue ‘Linking the mitochondrial genotype to phenotype: a complex endeavour’. A given gene will manifest itself in different ways depending on the complex of other genes surrounding it. For this gene, that complex or genotype will comprise its genotypic environment, in which it expresses itself—Sergei Chetverikov, 1926 ,,27,Drosoila (mt) –124 is aila (mt)  and invoila (mt) –128. Owiila (mt)  phenotyp7.Drosophila using balancer chromosomes and tricks of fly genetics. In this process, we are circumventing the role of hybrid breakdown and avoiding the first contact between mitonuclear alleles and mtDNA haplotypes that may have been co-adapted and isolated over speciation timescales. To what extent cryptic genetic variation is exposed during hybridization or in the generation of novel mitonuclear genotypes is unknown. In wild or outbred populations of a species, any novel mtDNA polymorphism and nuclear combination(s) will always be tested in heterozygous nuclear backgrounds in the F1 offspring. In almost all cases, the female in the cross carries mitonuclear genetic combinations that are tried and tested in a fitness sense. Hybridization will conceal fitness-related mitonuclear epistasis because the effects of recessive mitonuclear interactions are more likely to be experienced by the individual only in the next (F2) generation. This effect is clearly illustrated in between-population hybrids of the intertidal copepod Tigriopus californicus. Mitochondrial sequence divergence exceeding approximately 18% between populations can be successfully introgressed producing fertile F1 hybrids [2 generation and associated with an incompatibility between mtDNA-encoded COX and the nuclear-encoded cytochrome c (Cytc) gene [2 segregation assays of nuclear SNPs in alternative mtDNA backgrounds are an effective means of assessing the degree of conflict and cooperation spread across the nuclear chromosomes [The terms ‘conflict’ and ‘cooperation’ represent two ends of a fitness continuum and we generally consider conflict as a deleterious outcome and cooperation as competent or high fitness state, with zero, negligible or positive phenotypic effects. However, how these phenotypes are generated and maintained in natural populations is poorly understood. For example, we can artificially introgress mtDNAs across species barriers with no obvious fitness consequences in  hybrids . Hybrid tc) gene . Recent omosomes ,133.The macroevolutionary aspects of conflict and cooperation in mitochondrial evolution focus on competition or conflict among different mitochondria (or mtDNAs) within early proto eukaryotic cells. The assumption is that the competition would be deleterious to the ‘host’ nucleus and evolution would favour the elimination of this cytoplasmic polymorphism. There is some experimental evidence that is consistent with this deleterious-heteroplasmy scenario in a mouse model . A more 8.Chetverikov's vision of a genotypic environment in 1926  forecastFST for both nuclear and mtDNA), each mtDNA haplotype might experience only a few alternative nuclear genotypes with which to interact. Likewise, a strong population structure might limit the range of external environmental factors that any mitonuclear genotype would experience . In species with strong population structure, we might see stronger opportunities for mitonuclear coadaptation, and its breakdown by mitonuclear ‘transplant’ experiments. In species with little population structure and high allelic variation, mtDNA and nuclear variants would experience multiple different ‘backgrounds’, and the opportunity for tight coadaptation may be limited; however, the opportunity for epistasis and sex-specific mitonuclear effects could be greatly enhanced. This population structure-mitonuclear coadaptation hypothesis could help explain why the breakdown of mitonuclear coadaptation is so evident in Tigriopus, which has highly structured inbred populations, while this is not seen in large outbred species such as Drosophila.An important caveat for this view of mtDNA effect size is the strength of the population structure. In species with a strong population structure (high It is easy to construct mitochondrial genotypes that might generate phenotypic effects that can appear supportive of broad evolutionary trends in single publications, but interpreting these effects as proof that mitochondria have driven the origin of sex, speciation, pervasive genomic conflict, Mother's Curse or other interesting ideas needs to be based on a broad set of studies that fully examine context-specific effects. Clearly, there is much still to learn.


Fusarium keratoplasticum (Fk) and F. falciforme (Ff), have been implicated as the causative agents of STEF . Ou. OuFusar species ; however systems  13,14].,14.FusarFusarium infections and hospital water systems, finding evidence that these opportunistic fungi reside in certain parts of the hospital environment, such as showerheads and sink drains. Molecular evidence later reinforced these findings [Fk, initially referred to as FSSC 2 [Fusarium diversity in bathroom sink drains across the eastern United States, found Fk to be a dominant species, but Ff was also present in low levels [Ff keratitis infections have been attributed to traumatic introduction of soil particles or plant debris into the eye, consistent with this species’ widespread association with soil environments [Anaissie and colleagues  formallyfindings ,16. Fk, s FSSC 2 , was lats FSSC 2 . A subsew levels , furtherronments .Fusarium species have been isolated from the eggshells and embryonic tissue of failed sea turtle eggs for several decades, with hypotheses concerning their ecological role ranging from decomposition to pathogenicity [F. oxysporum species complex [Fk and Ff, originally identified as F. solani, were associated with mass mortalities in the nests of loggerhead sea turtles (Caretta caretta) in Boa Vista, Cape Verde [Fk isolated from eggs in Boa Vista provided evidence supporting Fk as a causative agent of STEF [Ff. Following this discovery, a worldwide survey of sea turtle nesting sites revealed that Fk and Ff were isolated from infected eggs from six of seven sea turtle species at major nesting sites in the Atlantic, Indian, and Pacific Oceans, as well as the Caribbean Sea [Fk and Ff on nesting sites and the factors that play a role in disease development.genicity . Previou complex . Artific complex . In 2010pe Verde . Koch’s  of STEF . Koch’s bean Sea ,20. DespFusarium include the presence of atypically colored areas  on the eggshell, with more severe infections showing gray hyphal mats on the outside and the inside of the eggs and on the embryos [Fk and Ff have also been isolated from asymptomatic eggs, suggesting that the presence of the pathogen may not be sufficient for disease to occur. Factors such as changing environmental conditions have been shown to influence hatching success and may also influence disease development [Symptoms indicating that sea turtle eggs might be infected with  embryos . Howeverelopment . Resultselopment .Fk and Ff, as highlighted in Fk and Ff native to sandy beach environments? Is their association with inundated nests indicative of a marine or sand-borne origin? Or are these pathogens introduced into the nest environment via runoff from the built environment, where they are associated with plumbing systems and opportunistic human infections? Distinguishing among these potential sources has important implications regarding the epidemiology and etiology of STEF and for formulating informed approaches focusing on prevention and management. The known global distribution of these opportunistic pathogens, in addition to the role environmental conditions play in disease incidence and severity, suggest an endemic nature, but more evidence is needed to support this hypothesis. Future research on this topic should include studies testing the association of Fk and Ff presence with hatch success in sea turtle nests, as well as experiments to determine the influence of environmental factors on Fusarium pathogenicity on sea turtle eggs.There is still much we do not know about STEF, including the source(s) of Fusarium diseases of humans [Fk. By analyzing Fusarium populations from sea turtle eggs and nesting environments in the context of these studies, this method could be used to relate STEF to the known diversity of Fk, which is based mostly on human clinical and plumbing environments, and Ff, which is based mostly on human clinical and soil environments.Population genetic data provide a useful means for differentiating source and migrant populations of an emerging pathogen. Multilocus sequence-based genotyping has been used to determine the novel or endemic nature of fungal diseases, including white-nose syndrome , chytridf humans . The latFk isolates (n = 231) from human infections and other sources, with the majority isolated from plumbing environments. This analysis also revealed that the Fk 2-d sequence type was prevalent in human infections and sink drains. Fk 2-d was one of the two most common sequence types recovered from the 2005 and 2006 contact lens–associated outbreaks of mycotic keratitis [Fk 2-d across a diverse array of genotypes. These results suggest a mixed reproductive model, involving sexual and asexual reproduction, allowing for adaptation to a changing environment and rapid spread of successful genotypes through clonal expansion [Ff appears to be even more diverse than Fk [Ff has not been conducted to assess whether it possesses a dominant expanding clone complex associated with human infections.Short and colleagues  used a 9eratitis . On top xpansion . Based o than Fk . HoweverFk and Ff derived from human infections and other environments include the following: (1) Is Fk 2-d disproportionately represented in STEF, as it is in human fusarioses and in plumbing systems? (2) If so, were the introduction and subsequent establishment of Fk 2-d in sea turtle nests due to anthropogenic influence? (3) Or does STEF-associated Fk diversity reflect a pattern we currently infer in Ff, in which diverse local genotypes cause opportunistic infections? And (4) do Fk and Ff isolated from sandy beach environments reflect an endemic source or migrant populations with regard to disease occurrence?Key questions raised by our current level of knowledge regarding Fk and Ff.The answers to these questions should provide a framework for designing management strategies. Because of bottlenecks and clonal selection, novel pathogens of sea turtle eggs are expected to exhibit reduced allelic variation and increased association among loci when compared with non-STEF-associated populations . If they are endemic, we predict that isolates from diseased sea turtle eggs and non-STEF-associated sources will not exhibit population subdivision . More stFk and Ff from the built environment and clinical sources to which STEF-associated isolates can be compared. In addition, a validated sequence-based typing system is available for rapid genotyping and assessment of population structure. These tools, combined with more intensive sampling of sea turtle nesting sites for Fusarium species, are key to understanding the current and future impact of STEF worldwide.As fungal diseases of wildlife become more common, it is imperative that the biological and ecological factors that contribute to the emergence and severity of outbreaks be identified. Establishing whether a pathogen is endemic or novel is an essential first step toward informed management and control of a newly emergent fungal disease . Populat


Forest tree breeding has been successful at delivering genetically improved material for multiple traits based on recurrent cycles of selection, mating, and testing. However, long breeding cycles, late flowering, variable juvenile-mature correlations, emerging pests and diseases, climate, and market changes, all pose formidable challenges. Genetic dissection approaches such as quantitative trait mapping and association genetics have been fruitless to effectively drive operational marker-assisted selection (MAS) in forest trees, largely because of the complex multifactorial inheritance of most, if not all traits of interest. The convergence of high-throughput genomics and quantitative genetics has established two new paradigms that are changing contemporary tree breeding dogmas. Genomic selection (GS) uses large number of genome-wide markers to predict complex phenotypes. It has the potential to accelerate breeding cycles, increase selection intensity and improve the accuracy of breeding values. Realized genomic relationships matrices, on the other hand, provide innovations in genetic parameters' estimation and breeding approaches by tracking the variation arising from random Mendelian segregation in pedigrees. In light of a recent flow of promising experimental results, here we briefly review the main concepts, analytical tools and remaining challenges that currently underlie the application of genomics data to tree breeding. With easy and cost-effective genotyping, we are now at the brink of extensive adoption of GS in tree breeding. Areas for future GS research include optimizing strategies for updating prediction models, adding validated functional genomics data to improve prediction accuracy, and integrating genomic and multi-environment data for forecasting the performance of genetic material in untested sites or under changing climate scenarios. The buildup of phenotypic and genome-wide data across large-scale breeding populations and advances in computational prediction of discrete genomic features should also provide opportunities to enhance the application of genomics to tree breeding. Forest tree breeding encompasses a number of steps to increase the frequency of advantageous alleles for several traits concurrently in a target population. Recurrent cycles of selection ultimately result in genetically improved planting material by maximizing genetic gain per unit time in the most cost-effective way  and association mapping is first presented. It provides a quick historical perspective on how and why we reached the point of convergence between quantitative genetics and genomics. Furthermore, it also serves to substantiate the fact that reductionist “genetic dissection” approaches or attempts to use single candidate genes or diffuse, indirect information from transcriptomics, have not proven useful for breeding practice and are therefore not discussed further. We focus on the factors that affect and the challenges that remain to fully integrate genomic data in tree breeding in light of the recent promising results of whole-genome prediction. Although making predictions is difficult “especially about the future” as Niels Bohr and others once amusingly said, we attempt to look at the near future of tree breeding when genotyping, whole genome sequencing and computational prediction of genomic features for thousands of trees will not be limiting. We anticipate a future where the progressive advances made possible by routine genomic selection (GS) in multiple large populations will provide a more powerful platform to revisit the discovery of discrete genomic elements that may further enhance whole-genome phenotype prediction and eventually allow direct discrete interventions at the DNA sequence level.The prospects of MAS for forest trees was properly doubted early on, limiting its potential value to specific genetic backgrounds resulting from linkage equilibrium of forest tree populations, QTLs interacting with environments and changes of allele frequencies across generations , whose effects on the phenotype are estimated in a “training” population. In forest trees, such a training set is usually composed by sampling one to a few thousand individuals in progeny trials derived from mating a few dozen parents that constitute the target elite germplasm bred. SNPs are used to build prediction models to be later applied to “selection candidates” for which only genotypes are gathered and phenotypes are predicted by the genomic data. The prediction models are cross-validated against a “validation” population, a set of genetically related individuals to the training set but that did not participate in the estimation of marker effects. A prediction model that delivers a high correlation between the observed and predicted breeding values is subsequently used in the breeding phase to calculate the genomic estimated breeding or total genotypic values of the selection candidates Figure . GS fundirσA/L)  because the phenotypes of a much larger number of seedlings in the nursery can be predicted with marker data compared to the number of trees that can be tested in conventional field progeny trials. Additionally, the use of realized genomic relationships is associated with increased accuracy in estimating σA and breeding values (r) . Phenotypes of the selection candidates can be predicted at very early ages, for example, when the seedlings are a few weeks old. GS not only could preclude or at least enhance the efficiency of progeny testing but would also optimize clonal testing phases by advancing a smaller number of pre-selected trees to be assessed in multi-site expanded clonal trials  genetic covariance matrix that combines the pedigree-based average numerator relationship matrix (A) with the marker-based relationship matrix (G). HBLUP increases the precision of the genetic parameters generated from traditional pedigrees as shown in recent studies with forest trees , age-age correlations, the long term models performance and cost and quality of DNA marker data. All these have been the subject of research and reviewed in detail in the context of tree breeding by Grattapaglia , with marker effects treated as random, normally distributed with common variance, has been very efficient. RR-BLUP has been equivalent to Genomic BLUP (GBLUP), providing the best conciliation between prediction efficiency and fast computation, while also revealing that essentially all major traits in forest trees fit the infinitesimal model . Model updating strategies will therefore be crucial to counteract the decay of relatedness and LD between the original training set and selection candidates as generations of breeding advance, as shown by simulations for eucalypt breeding . The use of a common SNP genotyping array across breeding programs of different organizations will be a key issue to provide the necessary economy of scale to integrate genomics into breeding.In the past 2 years, a number of additional experimental GS studies have been reported (Cappa et al., Eucalyptus clones (Marcatti et al., With easy access to SNP genotyping and positive results in essentially all major forest trees, we are now at the brink of widespread adoption of genomic prediction data, thus realizing the early promises of MAS in forest tree breeding. In addition to the outstanding research challenges discussed above, a promising area to enhance the value of genomic data will involve the inclusion of environmental co-variables in GS models as already shown in crops (Jarquin et al., Another area that will demand research comes from the evolution of sequencing technologies in moving from sparse SNP data to sequence data for GS. Apart from the challenge and cost of managing massive next generation sequencing data sets for large numbers of individuals in a breeding program framework, in theory, if sequence data were used instead of dense SNPs, accuracy should increase because rare causal alleles would be better captured in predictive models. Until now, however, simulation and experimental studies in domestic animals have shown that whole-genome sequence data does not increase accuracy when LD has a slow decay pattern (Macleod et al., Eucalyptus (Müller et al., Eucalyptus (Hussey et al., Populus (Zinkgraf et al., The success of whole-genome prediction and the poor outcome of dissection approaches in identifying functional quantitative trait nucleotides, have contributed nevertheless to an exciting new perspective for the study of complex trait variation. A clear pattern has emerged in annual plants indicating that the association signal of common variants in large sample sizes, although spread across the entire genome, is heavily concentrated in regulatory DNA in open chromatin marked by deoxyribonuclease hypersensitive sites (Sullivan et al., DG drafted the first version of the manuscript and all co-authors subsequently contributed to it by editing and formatting the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tunnelling nanotubes (TNTs), also known as membrane nanochannels, are actin-based structures that facilitate cytoplasmic connections for rapid intercellular transfer of signals, organelles and membrane components. These dynamic TNTs can form de novo in animal cells and establish complex intercellular networks between distant cells up to 150 μm apart. Within the last decade, TNTs have been discovered in different cell types including tumor cells, macrophages, monocytes, endothelial cells and T cells. It has also been further elucidated that these nanotubes play a vital role in diseased conditions such as cancer, where TNT formation occurs at a higher pace and is used for rapid intercellular modulation of chemo-resistance. Viruses such as HIV, HSV and prions also hijack the existing TNT connections between host cells for rapid transmission and evasion of the host immune responses. The following review aims to describe the heterogeneity of TNTs, their role in different tissues and disease conditions in order to enhance our understanding on how these nanotubes can be used as a target for therapies. Tunnelling nanotubes (TNTs) are intercellular cytoplasmic channels that enable direct physical interactions, facilitating the transport of cellular cargo between a range of different cell types. These have been referred to in recent literature by a number of names including “nanochannels”, “cytoplasmic extensions”, “intercellular bridges”, “nanotubular highways” or “epithelial bridges”. These are generally fine, long, non-adherent and are composed of F-actin-based extensions and function to promote intercellular cargo transfer between proximal and distant cells . GeneralWhile similar actin-based extensions such as lamellipodia, invadopodia and filopodia exist, they differ in their properties such as diameter, adherence and cell-cell cytoplasmic interaction . FurtherStudies have demonstrated the transfer of everything from prions , 9, retrThis review highlights the functional role of TNTs in cancer development, progression and metastasis, along with its role in viral infection.These nanotubes play a vital role in the intercellular communication in cancer cells and have been characterized in multiple cancer subtypes as a facilitator of cancer cell to cancer cell communication, as well as cancer cell to tumor matrix communication. It has been postulated that this intercellular communication with the tumor matrix may play a direct role in the progression and development of solid tumors . TNTs haNanochannels between cancer cells were first described in 2004 paper using imaging techniques such as SEM (scanning electron microscopy) and TEM (transmission electron microscopy) where organelle transport was evidenced in HEK, NRK and PC12 cell lines . A studyOne study identified these nanochannels as facilitators of cross-talk between tumor cells and endothelial cells during the process of metastasis. The cancer cells were observed to regulate the endothelium through the transfer of microRNAs, which was evidenced by a higher expression of pathogenic endothelium markers that was reversed upon pharmacological inhibition of nanochannel formation . The resA study on nanotubular membrane extensions in colon cancer concluded that these TNTs are extremely sensitive to mechanical stress, but also very dynamic in nature. They have the ability to retract and form channels again through extension of filopodia-like structures that make contact with the neighbouring cells . The elaStudies have also demonstrated that malignancy can be induced by cancer cells in neighbouring healthy cells using nanochannels through the transfer of microRNAs between them. TNTs were observed to transport oncogenic microRNAs which have been identified to be upregulated in cancer, specifically miR-19a in osteosarcoma and miR-199 in ovarian cancer, to non-malignant cells . ExosomeThe propagation of chemo-resistance through nanotubes between cancer cells is a compelling biological phenomenon that has been observed in few studies. This hypothesis of chemo-resistance development by cancer cells through intercellular transfer was first put forward in 1991 in Frankfurt et al., but TNTs as conduits were not implicated until much later . The forStudy published in 2013 further investigated the role of nanochannels between cancer and stromal cells in chemo-resistance cancer. They studied the TNTs formed in vitro between anchorage dependent spheroids as well as tumor explant cultures. In particular, they highlighted the transport of mitochondria through nanotubes between endothelial and cancer cells (ovarian and breast cancer cells) that corresponded with an acquired chemo-resistance in the recipient cancer cells . The stuMembrane nanochannels have been directly observed to be present between a variety of cell types within our body. Recent research has also identified the key role played by nanochannels during viral infections –33. StudAccording to a study published in Nature Cell Biology , HIV traAnother paper published also implicated the Env viral protein in playing a key role in the process of filopodial-bridge between an infected and an uninfected target cell . The viralphaherpesvirinae proteins also revealed that the viruses in question are able to induce cytoskeletal rearrangements to form TNTs which they subsequently used for intercellular transmission. This viral subfamily includes the herpes simplex virus (HSV), varicella-zoster virus (VZV), porcine pseudorabies virus as well as other important animal virus that are responsible for a number of diseases. Intercellular viral spread was monitored in the presence and absence of nanochannel inhibitors and this pathway for transmission was determined to enhance the spread of the virus significantly [In addition, Herpes Simplex Virus (HSV) was also observed to spread through thin intercellular cytoplasmic bridges up to 100 μm long, whose physical characteristics resemble TNTs. The study tagged an essential viral protein VP16 with a GFP tag and tracked the transmission of the viral particles through nanotubes, going from infected cells toward adjacent target uninfected host cells . A studyficantly ..A study in 2015 on Influenza A determined that the viral proteins could be transmitted through intercellular TNTs . NeuramiA recent study published in 2017 also highlights the role of TNTs in the spread of influenza virus. Lung epithelial cells are physically connected to each other through extensive nanochannel networks, which was exploited by the influenza virus for efficient cell-cell transmission . ConfocaTNTs play a very big role in the cellular communication between a number of cell types in the human body, but nanochannel communications between cells of the immune system in particular have been widely studied in recent times , 42, 43.Multiple immune cells can be connected simultaneously by these TNTs, which in turn establishes a complex intercellular network between immune cells. The same group published another paper describing the two distinct types of nanotubes that were formed between macrophages based on their physical properties . Thin naMultiple studies also determined the relationship between the specific types of TNTs with directionality. Thin nanotubes containing only actin as the structural component were observed to have unidirectional transfer of intracellular cargo, whereas thicker nanotubes with both actin and microtubule elements as structural components were observed to have bi-directional transfer . AnotherAnother study determined the role of nanotubes in early events associated with BCR signal transduction. They demonstrated the dynamic nature of TNTs and their ability to move laterally on the surface of the cell, which had been also characterized by early experiments on nanochannels in cancer cells. They also identified CD45 and Syk as molecular markers for induction of TNT formation in B cells as evidenced by their inducible co-localisation with the nanotubes . A 2001 TNTs are also known to play a significant role in maintaining the function of endothelial cells along with their surrounding tissues. Endothelial cells that are present in the extra-cellular matrix surrounding tumor tissue was found to form extensive TNTs with the tumor cells, and transfer of organelles between the different cell types modulate chemo-resistance in tumor cells, as well as endothelial phenotype , 30. In Yasuda et al. also discovered the effect of direct mitochondrial and lysosomal transfer between endothelial cells through TNTs and identified it as the primary driving force behind robust functional improvement in vasculopathies beyond endothelial progenitor cells . Endothevaricella) and porcine pseudorabies [TNTs are one of the most novel methods of mechanical intercellular communication over long distances, and research over the last two decades has demonstrated TNTs to be increasingly ubiquitous among different cell types. TNTs are used by cells for intercellular communication through calcium fluxes, proteins, miRNAs and organelles such as mitochondria, endosomes, and lysosomes. These components are transported through nanochannels along F-actin or microtubules by motor proteins, and hence require ATP. TNTs between immune cells, endothelial cells and stem cells are used to reverse stress and diseased conditions through intercellular mitochondrial and lysosomal transfer , 53. TNTdorabies . CellulaWith the growing knowledge about TNTs, it is now well known that they are involved in different functions which includes cellular communication to disease progression. However, due to their recent discovery, there are many aspects which still need to be explored. As discussed previously, TNT’s are formed through actin driven de novo process, however, there are certain alternative mechanism also through which they are formed. Detailed knowledge about these alternative and de novo mechanisms which includes “what triggers them to form” especially in case of cancer cells can proved to be useful from therapy point of view. Additionally, they are known to be involved in microbial and viral infection. Detailed overview of all the receptors present on them can provide much deeper insight to the mechanism of disease progression. Further, therapeutic resistance which has been linked with TNT’s can also be explored thoroughly in order to circumvent it. One of the major challenge in this field is presence of different types of TNTs which are cell specific. Transcriptomics and proteomics based studies can further give us more insight about these nano-level interaction. Finally, there is need to explore the role of TNTs in connection with reactive oxygen species (ROS) which may further help in finding their role in complex diseases such as diabetes, Alzheimer’s and Parkinson’s.


One of the challenges during chemotherapy and radiotherapy is to complete the planned cycles and doses without dose-limiting toxicity. Growing evidence clearly demonstrates the relationship between dose-limiting toxicity and low muscle mass. Moreover, malnutrition leads to low performance status, impaired quality of life, unplanned hospital admissions, and reduced survival.In the past, the lack of clear and authoritative recommendations and guidelines has meant that oncologists have not always fully appreciated the importance of nutritional therapy in patients receiving anticancer treatments. Therefore, collaboration between oncologists and clinical nutrition specialists needs to be urgently improved.Recent guidelines from scientific societies and practical recommendations by inter-society consensus documents can be summarized as follows: 1) timely nutritional therapy should be carefully considered if patients undergoing anticancer treatments are malnourished or at risk of malnutrition due to inadequate oral intake; 2) if oral intake is inadequate despite counseling and oral nutritional supplements, supplemental enteral nutrition or, if this is not sufficient or feasible, parenteral nutrition should be considered; 3) home artificial nutrition should be prescribed and regularly monitored using defined protocols developed between oncologists and clinical nutrition specialists; 4) appropriate nutritional management in the context of simultaneous care should become a guaranteed right for all patients with cancer.The purpose of this review is to provide oncologists with an overview of the aims and current evidence about nutrition in oncology, together with updated practical and concise recommendations on the application of nutritional therapy in cancer patients receiving chemoradiotherapy. In 1998, Andreyev et al. first reported that cancer patients with weight loss had worse outcomes when undergoing chemotherapy (CT) In the last 10 years, many studies assessing outcomes in cancer patients undergoing CT have clearly shown that lean body mass loss is an independent risk factor for dose-limiting toxicity, hospitalization, and survival. Moreover, patients who are malnourished at the start of treatment experience a further nutritional decline during CT Despite all these reports, a 1-day prevalence survey carried out in 154 French hospital wards on 1903 cancer patients showed that a high rate (42%) of malnourished patients were still not receiving nutritional therapy Although it is recognized that weight loss is not simply an irreversible marker of cancer patients with expected negative outcomes, the literature suggests that in patients receiving CT the benefit of nutritional support, either enteral or parenteral, has not been adequately considered by oncologists.In 2006, a survey carried out in the United Kingdom suggested that specialist oncological trainees lack the ability to identify factors that place cancer patients at risk of malnutrition Similarly, in 2016 another survey reported the lack of awareness and consideration of nutritional issues among Italian oncologists In the same year, Martin et al. carried out a qualitative study through interviews at 5 European centers  investigating the barriers to the implementation of nutritional care in patients with head and neck or esophageal cancers In the past, there has been insufficient appreciation of the clinical benefits of nutritional therapy during anticancer treatments, due to a lack of clear, authoritative, and shared recommendations or guidelines on the issue. Collaboration between oncologists and clinical nutrition specialists has been poor with oncologists struggling to identify patients at risk of malnutrition due to deficiencies in training and lack of time.The development of standardized nutritional care pathways is complex and requires nutritional care to be fully integrated as part of the multimodal care process in cancer patients. Improving the nutritional care process requires the involvement of several healthcare providers across different disciplines , together with an explanation of roles and responsibilities involved in the provision of nutrition throughout the continuum of care. This message has to be disseminated both to professionals For this purpose, a working group was established, with members coming from the Italian Association of Medical Oncology (AIOM), the Italian Society of Artificial Nutrition and Metabolism (SINPE), and the Italian Federation of Volunteer based Cancer Organizations (FAVO), with the objective of initiating a structured collaborative project named “Integrating Nutritional Therapy in Oncology” (INTO) This working group in 2016 released an inter-society consensus document detailing appropriate nutritional support in cancer patients Recently, the working group also elaborated a “Cancer Patients' Bill of Rights for appropriate and prompt Nutritional Support” In 2016, the European Society for Clinical Nutrition and Metabolism (ESPEN) released a new version of the guidelines on nutrition in cancer patients A crucial issue is also the timing of nutritional interventions. A window of anabolic potential seems to exist when survival is greater than 90 days, creating a chance for nutritional intervention to stop or reverse cachexia Similarly to pain management and palliative care There is agreement that unconditional artificial nutrition in all patients undergoing anticancer treatments is not indicated, but also that nutritional therapy should be carefully considered if these patients are malnourished or at risk due to an inadequate food intake Evidence of the benefits of nutrition in cancer patients from clinical trials is extremely necessary. Indeed, the collection of data supporting nutritional interventions could be one of the main actions that overcome the barriers hampering the provision of nutrition in these patients Of course, in long-term studies involving patients with chronic insufficient oral intake it would have been ethically unacceptable to have the control arm not receiving any nutritional support. So, randomized controlled trials (RCTs) on the effectiveness of EN or PN supplementation are ruled out Nowadays, there is a wide agreement that early consultation with a professional (physician and dietician) with documented skills in clinical nutrition, specifically for oncology patients, is beneficial for cancer patients receiving anticancer treatments Dietary counseling, including the use of ONS, should be the first-step toward increasing the oral energy and protein intake with the aim of improving clinical outcomes In patients undergoing adjuvant RT there is good evidence that dietary counseling improves oral intake, body weight, and some aspects of QoL as well as helping to reduce the incidence and severity of toxicity, thereby avoiding treatment interruptions Despite the small number of patients enrolled in the RCT (i.e. 37 vs. 74), Ravasco et al. demonstrated an improved survival (median follow-up of 6.5 years) in colorectal cancer patients who received early individualized dietary counseling during RT Cereda et al. performed a RCT in head and neck cancer patients undergoing RT or RT plus systemic treatment and receiving nutritional counseling In case of oral nutrition remains inadequate despite counseling and ONS, and in presence of normal gut function, total or supplemental EN should be considered Prospective and retrospective observational trials in patients with obstructing head and neck cancers undergoing RT or CT demonstrated that EN (better if early) compared with oral feeding reduces weight loss, frequency, and duration of treatment interruptions and the rate of hospital admissions Miyata et al. carried out a RCT in non-malnourished patients with esophageal cancer receiving neoadjuvant CT and supplemental artificial nutrition , and showed that EN during CT reduces CT-related adverse hematological events A cohort trial in colorectal cancer patients undergoing CT reported a longer survival (19.1 vs. 12.4 months) in 315 patients receiving counseling, ONS, and megestrol acetate compared with 313 patients without nutritional support When EN is not feasible, insufficient or contraindicated, supplemental or total PN ensures that cancer patients receive adequate nutritional therapy In a prospective study of over 51000 catheter days in cancer patients (45% receiving chemoradiotherapy) on home parenteral nutrition (HPN), the incidence of catheter-related bloodstream infections was 0.6/1000 HPN-days For many years, the role of HPN in advanced incurable cancer patients has been a controversial topic In particular, in a prospective study in 111 advanced cancer patients on HPN, those receiving anticancer treatments (65%) showed higher QoL scores than patients with no treatment The indication for HPN in incurable patients who are unable to eat mainly for malignant intestinal obstructions is extremely controversial. However, even in aphagic patients with advanced cancer receiving no anticancer treatment, HPN was shown to improve QoL In a Swedish observational study of the prevalence and use of artificial nutrition in palliative patients, 22% of inpatients and 11% of those in home care services received artificial nutrition, with PN being most common The start of HPN should be carefully considered together with the patient, taking into account his/her wishes, expected benefits on QoL, and prognosis as well as the likely burden for patients and caregivers Several studies have highlighted the importance of assessing body composition in cancer patients Two recent prospective studies have investigated the effects of PN in cancer patients receiving chemoradiotherapy using also BIVA After 90 days of HPN, malnourished cancer outpatients experienced significantly improved nutritional status , performance status (KPS), prognostic score (modified Glasgow prognostic score), and some BIVA measures Fasting periods have emerged as promising strategies to target clinical parameters that constitute the foundation for metabolic syndrome, cardiovascular disease, cancer, and neurodegenerative diseases. Although promising, these approaches are still experimental in nature and should not be initiated without medical supervision The evidence provided by human studies regarding the benefit of fasting and calorie restriction before and during CT is still very limited A recent review reported that several recent studies in cancer outpatient populations found that higher amounts of muscle mass were associated with improved survival One of the challenges during chemotherapy and radiotherapy is to complete the planned cycles and doses without dose-limiting toxicity. The relationship between dose-limiting toxicity and muscle mass loss has been clearly recognized. Similarly, malnutrition is a significant determinant of performance status and QoL.In conclusion, the crucial question is: should we need stronger recommendations to act for improving outcomes? Well, the answer is no, but we certainly need more robust clinical data to convince the entire international Oncology community as soon as possible. Oncologists should be aware that nutritional therapy must be timely considered and prescribed, when indicated, to all malnourished or at-risk-of-malnutrition cancer patients receiving chemoradiotherapy, as any delay in nourishing these patients might compromise the potential benefits of nutritional therapy.


Safe, effective vaccines are given to pregnant women to protect their infants and/or themselves against certain infectious agents; however, apart from tetanus vaccination, maternal immunization in low- and middle-income countries (LMICs) remains low. Tetanus toxoid vaccine is integrated into antenatal care services in Malawi with high coverage and provides an opportunity to identify factors that facilitate successful immunization delivery to pregnant women in LMICs.PATH and the University of Malawi’s Centre for Social Research conducted a mixed-methods study in 2015 to document community perceptions of maternal immunization, using tetanus vaccine as an example, and to identify factors perceived to be important to successfully introducing other maternal vaccines, such as influenza vaccine, in Malawi. We conducted 18 focus group discussions with pregnant and recently pregnant women and their family members and 76 semi-structured interviews with pregnant and recently pregnant women, community leaders, health workers, public health program managers, non-governmental partners, and policy makers.We identified factors perceived to support the introduction of new maternal vaccines, including strong maternal vaccine acceptance in the community, an existing strategy for maternal tetanus vaccine delivery, and positive health workers’ views about the introduction of additional maternal vaccines. Potential challenges to adoption and acceptance included identifying and tracking the target population and monitoring adverse events, and the need to ensure operational capacity of the health system to support the introduction and wide-scale use of an additional vaccine. For influenza vaccine specifically, additional challenges included limited awareness of influenza disease and its low prioritization among health needs.Lessons from the successful delivery of maternal tetanus immunization in Malawi may be informative for similar countries considering new vaccines for pregnant women or striving to optimize the delivery of those currently provided. We sought to conduct studies in one middle-income country in Latin America with high maternal influenza immunization coverage and one lower-income country in sub-Saharan Africa with high maternal tetanus immunization coverage. We sought advice from WHO, WHO Regional Offices, and immunization experts to identify countries that would be amenable to our research and whose experiences would be informative for other countries in their sub-regions and regions. The results from our study in El Salvador have been published When this study was conducted in 2015, Malawi provided TTV to pregnant women through routine antenatal care (ANC) services; tetanus-diphtheria vaccine replaced TTV in 2017. Tetanus vaccine is provided by nurses in public and private health facilities and by lay community health workers through community outreach, commonly located in churches, schools, and market places. The 2015-16 Malawi demographic health survey reports 73% of women received two or more doses of TTV in their last pregnancy and 90% of women’s most recent births were protected against neonatal tetanus by routine childhood immunization and immunization during pregnancy The objectives of this study were to determine community perceptions of disease transmission and prevention, vulnerability, and health priorities of pregnant women in Malawi; to understand factors associated with acceptance of maternal immunization, health care decision-making for pregnant women, and facilitators and challenges to receiving ANC, including vaccination; and to identify programmatic and operational factors perceived as important to the introduction and uptake of new maternal vaccines, specifically seasonal influenza vaccine, which is currently not routinely provided in Malawi, in an effort to inform future country-level maternal immunization implementation.22.1We conducted this study in one district in each of the three regions of Malawi: Rumphi (Northern Region), Dowa , and Zomba (Southern Region) in 2015 . We sele2.2This was a mixed-methods, cross-sectional, descriptive study. The study protocol and data collection materials were developed with experts in survey and qualitative study design. We systematically collected qualitative data through separate focus group discussions with pregnant women and women pregnant within the last year (hereinafter referred to as “pregnant women”); male family members; and women likely to influence a pregnant woman’s health care decisions   . We coll2.3The study met international ethics requirements and was approved by the ethical review boards of the Malawi National Health Science Research Committee  and the PATH Research Ethics Committee . We obtained written informed consent from all participants prior to interviews and group discussions.2.4Lay community health workers and nurses providing services to pregnant women in study areas were included. Community leaders and health workers identified pregnant women and family members for participation. We identified pregnant women who did not use immunization services through pregnant women and health providers participating in the study. We consulted public health stakeholders to identify national policy makers and non-governmental development partners. Focus group sample sizes were set with the expectation of reaching saturation, where no new issues emerge from additional respondents 2.5Chichewa or Chitumbuka). We audio recorded and transcribed interviews and discussions, and translated those conducted in local languages into English. Researchers collectively developed an analysis codebook based on interview guide themes and themes identified through an initial review of transcripts. We based our final coding structure on descriptive and interpretive codes using NVivo  for thematic analysis. The results presented include themes and responses commonly expressed by participants; individual sentiments are identified when applicable.We conducted semi-structured interviews with national public health managers and policy makers in English; we conducted all other interviews and group discussions in local languages , we found high variability in understanding of its cause and transmission, with some participants identifying unhygienic birth practices and others suggesting sexual transmission. In all focus groups, there was a general understanding that tetanus vaccines can protect both a mother and her infant from disease. A typical explanation from one pregnant woman in Dowa suggested that she would be interested in being vaccinated during pregnancy, “In discussions about influenza, we found high variability at the community level regarding knowledge of both the disease and its vaccine. There is no direct translation of the word “influenza” in either of the study areas’ primary local languages, however recognition of “flu” was widely expressed by focus group participants after the term was introduced by facilitators. Facilitators subsequently linked “flu” to influenza. While community members were initially unable to distinguish influenza from a common cold, most respondents recognized influenza symptoms once described and the concept of airborne transmission of disease was well understood. In subsequent questioning, “flu” was the fifth most frequently mentioned condition by community members when asked to identify the most important health issue affecting pregnant women and infants . Pneumonia was the second most frequently mentioned for infants. It was widely understood across focus groups that pregnant women and young children were more susceptible to disease, and participants frequently attributed this to having “lower immunity.” Suggestions from focus group participants for preventing the spread of influenza included keeping the house and environment clean, avoiding coughing on others, hand washing, and keeping homes well ventilated by opening windows.3.3so that our immunity will increase and the disease should not attack us” and because “you want to protect the unborn baby”, which were typical responses. Of pregnant women interviewed while leaving health facilities after ANC visits, 95% (38/40) reported “no concern” regarding the safety of receiving a vaccine during pregnancy. In contrast to vaccines, traditional medicines and medicines sold informally or through unregulated “hawkers”, as opposed to those purchased through a health facility, were widely discredited as being unsafe for pregnant women.We asked pregnant women, male and female family members, community leaders, and health workers about their perceptions of the safety and importance of maternal immunization. Citing their experience and knowledge of tetanus and TTV, pregnant women in focus group discussions across study sites expressed an overall positive view of maternal immunization. When asked if they would be interested in receiving an influenza vaccine when they were pregnant, two women explained that they would accept the vaccine “When asked to identify concerns around vaccines administered to pregnant women, male and female family members, health workers, and managers suggested that new vaccines could be misperceived as contraceptives or may cause an abortion. A health worker explained that Depo-Provera is the most common injectable contraceptive used in Malawi and it is distributed through community health workers, like TTV, leading to concern that these similarities could cause confusion among community members. Managers speculated that other community members, particularly a woman’s male partner, may question why an important vaccine was not available to a broader population. Male family members also expressed dissatisfaction with the lack of vaccines specifically offered to men.Pregnant women in focus group discussions across study sites generally did not express concern about receiving two vaccines at the same time during pregnancy; however, several women noted the importance of receiving information about the vaccines before being immunized. Despite general acceptance, some pregnant women described experiencing an adverse event following prior immunizations  and questioned whether symptoms would be more severe if two vaccines were given at the same time. A preference for receiving a combination vaccine instead of multiple single vaccine injections was a recurrent theme expressed. While not a common concern across focus groups, several pregnant women questioned the need for multiple doses of the same vaccine, indicating that the five-dose regimen of TTV for women of childbearing age was excessive.3.4We asked pregnant women to identify whom they sought for advice about receiving vaccines during pregnancy. In individual interviews, 46% of pregnant women identified community health workers as their main source of information on whether to receive vaccines, followed by friends or neighbors (34%) and doctors or nurses (29%). Across focus groups, pregnant women and female family members generally expressed strong support and respect for community health workers as trusted health care advisors. During focus group discussions with pregnant women, community leaders were also identified as important advisors for health care, and radio and newspapers were cited as sources of health care information.When we asked male and female family members about whether they needed to provide “permission” in order for a pregnant family member to received health care, including vaccination, we found that it was commonly expected that a pregnant woman would have a discussion with her husband or male partner, or inform him of her intent to seek care, rather than formally request permission. Similarly, female family members described that while they participate in making decisions about the health care a pregnant daughter or daughter-in-law receives, they do not have the right to prohibit her from receiving health services.3.5When asked about what would encourage pregnant women to attend ANC, pregnant women in focus groups across sites regularly cited receiving general guidance around their pregnancies and specific information on the health of their babies and themselves. Receiving supplements, such as iron, was also mentioned by several women. Women frequently suggested that more respectful treatment by health care providers would encourage them to seek health care during pregnancy. Support from village leaders and community health workers was also mentioned as encouragement to seeking care.[It] is important to counsel the people the way they do with cervical cancer. They first call the parents and give them counseling so that they should make a decision as to who wants to vaccinate her child. Maybe some adults who are pregnant will not take part. It’s their freedom to do that.”We also asked family members how health services, including immunization services for pregnant women, could be improved. Common responses across sites included more respectful health providers and receiving clear information on the relevant diseases and the purpose of the vaccines, including their positive and negative attributes. A mother of a pregnant woman from Zomba described the importance of receiving information before receiving a vaccine, recommending a similar approach to the counseling that parents received around human papillomavirus virus (HPV) vaccine for their school-aged daughters: “We asked pregnant women and family members about challenges pregnant women experience in receiving health care and vaccination during pregnancy. All focus groups cited similar issues, none of which were specific to maternal vaccines. Challenges included health staff shortages, vaccine stock-outs, anxiety around HIV testing (which is routinely conducted during ANC), lack of respect for confidentiality on the part of health workers, lack of transportation, and poor treatment by health staff. Although not commonly mentioned across focus group discussions, several pregnant women cited a fear of the pain associated with receiving injections, indicating this was a motivation to avoid immunization. Several health care workers also mentioned pain from injections as a reason their patients do not attend ANC visits.“… [Members of my church] don’t take medication. So for all my ten children, I have not gone to receive this vaccine (TTV). Neither the children nor I have received the vaccine.”Non-users of immunization services were specifically sought in this study, and three women were identified and interviewed. All three attributed their non-use to membership in a religious group that they reported does not allow members to receive medical care from health facilities. One woman in Dowa explained, 3.6We asked health workers to describe programmatic factors of Malawi’s maternal health system that could support the delivery of new maternal vaccines. Health workers stated that the ANC system was designed to encourage family support of women receiving health care during pregnancy, including vaccination. Health workers explained that their clinics incentivized partner involvement in ANC by allowing women escorted by their male partners to be seen preferentially as compared to unaccompanied women. Health care workers also explained that they require women to receive TTV before they are offered any other ANC services, describing this as a key factor in promoting high immunization coverage. A common narrative across focus groups of pregnant women and male and female family members described community efforts to increase coverage of health services for pregnant women, including local laws requiring tetanus vaccination, ANC attendance, and delivery in a health facility. Pregnant women also described local government penalties if they missed ANC visits, including financial fines.Study participants anticipated a number of operational challenges to introducing new maternal vaccines in Malawi. In discussions about the feasibility of adding a new vaccine to current vaccination coverage tracking systems, health workers and managers explained that while vaccination coverage is currently documented at the level of the community and health facility, information is not shared between healthcare sites, making it difficult to track vaccination status if a woman visits multiple health facilities during her pregnancy. Managers felt the current health information system had the capability to be expanded to capture coverage of a new maternal vaccine, although both health workers and managers voiced concern about the additional work required to track and report coverage monthly. Another concern raised at both district and national levels was the cost of reprinting health cards and registers to include dedicated space for tracking a new vaccine. Since influenza virus circulation is not routinely monitored outside of a few research sites in Malawi, accurate projections of the influenza season are not available. Without information on the duration of influenza season, maternal health care providers and immunization managers predicted difficulties in accurately estimating the number of women pregnant during, or just before, the influenza season for vaccine forecasting.“We do not have a [formal] system in place to record any adverse events, but women are advised that if they react to TTV, they should inform the TTV service providers at their local health facility.” A ministry of health manager reported that Malawi did have a system for monitoring adverse events, but regarded it as “not aggressive,” citing a total of four serious adverse events reported to the national level from the district level in the previous three years.When we asked whether a formal system for tracking adverse events following immunization (AEFIs) was in place in the country, we received varied responses between sub-national and national stakeholders. Health workers reported that AEFIs were not systematically monitored for any vaccine, and one maternal health district manager explained that In discussing anticipated challenges specific to maternal influenza vaccine introduction, public health managers noted concerns about the relatively short shelf life of seasonal influenza vaccine and the additional support required for proper disposal of unused vaccines at the end of each influenza season. A national-level manager recommended requiring vaccine suppliers to collect and properly dispose of expired and/or unused vaccines, citing the lack of government resources to do this on a national scale.3.7When presented with the hypothetical scenario of introducing seasonal influenza vaccine to pregnant women, maternal health workers identified several factors that they perceived could affect successful introduction. Adequate training was noted by health workers, district managers, and national policy makers as a key component of successful vaccine introduction in general, tying proper education and social mobilization for community members to increased vaccine acceptance and minimized misperceptions of vaccines. Education about seasonal influenza disease and the specific vaccine introduced was noted as particularly important, since influenza is not currently perceived as a leading disease priority of health workers or community members. A recurrent theme expressed by health workers was concern that sensitizing and educating the community about a new vaccine could lead to an increased workload for them. One health worker felt the additional tasks would negatively affect the quality of maternal health services, citing a perceived negative performance of community health workers after the introduction of HPV vaccine and a subsequent decline in overall vaccination coverage. Managers had differing opinions on the best approach to introducing a new vaccine. Some preferred staging the introduction to allow for applying lessons learned as services are nationally scaled and others preferred a nationwide introduction to avoid potential community perceptions of preferential treatment or discrimination.We asked immunization managers at the district and national levels to identify the most important lessons learned from recent vaccine introductions in Malawi, including pneumococcal vaccine (2011), rotavirus vaccine (2012), and HPV vaccine demonstration projects in Zomba and Rumphi districts (2014). Sufficient long-term funding to implement the immunization program was a common priority for respondents, and adequate support for training health workers and social mobilization was specifically mentioned.We asked health policy makers at the national level to provide recommendations for future introductions of new vaccines targeting pregnant women. Respondents highlighted the importance of advocacy and communication, and stakeholders felt that for maternal vaccines against influenza, communication efforts should focus on advocating the benefit of the vaccine to both the pregnant woman and her infant, instead of prioritizing one beneficiary over the other. Policy makers recommended simple, direct communication messages, noting the difficulty of translating more complex, nuanced messages into local languages. Health workers also cited the importance of having clear information on the side effects of any new maternal vaccine and were particularly interested in the safety of concomitant administration of any new vaccine with TTV.With respect to decision-making around new maternal vaccines, respondents cited knowledge of local disease burden as an important consideration for prioritizing vaccines. Although preferred, policy makers reported that country-specific vaccine efficacy, safety, and cost effectiveness data were not required for decision-making, provided quality evidence from nearby countries was available. Respondents noted that decision-makers in Malawi have relied on data generated from countries with similar geographies and environmental and socio-economic indicators to inform previous public health decisions.4This formative research study describes perceptions of maternal immunization from a variety of stakeholders in three districts of Malawi. It also identifies programmatic factors perceived to be important to the introduction and uptake of new maternal vaccines in the country and includes recommendations for maternal immunization planning at the country level. The information generated through this study can be used to assist public health decision-making and inform future vaccine introduction planning in similar contexts.We identified several factors that could support the successful expansion of maternal immunization in Malawi, the first being that there is high health worker acceptance and community trust in vaccines in the study districts, including vaccines administered in pregnancy. This is advantageous for potential future introduction of maternal vaccines, given the importance of community acceptance to vaccine uptake With regard to influenza disease specifically, however, we found limited awareness and low priority of its prevention by pregnant women and health workers, indicating a strong need to educate the community, including preferred health advisors, on the disease and to sensitize stakeholders to the importance of the vaccine and its reassuring safety profile In addition to community education, generating buy-in of key decision makers in Malawi will be valuable to the introduction of new maternal vaccines, but may be challenging for influenza given its currently low perceived priority compared to other diseases such as malaria and HIV. Participants in this study called for the strengthening of disease surveillance efforts to inform understanding of country-specific disease epidemiology, including identifying high-risk populations. The need to add influenza surveillance to the Malawi health information system was commonly mentioned by district and national respondents and is a key first step to gathering data needed to inform priority setting and awareness building. While there is a lack of detailed influenza epidemiology data in Malawi, limited surveillance is being conducted on influenza-associated hospitalized illness, influenza incidence in different populations, including pregnant women, and potential risk factors (such as HIV infection) for hospitalized disease Surveillance systems for AEFIs are key to monitoring vaccine safety and addressing safety concerns in a timely manner Although TTV is currently integrated into ANC services in Malawi, additional research is needed to identify ways to minimize the strain that adding a new vaccine presents to health workers and health systems—concerns not specific to maternal vaccines, but raised by health workers and managers in our study—and whether it could provide benefit to ANC, as new resources or practices are brought in. This will be especially important to consider for maternal vaccines provided seasonally, such as influenza vaccines, as they require a short-term surge in the number of vaccines delivered and health system workload. The WHO updated guidelines for ANC services in 2016 Our study has limitations. Although participants came from a variety of geographic areas in the country, the results and perspectives may not be representative of Malawi as a whole, or of other LMICs. However, we identified some similar themes in our study in a disparate population in Central America, particularly the high trust pregnant women place in community health workers and the importance of their advisory role in health care decision-making. Our sampling strategy, based on identifying pregnant women through community health workers providing maternal care and women leaving ANC clinics, captures only the experiences of women with access to health care. In addition, we were only able to identify three women in our study areas who refuse to use ANC services. Our results therefore may not be representative of women who chose not to access healthcare services or experienced significant barriers to healthcare access. However, we did attempt to identify respondents outside of ANC, a strength of our approach. Exploring experiences of populations with heightened challenges to healthcare access and potential strategies for outreach to them would provide additional valuable insight for vaccine introduction and delivery.While Malawi’s experience achieving high maternal TTV coverage provides key lessons learned, additional information will be required to guide decision-making and priority setting around the introduction of new maternal vaccines, including influenza vaccine. Results of this study have been shared with Malawi’s Ministry of Health and can help inform both future vaccine introduction in Malawi and efforts to introduce new maternal vaccines or increase vaccination coverage in other LMICs.JAF, KMN, and NB received a sub-grant award from WHO to conduct this work; AM, BN, JK, MJP, and MS received support from PATH; and JO, PL and JH received grant funding from the Bill & Melinda Gates Foundation. KMN is a member of SAGE.WHO disclaimer: Joachim Hombach and Philipp Lambach are staff members of the World Health Organization. The views expressed are those of the authors and not necessarily those of the World Health Organization.


Climate change is a global phenomenon, with particularly rapid changes observed in many mountain ranges, the arctic, and the Antarctic Peninsula, where temperatures have risen at twice the global average or more  to establish their influence on indirect (biotic) climate effects. They concluded that differences exist especially regarding the rate of colonization by new species, which is more rapid in whole-community transplantation studies compared to experiments employing open top chambers (OTCs). Yang et al. suggest that combined approaches therefore provide opportunities in disentangling direct effects of warming from indirect effects mediated through novel plant-plant interactions.Experiments are powerful tools for establishing cause-effect relationships and testing specific hypotheses regarding climate change impacts, but researchers should be aware of the limitations of different methods and techniques . Their results show that temperature increases can lead to a reduction in freezing tolerance, highlighting the complexity of warming effects and the importance of phenotypic plasticity in coping with climate change. In a transplantation experiment, Henn et al. demonstrated that leaf trait plasticity enabled alpine plants to persist within communities exposed to warming. This encompassed mostly convergence toward optima found in the local, pre-adapted plants when transplants were moved to warmer conditions. The authors note that the extent and importance of trait plasticity is species-specific, as was also reported in the study of Cui et al. who found low plasticity in leaf area of Viola biflora var. rockiana. For this species, the key to survival under climate warming seems to hinge on being able to avoid competitive effects from the surrounding community. Both studies demonstrate that climate change is likely to lead to range contraction and extinction especially for those Himalayan alpine species that are unable to adjust important traits, unless competitive exclusion is avoided by management intervention . The declining species richness in response to warming observed by Yang et al. is a further illustration of the risk of biodiversity loss in mountain ecosystems, with alpine-restricted species seemingly more sensitive than wide-ranging species according to Winkler et al.Liu et al. investigated the variation in non-structural carbohydrates and nutrients in Quercus variabilis along a 1,500 km north-south transect in China. They conclude that carbon or nutrient availability are likely not the main driving factors of range limits of this species, while recruitment limitation seems more important at the species' northern edge , while promoting especially late-successional and ruderal species in woodland and grassland. As the habitat suitable to lupine could double the next 50 years due to climate change, its role in altering competitive interactions and facilitating succession implies that it may further accelerate climate change-related ecological transition in cold biomes  vegetation to potentially harmful low temperatures , both in the short . The finding that a climate extreme led to legacy effects regarding plant cover and community composition years after the original event, highlights that climate change can cause significant impacts in cold biomes other than through long-term changes in mean conditions.The studies discussed so far addressed shifts in baseline (average) temperatures, yet climate warming also leads to a higher incidence of unusual short-term warm spells. Li et al. Their experimental study on the Qinghai–Tibetan Plateau showed that responses to warming very much depended on concomitant changes in precipitation: drier conditions led to a general deterioration of the grassland , while wetter conditions increased biomass production while preserving forage quality. It is thus critical for sustainable management of these systems that local precipitation trends and variability are taken into consideration in decision-making . In cold biomes, snowmelt timing is highly important for the onset of the growing season  were exposed to warming, one-time NPK fertilizer addition and/or interaction with the dominant shrub Rhododendron ferrugineum. Interestingly, they find that the nature of interactions with this shrub changed as the experiment went on, switching from facilitation by the shrub to competition and ultimately competitive release. Moreover, the impacts of warming and fertilization (including their interactions) differed depending on presence or absence of R. ferrugineum, highlighting the importance of considering species interactions in studying global change impacts.To elucidate these and other climate change related issues, long term studies are essential  species. Short and longer term responses may differ, which is why more experiments are needed that monitor communities for multiple years. This is challenging in these often remote locations, and made more difficult as the baseline climate is shifting so rapidly. Understanding, disentangling and predicting the longer-term outcomes of global change impacts in cold biomes will therefore likely remain a frontier in plant ecology.HD wrote the first draft of the editorial, with all co-authors jointly editing the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cardiac disease is a well-known complication of antiphospholipid syndrome (APS), with many patients presenting with valvular thickening or vegetations, referred to as Libman–Sacks endocarditis (LSE). Because cases of APS with cardiac involvement are relatively rare, paucity of large clinical trials studying this complication has made management challenging. In the absence of acute heart failure and embolic events, a medical approach is usually selected, consisting of anticoagulation and possibly corticosteroids when another underlying autoimmune disease is present. However, the role of various anticoagulant classes and the duration of steroid therapy continue to be debated. Here, we present a 45-year-old woman who developed two vegetations in the setting of secondary APS while taking rivaroxaban before experiencing marked improvement with the use of enoxaparin and steroids. This ultimately manifests clinically with clotting in the venous and/or arterial systems. APS is often secondary to another autoimmune disease such as systemic lupus erythematosus (SLE), or may be a primary phenomenon in the absence of other immunological conditions. In order to make a diagnosis, clinical features must be accompanied by the presence of at least one of three antibodies: anticardiolipin, anti-In some cases, clotting can result in the formation of vegetations on cardiac valves, a form of nonbacterial thrombotic endocarditis (NBTE) called Libman–Sacks endocarditis (LSE). The disease is characterized pathologically by the presence of small, warty lesions of platelets and fibrin usually measuring about 1–5 mm, occurring mainly on the mitral valve, followed by the aortic valve in frequency .Treatment of LSE has been controversial, largely due to the lack of adequate randomized clinical trials. A surgical approach is typically used when criteria similar to those for infectious endocarditis (IE) are met –5. When Here, we describe a case of LSE occurring in the setting of secondary APS with SLE in which an abnormally large vegetation formed on the aortic valve concurrently with a smaller vegetation on the mitral valve.The patient is a 43-year-old female who presented with sharp, substernal, nonexertional chest pain, and shortness of breath. Her past medical history included secondary APS (lupus anticoagulant positive) in the setting of SLE, with multiple, recurrent deep venous thromboses of the lower extremities. She was diagnosed with myopericarditis during a hospitalization one year prior when she presented with similar symptoms and an elevated troponin, after which a coronary catheterization demonstrated no significant coronary artery disease. Examination revealed a 2/6 systolic ejection murmur, without any radiation, gallops, rubs, or jugular venous distension. Auscultation of the lungs revealed clear breath sounds.An echocardiogram was ordered when she was found to have an elevated troponin level of 0.209 ng/ml without any evidence of acute ischemic pathology on her electrocardiogram. Subsequently, a 2 cm mass was seen on the aortic valve with evidence of obstructive pathology. A follow-up transesophageal echocardiogram redemonstrated this mass, along with a smaller mass on the mitral valve associated with mild mitral regurgitation . The aorThe patient's chest pain improved with colchicine, but her shortness of breath remained and was presumed to be from partial obstruction of the aortic valve by the large mass. After consultation with cardiothoracic surgery, medical therapy was initiated with a goal to avoid surgery if there were signs of improvement. She was already taking rivaroxaban when she originally presented, given that she had failed warfarin therapy in the past with persistently subtherapeutic INRs and recurrent DVTs, so she was started on therapeutic enoxaparin and aspirin. After a rheumatology consultation, hydroxychloroquine and prednisone were initiated as well. The patient remained stable with no new symptoms or signs of embolic events during her follow-up visits, and repeat transesophageal echocardiograms at 12 and 24 weeks demonstrated improvement in the size of both vegetations . Her preThis case highlights many of the challenging aspects of managing patients with cardiac involvement in APS. The optimal treatment approach continues to be poorly defined, largely due to the fact that many recommendations regarding management originate from expert opinion and small observational case studies rather than randomized trials.A treatment strategy begins with assessing the necessity of a surgical approach. Symptoms of acute heart failure, acute valvular regurgitation/stenosis, or recurrent embolization when recognized are considered to be relatively straightforward indications to consider surgical evaluation, in addition to patients who have failed medical management. Surgery is often offered to patients with mobile, left-sided vegetations greater than 1 cm for infective endocarditis, but this approach is not generalizable for cases of NBTE , 5. GiveAfter a commitment is made to a medical approach, a decision about the best form of anticoagulation is imperative. Warfarin with an INR goal of 2-3 has been considered the gold standard in patients with newly diagnosed APS presenting with their first thrombosis –8. GivenAdditionally, the proper anticoagulant to use in the setting of warfarin failure continues to be perplexing. Various approaches include using high intensity anticoagulation with an INR goal of 3-4, the addition of aspirin or hydroxychloroquine to a moderate intensity anticoagulation regimen, or switching to a new anticoagulant class despite the lack of evidence to support any benefit of one class over vitamin K antagonists –14. GiveWhen another underlying autoimmune process is present, steroids are frequently administered despite controversy over their benefits. Several case reports have demonstrated the ability of steroids to acutely decrease the amount of inflammation and disease activity, leading to rapid improvement in symptoms and imaging of the valves –17. DespThis case demonstrates the challenges in managing a complicated presentation of Libman–Sacks endocarditis in the setting of warfarin and rivaroxaban failure. Anticoagulation with enoxaparin and aspirin in combination with prednisone ultimately lead to dramatic improvement, although questions still remain about whether or not this approach could be extrapolated to similar cases with reliable outcomes.


In the past years, evidence has emerged that hallmarks of human metabolic disorders can be recapitulated in zebrafish using genetic, pharmacological or dietary interventions. An advantage of modeling metabolic diseases in zebrafish compared to other “lower organisms” is the presence of a vertebrate body plan providing the possibility to study the tissue-intrinsic processes preceding the loss of metabolic homeostasis. While the small size of zebrafish is advantageous in many aspects, it also has shortcomings such as the difficulty to obtain sufficient amounts for biochemical analyses in response to metabolic challenges. A workshop at the European Zebrafish Principal Investigator meeting in Trento, Italy, was dedicated to discuss the advantages and disadvantages of zebrafish to study metabolic disorders. This perspective article by the participants highlights strategies to achieve improved tissue-resolution for read-outs using “nano-sampling” approaches for metabolomics as well as live imaging of zebrafish expressing fluorescent reporter tools that inform on cellular or subcellular metabolic processes. We provide several examples, including the use of reporter tools to study the heterogeneity of pancreatic beta-cells within their tissue environment. While limitations exist, we believe that with the advent of new technologies and more labs developing methods that can be applied to minimal amounts of tissue or single cells, zebrafish will further increase their utility to study energy metabolism. Danio rerio) have evolved from being a model organism primarily used for studies of vertebrate development to a widely applied research tool, including its use in behavioral research, genetics, physiology, disease modeling, toxicology, and drug discovery (Zebrafish (iscovery . The groiscovery . The coniscovery .1) was dedicated to discuss novel solutions to remaining obstacles that prevent a wider and more impactful use of zebrafish for metabolic studies. As a result of this discussion, we summarize recent progress in “nano-sampling” approaches for metabolomics studies that provide possibilities to quantify metabolites with tissue-resolution in zebrafish larvae. Furthermore, we outline strategies using reporter tools and live imaging to study heterogeneity of cellular function within the tissue environment, an avenue that holds great promise to investigate the early cellular events leading to metabolic disease with clear advantages of using translucent zebrafish.Metabolic diseases are systemic disorders driven by the failure of single or multiple tissues to maintain homeostasis. Loss of metabolic homeostasis occurs when specialized tissues have lost their reserve capacity to react to daily metabolic challenges. For example, the diagnosis of diabetes is preceded by years of compensation during which a reduced number of functional beta cells maintains normoglycemia after meal intake. With further decline of beta-cell function during persistent metabolic stress, a minimum threshold of functional beta-cell mass is reached, causing elevated fasting and post-prandial glucose excursions and ultimately leading to diabetes . ChallenMetabolomics technologies have facilitated systematic studies of energy substrates and their intermediates in response to different metabolic states, as well as the contribution of pathologically elevated metabolites to disease onset and progression. Over the past years, metabolomics methods have also been increasingly applied to zebrafish. Typically, samples from pooled embryos/larvae and from adult tissues have been analyzed by nuclear magnetic resonance (NMR) spectroscopy as well as chromatographic or mass spectrometric (MS) methods .  and XenRecent technical advances in single cell metabolomics with cultured cells demonstrate the feasibility of reaching cellular resolution also for cells smaller than early embryonic blastomeres with subcellular sampling on the horizon ; reviewein situ metabolite distribution. For example, An alternative approach to nano-sampling is mass spectrometry imaging, which may achieve even higher spatial resolution and give snapshots of in vivo will be required. In vivo Nuclear Magnetic Resonance (NMR) spectroscopy is a promising approach for such metabolic monitoring, and a few pioneering studies have followed metabolite changes during development and in response to hypoxia or herbicide exposure in medaka embryos  and their wild-type siblings. Combining such tracer studies with the cellular and sub-cellular analysis methods currently being developed should provide unprecedented insight into metabolic pathways and their (patho-)physiological changes in vivo. Another strategy to examine metabolism beyond steady state levels is the application of tracer technologies to assess flux rates through different pathways. in vivo are being developed, a number of studies have already revealed altered metabolism in a zebrafish model of glioma progression, including changes in glycolytic rate as well as lipid and nucleotide metabolism  has been generated  levels and the redox potential of glutathione (EGSH) in specific subcellular compartments  in zebrafish and that foxp3 mutants display systemic inflammation, suggesting an involvement of these cells in the maintenance of immune tolerance (FOXP3 predispose to multi-organ autoimmunity (Moreover, the interactions between the immune system and beta-cells play a critical role in diabetes pathogenesis, yet these processes have not been modeled in the zebrafish pancreas. Implementing models of beta-cell inflammation and auto-immunity would allow one to study how these interactions are controlled in response to metabolic stress and aging . A recenferation . This woolerance . These rimmunity . In the immunity , and wilBeing able to assess beta-cell activity under metabolic and inflammatory stress is critical to identify small molecules that prevent the loss of its function in diabetes. We propose that some of the above-mentioned tools allowing to quantify the rate of ROS production or the metabolic state of cells can be applied to beta-cells as well. These tools, in conjunction with small molecule screening, can facilitate the discovery of novel therapeutic interventions that intervene at different levels in the cascade responsible for beta-cell stress and dysfunction.Progress has been made to exploit the advantages of zebrafish for studying the control of energy metabolism at tissue, cellular and subcellular resolutions. Achieving this level of resolution is critical considering the specialization of metabolically active tissues that often show different, and in some cases even opposite, homeostatic responses to metabolic challenges. Performing whole-larval transcriptomics metabolomics or proteomics analyses therefore provides limited information. The community should apply nano- or micro-sampling approaches wherever possible, facilitated by an active exchange of protocols and access to state-of-the art technologies. The same is the case for the use of reporter tools that often require experience and an optimized set-up, but once implemented provide powerful technologies to perform metabolic studies within the context of a live organism in physiological or pathological states.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.PG is an employee of Nestlé Research. The remaining 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.


Mechanosensing is increasingly recognised as important for tumour progression. Tumours become stiff and the forces that normally balance in the healthy organism break down and become imbalanced, leading to increases in migration, invasion and metastatic dissemination. Here, we review recent advances in our understanding of how extracellular matrix properties, such as stiffness, viscoelasticity and architecture control cell behaviour. In addition, we discuss how the tumour microenvironment can be modelled in vitro, capturing these mechanical aspects, to better understand and develop therapies against tumour spread. We argue that by gaining a better understanding of the microenvironment and the mechanical forces that govern tumour dynamics, we can make advances in combatting cancer dormancy, recurrence and metastasis.The online version of this article (10.1007/s12551-018-0466-8) contains supplementary material, which is available to authorized users. One of the biggest challenges in the treatment of cancer is to develop better ways to predict, detect and eradicate the spread of tumour cells to distant tissues. Cancer cells interact dynamically with their surrounding environment and not only remodel the nearby extracellular matrix but also affect immune cell infiltration, local fibroblasts and distant tissues. Pancreatic ductal adenocarcinoma (PDAC) provides an example of a cancer that is characterised by aggression fuelled by the microenvironment. PDAC tumours are often highly fibrotic with excessive deposition of extracellular matrix (ECM) molecules, including fibrillar collagen. Excess matrix deposition not only contributes to the aggressiveness of the malignancy but also poses major constraints on the delivery of chemotherapeutic reagents to the tumour  motifs. Binding and tension against the substratum cause integrins to undergo a conformational change promoting their activation and clustering, triggering adhesion and proliferation Schwartz . The conIntegrin adhesions not only control proliferation but also motility, via direct connections with the actin cytoskeleton. Vinculin and talin are mechanosensitive proteins that couple actin to integrins at focal adhesions. They form what is termed a molecular clutch Fig.  whereby Do cancer cells sense rigidity? Or have they lost this control? A recent study demonstrates that some cancer cells can maintain high proliferation rates even on low adhesion environments , a developmental process that goes awry in cancer and is linked to progression of epithelial cancers such as PDAC . The LINC complex regulates cell cycle progression in response to stress, for example in Drosophila melanogaster muscle  surfaces or hydrogels usually composed by acrylamide. The latter can be mechanically tuned by varying the crosslinker concentration to modulate the stiffness and incorporate RGD adhesive peptides to facilitate cell adhesion  not only allows decellularization of whole organs but also seems to leave ECM architecture intact  microscopy