Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences. Phage display is a selection technique in which a peptide or protein is fused with a bacteriophage coat protein and displayed on the surface of a virion. This technology was first described by George P. Smith in 1985, when he demonstrated the display of peptides on filamentous phage by fusing the peptide of interest to gene III of filamentous phage [Peptides binding to individual targets can be identified by affinity selection . For biopanning, a display library is incubated with an immobilized target, followed by extensive washing to remove nonreacting phages. Binders are usually eluted using acid or high salt and are enriched by amplification in the appropriate host cells. Three to five rounds of biopanning are usually performed in order to obtain targets that bind with high affinity Fig. . The priPhage-displayed peptide library can be used in B-cell and T-cell epitope mapping, selection of bioactive peptides bound to receptors or proteins, selection of disease-specific antigen mimics, selection of peptides bound to non-protein targets, selection of cell-specific peptides, selection of organ-specific peptides, and development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage-displayed peptide libraries have potential use in basic research and translational medicine. In this review paper, we discuss in detail each of the applications of the phage-displayed technology platform in the biomedical sciences.Upon encountering antigen, host humoral immunity activates and triggers production of antibodies directed against foreign protein epitopes. Knowledge of these protein epitopes is pivotal in understanding the pathogenesis of pathogen infections and in developing diagnostic reagents, therapeutic antibodies, and effective vaccines. An epitope (known as an antigenic determinant) is recognized by components of the immune system, including antibodies, B cells, and T cells. The epitopes of antigens, which are dependent on their structural properties, can be either linear or conformational . Linear Vibrio cholerae, can enter host cells by binding to GM1, a monosialoganglioside, and would result in acute diarrhea. Biopanning with cholera toxin B (CTB) subunit to select CTB-binding peptides that structurally mimic GM1 could serve as novel agents to block CTB binding on epithelial cells and prevent the ensuing physiological effects of cholera toxin [The considerable potential of phage display peptide libraries lies in their capacity to identify some peptide molecules that mimic epitopes (named mimotopes). Mimotopes have fewer similarities to primary amino acids of antigens and always show discontinuous sequences, but they can elicit an identical or highly similar antibody response to that of the native epitope. The development of diagnostic or preventive reagents requires screening phage-displayed peptide libraries for disease-specific epitopes/mimotopes in serum or cerebrospinal fluid samples obtained from patients with viral infections , 23, rhera toxin .Mimotopes can also be used to characterize unknown initiating events and provide clues toward disease pathogenesis. Biopanning with antibodies from systemic sclerosis patients was used to screen an immunopositive peptide that had a high degree of similarity to autoantigens, including heterogenous ribonucleoproteins (hnRNP), cytochrome c, fibrillarin, and late protein UL94 of human cytomegalovirus (CMV) . ImmunopMembrane receptors are pivotal for cell-cell biochemical and electrical signaling in essential physiological functions. Therefore, the pharmaceutical industries tend to focus on developing drugs targeting membrane receptors , 36. NovPhage display is also used to isolate receptor-independent peptide modulators, such as G protein-coupled receptors. Some selected peptides from G proteins can interact with the Gαi subunit, leading to an elevated sensitivity of guanine nucleotides to bind to A1 adenosine receptors  and to GThe pathogenesis of some diseases occurs through the expression of abnormal enzymes, which can serve as potential targets for developing inhibitors as new drugs to block the activities of these enzymes. Phage display has been used to identify the peptide substrate inhibitors that modulate enzyme activities , 49 Fig. BecauseIn cells, protein-protein interactions regulate the mechanisms of several important normal physiological functions. Phage display is a potent and versatile method for studying protein-protein interaction –58. It cMany different types of bacteria trigger protective immune responses through cell surface non-protein antigens, such as polysaccharides. Many tumor antigens are carbohydrates. Epitopes mimicking low immunogenic polysaccharides or carbohydrate antigens can be screened and identified by phage-displayed random peptide libraries , 64 Fig. These iThe phage display technique has also allowed us to identify novel peptides against RNA of interest by screening random peptide libraries , 68. Sel2, and SiO2, as well as magnetic materials such as Fe2O3 and Fe3O4 [A few studies have successfully employed peptide phage display to select binders of small molecules, such as fluorophores , microcynd Fe3O4 –79. Peptnd Fe3O4 . Moreovend Fe3O4 –84. SuchPeptide phage display through whole-cell panning offers a high-throughput approach for identifying peptides that bind specifically to a single cell type. Johnston and co-workers were the first to describe the use of peptide-displayed phage to identify peptide binding to several different cell types . The mosDepending on the applications of the ligand, selection can be performed with adherent or fixed cells. The experimental approach can be modified to isolate phages, which bind to the cell surface or peptides, thereby triggering the cellular uptake of the peptides. Peptide-displayed phage libraries are incubated with the cells for a defined period of time. The cells are subsequently washed to remove non-specific and weakly bound phage. In order to reduce the cross-reactivity of the peptide or the phage, blocking agents such as BSA are occasionally used. Removing unbound phage is required to obtain phage clones with strong binding to the desired target, and remove non-specific binding from the background. In general, the washing processes are relatively gentle; however, more stringent washes may increase the affinity of selected phage clones. In some cases, negative selection is performed to avoid the aforementioned problem. In general negative selection is not essential.Phage bound to the target is recovered using several elution strategies, including the use of acidic buffers, Dithiothreitol, and high ionic strength, which tend to decrease the interaction between the peptide and the target. Most commonly, acidic buffer is sufficient for the elution of target bound phage. However, in the case of strong peptide-target interactions, these elution procedures may only partially break peptide-target interactions, thereby resulting in loss of the high-affinity phage clones. To circumvent this problem, Strukelj and co-workers used a modified method, in which ultrasound was applied during acidic buffer elution to release target-bound phage and enable the selection of high-affinity phage clones . In caseUsing whole cells instead of purified proteins as target for in vitro biopanning has several advantages. The cellular receptors expressed on live cells can retain their native states , and their biological functions and activities. Biopanning with modified protocols can be used for the isolation of peptides that mediate specific cellular functions. For example, selection can be aimed at isolating surface-bound or internalized peptides. Direction elution of phage enables isolation of surface-bound phage. If surface-bound phages are removed by low-pH washes or through treatment with a protease, phage with internalizing characteristics can be isolated. In addition, the use of whole cells for biopanning enables the identification of cell surface molecules with unknown biological functions. This can be used to characterize cell surface profiles and provide information on molecular changes  between normal and disease cells.Although numerous cell-binding peptides have been successfully isolated using in vitro panning against cultured cells, several challenges still remain . In partOrgan-specific peptides can be isolated from phage display peptide libraries by performing the selection in a living animal. In vivo phage display technology was first described by Ruoslahti and co-workers in 1996 . They aiAfter removing the unbound phage in circulation, the desired organs are collected and homogenized. The organ-associated phage is recovered from the homogenized tissue. A fraction of the organ lysates are used to infect bacteria for phage amplification and for subsequent rounds of selection in another animal. Another fraction is used for phage titering to measure the amount of recovered phage. After 3–5 rounds of in vivo biopanning, the phage titer recovered from the target tissue should increase , 97, 100This approach has been applied to a number of different organs, including brain, kidney, lungs, liver, uterus, muscle, pancreas, thymus, and mammary gland . VariousUsing in vivo phage display technology to identify specific homing peptides is not without pitfalls. Peptides isolated from animal models using this method may not translate to human because of differences in the vasculature and peptide binding between species. Such limitations can be overcome by performing biopanning in humans. The phage library can be injected into terminal human patients and retrieved from human tissues to identify organ specific peptides . AnotherThe delivery of anti-cancer drugs to solid tumors is limited by physical transport barriers within tumors, and such restrictions directly contribute to decreased therapeutic index and the emergence of drug resistance. Drug delivery systems designed to precise spatiotemporal control have demonstrated potential to enhance drug delivery in animal models. Many nanoparticle delivery systems for anticancer drugs have entered the clinic, where they have been shown to exert anticancer effects by improving the pharmacokinetic and pharmacodynamic properties of their associated drugs . LiposomPeptide-mediated liposomes include three main components: an anticancer drug, a liposome carrier, and targeting ligands Fig. . Remote The use of peptides as targeting ligands offers several advantages, including ease of synthesis, structural simplicity, low cost, low immunogenicity, small size, ready diffusion, and simple targeted formulation assembly, as compared to larger biomolecules, such as antibodies. Previous studies showed that using larger biomolecules as targeting ligands may increase the clearance of antibody-modified nanoparticles from the blood –117. ThiColorectal cancer is one of the most commonly diagnosed cancers and a leading cause of cancer death worldwide. Traditional chemotherapy only has limited therapeutic efficacy due to non-specific delivery to tumor and non-tumor cells, and the development of drug resistance by cancer cells. Therefore, there is an urgent need to develop more tumor-specific targeted drug delivery systems that can more accurately and effectively deliver the anti-cancer chemotherapeutic drugs to the tumor sites. In a recent study, Wu et al. used phage display technology to identify three peptides that could bind to colorectal cancer cells with high specificity and binding affinity . The autAdvances in nanotechnology have facilitated a multidisciplinary approach toward the development of an ideal “smart nanodrug delivery system,” which can be decorated with a targeting ligand for diagnostic or imaging uses, in addition to therapeutic agents of interest Fig. . The avaCombinatorial phage-displayed random peptide libraries are very valuable tools for studying the interaction between peptides and other substances . In the past, scientists have been focused on identifying B/T cell epitopes –19, diseCancer cells, different from normal cells, are often found to overexpress certain antigens. Molecules which can recognize these tumor antigens with high specificity are suitable candidates as potential agents for guiding cancer chemotherapy to target tumor sites. The therapeutic monoclonal antibody, antibody-drug conjugates (ADCs), peptide-drug conjugates (PDCs) and peptide-mediated drug delivery systems have a major impact on the field of cancer therapy. Therapeutic monoclonal antibody based therapies have achieved remarkable clinical success and become one of the most important strategies for treating patients with hematological malignancies and solid tumors. The direct action of antibodies by receptor blockade or agonist activity may have limited therapeutic activity. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) have been demonstrated to have a major role in antibody efficacy , 129. UnPDCs have similar promise as ADCs but they differ in their pharmacological outcomes. Compared to ADCs, PDCs have additional advantages of having smaller molecular mass, higher tissue penetration, enhanced flexibility and well-defined conjugation chemistry, and faster and easier synthesis process when prepared in a homogenous form . SeveralNeither ADCs nor PDCs allows for high drug payloads on the conjugates molecule. The major challenges facing most ADCs and PDCs are their relatively low capacity for drugs. Hence, more effective payload strategies using different carriers are urgently needed. In addition to ADCs and PDCs, nanoparticles modified with targeting ligand are more effective payload strategies. Nanoparticles as potential vehicle for encapsulating chemotherapeutics can achieve higher drug-loading efficiency. Nanoparticles have been shown to increase the drug-loading capacity by approximately a thousand-fold compared to ADCs and PDCs . Much en1, 2-diaminocyclohexane) platinum (II)-loaded and hyaluronic acid polymer-coated nanoparticle (DACHPt/HANP) by possibly inhibiting peritoneal clearance and impairing the phagocytic activity of Kupffer cells [Despite having the advantages of increased therapeutic efficacy, many challenges still remain for nanoparticles, including potential for off-target effects, ligand stability, immune responses triggering and drugs delivery efficiency in the cytoplasm of target cells. In particular, the rapid clearance of these nanodrugs by the cells of the reticuloendothelial system (RES)/mononuclear phagocyte system (MPS), especially liver and spleen, can lead to increased toxicity to the off-target organs and reduced therapeutic efficacy. However, a recent publication suggested that such off-target effects can be reduced by Intralipid 20 % , which could decrease the toxicities in liver and spleen and increase the bioavailability of dichloro  requirements.


This condensed review gives an overview about two methodological approaches to study the impact of physical activity on cognition in elderly, namely cross-sectional studies and randomized controlled intervention studies with pre- and post-measures. Moreover, this review includes studies investigating different types of physical activity and their relation to cognitive functions in older age. Behavioral data are considered but the main focus lies on neuroscientific methods like event-related potentials (ERPs) and functional magnetic resonance imaging (fMRI). Healthy aging is associated with a decline in sensory, motor and specific cognitive functions. However, such declines depend on various factors, such as genetics and lifestyle . In partCross-sectional studies overall show a positive association between regular physical exercise and cognitive functions. However, in this type of studies the causal relationship between physical activity and cognitive functions cannot be unequivocally drawn. Nevertheless, meta-analyses of behavioral and neuroscientific studies showed stable associations between physical fitness and cognitive, mainly executive functions in older age . RandomiThe aim of the next two sections is providing an overview about cognitive changes associated with physical activity in elderly individuals across electrophysiological and fMRI studies using cross-sectional and randomized-controlled trial designs.Structural MRI studies revealed a consistent positive relationship between cardiorespiratory fitness and both brain volume and functional activation in cortical regions including anterior cingulate, lateral prefrontal, and lateral parietal cortex , 11. AlsEvent-related potentials derived from the electroencephalogram (EEG) during performance of cognitive tasks offer insights in the mechanisms underlying sensory and cognitive functions. As the ERPs have excellent time resolution, there is the possibility to analyze each process separately. Different ERP components reflect specific sensory, cognitive and central motor functions and can thus help pinpointing the origin of behavioral effects. However, due to a low spatial resolution of this method the spatial aspect is less informative. Instead, more informative are temporal and functional properties of the neurobehavioral gains due to physical activity. It has been shown that not only performance in executive tasks and fMRI activity pattern but also some ERP components are modulated by physical activity , 20. UsiAs in the review of Desjardins-Crépeau  in the rFinally, a recent review by Prakash et al.  providesAs shown, most of the cross-sectional studies reveal good evidence for the positive association between physical exercise and cognition, particularly executive functions in older population. However, given the correlative nature of these studies, causation cannot be established. Therefore, a number of randomized, controlled intervention studies with pre- and post- measures have been conducted with older adults. The duration of such trainings was very different. Berryman et al.  comparedColcombe and Kramer  conducteA more recent meta-analysis including randomized controlled studies from 1966 until 2009 with a total number of 2,049 participants showed modest improvements in processing speed, attention and executive functions, whereas memory effects were less consistent . A meta-Several recent studies investigated different types of physical training. Voelcker-Rehage et al.  comparedPhysical training interventions result in various effects on brain structure and function. Gomez-Pinilla and Hillman  proposedIn a recent study Kleemeyer et al.  investigIn a randomized intervention study Gajewski et al. , 60 compAs with all interventions which require effort and extra time, motivation has to be maximized to yield high compliance of older trainees. Hence, virtual-reality enhanced types of exercise (“exergaming”) may increase motivation and also success. Anderson-Hanley et al.  investigA similar but more everyday-like approach is natural physical activity which consists not only of aerobic and strength but also of coordinative and cognitive exercise. Such a multilevel exercise is dancing. Effects of dancing on cognitive performance as well as on brain structure and functions have been found , 64. KatApart from physical activity, cognitive activity appears to improve cognition like switching between tasks, visual search or working memory in older adults, as also clearly seen in the above-mentioned study , 60; seePhysical activity certainly enhances physical fitness but also cognitive fitness. Physical activity is related to unspecific and specific brain changes, the latter depending on the type of activity. Such brain changes are accompanied by improved cognitive functions. Higher-level functions such as executive functions are more improved than lower-level functions. Combined programs which embrace both aerobic, force and coordination training are more favorable since the different aspects of such training induce different brain and behavioral changes. It is probably even more effective to combine complex physical training with cognitive training. Insofar, natural exercises such as regular dancing which affect physical, coordinative and cognitive functions, offer the maximum benefit to preserve and even improve physical and mental fitness in advanced age. In future studies such combined or natural activities should be explored in intervention trials with adequate active control conditions. From a scientific perspective such studies should not only use subjective and behavioral measures but also non-intrusive neurophysiological methods such as electroencephalography.


Vaccine-based cancer immunotherapy has generated highly variable clinical results due to differing methods of vaccine preparation and variation in patient populations among other lesser factors. Moreover, these clinical responses do not necessarily correspond with the induction of tumor-specific cytotoxic lymphocytes. Here, we review the participation of natural killer (NK) cells as alternative immune components that could cooperate in successful vaccination treatment. NK cells have been described as helper cells in dendritic cell-based cancer vaccines, but the role in other kinds of vaccination strategies  is poorly understood. In this article, we address the following issues regarding the role of NK cells in cancer vaccines: NK cell anti-tumor action sites, and the loci of NK cell interaction with other immune cells; descriptions of new data on the memory characteristics of NK cells described in infectious diseases; and finally phenotypical and functional changes after vaccination measured by immunomonitoring in preclinical and clinical settings. NK celle (NCAM) –6. Appromulation . Unlike mulation . In factmulation . Moreovein vitro , 11. NK in vitro . For insin vitro . Another in mice . However in mice . On the  in mice .In humans, NK cells play an important role in tumor immunosurveillance alongside specific T lymphocytes. In an 11-year follow-up survey of a Japanese cohort study, it has been shown that low peripheral NK cell activity is associated with increased cancer risk . Other cConsidering the important role that NK cells have an immunosurveillance, it is desirable to focus the development of cancer therapies to augment NK cell killing and helping efficacy because it could aid in the induction of an optimal adaptive immune response against cancer.in vitro experiments and animal models, they have a limited capacity to traffic to tumor sites. Of note, in humans, factors regulating NK cell recruitment into neoplastic tissues are highly influenced by the tumor type and by the chemokine profile of the tumor microenvironment. A recent study suggested that CD56+ NK cells could scarcely infiltrate melanomas, hepatocellular carcinomas, breast cancers, and renal cell carcinomas  group had increased NK activity. Anti-CEA T cell response was enhanced in all of the nine pts analyzed, but it was not significantly different between groups. Thus, NK responses following DC vaccination may correlate more closely with clinical outcome than do T cell responses  is probably the most thoroughly evaluated activity performed by NK cells during treatment with monoclonal antibodies. NK cells can trigger ADCC to lyse IgG opsonized target cells. Several authors have reported a correlation between NK function and response to treatment with Cetuximab, Rituximab, and Trastuzumab in animal models and cancer pts , 82. NotIn summary, although immunomonitoring demonstrated NK cell relevance in DC immunotherapeutic approaches and a correlation with pt response in several clinical trials, our understanding of their role remains incomplete. In fact, doubts remain about whether NK cells function primarily through tumor cytotoxicity or if NK cells also exert a relevant helper function through their interaction with DCs, T cells, and other immune cells. This second type of interaction remains the source of much discussion. Consequently, it is important to monitor NK cell numbers as well as phenotypic and functional changes, such as the ability to exert tumor cell lysis and the production of immunomodulatory cytokines during the course of immunotherapy. Furthermore, addressing the capacity of treatment to generate the kind of NK memory-cell described in the prior section  could contribute to the long-term protection expected from cancer vaccines.1-polarizing DCs, providing DCs with antigenic material for presentation and by killing inappropriately matured DCs. On the other hand, DCs stimulate NK cells by both soluble and contact-dependent activators, thereby enhancing their cytokine production, proliferation, survival, and cytotoxicity. Although this DC–NK cell interaction has been demonstrated in vitro and in animal models, a review of the literature found very few clinical trials that performed NK cells immunomonitoring. The majority of these DC cancer vaccine clinical trials showed a correlation between NK cell number/percentage and/or activity augmentation and pt outcome. Surprisingly, to the best of our knowledge, NK cell analysis has not been carried out in trials for other kinds of tumor vaccines. To gain more knowledge about the role of NK cells in immunotherapeutic cancer vaccines, NK cell monitoring must be systematically incorporated into clinical vaccination trials. This could lead to a better understanding of the real impact of NK cells in the vaccine field.This review ventured beyond a description of the plurality of NK cell activities into the changes that therapeutic cancer vaccines can affect in NK cells, and how these lymphocytes can potentiate the immune system through DC vaccination. In this bidirectional crosstalk, NK cells hold the capacity to control and enhance DC-mediated anti-tumor immune responses by inducing the maturation of THThe 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.


Zaire ebolavirus (rVSV-ZEBOV) vaccine [The catastrophic West Africa Ebola epidemic that resulted in approximately 28,200 cases and 11,300 deaths over 22 months has been reduced to intermittent and, thus far, rapidly controlled flare ups in Liberia, Guinea, and Sierra Leone. During the period of most widespread and intense transmission, extraordinary efforts to develop, produce, and field-test an Ebola vaccine in the outbreak setting generated promising results. The combined findings of four parallel Phase I studies on healthy adults in Gabon, Kenya, Germany, and Geneva demonstrated safety and immunogenicity of the recombinant vesicular stomatitis virus (rVSV)-vectored  vaccine . Additio vaccine . Largely vaccine –8. Acros vaccine , large-sEbola emergence in human populations has become a sporadic yet recurring event. Thirteen African countries constituting over one-fifth of the continent have been affected by at least one documented outbreak since 1976 . AlthougPhased vaccination of health care workers in highest risk countries could protect otherwise fragile health systems in the event of Ebola emergence. Approximately 880 health care workers were infected in the West Africa outbreak and nearly 60% died . In LibeThe term health worker is typically used to refer to doctors, nurses, and midwives –17. BaseAdministration across high-risk countries could be conducted according to a prioritization scheme determined by a combination of indicators. Such indicators would account for recency of the last known transmission event as well as capacity for maintaining and distributing the vaccine supply, monitoring of safety concerns, and evaluating effectiveness or duration of protective immunity. It is recognized that involvement of an international authority, such as WHO, would be required to obtain and allocate the vaccine among countries, while individual countries would establish regional policies for implementation of a prescribed vaccination strategy. National policy would accordingly specify efforts to maximize participation and ensure adequate monitoring and evaluation based on international protocols.Phased implementation at the country level could involve randomly predetermined clusters of health facilities vaccinated sequentially as part of a stepped wedge design . FacilitThe approach of vaccinating health workers is potentially more feasible than other mass vaccination campaigns because it targets a relatively well-defined and receptive population. Although suspended in the absence of active transmission, vaccination of 6,000 health care and other frontline workers in seven urban and rural areas of Sierra Leone had been undertaken as part of the STRIVE trial. The recruitment strategies and enrollment milestones designed for this study could inform the development of routine vaccination protocols. A convenient administration strategy could be the vaccination of nurses and physicians during their training. Because duration of health care worker occupation is typically several decades, boosters may be necessary to sustain protection in the event of waning efficacy. In countries with little turnover within the profession, high coverage rates would be maintained without requiring large-scale “catch up” campaigns. Furthermore, the health care workforce is expected to have higher vaccine acceptance. During the Ebola June 2015 resurgence in Monrovia, physicians and health workers from hospitals where cases were being treated and/or contacts were being quarantined have opted to receive the vaccine .Despite the advantages of using health care workers as a targeted population for mass administration of the Ebola vaccine, inter-country variation in the stability of the population presents some potential drawbacks. The persistent shortage of health workers in many sub-Saharan African countries has been attributed to insufficient national investment in training systems, as well as out-migration due to wages, transition to different professions, and fear of occupational transmission, particularly of blood-borne pathogens such as HIV and hepatitis B . In counOther mass vaccination strategies intended to reduce secondary transmission events may be complementary. Through an initiative headed by WHO, Guinea is currently preparing to vaccinate contacts of survivors in an effort to safeguard against the flare ups that have been rattling West Africa since July 2015 . Similarp-values that did not account for α-spending rules, has advised caution in interpreting the reported efficacy [Due to the combination of behavioral change, active epidemiological surveillance , expandeefficacy . Prior tefficacy . Howeverefficacy .Extraordinary scientific and humanitarian momentum was garnered during the 2014–2015 Ebola outbreak. Routine vaccination of health care workers in non-epidemic but high-risk settings could be an effective approach for sustaining this momentum. We recognize that the approach includes challenges, such as possible inter-epidemic waning of protection, a large population of health care workers who are not nurses, midwives, or physicians, and poor coverage among health care workers in more rural areas or regions less accessible due to political unrest. In addition, criticism of the ring vaccination trial conducted in Guinea has prompted uncertainty regarding the accuracy of the estimated efficacy . Nonethe


Math anxiety is a common phenomenon which can have a negative impact on numerical and arithmetic performance. However, so far little is known about the underlying neurocognitive mechanisms. This mini review provides an overview of studies investigating the neural correlates of math anxiety which provide several hints regarding its influence on math performance: while behavioral studies mostly observe an influence of math anxiety on difficult math tasks, neurophysiological studies show that processing efficiency is already affected in basic number processing. Overall, the neurocognitive literature suggests that (i) math anxiety elicits emotion- and pain-related activation during and before math activities, (ii) that the negative emotional response to math anxiety impairs processing efficiency, and (iii) that math deficits triggered by math anxiety may be compensated for by modulating the cognitive control or emotional regulation network. However, activation differs strongly between studies, depending on tasks, paradigms, and samples. We conclude that neural correlates can help to understand and explore the processes underlying math anxiety, but the data are not very consistent yet. Math anxiety is important in psychological research due to its consequences: avoidance of future mathematics related career  and courMath anxiety is considered a multidimensional construct. One of the most well-known questionnaires – the mathematics anxiety rating scale (MARS) – differentiates between math test anxiety and numerical anxiety factors . BesidesMath anxiety shares properties and mechanisms with test anxiety and general anxiety, but can also be distinguished from them . Like otFigure 1). We will first address the neural correlates of the affective component of math anxiety and its regulation. Then, we turn to the neural correlates of the cognitive components of math anxiety, in particular processing efficiency. Finally, we outline how math anxiety and its neural correlates are related to math performance and finish with future perspectives.In summary, math anxiety is a common phenomenon which has considerable impact on the performance in math tasks e.g., . A solidThe affective component of math anxiety addresses the actual feelings and physiological reactions elicited by a math task in high math-anxious individuals. Thus, individuals with math anxiety reported negative attitudes such as dislike toward mathematics , negativper se but reflected similar emotionality. The pain-related activity was observed when high math-anxious individuals faced a math task but not during the math task itself, explaining that high math-anxious individuals try to avoid math. Additional analyses confirmed that math anxiety and not differences in math performance was responsible for the affective component.Regarding the first, math anxiety elicited increased activation in the pain perception network including the bilateral dorso-posterior insula and mid-cingulate cortex . The insRegarding the fear network, high math-anxious children showed hyperactivity and abnormal effective connectivity in the right basolateral amygdala . Since tIn summary, the affective component of math anxiety is associated with pain-related activity before math tasks and fear-related activity during math tasks, independent of trait anxiety. However, are both networks active in the same math-anxious individual or is this age-dependent? Since evidence for pain-related activity was found in adults and evidence for fear-related activity was found in children, the data point to an age-dependency of recruited networks which should be systematically studied with the same paradigms.Since math anxiety elicits negative emotional responses to math, high math-anxious individuals need to process and regulate these emotions which lead to cognitive consequences . ConsequOn the neural level, brain connectivity and brain activity patterns are altered by math anxiety due to emotion regulation. For instance, high math-anxious children showed a greater coupling of the hyperactive amygdala with cortical regions involved in processing and regulating negative emotions during the math task . This leControlling math-related emotions in math-anxious individuals does not automatically result in performance impairment. Emotion regulation can even help prevent or at least minimize the impact of an anxiety-caused performance deficit. The dorsolateral prefrontal cortex (DLPFC), as one part of this fronto-parietal network , seems cTo sum up, math anxiety elicits a negative emotional response to the math task, usually leading to impaired performance because of the additional involvement in emotion regulation. However, by enhancing the capacity for emotion regulation or cognitive control, high math-anxious individuals can compensate for their math-specific performance deficit. This influence of math anxiety on performance in arithmetic tasks can be conceptualized in a more general theoretical framework addressing the impact of anxiety on processing efficiency.Anxiety is hypothesized to have a general influence on processing efficiency . So far, this can be explained by differences in assessment, paradigms, and samples. Future research may address these issues by systematically manipulating methods, tasks, and samples to ensure that different results in different studies are due to methodological differences. Research on developmental trajectories could especially help identify the age-related neurocognitive correlates of math anxiety.Investigating the neurocognitive foundations of math anxiety can help explain the mechanisms that lead to performance deficits, detect anxiety-related differences in brain function, also in the absence of behavioral differences, and identify physiological markers of the emotional response to math. However, the few studies focusing on the neural correlates of math anxiety vary greatly in their methods , tasks , and samples . The methods differ in the investigation of correlational and causal structure–function relationships, the complexity of numerical tasks determines the degree of involvement of working memory resources and math ability levels depend on development. This leads to highly inconsistent results with little overlap between studies (cf. 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.


Colonic atresia and anorectal malformation are rare congenital anomalies individually. Few reports of the conditions combined in a single patient have been published in the literature. Neither colonic atresia, anorectal malformation or a combination of the disorders has previously been reported in the Haitian population.A 5-day-old female presented with feculent emesis, failure to pass stool since birth and an imperforate and stenotic anus. Exploratory laparotomy revealed colorectal atresia distal to a malformed cecum and a Wingspread low subtype anorectal malformation without any associated urogenital fistulae. Temporizing percutaneous ileal drainage was followed by second-stage anal perforation and dilation, ileal J-pouch and pull through.in utero have been implicated as the etiology of colonic atresia, simultaneous presence of anorectal malformation suggests a multifactorial cause. Investigation for multisystem abnormalities is warranted. Two-staged operative correction is considered the best treatment; however, long-term postoperative outcomes are uncertain.This is the first reported case of colonic atresia, anorectal malformation or the combination of the disorders among the Haitian population and one of only a handful of such cases reported worldwide. Although vascular accidents The coexistence of colonic atresia and anorectal malformation is a very rare occurrence and presents unique clinical and operative challenges. Investigation for additional congenital abnormalities is appropriate, and although two-stage operative correction is considered the best treatment, long-term outcomes are uncertain. CA represents 5–15% of all intestinal atresias, with an incidence as low as 1 in 66,000 live births  is a congenital anomaly in which part of the colon has not formed Worldwide, coexistence of these anomalies in a single patient has been reported only a handful of times  and moderate dehydration. Her abdomen was grossly distended without signs of peritonitis.In hospital, the patient presented with malnourishment (2.5 kg, 3Because external genitalia appeared anatomically correct; the anus was imperforate; and by using a probe, we could distinguish a stenotic anal canal, we classified the child as Wingspread low subtype ARM Wakhlu .Abdominal radiograph demonstrated extensive bowel dilatation. Attempt at barium enema revealed no recto-anal fistula.Cardiovascular examination revealed no evidence of cardiac anomalies with no murmurs, equal femoral pulses and appropriate pre- and post-ductal oxygen saturations.Electrolytic and fluid balance was optimized for surgical intervention and parenteral antibiotics were administered.Exploratory laparotomy revealed grossly intact gastrointestinal system from proximal stomach to patent ileocecal valve. The length of the small bowel was grossly adequate for nutrient absorption.However, distal to the ileocecal valve, five centimeters of a blind-ending colon existed with a fully-formed appendix and some bulbous anatomical features of a cecum. But there was no ascending, transverse, descending or sigmoid colon. We visualized the superior mesenteric artery but were unable to visualize the inferior mesenteric vessels. The colonic segment was not attached to the posterior abdominal wall and floated freely in the abdominal cavity Figure .Figure 1There was no rectum, no evidence of no intestinal-urinary or genitor-urinary fistulae and no gross anal musculature atresia—the pelvic bowl was an empty cul-de-sac at the base of which, the imperforate, stenotic anus could be palpated, confirming Wingspread low subtype ARM. There was no evidence of malrotation, other urogenital abnormality or abdominal wall defect.A percutaneous drainage tube was placed into the colonic segment and through the ileocecal valve into the terminal ileum. Post-operatively, the patient remained hemodynamically stable and received enteral nutritional support for two months.She then underwent a second-stage exploratory laparotomy, J-pouch creation, perforation and dilation of the anal opening and anal pull-through.The patient recovered from the second-stage operation without complications and is defecating effectively with plans for long-term monitoring and follow-up.Coexistence of CA and ARM has been reported only a handful of times in the literature (Goodwin et al. in utero are most commonly implicated in the etiology of CA; whereas, the etiology of ARM is widely considered multifactorial and may include participation of recessive genes (Nazer et al. Vascular accidents occurring Our patient’s mother did not smoke or use legal or illicit vasoactive drugs of any kind and had no family history of congenital anomalies. She denied first trimester menorrhagia and consanguinity. An impoverished woman, she had no access to adequate nutrition or prenatal medical care. As with previous cases of combined CA and ARM, etiology of our case remains unclear and is considered multifactorial.Presentation of CA can be delayed by a number of factors, including restricted access to care and lack of adequate diagnostic tools or clinical experience with the disorder (Levard and Boureau Presentation of the combined disorder of CA and ARM is unclear. However, as in our case, the classical features of bowel obstruction in neonates should raise suspicion for either or both underlying causes. Cardinal signs and symptoms of neonatal bowel obstruction include maternal polyhydramnios, bilious emesis, failure to pass meconium on first day of life and abdominal distension (Juang and Snyder Our patient’s abdominal radiograph revealed distended bowel loops consistent with intestinal obstruction. Although she was hemodynamically stable, the severity of imaging studies and obvious imperforate anus necessitated urgent operative intervention.in utero vascular accident of the inferior mesenteric artery may help explain the extensive CA; whereas, the ARM may be related to a genetic failure of anus formation.Laparotomy revealed CA and confirmed Wingspread low subtype ARM. While Wingspread high subtype ARM may involve rectal atresia, our case had no evidence of fistulae, presacral mass or anal musculature atresia also commonly associated with high Wingspread classification (Hamrick et al. Optimal surgical correction of combined CA and ARM is two-stage with an ostomy for decompression and evacuation of bowel content, followed by nutritional optimization before definitive correction Wakhlu . DependiIn a stable child with CA but without other abnormalities, complete recovery of bowel function can be expected (Mansoor et al. The coexistence of CA and ARM is a very rare occurrence and presents unique clinical and operative challenges. Investigation for additional congenital abnormalities is appropriate, and although multi-stage operative correction is considered the best treatment, long-term outcomes are uncertain.Written consent was obtained from the patient’s mother for the publication of this report and any accompanying images.


Aphids are economically important pests that cause extensive feeding damage and transmit viruses. While some species have a broad host range and cause damage to a variety of crops, others are restricted to only closely related plant species. While probing and feeding aphids secrete saliva, containing effectors, into their hosts to manipulate host cell processes and promote infestation. Aphid effector discovery studies pointed out parallels between infection and infestation strategies of plant pathogens and aphids. Interestingly, resistance to some aphid species is known to involve plant resistance proteins with a typical NB-LRR domain structure. Whether these resistance proteins indeed recognize aphid effectors to trigger ETI remains to be elucidated. In addition, it was recently shown that unknown aphid derived elicitors can initiate reactive oxygen species (ROS) production and callose deposition and that these responses were dependent on BAK1 (BRASSINOSTERIOD INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1) which is a key component of the plant immune system. In addition, BAK-1 contributes to non-host resistance to aphids pointing to another parallel between plant-pathogen and – aphid interactions. Understanding the role of plant immunity and non-host resistance to aphids is essential to generate durable and sustainable aphid control strategies. Although insect behavior plays a role in host selection and non-host resistance, an important observation is that aphids interact with non-host plants by probing the leaf surface, but are unable to feed or establish colonization. Therefore, we hypothesize that aphids interact with non-host plants at the molecular level, but are potentially not successful in suppressing plant defenses and/or releasing nutrients. Plants are facing constant threats from pests and pathogens. Nearly half of the one million known insect species feed on plants , includiAcyrthosiphon pisum (pea aphid) can only colonize plant species in the family Fabaceae. In contrast, Myzus persicae (green peach aphid) can infest over 400 plant species, mostly dicotyledonous plants, in over 40 families  legislation due to their negative impact on the environment. Another major issue is the emergence of new aphid genotypes that have acquired resistance to many of the different types of available insecticides. Different mechanisms can underlie the development of resistance including changes in the cuticle , point mTraditionally, plant–aphid interactions research has been focused on the plant side, which has provided insights into some of the plant defense mechanisms effective against aphids. These include preformed barriers, constitutive chemical defenses, as well as direct and indirect inducible defenses . Recent In this review, we will discuss the latest progress in understanding the molecular basis of plant–aphid interactions. More specifically, we will discuss the latest findings regarding plant immunity and aphid effectors, and highlight the potentially common concepts of plant–pathogen and –aphid interactions. In our discussion we will take a comprehensive view on potential molecular mechanisms involved in both host- and non-host-interactions. In addition, we will discuss the translational opportunities that may arise from our understanding of the plant immunity system and how new approaches could help achieve broad-spectrum and durable aphid resistance in crops.M. persicae has an exceptionally broad host range, its close relative, M. cerasi, will only infest a small number of herbaceous plants. The mechanisms underlying such differences in host range remain elusive and most research to date has been focused on understanding the interaction with compatible hosts.The host range of aphid species varies widely, and even among aphid species within the same genera . For exaFigure 1A).There are several steps involved in the initial contact between winged aphids and plants, prior to any feeding attempts. As detailed by Although recent work has started to unravel some potential mechanisms involved in (non)host resistance to aphids, this area of research is currently understudied with many questions remaining unanswered. For example, how do crops actually respond to aphids in incompatible interactions? What type of plant defenses are activated, and how are they activated? And are similar defenses involved across different crops? On the aphid-side of the interaction, what genes contribute to aphid host range, and how? Addressing these questions is essential for crop improvement and long-term protection against aphids, and potentially other insect pests.Figure 1). In some pathogen/pest interactions these barriers may be sufficient to confer resistance. However, it is likely that in many interactions complex series of molecular events are involved. Most studies of plant defense signaling in plant–aphid interactions have focused on the model system Arabidopsis thaliana-Myzus persicae. This revealed the contribution of several signaling pathways involved in activating defenses. For example, M. persicae feeding was found to induce the expression of SA-signaling pathway marker genes, such as PR-1 -signaling genes were induced, while JA-responsive genes were repressed. It has been speculated that the activation of SA-signaling pathways by aphids counters the activation of JA-dependent defense responses that are effective against aphids . However, elicitor activity has been detected in both aphid saliva and whole aphid extracts. For example, an unknown elicitor present in the 3–10 kDa fraction of saliva from the aphid species t aphids . The actM. persicae activated PTI-like responses in Arabidopsis , as bak1 mutants showed an increased survival rate of the pea aphid on Arabidopsis (non-host interaction), and an increased susceptibility to M. persicae (host interaction). In addition, induced resistance required PAD3, indicating at least two separate signaling pathways are involved. It is possible that the elicitors responsible for the observed activation of PTI-like responses are derived from the aphids themselves or any of the organisms that are associated with the aphids, such as bacteria. Indeed, work by Macrosiphum euphorbiae they showed the presence of the chaperonin GroEL from the aphid primary endosymbiont Buchnera aphidicola. These endosymbionts are located in specialized compartments called bacteriocytes in the hemocoel of the insects and contribute to the production of amino acids for their host. Although these bacteria strictly reside within aphids, it is interesting they are able to secrete proteins that are delivered with aphid saliva inside plants and activate defenses. Infiltration of purified GroEL protein resulted in the induction of PTI-like responses in Arabidopsis. More specially, GroEL induced callose deposition and ROS in a BAK1 dependent manner. Also, overexpression of GroEL in tomato and Arabidopsis reduced aphid fecundity. Overall, these recent findings suggest that the co-receptor BAK1, an important player in PTI mediated by plant pathogenic bacteria, is involved in perception of aphids and/or aphid-associated microbes. Interestingly, not only the saliva, but also the honeydew of aphids can alter plant defenses .Another form of plant immunity, ETI, is activated upon recognition of parasite effectors by resistance genes. Gene-for-gene type interactions have been found in several aphid-host systems. For example, extensive variation in the ability of observed . Also, bc manner . Other s-lettuce  and Aphi-soybean . SimilarR-gene Mi-1.2 belongs to the class of NB-LRR proteins and confers resistance to specific Macrosiphum euphorbiae (potato aphid) genotypes .Aphid effectors are most likely expressed in salivary glands and secreted into saliva, which is delivered inside the host during feeding and probing . Recent N. benthamiana . Both Mp10 and Mp42 were identified from a functional genomics screen, and reduced aphid virulence upon overexpression in thamiana . Only Mpthamiana . For ins capsici . HoweverA. pisum, is an abundant salivary protein that is essential for aphid feeding (M. persicae (MpC002) showed this protein promotes aphid virulence upon overexpression in N. benthamiana leaf discs .Other d PIntO2 . Mp1/PInm saliva  and receApPIntO2 . This su species . In addiractions . Both apArabidopsis lines as well as transient over-expression systems in both N. benthamiana and tomato have been successfully used to identify effector activities. In addition, more recently developed transient expression systems, such as the P. fluorescens Effector-to-Host Analyzer (EtHAn) system  system , where nHigh-throughput screening for relevant effector activities is an effective approach to prioritize effectors for functional characterization to understand their role in promoting susceptibility. The next step is to move forward toward host target identification, which can be achieved using several protein-protein interaction approaches, such as Yeast-two-Hybrid screening and immunoprecipitation of protein complexes followed by mass spectrometry. Identification of host targets and characterizing their role in plant–aphid interactions, and potentially in aphid host range, will be an important step forward toward developing novel control strategies against aphids.Another interesting aspect is variation of aphid effector repertoires. Interestingly, work by  suggesteIn addition to effectors, there are likely to be other classes of aphid proteins that are involved in plant infestation and potentially impact aphid host range. These could include digestive enzymes that help determine whether aphids can successfully feed from a particular host plant, or proteins involved in olfaction. Moreover, primary and secondary symbionts, viruses, and even plant pathogenic bacteria associated with aphids could add another level of complexity to plant–aphid interactions. There is the possibility that such organisms are involved in suppression and/or activation of a range of host responses during plant aphid interactions and affect susceptibility or possibly host range.As mentioned above, current aphid control heavily relies on the use of insecticides. Traditionally the control of pest insects has focused on mortality as a measure of efficiency. However, if we consider aphid fitness, there are many aspects by which we can measure the efficiency of control methods. Growth and development, reproduction, nutrient acquisition and survival are all vital factors determining the success of an aphid population. Negatively affecting one or many of these factors will likely reduce the overall fitness of an individual or population. Effective control does not necessarily have to rely on aphid mortality, especially if being used in the context of integrated pest management (IPM). In general, IPM relies on the use of multiple control methods simultaneously . By applying multiple measures to control a pest, it is less likely that new strains, biotypes or isolates may occur that overcome crop resistance, or that develop pesticide resistance. Research aiming to decipher the mechanisms by which pests interact with their hosts plays a crucial role in IPM strategies.One alternative to the application of insecticides to control aphid population growth is the deployment of biological control agents. Aphids have a range of natural predators that can help bring down population size or prevent populations from expanding. Predators used for biocontrol of aphids include predators like lady beetles (Coleoptera: Coccinellidae), parasitic wasps (Hymenoptera: Braconidae), and entomopathogenic fungi.R genes has been successful in preventing infestations in the field. Resistances have been effectively used for years in many crops including soybean, lettuce, melon, tomato, wheat, barley, maize, legumes, as well as fruit trees  it trees . Howeverit trees . Howeverappeared , indicatThe increasing availability of aphid genomics resources generates the opportunity to look for (aphid-specific) genes that may be suitable targets for the development of novel chemical control strategies that act specifically on the target organism. Alternatively, such targets may be used in control strategies based on RNAi. RNAi-mediated gene silencing was achieved in the laboratory by both injection of dsRNA directly into the aphid hemolymph and by allowing insects to feed on an artificial diet containing dsRNA . NeitherM. persicae.A highly underexplored area where potential novel sources of aphid resistance may be identified is non-host resistance. Evidence is only now emerging of plant genes that contribute to non-host resistance, and as described above such genes may be involved in activation of PTI or unknown defenses against aphids. With extensive host range variation among different aphid species, it will be interesting to investigate whether and how plants limit aphid host range and whether specific aphid genes, such as effectors, contribute to host range in the case of aphids with broad host ranges. Such insights will be particularly important to understand some of the most economically important aphid species, which have exceptionally broad host ranges, such as With aphids causing significant damage to economically important crops and issues with insecticide resistance and restrictions on their use, there is a need for the development of novel and sustainable aphid control strategies. However, to be able to develop such strategies there is a pressing need to gain a better understanding of the molecular basis of plant–aphid interactions and thus a need for fundamental research on the molecular mechanism involved.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.


For over 100 years, vaccines have been one of the most effective medical interventions for reducing infectious disease, and are estimated to save millions of lives globally each year. Nevertheless, many diseases are not yet preventable by vaccination. This large unmet medical need demands further research and the development of novel vaccines with high efficacy and safety. Compared to the 19th and early 20th century vaccines that were made of killed, inactivated, or live-attenuated pathogens, modern vaccines containing isolated, highly purified antigenic protein subunits are safer but tend to induce lower levels of protective immunity. One strategy to overcome the latter is to design antigen nanoparticles: assemblies of polypeptides that present multiple copies of subunit antigens in well-ordered arrays with defined orientations that can potentially mimic the repetitiveness, geometry, size, and shape of the natural host-pathogen surface interactions. Such nanoparticles offer a collective strength of multiple binding sites (avidity) and can provide improved antigen stability and immunogenicity. Several exciting advances have emerged lately, including preclinical evidence that this strategy may be applicable for the development of innovative new vaccines, for example, protecting against influenza, human immunodeficiency virus, and respiratory syncytial virus. Here, we provide a concise review of a critical selection of data that demonstrate the potential of this field. In addition, we highlight how the use of self-assembling protein nanoparticles can be effectively combined with the emerging discipline of structural vaccinology for maximum impact in the rational design of vaccine antigens. Through their power to prevent or reduce the burden of infectious diseases they make an enormous global impact by improving the life quality of both humans and animals. Vaccines may save up to three million children’s lives and up to six million total lives each year However, there are many important pathogens against which vaccines do not yet exist, and some current vaccines could be improved. For example, some vaccines do not protect against all circulating strains of a pathogen because many microbes have developed sophisticated mechanisms to escape the host immune system. Mutations on the antigens of microbes such as influenza (flu), human immunodeficiency virus (HIV), or meningococcus constitute a rapidly changing ‘disguise’ to avoid recognition by trained immune cells that might otherwise prevent infection or disease. Further, some vaccine antigens do not elicit sufficiently durable or potent immunity. In addition to this, the rise of drug-resistant pathogenic entities such as those causing shigellosis demands our attention in the search for proficient vaccines Many effective vaccines developed in the past used live-attenuated strains of a pathogen, or inactivated killed pathogens One of the earliest examples of a self-associating protein particle was reported in the 1950s: a protein extracted from the tobacco mosaic virus (TMV) was found to form rod-shaped particles, which morphologically resembled the original TMV but which did not contain genetic material Escherichia coli (E. coli) or eukaryotic systems, nanoparticles are at the frontline of new vaccine therapeutics.What are the advantages of nanoparticle antigens? Key parameters governing the elicitation of an efficient immune response to a microbe include both antigen density and distribution on the pathogen surface A variety of naturally occurring proteins can self-assemble into nanoparticles that are highly symmetric, stable, and structurally organized, with diameters of 10–150 nm de novo engineering of protein assemblies is challenging. Early proposals to rationally perform atomic and molecular manipulations to generate interesting new materials were mentioned by Richard Feynman in 1960 While many natural proteins have acquired self-assembling properties during evolution Saccharomyces cerevisiaeSince the emergence of nanoparticle vaccine antigens in the 1970s, numerous attempts to generate plain or chimeric nanoparticles with scaffolds from many origins have been described. Some of these are virus-like particles (VLPs) composed of single or multiple viral antigens, in some cases anchored in a lipid bilayer. Structural proteins from different microorganisms have served as templates for the production of such nanoparticles and for the presentation of immunogenic epitopes: the protein pIII of the filamentous phage f1 22.1Many viruses encode proteins that form stable nanoparticle structures, which self-assemble in infected host cells in order to package the viral genomes as a pre-requisite for propagation. However, if scaffold proteins are assembled in the absence of genetic material, then non-infectious non-replicating virus-like particles (VLPs) that closely resemble intact virions can be obtained. Consequently, viral nanoparticles or VLPs composed of one, or a few, recombinant self-assembling proteins were among the first antigen-nanoparticle candidates, some of which have indeed been developed into successfully marketed vaccines 3 capsid protein of the foot-and-mouth disease virus (FMDV), which was demonstrated to be a safe, stable and effective polypeptide vaccine for cattle and swine in vitro-produced vaccine to protect humans against HBV. In 1986, licensure was obtained for the first human vaccine based on a recombinant protein subunit antigen (HBsAg), which self-assembles into nanoparticles In the second half of the 20th century, the first genetically engineered vaccine was developed by cloning the VPPerhaps the first real VLP human vaccine is the successful story of two similar vaccines protecting against HPV infection, a major cause of anogenital disease and, especially, cervical cancer Several high-resolution crystal structures of different HPV L1 proteins have been determined, revealing the molecular basis for their oligomerization into the pentameric assembly unit of the viral shell In addition to the research and clinical development of the HPV nanoparticle vaccines, numerous other viruses  Plasmodium falciparum  malaria parasite genetically fused either to HBcAg P. falciparum NF54, is the key component of the RTS,S (Mosquirix) vaccine for which in mid-2015 the European Medicines Agency expressed a positive scientific opinion, following large-scale safety and efficacy data from phase III clinical trials 1–6 epitope was conjugated for display as an immunotherapy against Alzheimer disease Indeed, in the long search for a vaccine against malaria, successful clinical trials have been performed using chimeric nanoparticle antigens containing epitopes from the circumsporozoite protein (CSP) of the E. coli and which exposes a reactive Lys genetically engineered into the major immunodominant region of HBcAg Interestingly, HBcAg was recently used in successful preclinical studies as a nanoparticle scaffold presenting a structurally optimized antigen against respiratory syncytial virus (RSV), a leading cause of severe respiratory tract disease in children worldwide Hepatitis E virus (HEV) constitutes another successful example of the generation of recombinant VLPs with efficacy in preventing the progress of the infection. While insect cells have been used to prepare HEV particles of a truncated version of the HEV viral capsid protein that yielded an antigenicity similar to that of original HEV viral particles The need for next-generation vaccines against influenza has been one of the biggest drivers of research into novel VLP antigens. For several decades, influenza vaccines have been successfully produced using embryonated chicken eggs Nevertheless, a novel approach for the prevention of influenza in individuals aged 18 and older has been achieved with the development of Flublok, the first recombinant influenza vaccine to have been licensed. This trivalent vaccine contains recombinant HA proteins of three strains of seasonal influenza virus E. coli as a suitable low-cost scalable production system for VP2–VP6 rotavirus VLPs that elicited a strong antibody response and protection against RV-induced diarrhea in mice While influenza is one of the most promising targets for recombinant VLP technology, other viral targets have proved more challenging, especially when several viral proteins are required for VLP assembly Attempts to make HIV vaccines have also included various different VLPs, mostly involving lipid-enveloped assemblies where the internal HIV Gag p24 provides support for exposure of trimers of Env, the crucial HIV target antigen for neutralizing antibodies. A number of preclinical studies have shown the potential of this approach to efficiently induce humoral and cellular immune responses Human norovirus (NoV) causes endemic viral diarrheal disease and may cause up to half of all gastroenteritis outbreaks worldwide, and therefore is gaining increasing attention as another pathogen causing a globally significant healthcare burden The viral examples described above clearly demonstrate the success, and future promise, of using highly purified non-enveloped viral capsid proteins as plain or chimeric antigen nanoparticles. However, enveloped VLPs, due to the inclusion of a lipid bilayer in addition to the structural protein antigens, represent a more complex challenge that is now being partly overcome via breakthroughs in mammalian and insect cell expression technologies. Unlike the simpler non-enveloped viral protein nanoparticles, enveloped VLPs have more inherent safety issues due to potential contaminations from the expression system used, and may be more challenging to formulate with long-term stability profiles. Nevertheless, several enveloped VLP candidates are now in clinical trials.2.2Helicobacter pylori ferritin is made of 24 identical protomers In addition to viral nanoparticles or VLPs, there are many other naturally occurring self-assembling protein nanoparticles that have been identified from a wide variety of sources Ferritin self-assembles into nanoparticles with robust thermal and chemical stability. Hence, the ferritin nanoparticle is potentially well-suited to carry and expose immunogens. Moreover, since ferritin is composed of eight units each with three-fold axis symmetry, it is a convenient scaffold for the presentation of trimeric antigens. Indeed, Nabel and co-workers reported an elegant structure-based design strategy to generate ferritin nanoparticles genetically engineered to present a multivalent array of the flu virus hemagglutinin (HA) with its native trimeric conformation intact. HA is the key antigenic component of flu vaccines, and immunization of mice with these HA-ferritin nanoparticles yielded a very promising outcome: compared to a current commercial vaccine, the animals responded with a more potent immune response, as illustrated by the notably higher number of neutralizing antibodies, enhanced breadth of coverage against unmatched H1N1 viruses and increased generation of neutralizing Abs against two H1N1 highly conserved, yet independent, flu epitopes Another similar study was also reported by the Nabel team, wherein the conserved receptor-binding domain (a site of vulnerability) of the gp350 antigen from Epstein–Barr virus was presented in a structurally optimized orientation on nanoparticles of ferritin (24 subunits) or encapsulin (60 subunits). In preclinical studies, the chimeric nanoparticle-gp350 antigens elicited 10- to 100-fold more potent virus-neutralizing antibody titers than the soluble gp350 antigens alone in silico studies to optimize molecular scaffolds for epitope presentation and leading to the generation of recombinant ferritin nanoparticles displaying epitope-scaffolds harboring E1 or E2 epitopes from hepatitis C virus, promising candidates for preclinical studies in the quest for an HCV vaccine Also, very recent is the work by He et al., describing Ferritin has also been used as antigen support in the search for potent and safe vaccine tools against HIV http://www.avert.org/worldwide-hiv-aids-statistics.htm), the lack of a vaccine is an enormous unmet medical need. A key challenge in designing an anti-HIV vaccine is the high mutagenic capability of the virus and the unfeasibility of administering attenuated or killed virus because of safety issues. An additional hurdle is the negligible recognition potential of germline precursors of bNAbs, such as VRC01, against the wild-type gp120, the major immunogenic component of the HIV virus envelope. One way to overcome this obstacle was recently reported by Schief and co-workers Lumazine synthase (LS) represents another example of the inclusion of a bacterial particulate base for the optimization of vaccine candidates, as reported recently by Jardine et al. in their attempts to enhance the immunoreactivity of recombinant gp120 against HIV infection LS, which is responsible for the penultimate catalytic step in the biosynthesis of riboflavin, is an enzyme present in a broad variety of organisms, including archaea, bacteria, fungi, plants, and eubacteria Aquifex aeolicus as a nanoparticle platform for epitope display, Jardine et al. succeeded in increasing the potency of the immune response and breadth of coverage against HIV. The envelope (Env) glycoprotein is the only HIV surface protein targeted by neutralizing antibodies; it is made of three gp160 precursors that trimerize and are each then cleaved into gp120 and gp41 subunits. Jardine et al. engineered LS to display an optimized sub-component  of the wild-type gp120 antigen from the Env trimer Using LS from the thermophilic bacterium 2.3A method to obtain protein micelles from full-length amphiphilic membrane proteins was developed and used to prepare viral surface proteins as water-soluble particles with a hydrophobic interior and a polar exterior, of relatively homogeneous size: approximately 20–30 nm diameter, depending on the protein The micellar nanoparticle approach has also been exploited in the search for vaccines against the Coronaviridae virus (CoV) family, which represents an important group of emerging human pathogens, as witnessed in the severe acute respiratory syndrome SARS-CoV and Middle East respiratory syndrome MERS-CoV outbreaks of 2003 and 2012, respectively. Recombinant full-length forms of the major immunodominant CoV antigen—the amphiphilic spike glycoproteins—both from SARS-CoV and MERS-CoV were successfully obtained via non-ionic detergent-extraction from Sf9 cells. The purified spike proteins assembled into nanoparticles of ~ 25 nm diameter that, in adjuvanted formulations tested in mice, were capable of raising high-titer neutralizing antibody responses against the homologous virus 2.4de novo-designed trimeric subunit domain. Both subunits present oligomeric coiled-coil conformations and importantly, the resultant synthetic molecule was shown to refold and self-assemble into nanoparticles with polyhedral symmetry. Alternative oligomerization motifs such as the trimeric foldon domain from fibritin have also been used in such designs Proposals to perform molecular manipulations that, by exploiting chemical forces in a repetitious fashion, could lead to the production of interesting materials that date back at least to the 1960s Plasmodium berghei elicited a long-lasting immune response Indeed, using the polypeptide approach, the Burkhard team fused the C-terminal heptad repeat (HRC) region of the SARS-CoV spike protein in its pre-fusogenic state in frame with the nanoparticle scaffold in silico and crystallographic studies have further developed nanostructure design strategies, including the generation of particles over 22 nm in diameter ab initio design of new protein-protein interfaces with defined symmetry, geometry, and complementary packing arrangements, and the increasing number of protein structures in the PDB, it is speculated that additional achievements in the field of self-assembling protein design will be possible Early in the 21st century, Yeates and co-workers developed the nanohedra protein-design method, which was subsequently extended by Noble and co-workers 3Despite many successes in the field of vaccinology, new breakthroughs are still needed to protect humans from several important life-threatening diseases. Here, we have reviewed how a variety of non-infectious biological nanoparticles can offer solutions. For example, some plain nanoparticles (e.g. HBsAg or the HPV L1 protein) are simple molecular self-assemblies that are safe and efficacious vaccine antigens licensed for human use. Or, more complex chimeric nanoparticles can be platforms on which pathogen-derived immunogenic motifs can be presented to the host immune system. These biological scaffolds range from synthetic polypeptides to native macromolecules such as ferritin, lumazine synthase or VLP-forming antigens or lipid-enveloped VLPs. For some chimeric nanoparticles, there is evidence that the immunogenicity of the platform carrier itself is negligible or low compared to that of the mounted immunogen being presented ab initio design of self-assembling molecules is becoming ever more possible, thus enriching our molecular repertoire of nanoparticle scaffolds. Consequently, the design of plain or chimeric nanoparticle antigens, and the ability to manufacture these recombinantly in prokaryotic or eukaryotic systems, to make safe and effective immunogens, is becoming a reality. We have shown that vaccination clinical trials against a broad range of diseases are ongoing. While there are established examples of successful vaccines against diseases of viral and parasitic origin , the next decade may consolidate the use of multivalent nanoparticles as a therapeutic tool for the future against infectious diseases of other origins  but also against cancers and other disorders such as hypertension, asthma, or addictions (e.g. smoking). These amazingly versatile tools may also get us closer to universal vaccines against highly variable pathogens such as HIV, influenza, or meningitis. Continued efforts are still needed, but there is a well-founded optimism that further studies of nanoparticles and VLPs together with the implementation of structural vaccinology, facilitated by emerging B-cell cloning and antibody production technologies, will be translated into new and second-generation vaccines that will contribute to saving more lives worldwide.Recent preclinical studies have demonstrated how computational and structural biology can be combined for the rational design of well-oriented arrays of the most protective epitopes of a pathogen, in a manner suitable to raise the most desired immune responses. Further, the computational The authors are employees of GlaxoSmithKline Vaccines S.r.l., via Fiorentina 1, 53100 Siena, Italy.


Accumulation of soft tissue or dentinal remnants in the apical region is a common event that can cause blockage of root canals. This event can be avoided if apical patency is performed during the root canal shaping procedures. However, there is no consensus on the role of apical patency in relation to the success of endodontic treatment of necrotic teeth with apical periodontitis. Therefore, the purpose of this paper was to conduct a brief review on the role of apical patency in guaranteeing the success of endodontic treatments of necrotic teeth with apical periodontitis considering two other key points; the root canal anatomy and microbiology.  Blockage of the root canal in the apical region by remnants of dental hard and soft tissue debris, may cause procedural errors such as apical transportations, ledge and perforation. These debris may also contain bacteria capable of maintaining or inducing periradicular disease -4. For tThe most common method for performing this procedure is to use a so-called patency file during root canal instrumentation. This file can be defined as a small flexible K-file, which is moved passively through the apical foramen without widening it . In addiWhereas most studies are merely speculative regarding the advantages and disadvantages of performing or not apical patency -9, the pAnatomical considerationsAn unchangeable high-impact factor that remains in endodontics is the root canal anatomy. The complexity of this factor has been verified by several methods over the years such as diaphonization, microscopic investigation, radiographic methods, scanning electron microscopy (SEM), cone-beam computed tomography (CBCT) and computed micro-tomography (micro-CT) -26. Thiset al. [et al. [Morfis et al.  found noet al.  found a  [et al.  on the p [et al. , 30. How [et al. . Even in [et al. , 27-32.MicrobiologicalconsiderationsThe endodontic treatment of vital teeth is essentially a prophylactic treatment, because the pulp space is usually free of microorganisms, and the prevailing rationale for treatment is to prevent probable infection and consequent periradicular disease -39. On ti) when they are able to survive without nutrients; ii) when they resist treatment-induced disturbances in the ecology of the bacterial community, ; iii) when they reach a climax population density (load) high enough to inflict damage on the host; iv) when they possess attributes of virulence expressed in the modified environment and reaching concentrations strong enough to directly or indirectly induce damage to the periradicular tissues and v) when they have unrestrained access to periradicular tissues through apical/lateral foramina [Persistence of bacteria can influence the outcome of the endodontic treatment in five possible ways: foramina . Specifically related to the last factor mentioned above, apical patency cannot provide any advantage, because there is no real possibility of reaching microorganisms in lateral canals and apical deltas using the techniques currently known for this procedure.Can apical periodontitis heal after endodontic treatments without performing apical patency?Many investigators have retrospectively evaluated the influence of various factors that may affect the outcome of root canal treatments or retreatments -54. In tApical patency is not always achieved. Severe curvatures, broken instruments, miscalculation of canal length, development of ledges, obliteration and the anatomy of the root canals are among the most common reasons -64. HoweFailure of nonsurgical root canal treatment or retreatment is usually related to residual bacteria (persistent infection) or reinfection of an already disinfected root canal environment (secondary infection) , 65-70. a) a high bacterial load diminishes substantially after correct instrumentation of the cervical and middle thirds; b) in several situations, even when apical patency is not achieved using files, chemical apical patency may occur using irrigating solutions, medications and sealers that are able to "disrupt microbiologically" this region; and c) only the main canal can be subjected to the effective action of apical patency files, whereas a large number of lateral and smaller canals cannot be reached. The endodontic literature confirms these statements, whereas most cases of endodontic failure, evidenced by histological findings, are associated with microorganisms in lateral canals and apical deltas and/or with extra-radicular biofilms [Therefore, the postulation of periradicular disease perpetuation simply because apical patency was not obtained, is a very questionable inference. This statement is mainly based on the following considerations: biofilms , 67, 69.This paper demonstrated that apical patency may not be strictly necessary However, there are no robust clinical or scientific evidences showing a direct correlation between apical patency and success of endodontic treatments of necrotic teeth with apical periodontitis.


Radiation therapy has been evolving with technological advances in accelerator, computer, and imaging. As the planning target volumes (PTVs) are made increasingly conformal with the advent of three-dimensional conformal therapy (3D CRT) and intensity-modulated radiation therapy (IMRT), the requirements of precise PTVs localization and its dosimetric coverage for each treatment become more stringent. In 3D CRT, based on 3D anatomic information, treatment planning is designed to deliver dose distribution that conforms as closely as possible to the target volume and minimizes the dose to the critical organs. The advantage of IMRT is to treat a patient from a number of beams of different directions (or continuous arcs) with nonuniform fluences, which have been optimized to deliver a high dose to the target volume and an acceptable low dose to the surrounding normal tissue. The treatment planning program divides each beam into a large number of beamlets and determines their optimum fluences. This optimization process involves inverse treatment planning. Prediction of normal tissue complication probability (NTCP) after external beam radiotherapy (EBRT) is an important issue in the optimization of a treatment plan and should be considered because during EBRT a considerable volume of normal tissues receives radiation dose along with the tumor. Normal tissue complication probability (NTCP), in a normal tissue, is a function of delivered dose and irradiated volume of the normal tissue. Image-guided radiation therapy (IGRT) procedures employ imaging technology to help patient setup and target localization before and during treatment. Difficulties and problems associated with target localization may arise from inter- and intracranial variations in patient setup and anatomy, including shapes and volumes of treatment target and surrounding normal tissues. Some of current IGRT image guidance technologies include portal (MV) and radiographic (kV) imagers, in-room CT scanner, kV cone-beam CT, MV cone-beam CT, helical tomotherapy MVCT, and ultrasound.Dose verification for complex treatment techniques such as IMRT, SRS/SBRT, and brachytherapy is crucial. These complex treatments present high dose gradient regions in the boundaries between the target and surrounding critical organs. Dose accuracy in these areas can be critical and may affect treatment outcome. A dose verification phantom designed for advanced technology in radiation therapy clinical trials can serve as a tool for quality assurance program.Respiratory motion affects all tumor sites in the thorax and abdomen, although the disease of most prevalence and relevance for radiotherapy is lung cancer. Studies have shown that lung tumors can move several centimeters in any direction during irradiation. If respiratory motion is not accounted for, it causes artifacts during image acquisition. These artifacts cause distortion of the target volume and incorrect positional and volumetric information , 2. A prMost radiation therapy treatment planning systems now incorporate three-dimensional anatomy information attained by computed tomography (CT) images for registration. These images can be fused with magnetic resonance imaging (MRI) or positron emission tomography (PET) images for better delineation of the target volume. MRI is considered superior to CT in soft-tissue discrimination such as central nervous system tumors and abnormalities in the brain. MRI is also used in imaging head and neck cancers, sarcomas, the prostate gland, and lymph nodes. On the other hand, CT imaging is more sensitive to bony structures and calcification.Stereotactic radiosurgery (SRS) is a single-fraction radiation therapy procedure for treating intracranial lesions or a target volume close to critical structure, using a stereotactic apparatus and multiple small-field beams. The same procedure when used for delivering hypofractionated radiation treatment is called stereotactic radiotherapy (SRT). SRS and SRT involve three-dimensional imaging to localize the lesion and delivering a concentrated dose to the target volume while sparing as much as possible the normal tissues. Two common SRS techniques are available: linac-based X-ray knife and the Co-60 gamma-knife. Light ion  beams have also been used for SRS and SRT . Local rclinical trials and outcome research;new technologies development and implementation;treatment delivery techniques;disease specific treatment discussion;radiation dosimetry analysis;radiation protection, shielding, and design;clinical therapy physics review and applications;molecular imaging application in radiation therapy;medical imaging;professional issues in medical, clinical, and biomedical physics;radiobiology;quality control and assurance;computing algorithm and optimization;quality of life analysis;radiation safety.This editorial provides comprehensive introduction to technological advancements in radiation therapy and many important topics associated with these treatment technologies. Clinical data on these new technologies are provided by many institutions.This special issue is focusing on the new development in cancer therapy, quality control, radiotherapy techniques, radiation dosimetry, and clinical outcome studies. This special issue included various topics which have been discussed from researchers of the following:Tsair-Fwu LeeJack YangCheng-Shie WuuAn LiuFu-Min FangShyh-An Yeh


Paenibacillus dendritiformis produces a lethal protein (Slf) that is able to induce cell death in neighbouring colonies and a phenotypic switch in more distant ones. Slf is derived from the secreted precursor protein, DfsB, after proteolytic processing. Here, we present new crystal structures of DfsB homologues from a variety of bacterial species and a surprising version present in the yeast Saccharomyces cerevisiae. Adopting a four-helix bundle decorated with a further three short helices within intervening loops, DfsB belongs to a non-enzymatic class of the DinB fold. The structure suggests that the biologically active Slf fragment may possess a C-terminal helix rich in basic and aromatic residues that suggest a functional mechanism akin to that for cationic antimicrobial peptides.Bacteria have developed a variety of mechanisms for surviving harsh environmental conditions, nutrient stress and overpopulation.   •Crystal structure of DfsB reveals a non-enzymatic variant of the DinB fold.•Structural features are conserved in prokaryotic and eukaryotic DfsB homologues.•The sibling lethal factor processing site is highly solvent exposed and within a flexible loop.•The mature sibling lethal factor resembles a cationic antimicrobial peptide. Paenibacillus are exemplary in this regard, forming a variety of morphotypes on semi-solid agar: chiral branches, swirls and vortices Paenibacillusdendritiformis adopts two distinct growth patterns termed Chiral and Branching (tip splitting). Moreover, when the same stock of cells is spotted onto an agar plate a few centimetres apart, the colonies grow outwards evenly in all directions but minimally in the direction of the opposite colony PaenibacillusBacillus subtilis cells were immune to its effects, which may suggest a degree of specificity for Slf action.Bacteria possess a variety of mechanisms for survival under harsh environmental conditions, nutrient stress and overpopulation. Furthermore, some bacteria display a remarkable ability to grow into complex patterns dfsB gene is widely distributed across Gram-positive and to some extent Gram-negative bacterial kingdoms (PFAM entry DUF1706). Moreover, the presence of a close homologue of dfsB in Saccharomyces cerevisiae hints that yeast can influence the viability of co-cultured bacilli.The precursor PdDfsB is cleaved by subtilisin to yield biologically active Slf, and the chemical gradients of both proteins between two growing colonies could be accurately modelled. In summary, the two advancing colony fronts release subtilisin and DfsB, and as they approach one another, the concentrations of these proteins exceed a threshold and the subsequent formation of Slf induces the death of nearby cells and sporulation in those further away. Given the specificity and pattern-inducing phenotype associated with Slf, one might expect it to be limited to species that display these complex growth behaviours. However, remarkably, the B. subtilis protein DinB (DNA-damage inducible) and the ability to coordinate a metal via conserved histidines  within the yeast genome. We explored the phylogenetic distribution of these DUF1706 family sequences, using CLANS to perform pair-wise alignments and visualise the various clades . The overall structures of CdDfsB, EcDfsB and IRC4 are identical with that of PdDfsB, with strong conservation of overall topology and surface charge, particularly in the C-terminal helix .We sought to characterise a selected subset of DfsB homologues structurally, focussing on the three main clades shown in revisiae . For theStreptococcus DfsB homologues , both monomers and stable dimers could be identified (S. agalactiae DfsB dimer (SaDfsB; During purification of the two entified . We atte SaDfsB;  by molecStreptococcus mutans, do not possess the full DfsB precursor but, instead, express a shorter polypeptide that is essentially the isolated Slf fragment. Although it remains possible that these are pseudogenes, it is intriguing to consider the fact that DfsB/Slf is highly specific towards its own species and that many bacteria—including those that are human pathogens—possess a DfsB homologue. The most strongly conserved region of PdDfsB is the C-terminal helix, which is present within the mature Slf sequence. The pattern of conserved positive charge and aromatic positions (We found that some species, for example, ositions f is remi5CIV, 5COM, 5CQV, 5COF and 5COG.Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers