Peptides and peptidomimetics have attracted revived interest regarding their applications in chemical biology over the last few years. Their chemical versatility, synthetic accessibility and the ease of storage and management compared to full proteins have made peptides particularly interesting in diagnostic applications, where they proved to efficiently recapitulate the molecular recognition properties of larger protein antigens, and were proven to be able to capture antibodies circulating in the plasma and serum of patients previously exposed to bacterial or viral infections. Here, we describe the development, integration and application of strategies for computational prediction and design, advanced chemical synthesis, and diagnostic deployment in multiplexed assays of peptide-based materials which are able to bind antibodies of diagnostic as well as therapeutic interest. By presenting successful applications of such an integrated strategy, we argue that they will have an ever-increasing role in both basic and clinical realms of research, where important advances can be expected in the next few years.  Peptides are extremely versatile molecules with a wide range of applications. Peptides are attractive for chemists and chemical biologists thanks to the rich diversity of chemical functionalities they can potentially display. Indeed, amino acid building-blocks recapitulate all of the main traits of organic chemistry reactivities through their side chain groups, which range from hydrophobic and aromatic, to hydrophilic, acid, basic, and charged. The synthetic accessibility of linear chains through solid-phase methods, combined to the possibility to further derivatize side chains, opens access to a wide spectrum of chemical diversity and reactivity for the products. It comes as no surprise that the use of peptides and peptidomimetics in drug discovery in the development of peptide-based materials for medical devices, and even in vaccinology, has recently met with renewed enthusiasm. Indeed, peptides play key roles in a number of biochemical pathways in and outside cells, making the discovery of artificial sequences (or mimics) which are able to either interfere or synergize with the respective natural processes an attractive strategy towards the production of new materials: as an example, the advantages they can offer as drugs are specificity, potency, and low toxicity. However, practical hurdles such as their poor stability, short half-life, and the fact that they are targeted by proteases have somewhat limited the realization of their full potential in medicinal chemistry. While these limitations still call for significant efforts in the pharmacological setting, peptides and peptide-based derivatives can certainly offer great opportunities in the field of probe development for disease diagnostics.In this framework, peptide microarrays are appealing tools as they allow the simultaneous screening of hundreds to thousands of different sequences  immobilized in spatially ordered patterns of spots on solid supports ,2. AfterPeptide microarrays are increasingly being used in the biomedical area for the screening of enzyme function and inhibition  and to pDespite great improvements in the time-to-result and the excellent sensitivity and specificity provided by nucleic acid-based approaches to test for infectious diseases  ,7, serolIn this context, it is now well appreciated that, in many disease conditions, antibody-mediated responses may precede the manifestation of actual symptoms, sometimes even for a long time. This holds true for bacterial and viral infections. If the immune system has responded to a threat with a B-cell response, it is reasonable to assume that circulating antibodies, elicited specifically against molecular targets on pathogenic factors, could aptly represent efficacious biomarkers of the disease. The capture and detection of such antibodies expectedly requires the availability of their respective antigens as probes. To this end, the use of whole (attenuated) pathogens poses relevant problems as micro-organisms may be hard to culture in vitro, and therefore their production is impractical. The advent of reverse vaccinology has given a huge boost to the discovery of tools that are more manageable and amenable to use as molecular probes. Reverse vaccinology applies bioinformatic methods to mine pathogen genomes to identify protein antigens (Ag) that are typically expressed on the outer membrane surface, and are thus most prone to be recognized by molecules of the immune system ,9,10,11.A plausible way to overcome these obstacles would be to identify the regions of the antigen that are specifically recognized in the course of an immune response. Chemical and structural information on such regions, called epitopes, could then be used as template to develop mimics that bind specifically to the antigen-binding pocket of the desired disease-relevant antibody. Peptides and peptidomimetics represent the ideal chemical tools to realize small epitope mimics that recapitulate the essential determinants of immune-reactivity and Ab-recognition of the cognate full-length antigen.At this point, two main problems need to be tackled. The first lies in the identification of the epitope region on an antigen, most often in the absence of detailed X-ray information on the relevant antigen–antibody complex. In this context, sequence and structure-based computational methods have played an increasingly relevant role in recent years, facilitating the identification of molecules which are able to efficiently engage disease biomarkers. The second concerns the optimization of the presentation of the synthetic immunoreactive molecule(s) on diagnostic devices to efficiently capture the desired Abs. Circulating antibodies produced against disease-specific antigens can in fact be detected by passing patient fluids  over an immobilized bait and then measuring the amount of antibody retained with a labeled secondary antibody. Novel methods of oriented display, as well as the use of optimized surfaces for the attachment of the probes, can aptly provide a significant increase in the performances of novel diagnostic tools. From this perspective, we will present and discuss the work, views and methods we have developed and integrated in our laboratory to discover, design, synthesize, and display novel peptide-based molecules with the aim of realizing the potential of epitope-mimicry in the development of efficient diagnostic methods for the detection of disease biomarkers. The initial condition to start the design of small peptide-based mimics of the immunoreactive regions of antigens depends on the capability to correctly locate the privileged substructures that are preferentially engaged in antibody binding. In this context, computational approaches have played a constantly increasing role over recent years. Sequence-based methods and algorithms based on the combination of descriptors reporting on solvent exposure, hydrophobicity profiles, and geometrical properties have provided important results ,19,20,21However, it has become increasingly clear that a combination of factors spanning the sequence, structure, dynamics and location of specific protein regions determines the cross-talk between the antigen and the antibody. In principle, the identification of (surface-exposed or exported) antigen substructures that display peculiar sequence and dynamic properties is the basis for the design of peptidic interactors as probes for Ab detection. In this framework, epitope sequences tolerate mutations, suggesting that they are not involved in the stabilization of the antigen fold. These sites continuously evolve to escape recognition by the immune system of their host, without disrupting the global 3D fold required for the function of the protein they belong to, which would ultimately be detrimental for the survival of the pathogen. Furthermore, epitopes are in general flexible and support extensive conformational fluctuations which underlie the conformational selection mechanisms that underpin antibody binding. Finally, epitopes define large exposed patches on the protein surface. N residues, the N × N matrix (ijM) of average nonbonded interactions between pairs of residues can be built by averaging over the structures visited during an MD trajectory. The resulting energy matrix is then simplified through eigenvalue decomposition. The analysis of the N components of the eigenvector associated with the lowest eigenvalue was shown to identify residues that behave as maximally strong or maximally weak interaction centers. The principal eigenvector can be used to reconstruct a simplified version of the energy matrix, which recapitulates the residue pairs with maximal and minimal couplings with the native structure of the protein. The simplified map is next filtered with topological information to identify patches of local couplings characterized by energetic interactions of minimal intensities. Low-intensity couplings between distant residues in the structure are a trivial consequence of the distance-dependence of energy functions. However, low-energy couplings between residues that are proximal in the folded 3D structure of the protein identify those sites which are particularly frustrated (non-optimized), which can therefore be regarded as the preferential perspective points of interaction with binding partners. Moreover, such regions would tolerate mutations without dramatic consequences on the antigen three-dimensional structure [To put these considerations in a quantitative predictive model, we introduced MLCE (matrix of low coupling energies) ,23. The tructure ,25,26. Because it is based on a preliminary characterization of the structural ensemble of the antigen by molecular dynamics (MD) simulations, the method naturally takes into account information on the conformational transitions that underlie the adaptation to the respective antibody to form a complex. Overall, MLCE showed the unique ability to provide information on the epitope sequence, structure and flexibility. A pictorial scheme of the method is reported in The performances of the method were first benchmarked on a set of proteins whose complexes had been solved by X-ray crystallography. We then extensively applied it to the prediction of initial candidate probes for the early diagnosis of bacterial and viral infections. Burkholderia pseudomallei infections. Successful application was reported for the identification of new peptide-based diagnostic probes to detect Burkholderia pseudomallei is the etiological agent of melioidosis, a pulmonary infection that is widely diffused in South East Asia and Northern Australia [ustralia . The bacIn this context, we started from the knowledge of the 3D structure of a number of protein antigens and applied the MLCE approach to predict the sequence, structures and location of candidate epitopes ,32,33,34B.pseudomallei-infected patients. In one particular case , an appaThe MLCE approach was subsequently applied to the problem of developing diagnostic probes for ZIKA virus (ZIKV) infections. We focused on ZIKV non-structural 1 (NS1). In this case, MLCE identified a putative antigenic region of NS1. In this case, the approach returned a region of the protein encompassing different secondary structure motifs. Once realized and combined for co-presentation to patient sera, several candidates were found to be effective in discriminating ZIKA-positive vs. healthy samples .It is worth noting here that other computational and experimental approaches have been proposed and shown to perform with good success.Fiorucci and Zacharias introduced the electrostatic desolvation penalty (EDP) method . This apEllipro represents another method that does not require training . The worThe computer method SEPPA  makes usPEPITO combines propensity scales with surface exposure information ,37 and uSela-Culang and coworkers introduced a mixed computational experimental method to B-cell epitope identification . Using tOther exquisitely experimental approaches to epitope identification/characterization involve mass spectrometry (MS) approaches based on hydrogen/deuterium exchange (HDX), phage display based methods, and high-throughput synthesis and analyses. HDX-MS for epitope discovery is generally coupled to limited proteolysis approaches in epitope mapping problems: specifically, an antigen is digested by an array of proteases in the presence or absence of its antibody. The fragmentation patterns resulting from proteolysis are compared ,40,41. TPhage display represents another method to select linear and discontinuous epitopes. The latter can be selected using purified antibodies or preparations of polyclonal serum. In this approach, linear epitopes as well as structurally organized ones, constrained by disulphide bonds, can be displayed on very large phage libraries. Antibodies are subsequently used to pan the epitopes. The targets that get recognized can be sequenced to determine the sequences. Further popular strategies for epitope mapping consist of testing the binding of antibodies against multiple peptide sequences that are prepared with medium to high-throughput synthetic techniques. In some cases, overlapping sequences spanning the whole antigen sequence are tested for their antibody-binding capability; peptides found to bind are used to define an epitope. One potential drawback of this approach is that it may miss the recognition of discontinuous conformational epitopes. Based on these concepts, Kodadek and coworkers have shown impressive results in the identification and optimization of chemical tools to manipulate antibody recognition and capture pathogenic antibodies related to chronic lymphoid leukemia (CLL) or autoimmune disorders with high selectivity. To this end, they exploited a high-throughput screening protocol that used a large library of bead-displayed oligopeptides, generated via split-and-pool synthesis. This approach first concentrated on removing ligands, as the antigen-binding sites of antibodies that are not disease-related are first removed from the pool. Next, to identify ligands to disease-related antigen-binding sites, the remainder of the library was incubated with a soluble version of a patient-derived CLL. For an antibody in the presence of a large excess of competitor proteins, the antibody-binding beads were identified by the binding of a secondary antibody linked to a fluorescent probe. These active binding compounds were thus isolated and characterized with mass spectrometry. With this approach the authors are able to identify and further develop sequences or peptidomimetics that bind in the low nanomolar range. These approaches and their subsequent application in therapy and diagnostics are described at length in Doran et al. The structure-based prediction of potentially reactive sequences is a necessary first step in any project aimed at developing new molecules with interesting applications. Complementary to this, the presentation/display of designed sequences in the best spatial orientation for immunoreactivity with patients’ samples represents a fundamental step in the quest for devices with optimal performances. Peptide microarrays are composed by a variable number of peptides either in situ synthesized step by step on a suitable flat support or printed after individual synthesis and purification. In situ methods, based on spot synthesis of peptides (SPOT), particle-based synthesis and photolytographic methods, generate high-density arrays of thousands of peptides, potentially providing a huge amount of information by the linear scanning of aminoacidic sequences. This approach, reviewed elsewhere , is espeBurkholderia pseudomallei protein antigen PalBp [The conformational control of peptides is generally performed by stapling and cyclization techniques that are based on the formation of intramolecular covalent bonds or on the use of anchoring macrocyclic rigid scaffolds to thermodynamically favour a limited set of peptide conformations ,49. In oen PalBp . We compIn this direction, limiting heterogeneous bioprobe presentation onto analytical surfaces is crucial to guarantee consistent device performances and reliable diagnostic outcomes B.We have deeply investigated this aspect and developed new chemical strategies to gain optimal epitope display in our peptide-based bioassays. Our approach for surface modification is based on the use of polymeric coatings that rapidly self-adsorb on the analytical surface from a diluted water solution, forming an anti-fouling and hydrophilic layer bearing functional groups for the covalent immobilization of bio-probes ,51. BesiClick chemistry reactions in fact have exquisite chemoselectivity towards common functional groups that can be easily introduced into peptides providing a high-yielding and cost-effective immobilization strategy. Moreover, benign reaction conditions and fast reaction kinetics that commonly characterize this class of reactions address the need for fast immobilization well, which is particularly relevant to overcoming the intrinsically slow reactivity at the solution–solid support interface . Burkholderia infections in cystic fibrosis patients [A number of click-type immobilization strategies were compared in the frame of the peptide-based sero-diagnostics of patients . In thisOur results highlighted the challenges of real-case serological assays, where the in vivo response to immunogenic epitopes derived from infections is associated to a complex antibody repertoire that targets several conformers of the same epitope. In this type of assays, the polyclonality of patient’s response enhances non-specific interactions while the probe discrimination potential relies on a narrow border between signal detection and background noise. In this context, to gain optimal specificity and sensitivity, the whole dynamic distribution of peptide epitope conformers must be fully accessible to the range of antigen-directed elicited antibodies. To this end, any strategy targeted at enhancing the affinity of the immobilized probes for disease-specific antibodies, including space-oriented display of epitopes, fine control of probe density and multivalent presentations, is beneficial to the delivery of robust clinical tests . A fine-tuning of the probe density and orientation may be particularly useful to discriminate infections that present a high degree of antibody cross-reactivity, such as in the case of arboviruses. In this frame, the combined use of multiple peptides could provide superior discriminative capacity than a single probe by the generation of an immune-signature. In the case of Zika virus NS1 protein, we found that antigenic response is determined by discontinuous epitopes arising from spatial arrangements of non-contiguous linear peptide sequences. This could represent a severe limitation to the applicability of peptide microarrays in diagnostic tests. To address this limitation, starting from the computationally-guided identification of a surface antigenic NS1 region, we functionally mimicked the native antigen fold through the oriented and spatially controlled co-immobilization of proximal peptide sequences forming the putative antigenic region . This straightforward method enhanced the discriminative performance of single linear peptides, expanding the toolbox for immune-diagnostic peptides to the mimicking of antigenic surfaces .As biomolecule 3D properties are so intimately linked to their analytical performances, a further and innovative step to preserve their native structure and function upon surface immobilization is to gently encapsulate them within hydrogel matrices under solution mimetic conditions. This strategy is associated with additional advantages including increased loading capacity, reduced nonspecific binding and an enhanced signal-to-noise ratio. Most of the reported hydrogels for microarrays are based on polymeric and biopolymeric matrices. However, it is often not trivial to match analyte diffusion rates through the gel matrix with stable bioprobe entrapment. In addition, laborious strategies may be required for hydrogel chip fabrication due to the viscosity of common hydrogels. We tackled these limitations by developing a nanostructured self-assembling peptide hydrogel (YF-Q11) characterized by favorable properties for 3D microarrays . These iOverall, we obtained a stable and yet highly permeable semi-wet matrix to run ultrafast (<10 min) fluorescence immunoassays under solution-like conditions. Importantly, the hydrogel matrix did not show nonspecific interactions even in the presence of complex biological samples such as sera. Further work is currently ongoing in our laboratories to consolidate and expand these results and to apply this strategy in highly challenging assays.The use of peptide-based materials for diagnostic purposes holds a great deal of potential for further development and applications. It is tempting to suggest that methods for the de novo prediction of interacting sequences can be used not only to mimic antigen–antibody interactions but complex  interactions in general ,60,61,62In this context, the improvement in multiscale methods will permit us to simulate events such as the recognition of multiple receptors on host-cells, virus entry or bacterial invasion with a level of detail that can be directly translated into new design rules for the development of biomimetic systems ,64,65. M


Transcriptomic approaches revealed thousands of genes differentially or specifically expressed during nodulation, a biological process resulting from the symbiosis between leguminous plant roots and rhizobia, atmospheric nitrogen-fixing symbiotic bacteria. Ultimately, nodulation will lead to the development of a new root organ, the nodule. Through functional genomic studies, plant transcriptomes have been used by scientists to reveal plant genes potentially controlling nodulation. However, it is important to acknowledge that the physiology, transcriptomic programs, and biochemical properties of the plant cells involved in nodulation are continuously regulated. They also differ between the different cell-types composing the nodules. To generate a more accurate picture of the transcriptome, epigenome, proteome, and metabolome of the cells infected by rhizobia and cells composing the nodule, there is a need to implement plant single-cell and single cell-types strategies and methods. Accessing such information would allow a better understanding of the infection of plant cells by rhizobia and will help understanding the complex interactions existing between rhizobia and the plant cells. In this mini-review, we are reporting the current knowledge on legume nodulation gained by plant scientists at the level of single cell-types, and provide perspectives on single cell/single cell-type approaches when applied to legume nodulation. Ulmaceae family) and rhizobia, soil bacteria capable to fix and assimilate the atmospheric dinitrogen. The establishment of nitrogen-fixing nodules requires two developmental programs, one leading to the formation of infection threads (plant-made structures through which rhizobia grow to reach the developing nodule) and one leading to nodule morphogenesis. Several molecular, physiological and cellular aspects of this biological interaction were characterized during the past two decades. For instance, the root and bacterial exudates used to initiate the recognition between the two partners are now well-characterized [e.g., plants flavonoids and iso-flavonoids  are well-documented based on their ease to be monitored under the microscope, the molecular response of the root hair cells remains poorly described, especially when considering the specific programs required at each step of the infection of the root hair cell . Having Despite this effort, the temporal regulation of the expression of the legume genes upon rhizobia inoculation was difficult to highlight. This is inherent to the nodulation process itself since new root hair cells are continuously infected and each infection is independently progressing from another. Hence, the lack of synchronization of the infection of the root hairs by rhizobia logically leads to the isolation of a heterogeneous plant material: a mixture of unresponsive root hair cells (those located outside of the susceptible zone of the root system), responsive but uninfected root hairs, and responsive and infected root hairs. The latter category could also be divided in unique populations of cells according to their stage of infection by rhizobia.M. truncatula, the inner cortex and pericycle actively divide upon rhizobia inoculation whereas, in Lotus japonicus, the outer cortex cells divide . Oppositely, in determinate nodules , the initially active nodule meristem will degenerate in mature nodules. As a consequence, the cellular organization differ between determinate and indeterminate nodules  as a major regulator of the transcriptional activity of nodule genes and transposable elements  coupled with droplet-based microfluidic systems  are emerAccessing single-cell transcriptomes is only a first step to fully understand legume nodulation. Additional avenues must be considered in order to develop a system-level understanding of legume nodulation including the integration of transcriptomic, epigenomic, proteomic, and metabolomics datasets. In addition, gene regulatory networks including the characterization of the binding sites of transcription factors controlling the nodulation process  should aML designed, wrote, and edited this mini-review.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The rumen is an important digestive organ that plays a key role in the growth, production performance and health of ruminants. Promoting rumen development has always been a key target of calf nutrition. Current research reveals that an early feeding regime and nutrition have effects on rumen development and the establishment of rumen microbiota. The effects may persist for a long time, and consequently, impact the lifetime productive performance and health of adult ruminants. The most sensitive window for rumen manipulation may exist in the postnatal and weaning period. Thus, the early feeding regime and nutrition of calves deserve further research. The establishment of the rumen bacterial community is a mysterious and complex process. The development of microbial 16S rDNA gene sequencing and metagenome analysis enables us to learn more about the establishment of rumen microbes and their interactions in host gastrointestinal (GI) tract development.Digestive tract development in calves presents a uniquely organized system. Specifically, as the rumen develops and becomes colonized by microorganisms, a calf physiologically transitions from a pseudo-monogastric animal to a functioning ruminant. Importantly, the development of rumen in calves can directly affect the intake of feed, nutrient digestibility and overall growth. Even minor changes in the early feeding regime and nutrition can drastically influence rumen development, resulting in long-term effects on growth, health, and milk yields in adult cattle. Rumen development in newborn calves is one of the most important and interesting areas of calf nutrition. This paper presents a comprehensive review of recent studies of the gastrointestinal (GI) tract development in calves. Moreover, we also describe the effect of the environment in shaping the GI tract, including diet, feed additives and feeding management, as well as discuss the strategies to promote the physiological and microbiological development of rumen. Rearing healthy calves is very important as it can have a significant impact on their growth and milk production performance in adult life. Adequate calf development is therefore crucially important for the entire dairy industry. Calves are challenged by a series of stress factors after they are born, including changes in their surroundings. Specifically, the living environment changes from the sterile uterus to natural outside conditions, in addition to changes in nutrition from that provided by the mother to the digestion and absorption of feed by calves themselves. However, due to the poor immunity and the incomplete development of the digestive system in young calves, any interference from the external environment or changes to the nutrition can drastically affect the development of calves . Some ofCompared with monogastric animals, the forestomaches of ruminants have a specialized structure and function, which results in differences in digestion and physiology between ruminants and monogastric animals. Moreover, calves have additional unique systems that are present in their digestive tract during their development. At birth, the rumen is not completely developed, and significant changes in rumen development have to occur first before the calves can digest dry feed to guarantee their own growth needs. The specific changes include the development of the rumen organ and rumen epithelium, and the establishment of rumen microbiota. Understanding rumen development in newborn calves is one of the most important focus areas of calf nutrition.The digestive system of young ruminants begins to develop during the embryonic period. For example, the stomach chambers are visible by day 56 in bovine embryos . At birtThe ruminal epithelium performs many important functions and plays the key role in rumen development, including absorption, transportation, short-chain fatty acid metabolism, and protection. The proliferation and growth of the rumen squamous epithelium promotes the growth of papillae length and width, and increases the thickness of the interior rumen wall . Work byNewborn calves have a smooth epithelium with no prominent papillae. Calves fed solely with liquid feed have been shown to have limited rumen development characterized by decrease in rumen weight, papillary growth, degree of keratinization, pigmentation and musculature development ,7. Of noDespite many studies indicating that VFA can promote the development of rumen epithelium in vivo, the in vitro results suggest the opposite. For example, butyrate treatment decreases DNA synthesis of rumen epithelial cells in culture , while tStreptococcus and Enterococcus are the early colonizers of rumen, which convert rumen to a fully anaerobic environment to promote the rapid establishment of strictly anaerobic bacteria [9 cells/mL with strictly anaerobic bacteria being predominantly found in the rumen of lambs [At birth, the GI tract of young ruminants is sterile. During the first hours of life, the forestomach becomes rapidly colonized with an abundant bacterial population. The neonates acquire bacteria from the dam, partners, feed, housing and environment. The early gut microbes of suckled lambs were mainly derived from the mother’s teats (43%) and ambient air (28%), whereas those of bottle-fed lambs were dominated by bacteria from the mother’s vagina (46%), ambient air (31%), and the sheep pen floor (12%)  . FacultaBacteroides, whereas older animals were mainly colonized with Prevotella [Bacteroides (15.8%) and Prevotella (15.1%), which may suggest that starter feed can propel rumen microbiome development to more mature status. The presence of cellulolytic and methanogenic bacteria was observed in lambs at three–four days of age, and the population of these bacteria reached a level similar to that observed in mature sheep within seven days of age [Compared to older animals, the abundance of phylum Bacteroidetes was significantly lower in one-day-old calves and was mainly composed of the genus evotella . Work byevotella  reporteds of age . Study bs of age  reporteds of age  indicateThe rumen bacterial population of two-week-old calves fed milk replacer (MR) was reported to contain 45 bacterial genera belonging to 15 phyla . SimilarThe composition of the rumen bacterial community varied significantly among individual calves, suggesting a strong host-microbiota specificity in the rumen ,23. SimiAdditionally, the bacterial composition was different among the gastrointestinal tract regions and between mucosa- and digesta-associated communities . ColonizStrategies to promote morphological structure and metabolic function of rumen in pre-ruminants are an ongoing issue which greatly attracts a lot of attention from the scientific community. Numerous studies and approaches attempt to modulate rumen fermentation and the microbial community in young ruminants to accelerate rumen development. These approaches include alteration of diet composition and physical forms, addition of new types of feed additives, and introduction of variables in the feeding management.Liquid feed may affect plasma concentration of hormones and growth factors, such as insulin and IGF-1, which play important roles in stimulating proliferation of rumen epithelial cells ,28. ColoSoybean protein can be used as an alternative to milk protein in formulating MR . PreviouFeeding readily fermentable carbohydrates to calves increases VFA production in the rumen, which is necessary to stimulate the development of rumen epithelium ,42. Calv3–N) but a lower proportion of total VFA and propionate [Rumen development can also be affected by the dietary nutrient level. Interestingly, lambs fed a high protein diet had a higher concentration of ammonia nitrogen  fingerprint of the 16S ribosomal RNA genes, and reduced the number of Ruminococcus albus in calves [Candida tropicalis in MR had no effect on the morphology of the forestomach and enzymatic activities of ruminal digesta [Aspergillus oryzae fermentation extract in MR and starter feed [However, feeding probiotics to calves has not always been shown to exert positive effects on the development of cellulolytic bacteria. For example, adding a mixture of n calves . In contn calves . Supplem digesta . Rumen ater feed . OverallVFAs are the primary products of rumen fermentation and contribute to rumen epithelium development in calves. Previous studies suggested that infusion of sodium propionate or sodium butyrate greatly promotes the development of the rumen papillae in calves ,42,87. SBranched-chain VFAs (BCVFA), such as isobutyrate, isovalerate and 2-methylbutyrate, are naturally derived from the catabolism of branched-chain amino acids. Adequate levels of BCVFA are essential for the growth of some cellulolytic bacteria and digestion of structural carbohydrates in the rumen ,92,93. SThere are many studies focused on evaluating the potential of plant extracts as alternatives to feed antibiotics and growth promoters in ruminant nutrition. Plant extracts have been shown to favorably affect rumen microbiota  and moduWeaning age can influence the development of rumen in pre-ruminants. For example, calves weaned at six weeks of age had longer and wider papillae compared to calves weaned at nine weeks of age . In earlIntensive feeding of milk or MR may decrease starter feed intake, thereby delaying rumen development  . Hence, To summarize, it is beneficial for rumen development for calves to be fed high-quality liquid feed rich in biologically active substances. Minimization of the use of soy protein or appropriate acidification of MR may alleviate gastrointestinal epithelium lesions. Feeding readily fermentable carbohydrates to calves to increase VFA production can stimulate rumen development. A pellet or texturized starter feed is superior to a finely ground meal. Providing calves with high-graded forage, such as alfalfa hay, can reduce the occurrence of rumen acidosis and papillae keratinization. Moreover, additives can be used in calf feed due to their potential advantages in rumen manipulation, however, the types and the optimal inclusion rate deserve further study. More importantly, there is no fixed pattern of calf feed. The diet compositions and nutrient specifications should be matched with the feeding program and management to better promote rumen development.The rumen is a unique part of the GI tract in ruminants. As the rumen develops and becomes colonized by microorganisms, a calf physiologically transitions from a pseudo-monogastric to a functioning ruminant. The development of rumen in calves can directly affect feed intake, nutrient digestibility and eventual growth of calves. Any changes in the early feeding regime and nutrition can influence rumen development, and thus, lead to long-lasting effects on subsequent growth, health, and milk production performance. Study by Moallem and colleagues (2010)  reportedAdditionally, an early feeding regime and nutrition can influence rumen development and rumen microbial composition, ultimately exerting an effect on the lifetime milk yield in cattle. Studies indicated that diets can modify the establishment of the bacterial community in lambs during weaning, which can persist for four to five months ,122. The


Current advanced transistor architectures, such as FinFETs and (stacked) nanowires and nanosheets, employ truly three-dimensional architectures. Already for aggressively scaled bulk transistors, both statistical and systematic process variations have critically influenced device and circuit performance. Three-dimensional device architectures make the control and optimization of the device geometries even more important, both in view of the nominal electrical performance to be achieved and its variations. In turn, it is essential to accurately simulate the device geometry and its impact on the device properties, including the effect caused by non-idealized processes which are subject to various kinds of systematic variations induced by process equipment. In this paper, the hierarchical simulation system developed in the SUPERAID7 project to study the impact of variations from equipment to circuit level is presented. The software system consists of a combination of existing commercial and newly developed tools. As the paper focuses on technological challenges, especially issues resulting from the structuring processes needed to generate the three-dimensional device architectures are discussed. The feasibility of a full simulation of the impact of relevant systematic and stochastic variations on advanced devices and circuits is demonstrated. Aggressively scaled transistors are affected by three kinds of process variations. Most frequently and since long discussed in the literature are statistical process variations which are caused by the granularity of matter, such as Random Dopant Fluctuations (RDF). However, as summarized earlier  a diversWhereas the impact of statistical variations such as Random Dopant Fluctuations (RDF), Metal Gate Granularity (MGG), and Line Edge Roughness (LER) have been frequently and since long discussed in the literature , thIn the following, the main processes available for the patterning of three-dimensional nanoscale transistors are discussed concerning their simulation and their variability, and a method to extract compact models which include both systematic and stochastic variations which may also affect the shape of the transistor is presented.This paper reports about results obtained in the cooperative project SUPERAID7 , funded Besides the physical properties of the semiconductor material used, which among others limit carrier mobility, the patterning of the transistors has increasingly become challenging. This is both due to the ever-smaller feature sizes needed and the complex three-dimensional geometries employed for nanoscale transistors in order to achieve good channel control. In the following, this development is outlined especially in view of process variations and design implications.For traditional bulk CMOS, mainly gates and their spacers had to be patterned. In corresponding simulations of lithography, etching, and deposition mainly, the resulting footprint of gate and spacer was relevant, because this defined the physical gate length L and width W. In turn, variations in these patterning processes led to variations of L and W, which could readily be considered during the extraction of compact models . In mostAnother important non-ideality which challenges aggressive patterning steps is the printing of defects. These include especially defects in the multilayer mirrors used for masks and instead of lenses in an Extreme Ultraviolet (EUV) lithography step, or defects of the mask structures . Equipme1·λ/NA, where k1 is the technology factor with minimum value of 0.25 for dense lines, λ is the wavelength of the light used, and NA is the numerical aperture, equal to the sine of the opening angle of the last lens times the refractive index of the medium between lens and wafer . With 193 nm being the minimum wavelength at which lenses are sufficiently transparent, a minimum half pitch for dense lines of about 34 nm results in case of water immersion lithography.Shrinking device dimensions have led to more complex device architectures in order to maintain sufficient electrostatic control of the channel, low leakage and high drive currents. In order to extend CMOS scaling to its limits, first buried oxide layers were introduced for the so-called SOI (silicon-on-insulator) transistors. This has been followed by transistor architectures such as FinFETs, (stacked) nanowires and (stacked) nanosheets. These small and truly three-dimensional structures lead to considerable challenges for the patterning processes and for their simulation. As outlined below, they also generate different problems in terms of variability and impact on design. The problem starts from the basic limitation of optical lithography, where the minimum half pitch printed is given by d = kThe favorite industrial approaches for the patterning of nanoscale devices smaller than that limit are various kinds of double or even quadruple patterning. In Litho-Etch-Litho-Etch (LELE)  and LithA different situation holds for Self-Aligned Double Patterning (SADP): Here, first a carbon layer is patterned by optical lithography. Then, spacers are created by deposition followed by back etching, see For current FinFET transistors, usually SADP is applied for the patterning of the fins as dense lines, whereas LELE is used for gate patterning, because it is more flexible in terms of the layouts.Another design issue appears in case of EUV. Here, soft X-rays with 13.5 nm wavelength are used instead of laser light. Because lenses are not sufficiently transparent for light below 193 nm, in EUV instead of refractive optics, reflective optics are used, consisting of multilayer mirrors and new absorber materials . WhereasIn summary, various kinds of systematic, layout-dependent and defect-induced variations influence the patterns generated in a lithography or multiple patterning process. There are major even qualitative differences in terms of variability between optical lithography, SADP and other double patterning processes. Etching and deposition, which are key components of SADP, are discussed in the next chapter.In semiconductor technology, a large variety of deposition and etching processes is employed. Deposition processes differ among others in terms of the properties of the deposited layers and the layer conformality. The latter for instance critically influences the filling of contact holes and trenches. Etching processes differ, e.g., in terms of selectivity concerning the materials to be etched, etch-induced damage, and the degree of isotropy and anisotropy. Equipment simulation tools such as CFD-ACE+  and Q-VTDeposition and etching processes are subject to several sources of systematic variability. Depending on the process in question, these include the inhomogeneity of gas flow and temperature in the etching or deposition reactor, the non-homogeneous emission of metal atoms, e.g., from a sputter target, or the finite size of a sputter target. These sources lead to variations of deposition and etching rates across the wafer. Furthermore, non-vertical incidence of contributing species can lead to layout-dependent deposition or etching rates, as discussed above for the SADP process. An overview of sources of variability in deposition and etching processes is given elsewhere .DEP3D and ANETCH allow the simulation of these deposition and etching processes, provided that physical/chemical models suitable for the process in question have been implemented, parameters have been calibrated, and the required boundary conditions above the wafer are known, e.g., from equipment simulation. As an example, As outlined above, advanced nanoscale devices generally employ truly three-dimensional geometries. Furthermore, the approximation of these structures using rectangular shapes is no more possible, because the electrical performance depends on details, such as taper angles of FinFETs or corner rounding. Moreover, as discussed above already basic features such as line width depend on the three-dimensional interim structures generated during the patterning flow, e.g., in the form of non-vertical and/or non-straight resist edges. In turn, one of the key tasks in the SUPERAID7 project has been the development of an integrated three-dimensional topography simulator, which combines Dr.LiTHO , ANETCH In terms of process simulation and systematic process variations, the SUPERAID7 project has focused on topography simulation and the variations affecting the topography processes lithography, etching, and deposition: These processes and variations raise new challenges especially for three-dimensional nanoscale transistors as addressed in SUPERAID7 and in this paper.For the simulation of the other process steps, especially ion implantation and dopant activation/diffusion, the well-established process simulator Sentaurus Process  has beenBecause conventional compact models are not suitable for the three-dimensional devices as studied in SUPERAID7, a novel compact model (LETI-NSP) based on surface potential has been developed by CEA/Leti and used for the compact model extraction work in SUPERAID7. This model has among others been presented at IEDM 2016 .A of the electrical device data is then given as:Within SUPERAID7 the hierarchical approach developed at GSS (now Synopsys) to extract statistical compact models  has beenHere, in general A is a vector, because various electrical properties are considered. As soon as more than one statistical variation occurs, also r is a vector. It should be noted that in case of more than one statistical variation, the components of r should be statistically independent. Otherwise it would be necessary to find the root cause of their dependence, and express this by relating them to some (hidden) parameters which cause the  correlation.Since one statistical variation may influence several process results , s(r) is generally a matrix. This also allows the proper description of correlations, which arise in the case that an input parameter r influences more than one  result parameter. The same holds for P.In examples studied earlier , proces1 which influence process corners s, and varying process parameters r2 which do not influence process corners s. The probability distribution of an electrical device performance parameter A is then given by1(r1) and f2(r2) are the probability distributions of the varying process parameters r1 and r2, respectively.The mixed case where both process corners s and statistically varying process parameters r are used in the extraction of the extended compact model must be treated with some care, because only variables can be used in the compact model extraction which do not depend on each other and can therefore be treated independently. In turn, it must be differentiated between varying process parameters rThe selection of the process corners and/or the varying process parameters to be considered in the extraction of the group I parameters see  depends Generally, several different systematic variations may influence a device. Tracing all their combinations in parallel through the full simulation of the process sequence would require far too much effort. In turn, suitable design-of-experiment approaches must be used to limit the number of simulation splits. A simple and efficient approach is to first disregard variations which are due to expert knowledge already known to have negligible impact. Next, the impact of the remaining variations should be considered one by one, to identify a small number of most important variations. These variations could then either be considered in parallel, or more refined design-of experiment techniques could be employed to skip some or several elements of the full matrix of simulation splits.Ge which influences the etching of the inner spacers; (2) the fin SADP deposition factor dsadp which gives the relative variation of the deposition thickness from the nominal thickness in the SADP spacer creation process; (3) the defocus FGate of the lithography step used to pattern the gate. Both for n-type NMOS and p-type PMOS MOSFETs, Fgate had the largest impact on both saturation current and leakage current.Ge is varied whereas dsadp and Fgate have their nominal values. The V-shape for the impact of Fgate is an intrinsic feature of the dependence of CD on defocus in lithography, discussed above in connection with sadp a technologically appropriate variation was assumed. Its impact was, however, smaller than the impact of numerical discretization errors. In turn, in further work, a larger variation will be assumed here for the extraction of the compact model, whereas the realistic value can and will then be introduced via the probability distribution f(dsadp) in Equation (2). Based on these variations, the extended compact model was extracted, using the Mystic [Ge and the final extraction of the statistical compact model, as indicated in Coupled process and device simulation was then carried out for the process corners of these three statistical variations. e Mystic  tool froEspecially for three-dimensional nanoscale transistors, the details of the geometries generated during device fabrication are important both for the nominal device performance and for its stability against systematic variations caused by process equipment and/or by layout effects. Other than for statistical variations which can be simulated with stand-alone statistical device simulation, there is a huge variety of potential systematic variations, which must be addressed with various equipment simulation tools and process simulation modules. In turn, it is indispensable for optimized device and circuit manufacturing to well identify and characterize the sources of systematic variability, to sort out those with the largest impact, depending on the device and circuit in question, and to quantify their joint impact on the final nanoelectronic product. The impacts of systematic and statistical variability must be simulated and minimized in parallel, by adapting process flows, but potentially also device architectures and even fabrication equipment. In this paper, a holistic approach to use coupled equipment, process and device simulation combined with the extraction of variation-aware compact models has been presented together with some examples. The approach is partly based on well-established tools used in industry, partly on additional modules and extensions which are compatible to such standard frameworks. The suggestions made are intended to support industry in their efforts to optimize the stability of their products against all kinds of process variations.


Troponin is a key regulatory protein in muscle contraction, consisting of three subunits troponin C (TnC), troponin I (TnI), and troponin T (TnT). Calcium association to TnC initiates contraction by causing a series of dynamic and conformational changes that allow the switch peptide of TnI to bind and subsequently cross bridges to form between the thin and thick filament of the sarcomere. Owing to its pivotal role in contraction regulation, troponin has been the focus of numerous computational studies over the last decade. These studies elegantly supplemented a large volume of experimental work and focused on the structure, dynamics and function of the whole troponin complex, individual subunits, and even on segments of the thin filament. Molecular dynamics, Brownian dynamics, and free energy simulations have been used to elucidate the conformational dynamics and underlying free energy landscape of troponin, calcium, and switch peptide binding, as well as the effect of disease mutations, small molecules and post-translational modifications such as phosphorylation. Frequently, simulations have been used to confirm or explain experimental observations. Computer-aided drug discovery tools have been employed to identify novel potential calcium sensitizing agents binding to the TnC-TnI interface. Finally, Markov modeling has contributed to simulating contraction within the sarcomere on the mesoscale. Here we are reviewing and classifying the existing computational work on troponin and its subunits, outline current gaps in simulations elucidating troponin's role in contraction and suggest potential future developments in the field. Troponin (Tn) is a three-subunit protein complex that resides on the thin actin filament in muscle cells. Its three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT) have separate roles in facilitating muscle contraction  simulations and helped elucidate the functional importance of these motions. Molecular dynamics numerically integrates Newton's equations of motion and allows for the simulation of trajectories of biomolecular atoms and molecules  and fast-skeletal troponin C (sTnC) have been simulated using molecular dynamics. The Li group simulated a full troponin complex and demonstrated that the inter-linker region of sTnC was flexible in simulations, in contrast to what the static model suggested  and dilated (DCM) cardiomyopathy. A key use of MD is to study the dynamics of the cTn complex and its subunits in the presence of these mutations. HCM- and DCM-associated mutations that exist on the regulatory domain of cTnC have been in the focus of various conventional MD simulations. Early short simulations were run for 5 ns on the DCM-associated mutation D75Y in calcium-free cNTnC and showed that the D75Y mutation would lead to a reduction in contraction through stabilization of the closed state  on clusters derived from MD simulations were performed on cNTnC. This technique was able to identify a calcium-sensitizing compound, NSC147866, from the NCI II diversity set , challenging simulations to correctly account for those differences. In general, design of additional computational experiments that are verifiable All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Maternal diabetes is one of the most common and dangerous risk factors during pregnancy, as often there are no generalized signs. Diabetic fetopathy is a severe, poorly defined complication of gestational diabetes or preexisting maternal diabetes mellitus, with an ill-defined histological spectrum of changes. Herein we report a case of severe diabetic fetopathy diagnosed upon autopsy of a recently miscarried fetus. On histology, the liver revealed severe generalized macrovesicular steatosis and number of small cysts. The pancreas revealed not only Langerhans isle hyperplasia, but also Langerhans amyloidosis, evident of the severity of maternal diabetes and fetal hyperglycemia. The adrenal glands revealed hyperplasia in zona glomerulosa, due to aldosterone overproduction, evident of fetal hypertension. The current case is an extreme example of an undiagnosed and untreated gestational diabetes mellitus. The severity of histological changes, in this case, is suggestive of further extension of the diagnostic criteria of diabetic fetopathy to include more subtle changes that can be observed clinically and even a combination of maternal-newborn factors. Diabetic fetopathy is a severe fetal complication of gestational diabetes or preexisting maternal diabetes mellitus ,2. The cThe range of histological changes is ill-defined as with the stabilization of the condition of the fetus postpartum, most of them tend to regress ,4,5.Herein we report a case of severe diabetic fetopathy diagnosed upon autopsy of a recently miscarried fetus.A 40th gestational week female miscarried fetus was presented for autopsy in the Department of General and Clinical Pathology, St. Marina University Hospital, Varna, Bulgaria. The mother, 23 years old and otherwise healthy, had one previous pregnancy which concluded with elective abortion. There was no rhesus factor incompatibility. Prior to the miscarriage, the mother had not felt any movement of the fetus for 24 hours. Clinically during the miscarriage, a wrapped and tightened umbilical cord was observed around the neck of the fetus. The autopsy was carried out 24 hours after the miscarriage, a total of 48 hours after the fetal death, no placental specimen was sent for investigation.Upon observation, the fetus was with skin maceration and epidermal detachment from the dermal layer of 70% of the body surface and pathognomonic facial features Figure . The fetOn histology, the liver revealed severe generalized macrovesicular steatosis and a number of small cysts Figure . The panUpon request of additional medical history of the mother, no preexisting diabetes or signs of diabetes were established prior to the pregnancy. However, upon admission for the miscarriage her blood sugar levels were 17 mmol/l and within the following week dropped to 10 mmol/l, without anti-diabetic treatment.Based on the clinical and pathological findings the cause of death was determined as diabetic fetopathy with macrosomia, complicated with fetal malrotation and umbilical cord wrapped around the neck, resulting in intrauterine asphyxia.Maternal diabetes is one of the most common and dangerous risk factors during pregnancy, as often there are no generalized signs. Gestational diabetes is defined as a new onset of impaired glucose tolerance developing during pregnancy as a result of insulin resistance . ScreeniThe current case is an extreme example of an undiagnosed and untreated gestational diabetes mellitus. The changes in the fetal internal organs, such as the Langerhans amyloidosis and the macrovesicular hepatosteatosis, are normal complications of long-lasting diabetes and have so far not been reported in diabetic foetopathy and are evident of the range of fetal changes and fetal impairment of glucose and triglyceride metabolism . In addiThe severity of histological changes in this case is suggestive of further extension of the diagnostic criteria of diabetic fetopathy to include more subtle changes that can be observed clinically and even a combination of maternal-newborn factors, such as blood sugar levels measured every day for the first week and compared between the mother and fetus to establish a dynamic, as well as a similar dynamic with blood pressure, liver enzymes, low-density lipoproteins (LDL), high-density lipoproteins (HDL), potassium, uric acid, creatinine, urea and even pro-inflammatory adipokines.Such tests with the establishment of maternal-newborn dynamics would be helpful in determining even the slightest postpartum changes and evading further complication to the fetus.As for histological criteria for the diagnosis of diabetic fetopathy, an argument should be made that the changes sought in miscarried fetuses and deceased newborns should be the same as those sought after in adult patients with long-lasting diabetes.The present case illustrates the wide range of severe fetal changes in undiagnosed and untreated maternal diabetes. Although such severe changes rarely develop, their correlation with clinically observed phenomena, suggests the need for expansion of maternal monitoring in cases of gestational diabetes and introduction of new markers and maternal-fetal correlation of such markers postpartum.


Several molecular technologies aimed at regulating gene expression that have been recently developed as a strategy to combat inflammatory and neoplastic diseases. Among these, antisense technology is a specific, rapid, and potentially high-throughput approach for inhibiting gene expression through recognition of cellular RNAs. Advances in the understanding of the molecular mechanisms that drive tissue damage in different inflammatory diseases, including Crohn’s disease (CD) and ulcerative colitis (UC), the two major inflammatory bowel diseases (IBDs) in humans, have facilitated the identification of novel druggable targets and offered interesting therapeutic perspectives for the treatment of patients. This short review provides a comprehensive understanding of the basic concepts underlying the mechanism of action of the oligonucleotide therapeutics, and summarizes the available pre-clinical and clinical data for oligonucleotide-based therapy in IBD. The two major therapeutic approaches in this field are the antisense oligonucleotides (ASOs) that inhibit mRNA translation and the oligonucleotides, which function  pathway . Synthet pathway .In the last decades, the advent of new techniques of molecular and cellular biology has advanced our understanding of the factors/mechanisms that promote tissue damage in several chronic inflammatory diseases, including Crohn’s disease (CD) and ulcerative colitis (UC), the two major inflammatory bowel diseases (IBDs) in humans . This haAn antisense oligonucleotide (ASO) is a single-stranded deoxyribonucleotide, which is complementary to the mRNA target. The goal of the antisense approach is the downregulation of a molecular target, usually achieved by induction of RNase H endonuclease activity that cleaves the RNA-DNA heteroduplex with a significant reduction of the target gene translation . Other AThe recent developments in the human genome sequencing, the possibility of a rational design of oligonucleotides and the theoretical simplicity, and relatively cheap costs of these compounds led to their use as either therapeutic agents or tools for assessing gene function. Although, the researchers usually select the ASO candidate by testing the activity of few oligonucleotides that specifically regulate the target gene expression, it would be recommendable to identify the ideal ASO through an accurate evaluation of a panel of putative oligomers . It is cSince in their “naïve” form, ASOs could be rapidly digested, thus limiting their bioavailability , most ofIncreased ASO binding affinity and biostability have also been obtained using oligonucleotides with ribose modifications [i.e. substitution of the hydrogen at the 2-position by an O-alkyl group and locked nucleic acid technology (LNA)] that reduce conformational plasticity . HoweverASOs have been already used in various human pathologies. For instance, in 1998, FDA approved the use of fomivirsen, a compound that inhibits the translation of the mRNA encoding for the major immediate early region proteins of cytomegalovirus, for the treatment of cytomegalovirus-induced retinitis . In 2013IBD are chronic, immune-mediated diseases of the gastro-intestinal tract, which are characterized by tissue damage and development of local and extra-intestinal lesions . One of In inflamed gut of CD patients and UC patients, there is an enhanced expression of ICAM-1, a transmembrane glycoprotein constitutively expressed on the surface of intestinal epithelial cells and vascular endothelial cells . KnockdoAn alicaforsen-containing enema formulation was developed for patients with UC or patients with pouchitis, an inflammatory condition of the ileal pouch reservoir, which can develop in UC patients undergoing colectomy and ileal pouch-anal anastomosis. In mild to moderate left-sided UC patients, alicaforsen enema had no significant effect on the course of the disease . AfterwaNF-κB is a transcription factor composed of two proteins (p50 and p65) regulating the expression of many inflammatory and anti-inflammatory genes . It was IBD is believed to be triggered by complex interactions among host genetic susceptibility and many environmental factors, which lead to a sustained activation of inflammatory pathways and defects in counter-regulatory mechanisms in the gut . In inteLater on, a pharmaceutical compound, which contains a Smad7 ASO targeting the RNA encoding by the 107–128 DNA region, was developed for CD therapy. The drug, named mongersen , , was forAnother strategy to inhibit the expression of mRNA is represented by RNAi. RNAi is mediated by many endogenous RNAs . Once iAnother strategy is to conjugate the silencing RNAs with ligands of the target molecule. An example is the addition of N-acetylgalactosamine  to the RNA, thus enhancing asialoglycoprotein receptor (ASGR)-mediated uptake into liver hepatocytes . The majThe late stage of inflammation in IBD is characterized by the fibrotic process, which derives from an altered balance between matrix deposition and degradation . CarbohyThe rationale for the use of antisense-based therapies in IBD is supported by the benefit seen in preclinical models and initial clinical studies, together with the safety profiles of the compounds. Unfortunately, however, large clinical trials have not confirmed the promising results obtained with ASOs in preclinical models. Although, it is unclear why these treatments failed in patients, it is conceivable that some factors either related to the target or route of administration may have contributed to these negative results. For example, the negative results of alicaforsen can, in part, rely on the fact that ICAM-1 is just one of the various molecules involved in leukocytes trafficking, and therefore, even in the absence of ICAM-1, other integrins could promote recruitment of activated leukocytes in the gut. Another possibility is that systemic administration of ASO could be not ideal for allowing optimal concentration of the drug within the gut tissue, where there is the main expression of the target. This hypothesis is supported by the demonstration that rectal administration of alicaforsen is of benefit in patients with distal UC and in patients with pouchitis. While STNM01 is the only siRNA currently tested in IBD, there is sufficient evidence to believe that RNAi technology can represent a new and valid approach to regulate the expression of disease-related genes. Some issues in the design and development of these compounds, such as correct identification of target mRNAs, stability, and delivery to the site of interest remain to be solved.All datasets generated for this study are included in the manuscript.DF and VD wrote the paper. MF, IM and BR contributed to supervise parts of the paper. GM designed and drafted the paper.GM has filed a patent related to the treatment of IBD with Smad7 antisense oligonucleotides.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Microsystems with limited power supplies, such as electronic skin and smart fuzes, have a strong demand for self-powered pressure and impact sensors. In recent years, new self-powered mechanical sensors based on the piezoresistive characteristics of porous electrodes have been rapidly developed, and have unique advantages compared to conventional piezoelectric sensors. In this paper, in order to optimize the mechanical sensitivity of porous electrodes, a material preparation process that can enhance the piezoresistive characteristics is proposed. A flexible porous electrode with superior piezoresistive characteristics and elasticity was prepared by modifying the microstructure of the porous electrode material and adding an elastic rubber component. Furthermore, based on the porous electrode, a self-powered pressure sensor and an impact sensor were fabricated. Through experimental results, the response signals of the sensors present a voltage peak under such mechanical effects and the sensitive signal has less clutter, making it easy to identify the features of the mechanical effects. In recent years, self-powered mechanical sensors have attracted wide attention for their applications in wearable smart devices and intelligent microsystems with limited energy supply ,2,3,4,5.Traditional self-powered mechanical sensors applied in measurements of static pressure and dynamic impact are mainly piezoelectric sensors ,11,12, sIn recent years, the dual functions of energy storage and piezoresistive sensitivity in porous electrodes, such as activated carbon and carbon nanotubes, have been investigated , and havFor these self-powered mechanical sensors, the material properties of porous electrodes have a decisive influence on their energy storage density and mechanical sensitivity ,20,21. HIn this paper, an enhancement method for pressure sensitivity of porous electrodes is proposed. An elastic polymer is added to the porous energy storage material, which forms a spatial network skeleton structure at the microscopic level by a shearing process. On the one hand, the micro-elastic skeleton structure can improve the elasticity of the electrode, so that the electrode can withstand greater stress; on the other hand, the deformation effect of the elastic polymer skeleton under mechanical effects results in a more significant change in the contact state between the conductive particles, and thus enhances the mechanical sensitivity of the electrode. Finally, based on the proposed high elastic piezoresistive film, a self-powered mechanical sensor was fabricated, and its superior sensing performance was experimentally verified. In general, the preparation process of the porous electrode proposed in this paper achieves effective enhancement of pressure sensitivity, and promotes practical applications of the self-powered impact sensor, based on the piezoresistive effect of the porous electrode.As shown in On the one hand, the porous electrode enables energy storage and provides energy supply during impact, as shown in On the other hand, the mechanical sensitivity of the device is achieved due to the piezoresistive effect of the porous electrode. As shown in Therefore, enhancing the piezoresistive performance of porous electrodes is the key to realizing self-powered mechanical sensors, and the enhancement can be achieved through the optimization of the micro-morphology of the porous electrode. Previous literature  has studMix activated carbon powder and a binder powder  evenly by concussion and grinding. The mass ratio of the activated carbon powder and the binder powder is 85:15.The mixed powder is lightly pressed under high temperature heating F(ω)−The porous carbon electrode has a small equivalent stiffness coefficient and a large viscous damping coefficient , which can effectively suppress high frequency components in the response signal according to Equation .This paper presents a preparation process that can enhance the piezoresistive characteristics of porous electrodes. The experimental results show that by applying a shearing force along the surface of the electrode film, the binder is stretched to form a microscopic network skeleton structure. This has an envelop effect on porous particles such as activated carbon, and significantly enhances the piezoresistive characteristics and elasticity of the electrode. In addition, by adding a highly elastic rubber component, another more robust microscopic network skeleton structure is formed by the rubber under the shearing process, and a flexible porous electrode with superior piezoresistive characteristics and elasticity can be more effectively obtained. Based on such porous electrodes, both a self-powered flexible pressure sensor and a self-powered impact sensor were realized, with response signals of voltage peaks under pressure and high acceleration impact stimuli. Moreover, the sensors have superior ability to recognize features of mechanical processes, such as the number of consecutive pressing processes. These superior self-powered sensing characteristics provide new technological approaches for microsystems with limited power sources, such as electronic skin and smart fuzes.


Cancer mortality rates are undergoing a global downward trend; however, metastasis and relapse after surgery and adjuvant treatments still correlate with poor prognosis and represent the most significant challenges in the treatment of this disease. Advances in genomics, metabolomics, and proteomics are improving our understanding regarding cancer metabolic diversity, resulting in detailed classifications of tumors and raising the effectiveness of precision medicine. Likewise, the growing knowledge of interactions between nutrients and the expression of certain genes could lead to cancer therapies based on precision nutrition strategies. This review aims to identify the recent advances in the knowledge of the mechanistic role of bioactive phytochemicals in foodstuffs in tumor progression, metastasis, and chemo-resistance in order to assess their potential use in precision nutrition therapies targeting relapse in lung, breast, colon, and prostate cancer, and leukemia. A considerable number of bioactive phytochemicals in foodstuffs were identified in the literature with proven effects modulating tumor growth, progression, and metastasis. In addition, the use of foodstuffs in cancer, and specifically in relapse therapies, is being reinforced by the development of different formulations that significantly increase the therapeutic efficiency of these products. This can open the possibility for testing combinations of bioactive phytochemicals with cancer relapse treatments as a potential prevention strategy. Lung, breast, prostate, and colon cancers have the highest overall incidence and represent 36.4% of the total diagnosis according to GLOBOCAN 2018, a report that gathers data from the International Agency for Research on Cancer of the World Health Organization [In the last five years international scientific production related to new basic and translational discoveries on cancer has grown by 5% annually. Lung, colon, breast, prostate, and leukemia cancers account for 51% of scientific publications on cancer in this period . Many papers address the association between gene regulation and cancer progression or metastasis, still representing the main approach in cancer research. More recently, an increasing number of investigations based on genomics and epigenomics are focusing on the improvement of tumor classification, the relationship between cancer and microbiota, or the discovery of new therapeutic targets in metabolism, in order to develop specific therapies in the context of precision medicine.Survival is the foremost concern in lung cancer due to its progressive metastasis and resistance to therapy and, furthermore, the molecular hallmarks of its malignant properties are still insufficiently known. Recently, a 17-gene panel involving critical cellular processes such as hypoxia-mediated epithelial-mesenchymal transition (EMT) or epigenetic modifications was identified as a potential biomarker tool for prediction of metastasis and prognosis in patients affected by non-small-cell lung cancer (NSCLC) . Other eRegarding breast cancer, tumor heterogeneity makes patient classification according to their risk of metastatic relapse essential in order to guide decisions in adjuvant chemotherapy. However, in the era of personalized medicine, genomics alone does not provide enough knowledge to apply precision treatments to specific tumors. Therefore, complementary multi-omic studies are emerging as the most promising approaches addressing breast cancer complexity . For insThe application of new technologies now allows progressing in the understanding of the molecular basis of prostate cancer pathogenesis. Microarray-based transcriptomic analyses and next generation sequencing technology are examples of powerful tools for studying gene expression and changes in gene structure at the transcript level. Recent publications described new mechanisms regarding tumor suppressors in this type of tumors  and new Deregulated cellular energetics is another of the hallmarks of cancer. In this sense, cooperative lipid metabolism-related genes involved in colorectal cancer progression have been recently identified. The acyl-CoA synthetase/stearoyl-CoA desaturase (ACSL/SCD) lipid network fuels migratory and invasive properties through EMT induction and is associated with an increased risk of relapse in colorectal cancer patients . Other mRecent advances in leukemia genomics and epigenomics have facilitated the study of clonal populations and their genetic-epigenetic evolution, changing the classic view of leukemia into a complex heterogeneous disease aggravated by clonal evolution ,16. ThesCancer relapse worsens the prognosis of patients and is a factor that contributes significantly to mortality. However, only 2% of cancer publications in the last five years deal specifically with relapse . Cancer recurrence involves many biological interactions, such as genetic, transcription, environmental, endocrine signaling, and metabolism. These interactions add another layer of complexity in the understanding of cancer recurrence and metastasis, delaying progress in therapeutic opportunities . Lung caLung cancer has a recurrence rate that reaches 50% of patients. Despite having noteworthy advances in recent years on the knowledge of the disease biology and mechanisms of tumor progression, the survival rates remain low and more research is necessary on molecular aspects that give rise to more effective therapies . DiscovePRPS1), encoding a purine biosynthesis enzyme, were associated a few years ago with chemotherapy resistance and relapse [NT5C2) has recently been described regarding clonal evolution and chemotherapy resistance, with important implications in the development of new targeted inhibition therapies [In respect of leukemia, the high number of relapse-specific mutations acquired by patients affecting genes involved in different functions, leading to clonal evolution and resulting in chemotherapy resistance, points to the high molecular complexity of this process ,25. Unde relapse . In addiherapies ,28.In essence, the recent discoveries in relation to drug resistance and previously unknown molecular mechanisms associated with metastasis and recurrence demand alternative therapeutic strategies, among which metabolism could emerge as a new cancer therapy support. Although cancer relapse is due to a multitude of genetic mutations and biochemical processes, advances in genomics, metabolomics, and proteomics allow better understanding of the metabolic diversity due to genetics and microbiome variation, as well as a detailed classification of tumors, which provide precision medicine with individual treatments. Likewise, the best understanding of the metabolic variation allows us to know the interactions between nutrients, metabolism, microbiota, and related genes, facilitating the development of adjuvant cancer therapies based on precision nutrition strategies .Over the years, numerous epidemiological studies have been carried out to link the diet with cancer, either from a preventive approach or by associating the consumption of certain food products with tumor generation and growth. However, in parallel to the development of precision therapies in medicine, precision nutrition is an emerging science that relies on well-established factors such as genetic and epigenetic variation  and the Epidemiological studies triggered further research in terms of molecular mechanisms, which have significantly improved the effectiveness of phytotherapy, entering the context of precision nutrition . Recent The biological activity of food polyphenols, a broad family of compounds with representatives in virtually all foods, has been specially studied for decades since they have in common an intense antioxidant activity that suggests other potential health outcomes, for example in breast cancer . RecentlThe objective of this review is to identify the recent advances in the knowledge of mechanisms that support the effects of bioactive phytochemicals in foodstuffs associated with molecular targets of tumor progression, metastasis, or chemo-resistance, in order to assess the potential use of these products in precision nutrition therapies addressing the prevention of relapse in lung, breast, colon, and prostate cancer, and leukemia.A literature search was carried out on September 30th 2019 in Web of Science using the following databases: Web of Science Core Collection (WOS), Current Contents Connect (CCC), Derwent Innovations Index (DIIDW), Korean Journal Database (KJD), MEDLINE, Russian Science Citation Index (RSCI) and Scientific Electronic Library Online (SCIELO). In order to capture only the most recent advances, articles published between 2017 and 2019 were considered. The language filter was English. The same search strategy was carried out in SCOPUS. PUBMED was not used since it is a search engine in MEDLINE, already included in WOS.Search a strategy included three layers of keywords: (1) types of cancer targeted; (2) the most studied bioactive foodstuffs in the literature cited in the introduction, together with keywords indicating the characteristic factors of precision nutrition; (3) keywords identifying cancer relapse trying to cover all synonyms. In order to open the search, asterisk-terminated keywords were introduced. The search for the first indicator (the five target types of cancer) was performed on the “title” field of the search engine. However, the search for the second indicator, the one related to bioactive foodstuffs and the third one related to cancer relapse and its synonyms, were searched in the “topic” field, which includes title, keywords, and abstract; in order to include all types of research approaches. The keywords were combined using boolean operators “AND”, “OR”. The detailed search strategy is presented in The first field included general descriptors of cancer and its scope before “AND”. The second part identified the five types of cancer that are the subjects of this review. The first part of the second field included keywords on bioactive foodstuffs associated in the literature to positive effects on tumor progression or metastasis: Phytochemicals and plant extracts, phenolic compounds of various chemical structures, omega-3 polyunsaturated fatty acids (mainly docosahexaenoic acid (DHA)), and curcuminoids that are being studied in the field of cancer therapies in recent years ,56. The Publications that met the criteria of the search strategy were downloaded in full text and reviewed by the authors. Articles referring to dietary indices for prognosis, epidemiological studies, articles without identification of the specific molecules of the products studied, articles related to food groups and all those not containing a mechanistic explanation of the effect of the bioactive studied, were excluded.In the initial screening of titles, abstracts, and keywords, no records were excluded since the 303 identified papers met the search criteria. However, the strict evaluation of the full-text articles revealed that most of them did not contain adequate studies for the purpose of this review. Consequently, just over 10% of the papers identified were included for qualitative synthesis. Only research papers including molecular mechanistic knowledge were accepted, according to the objective of this review. Nevertheless, certain research on well-characterized bioactive extracts of plant, fruits, vegetables were also accepted although they did not include new mechanistic contributions.Twenty of the original research studies finally selected for qualitative analysis dealt with polyphenol foodstuffs, and three with lipids, mainly omega-3 and more specifically DHA. In addition, 12 articles on bioactive plant extracts were identified. All compounds in Regarding research on anticancer effects of food extracts, the knowledge of mechanisms of action is far more limited compared to polyphenol foodstuffs, due to the integration of several compounds in each extract. Despite this, they have been considered in this study since they may tentatively constitute ingredients of nutritional supplements for the treatment of different cancers. The inclusion of natural bioactive foodstuffs in therapies for different types of cancers is increasingly being accepted in the clinical setting. Potentiating the use of these bioactive compounds in cancer treatment requires improving its bioavailability. Therefore, a significant activity formula development is being carried out in this regard. In this sense, the case of curcumin stands out, since it is one of the most studied bioactive foodstuffs in terms of its potential anticancer effect. Various curcumin nano-preparations have recently been developed. Formulations aiming to increase the bioavailability of bioactive natural foodstuffs are essential for the application of these products in cancer therapies since, as we have seen, most of the phytochemicals reviewed in this work are generally hardly bioavailable polyphenols. Development of new nano-formulations of bioactive foodstuffs is key to widespread use of these products in cancer therapy.Since the same factors that are successfully driving precision medicine in cancer serve to design precision nutritional therapies, it is foreseeable that a new era in the treatment of cancer can be opened in coming years. Integration of nutritional strategies may be of special interest for patients treated with adjuvant therapy, in order to enhance therapy effects and to prevent cancer relapse. The knowledge regarding several bioactive foodstuffs mechanisms of action, and the fact that most of them address well established molecular targets, allows the transition into a potential clinical use. In the last decade, a growing number of research works have investigated the anticancer effects of bioactive natural products. However, only very recently have the molecular mechanisms by which nutrients may prevent relapse been explored.HSPA5, SEC61B, G6PD, HMOX1, and PDE3B, affecting essential pathways like DNA replication or the cell cycle. On the other hand, the synergy of curcumin and oligomeric proanthocyanidins emerges as an opportunity to develop effective therapies, since both compounds share similar molecular mechanisms [Curcumin is another bioactive food product that has been the subject of former numerous studies. Its anti-cancer effects, due its ability to modulate critical anti-apoptotic effectors such as Bcl-xl and NF-κB, are of prominent interest in potential relapse prevention treatments. Moreover, curcumin synergizes with vesicular stomatitis virus (VSV)-based oncolytic treatments modulating antiviral responses and components of the intrinsic apoptotic pathway in a prostate cancer cell model . Furtherchanisms . Recentlchanisms .d-glucoside has inhibitory effects on breast cancer cells growth [Some lipid character food bioactives also show anti-cancer effects with mechanisms of action interesting for cancer relapse prevention. In this sense, docosahexaenoic acid (DHA) modulates the growth of colorectal cancer cells and induces expression of genes related to apoptosis ,64. Alsos growth .The studies mentioned above explain many of the effects on cell growth, tumor progression and metastasis of food bioactive products, opening promising opportunities for the application of these phytochemical foodstuffs in therapies for cancer relapse in breast, prostate, lung, and colon cancer. However, no studies of individual food products were found that applied to leukemia relapse. Regarding this type of cancer, only one paper was recovered, suggesting that additional research should be made in this sense. The cited article analyses the effects of a ginger extract rich in gingerols, demonstrating that this extract exerts a synergistic interaction with methotrexate with high antiproliferative impact in the drug-resistant leukemic sub-lines . NeverthOne of the barriers to be overcome in the application of bioactive phytochemical foodstuffs in cancer therapies is the frequent lack of bioavailability of these products. In this sense, new formulation strategies are being developed, such as bioactive carrier lipids in which the carrier not only increases bioavailability, but also provides a synergistic biological activity with the active ingredient. Interestingly, it has been reported that the use of shark liver oil rich in alkylglycerols as a bioactive lipid vehicle for rosemary extract shows synergistic effects in the expression of genes associated with immune modulation, inflammation, oxidative stress, lipid metabolism, and tumorigenesis in colorectal cancer . Besidesd-glucoside lipids. Among the targeted cancers included in this review, lung and colon cancer are being widely studied, and prostate and breast cancer have concentrated the largest number of applications. However, there is a great lack of studies related to the use of phytochemical foodstuffs in leukemia therapies and more studies are needed in this disease. The possibilities of using foodstuffs in cancer treatments and more specifically anti-relapse therapies, are being reinforced with the development of formulation technologies that significantly increase the efficiency of these products.Although, in the context of cancer research, studies that refer to nutritional therapies based on the use of bioactive foodstuffs in adjuvant treatments are still limited, current results are encouraging since there are several phytochemical bioactive foodstuffs with proven modulating effects of tumor growth, progression, and metastasis, and therefore can be tested in humans with a reasonable probability of success if they are applied in cancer relapse treatments . In thisCancer heterogeneity is one of the features of this disease that makes its treatment particularly challenging. Innovative therapies for patients are continuously being tested and progress has been made in the last years developing early diagnosis protocols and improving patient prognosis. However, despite these efforts, a high percentage of patients relapse after surgery or initial therapy. Cancer relapse involves a great number of different molecular mechanisms that vary form one patient to another. This points to precision medicine as a key element to personalize cancer treatment and prevent relapse, and suggest the value of new effective and safe compounds that potentiate the effects of already-known chemotherapy agents. In this sense, the growing number of studies regarding the mechanisms of several bioactive foodstuffs in the treatment of different types of cancer, open a new layer in precision cancer therapy. Its association with specific genetic targets or different molecular pathways inhibiting tumor growth and metastasis constitutes an important customization component. Moreover, it shows high synergism with several chemotherapy drugs, acting as enhancers of these anti-tumor effects or even sensitizing and reverting chemotherapy resistance. In essence, these products are emerging as novel complementary agents that can be useful in precision nutrition therapies addressing relapse prevention in treatments of cancerous processes.


People living in conflict-affected areas incur physical and psychological trauma. They are also vulnerable to disease outbreaks and disruptions in the supply of food and water, medicines and health services. High-quality, accessible medical care is very difficult to provide in such settings. The Geneva Conventions hold warring parties accountable for the provision of care to civilians and combatants. However, these parties may not be willing or able to provide medical assistance to conflict-affected populations. Recent major conflicts, such as in Mosul, Iraq and Al Raqqa, Syrian Arab Republic, have moved health and humanitarian assistance closer to the frontline and challenged the risk-averse approach of traditional responders.Accountability to affected populations, as defined by the Inter-Agency Standing Committee,,The recent experiences of humanitarian responders show that operational guidance on the provision of trauma care in conflict settings is needed. These experiences also constitute valuable lessons, from which best practices can be identified and future responses improved. An assessment of response in Mosul found that the “WHO-coordinated efforts helped address critical needs in the provision of trauma care for wounded civilians and saved lives.”Most research on trauma care in conflict settings has been done in the context of symmetric warfare, when humanitarian agencies have equal access to all warring parties and the wounded, and where the military is the main provider of care. This research led to changes in care provision at the frontline, where the risk of functional impairment is usually highest. Rapid evacuation from the location of injury to the care facility is needed to save lives and reduce disabilities. Despite hard-won experience gathered by many military, United Nations, nongovernmental, civilian and humanitarian actors, there is very limited research on health-care provision and civilian protection in asymmetric warfare, where humanitarian agencies have no access to one or more warring party.Bulletin of the World Health Organization encourages submissions to its research, policy-and-practice and lessons-from-the-field sections to shape policy and improve health outcomes in conflict settings.To build the evidence base for future responses, the